Herbal

Drug Technology

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 Herbal
Drug Technology
SECOND EDITION

S S Agarwal
PhD (AIIMS), DSc (RGTU), FIPS, FIC, FISHR
Professor (Pharmacology) and Director, Amity Institute of Pharmacy
Pro Vice Chancellor and
Director General (Academic, Innovation and Research Coordination)
Amity University, Uttar Pradesh

M Paridhavi
M Pharm, PhD, FABP
Principal, Rajiv Gandhi Institute of Pharmacy
Kerala
HERBAL DRUG TECHNOLOGY

Universities Press (India) Private Limited

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© Universities Press (India) Private Limited 2007, 2012

First published 2007
Reprinted 2009
Second edition 2012

eISBN 978-81-7371-789-5

e-edition: First Published 2017

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 Contents

Cover
Title Page

Second Message
First Message
Foreword
Preface to the Second Edition
Preface to the First Edition
1 Introduction to Medicinal Plants
1.1 Illustrative Examples of Plants Used as Drugs
2 Indian Systems of Medicine
2.1 Ayurveda
2.2 Unani System of Medicine
2.3 Homeopathic System of Medicine
2.4 Siddha
2.5 Yoga and Naturopathy
3 Herbal Therapeutics: From Ancient Times to the 21st Century
3.1 The Ancient World
3.2 The World of Greece and Rome
3.3 The Long Interregnum
 3.4 Twentieth Century
3.5 Prospects for the Twenty-first Century
4 Essentials of Crude Drugs
5 Medicinal Botany
5.1 Plant Taxonomy
5.2 Morphological Considerations of Crude Drugs
5.3 General Considerations
5.4 Histology
6 In vitro Culture of Medicinal Plants: Tissue Culture
6.1 Requirements for Plant Tissue Culture
6.2 Setting Up a Tissue Culture Lab
6.3 Basic Laboratory Procedures
6.4 Processing of Plant Tissue Culture
6.5 Growth Profile
6.6 Growth Measurement
6.7 Plant Tissue Culture Methods
6.8 Root Culture
6.9 Shoot Tip Culture and Leaf Culture
6.10 Culture of Flowers, Ovaries and Ovules
6.11 Seed Culture
6.12 Nucellus Culture
6.13 Embryo Culture
6.14 Anther and Pollen Culture
6.15 Callus Culture
6.16 Types of Tissue Culture
6.17 Tissue Culture of Medicinal Plants
 6.18 Applications of Plant Tissue Culture
7 Systematic Examination of Powdered Drugs
7.1 Examination of Organised Drugs
7.2 Examination of Unorganised Drugs
Part 8.1 Application of Chromatography and Spectroscopy in Plant
Drug Analysis
8.1 Chromatography
8.2 Infrared Spectroscopy
8.3 Ultraviolet Spectroscopy
8.4 Differential Scanning Calorimeter
8.5 NMR Spectroscopy
8.6 Mass Spectroscopy
8.7 Spectral Properties of Herbal Products
Part 8.2 Extraction, Isolation and Analysis of Phytopharmaceuticals
8.8 Infusion
8.9 Decoction
8.10 Digestion
8.11 Maceration
8.12 Percolation
8.13 Successive Solvent Extraction
8.14 Supercritical Fluid Extraction
8.15 Steam Distillation
8.16 Headspace Techniques
8.17 Sepbox
8.18 Selection of a Suitable Extraction Process
8.19 Carbohydrates
8.20 Proteins
 8.21 Alkaloids
8.22 Glycosides
8.23 Carotenoids
8.24 Tannins
8.25 Oleoresins
8.26 Essential Oils
9 Screening Methods Used for Herbal Drugs
9.1 Introduction
9.2 Screening Methods for Anti-fertility Agents
9.3 Screening Methods for Anterior Pituitary Hormones
9.4 Screening Methods for Anti-diabetic Drugs
9.5 Screening Methods for Thyroid and Anti-thyroid Drugs
9.6 Estimation of Various Hormones Involved in Reproduction by
ELISA
9.7 Cardiovascular Screening Methods
9.8 Selection of an Animal Model for Cardiovascular Screening
9.9 Screening Methods for Anti-anginal Drugs
9.10 Screening Methods for Anti-hypertensive Drugs
9.11 Indirect Blood Pressure or Non-Invasive Blood Pressure (NIBP)
9.12 Screening Methods for Anti-arrhythmic Drugs
9.13 Screening Methods for Cardiac Glycosides
9.14 Langendorff Heart System
9.15 Screening Methods for Diuretics
9.16 Neuropharmacology
9.17 Screening of Anti-epileptic Agents from Herbal Sources
9.18 Screening Methods for Analgesic Activity
9.19 Screening of Herbal Drugs for Antipyretic Activity
 9.20 Screening Methods for Anti-cataract Agents
9.21 Screening Methods for Anti-glaucoma Agents
9.22 Screening of Herbal Drugs for Anti-cancer Activity
9.23 Screening Methods for Evaluation of Hepatoprotective Agents
9.24 Screening Methods for Anti-inflammatory Drugs
9.25 Screening Methods for Anti-ulcer Drugs
9.26 Screening of Anti-asthmatic Drugs
9.27 Screening Methods for Anti-lithiotic Drugs
9.28 Development and Evaluation of Transdermal Drug Delivery
Systems (TDDS) of Herbal Drugs
9.29 Development and Evaluation of Intra-uterine Contraceptive
Devices (IUCDs) for Herbal Drugs
9.30 Toxicity Studies
9.31 Committee for the Purpose of Control and Supervision on
Experiments on Animals (CPCSEA)
9.32 Clinical Trials
10 Standardisation of Herbal Drugs
10.1 Importance of Standardisation and Problems Involved in the
Standardisation of Herbs
10.2 Standardisation of Single Drugs and Compound Formulations
10.3 WHO Guidelines for Quality Standardised Herbal Formulations
10.4 Estimation of the Parameter Limits Used for Standardisation
10.5 Herbal Extracts
11 Herbal Formulations: A Comparative Study of Ayurvedic and
Modern Dosage Forms
11.1 Classification
11.2 General Considerations
11.3 Different Stages of Herbal Formulation
 11.4 Dosage Forms
12 Herbal Cosmetics
12.1 Source, Historical Background and Present Status
12.2 Raw Materials: Oils, Waxes, Gums, Hydrophillic Colloids,
Colours, Perfumes, Protective Agents, Bleaching Agents,
Preservatives, Antioxidants and Other Auxillary Agents
12.3 Formulation Aspects of Incorporating Herbal Extracts in Various
Preparations
13 Nutraceuticals: A Modern Approach
13.1 Introduction and Classification
13.2 Nutraceuticals and Diseases
13.3 Immune Boosters and Anti-inflammatory Agents
13.4 Inflammatory Disorders
13.5 Degenerative Diseases
13.6 Current Status and Legal Aspects
14 Chemotaxonomy
14.1 Chemotaxonomy
14.2 Origin and Chronological Development of Chemotaxonomy
(History)
14.3 Phytoconstituents Useful in Chemotaxonomy
14.4 Plant Serology and Serotaxonomy
14.5 Chemotaxonomy in Lower Plants
14.6 Prospects and Problems of Chemotaxonomy
15 The Role of Literature Search in Medicinal Plant Research
15.1 Definition
15.2 Purpose of Literature Search
15.3 The Internet as an Information Search Tool in
Phytopharmacological Research
 15.4 Planning a Search
15.5 Electronic Databases
15.6 Some Plant-related Databases
15.7 Searching Techniques
16 Patenting of Herbal Drugs
16.1 Definition
16.2 Benefits of Patent Protection
16.3 Patent Application
16.4 Plant Breeders' Rights
16.5 Important Points to be Kept in Mind while Drafting and Filing a
Patent
16.6 Indian and International Patent Laws and Proposed Amendments
16.7 The Trade-related Aspects of Intellectual Property Rights (TRIPs)
Glossary
Glossary of Botanical Terms
Glossary of Medical Terms
Bibliography
SECOND MESSAGE
Professor S S Agrawal (Pro Vice Chancellor, AUUP, Director, Amity Institute of
Pharmacy) along with Dr M Paridhavi, Principal, Rajiv Gandhi Institute of
Pharmacy, Trikaripur, Kasaragod, Kerala, have written the book entitled Herbal
Drug Technology for graduate and postgraduate students of pharmacy,
particularly those studying herbal drug technology.
In this second edition, there are a large number of additions.
The language used is very simple and lucid. The style of presentation is
impressive; the authors have worked hard to bring out the book.
I congratulate the authors for an excellent piece of work in terms of revision
and I hope that the book will be very useful.

I wish all success to the authors in their endeavour.

(Dr B Suresh)
President
President Pharmacy Council of India
FIRST MESSAGE
Prof S S Agrawal and Dr M Paridhavi have written meticulously the book
entitled Herbal Drug Technology for graduate and postgraduate students of
pharmacy particularly those studying herbal drug technology.
The language used is very simple and lucid. The style of presentation is
impressive; the authors have worked hard in bringing out the book.
I congratulate the authors for an excellent piece of work and hope that the
book will be very useful.

I wish all success to the authors in their endeavour.

(Dr B Suresh)
President
President Pharmacy Council of India
Preface to the Second Edition
The revised edition of Herbal Drug Technology is in your hands. This edition
has been brought out after a thorough revision of the earlier text. Several topics
have been added on the basis of developments in the field since the book was
written. We believe that the book now covers as far as possible the latest
technology followed all over the world.
The topics included are herbal cosmetics, nutraceuticals, chemotaxonomy,
methods of literature search and patenting of herbal drugs. The recent changes in
in vivo anti-cancer screening models and screening of cardiac glycosides have
also been added.
We are thankful to many professionals and colleagues for their help during the
preparation of the second edition. In particular, we wish to thank Dr K S
Muralikrishna, D H N Aswatharam and Dr E N Gaviraj.
We are also grateful to Preethimol Francis and Sujitha C for their valuable
help.
We thank the publisher, Universities Press (India) Private Limited, and the
staff members of Orient BlackSwan for their cooperation in bringing out this
book.
S S Agrawal
M Paridhavi
Preface to the First Edition
Herbal Drug Technology is written according to the curriculum of various
universities for both bachelors’ and master’s courses in pharmacy and allied
sciences conducted globally. It contains detailed text on Indian systems of
medicine, herbal therapeutics, crude drugs and medicinal botany—plant
taxonomy, morphological considerations of crude drugs and fundamental
considerations of histology. The book also discusses in vitro culture of medicinal
plants, nutritional requirements, processing, techniques, and establishes
guidelines for setting up an in vitro culture laboratory. It explains in detail the
systematic examination of crude drugs, their extraction, isolation and analysis
using various modern techniques like TLC, PC, HPLC, HPTLC, GLC, UV, IR,
1H-NMR, 13C-NMR, MS, GC–MS, DSC and Sepbox.

A special emphasis has been laid on screening of herbal drugs for

pharmacological activity like anti-fertility, anti-diabetics, anti-cancer, anti-
anginal, anti-thyroid and many others. There is a brief discussion on estimation
of hormones using ELISA, transdermal delivery systems, toxicological studies
of herbal drugs and pharmocovigilance of traditional medicines. Guidelines for
clinical trials and good clinical practices are also mentioned.
Standardisation of herbal drugs has been gaining importance. This book
discusses the WHO protocol, different methods used for standardisation, QC
standards for herbal extracts and validation of herbal products. It includes a
comparative study of Ayurvedic and modern dosage forms like churna, bhasma,
kwatha, taila, tablets, injections and ointments.
All recent scientific innovations/advances have been incorporated in a simple
and lucid manner. The book will be useful for students, academicians and
professionals of pharmacy, Ayurveda, Unani and Siddha.
The authors thankfully acknowledge the gracious permission accorded by the
Honourable President of India, Dr A P J Abdul Kalam and Distinguished
Scientist and Chief Controller (R&D) Dr A Sevathanu Pillai, for reproducing the
photograph of a typical herbal garden layout from their book Envisioning an
Empowered Nation.
The authors are thankful to Prof. Manjunatha V Jali, Principal, TVM College
of Pharmacy, Bellary and Dr E N Gaviraj for their valuable support.
The authors are also grateful to Ms Sibi P I, M Alvin Jose, Sachin Nain, Alok
Arya, Niranjan Galpalli, Kalai Selvan, Pragya Gupta, Ginpreet Kaur and Dr
Renu Agrawal for their valuable help.
S S Agrawal
M Paridhavi
 Foreword

Herbal drug technology has become a very important instrument for converting
botanical materials into therapeutically useful products and medicines. Thus, the
area will gain increasing importance, which of course is obvious and need no
further emphasis. However the countries which desire to bring herbal products
into the global market and become successful need to make continuous use of
modern scientific techniques and integrate it with traditional knowledge and
biodiversity. As India is trying to work towards achieving this goal, integration
of herbal drug technology and undertaking research specifically in this area,
assumes special significance.
The present book, written by two authors who have vast experience in the
modern area of experimental science and who have joined together to bring out
this publication, is one such honest effort in this direction. I believe that it will
fill the current information gap and also meet the educational need of
undergraduate and postgraduate pharmacy students who are being trained in the
area of herbal therapeutics for whom this book has been especially designed.

(N K Ganguly)
Director General
Indian Council of Medical Research
Pharmacy Council of India
 1

Introduction to
Medicinal Plants

Man has been using herbs and plants products for combating diseases since
times immemorial.
The Indian subcontinent is enriched by a variety of flora—both aromatic and
medicinal plants. This is due to the wide diversity of climatic conditions in India
ranging from deserts to swamplands. Numerous types of herbs have been well
recognised and catalogued by botanists from the high ranges of the Himalayan
tract up to the sea-shore of Kanyakumari. This extensive flora has been greatly
utilised as a source of many drugs in the Indian traditional system of medicine.
In India, the earliest mention of the use of medicinal plants is to be found in the
Rigveda which was written between 4500–1600 BCE. A detailed account of the
world’s first symposium on medicinal plants is given in the first chapter of
Vrihat Samhita and since 1600 BCE the amount of literature on this subject is
boundless. The traditional system of medicine is so engrained in our culture that,
even now 75% of the Indian population depend on this indigenous system for
relief. With such a huge section of an ever-increasing population relying on
herbal remedies, it is imperative that the plant products which have been in use
for such a long time be scientifically supported for their efficacy.
The World Health Organization is now actively encouraging developing
countries to use herbal medicine which they have been traditionally used for
centuries. They have identified 3000 plants from the forests of India and other
tropical countries which can be used as medicine. The active ingredients from
these plants are worth nearly Rs. 2000 crores of rupees for the US market alone
and nearly 8 times that for the world market (Muggenburgh H. 1983; Rustogi
R.P. 1980). Only with the scientific advancements in the fields of pharmacology
and toxicology in the western hemisphere, has drug development based on
natural products gained intensity in Europe and USA. The importance of such an
investigation, in India was realised long back and the first systematic study with
these aims was started by Sir Ramanath Chopra at Calcutta about 45 years back.
In the early stages, the science of medicine developed around those plants
which had curative properties. A continued search for medicinal plants during
the last several centuries has given rise to a long list of plants which are of great
use in the treatment of diseases, and for promoting health. It can be stated, more
or less truthfully, that every disease has a cure in a plant growing in nature.
Recently, Moose has described a number of vegetable drugs that can be used as
single drug remedies.
Drugs used in medicine today, are either obtained from nature or are of
synthetic origin. Natural drugs are those obtained from plants, animals, microbes
or minerals. Those obtained from plants and animals are called drugs of
biological origin and are produced in the living cells of plants or animals.
Until now, only 6000 plant constituents have been isolated and studied. The
flora on this earth, representing an inexhaustible source of medicinal plants,
remains incompletely explored. This unexplored world provides the most
challenging aspects of pharmaceutical and medical science to scientists in search
of new and more potent drugs with marked therapeutic virtues and negligible
side effects. During the last few decades, tremendous progress has been made in
the study of phytochemicals.
Natural products, as a basis for new drugs, have great promise and it is
gratifying to note that the World Health Organization have shown an abiding
interest in plant-derived medicines, described in the folklore of various
countries.
Plants have been one of the important sources of medicines since the dawn of
human civilisation. For instance, the Chinese drug Mahung was in use for over
5000 years for the treatment of different types of fever and respiratory disorders.
Cinchona sp was in use in Peru even in 1825, primarily for controlling malaria.
In spite of the tremendous development in the field of synthetic drugs and
antibiotics during the 21st century, plants still contribute one of the major
sources of drugs in modern as well as traditional medicine throughout the world.
One-third of the world’s population treat themselves with traditional medicines.
Some of the compounds now commonly used in medicine were isolated from
plant sources and used as early as in the 19th century. Examples are morphine
(1803), quinine (1812), atropine (1831), papaverine (1848), cocaine (1860),
digitoxin (1865), and pilocarpine (1875). Examples of some important
compounds isolated in the 20th century include ergotmine (1518), labeline
(1921), digoxin (1930), reserpine (1931), tubocurarine (1935), diosganin,
vincristine (1961), and vimblastine (1963).
Plants are the only economic source of a number of well-established and
important drugs. In addition, they are also the source of chemical intermediates
needed for the production of some drugs.
As stated before, about 75% of the Indian population relies heavily on the use
of herbal drugs for the treatment of diseases. The factors responsible for the
continued and extensive use of herbal remedies in India are their effectiveness,
easy availability, low cost, comparatively less toxic effects and the shortage of
practitioners of modern medicine in rural areas. There is a growing appreciation
in India, as in many other developing countries, of the need to make greater use
of traditional remedies in order to be able to provide medicine for primary health
care.
Although use of traditional remedies is advantageous, it does suffer some
limitations. The main limitation is the lack of standardisation of raw materials, of
processing methods and of the final products, dosage formulation, and the non-
existence of criteria for quality control. Research has to be directed to the use of
modern scientific methodology and techniques to standardise all these different
steps and for quality control.
Before Independence, the production of plant-based drugs in India was
confined mainly to cinchona and opium alkaloids, galenicals (i.e., medicine
extracted from plants) and tinctures. In the last three decades, bulk production of
plant drugs has become an important aspect of the Indian pharmaceutical
industry. Some of the drugs which are manufactured today include morphine,
codeine, papaverine, thebaine, emetine, quinine quinidine, digoxin, caffeine,
hyoscine, hyocyamine, atropine, xanthotoxin, sennosides, colchicines, berberine,
vinblastine, vincristine and ergot alkaloids, papaine, nicotine, strychnine, brucine
and pyrethroids.
In India, there are about 20 well-recognised manufacturers of herbal drugs, 140
medium or small-scale manufacturers, and about 1200 licensed small
manufacturers on record, in addition to many vaidyas having small
manufacturing facilities. The estimated current annual production of herbal
drugs is around Rs.100 crores. The demand for herbal remedies is ever-
increasing. Herbal medicines represent an estimated $60-billion a year global
market, about 20 per cent of the overall drug market, according to the United
Nations agency. There are 1650 herbal formulations in the Indian market and
540 major plants involved in their formulations.
During the last two decades, over 3000 plants have been screened in India for
their biological activities. As a result, a number of new drugs have been
introduced in clinical practice and some are in the advance stages of clinical
development.
There are well-documented scientific data on a good number of medicinal
plants that have been investigated. In spite of all these efforts, very few drugs of
plant origin would reach stage I of a clinical trial or gain enough creditability for
clinical use by the practitioners of modern medicine (Gupta S. S. 1994).
This is because herbal drugs are sometimes considered dubious and its
practitioners considered as quacks. The reasons are many. Doubts have been
raised on the use of herbal drugs for the following reasons: 1. Herbs of different
origin are often known by the same popular name.
2. Plants growing in different climatic and seasonal conditions do not have
identical chemical constituents or therapeutic effect.
3. The process of collection (fresh, shade or sun-dried), extraction, processing
and storage of herbal medicines cause variation in potency and safety.
4. The lack of specific standards for herbal medicines in suitable dosage form
creates difficulty in administration.
These shortcomings have delayed the integration of some of the better known
Ayurvedic and Unani principles with the modern system of medicine.
But things are looking up with the gradual acceptance of Ayurvedic medicine.
Further, detailed investigation on the mode of action of herbal drugs has revealed
that they are involved in enzymatic, endocrine and immunomodulating
functions. These have helped to widen their profile of activity and opened new
vistas of therapeutic applications.

1.1 ILLUSTRATIVE EXAMPLES OF PLANTS USED AS DRUGS THE

IMPORTANCE OF PLANTS AS A SOURCE OF USEFUL ANTI-
HYPERTENSIVE DRUGS WAS SUPPORTED BY THE ISOLATION OF
RESERPINE FROM RAUWOLFIA SERPENTINA, BY MULLER ET AL.
IN 1952. VERATRUM ALKALOIDS ARE OTHER USEFUL ANTI-
HYPERTENSIVE AGENTS OBTAINED FROM A PLANT SOURCE.

Allium sativum, zingiber officinale etc., have been mentioned to be useful in

cardiovascular ailments in classical testbooks on ancient medicine.
Plant products have contributed several novel compounds possessing
promising anti-tumour activity. For example, podophyllotoxin, alpha and beta
pelatin were found to be capable of inflicting considerable damage on
experimental tumours.
Various herbal medicines having a role in the treatment of diabetes have been
described in classical Ayurvedic literature. Mention has also been made of
different plant extracts used for anti-diabetic activity.
Aconitan A, isolated from Aconitum carmichaellii (family: Ranunculaceae)
when administered i.p. to alloxan-induced hyperglycemic mice, exhibited blood
sugar lowering action.
Quinquefolans A, B and C isolated from Panax quinquefolin (family:
Arliaceae) had a hypoglycemic effect in normal mice. Quinquefolan A on i.p.
administration alone, in alloxan-induced hyperglycemic mice produced a
hypoglycemic effect.
Some of the other extensively investigated medicinal plants are given Table
1.1. Table 1.2 lists some plants used for central nervous system (CNS) drugs in
Indian medicine.
Table 1.1 Examples of extensively investigated medicinal plants

Table 1.2 Herbal CNS drugs used in Indian medicine

Alstonia scholaris Nardostachys jatamanasi

Apium graveoleus Nymphaes lotus

Boerrharvia diffusa Rauwolfia serpentina

Cardus diodara Santalum album

Hydrocytyle asiatica Vaneriana wallichii

Melia azadirachta Vinca rosea

 Amongst the several plants investigated for anti-asthmatic effects, saponins
isolated from Clerodendron serratum, Gardenia turgida, Albizzia lebbek and
Solanum xanthocarpun were found to accord protection to sensitised guinea pigs
against histamine as well as antigen (egg albumin) micro-aerosols. The
protective effect of C. serratum saponin was found to be associated with the
augmentation of anti-allergic activity in the lung tissues—lung extracts from the
treated animals inhibited histamine and SRS-A responses on guinea pigs ileum
to a greater extent and for a longer period as compared to the extracts from
untreated control animals. Saponins from A. lebbek have also been demonstrated
to modulate immune responses through synthesis of reagenic antibodies. The
alcoholic extract of Tylophora asthmatica has been reported to prevent egg
albumin-induced anaphylaxis in guinea pigs and horse serum-induced
bronchoconstriction in sensitised rat lung. Chewing of leaves of T. asthmatica
for 6 days has been demonstrated to give protection to 71% cases of bronchial
asthma on antigen challenge. The plant saponins from C. serratum and A. lebbek
as well as the alkaloidal fraction of S. xanthocerpum and T. asthmatica have
been shown to protect sensitised mast cells from degranulation on antigen shock,
thus confirming the immuno-suppressive and membrane-stabilising effect. T.
asthmatica as well as saponin of A. lebbek have also been found to potentiate
bronchodilator beta-adrenergic activity, which is considered to be helpful for
relieving bronchospasm in asthmatic patients. The anti-allergic action of O.
sanctum has been found to be associated with significant production of IgE
antibodies.
Search for a potent hypolipidaemic agent based on ancient insight following
the Ayurvedic system, has been rewarding with the isolation of the oleoresin
fraction from Commiphora mukul and Guggul having hypolipidaemic activity,
comparable to Clofibrate with more favourable HDL–LDL cholestrol ratio. It
also decreases platelet adhesiveness and increases fibrinolytic activity necessary
for the prevention of myocardial infarction.It has been reported that the
hypolipidaemic and cardio-protective effect of C. mukul in combination with
Terminalia arjuna and Inula racemosa is comparable to Genfebrozil. The
hypocholesterolaemic effect of Pterocarpus marsupium associated with
hypoglycaemic activity, is of clinical significance as hypercholesterolaemia is
often associated with diabetes.
Medicinal plants commonly included in Ayurveda for liver ailments have
drawn much attention as there is no reliable hepato-protective drug available in
modern medicine. The hepato-protective effect of some liver protectives like
Picorrhiza kurrooa, T. cordifolia, Tephrosia purpurea against carbon
tetrachloride and galactosamine-induced hepatic injury have been confirmed
experimentally by various workers. In biliary ailments, plants such as
Andrographis paniculata, Lyffa ectinata and Ficus hispida have been found to
increase bile flow with reduction in serum bilirubin and SGPT levels.
Phyllanthus niruri and Eclipta alba have been reported to eliminate hepatitis B
surface antigen.
Plants have also been the source of anti-cancer compounds and anti-viral
interferons. An interferon stimulator derived from Glycyrhiza glabra has been
reported to give protection to patients with sub-acute hepatic failure known to be
fatal in majority of cases.
The possibility of developing a herbal drug for the treatment of autoimmune
diseases like multiple sclerosis and the dreaded AIDS cannot be ruled out.
Vegetables and cancer Various systems of tribal medicine, not only of India,
but also of China, Indonesia, Sri Lanka, Mexico, Malaysia, Philippines etc.
have identified a number of food materials of vegetable origin which are not
only anti-cancerous in nature but are also generally good for health. The
following are some of the plants traditionally used in this way (Madhavan
Kutty K. 1992): 1. Citrus aurantifolia 2. Allium cepalinn 3. Zingiber officinale 4.
Anacardium occidentale 5. Coccinia grandis 6. Piper nigrum 7. Garcinia
cambogia 8. Momordica charantia 9. Saccharum officinarum 10. Curcuma
longa 11. Muring oleifera 12. Emblica officinalis 13. Tamarindus indica 14.
Allium sativum 15. Piper betel Many of these have been used as food from
times immemorial and perhaps have now given way to more fashionable
vegetables (Jain S. K. and Shastry A. R. K. 1980).
The objective of producing inexpensive, potent and safe drugs of plant origin
can be met to some extent by promoting compound formulations of plant
medicines in their natural or semi-processed form as used in traditional
medicine. Proper standardisation and dosage formulation in due consideration of
the therapeutic–toxicity ratio will, however, be necessary with controlled clinical
trials to prove their efficacy.
 2

Indian Systems
of Medicine

Indian systems of medicine have a deep root in our cultural heritage and cater to
the medicare of large sections of our population. These systems mainly use
herbs. In recent times there has been a market shift towards herbal cures because
of the pronounced cumulative and irreversible ill effects of many modern drugs.
Indian systems of medicine (viz. Ayurveda, Unani, Siddha, Yoga and
Naturopathy) have developed over a long period of time. If we dwell for a
moment on our hoary past, the Rigveda, one of the oldest repositories of human
knowledge, mentions the use of 67 plants for therapeutic use, the Yajurveda
enlist 81 plants whereas the Atharveda written during 1200 bce describes 290
plants of medicinal value. Charak Samhia (990 bce) describes 341 medicinal
plants. The next landmark in Ayurveda was when Sushruta Samhita (600 bce)
mentioned 395 medicinal plants. Dhanwantari Nighantu mentions 750 medicinal
plants, 450 are mentioned in the Bhavaprakash, 480 in the Madanapala
Nighantu and 450 in the Kaiyadeva Nighantu. Idn Baiter (1197–1248) in his
book Al Jami li Mufradat has described about 1400 drugs, most of which are of
herbal origin. Avicenna (980–1037) in his book Al Quanoon has included about
700 drugs, out of which almost 200 drugs of herbal origin have been identified
as exclusively used in the Unani system of medicine (Joshi G.C. 1992). India
unquestionably occupies the top position in the use of herbal drugs. It is one of
the foremost countries exporting plant drugs and their derivatives. It also excels
in home consumption. It is not at all surprising that herbal drugs are so prevalent
in India given the great biodiversity and abundance of flora and the variety of
geographical conditions which allows the most exotic medicinal plants to be
grown here.
2 .1 AYURVEDA AYURVEDA IS AN ORIGINAL HOLISTIC SYSTEM
OF MEDICINE WHOSE PRINCIPLES OF THERAPEUTICS ARE
APPLICABLE UNIVERSALLY. A MODEL FOR COMPREHENSIVE
CLINICAL MANAGEMENT HAS BEEN DEVELOPED BASED ON A
SCIENTIFIC AYURVEDIC APPROACH.

The name Ayurveda is derived from two words: Ayur meaning “life” and Veda
meaning “knowledge or science” i.e., the “Science of Life”. Ayurveda evolved
over 5000 years ago, in the far reaches of the Himalayas, presumably from the
deep wisdom of spiritually enlightened prophets or Rishis.
Ayurveda is based on the interaction of body, mind and spirit.

2.1.1 Panchamahabhutas According to Ayurveda, all the objects in the

universe, including the human body, are composed of five basic
elements (Panchamahabhutas) namely, earth, water, fire, air and
vacuum (ether). These elements in different proportions are in a
balanced state to suit the needs and requirements of different
structures and functions of the body matrix and its parts. The growth
and development of the body matrix depends on its nutrition, i.e., on
food. Food, in turn, is composed of the five elements, which replenish
or nourish the like elements of the body after the action of bio-fire
(Agni). The tissues of the body are the structural entities whereas
humours are physiological entities, derived from the different
combinations and permutations of the Panchamahabhutas.
Doshas The Indian medical science attributes all morbid phenomena to the
disordered condition of the three principal humours in the body, called
doshas namely wind, bile and phlegm. These fluids pervade the whole
microcosm of man. So long as these are in their normal condition the body
remains healthy. If they are deranged, they subject the whole body to all
sorts of disorders. For perfect digestion, the three humours must be in their
proper proportion. If wind is predominant, the bowels become costive;
when bile is in excess, they became loose; when phlegm predominates, the
bowels remain in their normal condition. The five elements combine to form
the three basic forces, tridoshas, which exist in everything in the universe
and influence all mental and physical processes. From earth and air, the air
principle Vata is created; fire and water yield the fire principle Pitta; and
earth and water produce the water principle Kapha. All of us are born with
a particular balance of doshas. The proportions are largely determined by
the balance of doshas in our parents at the time of conception. Our body
type, temperament and susceptibility to illness are largely governed by the
predominant doshas.
Prana Illness may also result if the flow of energy, Prana, around the body is
interrupted. The flow is relayed via the seven Chakras (psychic energy
centres) which are situated at various points along the spinal column, from
the crown of the head to the tailbone. Usually the movement of the body
depends on the vata, which is of five kinds according to the functions they
perform in the organism. They are udana, prana, samana, apana and vyana.
Pitta helps in maintaining body heat. It is usually hot, liquid, yellow, bitter,
but acidic when vitiated, light and oily. It is of five kinds namely pachaka,
ranjaka, sadhaka, alochaka and bharajaka. Kapha is associated with energy
and is a white, heavy oleaginous, viscid, cooling liquid.
It is usually sweet, but becomes salt when defective. It is also of five kinds
namely kledana, bhodaka, terpaka, sleshmaka and avalambaka.
Besides the three humours mentioned above, there are seven more essential
parts of the body, called Dhatus. They are rasa (lymph), rakta (blood), mansa
(flesh), medas (fat), asthi (bone), majja (marrow) and shukrana (semen).

2.1.2 Treatment
Treatment of the disease consists in avoiding causative factors responsible for
the disequilibrium of the body matrix or of any of its constituent parts through
the use of Panchakarma procedures, medicines, suitable diet, activity and
regimen for restoring the balance and strengthening the body mechanisms to
prevent or minimise future occurrence of the disease.
Panchakarma therapy is a therapeutic procedure in the Ayurvedic system of
medicines. According to Charaka, the following procedures can be studied under
the Panchakarma therapy: (1) Vaman; (2) Virechan; (3) Niruha (asthapana); (4)
Anuvasna and (5) Sirovirechana (nasya). The panchakarma therapy is generally
done in three stages: the poorva karma, the pradhana karma and the paschata
karma.
Poorva karma
It is the first stage of the panchakarma therapy and it involves preliminary
procedures like the preparation of the patient, medicines and equipments
necessary. It is of the following three types: 1. Pachana (digestion therapy): It is
carried out by using general deepana and pachana drugs like digestive peya,
choorna, quatha, vati etc.
2. Snehana (oleation therapy): It includes abhyanga and bahya.
3. Svedana (sudation therapy): It is of two types namely angani and sagni
svedana.
Pradhana karma
It is the second stage of the therapy and involves the actual administration of the
drug. It is of the following types: 1. Vamana (emesis): Excretion of doshas from
the mouth.
2. Virechana (purgation): Excretion of doshas from the anus.
3. Basti (enema): The urinary bladder is called basti. Administration of drugs
though the basti into the body is known as Basti karma.
4. Nasya or Shirovirechana (nasal insufflation): Drugs which are administered
through the nostrils are known as nasya or shirvirechana.
5. Raktamokshana (blood-letting): It is also known as asravisruthi.
Paschata karma
The procedures adopted after the pradhana karma are known as paschata
karma. These are generally the adoption of rehabilitative measures after the main
treatment like diet, medicines and changes in the daily routine. It is of three
types.
1. Samsarajana karma: Restoration of digestive powers by advocating a specific
diet.
2. Rasayanadi karma: Administration of rasayana and vajeekarana drugs.
3. Shamana prayoga: Administration of medicines required to treat the
particular disease after the process of elimination.

2 .2 UNANI SYSTEM OF MEDICINE THE UNANI SYSTEM OF

MEDICINE OWES ITS ORIGIN TO GREECE. IT WAS THE GREEK
PHILOSOPHER-PHYSICIAN HIPPOCRATES (460–377 BCE) WHO
FREED MEDICINE FROM THE REALM OF SUPERSTITION AND
MAGIC AND GAVE IT THE STATUS OF A SCIENCE. THE
THEORETICAL FRAMEWORK OF UNANI MEDICINE IS BASED ON
THE TEACHINGS OF HIPPOCRATES. HE WAS FOLLOWED BY A
NUMBER OF OTHER GREEK SCHOLARS WHO ENRICHED THE
SYSTEM CONSIDERABLY. OF THESE, GALEN (131–210 CE)
STANDS OUT AS THE ONE WHO STABILISED THE FOUNDATION ON
WHICH ARAB PHYSICIANS LIKE RHAZES (850–925 CE) AND
AVICENNA (980–1037 CE) CONSTRUCTED AN IMPOSING
EDIFICE. UNANI MEDICINE WAS ENRICHED BY IMBIBING WHAT
WAS BEST IN THE CONTEMPORARY SYSTEMS OF TRADITIONAL
EGYPT, SYRIA, IRAQ, PERSIA, INDIA, CHINA
MEDICINE IN AND
OTHER MIDDLE EAST AND FAST EAST COUNTRIES.

In India, the Unani system of medicine was introduced by the Arabs. When the
Mongols ravaged Persian and Central Asian cities like Shiraz, Tabrez and Galan,
scholars and physicians of Unani medicine fled to India. The Unani system soon
took firm roots in the Indian soil.
The Unani system is based on the Hippocratic theory that a perfect balance of
“Arkhan” (elements), “Akhlat” (humour) and “Mijaz” (temperament) helps to
keep the body and mind healthy. Every individual has an inherent power of self-
preservation called the “Quwat-E-Modabira”. The theory pre-suppose the
presence of humours in human body—“Dum” (blood), “Bhalgam” (phlegm),
“Safra” (yellow pile) and “Soada” (black pile). The Unani system aims at
restoring the equilibrium of various elements and faculties of the human body. It
has laid down six essential prerequisites for the prevention of diseases. These
essentials known as “Asbabe-Sita-Zarooriya” are air, food and drinks, bodily
movements and response, sleep and wakefulness, excretion and retention. The
Unani system lays great emphasis on the maintenance of proper ecological
balance on one hand, and on keeping water, food and air free from pollution on
the other. Treatment in the Unani system of medicine is done mainly through diet
control for a simple disease in the initial stages followed by the administration of
a single drug, failing which a compound preparation may be administered. The
various treatments include “llajbit-tadbeer” (regimental therapy), “llajbit-ghiza”
(dietotherapy), “llajnit-dawa” (pharmocotherapy) and “jarahat” (surgery). In
pharmocotherapy, the physicians use naturally occurring drugs, mostly herbal.
The advantage with naturally occurring drugs is that the side effects/toxic effects
are either comparable or less than the allopathic system of medicine.

2 .3 HOMEOPATHIC SYSTEM OF MEDICINE THE WORD

“HOMEOPATHY” IS DERIVED FROM TWO GREEK WORDS,
HOMOIS MEANING “SIMILAR” AND PATHOS MEANING
“SUFFERING”. HOMEOPATHY SIMPLY MEANS TREATING DISEASES
WITH REMEDIES PRESCRIBED IN MINUTE DOSES, WHICH ARE
CAPABLE OF PRODUCING SYMPTOMS SIMILAR TO THE DISEASE
WHEN TAKEN BY HEALTHY PEOPLE. IT IS BASED ON THE
NATURAL LAW OF HEALING, “SIMILIA SIMILIBUS CURENTUR”
WHICH MEANS “LIKE ARE CURED BY LIKES”. DR. SAMUEL
HAHNEMANN (1755–1843) GAVE IT A SCIENTIFIC BASIS IN THE
EARLY 19TH CENTURY. IT HAS BEEN SERVING HUMANITY FOR
OVER TWO CENTURIES, WITHSTOOD UPHEAVALS OF TIME AND
HAS EMERGED AS A TIME-TESTED THERAPY. THE SCIENTIFIC
PRINCIPLES PROPOUNDED BY HAHNEMANN ARE NATURAL,
WELL-PROVEN AND CONTINUE TO BE FOLLOWED WITH SUCCESS.

2.3.1 Principles Homeopathy is the system of treatment based on

demonstrable laws and principles. The laws are discussed below.
Law of Similars It is also called the Law of Cure. This law demonstrates that the
selected remedy is able to produce a range of symptoms in a healthy person
similar to that observed in the patient, thus leading to the principle of Simila
Similibus Curenthur i.e., let like be treated by likes.
Law of Single Remedy This law directs the practitioner to choose and administer
such a single remedy which is most similar to the symptom complex of the sick
person at a time.
Law of Minimum Dose The similar remedy selected for a sick person should be
prescribed in minimum dose, so that when administered there is no toxic effects
on the body. It just acts as a triggering and catalytic agent to stimulate and
strengthen the existing defense mechanism of the body. It does not need to be
repeated frequently.
The key features that characterise the Homeopathic system of medicine are
given below:
1. It has two basic principles:
a. Treatment of like with like b. Use of the minimum effective dose.
2. Homeopathic drugs are called remedies.
3. A process of multiple dilutions and succussions are used to prepare
homeopathic remedies. It is known as potentisation.
4. The greater the number of stages of potentisation, the greater the therapeutic
potential of the remedy.
5. Common scales of potencies includes:
a. Decimal b. Centesimal with 1000 C represented as 1M and c. LM ( 1 in
50,000) 6. The remedies are prepared mainly by two methods: a. Multiple
vial method.
b. Single vial mehtod.
7. The starting solution is called the mother tincture.
8. The insoluble substances are made soluble by trituration, grinding and mixing
with lactose powder.
9. The potency selection is related to a number of axes: a. Acute/chronic b.
Physical/emotional c. The vitality of the patient d. The prescriber’s fear of an
aggravation.
e. The confidence of the prescriber 10. The process of cure has mainly four
axes: a. From most important organs to less important ones b. From inside to
outside c. From top to bottom d. Disappearance of symptoms in reverse
chronological order of their appearance 2.4 Siddha The Siddha system is
one of the oldest systems of medicine in India. The term “Siddha” means
achievement and “Siddhars” were saintly figures who achieved results in
medicine through the practice of Yoga. Siddha system’s literature is in Tamil
and it is practiced in the Tamil speaking parts of India. The system is also
called the Agasthyar system after its famous exponent Sage Agasthya. The
Siddha system is largely therapeutic in nature.
The principles and doctrines of this system, both fundamental and applied,
have a close similarity to Ayurveda, with specialisation in latro-chemistry.
According to this system, the human body is the replica of the universe and so
are the food and drugs, irrespective of their origin.
Like Ayurveda, this system believes that all objects in the universe, including
the human body, are composed of five basic elements namely, earth, water, fire,
air and sky. The food, which the human body takes and the drugs it uses are all
made of these five elements. The proportion of the elements present in the drugs
vary and their preponderance or otherwise is responsible for certain actions and
therapeutic results.
As in Ayurveda, this system also considers the human body as a
conglomeration of three humours, seven basic tissues and the water products of
the body such as faeces, urine and sweat. Food is considered to be the basic
building material of the human body which gets processed into humours, body
tissues and waste products. The equilibrium of humours results in a healthy body
and its disturbance or imbalance leads to disease or sickness.

2 .5 YOGA AND NATUROPATHY YOGA, WHICH IS ROOTED IN

HINDU RELIGIOUS PRINCIPLES, HAS BEEN IN PRACTICE FOR THE
LAST 500 YEARS. DERIVED FROM THE SANSKRIT WORD, YOGA
MEANING “UNION,” IT ENCOMPASSES A VARIETY OF DISCIPLINES
DESIGNED TO ULTIMATELY BRING ITS PRACTITIONERS CLOSER TO
G OD . DYNANA YOGA, FOR INSTANCE, SEEKS UNION THROUGH
MEDITATION, WHILE JANANA YOGA ENTAILS THE STUDY OF
SCRIPTURES AND KARMA YOGA CALLS FOR SELFLESS SERVICE TO
GOD AND MANKIND. YOGA OFFERS A SIGNIFICANT VARIETY OF
PROVEN HEALTH BENEFITS, EVEN THOUGH IT IS NOT A CURE
FOR ANY MEDICAL AILMENT.

Yoga exercises increase the efficiency of the heart and slow the respiratory rate,
improves fitness, lowers blood pressure, promotes relaxation, reduces stress, and
anxiety. It also serves to improve coordination, posture, flexibility, range of
motion, concentration, sleep, and digestion. It is a supplementary therapy for
conditions such as cancer, diabetes, arthritis, asthma, migraine, and AIDS. It also
helps to combat addictions like smoking.
 3

Herbal Therapeutics:
From Ancient Times to
the 21st Century

Man from the very beginning has been aware of the problems of life and for a
very long time has been taking care of his health through diet and drugs. Plants
were used extensively for cures and general well-being. But it was only in the
period of the Ayurvedic Samhitas that there were serious attempts in studying
plants scientifically.
In the course of evolution, consciousness was first manifested in plants. Long
before Jagdish Chandra Bose demonstrated the sign of life in plants by his
scientific experiments, the Vedic seers realised it through self-identification. The
Chhandogya Upanishad (6–11.1) says, “As the plant is possessed by Jivatman
(conscious self), it stands joyfully and if injured in bark a liquid oozes out which
is absent in lifeless (dried) plants.”
Man comes last in this ladder of evolution and has been in contact with plants
from the very beginning. In excavations of Mohenjodara and Harappa, remnants
of trees are found which indicates the use of plants in those days and also the old
tradition of tree-worship which still continues in the Indian community. It is not
improbable that the primitive man had easy recourse to plants in cases of illness.
He must have used them in various forms as amulets and drugs. In the Atharveda
one finds mention of a number of amulets made from plants such as asvattha
(Ficus religiosa linn), darbha (Imperata cylindrica), varuna (Crataeva nurval),
satavari (Aspargus racemosus) etc. It is not difficult to imagine that even in the
Stone Age, man used plants as drugs by crushing them on stone and extracting
their juice. In the present chapter, an overview of the development of therapeutic
agents from herbal sources, ranging from the traditional period to the modern
era, has been discussed.

3.1 THE ANCIENT WORLD

3.1.1 Egypt
The most important document that is known with regard to the history of the
materia medica of ancient Egypt and of the east Mediterranean is the most
famous papyri, discovered in 1873 in Egypt by G.Ebers from which the name
“papyrus” originates. This papyrus, according to the Arab who sold it to Prof.
Ebers, had actually been discovered in Thebes in 1862. It starts with the
following words: “Here begins the book on the preparation of medicines for all
parts of the human body”. This papyrus which might be considered as one of the
most ancient pharmacopoeias and principal source of information on Egyptian
medicine, reports numerous medicaments of mineral and animal origin (lead,
alum, zinc carbonate, gold, honey, ox fat and bile, fat of marten, etc.) besides a
considerable number of vegetable drugs, many of which are still in use. It has
been possible to identify as many as 160 vegetable drugs and it is certain that
Ancient Egyptians were acquainted with the use of senna, colocynth, poppy,
castor oil, acacia gum, squill, thyme, turpentine, linseed, henbane, and Indian
hemp. Examples of the means adopted to cope with diseases, can be taken from
Eber’s Papyrus (about 1550 BCE). The text starts with three general “recitals” or
incantations to increase the efficacy of the remedies.
It is documented by the paintings at the Valley of the Queens, in Luxor, a short
time after the compilation of Eber’s Papyrus, that Queen Hatshepsut (1504–1483
BCE) of the 18th dynasty, in 1493 BCE sent an expedition in which even some

doctors took part, to Africa, from where various drugs were brought back—alum
and soda, essential for embalming and also present in many prescriptions; durra
(sorghum), the mucilaginous extract of which was often given to fight
indigestion, myrrh gum or red sandalwood from Nubia, used as astringents,
Ammi majus (Bishop’s weed), the root of which was eaten by caravaneers to
protect themselves from the burning sun. Modern science has discovered that 8-
methoxypsoralen, present in Ammi majus stimulates pigment formation in the
skin and produces an artificial suntan. Kesso (Japanese Valerian) blossoms one
of the most antique remedies for tapeworm infection originates from Abyssinia.
A wad of fibres impregnated by Acacia spikes, finely ground dates and honey,
was used as a contraceptive. Thousands of years later it was found that the
Acacia spikes contain a gum that when dissolved in fluid forms lactic acid.
Many modern contraceptive gels contain lactic acid as one of their primary
components. Pomegranate root and bark remained in the British Pharmaceutical
Codex (BPC) of 1934. The dried bark and stem or the root of the plant (Punica
granatum) provides the toxic mixture of tannates and alkaloids, known as
pelletierine tannate (British Pharmacopoeia 1948). This substance has a
specified action on tapeworms, whilst it is ineffective against most of the other
intestinal parasites. The Ancient Egyptians came, on this occasion, fairly close to
the knowledge of the modern pharmacopoeias.

3.1.2 Mesopotamia
During the construction of the Pyramids, the slaves inducted for the occasion
were given enormous quantities of radishes, garlic and onions justifiable only for
medical reasons. In 1948, a substance called raphanin was isolated from radish
seeds. This substance has distinct antibiotic properties against a number of
bacteria including cocci and coli; at about the same time, allicin and allistatin,
both powerful antibiotics, were extracted from garlic and onions.
In the meantime in Mesopotamia, another branch of medicine was running
parallel. Amongst the cuneiform tablets of Nineveh, that go back to 2000 years
BCE and were copied by the scribes around the 7th century BCE by the will of King

Assurbanipal in order to hand down the Babylonese culture to his descendants,

there are many tablets that report medical prescriptions. The essential features of
ancient Mesopotamian medicine were the use of incantations, the resort to a vast
number of magical rites, the persistent practice of divination, and the
unimportant place given to drugs. Of their belief in magical beings and their
ability to influence disease and the afterlife, the Assyro-Babylonian bronze
amulet is an example.
One plant which kept its Assyrian magical significance into medieval times
was the mandrake, Atropa mandragora. It was famous in the Bible and still
famous in the herbals of the First Elizabethan reign. Belladonna was used in
Mesopotamia to counteract bladder spasms, persistent coughing, asthma and
excessive salivation. The cuneiform tablets provide evidence that the slaves were
used as guinea-pigs and poisonous substances were first tried on them in order to
establish the dose before administering the medicines to the relatives of the king.
 Both in Egypt and in Mesopotamia, the remedies were prepared with a greatly
developed pharmaceutical and technical competence by the priest’s caste that
guarded the secrets of their preparations in the temples; the exchange of
unguents, perfume and medicated oils was held in the shadow of the porticoes of
the temples. Mystery and magic were however intimately mixed with rational
remedies, the identity of which was often veiled by fantastic names.
To conclude, the most ancient civilisations enriched our history and general
literature, far more than they enriched our medicine and its pharmacopoeia.

3.1.3 Judea
As far back as 1990 BCE, at the time of Abraham and Jacob, street vendor
pharmacists roamed about the villages selling spices, gums, balsams and
different medicines. The Reformation awoke interest in Biblical study and the
first systematic accounts of the medicine of the Old Testament were published in
the seventeenth century. Julius Preuss (1861–1913) was responsible for a
massive contribution to the subject. Preuss’ book “Biblisch-talmudische
Medizin” of 1911, is still of fundamental importance.
The Hebraic monotheists envisaged a God who could be both a giver of disease
and a source of healing and the Mosaic code of mental and physical cleanliness
had, as its central aim, a fitness to be in the divine presence.
As an example of a modern drug, with a history which reaches back to the
Biblical texts, we can take what the book of Leviticus (23:40) refers to as “the
boughs of goodly trees …. and willows of the brook”. Willow bark, the bark of
Salix alba and related species, was still the subject of a monograph in the BPC of
1934. The same compendium also describes salicin, the crystalline glycoside
obtained from willow bark. From salicin, it is but a short step to the synthesis of
salicylic acid, and then to acetylsalicylic acid—which is aspirin, the most widely
used drug today.

3.1.4 America
In the various Indian tribes of the American continent (Dakota, Winnebago,
Pima, Apache, Comanche, Mescaleros etc.) the following remedies, among
others were used:

Decoctions of the leaves and bark of the willow, of dogwood and cinchona
or of the quaking aspen as febrifuges;
 Decoctions of the leaves of holly spurge, iris, blood-wort as emetics;
Cellulosic material, magnesium salts, sacred bark (Cascara sagrada),
American aloe, may-apple, jalap etc. as laxatives;
Red oak, cuckoo-pint, Senega snake root, Oswego tea, etc. as anti-
dysenterics;
Lobelia in asthma, sticky head in tuberculosis,
Red cedar, bushy-weed, flax, basil, holy plant etc. in various respiratory
affections;
Mesquite, wild verbena, clematis etc. as intestinal antispasmodics;
Sarsaparilla, juniper, magnolia, wintergreen etc. as diuretics;
Powdered rattlesnake, cedar sprouts etc. to speed labour as abortives;
Cartilage of deer’s heart in hydrophobia.

3.2 THE WORLD OF GREECE AND ROME

It has long been thought that Greece was the source of every medical practice
but when in the last century, archaeologists started to research and interpret the
traces of past civilisations, they found precise documentation with regard to
preparations based on natural drugs used by doctors of ancient times for the cure
of various sicknesses, hundreds of years before the first Greek doctor. Greek
civilisation reached its zenith in Athens in the 5th century before Christ—the
Golden Age of Pericles—an age which witnessed the life of Hippocrates (460–
370 BCE), the father of medicine. Folk medicine, temple medicine and the
Hippocratic tradition form the three main themes of ancient Greek medicine.
Hippocrates and his followers retained a number of the old ideas and the
doctrine of the four humours (black bile, blood, yellow bile and phlegm). All
their properties and qualities carried Chaldean (or neo-Babylonian) number-lore
into the pattern of Greek thinking, and so onwards to beyond the Renaissance
period. The first great specific achievement in the history of medicine was the
clinical records of 42 cases (of which 25 were fatal) written by Hippocrates, and
included in the authentic corpus of the Hippocratic collection. This collection
represents the remains of the medical library of the school of medicine on the
island of Cos, the home of Hippocrates.
 Hippocrates was extraordinarily practical and some of his methods of dealing
with fractures and dislocations can be used even today. His freedom from
superstition enabled him to describe epilepsy, a symptom which previous ages
regarded as the most characteristic feature of the illness caused by demonic
possession, as being a mental disorder and not a curse from the gods or a gift of
prophecy. His sense of the ethics of medicine led to the elaboration of the
Hippocratic Oath, and his “aphorisms” provide gems which sparkle even
through the distance of time.
But he used singularly few, and mostly simple drugs. Therapeutics, as we know
it, began with Hippocrates.
Besides his factual, clinical cast of mind, his scientific method of enquiry, and
his ethical ideals, Hippocrates was also known for his discovery of the Vis
Medicatrix Naturae—the body will frequently heal itself if the patient and
physician give it half a chance.
One who was born within a very few years of the death of Hippocrates, and
who attempted to describe some of the remedies for diseases, was Theophrastus
of Eresos, a beloved pupil of Aristotle, and the pupil to whom, on his death,
Aristotle left his botanical garden and library. Theophrastus produced the
remarkable “De Historia Plantarum” the first extensive and precise herbal,
describing 455 plants and summarising all that was known at that time of the
medicinal properties of each; this contrasts with the 236 plants mentioned in the
Hippocratic writings.
An example of plant-lore which has survived from the writings of the Greeks to
our own times is the male fern (Dryopteris filix-mas).

3.2.1 Greek Medicine in Rome

As the empire of Greece eclipsed, medicine—still intellectually Greek medicine
—moved onwards through Alexandria to Rome. There, the medicine of the first
two centuries of the Christian era became dominated by the writings of three
great figures: Celsus, Discorides and Galen, each of whom continued the search
for new and important remedies.
However, an edict of Charlemagne in about 800 CE recommended the
cultivation of plants useful for medicine, including aniseed, coriander, fennel,
mint, rosemary, sage etc. During this period, if progress in Europe was
insignificant, the Arab world traversed a period of great scientific activity. Many
new drugs spread together with Arabic medicine: areca nut, cinnamon, musk,
lemon, nutmeg, manna, Nux vomica, senna, tamarind and camphor are among
the most important ones. Even though the medicine of Rome was almost entirely
in Greek hands, the best account of it is available in the “De medicina” of Celsus
(fl. CE 10–37). It is the remains of what was probably a great encyclopedia. Book
V and VI are vital ones from the point of view of therapeutics for they deal
directly with drug therapy.
Celsus gives a good account of the Roman pharmacopoeia. This
pharmacopoeia included many plant remedies from which we have now isolated
potent plant principles or alkaloids. An example is the henbane (Hyoscyamus
niger), which Celsus used as a hypnotic and from which the alkaloids,
hyoscyamine and hyoscine, can be extracted. Celsus showed much clinical
acumen and in ascites advises daily measurement of the abdominal girth, and
fluid intake and output. For dropsy, he advises, “It is useful also to suck a boiled
squill bulb”. This is the white squill of the BPC 1934. It contains potent
glycosides having a digitalis-like action on the heart and it is notable that the
practitioners of the days of Celsus knew of, and used appropriately, an effective
cardiac glycoside. The first century world also knew other useful drugs and,
amongst these, opium remains important today.
Thus, today’s doctor would not be totally helpless if he had the 1st century
pharmacopoeia. He would probably be glad to have travelled with Celsus and to
have gathered the sweet smelling Cretan thyme, Thymbra capitata, a source of
the antiseptic oil of thyme, which has properties approaching the antiseptic used
by Lister.

3.3 THE LONG INTERREGNUM

Vedic literature, written according to some around 2000 BCE, and in particular, the
Atharveda, contained medical references to the cure of rheumatism, neuralgia,
jaundice, tumours, bronchitis, skin diseases etc. We now traverse the vast period
of about 1500 years, between the death of Galen (CE 131–201), at the end of the
2nd century after the birth of Christ and about 200 years ago. We can divide this
period into the Middle Ages, ending with the capture of Constantinople by the
Turks in 1453, the Renaissance, carrying us to about CE 1600, and then the
separate two centuries that followed.

3.3.1 Middle Ages

With the death of Galen, original and experimental scientific enquiry in the
fields of medicine virtually ceased for over 1200 years. During this long period
of the Dark Ages, learning continued in the Muslim West. Thus, while Europe
lay an intellectual and material waste, the medical texts of Greece were
translated as they were carried eastward by the rise of the Nestorian Church and
then into the possession of Mohammedan physicians following the vast
expansion of the Arabian Empire after the birth and conquests of Mohammed
(CE 570–632).
In the mean time, new exotic drugs made their appearance in Europe: cinchona,
cocoa, ipecacuanha etc. Scientific missions, such as those of Condamine, Ruiz
and Pavon brought knowledge of new plants to the West, opening new avenues
for new investigations. A more precise knowledge of the vegetable world was
beginning. Europe in the Middle Ages, learnt its medicine from Latin
translations of the huge, unwieldy cannon of Iban Sina (CE 980–1037), the Prince
of Physicians, known in the Latin West as Avicenna. It learnt its materia medica
from the “Grabadin medicinarum particularium” usually attributed to Mesue the
Younger (died c. 1015). This book which taught Latin Europe the pharmacy and
therapeutics of the Arabs was enormously important and popular. Francis Adams
says “The Arabians added camphor, senna, musk, nux vomica, myrobalans,
tamarind and a good many other articles to the materia medica, but upon the
whole, they transmitted the science to us in much the same shape as regards
arrangement and general principles as they received it from their Grecian
master”. Adams’ statement does seem a fitting summary of a period whose
principal contribution was custodial. Perhaps, the most important fact that we
would wish to add to what Adams remarked is that the Arabians also made
medicinal use of mercury, prior to the discoveries of Ehrlich. Just before the
outbreak of World War I, scientific medicine recognised only three or four drugs
as being truly specific. Of these one was mercury, and was used in the treatment
of syphilis.
In the West, the Dark Ages were broken by little with the exception of the
foundation and growth of the School of Medicine in Salerno. Until 1000 CE,
classical learning persisted in the southern parts of Italy and where Greek was
spoken.
Of the many books written in the Medical School of Salerno, the most
important was the “Antidotarium” by Nicolaus Salenitanus (fc. 1140 CE). This
important book was the first work of its kind to be printed once the means of
doing so had been discovered. In England, medicine, before the Norman
conquest of 1006, was in the hands of the Sanon leeches, the ‘wise women’ of
the towns and villages, and the totally ignorant. All were at the mercy of a
mixture of herbal potions, charms, spells and gross superstitions.

3.3.2 Renaissance
Throughout the Middle Ages, the learned in Europe acknowledged theology as
their master. But, from about 1450, a group of events conspired to break the old
mould. These included the invention of printing, the fall of the Byzantium
Empire, the final proof to the world of heliocentric astronomy, the great voyages
of discovery like those of Christopher Columbus (1415–1506) and the discovery
of gunpowder.
The fall of Byzantium (later Constantinople and eventually modern Istanbul) to
the Turks in 1453, and the flight of the Byzantine scholars with their precious
manuscripts to Italy and other centres of learning in Europe, was one of the chief
means of the Renaissance. Byzantine documents were much closer to their
classical originals than anything else previously available in the West. A
beginning was made on what gradually became the main source of medical care
for the everyday people: Grocer-apothecaries, often called “pepperees”, arose
from the grocers and their guild, and began to specialise in herbs, their
importation and the drug trade.
In other hands, the breaking of the old mould was forceful and pragmatic,
rather than scholarly. Most notable was Philippus Aureolius Theophrastus
Bombustus von Honenheim (1493–1541), who called himself “Paracelsus”.
Garrison calls him the precursor of chemical pharmacology and therapeutics and
“the most original thinker of the sixteenth century…” Osler calls him “the
Luther of Medicine”. He introduced laudanum, tincture of opium and provoked
interest in the medicinal applications of chemistry.
The Renaissance still promoted interest in plant medicines, and this interest led
to the vast growth of herbals, some of which were of monumental proportion.
The most beautiful of the early German herbals was the “De historia stirpium
commenterii insignes” (1542) of Leonhart Fuchs (1501–1566). The best
remembered of the English herbalists was John Gerarde (1545–1612) whose
“The Herball or General Historie of Plantes”, first published in London in 1597,
became renowned.
 On the founding of the colonies in the New World, many new plants were
brought back, and these included ipeacac, cinchona, tobacco and a number of
other new drugs. The period also saw the appearance of the early
pharmacopoeias—including “Pharmacopoeia Londinensis”, of 1618 issued by
the College of Physicians in two editions; this was substantially later than the
Renaissance masterpiece, the “De humani corporis fabrica libri septum” (1543)
of Andreas Vesalius (1514–1564) who established modern anatomy, and
therefore surgery. They were, however, only ten years earlier than the equally
epoch-making “Exercitatio anatomica de motu cordis et sanguinis in
animalibus”(An Anatomical Exercise on the Motion of the Heart and Blood in
Animals) by William Harvey.
The litany of filth and rubbish clogging the pharmacopoeias was of no
attraction to Thomas Sydenham (1624–1689); known as “the English
Hippocrates” on account of his careful, close observation of patients and his
records of those studies. He was a forceful advocate of fresh air in sickrooms
and the use of a cooling, hygienic regimen in the management of smallpox. He
brought laudanum into practical English medical use, was one of the first to use
iron in anaemia and, even more importantly, popularised the use of the Peruvian
bark, quinine, for treating malaria.

3.3.3 Eighteenth Century

The 18th century began as part of the 17th century pastoral world, with the
Renaissance and post-Renaissance periods already some distance into the past; it
ended with the Industrial Revolution fully established and the population of
Great Britain still at not much over ten million. In medicine and the related
sciences, the century saw the development of the binomial classification by Carl
von Linné; the establishment of the modern bedside method of teaching by
Herman Boerhaave. The 18th century was, of course, also dominated by the two
Hunters—William Hunters, the most sought-after and successful obstetrician of
his day, and John Hunter, a comparative anatomist, human anatomist, collector
of exhibits of pathology on a vast scale, and a surgeon who “found surgery a
mechanical art and left it an experimental science”. The conventional
therapeutics and medical practice of the end of the century can be represented by
the excellent “A Treatise of the Materia Medica”, a work of 1789 published by
William Cullen (1710–1790), effective founder of the fine school of medicine in
Glasgow.
 W Heberden’s “Commentarii de morborum historia et curatione” of 1802 was
the last really important treatise on medicine written in Latin.
The herbal drugs, which at the beginning of the century had gained
considerable impetus after the isolation of the active principles, slowed in order
to follow a particular trend of applicative study. After Matthias Jacob Schleiden,
a professor in the University of Jena, had demonstrated the importance of the
microscope in defining the recognisable characteristics of drugs, the study of the
anatomical characters of crude drugs gained prime importance. The end of 18th
century was marked by two events—William Withering’s discovery of digitalis
in 1785 and Jenner’s invention of the smallpox vaccine in 1798. The beginning
of clinical pharmacology and modern immunology can be traced to these events.
William Withering (1741–1799) explored the use of the digitalis plant in
dropsy using an old country tradition. Withering is also the pioneer of drug
standardisation as a basis for effective control of dosage; dose titration when the
therapeutic–toxicity ratio of a drug is narrow, and the scientific study of dose–
response relationships.
Withering was undoubtedly one of the finest medical botanists; he can also be
seen as one who not only made an important discovery but established principles
extending far beyond the drug he discovered.
Edward Jenner (1749–1823) apparently following a Turkish tradition that
contact with cows or cowpox would protect against smallpox, introduced his
smallpox vaccine. He published his findings in his paper “An inquiry into the
causes and effects of the variolae vaccinae” in 1798.

3.3.4 Nineteenth Century

The 19th century witnessed the establishment of modern pharmacology; its
medicine and surgery were transformed by the discoveries of inhalation
anaesthesia and antisepsis. The understanding of the nature and cause of many
diseases was revolutionised by the development of cellular and germ theories.
In France, Pierre Charles Louis (1787–1872) founded medical statistics, and
René Théophile Laënnec (1781–1826) correlated the pathology of disease with
its physical manifestations in the living, in a manner which dwarfed in
importance his invention of the stethoscope in 1816.
Jacob Henle (1809–1885), in his article “Von den miasmen and contagien” (On
miasmas and contagions) of 1840 postulated the relationships between micro-
organisms and disease.
The greatest of pathologists and one of the towering figures in the
establishment of modern medicine was Rudolf Virchow (1821–1900), who
became the founder of cell pathology. The drugs of the early, mid, and late 19th
century comprise the alkaloids and glycosides; the early inhalational
anaesthetics; and the early analgesics and barbiturates. Other drugs, also
representing the beginning of synthetic medicinal chemistry were also
discovered.
At the beginning of the 19th century, Derosne isolated narcotine and morphine
from opium; Pelletier and Caventou isolated strychnine from Nux vomica and
quinine from cinchona; Robiquet, cantharidin from cantharides and caffeine
from coffee. The first presentation of alkaloids in literature is the paper of 1802,
“Memoire Sur Fopium” by Derosne. Once the chemical method of isolating
alkaloids was understood, the preparation of quinine, emetine, strychnine,
atropine, codeine and a number of other alkaloids soon followed. The discovery
and isolation of the early glycosides proceeded in parallel with that of the
alkaloids. This led to the isolation of salicin, the active constituent of willow, and
digitoxin, the active principles of digitalis. Salicylic acid was produced from
salicin as early as 1839.
The first etherosides were isolated at the same time; salicin from willow
(Leroux in 1830), amygdalin from bitter almonds (Robiquet in 1830),
crystallised digitalin from foxglove (Nativelle in 1868). Few contributions to
therapeutics have ranked with the discovery of the inhalational anaesthetics,
which permitted the enormous advances of modern surgery and eliminated the
surgical horror of the centuries before. The first to use ether vapour successfully
as an anaesthetic was Crawford Williamson Long (1815–1878). Sir James Young
Simpson (1811–1870), professor of obstetrics at Edinburgh, became the first to
use ether in midwifery.
The discoveries made at the end of the century included the introduction in
1899 of aspirin and in 1903, the synthesis of barbitone. The best of the contents
of the British Pharmacopoeia of 1864 seem to be digitalis, opium, atropine,
quinine sulphate, ether, chloroform, ferrous sulphate, iodine, sodium
bicarbonate, salt and a few others household remedies. To set against these
useful substances, there have been some toxic plant and mineral ingredients in
the materia medica.
3.4 TWENTIETH CENTURY
The potential of medicinal plants are everyday becoming more and more
obvious and new remedies to tackle dreadful diseases are being discovered.
Taxol and vincristine have been isolated for treatment of cancer. The source
plant of taxol is Talispatra (Taxus baccata linn), which is sthauneyaka in
Ayurveda and locally known as thuner. Vincristine is obtained from the plant
periwinkle (Vinca rosea), commonly known as sadabahar and found
everywhere. Among aromatic plants, guggulu (Comminphora mukul) has been in
prevalent use since time immemorial as a fumigation agent but later on was
found to be effective against hypercholestrolaemia which is confirmed by recent
experimental and clinical studies.
In India, a number of governmental and non-governmental organisations and
institutions are at present engaged in the study of medicinal plants in a scientific
way. Notable among them are the Central Drug Research Institute (CDRI), the
Central Institute of Medicinal and Aromatic Plants (CIMAP), the National
Botanical Research Institute (NBRI) (all in Lucknow) and the Tropical Botanic
Garden and Research Institute (TBGRI) (Thiruvanthapuram). There has also
been considerable progress in standardisation of raw material and finished
products. In recent years significant work has been done on ethnomedico-
botanical studies not only in India but all over the world. The main interest in the
medicine of the present century has been in its social implications and the
recognition of preventive medicine, on the one hand, and the development of
many effective means of therapy, on the other.
Upto 1910, scientific medicine recognised only a few drugs as specific
remedies: quinine, the alkaloid from cinchona which could eradicate certain
malarial parasites; emetine, the alkaloid from ipecacuanha which could
exterminate the protozoan causing amoebic dysentery; and mercury, which was
more toxic to the spirochacete of syphilis than to the syphilitic.
By 1907, Ehrlich had synethesised and tested over 600 arsenical compounds.
One such compound, solarsan, was used against Treponema pallidum in 1911. It
was 1914, before the anti-coagulant capability of sodium citrate and its potential
for use in blood transfusion was demonstrated, but the modern technique of
delivering blood by continuous drip infusion dates only from 1935.
During World War II there was a forced reduction in importation of raw drugs.
At the end of the war there was only a partial resumption of the importation, in
view of the fact that, owing to the development of the pharmaceutical industry,
many drugs were substituted by more modern drugs produced synthetically. This
had an influence on the number of drugs recorded in the Britannica
Pharmacopoeia and consequently on the curriculum of pharmaceutical studies.
The prognosis in many infections was dramatically changed by the
sulphonamides and totally transformed by penicillin. Modern methods of blood
transfusion allowed much surgical progress and a number of other drugs were
discovered. The efficacy of Vitamin B12 in pernicious anaemia was first shown
in 1948. The range and number of the antibiotics rapidly increased, largely due
to academic effort and the energies of the pharmaceutical companies.
In 1942, when sulphonamides were being used in typhoid fever, it was
observed that hypoglycaemia sometimes resulted. A. Loubatieres then made the
important discovery that the compound showed no such effect in a
pancreatectomised animal. This led to the demonstration that the antibacterial
agent, carbutamide, was useful in the treatment of diabetes mellitus and, soon
after this, to the introduction of tollbutamide, heralding the extensive use of the
sulphonyl ureas. This gradually led to the development of benzothiazides and
finally diuretic and anti-hypertensive agents.
The wide acceptance of the receptor theory led to the development in 1962 of
pronethalol and then propranolol followed by antihistamines and H2 blockers.
Against these achievements, there have been a limited number of drug disasters
—a number of drugs have been withdrawn due to unexpected adverse effects
and there have been a few incidences of drug side effects and adverse reactions.
The disaster of thalidomide, ginseng, pondimin and the supplements containing
β-carotene are some examples in this regard.

3.5 PROSPECTS FOR THE TWENTYFIRST CENTURY

With the populace becoming more and more aware of the adverse effects of
allopathic drugs, the ritual “pill for every ill” might be displaced by a demand
that drugs be used only when essential and a greater reliance might be placed
upon Hippocratic or holistic medicine. This would, hopefully, reduce the
incidence of iatrogenic diseases. The education of the patient and the practitioner
needed to affect these attitudes might be a major part of the practice of the next
few decades.
 Secondly, preventive medicine must be greatly appreciated; it has always been
more effective to prevent rather than cure a disease.
Thirdly, we must expect to see a rationalisation of the means of therapeutics.
The concept of a selected drugs list has now entered general medicine practice in
many advanced countries.
Fourthly, we can expect intense efforts in the research fields related to
cardiovascular disease and cancer.
Fifthly, we can assume that therapeutics will, in the short-term future, make
some attempt to bridge the gap between iatrogenic disease as seen on the small-
scale (by the general practitioner, for example) and the epidemiological or large-
scale community hazard (as seen by the drug regulator bodies). It is estimated
that post-marketing surveillance studies would need to include 1000 patients and
10000 controls if they were to detect, with 95% confidence, events occurring
under favourable circumstances for investigation in 1 in 3000 patients. A general
practitioner would be most unlikely ever to see such an event. Yet one death in
3000 patients (333 deaths in 1 million patients) would be a national disaster and
many drugs are sold to more than a million patients. We have, as yet, no
adequate means of detecting rare, serious adverse effects which must lead to the
withdrawal of drugs. This again would look like a direction for the therapeutics
of the future.
Sixthly, there will surely be new drugs. Some will arise from present-day
pharmacology: the angiotensin-converting enzyme inhibitors offer promise, and
the concept has lead to the recent introduction of many useful remedies. The
forefront of current knowledge offers promise: the cloning of factor VIII, upon
which most haemophiliacs depend, represents a technical triumph without
parallel and was reported in November 1981. Most startlingly of all, it has
become fairly clear that genetic engineering and related technologies represent
the biggest single advancement in the life sciences since the advent of the
antibiotics. Genetic engineering has already led to the production of pure human
insulin and has provided human growth hormone for the treatment of pituitary
dwarfism. The expectation is that we will be able to make, and use, molecules
which could never have become part of practical therapeutics, if extraction from
natural sources had been the only way to obtain them.
The examination of the drugs used in traditional medicine in the various
countries of the world is one of the priority programme of the WHO. In fact
from 1973–1974, the WHO with the UNICEF, made a collaborative study of
world health with the aim that all the peoples of the world should have health-
care up to a target date fixed as the year 2000. As the cost of the general
diffusion of the health-care system of the developed countries was deemed too
high as to render the achievement of the aim within the prefixed time impossible,
it was proposed that alternative methods represented by traditional therapeutic
agents be used.
To summarise, medicinal and aromatic plants have great potentiality in the
amelioration of suffering in human and other living beings. Therefore, keeping
step with a global awareness in the field there should be a sustained effort to
study, cultivate and propagate these plants.
 4

Essentials of Crude
Drugs

Plants affect different facets of life such as cultural, economical, medical and
spiritual. Since time immemorial, plants have been extensively used by man for
the treatment of myriad illnesses. This has been discovered from the
hieroglyphics on clay tablets etched by man even before he was able to record
the medicinal value of plants on paprous parchment.
Plants have played a major role as the basic source in several key industries,
thus being responsible for stabilising and enhancing the economy of developing
countries like India. Many valuable compounds like morphine, quinine,
reserpine, vincristine, vinblastine etc. have been obtained from plant source.
Plants provide us with nearly 30% of the medicines and drugs that we use, so
much so, that many pharmaceutical companies have collecting centres
specifically set up in many forests of the third world and developing countries
for collecting plants used as drugs. The list of such drugs is endless: opium,
ginseng, cinchona, sandalwood, cascara, cinnamon, digitalis, drugs got from
olives, rauvolfia roots (sarpagandha), Madagascar periwinkle (vinca rosea), the
bark of arjuna, Indian pennywort (brahmi), the species of dioscorea, ergot fungi,
the species of ephedra etc. The occurrence and distribution of these important
taxa are the ultimate resultants of medicobotanical studies carried out in various
forests. In order to unearth more and more of these medicinally and
economically important compounds, intensive medicobotanical explorations
have been encouraged in the country. The practice of traditional herbal remedies
has been a hallmark of the indigenous medical traditions of India and especially
these of the tribal medicine of our country. It is interesting to note that folk
medicines have contributed to modern medicine in almost all the parts of the
world.
 In view of the significance of plants, it is worthwhile to document this
knowledge, as it will greatly help in the advancement of scientific work on
plants. Research work on these lines has already been carried out in different
countries where attempts have been made to utilise the immense plant wealth
available at arms reach. But is has to be intensified.
The data on different species of plants given in this chapter provide valuable
information for ethnobotanical and ethnobiological investigation. Attempts have
been made to delineate biological source, chemical constituents and the medical
attributes of the plant species which are being used in the various systems of
medicine for different diseases.

Common name: Acacia

Botanical name: Acacia senegal Family: Leguminosae Parts

used: Exudate from stems and branches Chemical constituents:
Carbohydrates; polysaccharides; arabin which on hydrolysis yields
galactose, arabinose, rhamnose and glucuronic acids.
Uses: Demulcent; emulsifying agent; binding agent.

Common name: Aconite

Botanical name: Aconitum napellus Family: Ranunculaceae Parts

used: Dried root Chemical constituents: Alkaloids: aconitine;
picroacontine; aconine; mesaconitine; hypoconitine; napelline;
neopelline and neoline in traces.
Uses: Tincture of aconite is used in neuralgia and rheumatism;
cardiac irritant; and for catarrhal ailments in homeopathy.

Common name: Agar

Botanical name: Gelidium amansii

lamouroux, G. cartilagineum; G. pristoides Family: Geleidiaceae
Parts used: Dried complete plant Chemical constituents:
Heterogenous polysaccharides (which consists of calcium salts of a
sulphuric ester of a carbohydrate complex): (a) agarose (70%), (b)
agropectin (an acidic sulphonated compound).
Uses: Bulk laxative; in preparation of vaginal capsules and
suppositories; to prepare nutrient media in bacteriological culture;
industrial applications like emulsions and sizing, silk textiles etc.

Common name: Ajwain

Botanical Name: Trachyspermum roxburghianum Family:

Apiaceae Parts used: Fruits Chemical constituents: Volatile oil:
limonene; α-terpinene; d-linalool; thymol Uses: Carminative;
digestive; tonic; anthelmintic; antidiarrhoeal; antirheumatic. The
drug also possesses antibiotic and diuretic properties.

Common name: Alginate

Botanical name: Laminara species Family: Laminariaceae Parts

used: The purified carbohydrate product from the complete plant
Chemical constituents: Sodium salt of alginic acid (alginic acid is a
polyuronide containing 1,4 linkage residues of D-mannuronic acid
and L-glucuronic acids).
Uses: In cosmetics; in dermatological preparations; in dental
preparations; in adhesive paste; in textile industry.

Common name: Aloe

Botanical name: Aloe barbadensis Family: Liliaceae Parts used:

Dried juice of the leaves Chemical constituents: Glycosides:
anthracene; barbaloin or aloin, isobarbaloin; aloinosides A and B
Uses: Laxative; used externally for ulcus cruris; eczema and burns;
used in cosmetics.
 Common name: American aloe,
century plant (Hindi: Banskeora)

Botanical name: Agave americana Family: Agavaceae Parts used:

Sap/juice Chemical constituents: Isoflavonoids; alkaloids;
coumarins; vitamins A, B1, B2
Uses: Demulcent; laxative; antiseptic; digestive; used in
tuberculosis.

Common name: Arjun

Botanical name: Terminalia arjuna Family: Combretaceae Parts

used: Dried bark Chemical constituents: Tannins; arjunglucosides;
phytosterols; organic acids; sugars etc.
Uses: Mild diuretic; astringent, tonic and febrifuge; beneficial
effects in ischaemic heart disease.

Common name: Artemisia

(sea wormwood)
Botanical name: Artemisia cina Berg and A. maritima Family:
Compositae Parts used: Unexpanded flower heads Chemical
constituents: Terpenoids: sesquiterpene, santonin, β-santonin;
volatile oil: cineole; artemisin.
Uses: Anthelmintic.

Common name: Asafoetida

Botanical name: Ferula asafoetida Family: Umbelliferae Parts

used: Oleo-gum resin obtained by incising the lining of the
rhizomes of roots Chemical constituents: Volatile oil: isobutyl
propanyl disulphide; resins (40–65%): free asaresinotannol in
combination with ferulic acid; gums.
Uses: Carminative; expectorant;
antispasmodic; laxative.

Common name: Ascatic storax

(liquid amber)

Botanical name: Liquidamber orientalis Family: Hamamelidaceae

Parts used: From the wounded stem Chemical constituents:
Resins; balsamic acids: cinnamic acid (16–24%); cinnamic acid
ester, storesinol (25%).
Uses: Pharmaceutical aid; antiseptic.

Common name: Asoka

Botanical name: Saraca indica. L.

Family: Leguminosae Parts used: Dried stem bark Chemical
constituents: Tannins and crystalline glycosides in traces.
Uses: Astringent; remedy for uterine disorders; internal piles and
dysentery.

Common name: Aswagandha

(Winter cherry)

Botanical name: Withania somnifera Family: Solanaceae Parts

used: Dried roots and stems Chemical constituents: Pyrazole
alkaloids: withasomnine, uschohygrine, anahygrine, anaferine;
steroidal. lactones: withanolides.
Uses: Sedative; tonic; aphrodisiac; toning up the uterus;
bacteriostatic and anti-tumourous drug.

Common name: Bael

(Indian quince)

Botanical name: Aegle marmelos Family: Rutaceae Parts used:

Entire unripe or half ripe fruit Chemical constituents:
Furocoumarines: marmelosin; tannins (20%); reducing sugars.
Uses: In the treatment of chronic diarrhoea; cooling effect.
 Common name: Balsam of Tolu

Botanical name: Myroxylon balsamum L.

Family: Leguminosae Parts used: Trunk (by incision) Chemical
constituents: Resins (80%); volatile oil (7.8%): benzyl benzoate;
organic acids: cinnamic acid (2.15%); benzoic acid (5%).
Uses: Expectorant; flavouring agent; antiseptic.

Common name: Bamboo

(Hindi: Tabasheer, Banse)

Botanical Name: Bambusa arundinacea Family: Bambusaceae

Parts used: Roots, leaves, sprouts, grains and siliceous deposits in
clumps.
Uses: Astringent; laxative; diuretic; tonic; antileprotic; anthelmintic;
antidiarrhoeal and a wound healing agent. The drug is also used in
haemorrhoides, inflammations and in general debility.

Common name: Banyan

(Hindi: Bat)
Botanical name: Ficus benghlensis Family: Moraceae Parts used:
Bark, leaves, buds, fruits and latex Chemical constituents:
Flavonoids; glycosides and leucocyanidin glycoside.
Uses: Astringent; refrigerant;
anti-inflammatory; anti-arthritic;
anti-diarrhoeal; anti-emetic and tonic.

Common name: Barley

(Hindi: Jaun)

Botanical Name: Hordeum vulgare Family: Poaceae Parts used:

Grains.
Chemical constituents: Aminoacids:
arginine; histidine; lysine; tyrosine;
tryptophan; phenylalanine; cystine;
methionine; leucine; isoleucine; glycine.
Uses: Astringent; emollient; diuretic; aphrodisiac; digestive and
tonic. The drug is also useful in asthma, fever, ulcers and anaemia.

Common name: Belliric myrobalan

(Hindi: Bahera)

Botanical name: Terminalia bellirica Family: Combretaceae Parts

used: Dried fruits Chemical constituents: Tannins: gallic acid,
ellagic acid; ethyl gallate; galloyl glucose; chebulagic acid;
mannitol; glucose; galactose; fructose and rhamnose; fixed oil:
palmitic, stearic, oleic and linoleic acids.
Uses: Astringent; tonic and antiseptic; treatment of diarrhoea and
dysentery; purgative; anti-diabetic.

Common name: Betelnut

(Hindi: Supari)
Botanical name: Areca catechu Family: Palmaceae Parts used:
Seeds, roots and leaves.
Chemical constituents: Kernel contains tannins; catechin; gallic
acid; oily matter; gum; glucosides (50–60%); alkaloids:
arecoline, arecaine, arecadine, guvacoline; lipids consisting of
lauric, oleic and
myristic acids.
Uses: Astringent, stimulant, anthelmintic. The ethanolic extract of
nuts has shown
remarkable oxytocic activity at a dose of 100 mg on isolated rat
uterus.

Common name: Bitter almond

Botanical name: Prunus amygdalus Family: Rosaceae Parts used:

Seeds Chemical constituents: Cyanogenetic glycosides:
amygdalin; fixed oil
(40–55%); protein, mucilage etc; volatile oil: benzaldehyde (80%);
HCN (2–4%).
Uses: An ingredient in cough
remedies; vehicle for oily injection;
a flavouring agent.

Common name: Black henbane

Botanical name: Hyoscyamus niger Family: Solanaceae Parts

used: Dried leaves and flowering tops Chemical constituents:
Tropane
alkaloids: hyoscyamine (60%),
scopolamine (40%); atropine;
apoatropine; cuskhygrine.
Uses: Mydriatic; anti-spasmodic;
anti-muscarinic effect; anti-sialogogue.
 Common name: Black-oil tree
(Hindi: Malkangni)

Botanical name: Celastrus paniculatus Family: Celastraceae Parts

used: Seeds, leaves and oil Chemical constituents: Oil; tannins;
glucosides; colouring matter and alkalaids like celopagine,
celophanigine and celapanine.
Uses: Tranquillizer; laxative; digestant; improves memory;
rubefacient and stimulant.

Common name: Black peppercorns

(Hindi: Kalimirch)

Botanical Name: Piper nigrum Family: Piperaceae Parts used:

Fruits Chemical constituents: The fruits contain volatile alkaloids –
piperine and chavicine. The essential oil contains phellandrene;
caryophyllene; caryophyllene oxide; sabenene; myrcene; limonene
α- and β-pinenes, α-benganotne, α-humulene, β-cymene and α-
selinene.
Uses: Digestive; anthelmintic; carminative and rubefacient. The
drug is also used as a bioavailability enhancer.

Common name: Bogbean

Botanical name: Menyanthes trifoliata Family: Gentianaceae

Parts used: Whole plant Chemical constituents: Gentianin;
scoupolitin; quercetin; rutin;
chlorogenic acid; caffeic acid;
ferulic acid.
Uses: In the treatment of headaches.

Common name: Burma mangrove (Hindi: Kankra)

Botanical name: Bruguiera gymnorhiza Family: Rhizophoraceae
Parts used: Bark and root Chemical constituents: Carbohydrates;
sterols: β-sitosterol, cholesterol, campesterol,
28–isofucosterol; α-amyrin;
β-amyrin; leupeol and ursolic acid.
Uses: In syphilis; cancer and diabetes.

Common name: Cacao

Botanical name: Theobroma cacao Family: Sterculiaceae Parts

used: Seeds Chemical constituents: Xanthines and fixed oil. The
seeds contain a very small amount of endorphins, which are
powerful pain killers that occur naturally within the body.
Uses: Heart and kidney tonic; nervous system stimulant; diuretic. It
is also used as a base for suppositories and pessaries.

Common name: Camphor tree

Botanical name: Cinnamomum camphora Family: Lauraceae

Parts used: Wood of stems and roots Chemical constituents:
Terpenoids: bicyclic menoterpene and ketone; borneol; terpineol; 1–
8 cineole; pinene; phellandrene; eugenol and safrole.
Uses: Topical anti-pruritic; mild
antiseptic and carminitive.

Common name: Caper spurge

Botanical name: Euphorbia lathyrus Family: Euphorbiaceae Parts

used: Seeds, latex Chemical constituents: Fixed oil; resin;
euphorbone present in the latex.
Uses: Purgative.
 Common name: Capsicum

Botanical name: Capsicum frutescens or C. annuum L.

Family: Solanaceae Parts used: Dried, ripe fruit Chemical
constituents: Pungent phenolic compounds; capsaicinoids, a
mixture of 5 isomeric acid amides:
capsaicin (70%), homocapsaicin,
dihydrocapsaicin; caroteneoids; ascrobic acid; fixed oil; flavonoids.
Uses: Carminative; nerve stimulant; increases capillarity of blood
vessels; source of vitamin C; in rheumatism
of joints.

Common name: Caraway

Botanical name: Carum carvi Family: Umbelliferae Parts used:

Dried, ripe fruits Chemical constituents: Volatile oil (3–7%); d-
carvone; d-limonene; carveol; dihydrocarvone; geraniol etc; fatty oil
(18–23%); protein (21–23%).
Uses: Spasmolytic; carminative, as a spice.

Common name: Cardamom

Botanical name: Elettaria cardamomum Family: Zingiberaceae

Parts used: Dried, nearly ripe fruit.
Chemical constituents: Volatile oil (3.5–7% in fruits and 8–9% in
seeds); 1,8 cineole, limonene; borneol; 1-terpineol; terpenylacetate;
starch and fatty oil.
Uses: Carminative; flavouring agent.

Common name: Carrot

(Hindi: Gajar)
Botanical name: Daucus carota Family: Apiaceae Parts used:
Seeds and roots.
Chemical constituents: The seed oil contains carotol, daucol,
bisabolene. The root oil contains terbineline, carryophenline and
sabinene.
Uses: In spice blends, for flavouring alcoholic beverages.

Common name: Cascara sagrada

(sacred bark)

Botanical name: Rhamnus purshiana DC.

Family: Rhamnaceae Parts used: Dried bark Chemical
constituents: Glycosides: anthracene glycosides, C-glycosides (80–
90%) comprising cascarosides
A & B, cascarosides C & D; barbolain; chrysaloin; O-glycoside (10–
20%): emodin oxanthrone glucoside, homodianthrones of emodin,
heterodianthrones like palmidin A, B and C.
Uses: Cathartic.

Common name: Cassia

(Chinese cinnamon)

Botanical name: Cinnamomum cassia Family: Lauraceae Parts

used: Dried stem bark Chemical constituents: Volatile oil:
cinnamic aldehyde (75– 90%); terpene aldehyde and esters.
Uses: Flavouring agent; mild astringent; powerful germicide.

Common name: Castor oil

Botanical name: Ricinus communis Family: Euphorbiaceae Parts

used: Seeds Chemical constituents: Lipids: fixed oils (44– 53%);
triricinolin (75%)
which on hydrolysis yield ricinolic acid.
Uses: Cathartic; in soap industry; lubricant.

Common name: Cedar

Botanical name: Cedrus libani Family: Pinaceae Parts used:

Leaves and wood.
Chemical constituents: Volatile oil: cedrene; atlantol and atlanton.
Uses: Antiseptic; expectorant, in tuberculosis; in diabetes.

Common name: Chaste tree

Botanical name: vitex agnus-castus

Family: Verbenaceae Parts used: Berries Chemical constituents:
Viticin; casticin; penduletin; chrysophenol-D; ancubin; agnuside;
cineol; pinene and castine.
Uses: Used in eye diseases and in menstrual problems. It is also
used for stomach aches.

Common name: Chaulmoogra

(Marothi tree) oil
Botanical name: Hydnocarpus kurzii Family: Flacourtiaceae Parts
used: Fresh ripe seeds Chemical constituents: Mixture of
glycerides; fatty acids: hydnocarpic acid, chaulmoogric acid, garlic
acid, oleic acid, palmitic acid.
Uses: Treatment of leprosy and tuberculosis.

Common name: Chenopodium

(wormseed) oil

Botanical name: Chenopodium ambrosioides Family:

Chenopodiaceae Parts used: Flowers and fruits Chemical
constituents: Essential oil; peroxide; ascaridol (65–70%); p-cymol;
limonene; camphor; α-terpineol; pinene.
Uses: Anthelmintic, in combination also for intestinal infections.

Common name: Chirata

Botanical name: Swertia chirata Family: Gentianaceae Parts

used: Flowers Chemical constituents: Bitter principles (1.4–1.5%);
ophelic acid; chiratin; amarogentin.
Uses: Bitter tonic; stomachic, febrifuge.
 Common name: Cinnamon

Botanical name: Cinnamomum zeylanicum Family: Lauraceae

Parts used: Dried inner bark of the shoots of coppiced tree.
Chemical constituents: Volatile oil: cinnamic aldehyde (60–75%);
phenol (chiefly eugenol); alcohols, etc.
Uses: Flavouring agent; as an astringent; powerful germicide.

Common name: Clitoria

(Hindi: Aparajita)

Botanical name: Clitoria ternatea Family: Fabaceae Parts used:

Roots, leaves and seeds Chemical constituents: 3-mono-glucoside,
3-rutinoside, 3-neohesperidoside, aparagitin and β-sitosterol.
Uses: Refrigerant; laxative, aphrodisiac; anthelmintic and in
bronchitis.

Common name: Clove

Botanical name: Eugenia caryophyllus Family: Myrtaceae Parts

used: Dried flower Chemical constituents: Volatile oil: phenol
chiefly eugenol, acetyl eugenol; humulen; α- & β caryophyllene;
tannins.
Uses: Stimulant; aromatic; and in dental preparations.

Common name: Club moss

Botanical name: Lycopodium clavatum Family: Lycopodiaceae

Parts used: Moss, spores Chemical constituents: Alkaloids (0.1–
0.2%); polyphenols; flavonoids and triterpenes.
Uses: Diuretic; sedative; anti-spasmodic and digestive.

Common name: Cnidium

Botanical name: Cnidium officinale Family: Apiaceae Parts used:

Rhizome.
Chemical constituents: Cindilide;
neocindilide; lingustilide; preglenolone; ferulic acid.
Uses: Sedative, anti-spasmodic, anti-bacterial.

Common name: Coconut

(Hindi: Nariyal)

Botanical name: Cocos nucifera Family: Palmae Parts used:

Fruit, oil, juice, kernel, flowers and roots.
Chemical constituents: Enzymes like oxidase, catalase. Fresh
kernel contains nitrogenous substances; fat, lignin. Palm sugar milk
in the coconut contains sugars; gums; albumin; tartaric acid and
mineral water. Coconut oil contains free caprylic acid; glycerides of
lauric acid; palmitic acid and stearic acids. Other constituents like
saccharose; myoinositol; sorbitol; diphenyl urea; aliphatic alcohols;
leucoanthocyanins; polyphenols; cocositol; α- and β-amyrin, and
alkalaids namely ligustrazine and 2, 3, 5 -trimethyl pyrazine.
Uses: Anthelmintic; nutrient;
antipyretic; diuretic; cooling and demulcent.

Common name: Colchicum

(autumn crocus)
Botanical name: Colchicum luteum Family: Liliaceae Parts used:
Corm and seeds Chemical constituents: Alkaloids: colchicines
(0.8% in seeds and 0.6% in corms ), demecolcine.
Uses: Relieves gout; demecolcine is
effectively used against epidermal cancer and myeloid leukaemia,
colchicine is used to induce polyploidy.

Common name: Colocynth

(bitter apple)

Botanical name: Citrullus colocynthis Family: Cucurbitaceae

Parts used: Dried pulp of unripe but
full grown fruit Chemical constituents: Resins;
tritepenoids; cucurbitacin E and I;
phytosteoid glycoides; bitter principles.
Uses: Powerful cathartic.

Common name: Colophony

(pine resin)

Botanical name: Pinus species Family: Pinaceae Parts used:

Residue, after distilling the oleoresin from various species of Pinus
Chemical constituents: Resin acids: abietic acid (90%); neutral
inert
substance (resenes); fats.
Uses: In ointments and medicinal
plasters; manufacturing of varnishes
and disinfectant liquids.

Common name: Common basil

(Hindi: Babul);
Sacred basil (Hindi: Tulsi)

Botanical name: Ocimum species like O. sanctum, O. basilicum.

Family: Labiatae Parts used: Fresh and dried leaves.
Chemical constituents: Essential oil: phenols (eugenol 70%), nerol,
eugenol; methyl ether; caryophyllene; terpinene 4-ol; α-selinene and
β-pinene; camphor and carvacol.
Uses: Expectorant; stomachic;
carminative; refrigerant and febrifuge; anti-bacterial; insecticidal
and
anti-protozoal; anti-fertility agent.

Common name: Common foxglove

Botanical name: Digitalis purpurea Family: Scrophulariaceae

Parts used: Leaf Chemical constituents: Digitoxigenin;
gitoxigenin; gitaloxigenin.
Uses: Cardiotonic; diuretic.

Common name: Common plum

(Hindi: Alubukhara)

Botanical name: Prunus domestica Family: Rosaceae Parts used:

Fruits Chemical constituents: Sugars:
D-galactose, D-mannose, L-arabinose, D-xylose, L-rhamnose,
glucuronic acid; flavonoids: kaemferol, kaempferide,
leucoanthocyanidin.
Uses: Laxative; refrigerant; stomachic and as tonic.

Common name: Coriander

Botanical name: Coriandrum sativum Family: Umbelliferae Parts

used: Dried, ripe fruits Chemical constituents: Volatile oil: linalool
(60–0%); terpenes (20%): geraniol, boeneol, citronellal; fatty oil and
proteins.
Uses: Carminative; stimulant and aromatic.

Common name: Cornflower

Botanical name: Centaurea cyanus Family: Asteraceae Parts

used: Flowers, seeds and leaves Chemical constituents:
Flavonoids; sesquiterpene lactones and coumarins.
Uses: Bitter tonic; stimulant; digestive and mild laxative.

Common name: Costus

Botanical name: Saussurea lappa Family: Compositae Parts used:

Dried roots Chemical constituents: Volatile oil (1.5–2.5%):
custanolide (a new sesquiterpene lactone); resinoids; alkaloids and
tannins Uses: Stimulant; in cough; asthma; fever; dyspepsia and
skin diseases;
in perfumes.

Common name: Cotton

Botanical name: Gossypium

herbaceum L.
Family: Malvaceae Parts used: Epidermal trichomes of the seeds
Chemical constituents: Carbohydrates: polysaccharide
cellulose (90%); wax; oil and fat in traces.
Uses: Main constituent of surgical dressings; filtering medium and
insulating material.
 Common name: Creat
(Hindi: Kalmegh)

Botanical name: Andrographis paniculata Family: Acanthaceae

Parts used: Dried or fresh entire aerial portion of the plant
Chemical constituents: Bitter
principles: andrographolides;
flavonoids: echidinin; wightin-serpyllin.
Uses: Bitter tonic and stomachic;
relieves griping, irregular stools and loss of appetite in case of
infants;
febrifuge; general debility.

Common name: Croton

(Hindi: Jamalgota)

Botanical Name: Croton tiglium Family: Euphorbianceae.

Parts used: Seeds and oil Chemical constituents: Seeds contain
fixed oil containing stearic, palmitic, crotonic
and tiglic acids; proteins (18%); glucoside–
crotonoside; alakaloids and the enzyme pipase.
Uses: Cathartic; digestive; febrifuge; anti-inflammatory;
expectorant; rubefacient and useful in bronchitis, fever, leucoderma,
convulsions, urolithiasis and psychological disorders.

Common name: Cumin

(Hindi: Safed Zeera)

Botanical name: Cuminum cyminum Family: Umbelliferae Parts

used: Fruits Chemical constituents: Fatty oil; resin; mucilage;
gum; proteins; essential oil. The characteristic odour of cumin oil is
attributed to the presence of seminaldehyde, 1, 3-p menthadien-1al
and 1, 4-p menthadien-7 al.
Uses: Carminative; aromatic; stomachic; stimulant; aphrodisiac;
astringent.
 Common name: Cutch tree,
catechu (black)

Botanical name: Acacia catechu Family: Leguminosae Parts

used: Heart wood Chemical constituents: Tannins:
catechins; catechutannic acid (25–60%); flavonoids; quercetin and
its derivatives.
Uses: Astringent; digestive; used in relaxed conditions of throat,
mouth and gums.

Common name: Deadly nightshade; Indian belladonna

Botanical name: Atropa belladonna; A. acuminata Family:

Solanaceae Parts used: Dried root and root stock Chemical
constituents: Tropane alkaloids; L-hyoscyamine (90%); D, L-
hyocyamine; scopolamine (10%); apoatropine (+); belladonine (+).
Uses: Mydriatic; anti-spasmodic;
anti-muscarinic; anti-sialagogue.

Common name: Desert Indian wheat

(Hindi: Isapgol)

Botanical name: Plantago ovata Family: Plantaginaceae Parts

used: Dried seeds Chemical constituents: Carbohydrates; mucilage
(pentosan + aldobionic acid); pentosan; aldobionic acid (upon
hydrolysis yields
galacturonic acid + rhamnose) Uses: Demulcent; in chronic
constipation; chronic dysentery of amoebic and bacillary origin;
chronic diarrhoea.

Common name: Dill

Botanical name: Anethum graveolens L.
Family: Umbelliferae Parts used: Dried fruit Chemical
constituents: Volatile oil (3–35%): carvone dihydrocarvone in
traces; d-limonene; phellandrene.
Uses: Carminative; stimulant and aromatic.

Common name: Dwarf cavendish banana

Botanical name: Musa nana lour Family: Musaceae Parts used:

Leaves and roots Chemical constituents: α and δ tropherol; β-
carotene; riboflavin; niacin; ascorbic acid; proteins; iron and
carbohydrates.
Uses: In constipation; painful urination, and
in haemorrhoids.

Common name: Embelia

(Hindi: Vayvidang)

Botanical name: Embelia ribes Family: Myrsinaceae Parts used:

Dried fruits Chemical constituents: Embelin (2.5%); embelic acid;
vilangin.
Uses: Anthelmintic; Anti-fertility agent.

Common name: Ephedra

Botanical name: Ephedra geradiana Family: Gnetaceae Parts

used: Dried young stems Chemical constituents: Alkaloidal
amines: ephedrine (30–90%);
pseudoephedrine; norpseudoephedrine.
Uses: Ephedrine is used in asthmatic conditions and hay fever.
 Common name: Ergot

Botanical name: Claviceps purpurea Family: Hypocreaceae Parts

used: Dried sclerotium Chemical constituents: Indole
alkaloids: water soluble-ergometrine group: ergometrine,
ergometrinine; water insoluble (ether soluble)
ergotamine group: ergotamine,
ergosine, ergosinine; ergotoxine
group: ergocristine, ergocorstinine,
ergocryptine, ergocryptinine,
ergocornine, ergocorninine.
Uses: Oxytocic (ergometrine group); anti-epinephrine activity
(ergotamine group); treatment of persistent
migraine; sympatholytic.

Common name: Eucalyptus

Botanical name: Eucalyptus globulus Family: Myrtaceae Parts

used: Leaves Chemical constituents: Volatile oil:
eucalyptol/cineole, piperitone;
phellandrene; eraniol; geranyl
acetate; citronellal; gallo-tannins.
Uses: Antiseptic; anti-bacterial and anti-tuberculosis; diaphoretic;
expectorant.
 Common name: Euodia

Botanical name: Euodia officinalis Family: Rutaceae Parts used:

Fruits Chemical constituents: Alkaloids: eudoamine; rutaecarpine;
higenamine; dehydroeuodiamine.
Uses: Analgesic; cardiotonic; anti-hypertensive.

Common name: Fennel

Botanical name: Foeniculum vulgare Family: Umbelliferae Parts

used: Dried, ripe fruits Chemical constituents: Volatile oil:
anethole; a phenolic ether: d-fenchone; a ketone: methyl chavicol;
terpineol etc; fatty oil (12–18%) and protein (14–22%).
Uses: Spasmolytic; carminative; stomachic; galactagogue; comforts
belly ache in infants; eye wash.

Common name: Galangal, India root (Hindi: Rasna)

Botanical name: Alpinia officinarum Family: Zingiberaceae Parts

used: Rhizomes Chemical constituents: Essential oil (0.5–1%):
cineole, α-pinene, eugenol; sesquiterpene; alcohol; alpinol (a resin);
phlobaphenes (tannins); kaempferide; galagin and alpinin
(flavonoids).
Uses: Stomachic; stimulant and carminative; in rheumatism and
catarrhal affections; anti-bacterial and anti-fungal.
 Common name: Gambier
or catechu (pale)

Botanical name: Uncaria gambier Family: Rubiaceae Parts used:

Leaves and shoots Chemical constituents: Tannins
(condensed tannins): catechins
(7–33%); catechutannic acid (22–50%); flavanoids: quercetin and a
fluorescent substance.
Uses: Astringent; used in cases of diarrhoea; used in dye and
tanning industries.

Common name: Garlic (Hindi: Lehsun)

Botanical name: Allium sativum Family: Liliaceae Parts used:

Fresh compound bulb Chemical constituents: Essential oil: allin, a
sulphur containing amino acid; allicin: allyl sulphide; polysulphides
responsible for the unpleasant smell
of the oil.
Uses: Flavouring agent; antibacterial;
in cases of hypertension and
artheosclerosis; treatment of malignant tumours; carminative; gastric
stimulant; as insecticide.

Common name: German chamomile

Botanical name: Chamomilla recutita Family: Asteraceae Parts

used: Flowers Chemical constituents: Volatile oil; flavonoids;
bitter glycosides; coumarins and tannins.
Uses: Anti-inflammatory;
anti-spasmodic and anti-allergic.
 Common name: Ginger

Botanical name: Zingiber officinale Family: Zingiberaceae Parts

used: Rhizomes Chemical constituents: Volatile oil: sesquiterpene,
zingiberene;
sesquiterpene alcohol; zingiberol; borneol; linalool; geraniol etc
aldehyde: citral; pungent principles (5–8%): gingerol, shogaol,
zingerone; resinous matter; starch; mucilage.
Uses: Carminative, stimulant,
flavouring agent.

Common name: Guar gum

Botanical name: Cyanopsis tetragonoloba Family: Leguminosae

Parts used: Refined endosperm of the seed Chemical constituents:
Carbohydrate; gums: guaran, the water soluble portion
of the gum which yields on hydrolysis galactose 35% and mannose
60–65%.
Uses: Protective colloid; binding and disintegrating agent in tablet
preparations; in bulk laxatives; in peptic ulcer therapy.

Common name: Gum benzoin

Botanical name: Styrax benzoin,

S. paralleloneurus Family: Styraceae Parts used: resin Chemical
constituents: Balsamic acids: free cinnamic and benzoic acids and
their esters; triterpenoid acids: siaresinolic acid; coniferyl benzoate
and cinnamyl benzoate.
Uses: Expectorant; antiseptic, tincture is used in cosmetic
preparations.
 Common name: Hawthorn

Botanical name: Crataegus pinnatifida Family: Rosaceae Parts

used: Fruits Chemical constituents: Amygdalin; quercetin;
chlorogenic acid; ursolic acid Uses: Digestive; anti-bacterial;
hypotensive; used in abdominal distension; pain; diarrhoea.

Common name: Himalayan may apple

Botanical name: Podophyllum hexandrum Family: Berberidaceae

Parts used: Dried rhizomes Chemical constituents: Resins (10–
18%):
lignan resin; podophyllin (a mixture
containing podophyllotoxin (40%); α peltain, β peltatin in traces);
quercetin derivatives (flavonoid).
Uses: In the treatment of Condylomata
acuminatium; anti-mitotic effect; violent
cathartic (American variety only) and
etoposide (synthetic derivative of
podophyllotoxin) is used to cure small
cell and testicular cancer.
 Common name: Honey

Botanical name: Apis dorsata,

A. indica, A. florea etc.
Family: Apidae Parts used: Sugary secretion deposited in the
honey comb Chemical constituents: Carbohydrates; invert sugars;
sucrose; enzymes; vitamins; micro elements and mineral substances
and organic acids.
Uses: Pharmaceutical aid; nutrient and demulcent, laxative; pill
excipient.

Common name: Horsetail

Botanical name: Equisetum arvense Family: Equisetaceae Parts

used: Aerial parts Chemical constituents: Silicic acid and silicates;
flavonoids; alkaloids and steroids.
Uses: Clotting agent; astringent; in rheumatic conditions.

Common name: Hyssop

Botanical name: Hyssopus officinalis Family: Lamiaceae Parts

used: Aerial parts Chemical constituents: Volatile oil:
pinocaphone, pinene, camphene, etc.
Uses: Expectorant; diaphoretic;
stimulant; carminative

Common name: Indian gooseberry (Hindi: Amla)

Botanical name: Emblica officinalis Family: Euphorbiaceae Parts
used: Fresh or dried fruit Chemical constituents: Tannins;
phyllembelin–gallic acid; ellagic acid and glucose; pectins and
vitamin C.
Uses: Liver tonic, as a purgative,
treatment of jaundice, dyspepsia and cough; cooling, diuretic, anti-
bacterial and anti-fungal; anti-spasmodic; used
in gastritis syndrome.

Common name: Indian hemp (Cannabis)

Botanical name: Cannabis sativa L.

Family: Cannabinaceae Parts used: Dried flowering tops
Chemical constituents: Resins
(2.5%–15%): tetrahydro-cannabinol; cannabidiolic acid.
Uses: Sedative; analgesic; hypnotic; anti-bacterial agent.

Common name: Indian long pepper (Hindi: Pipal)

Botanical name: Piper longum Family: Pipersaceae Parts used:

Dried fruits Chemical constituents: Essential oil (1–2.5%): α & β
pinene, phellandrene, dipentene etc; alkaloids (5–9%):
piperine (1–2%), piperetine and
chavicine; resins; starch.
Uses: Aromatic; stomachic and
carminative; in acute bronchitis.
 Common name: Indian lotus
(Hindi: Kanval)

Botanical name: Nelumbo nucifera Family: Nymphaeceae Parts

used: Leaves and seeds Chemical constituents: Alkaloids:
nuciferine, roemerine, nornuciferine; flavonoids: quercitin; proteins;
sugars and vitamins.
Uses: In spermatorrhea, insomnia, haemorrhage.

Common name: Indian pennywort

(Hindi: Brahmi)

Botanical name: Centella asiatica Family: Umbelliferae Parts

used: Fresh and dried leaves Chemical constituents: Saponin
glycosides: brahmoside, brahminoside; triterpacids: brahmic acid
and
isobrahmic acid; thankuniside.
Uses: Sedative, anti-protozoal, in case of epilepsy and other
neurological conditions; blood purifier; anti-bacterial.
 Common name: Indian senna

Botanical name: Cassia angustifolia Family: Leguminosae Parts

used: Dried leaves and pods Chemical constituents: Anthracene
and dianthrone glycosides (leaves: 3%; pods: 1.5–2.5%);
homodiantherone
glycosides: sennoside A (0.3–0.45%), sennoside B (0.3–0.5%);
hetero-dianthrone glycosides: sennoside C and sennoside D; rhein;
aloe emodin; flavonoids: kaempferol glycosides.
Uses: Laxative.

Common name: Indian squill

Botanical name: Urginea indica Family: Liliaceae Parts used:

Bulbs Chemical constituents: Cardiac
glycosides; bufadienolides.
Uses: In heart ailments; diuretic in cardiac edema; rodenticide.

Common name: Indian valerian

Botanical name: Valeriana wallichii Family: Valerianaceae Parts

used: Dried, stolons, roots and rhizomes Chemical constituents:
Valepotriates (0.5–3%); valtratum; essential oil (0.5–1.5%);
valeriosidat, alkaloids
in traces.
Uses: Sedative; remedy for hysteria.

Common name: Japanese cornelian dogwood

Botanical name: Cornus officinalis Family: Cornaceae Parts used:

Pseudocarp Chemical constituents: Iridoids: loganin; seeroside;
tannins and lactins.
Uses: Anti-asthmatic, anti-bacterial.

Common name: Japanese rose

Botanical name: Rosa rugosa Family: Rosaceae Parts used:

Flower and fruit Chemical constituents: Flavonol
glycosides; quercetin; apigenin;
7-O-glycoside; anthocyanidin; tannins: galic acid; essential oils:
linalool,
geraniol and sesquiterpenes.
Uses: In atherosclerosis, diabetes and diarrhoea.

Common name: Kokum butter tree (Indian butter tree)

Botanical name: Garcinia indica Family: Guttiferae Parts used:
Seeds Chemical constituents: Lipids; fatty acids: stearic (56.4%),
oleic (39.4%), palmitic (2.5%) and linoleic (1.7%).
Uses: Nutritive; demulcent, astringent and emollient;
pharmaceutical aid in preparation of ointments.

Common name: Kombe arrow poison

Botanical name: Strophanthus kombe; S. gratus Family:

Apocynaceae Parts used: Seeds Chemical constituents: Cardiac
glycosides; cardenolides: k-stropanthin.
Uses: In acute cardiac failure; diuretic in cardic edema.

Common name: Kurchi

(Tellicherry bark)

Botanical name: Holarrhena antidysentrica Family: Apocynaceae

Parts used: Dried stem bark Chemical constituents: Steroidal
alkaloids (2–4%); conessine;
nor-conessine; isoconessine; kurchicine; holarrhimine and
holarrhenine.
Uses: Treatment of dysentery; astringent and tonic; in the synthesis
of steroid hormones.

Common name: Land caltrops

(Hindi: Gokhru)

Botanical name: Tribulus terrestris Family: Zygophyllaceae Parts

used: Fruits Chemical constituents: Saponin glycosides; steroidal
sapogenins; protodioscin.
Uses: Diuretic, cooling, tonic and
aphrodisiac, in painful micturition,
and impotence.

Common name: Lanolin

Botanical name: Ovis aries Family: Bovidae Parts used: Wool

Chemical constituents: Lipids; fats; cholesterol; isocholesterol;
esters of fatty acids like lanopalmitic, lanoceric, caranubic, oleic,
myristic etc.
Uses: Pharmaceutical aid.

Common name: Lemon grass oil

Botanical name: Cymbopogon citratus Family: Graminae Parts

used: Leaves Chemical constituents: Essential oil; citral (65–
86%); geraniol; myrcin; methyl heptenone; dipentene;
citronellal; linaol; α-terpinol.
Uses: In the commercial production of citral; in the soap industry.

Common name: Leopard lily

Botanical name: Belamcanda chinensis Family: Iridaceae Parts
used: Rhizome Chemical constituents: Blemcandin; tectoridin;
shekanin and iridin.
Uses: Anti-inflammatory; expectorant and in dysmenorrhoea.

Common name: Lime

Botanical name: Citrus limon Family: Rutaceae Parts used: Outer

part of the ripe or nearly ripe fruit Chemical constituents: Volatile
oil; aldehydes like citral and citronellal; terpenes: limonene (90%)
etc;
flavanoids: neohesperidin and
naringin, vitamin C.
Uses: Pharmaceutical aid.

Common name: Linseed

Botanical name: Linum usitatissimum Family: Linaceae Parts

used: Dried ripe seed Chemical constituents: Fixed oil;
mucilage; protein; cyanogenetic
glycosides; linomarin (1–5%).
Uses: Externally as a poultice;
internally as demulcent; laxative in haemorrhoids and to extract
linseed oil.

Common name: Liquorice

Botanical name: Glycyrrhiza glabra Family: Leguminosae Parts
used: Dried, peeled or unpeeled root and stolon Chemical
constituents: Saponin glyosides; 18 β glycyrrhetic acid (aglycone);
flavonoids: liquirintin; liquirtigenin; isoliquiritin;
isoliquritigenin; coumarins: hermiarin and umbelliferone.
Uses: Demulcent and expectorant;
laxative in combination with other drugs; anti-inflammatory agent in
dermatological
preparations; spasmolytic agent.

Common name: Lobelia

Botanical name: Lobelia nicotianaefolia Family: Lobeliaceae

Parts used: Dried ariel parts Chemical constituents: Alkaloids;
piperdine type; lobeline; isolobeline Uses: In spasmodic asthma;
chronic bronchitis; in cases of resuscitation.

Common name: Machoti

Botanical name: Polygonam aviculari Family: Polygonaceae Parts
used: Aerial parts Chemical constituents: Tannins;
flavonoids; polyphenols; silicic acid
and mucilage.
Uses: Astringent; diuretic;
anti-diarrhoeal; anthelmintic,
to stop nosebleeds.

Common name: Malay tea

(Hindi: Bavchi)

Botanical name: Psoralea corylifolia Family: Leguminosae Parts

used: Dried fruits Chemical constituents: Tropane alkaloids (0.2–
0.5%): L-hyoscyamine; D, L-hyscyamine; scopolamine (10%);
apoatropine (D), belladonine (t).
Uses: Mydriatic; anti-spasmodic;
anti-muscarinic; anti-sialagogue.

Common name: Male fern

Botanical name: Dryopteris filix-mass Family: Aspidiaceae Parts

used: Rhizomes and frond bases.
Chemical constituents: Oleoresins (6.5–15%); phloroglucinol
derivatives; aspidinol, albaspidine;
filicic acid (filicin).
Uses: Anthelmintic.

Common name: Maypop

Botanical Name: Passiflora incarnata Family: Passifloraceae

Parts used: Leaves Chemical constituents: Alkaloids: harmine;
harmaline; vitexine; isovitexine and other constituents like
scopoletin and umbelliferone.
Uses: Used in cases of neuralgia,
insomnia, violent headache,
dysmenorrhoea. Also used as a sedative, hypnotic and
antispasmodic.

Common name: Mexican Pricklypoppy

(Hindi: Bhatbhamt, Satyanas)

Botanical name: Argemone mexicana Family: Papaveraceae Parts

used: Seeds, fresh root and milky juice.
Chemical constituents: Alkaloids such as allocryptomine,
berberine, cheilanthi foline, chelerythrine, dihydrosanguinarine,
morsanguinarine, oxyhydrastatine, sanguinarine, mexicanol, α- and
β-stylopine, isorhamnetin, isorhamnetin-3-glucoside and
isorhamnetin-3, 7-diglucoside.
Uses: Diuretic; hypnotic; laxative; nauseant; emetic; pectorant.
 Common name: Mint (Hindi: Pudina)

Botanical name: Mentha arvensis Family: Labiatae Parts used:

Leaves and flowering tops Chemical constituents: Volatile oil
(5%): menthol (40–60%), dementholised oil (50–60%); menthyl
acid acetate (24.4%); free menthol (44.8%); menthone (24.6%) and
hydrocarbons like pinene, limonene, caryophyllene and cademene.
Uses: Pharmaceutical aid; in cold and cough; rheumatism;
headache,
carminative; antiseptic and
anti-fertility agent.

Common name: Morning glory

Botanical name: Ipomoea orizabensis Family: Convolvulaceae

Parts used: Dried root Chemical constituents: Resins (15%);
glycosidal resins (6–18%); jalapin; ipuranol; ipurganol; orizabin;
volatile oil; scopoletin etc.
Uses: Cathartic.

Common name: Morning glory

(ivy leaved) (Hindi: Kaladana)

Botanical name: Ipomoea hederacae Family: Convolulaceae Parts

used: Dried ripe seeds Chemical constituents: Resin
glycosides (8%); fixed oil; saponins.
Uses: Hydrogogue, purgative.

Common name: Motherwort

Botanical name: Leonurus cardiaca Family: Lamiaceae Parts

used: Aerial parts Chemical constituents: Alkaloids;
iridoids; flavonoids; thyanenes.
Uses: Cardio tonic; anti-spasmodic;
sedative; stimulates the muscles of the uterus and is particularly
suitable for delayed periods; premenstrual tension.

Common name: Musk root

(Hindi: Jatamansi)

Botanical name: Nardostachys jatamansi Family: Valerianaceae

Parts used: Dried rhizomes Chemical constituents: Volatile oil
(0.3–0.4%); sesquiterpenes; valeranone.
Uses: Sedative; in spasmodic attacks of hysteria, palpitation of
heart; substitute for valerian.

Common name: Mustard

Botanical name: Brassica nigra Family: Cruciferae Parts used:
Dried ripe seeds Chemical constituents: Fixed oil
(20–30); glycerides of oleic; linoleic
linolinic acids; mustard oil; volatile oil obtained on hydrolysing the
isothiocyanate glycoside sinigrin (1–1.2%); proteins; mucliage etc
(30%).
Uses: Rubefacient; used as a condiment; emetic in large doses; in
the preparation of liniments and plasters.

Common name: Myrobalan

Botanical name: Terminalia chebula Family: Combretaceae Parts

used: Dried fruits Chemical constituents: Tannins: pyrogallol type,
chebulagic acid; ellagi tannins: chebulinic acid, chebulic acid,
ellagic acid, gallic acid, etc.
Uses: Externally, in the treatment of chronic ulcers; wounds, as a
gargle in stomatitis; laxative; relieves headache; it is the chief
ingredient of triphala.

Common name: Myrrh

Botanical name: Commiphora molmol Family: Burseraceae Parts

used: Stems Chemical constituents: Volatile oil: eugenol; cuminic
aldehyde: α- pinene; limonene and sesqutiterpees; resin: resin acids
like α, β, γ-commiphoric acids.
Uses: Antiseptic; stimultant, used in mouth wash of tooth paste; in
perfume industry.
 Common name: Nutmeg

Botanical name: Myristica fragrans Family: Myristicaceae Parts

used: Dried kernels of seeds Chemical constituents: Volatile oil:
myristicin (4–8%); pinene; sabinene; camphene; safrol; eugenol;
methyl eugenol; isoeugenol; methyl isoeugenol; methoxy eugenol;
fixed oil; saponins, etc.
Uses: Flavouring agent; carminative; digestive; abortifacient.

Common name: Opium

Botanical name: Papaver somniferum Family: Papaveraceae Parts

used: Latex obtained from unripe capsules Chemical constituents:
Phenanthrene type-alkaloids: morphine (10%), codeine (0.5%),
thebaine; benzylisoquinoline type: papaverine (1%), noscopine
(6%), narceine; all in combination with meconic acid (3–5%) Uses:
Analgesic; hypnotic and narcotic; in case of cough and bronchitis;
diarrhoea and dysentery.

Common name: Orange

Botanical name: Citrus chrysocarpa Family: Rutaceae Parts used:

Dried outer part of the pericarp of ripe or nearly ripe fruits
Chemical constituents: Volatile oil (2.5%): limonene, citral,
citronellal and terpineol; linalyl and geranyl acetate; vitamin C,
glycoside; flavonoid; glycosides (5–14% ): hesperidin;
neohesperidin.
Uses: Flavouring agent; bitter tonic; sources of vitamin C and A.
 Common name: Oriental water-plantain

Botanical name: Alisma orientalis Family: Alismataceae Parts

used: Corms Chemical constituents: Essential oils; sterols; resins.
Uses: Dysuria; enteritis; diarrhoea.

Common name: Papaya

Botanical name: Carica papaya Family: Caricaceae Parts used:

Fruits, latex Chemical constituents: Cryptoxanthin; β-carotene;
violaxanthin; cryptoflavin; neoxanthin and chrysanthemaxanthin.
Uses: Fruits are thermogenic;
digestive; anthelmintic; anti-fungal;
aphrodisiac and anti-inflammatory.

Common name: Peanut oil

(Hindi: Arachis ) oil

Botanical name: Arachis hypogaea Family: Leguminosae Parts

used: Seeds Chemical constituents: Lipids: oleic,
linoleic, palmitic, stearic, archidic, behnic and lignoceric acids.
Uses: Pharmaceutical aid; lubricant; in the preparation of soaps.

Common name: Peepal tree

(sacred fig)
Botanical name: Ficus religiosa Family: Moraceae Parts used:
Bark, leaves, tender shoots, fruits, seeds and latex.
Chemical constituents: Phenolic
glucoside; steroids: β sytosterol;
carbohydrates: arabinose; mannose; glucose.
Uses: Astringent; aphrodisiac;
anti-bacterial; purgative, digestive etc.

Common name: Peppermint oil

Botanical name: Mentha piperita Family: Labiatae Parts used:

Fresh aerial parts Chemical constituents: Volatile oil: menthol
(50%), menthone (10–30%), piperitone; menthylester; menthofuran
(5–10%).
Uses: Carminative; spasmolytic;
cholagogue; mild anti-diarrhoeal; aromatic; stimulant; in tooth paste
preparation.
 Common name: Periploca of the woods (Hindi: Gudmar)

Botanical name: Gymnema sylvestre Family: Asclepiadaceae

Parts used: Whole plant/leaves Chemical constituents: Gymnema
saponins I–V, gymenic acid.
Uses: Anti-diabetic, cardiotonic;
astringent; anthelmintic; anti-inflammatory.

Common name: Picrorhiza

Botanical name: Picrorhiza kurroa Family: Scrophulariaceae

Parts used: Dried seeds and rhizomes Chemical constituents:
Iridoid
glycosides: picrorhizin, picroside A, kutkoside, kurrin (0.5%),
kutkiol, kutkisterol.
Uses: Bitter tonic; febrifuge;
cholagogue; stomachic, mild laxative; in malaria associated with
constipation.

Common name: Pigweed

(Hindi: Gadahpurna)

Botanical name: Boerhaavia diffusa Family: Nyctaginaceae Parts

used: Fresh or dried plant Chemical constituents: Alkaloid
(0.04%): punarnavine; steroids:
β-sitosterol, α-sitosterol, palmitic acid, ester of β sistosterol,
teteracosanoic acid, hexacosanic acid, stearic and archidic acids,
allantoin.
Uses: Diuretic; extensively used in edema of ascites.

Common name: Prickly amaranth

(Hindi: Cauleyi)
Botanical name: Amaranthus spinosus Family: Amaranthaceae
Parts used: Whole plant Chemical constituents: Sterols:
β-cytosterol, stigmasterol,
campesterol, cholesterol,
α-spinasterol and glycosides.
Uses: Diuretic; stomatic; anti-pyretic; febrifuge in leucorrhoea, etc.

Common name: Purple loosestrife

Botanical name: Lythrum salicaria Family: Lythraceae Parts

used: Aerial parts.
Chemical constituents: Glycoside: salicarin; tannins; volatile oil;
phytosterols and mucilage.
Uses: Anti-diarrhoeal; wound healing; in ophthalmic diseases.

Common name: Pyrethrum

Botanical name: Chrysanthemum cinerariifolium Family:

Compositae Parts used: Dried, closed or half open flower.
Chemical constituents: Insecticides (0.4–2%): terpene esters;
pyrethrin I (0.64%), pyrethrin II (0.65%), cinerin I (0.15%), cinerin
II (0.23%), jasmolin I, jasmolin II, essential oil (0.3%).
Uses: Contact insecticide.
 Common name: Quassia (bitter wood)

Botanical name: Picrasma excelsa, P. qassioides.

Family: Simarubaceae Parts used: Dried stem wood Chemical
constituents: Bitter principles of terpenoid nature; quassin;
picrasmin (or isoqassin); neoqassin.
Uses: Bitter tonic; to expel thread worms.

Common name: Quillaia (soapbark)

Botanical name: Quillaia saponaria Family: Rosaceae Parts used:

Dried inner bark Chemical constituents: Triterpenoid saponins:
quillaic acid, galacturonic acid, glucuronic acid, galactose.
Uses: In soap industry as detergents, shampoo, etc; emulsifying
agent; in tooth and paste powder.

Common name: Quince

Botanical name: Cydonia oblonga Family: Rosaceae Parts used:

Fruits and seeds Chemical constituents: Tannins; pectin; mucilage;
cynogenetic
glycosides and fixed oil.
Uses: In diarrhoea; mouth ulcers; gum problems; sore throat;
bronchitis and
as bulk laxative.
 Common name: Quinine bark

Botanical name: Cinchona calisaya,

C. ledgeriana, C. officinalis, C. succiruba Family: Rubiaceae Parts
used: Dried bark Chemical constituents: Quinoline alkaloids:
quinine (50–60%), quinidine, cinchonine, cinchonidine; quinovin, a
bitter glycoside; tannins: phlobatannins.
Uses: Quinine is used in the treatment of malarial fever, in the
preparation of tonic water; quinidine is used as a cardiac depressant.
It is characterised as an anti-arrythmic and anti-fibrillatory drug.

Common name: Radish (Hindi: Muli)

Botanical Name: Raphanus sativus Family: Cruciferae Parts used:

Leaves, roots and seeds Chemical constituents: Seeds contain
antibacterial principles: raphanin; glycosinolates; enzymes; pectic;
anthocyanin; arabinogalactin and proteins. The leaves contain
vitamin C.
Uses: The leaves, seeds and old roots are used in the treatment of
asthma and other chest complaints. The juice of the fresh leaves is
diuretic and laxative.The seed is carminative, diuretic, expectorant,
laxative and stomachic. The root is antiscorbutic, antispasmodic,
astringent, cholagogue, digestive and diuretic. It is antibacterial and
antifungal. The plant also shows anti-tumour activity.

Common name: Rauwolfia

Botanical name: Rauwolfia serpentina Family: Apocynaceae Parts

used: Dried roots with the bark intact Chemical constituents:
Indole alkaloids (1.5–3%); reserpine group: reserpine, rescinnamine,
desperidine; ajmaline group: ajmaline, ajmalicine; serpentine group:
serpentine, serptentinene and alstonine.
Uses: Sedative (reserpine group);
stimulates central and peripheral systems (ajmaline group); anti-
hypertensive
(serpentine group); in snake bite.

Common name: Red sandalwood

(Hindi: Baragunci)

Botanical name: Adenanthera pavonina Family: Mimosaceae

Parts used: Bark, leaves, seeds and heartwood Chemical
constituents: Fatty acids:
palmitic, stereic, arachidic, lignoceric,
linoleic and eicosenoic; glucoside; polysaccharide; β-citosterol and
stigmasterol Uses: Astringent; aphrodisiac;
anti-emetic and febrifuge.

Common name: Rhubarb

Botanical name: Rheum emodi;

Rheum webbianum.
Family: Polygonaceae Parts used: Dried rhizome Chemical
constituents: Anthracene derivatives (2–3%); chrysaphanol;
aloe-emodin; rhein; emodin and physcoin; heterodianthrones like
palmidin A, B, C and rheidin A, B, C etc; tannins: glucogallin
tetrarin; catechin and epicatechin, resins, fatty oil, etc.
Uses: Laxative.
 Common name: Saffron (Hindi: Kesar)

Botanical name: Crocus sativus L.

Family: Iridaceae Parts used: Dried stigma Chemical
constituents: Carotenoids; crocin (a coloured glycoside);
picrocrocin (a colourless bitter monoterpenoid glycoside); volatile
oil; terpenes; terpene alcohols and esters.
Uses: Flavouring; colouring agent; stomachic and anti-spasmodic;
stimulant and aphrodisiac; nerve sedative; emmenagogue.

Common name: Screw tree

(Hindi:Marodphali)

Botanical name: Heclicteres isora Family: Steruculiaceae Parts

used: Roots, bark and fruits Chemical constituents: Phytosterols;
saponins; phlobotannins and sugars.
Uses: Expectorant; demulcent; anti-diabetic and anti-diarrhoeal.

Common name: Senega, snake root

Botanical name: Polygala chinensis Family: Polygalaceae Parts

used: Roots Chemical constituents: Triterpenoidal saponin;
polygallic acid; senegin (4%).
Uses: Expectorant; in case of bronchitis.
 Common name: Sensitive plant
(Hindi: Lajvanti)

Botanical name: Mimosa pudica Family: Mimosaceae Parts used:

Whole plant Chemical constituents: Gentisic acid; jasmonic acid
and D-panitol.
Uses: In urinary infections, haemorrhoids; dysentery; fever; syphilis
and venereal diseases.

Common name: Sesame oil

Botanical name: Sesamum indicum Family: Pedaliaceae Parts

used: Seeds Chemical constituents: Lipids (45–60%); sesamolina
lignan of the unsaponifiable portion of the oil.
Uses: Pharmaceutical aid; nutritive; laxative; demulcent; synergist
for pyrethrum insecticides.

Common name: Shark-liver oil

Botanical name: Hypoprion brevirostris Parts used: Livers of the

shark Chemical constituents: Vitamin A; glycerides of saturated
and unsaturated fatty acids.
Uses: Source of vitamin A; treatment of xerophthalmia; in
combination with Vitamin D; nutritive.
 Common name: Silver birch

Botanical name: Betula pendula Family: Betulaceae Parts used:

Leaves, bark and sap Chemical constituents: Saponins; flavonoids;
tannins and volatile oils.
Uses: In rheumatic conditions; diuretic; antiseptic.

Common name: Slender dwarf morning glory (Hindi:

Shankhpushpi)

Botanical name: Evolvulus alsinoides Family: Convolvulaceae

Parts used: Dried plant Chemical constituents: Alkaloids: betaine;
evolvine.
Uses: Bitter tonic; in dysentery; chronic bronchitis and asthma;
anthelmintic; anti-inflammatory; sedative.
 Common name: Spiked ginger lily (Hindi: Kapur Kachri)

Botanical name: Hedichium spictum Family: Zingiberaceae Parts

used: Roots and rhizome.
Chemical constituents: Volatile oil (4%): cineoe, limonene, γ-
terpenene, β-phellandrene, p-cymene; linalool; β-terpineol; β-
caryophyllene; ethyl-p-methoxy cinnamate; ethyl cinnamate; d-
sabinene.
Uses: Insect repellant; stomachic; carminative; tonic and stimulant,
expectorant; emmenagogue. In liver complaints; mild tranquillizer;
anthelmintic, treatment of pulmonary eosinophilia.

Common name: Spindle tree

Botanical Name: Euonymus europaeus Family: Celastraceae Parts

used: Seeds Chemical constituents: Euonymol; euonysterol;
homoeuonysterol.
Uses: Used in liver disorders, profuse diarrhoea with blood and
mucous.

Common name: Starch

(corn; rice; wheat; potato)

Botanical name: Zea mays; Oryza sativa; Triticum stivum;

Solanum tuberosum Family: Graminae; Solanaceae Parts used:
Grains and tubers of potato etc.
Chemical constituents: Polysaccharides (water soluble amylose
and water insoluble amylopectin) Uses: Dusting powder;
pharmaceutical aid; antidote for iodine poisoning; nutrient and
demulcent.
 Common name: Strychnine tree (Nux-vomica)

Botanical name: Strychnus nux-vomica Family: Loganiaceae Parts

used: Dried ripe seeds Chemical constituents: Indole alkaloids
(2.5–5%): strychnine; brucine; glycoside; loganin; fixed oil.
Uses: Spinal cord stimulant; in case of fatigue; bitter stomachic.

Common name: Sweet flag

(Hind: Bacc)

Botanical name: Acorus calamus Family: Araceae Parts used:

Dried peeled and unpeeled rhizomes Chemical constituents:
Volatile oil (1.5–3.5%): α-asarone and β-asarone (8–19%).
Uses: Stomachic; anti-spasmodic and carminative; sedative; anti-
fungal; anti-bacterial and insecticidal; in bronchospasm.

Common name: Tamarind

Botanical name: Tamarindus indica Family: Leguminosae Parts
used: Dried ripe fruits Chemical constituents: Organic acids (10%)
and their salts like tartaric acid; citric acid; maleic acid: sodium and
potassium tartarate (8%); invert sugars (30–40%) Uses: Mild
laxative.

Common name: Tanner’s cassia

(Hindi: Tarval)

Botanical name: Cassia auriculata Family: Caesalpinaceae Parts

used: Roots, bark, leaves, flowers and seeds Chemical
constituents: Emodin; chrysophanol and rubiadin.
Uses: Astringent; in leprosy and asthma; anthelmintic; diarrhoea and
in diabetes.

Common name: Tannic acid

from oak gall

Botanical name: Quercus infectoria Family: Fagaceae Parts used:

Nutgalls Chemical constituents: Tannic acid; mixture of esters of
glucose with gallic acid.
Uses: Astringent; externally in treatment of burns; in alkaloidal
poisoning.

Common name: Thorn apple

(Hindi: Datura)

Botanical name: Datura metel Family: Solanaceae Parts used:

Dried leaves and flowering tops Chemical constituents: Alkaloids
(2–3%); tropane alkaloids: hyoscyamine; scopolamine and
flavonoids.
Uses: Mydiatic; anti-spasmodic; anti-muscainic and anti-sialagogue.

Common name: Tragacanth, hog gum

Botanical name: Astralagus gummifer Family: Leguminosae Parts

used: Stem Chemical constituents: Complex polysaccharide (water
soluble tragacanthin + water insoluble bassorin).
Uses: Demulcent; suspending agent; binding agent; emulsifying
agent; laxative.

Common name: Turmeric

Botanical name: Curcuma longa Family: Zingiberaceae Parts

used: Dried rhizomes Chemical constituents: Curcuminoids:
curcumin; desmethoxy curcumin; bidesmethoxy curcumin volatile
oil (5%) sugars; bitter substances; fixed oils and acids.
Uses: Cholertic and cholagogue; anti-inflammatory agent; aromatic;
stimulant; tonic and carminative; in respiratory diseases; to lower
blood cholesterol level; anti-microbial and anti-fertility agent.

Common name: Turpentine oil

from long-leaf pine

Botanical name: Pinus palustris Family: Pinaceae Parts used:

Resin Chemical constituents: Volatile oil: α-pinene, β-pinene,
traces of dipentene, methyl charvicol, bornyl acetate.
Uses: Mild antiseptic; counter-irritant; insecticide; expectorant and
in bronchitis; in the preparation of synthetic camphor.

Common name: Vasaka, Malabar nut

Botanical name: Adhatoda vasica Family: Acanthaceae Parts

used: Fresh and dried leaves Chemical constituents: Alkaloids
(0.25%), a quinazoline type: vasicine, oxyvasicine, vasicinone;
essential oil: adhatodic acid; vasakin.
Uses: Expectorant; oxytocic; abortifacient.

Common name: Vinca, Madagascar periwinkle

Botanical name: Catharanthus roseus Family: Apocynaceae Parts

used: Whole plant Chemical constituents: Alkaloids: roots
(0.85%), leaves (0.67%), stems (0.31%); indole and indoline
alkaloids; ajmalicine; lochnerine; serpentine; dimeric indole
alkaloids; vimblastine and vincristine.
Uses: Antineoplastic; treatment of Hodgkin’s disease; treatment of
leukaemia in children.
 Common name: Violet
(Hindi: Banafsha)

Botanical name: Viola odorata Family: Violaceae Parts used:

Aerial parts Chemical constituents: Alkaloid: isoquinoline-type-
violine; volatile oil.
Uses: Expectorant; diaphoretic; antipyretic and diuretic; emollient,
demulcent and emetic.

Common name: Viper’s bugloss

Botanical name: Echium vulgare Family: Boraginaceae Parts

used: Flowers Chemical constituents: Pyrrolizidine alkaloids;
allantine; alkannins and mucilage.
Uses: Diuretic; in dry cough.

Common name: White dittany, (burning bush)

Botanical name: Dictamnus albus Family: Rutaceae Parts used:

Roots and flowers Chemical constituents: Volatile oil: estragol,
anethol; alkaloid: dictamnin.
Uses: Abortifacient, anti-spasmodic.
 Common name: White water lily

Botanical name: Nymphaea alba Family: Nymphaeceae Parts

used: Rhizomes and flowers Chemical constituents: Alkaloids;
glycosides; tannins and resins.
Uses: Astringent; antiseptic; in vaginal soreness and discharge;
insomnia and anxiety.

Common name: Wild asparagus

(Hindi: Satavari)

Botanical name: Asparagus racemosus Family: Liliaceae Parts

used: Tuberous roots Chemical constituents: Glycosides: saponin,
saponin A4, A5, A6, A7, A8.
Uses: Anti-oxytocic; galactagogue; anti-cancer; diuretic;
aphrodisiac and nutritive tonic.

Common name: Wild cherry

Botanical name: Prunus serotina Family: Rosaceae Parts used:
Dried bark Chemical constituents: Cyanogenetic glycoside:
prunasin; coumarin glycosides scopoletin.
Uses: Sedative.

Common name: Wild snake root

Botanical Name: Asarum canadensis Family: Aristolochiaceae

Parts used: Root and rhizome Chemical constituents: Asarol;
methyl eugenol; asarin and starch.
Uses: antibiotic; used for indigestion, cramps, colds, coughs.

Common name: Wild strawberry

Botanical name: Fragaria vesca Family: Rosaceae Parts used:

Leaves and fruits Chemical constituents: Flavonoids; tannins;
volatile oil: methyl salicylate; borneol.
Uses: Mild astringent; diuretic, anti-diarrhoeal; in sore throat.
 Common name: Worm killer
(Hindi: Kiramar)

Botanical name: Aristolochia bracteolata Family:

Aristolochiaceae Parts used: Roots and leaves Chemical
constituents: Aristolochic acid; fatty acids: myristic, palmitic,
stearic, lignosaric and oleic acids.
Uses: Anthelmintic; cathartic, anti-inflammatory, etc.

Common name: Yam

Botanical name: Dioscorea deltoidea; D. prazeri; D. floribunda.

Family: Dioscoreaceae Parts used: Tubers Chemical constituents:
Saponin glycoides; steroidal sapogenins; diosgenin Uses: Steroid
precursor.

Common name: Yellow gentian

Botanical name: Gentiana lutea Family: Gentianaceae Parts used:

Dried rhizome and root Chemical constituents: Bitter glycosides:
gentiopicrin, amarogentin, alkaloid gentianine; xanthone
derivatives: gentisin, gentioside; sugars: gentiobiose, gentianose.
Uses: Bitter tonic, in cases of anorexia; dyspepsia.
 5

Medicinal Botany

The last chapter brought out the fact that different parts of a plant can be used to
make drugs. Sometimes many plants within a species itself can be used, for
example, the dried bark of Cinchona calisaya, and even C. ledgeriana and C.
officinalis can be used for the treatment of malaria. Therefore taxonomy can be
very helpful in determining which plants can be used as drugs.

5.1 PLANT TAXONOMY

Taxonomy may be defined as a science that includes identification, nomenclature
and classification of objects and is usually restricted to objects of biological
origin; when limited to plants, it is often referred to as systematic botany. Plant
taxonomy is an interdisciplinary science which requires the input of various
botanical sciences like morphology and anatomy. Till today, the morphological
characteristics of plants constitute indispensable means of classification and
diagnosis. Recent researches show that most branch of botany such as anatomy,
polynology and embryology contribute a lot to systematics. The taxonomic
contribution of cytogentics has also been enormous.
Identification, nomenclature and classification which together constitute
taxonomy can be defined as follows: Identification is the determination of a
taxon as being identical with or similar to another and already known element.
Nomenclature is concerned with the determination of the correct name of a
known plant according to an already defined nomenclatural system. The correct
authentication of the plant source of various indigenous systems of medicine is a
vital parameter in developing pharmacopoeial standards for herbal drugs and
hence the nomenclature of a plant is a subject of international importance.
Classification is the sorting of a plant into groups or categories according to a
particular plan or sequence and in confirmity with a nomenclatural system.
Besides systematics, chemotaxonomy has also made a great contribution
towards classification and phylogeny. Computers have an obvious role in dealing
with the large number of plants involved not only in the aspects of storage and
retrieval of information, but also in the science of numerical taxonomy, which
will probably play an important role in the development of systematics.
Heywood’s Modern Methods in Plant Taxonomy throws more light on the
significance of systematics. Plant taxonomy is important for the following
reasons:
1. It helps in learning about the different kinds of plants on the earth: their
names; their distinctions and affinities; their distribution and habitat
characteristics; and the correlation of these facets of knowledge with pertinent
scientific data contributed by research activities in related fields of botanical
endeavour.
2. It helps in the demonstration of the tremendous diversity of the plant world
and its relation to man’s understanding of evolution.

5.1.1 Identification of the Plant Family

Plants can be grouped under two major families—gymnosperms and
angiosperms. A plant is a gymnosperm or an angiosperm depending on the
following features:
Gymnosperms
Gymnosperms include cycads, ginkgo, taxads, conifers, gnetales and some
extinct taxa. Flowers are absent; seeds are naked and not enclosed in any ovary.
The seeds are wedged between the scales of a woody or sometimes pulpy scale
cone (though rarely enclosed in the scale). They are sometimes solitary, in pairs,
or on the margin of reduced specialised leaves with the pollen produced in soft
corners. Existing genera of the gymnosperms are few. Evidently they are relics
of a much larger group of plants which were dominant a long time ago. Only the
conifers are of marked importance in modern vegetation and the relatively few
genera of even this group belong to a small number of well-differentiated
families. Their dominant position in the ecological systems of many temperate
forests is due to the great size of the individuals rather than to the number of
plants or species.
Angiosperms
Angiosperms are the most common seed plants. The plants are either
monoecious or dioecious. The ovules (probably apetalous solitary
megasporangia) are variously disposed (often in panicles). Flowers are present
including: stamens or pistils or both with sepals and petals; seeds are enclosed in
an ovary.
Angiosperms may again be classified as monocotyledons and dicotyledons.
Monocotyledons
Monocotyledons are plants whose members of a floral series are in one whorl, as
the calyx, corolla, etc. The characteristic features of a monocotyledon can be
listed below:
Embryo: 1 cotyledon; stem: closed bundles; leaves: parallel venation; flowers:
usually 3-merous.
Dicotyledons
Plants of this class are differentiated by their possession of two cotyledons,
having a pair of seed lobes. The characteristic features of a dicotyledon can be
listed below:
Embryo: 2 cotyledons; stem: open vascular bundles; leaves: nettled venation;
flowers: usually 5-merous.
Dicotyledons may again be classified as polypetalae, gamopetalae, or
monochlamydeae, depending on the features present.
Polypetalae Flowers with both calyx and corolla, having several distinct petals.
Polypetalae may further be classified as:
Thalamiflorae: Here the parts of the flowers are hypogynous, separately inserted
on the thalamus.
Discifloral: Sepals and calyx may be distinct or united. A conspicuous disc is
present at the base of the ovary. Androecium stamen: usually definite in number;
ovary superior.
Calyciflorae: Plants having their petals and stamens adnate to the calyx.
Flowers: perigynous, or epigymouse; disc: rarely present; ovary often inferior.
Gamopetalae Two distinct whorls of perianth and the corolla are fused i.e. the
plants have the petals united.
Monochlamyde Monochlamydes are a large division of phanerogams which
have only one set of floral envelopes which is usually sepaloid.
Plants are classified under different families. The characteristics of some
common families are given below.
Amaranthaceae
Herb, shrubs, or under-shrubs; flowers: mostly bracted and perianth lobes mostly
scarious with green or purple mid-ribs; placenta basal; single ovule or seed:
erect; stipules: none or, if present either scarious or persistent and sheathing the
stem; involucre: none; fruit: usually an indehiscent utricle. The flowers are uni-
or bisexual; the stamens are 1–5 in number, opposite perianth members; ovary is
unilocular with a solitary basal ovule. The flowers are obscured among many
dry, scarious, persistent, separate bracts which are often coloured; the sepals are
also often scarious. Example: Spiny amaranth (Amaranthus spinosus)
Annonacea
Aromatic trees; woody trees or shrubs with alternative simple exstipulate and oil
gland-dotted leaves; flowers: usually 3-merous; bisexual or rarely unisexual;
perianth: arranged in two whorls—three green sepals in the outer whorl and
petals in the inner whorl. Sepals and petals in the bud meet only at the margins—
they do not overlap. Stamens and carpels: usually many and spirally arranged;
anther: has four pollen chambers; fruits: an etaerio of berries or achenes; seeds:
large. Example: Nutmeg (Monodora myristica)
Apicaceae/umbelliferae
They are generally herbs with simple or compound flowers. Flowers: umbels,
bisexual, epigynous; calyx: 5; petals and stamens: 5; ovary: bilocular with a
single pendulous ovule in each locule; fruit: schizocarpic with one-seeded
mericarp. Example: Coriander (Coriandrum sativum)
Araceae
Rhizomatous or tuberous; they are mostly herbaceous terrestrial plants with
milky, watery or shortly pungent sap. The plants are sometimes epiphytic with
aerial roots but commonly terrestrial. Leaves: simple or compound, solitary or
few, basal or cauline and alternant, petioled with membranous sheathing base;
flowers: bisexual or unisexual; perianth: rare in unisexual flowers and present in
bisexual ones, of 4–6, free or connate segments; stamens: 2, 4, or 8, hypogynous;
anthers; mostly 2-celled, dehiscing by pores or slits, free or connate; fruit: a
berry; seeds: mostly with endosperm. Example: Sweet flag (Acorus calamus)
Asteraceae/Compositae
Herbs or shrubs; leaves: alternate or opposite, frequently radical, simple or
pinnately or palmately lobed, divided or compound frequently in basal rosettes;
inflorescence: arranged in head or capitula; flowers: regular or zygomorphic,
unisexual or bisexual (the plants are then usually monoecious or dioecious);
calyx: usually modified into pappus with the pappus being very diverse in form
and type; corolla or ray florets; disc florets with valvate spreading lobes; ovary:
inferior, unilocular with a solitary basal ovule. Example: Wormseed (Artemisia
cina berg)
Berberidaceae
Shrubs or perennial herbs; leaves: simple or pinnately or ternately compound,
deciduous or evergreen, alternate or sometimes wholly basal; flowers: bisexual;
sepals: 4–6, or rarely none; anthers: usually opening by 2 pores each with a flap-
like covering; carpel: one; ovules: one–several; stamens: twice as many as the
petals; petals: 6 or 9; leaves: peltate, palmately 7–9 lobed, large; fruits: usually a
berry with the ovary withering and leaving the 2 seeds naked. Example:
Himalayan mayapple (Podophyllum hexandrum)
Boraginaceae
Herbs, shrubs or tree, usually scabrous or hairy but sometimes glabrous; leaves:
opposite with cystoliths, usually roughly hairy; inflorescence: determinate,
corymbose, usually of scorpoid cymes; calyx: usually 5-cleft, persistent; corolla:
tubular, funnel-shaped, lobes usually 5; stamens: usually 5, entire corolla tube;
ovary: bilocular with 2 ovules in each locule or tetralocular with 1 ovule in each
locule; fruit: drupe or of 2–4 nutlets. Example: Viper’s bugloss (Echium vulgare)
Bromeliaceae
Usually short-stemmed, herbaceous epiphytes; leaves: mostly basal and rosette
forming, rarely cauline, spirally alternate, mostly stiffly lorate, often spiny,
serrated and trough-like; inflorescence: a terminal head, spike raceme or panicle
with the flowers in the axils of often brightly coloured bracts; flowers: bisexual
or rarely functionally unisexual, actinomorphic or weakly zygomorphic;
perianth: of 2 series—the outer series of 3 herbaceous, calyx-like segments
(sepals) and the inner of 3 corolla-like segments with petals free or variously
connate, often brightly coloured, in some groups with scales or prominent
nectaries within; stamens: 5, often borne on the corolla; fruit: a perianth,
sometimes syncarpous; seeds: with copious mealy endosperm and small embryo.
Example: Pineapple (Ananas comosus)
Caesalpinacceae
Tree or shrubs, rarely herbs or climbers; flowers: usually bisexual, usually barely
perigynous; calyx: 5-parted, spathe-like; petals: free, nearly equal or similar;
androcium: 10, free, sometimes staminodes are present with the functional
stamens reduced; leaves: usually pinnate to tri-pinnate, sometimes palmate to
simple; stipules: present or sometimes none, sometimes reduced to glands, often
falling away as the leaf matures. Example: Tanner’s cassia (Cassia auriculata)
Caryophyllaceae
Annual or perennial herbs; inflorescence: a dichasial cyme ending into
monochasial cyme; flowers: bisexual or infrequently unisexual, actinomorphic,
5-merous, hypogynous; sepals: 4–5; petals: 4–5; stamens: usually twice the
number of petals; ovary: unilocular with free central placentation; stems:
characteristically with swollen nodes; leaves: opposite, simple, mostly linear to
lanceolate, often connected basally by a transverse line or by a shortly connate
perfoliate base, decussate; stipules: scarious or more often absent. Example:
Soap wort (Saponaria officinalis)
Cyperaceae
Perennial or infrequently annual grass-like or rush-like herbs, often present in
damp boggy soil; roots: fibrous from a very short or elongated and creeping
rhizome, the later rarely tuber-like; stems: mostly solid, often triquetrous,
generally unbranched below the inflorescence, frequently leafless; flowers: very
minute, subtended by chaffy bracts, bisexual or unisexual, arranged in spike-lets
in spicate, racemose, paniculate or umbellate inflorescence types; fruit: nut-like,
indehiscent, when from a 2-branched style pistil, often more or less 2-sided or
lenticular and when from a 3-branched style often 3-sided, the achene sometimes
enclosed in a sac or by a partially enveloping and connate glume; seed: with
albumen. Example: Nutgrass (Cyperus rotundus)
Dioscoreaceae
Twining herbaceous vines; leaves: simple; flowers: minute unisexual in spike or
racemes; ovary: 3-celled ovules two in each, unisexual; leaves: natted-veined;
stem: below ground, a rhizome or a caudex; roots: often tuberous; fruit: a
capsule or a berry; seeds: with endosperm. Example: Wild yam (Diascorea
deltoidea)
Euphorbiaceae
Monoecious or occasionally dioecious herbs, shrubs or plants which have seeds
giving out a milky latex; perianth: of a single whorl or of two distinct whorls or
even absent; stamen: one or more; ovary: usually trilocular with 1–2 pendulous
ovules in each locule; fruit: generally a regma; leaves: mostly alternate,
sometimes opposite or whorled, simple or variously compound, usually
stipulate; fruit: usually a schizocarp capsule splitting often elastically into 3 one-
seeded cocci that dehisce ventrallly; seeds: with straight or bent embryo and
copious soft fleshy to fleshy endosperm. Example: Indian gooseberry (Emblica
officinalis)
Fagaceae (Papilionaceae)
Deciduous or evergreen monoecious trees and shrubs; leaves: alternate, simple,
cleft, lobed or entire, deciduously stipulate; flowers: functionally unisexual,
apetalous, solitary staminate, staminate flowers with a 4–6–7 imbricately lobed
perianth of sepals; stamens: 4–40; ovary: inferior and adnate to a 4–6 lobed
perianth of sepals; fruit: one-seeded nut subtended or enveloped by a cupule or
involucre which may or may not be muricate; seed: with a large embryo and no
endosperm. Example: Aleppo oak (Quercus infectoria)
Fumariaceae
Glabrous, herbaceous plants with watery sap; flower: zygomorphic; sepals: 2;
petals: 4; stamens: 6 in two phalenges with 1–2 nectaries at base; carpels: 2;
ovary: unilocular, parietal placentae; fruit: usually 1-seeded nut, transversely
septate caspsule dehiscing by valves; seeds: with a minute embryo and copious
soft watery fleshy endosperm; leaves: alternate, in basal rosettes or cauline.
Example: Drug fumitory (Fumaria officinalis)
Gnetaceae/Ephedraceae
Shrubs to about 1 feet high or much less, erect, decumbent or climbing, much
branched, the branches usually green for several seasons; leaves: mostly
deciduous, opposite or whorled, more or less connate, basally and usually
reduced to membranous sheaths; plants dioecious or rarely monoecious;
staminate flowers: in compact sub-globose to oblong form, compound; ovule:
has 2 integuments, the outer integument is represented by 4 basally coherent
bracts, the inner has 2 similar bracts that elongate considerably at the time of
pollination and becomes style-like in appearance; seed: has leathery integument,
globose to cylindric form, membranous or winged or forming a berry-like
syncarp, often red in colour, with 2 cotyledons. Example: Ephedrine (Ephedra
gerardiana)
Lamiaceae/Labiatae
Predominantly annual or perennial herbs, sometimes under-shrubs or shrubs,
usually aromatic; leaves: simple, opposite or whorled, exstipulate often with
epidermal glands, secreting volatile oils; ovary: biocular, becoming tetralocular
by false septum with l ovule in each locule; style: gynobasic; stems and twigs:
usually quadrangular; the flowers are bracteolate or not, and each cyme is
usually subtended by a foliaceous bract that may exceed the cyme; flowers:
bisexual, zygomorphic, calyx persistent, imbricate, typically 5-lobed, sometimes
bilabiate; fruit: composed of typically 4 nut-lets, distinct or cohering in pairs,
enclosed within the persistent calyx; seed: with scant fleshy endosperm that is
often absorbed by the developing embryo. Example: Peppermint (Mentha
piperita)
Lauraceae
Usually aromatic trees or shrubs; leaves: simple, entire, usually leathery,
evergreen; flowers: bisexual, relatively small, hypogynous or perigynous,
commonly in panicles; sepals: usually 4 or 6, greenish yellow, yellow or white,
coalescent basally; petals: none; stamens: commonly 12 in 4 cycles of 3; anthers:
with 2 or 4 pollen chambers, with 4 pores each covered by a flap, the two outer
cycles opening inward, the third outward, the fourth usually being sterile; carpel:
1; ovule: 1; fruit: a drupe or drupe-like; seed: large with no endosperm.
Example: Camphor tree (Cinnamomum camphora)
Liliacea
Annual, aquatic or marsh plants, stem-less, fibrous roots from a very short
rhizome; leaves: basal; crowded, alternate, linear, terete and spongy, sheathing
basally, the sheaths often with minute hyaline scales within; flowers: all without
perianth, usually subtended by a bract, basal spikes all with pistillate flowers of
single sessile pistil; the perfect flowers are mostly bracteate and consist of a
staminate flower adjacent or closely appressed to and slightly below a pistillate
flower; fruit: a ribbed caryopsis or a winged caryopsis. Example: Wild asparagus
(Asparagus racemosus)
Malvaceae
Tree, shrubs or herbs; leaves: alternate, simple, entire or variously lobed, usually
palmately veined, stipulate; flowers: solitary, axillary or terminal, actinomorphic,
5-merous, hyphogynous; sepals: 5; petals: 5, twisted; stamens: many,
monodilphous and the staminal tubs are adnate to the base of the petals; ovary:
two–many celled; ovule: 1 or inner in each placentation axile; fruit: typically a
loculicidal capsule or the mature carpels of the ovary separate from one another
and from the axile, sometimes a berry; seed: often pubescent or comose, the
embryo straight or curved, the endosperm mostly present and often oily.
Example: Cotton plant (Gossypium herbaceum)
Meliacea
Trees or shrubs, young parts and inflorescence pubescent otherwise glabrous;
inflorescence: a compound cyme; flowers: bisexual, actinomorphic, often in
cymose panicles; calyx: usually imbricate and small; sepals: 4–5 usually basally
connate; corolla: controlled or imbricate; petals: 4–5, distinct or connate or
adnate to the staminal tube and then valvate; stamens: 4–12; ovary: 2–5 celled,
axile placentation; leaves: usually alternate, pinnately compound, decompound,
or rarely simple, lacking pellucid dots, estipulate; fruit: a berry, capsule, or rarely
a drupe; seeds: often winged, endosperm fleshy or none. Example: Neem
(Azadirachta indica)
Mimosaceae
Tree or shrub; flower: crowded in globose heads, actinomorphic; calyx and
corolla: valvate in bud; androecium: 8, numerous, much longer than the corolla.
Example: Sensitive plant (Mimosa pudica)
Monimiaceae
Aromatic trees or shrubs or rarely vines; leaves: simple, evergreen, usually
opposite or whorled but sometimes alternate, flowers: bisexual or sometimes
unisexual, perigynous; sepals: usually present, 4–8; petals: present or absent;
stamens: numerous or sometimes few and in one or two series; anthers: splitting
length-wise; filaments: with or without glands; carpels: numerous; ovule: one;
fruits: enclosed by the floral cup. Example: Balsam of Tolu (Peumus boldus)
Myristicaceae
Usually aromatic trees or shrubs; leaves: simple, entire, evergreen, alternate;
flowers: unisexual, small, in clusters of various types; calyx: usually 3-lobed;
petals: none; stamens: 2–30; filaments: coalescent; anthers: splitting length-wise;
carpel: 1; stigma and usually the style are present; ovule: 1; fruit: drupe-like, but
splitting open; seed: with a coloured fleshy aril and partly or wholly enveloped
by it. Example: Nutmeg (Myristica fragrans)
Nymphaeaceae
Aquatic, annual or perennial herbs; stem: cauline or generally rhizomatous, erect
or creeping; leaves: alternate, simple, mostly capillarily dissected and immersed,
usually floating or emersed, peltate, milky latex often present; flowers: solitary,
axillary; perianth: differentiated in calyx and corolla; sepals: 3–5; petals: 3–5 or
many; stamens: many; carpels: 3 or more embedded torus; fruit: a follicle and
aggregate of indehiscent nut-lets or a leathery tardily dehiscent berry; seeds:
with straight embryo and the endosperm starchy. Example: Water lily
(Nymphaea alba)
Orchidaceae
Perennial herbs, terrestrial, usually succulent; roots: fibrous or tuberous or cord-
like; leaves: alternate or rarely opposite or whorled, simple, often distichous and
sometimes closely imbricated; flowers: terminal spike-like raceme; perianth: 6 in
two series; androecium: confluent with the style into a column, pollen grain in
waxy or powdery masses; ovary: unilocular with 3 parietal placentae; fruit: a
capsule, dehiscing by 3–6 hygroscopically sensitive valves which remain
apically connate; seeds: very abundant, minute, often fusiform, without
endosperm, the embryo undifferentiated. Example: Marsh orchid (Orchis
latifolia)
Papaveraceae
Herbaceous annuals or perennials, rarely shrubs and rarely trees; leaves:
alternate, entire to pinnately or palmately cleft; estipulate flowers mostly
solitary, bisexual, actinomorphic; perianth: biseriate or 3-seriate; calyx: of 2–3
distinct sepals; corolla: 4–6; petals: in 1– whorls, distinct, imbricate, often
crumpled in bud; fruit: a capsule, dehiscing by pores or values, rarely
indehiscent; seed with minute embryo and copious oily or mealy endosperm.
Example: Opium (Papaver somniferum)
Pinaceae
Trees, rarely shrubs; branches whorled or opposite or rarely alternate; leaves:
spirally arranged or fascicled, linear, needle-like, mostly persistent, sometimes
deciduous, monoecious; fruit: a woody cone with dry usually winged seed,
usually two on each scale, the embryo with 2–15 cotyledons. Example: Cedar of
Lebanon (Cedrus libani)
Piperaceae
Erect or scandent herbs, shrubs or infrequently trees, evergreen when woody, the
stems with vascular strands distinct and sometimes somewhat scattered as in
monocots, frequently succulent when herbaceous, nodes often jointed or
swollen; leaves: alternate, rarely opposite or whorled, petiolate, entire, the
stipules adnate to petiole when present; flowers: very minute, bracteate, usually
bisexual or in some species unisexual, generally in dense fleshy spikes or the
spikes umbellate; perianth: absent; stamens: 1–10, hypogynous, the filaments
usually distinct; anthers: each of 2 distinct or confluent cells, dehiscence by
longitudinal slits; pistil: one; ovary: superior, 1-loculed; carpel: 2–5; ovule:
solitary basal orthotropous; style 0–1; stigmas: 1–5; fruit: a small drupe; seed:
small with endosperm and minute embryo. Example: Black pepper corn (Piper
nigrum)
Poaceae/Gramineae
Annual or pernnial herbs; stem: terete or flattened, mostly fistular, often
composed of rhizomes below ground and of culms above, the culms hollow
except at the nodes or infrequently solid, with a central pithy tissue; leaves:
distichous with sheathing base, linear and greatly elongated or rarely broad,
alternate and fundamentally in two ranks on the stem; flower: uni- or bisexual,
bilaterally symmetrical but not obviously so, in spike-lets of imbricate bracts;
perianth: of 2–3 minute scales or none; stamens: 3 or rarely 6 or more; carpels:
3; styles and stigmas: 2 with the stigmas being feathery; ovary: superior,
unilocular with a single basal ovule; fruit: a caryopsis or rarely a nut or a berry
or a urticle; endosperm abundant. Example: Common barley (Hordeum vulgare)
Polygonaceae
Herb, rarely shrubs or small trees with alternating leaves having sheathing
stipules; inflorescence: a panicle; perianth: usually 3–6, often coloured,
imbricate, persistent; flowers: bisexual, zygomorphic; stamens: 4–8; ovary:
unilocular with basal ovule; fruit: a trigonous on biconvex nutlet; seed: often
hairy with a conspicuous micropylar aril or callosity, the embryo straight, axial,
the endosperm soft fleshy. Example: Knotweed (Polygonum aviculare)
Ranuneulaceae
Annual or perennial herbs or climbing shrubs; leaves: radical or alternate, most
generally palmate, mostly estipulate; flowers: uni- or bisexual, regular or
irregular; actinomorphic, zygomorphic; petals: absent or 3–5 or more; sepals: 5
or more, usually deciduous; stamens: indefinite; carpels: usually numerous and
free; fruit: typically a follicle, sometimes an achene, berry or rarely a capsule;
seed: with minute embryo and a copious generally watery fleshy endosperm.
Example: Monk’s hood (Aconitum napellus)
Rosaceae
Herbs or shrubs or trees; leaves: simple or compound, stipulate, alternate,
sometimes caducous or adnate to petiole; flowers: usually bisexual or
infrequently unisexual, actinomorphic; perianth: biseriate or one or both series
absent, generally perigynous, basal portions usually adnate into a hypanthium;
calyx: typically of 5 basally connate sepals; petals: usually present; stamens:
numerous and in one or several whorls of 5 stamens each, perigynous around the
gynoecium; anthers: small, 2-celled dehiscing longitudinally; fruit: an achene,
follicle, pome, drupe or aggregation of drupelets; seed: with small embryo and
usually without endosperm. Example: Wild strawberry (Fragaria vesca)
Rubiaceae
Herbs, shrubs or trees; flowers: regular, bisexual, actinomorphic or rarely
zygomorphic; calyx: limb 4–5 left; corolla: tubular or rotate; stamens: as many
as the corolla lobes; ovary: 2-many locular with axile placentation; leaves:
opposite or whorled, simple, entire or rarely toothed; fruit: a berry, drupe or
capsule; seeds: sometimes winged, the endosperm usually copious and fleshy or
rarely cartilaginous. Example: Yellow cinchona (Cinchona calisaya)
Rutaceae
Trees or shrubs, herbage covered with glandular dots; leaves: alternate or
opposite, simple or palmately or pinnately compound sometimes reduced to
spines, estipulate; flowers:bisexual, actinomorphic, in varying types of
inflorescence; perianth: typically biseriate, usually imbricate; sepals: 3–5 and
distinct or basally connate; corolla: usually present, hypogynous or perigynous;
petals: 3–5, rarely none; fruit: a valvate capsule or a leathery rind berry or
separating into mericarps or sometimes a winged berry or drupe; seed: with a
large straight or bent embryo, the endosperm present and fleshy or absent.
Example: Bael (Aegle marmelos)
Solanaceae
Herbs, shrubs or usually small tree; leaves: alternate, simple exstipulate; stems:
with bicollateral vascular strands; flower: bisexual, actinomorphic or slightly
zygomorophic, solitary or in cymes; calyx: often persistent, 5-cleft; corolla: 5
lobes; stamens: 5, on corolla tube; ovary: superior, bilocular with many ovules in
each locule; placentae: swollen, axile placentation; fruit: a berry or septicidal
capsule; seeds: smooth or pitted, embryo embedded in the fleshy and semi-
transparent endosperm. Example: Deadly nightshade (Atropa belladonna)
Zingiberaceae
Perennial aromatic herbs often with tuberous roots; stems: bracted and scapose
or leafy and then very short or elongated; perianth: in two 3-merous whorls
differentiated into calyx and corolla; stamens: basically 6, the posterior member
of the inner whorls is fertile and the other two are united to form the petaloid
labellum, the member of outer whorl is always absent and the other may be
absent or present as large leafy staminodes; ovary: 3 or 1-locular with numerous
ovules, inferior; leaves: basal or cauline, alternate, sessile or petiolate; fruit: a 3-
valved, loculicidal capsule or fleshy, indehiscent and berry-like; seeds: with
copious hard or mealy endosperm, the embryo straight. Example: Cardamom
(Elettaria cardamomum)
Taxaceae
Leaves: persistent, alternate, often 2-ranked, linear to linear-lanceolate; plants
dioecious or monoecious; staminate flowers: solitary or in small axillary cone-
like clusters of 3–14 stamens; anthers: 3–9 celled, borne on peltate or apically
thickened scales; seed: dry and nut-like, surrounded or enveloped by a soft
fleshy usually highly coloured arillus; embryo: with 2 cotyledons. Example:
Common yew (Taxus baccata)
Urticaceae
Monoecious or dioecious fibrous herbs or infrequently sub-shrubs or small trees,
the cystoliths usually present in epidermal cells, the sap watery; leaves: alternate
or opposite simple, stipulate; flowers: unisexual, regular, minute, mostly green;
perianth: present or absent, when present usually biseriate, or 4–5 distinct or
connate parts undifferentiated into calyx and corolla; staminate flowers: mostly
with 4 stamens; fruit: an achene or drupe, often enclosed by the persistent
perianth; seed: with straight embryo surrounded by oily endosperm. Example:
Olona (Touchardia latifolia).

5.2 MORPHOLOGICAL CONSIDERATIONS OF CRUDE DRUGS

Proper authentication of a drug depends almost entirely on morphological
characters. Most crude drugs are derived from a part of a plant e.g., bark, leaf,
stem, fruit, root, flower and bud. The morphological or macroscopical study of
the respective part is done by observing it with a naked eye or with the aid of a
magnifying lens.
The plant parts that have been considered for extraction of drugs are the (i)
bark, (ii) underground structure, (iii) leaves, (iv) flowers, (v) fruits and (vi)
seeds. For each group, a particular systematic examination should be carried out.

5.2.1 Bark
Terms used in systematic description:
Shape
(i) Flat; (ii) curved; (iii) recurved; (iv) channelled; (v) quill; (vi) double quill;
(vii) compound quill.
External characteristics
(i) Smooth; (ii) rugged; (iii) scaly; (iv) exfoliation; (v) rhytidoma.
Fracture
(i) Short; (ii) granular; (iii) splintery; (iv) fibrous.
5.2.2 Underground Structure
Underground structures include, roots, rhizomes and stolons (modified stems
with buds, scale leaves and scars). They have a central pith surrounded by a ring
of xylem tissues; bulbs (fleshy underground compressed leaves and stems);
corns (fleshy compressed stems with attached scaly leaves and buds).
Terms used in systematic description:
Direction of growth
(i) Horizontal; (ii) oblique; (iii) vertical.
Shape
(i) Cylindrical; (ii) conical; (iii) fusiform; (iv) napiform; (v) straight; (vi)
tortuous.
Fracture
(i) Brittle; (ii) hard; (iii) horny; (iv) mealy; (v) splintery.

5.2.3 Leaves
The leaf is comprised of all the photosynthetic organs arising from a node on a
stem. A leaf may be simple or may consist of many leaflets, in which case it is
termed as a compound leaf.
Terms used in systematic description:
Shape
(i) Aericular; (ii) linear; (iii) lanceolate; (iv) oblong; (v) tabulate; (vi) ovate; (vii)
sagittate; (viii) cordate; (ix) hasted; (x) lumata; (xi) reniform; (xii) spathulate;
(xiii) cuneate; (xiv) elliptical; (xv) rounded; (xvi) obovate; (xvii) obcordate.
Margin
(i) Entire; (ii) repent; (iii) serrate; (iv) biserrate; (v) retroserrate; (vi) dentate;
(vii) bidentate; (viii) crenate; (ix) bicrenate; (x) spiny; (xi) lobed.
Apex
(i) Acute; (ii) acuminate; (iii) obtuse; (iv) cuspidate; (v) mucronate; (vi)
tendrillar; (vii) truncate; (viii) retuse; (ix) emarginate.
Base
(i) Asymmetric; (ii) cordate; (iii) reniform; (iv) sagittate; (v) hasted; (vi)
decurrent.
Venation
(i) Parallel; (ii) reticulate-pinnate; (iii) reticulate-palmate.

5.2.4 Flower
A flower consists of four basic parts: calyx, corolla, androecium and gynoecium.
Their reproductive organs exhibit a wide variety of forms and in many cases, an
apparently single flower consists of many flowers forming an inflorescence.
Terms used in systematic description:
Inflorescence
Racemose
(i) Raceme; (ii) spike; (iii) spike-lets; (iv) catkin; (v) spadix; (vi) corymb; (vii)
umbel; (viii) capitulum (or head).
Cymose
(i) Monochasial cyme (helicoid, scorpioid and sympodial cyme); (ii) dichasia
cyme;
(iii) polychasial cyme
Special types
(i) Cyathium, (ii) verticillaster and (iii) hypanthodium
Flower
(i) Perfect; (ii) imperfect; (iii) sessile; (iv) sub-sessile; (v) actinomorphic; (vi)
zygomorphic.
Arrangment of receptacle
(i) Hypogynous; (ii) perigynous; (iii) epigynous.
Calyx and corolla
(i) Polysepalous; (ii) gamosepalous; (iii) polypetalous; (iv) gamopetalous; (v)
perianth.
Androecium
(i) Polyandrous; (ii) adelphous; (iii) syngenesious; (iv) didynamous; (v)
epipetalous; (vi) epiphyllous; (vii) gynandrous; (viii) basifixed; (ix) adnate; (x)
dorsifixed; (xi) versatile.
Gynoecium
(i) Apocarpous; (ii) syncarpous; (iii) placentation (marginal, axil, central,
parietal, basal, superficial); (iv) ovule (orthotropous, antropous, amphitropous,
campylotropous).

5.2.5 Fruit
Terms used in systematic description:
Shape
(i) Arcuate; (ii) globular; (iii) oblong; (iv) ellipsoidal.
Type

5.2.6 Seed
Seeds arise by the ripening of fertilised ovules and possess a hilum, a micropyle,
and sometimes a raphe.
Terms used in systematic description:
Shape
(i) Globular; (ii) oblong; (iii) oval; (iv) reniform; (v) plamocomex; (vi) spherical;
(vii) angularly ovate.
External character
(i)Testa; (ii) micropyle; (iii) hilum; (iv) chalaza; (v) raphe.

5.3 GENERAL CONSIDERATIONS

The contribution of the scientists, Robert Hooke (1635–1703), Leeuwenhoek
(1632–1723), Grew (1641–1712), Malpighi (1628–1694), Schleiden (1804–
1881), von Mohl (1805–1812), Nageli (1817–1891), De Bary (1831–1888),
Robert Brown (1773–1858), Strasburger (1844–1912), Weismann (1834–1914)
in the field of plant anatomy is highly significant. Discovery of the electron
microscope by Knoll and Ruska in 1932 resulted in exciting and revealing
pictures of cells.
A group of cells of the same type or of mixed type but having a common origin
and performing an identical function, is known as a tissue. Plant tissues are
basically classified into permanent and meristematic tissues. Permanent tissues
include simple, complex and secretory tissues. Meristematic tissues include the
epidermal tissue system. The following section gives an essential and relevant
explanation about the various tissues and their functions.

5.3.1 Parenchyma
The parenchyma consists of living cells of different shapes and with different
physiological functions. They play an important role in wound recovery and
regeneration. The larger portions of a plant such as the pith, all or most of the
cortex of the root and shoot, the pericycle, the mysophyll of the leaf and the
fleshy part of fruits consists of parenchyma. These cells also occur in the xylem
and phloem. Usually, the parenchymatous cells are polyhedral and their diameter
in different planes is more or less equal; but many other shapes are common like
elongated, parenchymatous cells, lobed cells and stellate cells etc. The cells may
be tightly packed and without intracellular spaces or it may have a well-
developed system of intercellular spaces. The development of intracellular
spaces is either schizogenous or lysigenous. The parenchyma cells which take
part in photosynthesis contain chloroplasts and the tissue they form is termed as
chlorenchyma. Parenchyma cells with relatively thick lignified secondary walls
are common, especially in the secondary xylem. Most parenchymatous cells
contain hemicellulose, which serve as reserve substance. Many cells contain
tannins, and such cells may be scattered throughout the plant or they may form
continuous systems. Most of the tannins are found in the vacuoles. Tannin-
containing cells retain the ability to divide and grow, as do parenchyma cells
devoid of tannins. Mineral substances can be found in various crystalline forms
in parenchyma cells. Some of these cells may remain viable after formation of
the crystals, but others die. Idioblastic parenchyma cells may contain various
substances such as the enzyme myrosin, mucilaginous and resinous substances.

5.3.2 Collenchyma
Collenchyma consists of living cells which are more or less elongated cells and
which generally have unevenly thickened walls. It functions as a supporting
tissue in young growing organs and in mature organs of herbaceous plants.
These cells, like parenchyma may contain chloroplasts and tannins.
Collenchyma may occur in stems, leaves, floral parts, fruits and roots. In the
latter, collenchyma is mainly developed when they are exposed to light.
Collenchyma is absent in the stems and leaves of many monocotyledons where
schlerenchyma develops at an early stage. Collenchyma usually forms
immediately below the epidermis but, in certain cases, one or two layers of
parenchyma occur between the collenchyma and the epidermis. These cells may
be short prisms or resemble the neighbouring parenchyma cells with their long
and fibre-like shapes with tapered ends, but all intermediate shapes and sizes
also occur. The longest collenchyma cells are found in the central positions and
shorter ones on the periphery of strands of collenchyma. These may be of
angular, lamellar or lacunar type depending upon the type of wall thickening.
The cells may contain pectin, hemicellulose and cellulose.

5.3.3 Sclerenchyma
Sclerenchyma tissue is composed of cells with thickened secondary cell walls,
lignified or not, whose principle function is support, and sometimes protection.
These cells exhibit elastic properties. There are two types of sclerenchymatous
cells viz.
1. Fibres
2. Sclereids
Fibres
Fibres are long, narrow cells with tapered and sometimes branched cells,
developed from meristematic cells which occur in different parts of the plant
body. Fibres are most commonly found among vascular tissues but in many
plants they are also well-developed in ground tissues. They may be xylary or
extra-xylary depending on their position in the plant body. Xylary fibres
constitute an integral part of the xylem and develop from the same meristematic
tissues as do the other xylem elements. Xylary fibres may be libriform or
tracheid, depending upon wall thickness, type and amount of pits. Another type
of fibre present in the secondary xylem of the dicotyledons is the gelatinous or
mucilaginous fibre which contains much of α-cellulose and is poor in lignin.
Besides being present in xylem elements, extra-xylary fibres also occur
elsewhere in the plant. They occur, for instance, in the cortex or they may be
closely related to the phloem elements. In the stems of many monocotyledons,
the extra-xylary fibres occur in an uninterrupted hollow cylinder in the ground
tissue. They may be situated at various distances inside the epidermis and may
even surround the outermost vascular bundles. Usually in monocotyledons,
fibres form sheaths around the vascular bundles. The mature fibres have well-
developed, usually lignified secondary walls which are sometimes so thick as to
obscure the lumen of the fibre.
Sclereids
Sclereids develop from parenchyma cells whose walls become secondarily
thickened. In many cases they occur as hard masses of cells within soft
parenchyma tissue. In many plants, sclereids appear as idioblasts i.e., as cells
which are readily distinguished from the surrounding cells of the tissue by their
size, shape and thickness of wall. Basically there are four types of sclereids, they
are:
1. Branchy sclereids or stone cells, which are more or less isodiametric in form;
such sclereids are usually found in the phloem, the cortex and the bark of
stems and in fruits.
2. Macrosclereids which are rod-shaped sclereids; they often form a continuous
layer in the testa of seeds.
3. Osteosclereids which are bone or spool-shaped sclereids, the ends of which
are enlarged, lobed and sometimes even somewhat branched; such sclereids
are mainly found in seed coats and also in the leaves of certain dicotyledons.
4. Asterosclereids which are vigorously branched and often star-shaped; such
sclereids are mainly found in leaves.

5.3.4 Xylem
Xylem comprises part of the vascular system and helps in the transport of water
and solutes. It is a complex tissue and consists of several types of cells. The most
important cells are the tracheary elements which are non-living cells that are
principally concerned with the transport of water and which also, to a certain
degree, have a supporting function. Fibres are present in the xylem where they
are mainly concerned with the strengthening of the plant body. Sometimes,
sclereids may also be present. Parenchyma cells which have storage and other
functions also occur in the xylem. The xylems of some plants contain laticifers.
The xylem produced by the procambium in the primary body is called the
primary xylem. In the primary xylem, the elements that are completed early i.e.,
the protoxylem, are distinguished from those completed later i.e., the
metaxylem. The xylem produced as a result of the activity of the vascular
cambium is called the secondary xylem. It consists of the following different
kinds of tracheary elements.
1. Tracheids
2. Vessels (tracheae)
3. Wood fibres
4. Wood parenchyma
Except for wood parenchyma, all other xylem elements are lignified, thick-
walled and dead.
Tracheids
Tracheids are elongated tube-like dead cells with hard, thick and lignified walls
and a large cell cavity. Their ends are usually tapering or oblique and their walls
are provided with bordered pits. They may also be annular, spiral, scalariform or
pitted. Tracheids give strength to the plant body but their main function is
conduction of water from the root to the leaf.
Vessels
Vessels are elongated tube-like dead cells placed end to end with their transverse
or end walls dissolved. They may be annular, spiral, scalariform, reticulate and
pitted depending upon the thickening nature of their walls. They are thick-walled
and lignified.
Wood fibres
Wood fibres are the sclerenchymatous cells associated with wood or xylem.
They occur abundantly in woody dicotyledons and add to the mechanical
strength of the xylem and the plant body as a whole.
Wood parenchyma
Wood parenchyma consists of parenchymatous cells associated with the xylem.
These are alive, thin-walled and generally abundant.
Protoxylem can be classified depending upon its position and the number of
protoxylem groups present. Depending upon the position of the protoxylem,
there are three types of primary xylem—Endarch wherein, the protoxylem
elements are situated close to the centre of the axis and the metaxylem develops
outwardly; Exarch wherein, protoxylem elements are directed away from the
centre of the axis; and Mesarch wherein, the protoxylem elements are situated in
the centre surrounded by metaxylem elements.
Based on the number of protoxylem groups present in the primary xylem, there
are three types—Diarch wherein, two groups of protoxylem are present; Triarch
wherein, three groups of protoxylem extend outward; and Polyarch wherein,
more than three groups of protoxylem extend outward.
5.3.5 Phloem
Phloem is also a part of the vascular system. It helps in conduction of food from
the leaf to the storage organs and the growing regions. The primary phloem
similar to the primary xylem develops from the procambium. The primary
phloem is divided into protophloem and metaphloem. The protophloem is one
which develops from the procambium during an early ontogenetic stage, and
matures before the organ in which it is located has ceased its longitudinal
extension growth. The metaphloem is one which develops from the procambium
but at a later stage of development. The important cells of the phloem are the
sieve elements which serve in conduction of photosynthetic products. Additional
to these elements, phloem also contains typical parenchyma cells in which
reserve substances as well as specialised parenchyma cells are stored, i.e., the
companion cells and albuminous cells which are connected with the functioning
of the sieve elements. Fibres, sclereids and sometimes laticifers and resin ducts
may also be found in the phloem tissue. The phloem is composed of the
following elements.
1. Sieve tubes
2. Companion cells
3. Phloem parenchyma
4. Bast fibres
Sieve tubes
Sieve tubes are slender tubular-like structures, composed of elongated cells,
placed end on end. Their walls are thin, made of cellulose and perforated in
nature; hence their names, sieve tubes or sieve plates.
Companion cells
Companion cells are associated with each sieve tube and connected with it by
simple pits. These cells are thin-walled and elongated. They may develop on
various sides of the sieve tube or form longitudinal rows on one side only.
Companion cells are strongly attached to the sieve tube members from which
they usually cannot be separated even by maceration. The walls between sieve
tube members and companion cells are thin or apparently possess many thin
areas, or sieve areas on the side of the sieve tube member and primary pit fields
on the side of the companion cells.
Phloem parenchyma
Phloem parenchyma, as the name indicates, are phloem where some
parenchymatous cells are present. These are often cylindrical in nature.
Bast fibres
Bast fibres contain sclerenchymatous cells. They are frequently found in
secondary phloem, but generally absent in primary phloem.
In the most primitive form, the sieve elements are parenchymatous cells that
have undergone modifications in connection with their function, followed by the
loss of the nucleus. This protoplasmic specialisation apparently resulted in the
development of the interdependencies of the sieve elements and the parenchyma
cells that retain their nucleus i.e., the albuminous cells in the gymnosperms and
the companion cells in the angiosperms.

5.3.6 Cambium
Cambium is a lateral meristem and is in the form of a continuous layer of cells
present between the xylem and phloem of the vascular bundles. The cells of
cambium are highly vacuolar and uninucleate. They have thin walls made up of
cellulose showing numerous pits. Based on the presence or absence of cambium,
the vascular bundles can be classified as follows:
Open vascular bundle Here, the cambium is present. It is the characteristic
feature of dicotyledons.
Closed vascular bundles Here, the cambium is absent. These are found in
monocotyledons.
Depending upon the arrangement of the xylem and phloem, the vascular bundles
are classified as follows:
Radial vascular bundle Here, the xylem and phloem form separate bundles and
are laid on different radii alternating with each other. These are the characteristic
feature of roots. They are typically exarch.
Conjoint vascular bundle Here, the xylem and phloem are combined to form
one bundle. These are of following types:
Collateral type: Wherein, the xylem (being internal) and phloem (being external)
present together on the same radius. Collateral vascular bundles are the most
common type in stems and leaves.
Bicollateral type: Here, the phloem and cambium occur twice i.e., once on the
outer side of the xylem and again on its inner side (i.e., the sequence is outer
phloem, outer cambium, xylem, inner cambium and inner phloem). It is always
of the open type.
Concentric type: It is always of the closed type. Here, one vascular element
totally surrounds the other vascular element. If the xylem is present at the centre,
surrounded by the phloem the condition is called hydrocentric. Similarly, if the
phloem is present at the centre, surrounded by the xylem, the condition is called
leptocentric.

5.3.7 Secretory Tissues

Secretion in plants is a common phenomenon. The secretion process involves the
formation of the cell wall and cuticle, suberisation, wax deposition and the
migration of specific substances from the cytoplasm into the vacuoles. In
addition to the secretion of these type of materials, there are cells—groups of
cells with more complicated structures—which secret specific substances. These
secretory structures occur in various plants or in different organs and tissues.
Conveniently, secretory tissues can be classified into two types, they are:
1. Laticiferous tissue
2. Glandular tissue
Laticiferous tissue
These tissues are generally long, thin-walled and profusely branched, containing
a suspension (milky juice) of many small particles in a liquid with a varying
refractive index known as latex. This liquid may occur either in a series of cells
or in single long cells. Both these specialised structures are termed laticifers.
However, latex may also occur in unspecialised parenchymatous cells. Laticifer
cells help in the storage of food and also act as translocatory tissues. The cell
wall of laticifers is entirely primary and may be as thick as or thicker than the
neighbouring parenchyma cells. The thick walls contain cellulose and a high
proportion of pectic substances and hemicellulose. These walls are highly
hydrated. Both, the thick walls and thin ones, which do not differ from the walls
of the neighbouring parenchyma cells, are very elastic. There are two types of
laticiferous tissues—latex cells and latex vessels.
Latex cells
These are also called non-articulated latex ducts. They develop from a single cell
which greatly elongates with the growth of the plant and which is sometimes
branched. Such laticifers are also termed laticiferous cells. They branch
profusely through the parenchymatous tissue of the plant, but without fusing
together to form a network. These cells are found in Catharanthus, Ficus,
Euphorbia etc. Non-articulated laticiferous cells are characteristic of various
species of the following families: Apocynaceae, Asclepiadaceae, Euphorbiaceae,
Moraceae and Urticaceae. Different forms of non-articulated laticifers exist, and
in certain mature plants, laticifers cells may develop into very large systems
which extend throughout the different shoot and root tissues.
Latex vessels
These are also called articulated laticifers or compound laticifers. They consist
of simple or branched series of cells which are usually elongated. The end walls
of such cells remain entire, become porous or disappear completely. Such
laticifers are also termed laticiferous vessels. The latex vessels are characteristic
of different species of the Compositae, Convolvulaceae, Papaveraceae,
Euphorbiaceae, Caricaceae, Sapotaceae, Liliaceae and Musaceae families.
Glandular tissue
These tissues are made up of glands, containing secretory or excretory
substances. Glands may consist of single isolated cells or small groups of cells
with or without a central cavity. It is of two types:
1. External glands
2. Internal glands
External glands
Present on the epidermis in the form of an outgrowth. It is of the following
types:
Hydathodes: Occurs on the leaves, mostly at the tips of marginal veins. Their
function is secretion of drops of water substances.
Glandular hairs: These consist of unicellular or multicellular stock and a head
composed of one or more secretory cells.
Stinging hairs: These secrete the poisonous substances that cause irritation of
skin.
Nectaries: These secrete sugar-like substances, generally associated with
different parts of the flower.
Digestive gland: These secrete protein-digesting enzyme to obtain nitrogen from
the bodies of insects.
Internal glands
These are found inside tissues and are of the following types:
Oil glands: Found in the mesophyll of the leaves and cortex of stem. Oil glands
are formed lysigenously.
Mucilage secreting glands: Found in leaves and secretes mucilage.
Resin ducts: Resin ducts are a common feature of conifers and are present in the
primary body. In the secondary body, they are more liable to be influenced by
external factors. The resin ducts are formed schizogenously. They appear tube-
like and are lined by small parenchymatous cells.
Gum ducts: The phenomenon termed gummosis is mainly a result of
metamorphosis of the organised cell wall material to unorganised amorphous
material, such as gums or resins. In extreme cases, gummosis leads to the
formation of lysigenous gum or resin cavities or ducts.

5.3.8 Epidermal Tissue System

The epidermal tissue system can be conveniently studied under the following
headings:
(1) epidermis; (2) stomata; (3) trichomes.
Epidermis
The epidermis constitutes the outermost layer of the cells of leaves, floral parts,
fruits, seeds, stems and even roots before they undergo considerable secondary
thickening. The term epiblema or rhizodermis is generally used for the root
epidermis. It gives mechanical protection to the internal tissues, checks loss of
water through transpiration, helps in gaseous exchange through stomata and also
helps in storage of water and metabolic products. The epidermal cells are
parenchymatous in nature with colourless cell sap. Depending upon the multiple
functions of the outer cells, the epidermis contains a wide variety of cell types.
The outer walls of the epidermis are often thickened and cutinised. The cuticle
checks evaporation of water and is of varying thickness in different plants. It is
usually thicker in plants growing in dry habits. The surface of the cuticle may be
smooth, rough, ridged, or furrowed. The cutin stains red with Sudan IV. The
cuticle consists either of multiple epidermis and bulliform cells. Multiple
epidermis originates due to periclinal division of the epidermal cells and may be
2–16 layers deep, situated just above the exodermis. Bulliform cells are thin-
walled, large and highly vacuolated. They are found in the upper leaf epidermis
of Poaceae and other monocotyledons.
Stomata
In the leaves and young green shoots, the epidermis possesses numerous minute
openings called stomata. The continuity of the epidermis is interrupted by these
minute openings, each of which is limited by two specialised cells, called the
guard cells. The guard cells, together with the opening between them, constitute
the stoma. The stomata are usually found on the aerial portions of the plant and
especially on leaves, ordinary stems and rhizomes. Stomata are absent in roots
and in the entire plant body of certain parasitic plants that lack chlorophyll. They
are present in a few submerged water plants. They are also found on petals,
staminal filaments, carpels and seeds, but these stomata are usually non-
functional. The following types of stomata have been distinguished in
dicotyledons on the basis of the arrangements of epidermal cells neighbouring
the guard cells:
Ranunculaceous stomata (anomocytic) In this type of stomata, the guard cells
are surrounded by a certain number of cells that do not differ in size and shape
from the other epidermal cells. They are generally found in members of the
Ranunculaceous, Cucurbitaceae, Scrophulariaceae and Papaveraceae families.
Cruciferous type (anisocytic) Here, the guard cells are surrounded by three
unequally sized subsidiary cells. Commonly found in the Cruciferae family.
Rubiaceous stomata (paracytic) Here, the longitudinal axis of the subsidiary
cells is parallel to that of the guard cells and aperture. Commonly found in the
Rubiaceae, Mimosaceae, Convoluvlaceae families.
Caryophyllaceous type (diacytic) Here, each stomata is surrounded by two
subsidiary cells, the common wall of which is at right angles to the longitudinal
axis of the stoma.
Actinocytic stomata Here, the stomata are surrounded by a circle of radiating
cells.
The following types of stomatal complexes have been noticed in
monocotyledons:
1. In case of Araceae, Commelinaceae, Musaceae, Zingiberaceae families, the
guard cells are surrounded by 4–6 subsidiary cells.
2. In case of Palmae, Pandanaceae and Cyclanthaceae families, the guard cells
are surrounded by 4–6 subsidiary cells of which two are roundish, smaller than
the rest and are situated at the ends of guard cells.
3. In case of the Flagellariaceae family, the guard cells are accompanied laterally
by two subsidiary cells, one on each side.
4. In case of the Dioscoreaceae family, the guard cells are not associated with
any subsidiary cells.
Trichomes
Epidermal appendages (both unicellular and multicellular) present in the plant
are called trichomes. Trichomes may persist throughout the life of an organ or
may soon fall off. The walls are thick due to cellulose deposition or may be
highly lignified. They are highly variable in form, structure and function.
Trichomes play an important role in protecting the plant organs from external
heat and thus, reduce the rate of transpiration. Broadly trichomes can be
classified into two types: non-glandular and glandular trichomes.
Non-glandular trichomes These are also called covering trichomes. They are of
the following types:
Unicellular: Contains a single cell and may be present on the stem, leaves and
roots.
Multicellular and branched: Which may be stellate.
Squamiforum hairs: Which are conspicuously flattened and multicellular.
Shaggy hairs: Which consists at least at the base of two or more contagious rows
of cells.
Glandular trichomes These are involved in the secretion of various substances,
example: salt solution, sugar solutions, terpenes and gums. The secreting
trichomes are often called glands. These are of the following types:
Trichome hydathodes: These are found, for example, in young leaves and stems
of Cicerarietinum and secrete an aqueous solution containing some organic
acids.
Salt-secreting trichomes: In these glands, the cytoplasm is dense, rich in
mitochondria, endoplasmic reticulum, and golgi bodies and has many vesicular
structures. The salt solution is actively secreted onto the surface of the secretory
cells.
Nectar-secreting trichomes: The vesicles mainly of endoplasmic reticulum origin
are involved in the secretion of nectar.
Mucilage-secreting glands: These glands secrete mucilage, mainly a
polysaccharide. The extruded mucilage accumulates in spaces between the cell
wall and cuticle.

5.4 HISTOLOGY
The histological study of an organised drug is an important parameter of
microscopical evaluation. The presence, type, arrangement and contents of
different cellular structures in transverse and longitudinal sections are revealed
by a suitable histological study of the crude drug using a microscope. Characters
like trichomes, stomata, stone cells, fibres, vessels, cork cells, vascular bundles,
palisade cells, etc. serve as diagnostic properties of organised drugs, e.g;
stratified cork in roots and rhizomes of Rauwolfia serpentina, spiral and annular
thickening vessels in Cassia angustifolia and collateral closed vascular bundles
in rhizomes of Zingiber officinale.
In general, the anatomical details of organised drugs comprising stem, leaf,
roots and fruits are enlightening.

5.4.1 Histology of Stems

Dicotyledonous stems basically contain the following histological features (Fig.
5.1a and b): epidermis; cortex; pericycle; vascular bundle (phloem, cambium
and xylem); medullary rays; pith.
Epidermis
The epidermis forms the outermost layer. It is cuticularised/non-cuticularised
and contains a single row of cells. Externally, the stem is bounded by the
epidermis which contains, apart from the typical epidermal cells, guard cells,
idioblasts and different types of trichomes.
Cortex
The stem cortex is the cylindrical region between the epidermis and the vascular
cylinder. It may comprise various cell types though it usually contains thin-
walled parenchymatous tissue. In other cases, the outer region of the cortex,
which borders on the epidermis may include collenchyma or fibres and the inner
region, parenchyma. The collenchyma or fibres may form a continuous cylinder
or they may be present in the form of separated strips. The stem cortex may
contain sclereids, secretory cells and laticifers. It is divided into three sub-zones:
External cortex called hypodermis containing collenchymatous cells.
General cortex or central cortex containing paranchymatous cells, which are
thin-walled, large, rounded or oval.
Endodermis or internal cortex almost invariably containing numerous starch
grains and known as the starch sheath. The endodermis is not well-differentiated
in aerial stems.
Fig. 5.1a Histological features of a dicotyledonous stem (sunflower)
Pericycle
Pericycle is present between the endodermis and the vascular bundles. The
pericycle can be identified by the presence of semi-lunar patches of
sclerenchyma and the intervening mass of parenchyma. Each patch is associated
with the phloem of the vascular bundle.
Vascular bundles
These are collateral, open and arranged in a ring. Each bundle is composed of
phloem/bast, cambium and xylem/wood.
Phloem This lies externally and is composed of cellulose-walled elements. It
consists of: Sieve tubes: slightly large cavities.

Fig. 5.1b Histological features of a dicotyledonous stem (xanthium)

Companion cells: smaller cells than the sieve tube.
Phloem parenchyma: small-celled parenchyma, other than the sieve tubes and
the companion cells.
Cambium It is a continuous layer of cells present between the xylem and the
phloem and contains a band of thin-walled tissue. It is roughly rectangular in
shape and arranged in radial rows.
Xylem/wood The xylem tissue is composed of the following elements:
Wood vessels: The most easily recognisable elements in wood are some large
thick-walled elements, arranged in a few radial rows; these are the wood vessels.
The smaller vessels, constituting the protoxylem lie towards the centre, and the
bigger ones constituting the metaxylem lie away from the centre. Protoxylem
consists of annular, spiral and scalariform vessels and the metaxylem of
reticulate, pitted vessels. Their walls are always thick and lignified.
Tracheids: Sorrounding the metaxylem vessels and lying in between them are
some small, thick-walled cells. These are called tracheids.
Wood fibres: These appear somewhat irregular and polygonal in section. They
are thick-walled and lignified.
Wood parenchyma: There is a patch of thin-walled cells occurring on the inner
side of the bundle surrounding the protoxylem. This is referred to as the wood
parenchyma.
Medullary rays
Medullary rays contain a few layers of cells which may be polygonal or radially
elongated, and which are present between two vascular bundles.
Pith
The pith is more or less a cylindrical body of tissue in the centre of axis,
enclosed by the vascular tissue. The pith consists of a rather uniform tissue
mainly parenchymatous, in which the cells are usually arranged loosely. Often,
thick-walled lignified parenchyma cells and sclereids are also present. Fibres
occur only rarely, and if so in the peripheral region where they are associated
with the primary vascular tissue. In some species, secretory structures occur in
the pith.

5.4.2 Histology of Leaf

A leaf can be considered as dorsiventral or isobilateral depending on the location
of the palisade parenchyma.
Dorsiventral leaf
Here, the palisade parenchyma occurs on one side of the leaf and the spongy
parenchyma on the other side. Such a leaf is also called a bifacial leaf. It exhibits
the following histological features (Fig. 5.2).
Upper epidermis Usually, it consists of a single layer of cells which is
cuticularised (thick). The epidermis of leaves (both upper and lower) of different
plants varies in the number of layers, its shape, structure, arrangement of
stomata, appearance and arrangement of trichomes. Chloroplasts and stomata are
usually absent.
Lower epidermis It also contains a single layer of cells which is cuticularised
(thin). The layer is interspersed with stomata and chloroplasts. In each stoma, the
respiratory cavity may also be present.
Mesophyll The mesophyll comprises the parenchymatous tissue internal to the
epidermis. It usually undergoes differentiation to form the photosynthetic tissues
and so contains chloroplasts. In many plants, especially among the dicotyledons,
two types of parenchyma can be distinguished in the mesophyll. They are the
palisade parenchyma and the spongy parenchyma.

Fig. 5.2 Histological features of a dorsiventral leaf

Palisade parenchyma The cells of a typical palisade parenchyma are elongated
and in a cross-section of the leaf, they are rod-shaped and appear to be arranged
in rows, while in a section parallel to the leaf surface, these cells seem rounded
and separated or only slightly attached to one another. The function of palisade
parenchymatous cells as a whole is to manufacture sugar and starch in the
presence of sunlight. The palisade cells are found immediately below the uni- or
multiseriate epidermis, but sometimes a hypodermis may be present between the
epidermis and the palisade tissue. The cells of the palisade parenchyma may be
arranged in one or more layers, and in the later case, the length of the cells in the
different rows may be equal or may become shorter towards the centre of the
mesophyll. The palisade tissue is usually found on the adaxial surface of the leaf.
In some cases, it may be found only on the abaxial side of the leaf while in other
cases, it may be present on both sides of the leaf.
Spongy parenchyma The cells of spongy parenchyma are variously shaped.
They may
resemble the palisade cells, have equal diameters or be elongated in a direction
parallel to the leaf surface. The cells contain a few chloroplasts and to some
extent manufacture sugar and starch.
Vascular bundles It comprises the xylem and the phloem. The xylem consists of
various kinds of vessels (particularly annular and spiral), tracheids, wood fibres
and wood parenchyma. Phloem consists of some narrow sieve tubes, companion
cells and phloem parenchyma. Around each vascular bundle, the bundle sheath,
which contains closely arranged thin-walled parenchymatous cells, is present.
Sometimes, the sclerenchyma occurs as a complete or incomplete sheath.
Isobilateral leaf
When the palisade parenchyma is present on both sides of the leaf, the leaf is
said to be an isolateral or isobilateral leaf.
The histological features are more or less similar from one surface to the other
(Fig. 5.3). Equal number of stomata (more or less), uniformly thickened and
cutinised cells are present on either side of the epidermis. Unlike the dorsiventral
leaf, the mesophyll is not normally differentiated into palisade and spongy
parenchyma, but mostly consists of spongy cells, in which the chloroplasts are
evenly distributed.
Fig. 5.3 Histological features of isobilateral leaf

5.4.3 Histology of Roots

The various histological features of the dicotyledonous root are (Fig. 5.4a and
b): epiblema or piliferous layer; cortex; pericycle; vascular bundle; conjuctive
tissue; pith.
Epiblema or piliferous layer
The epidermal cells of the roots are thin-walled and usually devoid of the cuticle,
although sometimes the outermost cell walls, including those of the root hairs
undergo cutinisation. It is involved in the absorption of water and solutes from
the soil. The root hairs increases the absorbing surface of the root.
Fig. 5.4a Histological features of dicotyledonous root (maize)
Fig. 5.4b Histological features of dicotyledonous root (chick pea)
Cortex
Usually, the cortex of the root consists mainly of parenchymatous cells. The root
cortex is usually wider than the stem cortex. Leucoplasts and starch grains are
present in the cortex. The innermost layer of the cortex constitutes the
endodermis, which occurs as a ring around the stele. The arrangement of the
cells of the cortex may be in radial rows, at least in the inner layer, or the cells of
two adjacent concentric layers may be arranged alternatively. The parenchyma
cells of the root cortex usually lack chlorophyll, although chloroplasts are found
in the roots of certain water plants and in the aerial roots of many epiphytes.
Secretory cells, resin ducts and laticifers are found in the cortex of different
plants.
Pericycle
It contains a single circular layer present internal to the endodermis. The cells
are very small, thin-walled and contain abundant protoplasm.
Conjuctive tissue
The parenchyma lying in between the xylem and the phloem bundles constitutes
the
“conjuctive tissue”.
Vascular bundles
As in dicotyledonous stem, the vascular bundles are arranged in a ring. The
arrangement of the vascular bundle is radial. The xylem is exarch. The number
of vascular elements (xylem or phloem) varies from 2–6 (di-, tri-, tetra-, penta-,
hexa-arch), very seldom are they more. The cambium makes its appearance only
later as a secondary meristem. The sieve tubes, companion cells and phloem
parenchyma are present in the phloem bundle. The xylem bundle contains
protoxylem and metaxylem.
Pith
These cells are present in a small area in the centre of the root. Very soon, the
cells become obliterated owing to the wood vessels, and meet in the centre.
Distinction between dicotyledonous and monocotyledonous roots
Table 5.1 lists the difference between a dicotyledon and a monocotyledon root.
Table 5.1 Distinction between dicotyledonous and monocotyledonous roots

Dicotyledonous Monocotyledonous

1. The xylem bundles are 2–6 in They are numerous in number

number; they are rarely absent or (polyarch).
more than this number.

2. The pith is usually very small or Large and well-developed cells are
absent. found.
 3. The pericycle undergoes The pericycle undergoes modification
modification to give lateral roots, to give only lateral roots.
cambium and cork-cambium.

4. The cambium appears as a Absent.

secondary meristem later.

5.4.4 Histology of Fruits

The histology of the fruit can be discussed under the following headings:
Pericarp
In the mature fruit, the pericarp is distinguished into the exocarp, the mesocarp
and the endocarp, sometimes only the exo- and the endocarps are
distinguishable. In many of the fruits, the exo- and endocarps may consist only
of epidermal tissues. In large fruits, vascular bundles are present. In dehiscent
fruits (follicle), the exocarp consists of thick-walled cells, the mesocarp is
parenchymatous in nature while the endocarp also consists of thick-walled cells.
The vascular bundles have sclerenchymatous sheaths. In case of legumes, the
exocarp consists of only epidermal cells, the mesocarp is comprised of relatively
thick parenchymatous cells and the endocarp is sclerenchymatous, on the inside
of which, vascular bundles are situated in the parenchyma of the mesocarp and
are accompanied by sclerenchyma. In case of siliqua, the exocarp and the
mesocarp consist of thin-walled cells and the endocarp is sclerenchymatous. In
case of capsule, the mesocarp is parenchymatous. The elongated thick-walled
cells may sometimes be present below the epidermis.
In case of indehiscent fruits (cypsela), the epidermis is discernible in the
pericarp. Five layers can be distinguished in the pericarp i.e., the outer
epidermis, the hypodermis, a zone of thin-walled cells, cross cells and tube cells.
In the case of cremocarp, the mesocarp is parenchymatous and its cells have
small papillae and finely striated walls. The oil ducts in the mesocarp may be
surrounded by polyhedral cells which become brown as the fruit ripens. The
slightly elongated cells of the innermost layer of the mesocarp are wider than
those of the endocarp. The endocarp consists of narrow cells of which the
longitudinal axis is parallel to the transverse axis of the mericarp.
Testa
In the testa of certain plants, a layer of radially elongated cells, which are
palisade-like but devoid of intercellular spaces may be present.
Characteristically, the walls are unequal, and may contain cellulose only or
lignin or cutin as well. The walls of certain epidermal cells may be transversed
by plasmodesmata. In certain seeds, the outer epidermis is covered by a cuticle.
With the development of the seed coat, a mucilageneous substance develops on
the inner side of the thick outer wall of the epidermal cells so as to almost
completely fill the cell lumen.
Endosperm
The endosperm may consist of thin-walled cells with large vacuoles which do
not contain reserve substances (in case of developing seed). In many other seeds,
the endosperm may consist of thin-walled cells and then the reserve material is
located within the cell or it may consist of thick-walled cells and then the walls
themselves constitute the reserve substances.
 6

In vitro Culture of
Medicinal Plants:
Tissue Culture

It was in 1832, that Theodor Schwann reported that cells could be cultured
outside the body of the organism, if proper external conditions are provided.
Wilhelm Rouse, in 1835 cultured embryonic cells of chicken in salt solution. In
1839, Reichinger said that fragments of cells thicker than 1.5 mm were capable
of growing in external conditions but fragments below this limit failed to grow.
He did not use any nutrient in his experiment.
Arnold in 1885 and Jolly in 1903 observed growth and cell division of
Salamander leucocyte cells in culture. American zoologist Granville Harrison in
1907 successfully cultured the nerve cells of frog in solidified lymph. Harrison is
known as the father of tissue culture.
In 1910 M J Burrows cultured embryonic tissue of chicken in plasma.
Mammalian cells were first cultured by Aleies Carrel. By repeated sub-culturing,
he was able to culture the tissue for 34 years. Organ culture was first done by D
H Fell in 1929, in England. He used solidified plasma and embryonic extract as
the nutrient medium.
Haberlandt, a German botanist, was the first person who attempted to culture
plant tissues in vitro. In 1898, he used cells from the palisade tissues of leaves,
cells from the pith, epidermis and epidermal hairs of various plants in a culture
media containing Knop’s solution, aspergine, peptone and sucrose. The cultured
cells survived for several months but the cells failed to proliferate. This may be
due to the following reasons:
1. use of very simple media,
2. culture of highly differentiated cells
3. and use of aseptic techniques.
Later, in similar experiments conducted by Winkler and Thielmann in 1902, the
cells remained alive for a long period but they failed to divide.
Culture of meristematic tissue was started in the early 20th century. Isolated
root tips were first cultured by Robbin in 1922. Robbin and Maneval cultured
roots and maintained the culture for 20 weeks by subculturing.
In 1934, White successfully cultured isolated tomato roots in a medium
containing besides other nutrients, sucrose, inorganic iron salts, thiamine,
glycine, pyridoxine and nicotinic acid. Gautheret noted that a cambium culture
from Salix caprea, Populus nigra etc. continued to grow for few months under
aseptic conditions. Later he supplemented the medium with β-vitamins and
indole-3-acetic acid. Several scientists recognised the importance of β-vitamins
and growth hormones in cell differentiation.
White in USA, Nobecourt and Gautheret in France made successful attempts in
culturing plant tissues on artificial medium continuously. For a carrot culture,
Gautheret used Knop's solution supplemented with Bertholot's salt mixture,
glucose, gelatine, cysteine hydrochloride and IAA.
White developed a culture of procambial tissue from young stems of hybrid
Nicotiana glauca and N. longsdorfii and noted unlimited and undifferentiated
growth. He also recorded development of leafy buds in a tissue culture of the
hybrid N. glauca and N. longsdorfii in a nutrient medium Subsequently, tissues
from various plants have been cultured successfully. It was noted that older
cultures show increasing degrees of organisation. The role of vitamins in plant
growth was also recognised.
Tissues of Sequoia semipervirens were cultured by Ball. Pollens of Taxus and
Ginko biloba were cultured by Tulecke. Conifer tissues were successfully
cultured by Harvey and Grashan.
Street et al. provided important information about root–shoot relationships.
Wetmore et al. worked on the factors controlling vascular tissue differentiation
from tissue culture studies.
Steward et al. studied the importance of coconut milk and 2,4-D as nutrient. It
was discovered that the stimulatory property of coconut milk is due to the
presence of zeatin. The potent cell division factor was found to be kinetin, which
is a 6-furfurylaminopurine. Cytokinin, a 6-substituted amino-purine compound,
was found to stimulate cell division in a culture of plant tissues. Monocot tissues
were successfully cultured on a medium containing coconut milk.
A group of scientists – Muir, Hildebrandt, Riker, Street, Shigomura, Torrey and
Reinert – worked on suspension culture. Callus obtained from Tagetus erecta
and Niotiana tabacum, when subjected to agitation with a shaker in the presence
of a liquid culture medium, produced a suspension of single cells or cell
aggregates. Such a cell suspension could be sub-cultured. In this method,
isolated single cells were put on a square filter paper placed on active nurse
tissues, which supplied the required nutrients to the growing single cell. Jones et
al. described the hanging drop method, wherein cells were suspended on a
hanging drop in a micro-chamber. Scientists also developed the agar plating
technique of single cell cloning, which involved separation of the cell fraction by
filtration, mixing with warm agar and then placing a thin layer in a petridish.
The possibility of regeneration of a whole plant in culture from a short tip of
angiospermic plants was also explored. Wetmore et al. obtained whole plants
from a culture of shoot apices having one or two leaf primordia.
Morgan in 1901 was the first person to coin the term “totipotent cell” – a cell
which can develop into a whole organism by regeneration. According to White’s
hypothesis, if all the cells of a multicellular organism are totipotent, then, in
isolated conditions, such cells can regain their dividing power and produce
whole plants.
Vasil and Hilderbrandt noted that single cells are able to produce new plants.
Guha et al. obtained haploid embryos from pollen and anther culture while
Nitsch developed a method of obtaining microspore culture from Nicotiana and
Datura. He was also able to double the chromosome number and obtain
homozygous diploid plants.
Cocking recorded the release of protoplasts from root tip cells by using fungal
cellulose in a 0.6 M sucrose solution. He was also able to culture isolated
protoplasts, which regenerated new cell walls and produced cell colonies and
ultimately plantlets.
By the late 1970s, it was evident that plant tissue culture technique could be
successfully used in various fields of agriculture, such as production of
pathogen-free culture, production of secondary products, clonal propagation,
mutant culture, haploid breeding and genetic engineering.
It was also discovered that secondary products could be synthesised in large
amounts from suspension cultures. Some of these substances were enzymes,
vitamins, food flavours, sweeteners, anti-tumour alkaloids and insecticides. In
Japan, in vitro propagation has been achieved at an industrial level.
Clonal propagation of orchids and several other ornamental and economic
plants which were naturally rare have been achieved by in vitro culture.
The dormancy period of seeds can be shortened by excising the seeds and
culturing its embryo in artificial medium (embryo culture). Abortive embryo can
be grown successfully by embryo culture.
Barton et al. stated that foreign genes with desirable characters attached to a
plasmid may be inserted into the naked protoplast by means of liposome. But,
the expression of the introduced gene in a mature plant is still doubtful.
The immobilisation of biocatalysts, including plant cells, has received much
attention in recent decades. It has got many possible advantages, the main being
that immobilisation can facilitate the use of continuous flow processes.
During the last two decades, the varied and continued experimentation in tissue
culture technique has extended the area of study greatly from merely the study of
totipotency of cells (Steward, Mapes and Mears, 1958; Steward, Mapes and
Smith, 1958), to the study of synthesis of plant constituents in suspension
cultures, single cell clones, meristem culture, plant tumours, pollen and anther
culture, biochemical mutants, protoplast culture and somatic hybridisation, etc.
To enlist the various developments, the following facets may be demarcated:
1. Micropropagation, including rapid clonal propagation;
2. virus elimination;
3. embryo culture;
4. haploid production;
5. protoplast culture for somatic hybridisation between genetically incompatible
parents
and genetic engineering or transgenesis;
6. cryobiology;
7. commercial production of pharmaceuticals.
Of these propositions, the first four are immediately feasible for major
economic applications, whereas the last three are at present mere aspirations.
Plant tissue culture can be defined as the in vitro culture of plant or animal
cells, tissue or organ on a nutrient medium under aseptic conditions, usually in a
glass container.
 It is also called a “sterile culture” or an in vitro culture. By this technique,
living cells can be maintained outside the body of the organism for a
considerable period.
According to Street, tissue culture refers to any multicellular culture with
protoplasmic continuity between cells and which grow on a solid medium or is
attached to a substratum and nourished by a liquid medium.
Plant tissue culture is used as a gross term for protoplast, cell, tissue and organ
cultures grown under aseptic conditions.
“Cell culture”, involves the growth or multiplication of any cell (may be
microbe or plant or animal cell), while the term “tissue culture” involves the
cultivation of a plant or mammalian cell which normally forms a “multicellular
tissue”.
Plant tissue culture allows the raising of new plants in an artificial nutrient
medium from very small parts of plants, such as shoot tip, root tip, callus, seed,
embryo, pollen grain, ovule or even a single cell. Whether the cultured tissue
develops into a plant or grows unorganised depends on the genetic potential of
the tissue and the chemical and physical environment.
The major advantages of plant tissue culture over conventional cultivation
techniques are as follows.
1. Useful compounds can be produced under controlled environmental
conditions independent of climatic changes or soil conditions.
2. Cultured cells are free of microbes and insects.
3. The cells of any plants – tropical or alpine – can be easily multiplied to yield
their specific metabolites.
4. Automated control of cell growth and rational regulation of metabolic process
would contribute to the reduction of labour cost and the improvement of
productivity.
5. The plant tissue culture technique can be used for the synthesis of those
therapeutic compounds which are too difficult to synthesise in the laboratory.
6. Uniform biomass can be obtained at all times, and can be managed under
regulated conditions.
7. It can be used to study the biogenesis of secondary metabolites. By feeding
labelled precursors to cell cultures, the particular metabolic pathway of the
desired compound can be interpreted.
8. The technique also offers the advantage that cultured cells can be
immobilised. Immobilised cells can be used for bio-transformation reactions.

6.1 REQUIREMENTS FOR PLANT TISSUE CULTURE

The important requirements of plant tissue culture are summarised below.
1. Aseptic conditions
2. Proper or optimum aeration
3. Equipments and
4. Suitable nutrient medium

6.1.1 Aseptic Conditions

Complete aseptic conditions are required for tissue culture. The tissues,
equipments, culture media and the room should be free from micro-organisms.
Usually dry heat, wet heat, ultrafiltration and chemicals are used for the
sterilisation process. The equipments are sterilised by the dry heat method in an
autoclave. Wet heat sterilisation used for glass wares involves autoclaving at
121ºC and 15 lb pressure for 15 minutes. Ultrafiltration is used for the
sterilisation of liquid media which are unstable at high temperature. The working
area and the instruments are sterilised by chemicals such as alcohol.
Surface sterilisation of materials
Materials such as seed, fruit, stem, leaf, etc need to be subjected to surface
sterilisation, to prevent microbial contamination since, they usually carry micro-
organisms on a normal basis. The following sterilisation agents are used:
a. 9–10% calcium hypochlorite for 5–30 minutes.
b. 2% sodium hypochlorite solution for 5–30 minutes. The materials need to be
washed thoroughly in double-distilled water, after sterilising in these solutions.
c. 10–12% of hydrogen peroxide solution for 5–15 minutes.
d. 1–2% bromine water, for 2–10 minutes.
e. 1% solution of chlorine water, mercuric chloride, silver nitrate or antibiotics,
etc, can also be used.
f. Absolute alcohol is used for hard tissues.
The sterilisation process of different plant materials is described below.
Seeds Absolute alcohol, 10% calcium hypochlorite or 1% bromine water are
usually used for sterilising seeds. The time required for the surface sterilisation
depends upon the rigidity of the seed coat. The seeds are submerged in absolute
alcohol for 10 minutes then 10% calcium hypochlorite for 20–30 minutes or 1%
bromine water for 5 minutes. The seeds are then subjected to washing with
sterile water 3–5 times and allowed to germinate in sterile water or on sterile
filter paper.
Fruits Fruits need to be washed with absolute alcohol and then submerged in 2%
hypochlorite solution for 10 minutes. They are then subjected to repeated
washing with sterile water. The seeds are obtained from such fruits and the
seedlings grown.
Stem At first, the stem pieces are washed in tap water, rinsed in absolute alcohol
and then immersed in 2% sodium hypochlorite for 15–30 minutes. The terminal
portion of the stem pieces are cut off and either planted vertically on solid agar
medium or is dissected out and the required tissue taken for the culture.
Leaf Young leaves need to be selected for this purpose. The surface of the leaves
are wiped carefully with absolute alcohol and then immersed in 0.1% mercuric
chloride for one minute. They are then subjected to repeated washing with sterile
water and are dried with sterile tissue paper.
Storage organ Such materials are first washed in running tap water and
immersed in 2% sodium hypochlorite for 20–30 minutes.
For plant materials having cutin, suberin or epidermal appendages, a small
amount of 0.05% detergent, such as tween 80, is added to the hypochlorite
solution.
The contaminated tissues, characterised by localised internal discolouration,
should be discarded.
Sterilisation of equipment
Chromic acid–sulphuric acid mixture, hydrochloric acid, nitric acid, strong
detergent solution, alcohol, incubator or autoclaves etc are used for this purpose.
a. Pyrene or borosilicate glasswares are sterilised by using chromic acid–
sulphuric acid mixture, and then washed in tap water at least 5–6 times, rinsed
in distilled water and finally with double-distilled water. Sterilised glass wares
are placed in a dust-free place and should be washed with double-distilled
water before being used.
b. Glass wares are also sterilised by dipping overnight in a strong detergent
solution and then washing with hot water followed by cold sterile water. To
overcome the corrosive nature of the chromic acid–sulphuric acid mixture,
detergent solutions are used.
c. Glass, metal or silicon rubber equipments can be sterilised by the dry heat
method (by hot air oven) at 180ºC for one hour. The materials are wrapped in
an aluminium foil before placing in an oven.
d. Autoclave can also be used to sterilise equipments, usually at 121ºC for 20–30
minutes at 15 lb pressure. De-mineralised water must be used in an autoclave.
Aluminium foil is not suitable for wrapping, as it is impermeable to steam.
Unwaxed papers are used for the purpose.
e. Metallic materials such as scalpel, scissor, forceps etc are sterilised by dipping
in alcohol, flamed and cooled before use. UV light can also be used for
sterilisation purposes.
f. Equipment and culture apparatus need to be wrapped in aluminium foil or
cellophane paper during transfer to the aseptic area.
Contaminated cultures are discarded by autoclaving for a short period of time
to kill the contaminants. The glass wares are then cleaned.
Sterilisation of the culture medium
Micro-organisms may be present at the inception of the culture. The presence of
sugar in nutrient media influences the rapid growth of micro-organisms, which
may destroy the tissue.
Usually, the culture medium is sterilised by autoclaving at 15 lb pressure at
121ºC for 15–30 minutes. The autoclaving time depends upon the volume of the
culture media e.g., 75 ml of liquid per vial requires a period of 20 minutes; 250–
5000 ml/vial requires 25 minutes and 1000 ml/vial needs 30 minutes. After
sterilisation, the medium should be used within14 days.
The gibberellins, vitamins and other chemicals used in culture media, undergo
degradation at high temperature during autoclaving. Sucrose also undergoes
partial degradation to d-glucose and d-fructose, during autoclaving; which may
have some inhibitory effect on the culture. Such chemicals or substances are
sterilised by passing them through a membrane filtration unit attached to a
graduated syringe (ultrafiltration). The sterilised liquid is added directly to the
autoclaved medium.
 Some substances in pure aqueous solution may be stable when autoclaved, but
undergo chemical modification in the presence of other substances of the
medium during sterilisation. Such substances are sterilised separately by
autoclaving and then added together.
Antibiotics are added to the medium to prevent the growth of the micro-
organisms.e.g potassium benzyl penicillin (100 mg/ml) which prevents the
growth of gram +ve bacteria, streptomycin sulphate (100 µg/ml) which prevents
the growth of gram –ve bacteria and gentamycin (100 µg/ml) which prevents the
growth of both gram +ve and gram –ve bacteria.
Usually, a large quantity of distilled water is required for plant tissue culture.
The container used for storing the distilled water should be frequently cleaned
and the distilled water not stored for a long time. The water may also be
sterilised by autoclaving for an hour in a pyrex glass flask provided with a non-
absorbent plug fitted to its mouth.
Sterilisation of the environment
a. The tissue culture room should be sterilised by the fumigation method.
b. The inoculation chamber is sterilised with UV rays before inoculation. The
UV germicidal lamp is turned on about two hours before initiation and should
be turned off before using the chamber. The chamber is kept free from micro-
organisms by a continuous flow of sterilised air. The surface of the inoculation
chamber must be wiped with ethyl alcohol or isopropanol (70% or 80%).
Before inoculating the tissue into the culture vessel, the neck of the vessel is
quickly flamed. Its plug is rapidly removed, the tissue inoculated and the plug
immediately replaced.
c. Before initiating the inoculation work, hands must be washed thoroughly with
soap and hot water for several minutes and the skin wiped with paper towels.
Before certain operations, such as removing seed coats by hands, the hands
and fingers may be dipped in ethanol.

6.1.2 Aeration
Adequate aeration is required for the cell to grow. Tissues which are cultured on
semi-solid media do not require any special method for aeration, but tissues
which are grown in suspension cultures, require special devices for aeration. The
aeration for submerged cultures can be provided by the following methods:
1. By placing the culture vessel with the liquid medium on an automatic shaker.
2. Filter paper bridge method can also be used i.e. the two ends of the filter paper
are dipped in a medium and the middle horizontal portion on which the tissue
is placed remains above the level of the medium.
3. Aeration can also be provided by passing sterilised air through the medium
and by stirring the medium.
The culture vessels are closed with non-adsorbent cotton covered in cheese
cloth. This process allows proper aeration but prevents the entry of micro-
organisms.

6.1.3 Equipment
The following instruments, glass wares and apparatus are required for plant
tissue culture:
1. Culture vessels: Usually borosilicate glass vessels are preferred, since they
can be easily sterilised and their mouths flamed during transfer processes. The
culture vessels include test tubes, conical flasks, bottles, special flat tubes.
Earlier even watch glasses were used for organ culture. Now, the common
vessels are 100 ml conical flasks or large (25 × 150 mm) test tubes.
2. Different types of glass wares like measuring cylinders, beakers, funnels, Petri
dishes, graduated pipette, conical flask etc. are required for the preparation of
the nutrient media.
3. Non-absorbent cotton plug, screw cap or polyurethane foam is required to
close the mouth of the culture vessel.
4. Aluminium foil is required to cover the exposed part of the plug. This
prevents the plug from becoming wet when autoclaved. Wet and contaminated
plugs are immediately discarded. Fresh aluminium foils are needed for the
second culture.
5. Spirit lamps are required to sterilise the mouth of the culture vessels.
6. Scissors, scalpels and forceps are needed for explant preparation from excised
plant parts and for their transfer.
7. An autoclave is required for sterilising the nutrient media and glass wares,
scissors, forceps, etc. prior to use.
8. An incubator is also required for maintaining the temperature conditions to
facilitate the culture of callus and its subsequent maintenance.
9. An inoculating chamber is a must, to conduct all aseptic transfers. The inner
surface of the chamber is sterilised by wiping out with ethyl alcohol and
allowing sterile air to pass through it.
10. A shaker is also needed for the suspension of cell culture.
11. A sensitive balance is required to weigh the chemicals and nutrient elements.
12. A refrigerator is needed for storing chemicals like growth hormones, etc. and
stock solutions.
13. A pH meter for adjusting the pH of the media, and other instruments like
magnetic stirrer and centrifuge are also needed.
14. Hand lens, dissecting microscope, compound microscope with
microphotographic facilities, and shelves for keeping the reagents, and also
labels, marking pencils, etc. are essential.

6.1.4 Nutrient Media

The isolated plant cells and tissues require a basal medium for their growth. The
medium generally contains inorganic salts of major and minor elements, carbon
source, growth hormones and vitamins. The composition of tissue culture media
involves the following ingredients:
Inorganic substances Macro-elements (in mmol/l) like Na, K, P, Ca, Mg and S
and micro-elements (in mmol/l) like Ba, Mn, Zn, Cu, Mo, Cl, Ni, Al, etc are
used. Ammonium nitrate or any ammonium salt serves as the nitrogen source.
KCl, KNO3 or KH2PO4 is the potassium supplement while CaCl2.2H2O, Ca
(NO3). 4H2O or its anhydrous form is used as the calcium source. Magnesium
and sulphur are added in the form of magnesium sulphate. Phosphorus is
obtained from NaH2PO4. H2O or KH2PO4. The stock solution of each inorganic
element having ten or hundred times the concentration actually required is
prepared, autoclaved and stored in a freezer.
Organic components Sugar, usually 2–4% of sucrose or glucose is used.
Vitamins and amino acids These are usually added in traces. The following
vitamins are usually used:
Thiamine hydrochloride (0.1–1 mg/l), pyridoxine (0.5 mg/l), nicotinic acid (0.5
mg/l), folic acid (0.5 mg/l), pantothenic acid (0.1 mg/l) and thiamine (vit B1).
The stock solution of all these substances at 100 times the required
concentration is prepared and stored in a refrigerator.
 Since, the vitamins are heat sensitive, they are filter sterilised and added with a
syringe or pipette to the warm unsolidified autoclaved medium.
The presence of amino acids like orginine, aspartic acid, glutamic acid,
glutamine or methionine is beneficial.
Growth hormones Growth hormones play a very important role in callus culture.
Auxins and cytokinins promote cell division, cell elongation, cell differentiation
and organ formation.
IAA (indole-3-acetic acid, 1–50 mg/l), NAA (naphthalene acetic acid, 0.1–10
mg/l) and 2,4-D (2,4-dichlorophenoxy acetic acid, 0.05–0.5 mg/l) are used as
auxin source. Kinetin (0.01–10 mg/l), zeatin and benzyl adenine are used as
cytokinins.
Coconut water, yeast extract, casein hydrolysate, malt extract are also added to
supplement the media when chemically defined media do not produce desired
results.
Carbon source Activated charcoal (the adsorbent) is also used in the media
because of the following reasons.
a. Removes unwanted substances or contaminants from the media
b. Controls the supply of endogenous growth hormones
c. Darkens the supporting matrix
d. Adsorbs the secondary products secreted by the culture tissue.
e. De-mineralises or double-distills water.
Agar is used as a solidifying or gelling agent, usually at a concentration of 0.6–
1% agar.
Table 6.1 represents the composition of nutrient media formulations used in
plant tissue culture.
Preparing stock solutions
The use of stock solutions reduces the number of repetitive operations involved
in media preparation and, hence, the chance of human or experimental error.
Moreover direct weighing of media components (e.g., micronutrients and
hormones) that are required only in milligram or microgram quantities in the
final formulation cannot be performed with sufficient accuracy for tissue culture
work. For these components, preparation of concentrated stock solutions and
subsequent dilution into the final media is a standard procedure. In addition,
concentrated solutions of some materials are more stable and can be stored for
longer periods than more dilute solutions.
It is common practice to make a stock solution 10 or 100x, depending upon the
solubility of the compound.
PROCEDURE
1. Weigh out the required amount of the compound and place it in a clean flask.
2. Dissolve it in a small amount of water, ethyl alcohol, 1 N NaOH, or 1 N HCl.
3. Slowly add double-distilled water to the flask, while agitating.
4. Continue this until the proper volume is reached and label.
5. The label should contain preparation and expiration dates, and the name of the
person who prepared the solution.
Certain solutions, e.g., IAA, must be prepared and stored in amber coloured
bottles to prevent photodecomposition. The concentrations at which auxins and
cytokinens are used to achieve various results are presented in Table 6.2.
Table 6.1 Composition of nutrient media formulations used in plant tissue
culture
Table 6.2 Some plant growth regulators commonly used in plant tissue culture
Macronutrients
Stock solutions of macronutrients can be prepared at 10 times the concentration
of the final medium. A separate stock solution for calcium salts may be required
to prevent precipitation. Stock solution of macronutrients can be stored safely for
several weeks in a refrigerator at 2–4ºC.
Micronutrients
Micronutrient stock solutions are generally made up to 100 times their final
strength. It is recommended that micronutrient stocks be stored in either a
refrigerator or freezer until needed. Micronutrient stock solutions could be stored
in a refrigerator for up to 1 year without appreciable deterioration. Iron stock
solutions should be prepared and stored separately from other micronutrients in
an amber storage bottle.
Vitamins
Vitamins are prepared as 100 or 1000x stock solutions and stored in a freezer (–
20ºC) until used. Vitamin stock solutions should be made up, each time it is
needed if a refrigerator or freezer is not available. It can be stored safely in a
refrigerator for 2–3 months only.
Growth regulators
Some common growth regulators used in plant tissue culture were listed in Table
6.2. The auxins NAA and 2,4-D are considered stable and can be stored at 4ºC
for several months; IAA should be stored at –20ºC. Auxin stock solutions are
generally prepared at 100–1000 times the final desired concentrations. Solution
of NAA and 2,4-D can be stored for several months in a refrigerator or
indefinitely at –20ºC. Generally IAA and 2,4-D are dissolved in a small volume
of 95% ethyl alcohol or KOH and then brought to volume with double-distilled
water; NAA can be dissolved in a small amount of 1 N NaOH or KOH, which
can also be used to dissolve 2,4-D and IAA.
Cytokinins are considered to be stable and can be stored at –20ºC. Cytokinins
stock solutions are generally prepared at 100 to 1000x concentrations. Many
cytokinins are difficult to dissolve, and a few drops of either 1 N HCl, 1 N
NaOH, in KOH or DMSO, are required to bring them into solution.
Storage of stock solutions
Storage conditions for most stock solutions have already been pointed out;
however, some additional points can be made. For convenience, many
laboratories prepare stock solutions and then divide them into aliquots sufficient
to prepare 1 to 10 litre of medium; these aliquots are stored in small vials or
plastic bags in a freezer. This procedure removes the inconvenience of having to
un-thaw a large volume of frozen stock each time medium is prepared. Some
have found that heating in a microwave oven is a satisfactory and quick method
of thawing concentrated medium.
Preparing plant tissues culture media from powders
The following procedure of media preparation is applicable if the constituents
are in packed powder form. The powdered media are extremely hygroscopic and
must be protected from atmospheric moisture. If possible, the entire contents of
each package should be used immediately after opening. Preparing the medium
in a concentrated form is not recommended as some salt added to the medium
may affect the shelf life and storage conditions. The basic steps for preparing the
culture medium are as follows:
PROCEDURE
1. Measure out approximately 90% of the final required volume of tissue culture
grade water, e.g. 900 ml for a final volume of 1000 ml. Select a container
twice the size of the final volume.
2. While stirring the water, add the powdered medium and stir until completely
dissolved.
3. Rinse the original container with a small volume of tissue culture grade water
to remove traces of the powder. Add to the solution in Step 2.
4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins,
auxins, cytokinins, etc).
5. Add additional tissue culture grade water to bring the medium to the final
volume.
6. While stirring, adjust medium to the desired pH using NaOH, HCl, or KOH.
7. If a gelling agent is used, heat until the solution is clear.
8. Dispense the medium into the culture vessels before or after autoclaving
according to the application. Add heat labile constituents after autoclaving.
9. Sterilise the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121ºC for
the time described under sterilisation of media.
10. Allow medium to cool prior to use.
NOTE: Heating may be required to bring powders into solution.
Storage Store dry medium at 0–5ºC. Deterioration of powdered medium may be
recognised by (i) colour variations; (ii) granulation, clumping or particulate
matter throughout the powder; (iii) insolubility; (iv) pH change or (v) inability to
promote growth when properly used.
Precipitation in plant tissue culture powdered media Precipitates are known to
occur, with time, in plant tissue culture media. They are composed of small, pale
yellow-white particles. Analyses of precipitates indicate a predominance of iron,
phosphate and zinc. The probable cause of the precipitates is the inevitable
oxidation of ferrous ions, when the solubility of ferric phosphate occurs. There
are no reports of detrimental effects on the growth and development in plant
tissue culture due to precipitates.
Preparation from basal salt solution
To avoid precipitation over long-term storage, several laboratories have
formulated two solutions which when mixed at the proper dilution make a
solution with the appropriate salt concentration. The basic steps for preparing 1
litre of culture medium are listed below.
PROCEDURE
1. Measure out approximately 700 ml of tissue culture grade water.
2. While stirring the water, add 100 ml of macro-nutrient solution.
3. Continue stirring the mixture while adding 100 ml of micro-nutrient solution.
4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins,
auxins cytokinins, etc.)
5. Add additional tissue culture grade water to bring the medium to the final
volume.
6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.
7. If gelling agent is used, heat until the solution is clear.
8. Dispense the medium into the culture vessels before or after autoclaving
according to the application. Add heat labile constituents after autoclaving.
9. Sterilise the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121ºC for
the time period described under sterilisation of media.
10. Allow the medium to cool prior to use.
Storage Store basal salt at 0–5ºC.
Vitamin preparation and use
Powdered vitamin mixtures are hygroscopic and must be protected from
atmospheric moisture. The entire contents of each package should be used
immediately after opening. The basic steps for preparing 1000% concentrated
solutions with vitamin mixtures are listed below.
PROCEDURE
1. Measure out 70% of the final required volume of de-ionised distilled water
(e.g., 70 ml for a final volume of 100 ml).
2. While stirring the water, add the powdered vitamin mixture. Stir until
completely dissolved.
3. Rinse the original container with a small volume of water to remove traces of
the powder. Add to the solution in Step 2.
4. Add additional water to bring the medium to the final volume.
5. The resulting 1000x concentrated solution should be used at a concentration
of 1 m/l of medium.
6. Follow the same steps to prepare a 100x concentrated solution and use at 10
ml/l of medium.
Vitamin solutions Vitamin solutions are sterile filtered through a double 0.2 mm
filtration unit and are ready for use. They should be added at a concentration of 1
ml/l of medium to prepare the final recommended concentration of vitamins in
the medium
Storage Store vitamin mixtures and solutions in a refrigerator at 0–5ºC.
Precipitation in vitamin solutions may occur during storage. They can be re-
dissolved by warming the solution in a water bath (35–37ºC) for a short period
of time.
Preparation of MS media
The following procedure can be adopted when packed powders are not available.
PROCEDURE
1. Weigh and dissolve macronutrients in 400 ml of double-distilled water.
2. Add 10 ml of micronutrient stock, 5 ml of iron stock and 1 ml of kinetin stock
solutions to the above solution with a pipette.
3. Add 100 mg of myo-inositol.
4. Add 100 mg of indole-3 acetic acid in few drops of sodium hydroxide.
5. Make up the volume to 800 ml with double-distilled water.
6. Adjust the pH to 5.7 by adding few drops of 1N NaOH or 1N HCl.
7. Make up the final volume to 1000 ml with double-distilled water.
8. Add 3 gm of sucrose and 0.8 gm of agar to 100 ml of MS medium in a 250 ml
flask.
9. Close the mouth with an aluminium foil. Subject to sterilisation by
autoclaving and allow to cool.
10. Add 1 ml of vitamin stock solution while the agar is still warm and gently
swirl the flask to mix the vitamin with the medium. Pour about 10 ml of
medium in each culture vial.

6.2 SETTING UP A TISSUE CULTURE LAB

Any laboratory, in which tissue culture techniques are performed, regardless of
the specific purpose, must contain a number of basic facilities. These usually
include the following.
1. A general washing area
2. A media preparation, sterilisation and storage area
3. An aseptic transfer area
4. Environmentally controlled incubators or culture
rooms
5. An observation/date collection area

6.2.1 Washing Area

The washing area should contain large sinks, some lead-lined to resist acids and
alkalies, draining boards, and racks and have access to de-mineralised water,
distilled water and double-distilled water. Space for drying ovens or racks,
automated dish washers, acid baths, pipette washers and driers, and storage
cabinets should also be available in the washing area.

6.2.2 Media Preparation Area

The media preparation area should have ample storage space for chemicals,
culture vessels and closures and glass ware required for media preparation and
dispensing. Bench space for hot plates/stirrers, pH meters, balances, water baths
and media dispensing equipments should be available. Other necessary
equipment may include air and vaccum sources, distilled and double-distilled
water, Bunsen burners with a gas source, refrigerators and freezers for storing
stock solutions and chemicals, a microwave or a convection oven, and an
autoclave or domestic pressure cooker for sterilising media glass ware and
instruments.
In preparing culture media, analytical grade chemicals should be used and good
weighing habits practiced. To insure accuracy, exact step-by-step routine should
be developed for media preparation and a complete checklist required for all
media preparers, even for the simplest media.
The water used in preparing media must be of the utmost purity and highest
quality. Tap water is unsuitable because it may contain cations (ammonium,
calcium, iron, magnesium, sodium etc), anions (bicarbonates, chlorides,
fluorides, phosphates, etc), micro-ogranisms (algae, fungi, bacteria), gases
(oxgen, carbon dioxide, nitrogen), and particulate matter (silt, oils, organic
matter etc). Water used for plant tissue culture should meet, at a minimum, the
standards for type II reagent grade water, i.e., be free of pyrogens, gases and
organic matter and have an electrical conductivity less than 1.0 µmho/cm.
The most common and preferred method of purifying water to type II standards
is a de-ionisation treatment followed by one or two glass distillations. The de-
ionisation treatment removes most ionic impurities, and the distillation process
removes large organic molecules, micro-organisms and pyrogens. Three other
methods that will produce water of type II purity are absorption filtration, which
uses activated carbon to remove organic contaminants and free chlorine;
membrane filtration, which removes particulate matter and most bacterial
contamination; and reverse osmosis, which removes approximately 9% of the
bacterial, organic and particulate matter as well as about 90% of the ionised
impurities.

6.2.3 Aseptic Transfer Area

Under very clean and dry conditions, tissue culture techniques can be
successfully performed on an open laboratory bench. However, it is advisable
that a laminar flow hood or sterile transfer room be utilised for making transfers.
Within the transfer area there should be a source of electricity, gas compressed
air and vaccum.
The most desirable arrangement is a small dust-free room equipped with an
overhead ultraviolet light and a positive pressure ventilation unit. The ventilation
should be equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-
µm HEPA filter of 99.97–99.99% efficiency works well. All surfaces in the room
should be designed and constructed in such a manner that dust and micro-
organisms do not accumulate and the surfaces can be thoroughly cleaned and
disinfected. A room of such design is particularly useful if large numbers of
cultures are being manipulated.
Another type of transfer area is a laminar flow hood. Air is forced into the unit
through a dust filter then passed through a HEPA filter. The air is then either
directed downward (vertical flow unit) or outward (horizontal flow unit) over the
working surface. The constant flow of bacteria-free filtered air prevents non-
filtered air and particulate matter from settling on the working surface.
The simplest type of transfer area suitable for tissue culture work is an enclosed
plastic box commonly called a glove box. This type of culture hood is sterilised
by an UV light and wiped down periodically with 95% ethyl alcohol when in
use. The glove box is used when relatively few transfers are required.

6.2.4 Culture Room

Tissue cultures should be incubated under conditions of well-controlled
temperature, humidity, air, circulation, and quality and duration of light. These
environmental factors may influence the growth and differentiation of the culture
directly during the process or indirectly by affecting their response in subsequent
generations. Protoplast cultures, low-density cell suspension cultures, and anther
cultures are particularly sensitive to environmental cultural conditions.
Typically, the culture room for growth of plant tissue cultures should have a
temperature between 15–30ºC, with a temperature fluctuation of less than ±
0.5ºC; however, a wider range in temperature may be required for specific
experiments. It is also recommended that the room have an alarm system to
indicate if the temperature is over or below preset high or low temperature
limits. A continuous temperature recorder to monitor temperature fluctuations is
also recommended. The temperature should be constant throughout the entire
culture room (i.e. there should be no hot or cold spots). The culture room should
have enough fluorescent lighting to reach an intensity of 10,000 lux; the lighting
should be adjustable in terms of quality and photoperiod duration. Both light and
temperature should be programmable for a 24 hr period. The culture room
should have fairly uniform forced air ventilation, and a humidity range of 20–
98% controllable to ±3%. Many incubators, large growth chambers, and walk-in
environmental chambers meet these specifications.

6.2.5 Basic Laboratory Equipment

Many tissue culture techniques require similar basic laboratory equipment. Table
6.3 lists the items that are commonly found in a laboratory for in vitro
propagation of plant materials.
Table 6.3 Equipment used in tissue culture techniques
6.3 BASIC LABORATORY PROCEDURES
The majority of laboratory operations involved in in vitro propagation of plants
can be easily learned. One mainly needs to concentrate on accuracy, cleanliness
and a strict adherence to details.
6.3.1 Weighing
The preparation of media requires careful weighing of all components. Even if a
commercially prepared medium is used, care must be taken.
Because of the diversity of laboratory balances in use, it is impossible to
review the details of their operation. The manufacture’s instructions should be
consulted before using any balance. The types of balances, often encountered in
the laboratory include top-loading single pan balance, triple beam balance,
double pan torsion balance, analytical single pan balance and top-loading
electronic balance. The last type has become quite popular in recent years due to
its accuracy, ease of use, and durability. With certain models of top-loading
electronic balances, even milligram accuracy is possible. Such accuracy
previously required the use of analytical balances.
Several precautions must be observed to obtain accurate weights. First, the
balance should be located on a hard, stable, level surface which is free from
vibrations and excessive air drafts. The balance or weighing area should
preferably be kept calm and the balance never overloaded. It is always advisable
to use a light weight weighing container or paper rather than placing the material
to be weighed directly on the pan surface.

6.3.2 Measuring Liquids

Calibrated glass ware (e.g., beakers, flasks and pipettes) are required for the
preparation of culture media. Graduated cylinders of 10-, 25-, 100-, and 1000-ml
capacities are used for many measuring operations, but volumetric flasks and
pipettes are required for more precise measurements. Measurement of solutions
with pipettes or graduated cylinders is only accurate when the bottom of the
curved air–liquid interface is aligned with the measuring mark.
Pipettes should be filled with a hand-operated device, called a pipettor, which
eliminates the hazards of pipetting by mouth. Pipetting by mouth should be
avoided as much as possible. Three types of pipettors are commonly available:
a. The first is a bulb type pipettor, which is controlled by a series of valves.
b. The second type of pipettor is operated simply by rotating a small wheel on
the side of the handle. Rotating the wheel upward creates a suction bringing
the liquid into the pipette while rotating the wheel in the opposite direction
releases the liquid.
c. A third type of pipettor utilises an electric air pump. Liquid is drawn into the
pipette by pressing a button and released by pressing another button.

6.3.3 Cleaning Glass Ware

The conventional method of washing glass ware involves soaking the glass in a
chromic acid–sulphuric acid bath followed by rinsing with tap water, distilled
water and finally double-distilled water. Due to the corrosive nature of chromic
acid, the use of this procedure has been eliminated except for highly
contaminated or soiled glass ware. Adequate cleaning of most glass ware for
tissue culture purposes can be achieved by washing in hot water (70ºC +) with
commercial detergents, rinsing with hot tap water (70ºC +), and finally rinsing
with distilled and double-distilled water. However, highly contaminated glass
ware should be cleaned in either a chromic acid–sulphuric acid bath or by some
other proven method such as ultrasonic cleaning, washing with sodium
pyrophosophate, or boiling in metaphosphate, rinsing, boiling in a dilute
hydrochloric acid solution, and then finally re-rinsing. Cleaned glass ware
should be inspected, dried at 150ºC in a drying oven, covered with aluminium
foil and stored in a closed cabinet.
The following general procedure is recommended for cleaning glass ware that
contains media and cultures after all necessary data have been collected.
a. Autoclave all glass ware with media and cultures still in it. This kills any
contaminating micro-organisms that may be present.
b. After the autoclaved media has cooled, but while it is still in a liquid state,
pour it into bio-hazard plastic bags or thick plastic bags, seal, then discard.
c. Wash all glass ware in hot detergent water using a suitable bottle brush to
clean the internal parts of the glass ware. Any glass ware that is stained should
be soaked in a concentrated sulphuric aid–potassium dichromate acid bath for
4 hours, and then rinsed 10 times before washing it with detergent water.
d. All glass ware should be rinsed three times in tap water, three times in de-
ionised water, three times in double-distilled water, dried and stored in a clean
place.
e. Wash all instruments and new glass ware in a similar manner.

6.3.4 Sterilisation
The most tedious parts of in vitro techniques are sterilising plant materials and
media and maintaining aseptic conditions once they have been achieved.
Bacteria and fungi are the two most common contaminants observed in cell
cultures. Fungi spores are light in weight and present throughout the
environment. When a fungal spore comes into contact with the culture media
used in tissue culture, conditions are optimal for germination of the spores and
subsequent contamination of the culture.
Sterilising culture rooms and transfer loads
Large transfer rooms are best sterilised by exposure to ultraviolet light.
Sterilisation time varies according to the size of the room and should only be
done when there are no experiments in progress. Ultraviolet light is harmful to
the eye. Smaller transfer rooms and hoods can also be sterilised with UV lights
or by treatment with bactericides and/or fungicides. Laminar flow hoods are
easily sterilised by turning on the hood and wiping down all
surfaces with 95% ethyl alcohol 15 minutes before initiating any operation under
the hood.
Culture rooms should be initially cleaned with a detergent-based soap and then
carefully wiped down with a 2% sodium hypochlorite solution or 95% ethyl
alcohol. All floors and walls should be washed gently on a weekly basis with a
similar solution; extreme care must be taken to avoid stirring up any
contamination that has settled. Commercial disinfectants such as lysol, zephiram
and roccal diluted at manufacturer’s recommended rates can be used to disinfect
work surfaces and culture rooms.
Sterilising glass ware and instruments
Metal instruments are best sterilised using glass bead sterilisers; these sterilisers
heat to approximately 275–350ºC and will destroy bacterial and fungal spores
that may be found on the instruments. The instruments need only be inserted into
the heated glass beads for a period of 10 to 60 seconds. They should then be
placed on a rack under the hood to cool until needed.
Metal instruments, glass ware, aluminium foil etc, can also be sterilised by
exposure to hot dry air (130–170ºC) for 24 hours in a hot air oven. All items
should be sealed before sterilisation but not in paper, as it decomposes at 170ºC.
Autoclaving is not advisable for metal instruments because they may rust and
become blunt under these conditions. Instruments that have been sterilised in hot
dry air should be removed from their wrapping, dipped in 95% ethyl alcohol,
and exposed to the heat of a flame. After an instrument has been used, it can be
dipped in ethyl alcohol again, re-flamed, and then re-used. This technique is
called as “flame sterilisation”. Safety is a major concern when using ethyl
alcohol. Alcohol is flammable and if spilled near a flame will cause an instant
flash fire. This problem is compounded in laminar flow hoods due to the strong
air currents blown towards the worker. Fires commonly start when a flamed
instrument is thrown back into the alcohol beaker. Limiting the supply of oxygen
can easily put out fires.
Autoclaving, a method of sterilisation with water vapour under pressure, can be
used to sterilise cotton plugs, gauze, labwear, plastic caps, glass ware, filters,
pipettes, water and nutriment. Nearly all microbes are killed by a 10–15 minute
exposure to the super-heated steam of an autoclave. All objects should be
sterilised at 121ºC and 15 psi for 15–20 minutes.
Sterilising nutrient media
Two methods (autoclaving and membrane filtration under positive pressure) are
commonly used to sterilise culture media. Culture media, distilled water, and
other stable mixtures can be autoclaved in glass containers that are sealed with
cotton plugs, aluminium foil or plastic closures. However, solutions that contain
heat labile components must be filter sterilised.
Generally, nutrient media are autoclaved at 15 psi and 121ºC. For small
volumes of liquids (100 ml or less), the time required for autoclaving is 15–20
minutes, but for large quantities (2–4 l) 3–40 minutes are required. The pressure
should not exceed 20 psi, as higher pressure may lead to the decomposition of
carbohydrates and other thermo-labile components of a medium.
Many proteins, vitamins, amino acids, plant extracts, hormones and
carbohydrates are thermo-labile and may decompose during autoclaving. For
such substances filter sterilisation can be used. The porosity of the filter
membrane should be no bigger than 0.2 µm. The empty glass ware that is to hold
the media must be sterilised in an autoclave before filter sterilisation.
Nutrient media that contain thermo-labile components has to be prepared in
steps. A solution of the heat stable components is sterilised in the usual way by
autoclaving, then cooled to 50–60ºC under sterile conditions; in a separate
operation, solutions of the thermo-labile components are filter sterilised. The
sterilised solutions are then combined under aseptic conditions to give the
complete media.
Sterile culture techniques
Successful control of contamination depends largely upon the operators
techniques in aseptic culture. The potential sources of contamination include
dust, hair, hands, pollen grains and cloth. Obviously hands should be washed,
sleeves rolled up, long hair tied back before starting a tissue culture technique.
Washing hands with disinfectant which has got 70% optimal bactericidal activity
should be done as a precautionary measure. Talking or sneezing while culture
material is exposed can lead to contamination. When transferring plant parts
from one container to another, do not touch the inside edges of either vessel. By
observing the location of contamination in a culture vessel, we may be able to
determine the source of contamination.
It is helpful to know why contamination is so detrimental to tissue growth. In
plant tissue culture, small pieces of plant tissue are placed on or in a medium
rich in nutrients and sugar. If bacteria or fungi come in contact with the plant
tissue or the medium, the culture becomes contaminated. The contaminants
(bacteria and fungi) will use the nutrients from the medium and the plant, which
quickly destroys the plant tissue. Our aim is thus to surface sterilise the plant
tissue and put it in a sterile growth medium without any bacteria or fungi getting
on the plant or medium. This is not easy because bacteria and fungal spores are
always present in the air.
When we see sunlight shining in through a window we can, from certain
angles, see dust particles in the air. There are hundreds of bacteria attached to
each dust particle. A horizontal laminar flow unit is designed to remove these
particles from the air. Room air is pulled into the top of the unit and pushed
through a HEPA (high-energy particle air) filter with a uniform velocity of 90
ft/min. across the work surface. The air is filtered by the HEPA filter so nothing
larger than 0.3 µm can pass through. This renders the air sterile. The flow of air
from the unit discourages any fungal spores or bacteria from entering. All items
going inside the unit should be sterile or spread with ethanol or isopropyl
alcohol. They will remain sterile unless contaminated again.
A transfer cabinet provides an enclosed environment that is not sterile but can
be sterilised. A cardboard box lined with aluminium foil or plastic, or a well-
cleaned aquarium, provides a shield to reduce contamination. A box that is 20–
24 inches wide, 20–24 inches high, and 12–16 inches deep provides a good work
area. Working inside any of these, though does not always guarantee success.
 The following precautions are necessary for all work areas: The room should
be swept and if possible mopped. Each work surface should be washed with a
10% lysol or other disinfectant solution. Doors and windows should be closed.
Air conditioners and fans should be turned off. If possible, each student must
have a work space that can be properly treated against contamination, for
example, the box or aquarium mentioned earlier, or a piece of poster paper lying
on the table to indicate the student’s sterile work space. Have spray bottles filled
with 70% ethanol or isopropanol (never methanol) placed so each student has
access to one bottle. Spray everything entering into the sterile area. Have a
central supply area so that all necessary items can be picked up and taken to the
workspace as needed. Items can be returned to the central supply area after being
used. Sterile instruments will be needed for each person. One way to accomplish
this is to have a ½ pint jar of 70% ethanol for scalpels and short forceps. When
tissue has to be positioned in a vessel, long 10-inch forceps are needed. The long
forceps need to be placed deep enough in alcohol so that any part of the forceps
that might come into contact with the vessel is sterilised. A l00 ml graduated
cylinder can be used to hold the alcohol and long forceps. A ½ pint jar of sterile
water is needed so that instruments can be dipped into it to remove residual
alcohol which might otherwise dry out plant tissues. A sterile work surface is
needed to trim the sterile tissue. The easiest thing to use is a sterile Petri dish.
Glass Petri dishes can be autoclaved and re-used. Pre-sterilised plastic dishes are
used and discarded. Spray the bag of dishes with 70% ethanol before opening it
and place the desired number of unopened dishes at each station. Each dish has
two sterile surfaces—the inside top and the inside bottom. Long hair should be
tied back or covered. Hands should be washed, not scrubbed (scrubbing dries
hands and creates flakes of skin that have bacteria) and sprayed with 70% ethyl
or isopropyl alcohol, or coated with isoproyl alcohol gel. Gloves and masks
provide extra protection. There should be no conversation while performing
sterile operations. Place a backrest on your chair. Try to work with your arms
straight. This position may feel awkward, but it will reduce contamination. Do
not pass non-sterile items over sterile areas or items. Make your movements
smooth and graceful so that you do not disturb the air more than is necessary.
Work quickly though, as the longer it takes to manipulate the tissues, the greater
the chance of contamination. Have only the necessary items in the sterile work
area. Remove items that are no longer needed, as quickly as possible. Act out
each step before beginning so that you understand what you are about to do.
 The cultures should be stored in a well-lit area (not in direct sunlight), and
stored at temperatures less than 80ºF. If they are placed under lights, fluorescent
light is to be used. The preferred schedule is 16 hours of light and 8 hours of
darkness. The temperature is to be checked prior to placing the cultures under
the lights because temperature will build even under fluorescent lights.
The cultures are checked every 3–5 days of contamination. Slimy areas mean
bacterial contamination while fuzzy areas are due to fungal contamination. The
contaminated containers should not be opened. The contaminants could be
disease-causing i.e. pathogenic. The only safe way to dispose of these is to
autoclave (or pressure-cook) them for 15 minutes at 15 psi. Contaminated plastic
dishes can be placed inside a large can or autoclavable bag to be sterilised before
discarding.

6.3.5 Determination of pH
The pH of a solution is a measure of the concentration of hydrogen ions in the
solution. The pH scale extents from very acidic (0) to very alkaline (14) with 7
being the “neutral” point. The pH of most culture media is adjusted to 5.7 ± 0.1
before autoclaving. The pH can influence the solubility of ions in the nutrient
media, the ability of agar to gel, and the subsequent growth of cells. Thus,
accurate determination and control of the pH of the tissue culture media are
necessary. Generally, pH is determined with a pH meter.

6.4 PROCESSING OF PLANT TISSUE CULTURE

The different steps involved in plant tissue culture is pictorially depicted in Fig.
6.1.
Fig. 6.1 Schematic representation of the process of tissue culture

6.5 GROWTH PROFILE

The growth profile of the plant tissue culture can be classified under two
headings—single cell culture and callus culture.

6.5.1 Single Cell Culture

The single cell culture exhibits various stages of growth (Fig. 6.2).
Fig. 6.2 Growth curve of single cell culture
Lag phase Here, the cells are exposed to a fresh medium; the cell starts or
regains the ability to divide and the tissue shows slow growth.
Exponential phase This phase is characterised by rapid cell multiplication. The
time of this phase varies according to the cell and its nutrient regime. Usually, it
is of short duration and lasts for 3–4 generations.
Linear phase In this stage, the growth follows a linear pattern with respect to
time.
Progressive deceleration phase Here, the rate of cell division decreases with the
aging of the culture.
Stationary phase In this stage no growth of cells occurs. The rate of production
of cells is equal to the rate of their death.
Senescent phase Here, the cells die due to unavailability of nutrients.

6.5.2 Callus Culture

The various stages of the growth profile of a callus culture (Fig. 6.3) are as
follows.
Fig. 6.3 Model growth curve of callus culture
Lag phase After inoculation of the explant into the nutrient media, there is a lag
time in which the cells undergo no growth; this is when the cell are trying to
adjust to the new nutritional environmental conditions.
Exponential phase Here, the cells undergo rapid multiplication and consume
nutrients from the medium leading to their depletion.
Decline phase Depletion of the nutrient elements in the media lead to the
starvation of some cells which in turn leads to the decline in the growth of callus
tissue.
Stationary phase From this stage onwards, no growth is evident. Sub-culturing
of the cells is required for further growth and development.

6.6 GROWTH MEASUREMENT

There are various techniques available for measuring culture growth:
1. Cell number
2. Packed cell volume
3. Fresh and dry weights
4. Nutrient uptake studies
5. Cell-viability measurements, like cytoplasmic streaming, membrane integrity
measurement and biochemical activity measurement.

6.6.1 Cell Number

This is a simple method which involves the direct counting of the cells. It gives
an accurate information about the number of cells. The cells are treated with
pectinase or chromic trioxide, and then counted directly.
Determination of the total cell-count by using a haemocytometer is described
below:
1. Prepare a cell suspension in a balanced salt solution containing approximately
2.5 × 105 cells per ml.
2. With the cover-slip in place, use a Pasteur pipette or any other suitable device
to transfer a small amount of cell suspension to both chambers of the
haemocytometer by carefully touching the edge of the cover-slip with the
pipette tip and allowing each chamber to fill by capillary action. Do not
overfill or underfill the chambers.
3. Starting with one chamber of the haemocytometer, count all the cells in the 1
mm centre square and four 1 mm corner squares. (Fig. 6.4a)
NOTE: Count cells on the top and left touching the middle line of the perimeter
of each square. Do not count cells touching the middle line at the bottom and
right sides. (Fig. 6.4b)
4. Repeat this procedure for the second chamber.
NOTE: If more than 10% of the cells appear clustered, repeat the entire
procedure making sure that the cells are dispersed by vigorous pipetting in the
original cell suspension. If less than 200 or greater than 500 cells (20–50 cells
per square) are observed in the 10 squares, repeat the procedure adjusting to an
appropriate dilution factor.
5. Cell counts: Each square of the haemocytometer, with the cover-slip in place,
represents a total volume of 0.1 mm3 or 10–4 cm3. Since 1 cm3 is equivalent to
1 ml, the subsequent cell concentration per ml (and the total number of cells)
will be determined using the following calculations:
Cells per ml = the average count per square × dilution factor × 104 (count 10
squares)
Example: If the average count per square is 45 cells, the cells per ml is
45 × 104 = 4.5 × 105 cells/ml
Total cells = cells per ml × the original volume of fluid from which cell sample
was removed
Example: 4.5 × 105 (cells per ml) × 10 ml (original volume) = 4.5 × 106 total
cells.
6. Withdraw a second sample and repeat the counting procedure to ensure
accuracy.
Fig. 6.4a Standard haemocytometer chamber. The circle indicates the
approximate area covered at 100x microscope magnification (10x ocular and 10x
objective).
Fig. 6.4b Corner square enlargement. Include cells on the top and left touching
the middle line (O). Do not count cells touching middle line at the bottom and
right (Ø). Count the 4 corner squares and the middle square in both chambers
(one chamber is represented here).

6.6.2 Packed Cell Volume

Here, a known volume of the suspension culture is subjected to centrifugation
and the cells allowed to settle at the bottom. Then, the total volume occupied by
the cells is calculated as a percentage of the total volume of suspension. This
method only gives approximate results.

6.6.3 Fresh and Dry Weights

This is the most widely used method of measuring culture growth. In the linear
phase, the cells prepare cell wall material and starch from the available
carbohydrates. At the progressive deceleration and stationary phases, the
available carbohydrates are metabolised by the cells. Automatically, the
synthesis of starch and its accumulation is stopped. Hence, there is a decline in
the dry weight corresponding to the metabolism of accumulated starch. But there
is also a progressive increase in fresh weight as the cells are larger and trap
culture medium on the filter bed.

6.6.4 Nutrient Uptake Studies

Here, the growth measurement is carried out by analysing a certain specific
nutrient element in the media like glucose, sucrose, inorganic phosphates, etc,
which indicates if a particular component is limiting at any stage in the culture
cycle. From the date of carbohydrate analysis, the carbon conversion efficiency
is calculated and the biomass subsequently measured.

6.6.5 Cell Viability Measurements

The ability of a cell to live and grow is called cell viability. Depending upon the
metabolic functions, the following viability tests can be performed.
Cytoplasmic streaming
This method is applicable only to cell suspensions, protoplasts and single clumps
of cells through which light can pass. It is a non-destructive method of
measuring the growth profile of the cells.
Measuring membrane integrity
This method can be applied to all types of cultured plant materials. Different
types of cells in tissues leak electrolytes at different rates. This electrolyte loss
can be measured from cells having contact with water.
Measuring biochemical activity
It involves the use of 2,3,5-triphenyl tetrazolium chloride (TTC). In the presence
of TTC solution, living cells turn red whereas the dead tissue remains
unchanged. The red complex precipitate is extracted from the cells with alcohol
and absorbance is measured spectrophotometrically.

6.7 PLANT TISSUE CULTURE METHODS

The following methods of tissue culture are in practice (Fig. 6.5).
1. Hanging drop culture
2. Double cover-slip method
3. Culture in stewards auxophyton
4. Culture in bottles, conical flasks
5. Culture in microchamber
6. Culture in Petri dish, watch glass, etc.

6.7.1 Hanging Drop Culture

This is the simplest method. It involves the following steps (Fig. 6.5a).
a. A tissue of approximately 1 mm diameter is taken and placed on a cover-slip
containing a drop of solidified medium.
b. A grooved slide of 1" × 3" size is taken and the cover-slip is placed over it.
c. The edges of the cover-slip (rim region) are sealed with wax.
In this type of culture, the viability of the tissue is very poor. The tissue can be
made viable for a longer period of time by frequently changing the culture
medium i.e., by the sub-culturing method. In sub-culturing, the tissue is divided
into smaller parts and each part is sub-cultured in a new grooved slide with fresh
nutrient medium.
Fig. 6.5 Diagrammatic representation of the different methods of tissue culture

6.7.2 Double Cover-slip Method

This method was developed by Torrey and Maximov for culturing pea root
callus on solidified medium. Since it involves the use of two cover-slips on a
grooved slide, it is termed as the double cover-slip method. It involves the
following steps:
a. A small round cover-slip containing a tissue and the nutrient medium is placed
on a larger square cover-slip.
b. The small cover-slip is attached to the larger cover-slip by a drop of water or
salt solution and the edges of the cover-slip are sealed with wax.
c. The culture medium must be changed frequently. For this, the small cover-slip
is detached from the larger cover-slip and washed with a fresh nutrient
solution. Again the cover-slip is attached to the larger cover-slip, placed in a
new grooved slide with the edges sealed (Fig. 6.5b).

6.7.3 Culture in Steward’s Auxophyton

This method was developed by Steward, Caplin and Miller, for the proliferation
of small explants from carrot root. The culture apparatus used for the purpose is
called auxophyton.
It involves the following steps:
a. Select a tube of 12.5 cm length and 3.5 cm diameter in size having a side neck
at the middle.
b. Introduce the medium (10 ml) and the explant into the tube, closed with a
cotton plug, which helps in gaseous exchange.
c. About 24 such tubes can be mounted along the edge of a disc which is rotated
at a slight angle to the horizontal at a speed of 1–2 revolutions per minute. It
allows the tissue to be exposed to air and the liquid medium, which flows
along the tube from end to end alternately (Fig. 6.5c).
The apparatus is kept in a temperature controlled room with a fluorescent
light. The culture tubes are uniformly illuminated. The medium generally
becomes turbid due to the release of free cells and cell aggregates from the
explants. The suspension itself may be used for the sub-culture.
6.7.4 Culture in Bottles, Conical Flasks
Here, conical flasks, bottles or Petri dishes are used as culture vessels (Fig.
6.5d). It involves the following steps:
a. A culture vessel of suitable size is selected and nutrient medium is poured into
it.
b. The tissue is inoculated on the media. In due course of time, the cells undergo
proliferation on the surface of the bottle or conical flask, forming a monolayer.
c. Frequent sub-culturing or changing the medium is necessary to maintain the
viability of the cells for a longer period of time.

6.7.5 Culture in Microchamber

Jone, Hildebrandt and Riker cultured the cells from a callus of the hybrid
Nicotiana tabcum and Nicotiana glutinosa in a microchamber which was sealed
with inert mineral oil (Fig. 6.5e). The cells can be minutely observed in such a
culture.

6.7.6 Culture in Petri Dish, Watch Glass

Organs may be cultured in a Petri dish or watch glass (Fig. 6.5f). The Petri dish
may be incubated in a humidified incubator. For culturing small number of cells,
the Petri dish is recommended.

6.8 ROOT CULTURE

The significance of root culture is as follows:
1. From the root culture technique, it is possible to produce genetically uniform
roots within a few days. Such genetically uniform root tips are called “clones
of isolated roots”. These can be used for further study.
2. From the root culture, it is possible to study the nutritional requirements of
roots. For example: Supply of thiamine (vitamin source) is required for the
unlimited growth of roots of some plants.
3. It is also suitable for the study of lateral root and bud formation, initiation of
cambial activity and nodulation.
4. The effect of shoot and root growth can be studied.
5. It is possible to study the factors and the conditions required for the
development of secondary vascular tissues.
6. It can be used for the commercial production of useful substances.
Method of root culture of tomato
The various stages involved in the root culture of say, tomato are as follows.
1. The seeds of the tomato are subjected to surface sterilisation with 80% alcohol
for one minute in a conical flask.
2. Subsequently, the seeds are treated with chlorine water for 10 minutes.
3. Afterwards, the seeds are rinsed thoroughly in double-distilled water.
4. About 5 seeds are transferred to a Petri dish lined with no. 1 Whatman filter
paper and moistened with 8–10 ml of double-distilled water.
5. The Petri dish is wrapped with an aluminium foil and placed in an incubator at
25ºC for about 5 days in the dark.
6. Sterile root tips are obtained by the germination of seeds and are cultured on
White’s medium. Some modifications of the White’s medium is found to be
beneficial i.e., use of iron in chelation form (NaFeEDTA) instead of ferric
sulphate is advisable, since prolonged culture use of ferric sulphate in the
medium leads to iron deficiency. 1.5–2% of sugar concentration is sufficient.
The requirement of vitamins varies according to the plant.
7. About 10 mm long root tips are taken and transferred to flasks containing
culture medium. The culture is incubated at 25ºC for 10 days. The root
develop and produce many lateral roots. The main root is subdivided into
sectors, each of which contains a portion of the main root and several lateral
roots. Each such sector is placed on a fresh medium. At regular intervals,
subculture is done. In this way, the culture can be maintained for several
weeks.
The steps involved are shown in Fig. 6.6.
Fig. 6.6 Schematic representation of root culture

6.9 SHOOT TIP CULTURE AND LEAF CULTURE

The main advantage of shoot tip culture is that pathogen-free clones can be
produced very rapidly. Therefore, it is a very important method of vegetative
propagation. It is used commonly for the mass-production of orchids.
The steps involved are shown pictorially in Fig. 6.7.
Method
1. The size of the shoot tip selected for culture is very important. About 10 mm
long shoot tips are removed from healthy plants.
2. The explants are subjected to surface sterilisation in hypochlorite solution for
about 15 minutes.
3. They are then washed in double-distilled water 2–3 times.
4. The washed explants are placed on a Petri dish lined with filter paper for
dissecting the shoot apex. The filter paper is moistened with double-distilled
water.
5. The terminal 0.5 mm of the shoot apex is carefully excised and is placed in the
depression of the filter paper bridge, which is folded in the form of the letter
M (Fig. 6.8).
6. About 3 ml of the Murashige and Skoog’s medium is placed in the culture
vessel. The arms of the folded filter paper are dipped in the liquid medium.
The culture vessels are plugged with non-absorbent cotton and covered with
aluminium foil.
7. Subsequently, the culture tubes are placed in a plant growth chamber at 27ºC
with an intensity of 1000 lux and a photo period of 16 hours at 70% relative
humidity.
Usually, depending upon the material, the lateral bud formation requires few
weeks to several months time. Potato shoot tip grows quickly and forms several
buds within 5–6 weeks. For root initiation, these are transferred to a medium
lacking BAP. After rooting, plantlets are transferred to a sterile soil mixture in
humid condition. The plantlets are transferred first to a green house and finally
to a field.
Fig. 6.7 Schematic representation of shoot tip culture
Fig. 6.8 Shoot tip placed on M-folded filter paper

6.9.1 Culture of Leaves

It involves isolation of the explant, its surface sterilisation and culturing it on
solidified agar medium (Fig. 6.9). The growth of leaves on the culture medium
depends upon the stage of the leaves during excision. It is observed that explants
from young leaves grow better than explants from older leaves.
Fig. 6.9 Culture of leaves

6.10 CULTURE OF FLOWERS, OVARIES AND OVULES

6.10.1 Culture of Flowers
For culture of flowers, the flowers are selected two days after pollination and
subjected to surface sterilisation with 5% hypochlorite solution. They are then
repeatedly washed with double-distilled water. Subsequently, the surface
sterilised explant is inoculated on White’s medium. When cultured, such flowers
produce fruits. Larger fruits are obtained on medium supplemented with
hormones (such as indole-3-acetic acid, gibberellin, cytokinin, etc.). Flowers
excised before pollination do not produce fruits.

6.10.2 Culture of Ovaries

Ovaries excised after pollination can produce fruits on a simple medium
containing mineral salts, sugar and vitamins. Ovaries taken from unpollinated
flowers fail to produce fruits on such a simple medium. Unpollinated flowers
may develop into seedless fruits on a medium supplemented with hormones.
Seedless fruits are obtained from Lycopersican by culturing unpollinated ovaries
on a medium supplemented with 2,4-D.
Culture of ovaries has the following advantages:
a. The physiology of fruit development can be studied.
b. Haploids can be produced.
c. Dormancy period of seeds can be reduced by ovary culture.
d. Rare hybrids can also be produced by ovary culture.

6.10.3 Ovule Culture

Culture of ovules excised at or after the globular stage produces mature seeds
easily. This has been noted in several plants, such as Allium cepa, Gynandropsis
and Nicotiana tabacum. Ovules excised soon after fertilisation but before
reaching the globular stage, usually fail to produce more seeds on culture. Such
ovules require a special medium for proper growth.
Ovule culture has the following advantages:
a. By ovule culture, it is possible to grow zygote or very young embryos which
cannot be cultured easily.
b. The various developmental stages of zygote or young embryo can be studied
in ovule culture.
c. The nutritional requirements of young embryo can be studied.
d. In some interspecific crosses where embryo culture fails to raise seedlings,
ovule culture may be the effective method to raise viable seedings.

6.11 SEED CULTURE

In seed culture of broomrape (Orobanche aegyptiaca), both monopolar and
bipolar seedling formation occurs depending upon the composition of the culture
media.
In a medium containing coconut milk or yeast extract, monoplar seedling
formation occurs. If the medium is supplemented with IAA or gibberellin or
kinetin, bipolar seedling is formed.

6.12 NUCELLUS CULTURE

Disease-free clones can be obtained by nucellus culture. In various species of
Citrus
nucellar, monoembryony and polyembrony occur. When nucellar tissue of a
polyembryony species of citrus, excised after pollination, is cultured on White’s
medium supplemented with casein hydrolysate, callus is formed. This callus
produces several pseudobulbils, most of which form embryoids and ultimately
produce seedlings.
Nucellar tissues excised from unfertilised ovules have no adventitive
embryoids. If cultured on a medium containing malt extract and adenine,
embryoids are formed but they are unable to germinate. When such embryoids
are excised and cultured on a medium containing gibberellin, plantlets may be
formed which can then be transplanted to soil.

6.13 EMBRYO CULTURE

Culturing embryo excised from seeds on sterilised culture medium is called
“embryo culture”. It has the following advantages:
a. By embryo culture, it is possible to study the nutritional requirements of the
embryos at various developmental stages.
b. It is possible to know the factors controlling differentiation of embryos.
c. Many interspecific hybrid plants can be produced by embryo culture.
d. The dormancy period of seeds can be shortened.
e. It is possible to propagate rare plants. For example, some coconuts instead of
producing endosperm, form solid soft tissue. Such nuts are rare and expensive.
The seeds of this variety fail to geminate normally. But they can be grown by
embryo culture.
f. By embryo culture, it is possible to know the viability of the seeds.
g. Haploids can be produced.
h. Disease-resistant varieties can be obtained with embryo culture.
Method
It involves the following steps:
1. The selected fruit is surface sterilised for 15 minutes with alcohol (or calcium
hypo-chlorite or sodium hypochlorite) and then washed thoroughly in double-
distilled water.
2. The fruit is cut and the ovule or seed is taken out with a sterile forceps and
placed on a sterile Petri dish.
3. The seed or ovule is cut open and the embryo is separated.
4. The excised embryo is immediately placed on the nutrient medium of a
culture vessel under aseptic conditions.
5. The planted embryos on tubes, flasks, bottles or other containers are kept at
room temperature in diffused light.
6. Seedlings are produced and these are transferred to a sterile growth
stimulating medium before transfer to field conditions.
The diagrammatic representation of embryo culture is shown in Fig. 6.10.

6.14 ANTHER AND POLLEN CULTURE

The schematic representation of anther culture is shown in Fig. 6.11.
Method of anther culture
Selection of flower buds of proper size in suitable developmental stages is very
important.
1. The selected flower buds are subjected to surface sterilisation with alcohol or
hypochlorite solution for about 10–20 minutes.
2. They are then rinsed repeatedly in sterile double-distilled water.
3. By using forceps and a dissecting needle, the anthers are carefully excised
from the flower buds. The filaments must be removed prior to culture,
otherwise callus may be formed at the cut ends.
4. The excised anthers may be cultured either on agar solidified culture medium
or placed on a filter paper bridge over a liquid medium, aseptically, at 25ºC in
the presence or absence of light. Light is essential after plantlets are formed.
Continuous illumination from a cool white fluorescent lamp of 300 lux is
satisfactory.
Fig. 6.10 Schematic representation of embryo culture
Fig. 6.11 Schematic representation of anther culture
The plantlets are formed after 4–5 weeks of inoculation. Many plantlets are
produced from a single anther. These plantlets are carefully separated quite early
and cultured on a fresh root-inducing medium containing 0.5% agar and all other
components in half strength to that of the anther culture medium. After
formation of a proper root system, they are transplanted to pots. These pots are
preferably kept in humid conditions for a few days.
Method of pollen culture
The first stages of pollen culture are similar to that of anther cultures.
1. Pollen grains are removed from the anther either mechanically or by natural
dehiscence. For removing pollen grains mechanically from one another, a
slight incision is made on the anther tissue with a scalpel and its carotenes are
gently squeezed into the medium.
2. Anthers are placed in a 5 ml liquid medium in a Petri dish.
3. Petri dishes containing the pollen grains in the culture media are sealed with
parafilm and incubated at 28ºC in dark for fourteen days.
After 14 days, the culture is kept in an illuminated chamber at 25ºC and a day
length of 12 hours. 3–8 weeks may be required to obtain haploid plantlets.
Significance
Pollen culture is of great importance in mutagenic studies. Many haploid plants
can be produced very rapidly. The homozygous diploid plants obtained by
doubling the chromosomes of haploids have great importance in plant breeding
and crop improvement.

6.15 CALLUS CULTURE

Undifferentiated cell mass is called callus. It is formed by the proliferation of the
parent tissue. The cells are parenchymatous, amorphous and unorganised.
Generally, it is formed as a result of injury at the cut ends of a stem or a root.
It may initiate from explants of any multicellular plant. These explants can be
from stem, root, leaf, flower or seed.
 Subculturing of the callus at frequent intervals is essential because of the
following reasons: (i) The nutrient may be exhausted; (ii) agar may be
dessicated; or (iii) the cell metabolites may accumulate and cause toxicity.
Repeated subculturing can be avoided by freeze preservation of the culture.
Plate 1a shows a callus cultured in a test tube.
Method
The callus development from a callus root culture is explained below (Fig. 6.12).
1. A fresh and healthy carrot root is selected. It is thoroughly washed in running
tap water. 1–2 mm of the external surface is scrapped. The upper 1 cm of the
carrot root is discarded and the remaining is cut into 0.5 cm thick slices, and
placed immediately in a beaker containing double-distilled water.
2. The slices are transferred to a beaker containing sodium hypochlorite solution
for 10 minutes. They are then washed successively in 3 beakers containing
double-distilled water, keeping the slices for 20–30 seconds in each. The slices
are kept in the third beaker.
3. A slice is taken and placed in a Petri dish. Numerous tissue cylinders are cut
out from the cambial region by a sterilised cork borer, and the tissue cylinders
are placed in a Petri dish containing double-distilled water.
4. A tissue cylinder is transferred to a Petri dish and its two sides are trimmed
with a sterile scalpel and discarded. The remaining cylinder is cut into
explants measuring 5 mm in diameter and 2 mm in thickness, and placed in a
Petri dish containing double-distilled water.
5. Explants are then transferred with a sterile forceps onto the surface of a filter
paper on a Petri dish. The upper and lower surfaces of each explant are
blotted.
6. One such explant is transferred to each culture tube containing the nutrient
medium.
7. Culture tubes are kept in a glass storage jar wrapped in aluminium foil and
placed in an incubator at 25ºC.
Fig. 6.12 Schematic representation of callus culture
After a few days, the surface of the explant becomes somewhat rough,
indicating the initiation of the callus. Callus can be maintained from few weeks
to months depending on the rate of growth. Generally, after 6–8 weeks the callus
is subcultured. The callus is divided into small parts of 100 mg approximately.
Each piece is transferred to a new flask containing 30 ml of culture medium and
subcultured at a temperature of 25ºC or above.
Significance
It is possible to rapidly determine the total amount of nucleic aids from the callus
culture. It is also possible to study the biosynthetic pathway of various metabolic
processes by using tracer elements in callus culture. Friable callus is suitable for
cells suspension culture.

6.16 TYPES OF TISSUE CULTURE

Tissue culture can be categorised into the following types.

Static culture
In static culture, cell proliferation takes place on solidified nutrient agar medium.
It is also called callus culture. The details of this culture have been discussed in
previous sections.
Suspension culture
It is the culture of cells or cell aggregates dispersed in a moving liquid medium.
Cells of a suspension culture often possess large nuclei, dense cytoplasm and
starch grains. They show hormone habituation, increase in ploidy level and loss
in totipotency. But cell suspensions from different sources vary considerably in
the expression of these characteristics.
Subculture
When the culture has reached the maximum cell density, subculturing should be
done. Otherwise, the cells may die and lyses occur. Subculturing is usually done
at the early stationary phase or during the progressive deceleration phase. In a
suspension culture, maximum cell density is usually reached within 18–25 days.
But in some cases, maximum cell density is reached within 6–9 days. Before the
first subculture, the culture is passed through a stainless steel filter or nylon to
remove cell aggregates and residual in culture. By repeated subculture, a cell
suspension culture can be maintained for a long time. The details of suspension
culture have been discussed in previous sections.
Batch suspension culture
The cells are grown in a fixed volume of medium. It is a closed system of
culture.
Continuous suspension culture
A constant flow of the culture medium is maintained in the culture vessel, that is,
there is a replacement of equal volume of culture medium after depletion. New
cell formation is more or less equal to the amount of old washed out cells. Thus,
the culture is maintained in a steady condition.
In an open system, fresh medium is added, the spent medium is withdrawn and
the cells are also harvested.
In a closed culture, all cells remain in the culture, thus the number of cells
increases until the stationary phase is reached. Carrot culture is usually done by
this method
Significance of suspension culture
It is possible to synthesise secondary plant products such as alkaloids,
glycosides, etc by suspension culture. It also solves many problems in applied
botany.

6.17 TISSUE CULTURE OF MEDICINAL PLANTS

Tissue culture of medicinal plants has been looked upon with great interest.
Research has been spurred by the need for large quantities of herbal drugs.
Emphasis has been laid on plants yielding raw materials for the production of
steroid hormones and oral contraceptives, namely, Dioscorea floribunda, D.
deltoidea, D. composita, Solanum khasianum, Costus speciosus. However,
morphogenetic work started with Rauwolfia serpentiana, the miracle drug plant
of India. Atropa belladonna, Solanum xanthocarpum, S. laciniatum and S.
elaeagnifolium, etc. are some other plants of which tissue culture has been done.

6.17.1 Dioscorea spp.

There are two lines of approach for developing tissue culture methods for rapid
propagation of plants, i.e., regeneration of plants through organ culture without
the intervening callus formation or their differentiation from callus cultures. The
former method affords clonal propagation, whereas in the latter, the plants
though formed at a much faster rate may be variable, because the callus in
culture is often genetically instable.
Clonal propagation
Plant established in aseptic culture through shoot apices or single-node segments
of a field-grown vine comprise the experimental material for this kind of
propagation. On an average, 40 single-node leaf cuttings are obtained from
aseptically grown plant and when cultured in a medium containing 0.5 mg/l α-
naphthaleneacetic acid (NAA) or 0.1 mg/l 2,4-dichlorophenoxy acetic acid (2,4-
D), all of them root within a period of 20 to 30 days, resulting in the formation
of as many plants. Furthermore, the same number of cuttings can be repeatedly
obtained, after every second month, from each flask by subculturing the
remaining rooted base of the plants. It is also significant that plants can be
produced all the year round and for several successive years from the same
culture without any decline in the regenerative potentiality. In this way,
enormous true-to-type planting material could be obtained in a short time. It
takes 40 to 50 days to obtain 5 to 6 leaved plantlets in potted soil from cuttings
taken from a single aseptically grown plant. Before transplantation in soil, the
plantlets need to be acclimatised by growing them in an inorganic salt solution.
There is 100% survival of plantlets in liquid culture and soil. Field trials have
been performed on thousands of in vitro-raised plants. They have all grown
vigorously and produced normal tubers. The method has special relevance for
initial multiplication of elite clones and may be very useful in breeding
programmes for speeding up the selection process.
Induced embryogenesis in somatic callus culture
The research work done on callus culture emphasises that the differentiating
cells of callus cultures of D. fluoribunda and D. deltoidea followed the
embryogenetic pathway, resulting in the formation of embryoids which later on
developed into plantlets. By closely investigating the histology or anatomy of
embryogenesis even the “single cell stage” in embryoid formation can be traced
in the proembryogenic tissue of both the Dioscorea spp. Embryogenesis can be
induced by treatments containing 0.25 to 0.5 mg/l BAP and 1 mg/l indoleacetic
acid (IAA) in both the calli of D. fluoribunda and D. deltoidea. Embryogenesis
can be augmented in the tuber callus of D. deltoidea by increasing the
concentration of IAA to 3 mg/l in the above pair, whereas in its leaf callus, it
could be induced, though to a lesser extent, by substituting zeatin (z) or 6 γγ-
dimethyl allylamino purine (DAP) for BAP.
In cultures showing optimum differentiation, even 50 plantlets or more were
formed per culture (Plate 1b). Further, several crops of plantlets were obtained
from a single differentiating culture by repeatedly excising the shoots and
subculturing the remaining portion. The callus-regenerated plants showed
variation in vigour, leaf shape and size and in their ploidy level, from diploids to
polyploids and neuploids. Sometimes, albino mutants to those having variegated
leaves were also isolated from differentiating leaf callus of D. flouribunda.
Plate 2 shows the shoot and roots of a regenerated plant.
Numerous tissue-regenerated plants of D. fluoribunda were acclimatised and
given field trials, where they grew vigorously and normally (Plate 3).
Regeneration of plants through callus, besides offering a much faster rate of
multiplication than that through stem cuttings, may also help in evolving new
strains or variants without resorting to breeding or mutation breeding.
Induced tetraploidy
An easy method for obtaining a large number of tetraploids of Diosorea
floribunda, in a short time is by the application of colchicine in an aseptic
culture. Seitz-filtered colchicine (0.05%) and soaked cotton wool was placed two
days onto the developing shoot buds from aseptically cultured rooted base of
plant prepared by excising all the visible shoots. Within an incubation period of
30 days, a number of shoots developed from such treated bases, which (shoots)
were more vigorous, with stout stems, large-sized, thicker leaves as compared to
the control. Such tetraploid shoots were rapidly mass-multiplied through the in
vitro culture of single-node leaf cuttings and a large number of tetraploid plants
of D. floribunda were obtained by growing them in soil. This process has great
significance for obtaining triploid D. floribunda, which may be expected to
produce bigger tubers with higher diosgenin content. Generally, the triploids are
valued for their superior fruits (seedless) but they may be superior in certain
vegetative characters too. Populus tremuloides, the triploid quaking aspen has
more desirable pulpwood characteristics as compared to its diploids.
Biosynthesis of diosgenin
It was generally believed that while the diosgenin is synthesised in the growing
shoots of Dioscorea, the tuber is only the storage organ for diosgenin and does
not synthesise it. However, dispelling this belief, scientists, for the first time
demonstrated with D. deltoidea that even tuber tissue could biosynthesise
diosgenin. Initially, the percentage of diosgenin produced was 0.7, which was
stepped up more than 2-fold, i.e. 1.6 by making certain changes in the
composition of the medium but without adding any precursors. This is perhaps
the highest percentage of diosgenin reported from Dioscorea callus cultures
without feeding precursors. The 20-fold increase in fresh weight of callus in 2
months obtained in this study is also the highest recorded so far. It may be
mentioned that earlier, diosgenin production from cultures obtained by callusing
the entire seedlings of several species of Dioscorea have been reported. High
content of diosgenin and fast rate of growth of callus in these experiments hold
promise for the large-scale cultivation and continuous production of diosgenin in
fermentors. Besides the tuber callus, the callus tissue of leaf, stem and root of D.
deltoidea were also grown satisfactorily. The leaf callus of D. floribunda showed
the highest rate of proliferation so far, i.e. 60-fold increase, in fresh weight in 2
months, whereas the tuber callus of D. prazeri was grown to a limited extent.

6.17.2 Atropa belladonna

Excised roots of Atropa belladonna established in a continuous culture showed
very good growth, particularly during the initial subculture and synthesised
about 0.4% of hyoscyamine/atropine. The atropine content was stepped up to
about 4-fold, i.e. 1.2% by incorporating 3 mg/l DLA =1 ornithine-HCl and 6
mg/l 4-phenylalanine in the medium. However, the excised roots showed
progressive decrease in their growth rate during their prolonged culture over a
period of 5 years accompanied by a gradual decrease in their atropine content
from 1.2% to 0.5%. A new concept of rejuvenation of the tissue culture has been
elucidated by scientists through restoring the initial growth rate and stepping up
the biosynethic potentiality of such excised root cultures. This is achieved by
inducing plantlet formation in segments of old root cultures and initiating fresh
cultures of the roots of the regenerated plants.

6.17.3 Rauwolfia serpentina

Comprehensive morphogenetic studies were pursued by scientists in the somatic
callus (particularly leaf and stem callus) cultures of Rauwolfia serpentina.
Complete histological details were worked out in respect to the various
ontogenetic pathways starting from a “single cell” (constituent of the
differentiating callus) leading to the formation of multiple plantlets, which
flowered and fruited normally in field conditions. Two distinct ontogenetic
pathways were observed in the regeneration of plants from the leaf callus of R.
serpentina:
a. Adventive embryogenesis in which some of the stages of zygotic
embryogensis were recapitulated; and
b. Differentiation of the bipolarised meristematic nodules in which shoot and
root growing points were established on opposite poles.
Both types of regenerants formed meristemoids which occurred as isolated
pockets of meristematic cells in the same proembryogenic tissue. The
proembryogenic tissue is formed in discrete regions in the mass of differentiating
callus. Other regenerants differentiated in the same callus were shoot buds
unaccompanied by root formation, or shoot buds formed from roots
differentiated from the callus. Generally, only one of the different kinds of
regenerants is reported to be formed in callus cultures of a particular part of the
plant. But there are reports where two kinds of regenerants were formed. Some
scientists reported the formation of all the regenerants in the same callus culture.
In further studies, differentiation of plantlets were obtained from stem, root and
hypocotyl callus as well. The tissue regenerated plants were found diploid (2n =
22). Regeneration of multiple plants were also obtained from root segments
taken from excised root culture as also by proliferating shoot apices, taken from
field-grown plants, in a combination of BAP and NAA. In certain cultures, more
than 50 shoots were formed from a shoot tip within a culture period of two
months.
Excised leaves of Rauwolfia serpentina have been successfully grown in an
aseptic culture in order to understand the nature of the leaf growth hormones.
Under different cultural conditions, prolific rooting of excised leaves have been
achieved, but adventitious shoot buds could not be differentiated. However,
propagation was effected by easily rooting tender stem segments in vitro.
Biosynthesis of reserpine
Mitra (1964) successfully established root callus and Chaturvedi (1968) leaf and
stem callus of Rauwolfia serpentina. Histogenesis of callus formation from
different organs of the plant was also studied in detail. In such studies, the
supplement of the medium was absolutely necessary in addition to kinetin or
adenine (Ad) or Ads used in combination with an auxin-2,4-D or NAA. Later on,
after elaborate nutritional studies, the different calli could be grown not only
without culture media but at a very prolific rate of growth showing a 60-fold
increase in fresh weight in 30 days. However, none of the calli contained
reserpine or any Rauwolfia-alkaloids. Nevertheless, such calli grown on a
synthetic defined medium present a good system for studying the biosynthetic
pathway of reserpine.

6.18 APPLICATIONS OF PLANT TISSUE CULTURE

The applications of plants tissue culture can be studied under following
headings:
1. In the production of useful phytoconstituents
2. Biochemical conversions
3. Clonal propagation
4. Plant improvement
5. Immobilisation of plant cells
6.18.1 Application of Tissue Culture in the Production of Useful
Phytoconstituents
The following metabolites have been isolated from plant cell cultures: alkaloids,
lipids, flavonoids, furanocoumarins, terpenoids, sugars and polysaccharides,
latex, oils, tannins, anthraquinones, plant growth hormones, flavones, chalcones,
napthaquinones, vitamins, proteins and peptides, anti-cancer agents, anti-viral
agents, and enzymes.
Table 6.4 lists the phytopharmaceuticals that have been successfully isolated
from plant tissue cultures.
Large-scale production of biomass containing useful metabolites have been
achieved, namely, digitalis glycosides, diosgenin-derived steroidal hormone
precursors, shikonin, rosamarinic acid, opium alkaloids (codeine and morphine),
ginsenosides, ajmalicine and other indole alkaloids including vimblastine and
vincristine.
Table 6.4 Phytopharmaceuticals isolated from tissue cultures

Name of the compound Culture type

Ajmalicine (Catharanthus roseus) Suspension culture

Codeine (Papaver somniferum) Suspension culture

Digoxin (Drosera lanata) Suspension culture

Morphine (P. somniferum) Suspension culture

Nicotine (Nicotiana tabacum) Suspension culture

Reserpine (R. serpentina) Suspension culture

Tropane alkaloids (Datura innoxia) Suspension culture

Xanthotoxin (Ruta graveolens) Suspension culture

Anthraquinones (Cassia angustifolia) Cell culture

 Caffeine (Coffea arabica) Cell culture

Diosgenin (Dioscorea composita) Cell culture

Papain (Carica papaya) Cell culture

Vimblastine (C. roseus) Cell culture

Berberine (Coptis japonica) Cell and suspension culture

Cardenolides (Digitalis purpurea) Cell and suspension culture

Indole alkaloids (Ipomoea violacea) Cell and suspension culture

Rosamarinic acid (Coleus blumei) Cell and suspension culture

Atropine (Atropa belladonna) Hairy root culture

Quinine and quinidine (Cinchona Root culture

ledgeriana)

Possible complex mixtures such as rose and jasmine oil have also been
produced on a large-scale by tissue culture. A red-coloured phenolic,
naphthoquininone compound used as a dye and astringent, called “shikonin” has
been produced from cell cultures of lithospermum erythrorhizon.
Some compounds, which are not found as normal constituents of a species, can
be found in the cell cultures of the species. e.g.:- (i) a new CNS-active indole
alkaloid percine and a new anti-inflammatory lignin; (ii) unusual ether lipids: 1
(3), 2-diacylglycerol 3(1)-O-L1-(N, N, N-trimethyl) homoserine have been
isolated from algae cultures of Chlorella fusca; (iii) rutacultin from a tissue
culture of Ruta graveolens.
The rate of production of secondary metabolites from tissue culture technology
was found to be remarkably higher than from the intact plant e.g. (i) shikonin
from lithospermum;
(ii) berberine from coptis; (iii) jatrorrhizini from berberis; (iv) rosmarinic acid
from coleus. Even the yield was higher. The yield of some important natural
products is given in
Table 6.5.
 Bioactive shoot cultures have been established for Belladonna, Solanum,
Dioscorea and Vinca.
Table 6.5 Yield of some important natural products

6.18.2 Biochemical Conversions

Creating new plant cell strains can bring about desired biotransformation
reactions in the substrate molecule. Chemically difficult reactions which are hard
even in the presence of enzymes or micro-organisms can be achieved easily by
plant cell cultures. The biotransformation pattern includes methylation,
demethylation, acetylation, reduction, glycosylation, hydroxylation, etc. These
biotransformation reactions by plant cell strains play an important role in the
production of compounds having medicinal value.
Biotransformation of steroids by micro-organisms as well as by plant cell
cultures has been of increasing interest during the last years. As an example, we
will discuss the biotransformation of digitoxin to cardiac glycosides by plant cell
cultures.
Digitoxin can be glycosylated to purpurea glyoside A by cell culture of
Digitalis purpurea as well as Digitalis lanata. Digitoxin and purpurea glycoside
A are hydroxylated at the C-16 site to gitoxin and purpurea glycoside B by cell
cultures of D. purpurea, whereas cell cultures of D.lanata hydroxylate purpurea
gluycoside A at the C-12 site to deacetylanatoside A. Furthermore, purpurea
glycoside A can be acetylated to lanatoside A, which is hydroxylated at C-12 to
lanatoside C.
Those cardenolides hydroxylated at C-12, the so-called C-glycosides, are of
important medicinal value. There is an increasing need for digoxin, whereas the
use of digitoxin is rapidly decreasing. Digitalis lanata plants, however, always
contain high amounts of the “A-glycosides”, which have to be extracted and
separated from the interesting and more medicinal C-glycosides during the
technical process of isolation.
Stereochemical structure of digitoxin

Biotransformation of cardiac glycosides by micro-organisms, though, turns out

to be of low effectiveness. Therefore, the study of biotransformation capacities
of different cell strains of various Digitalis species as well as of further
cardenolide-containing plants has been initiated. The main thrust is to find cell
strains which are able to hydroxylate an A-glycoside selectively at C-12 (i.e,.
finding a process by which it might become possible to transform A-glycosides
into C-glycosides on an industrial scale).
Materials used and methods
Cell strains
The following cell strains, initiated from plant stems, have been used in the
experiment.
D. lanata cell strain no. 72 D, 72 L, 285, 347 C3, B2, 287, 293, 364, E, 293 HL,
447, 228, 508, 216, 130.
D. leucophaea cell strain no. 30 625-10-15s.
D. lutea cell strain no. D. 1u-1 and D. 1u-1F.
D. mertonensis cell strain no. 9805-3.
Thevetia neriifolia cell strain no. S.
Culture media and cultural conditions
The basal medium of Heller without growth hormones, but supplemented with
30% coconut water, (deportenised by heating) and 2% sucrose (medium I) and of
Murashige and Skoog, supplemented with 2 mg/l kinetin, 1 mg/l indole-3-acetic
acid, 1g/l caseinhydrolyaste and 2% sucrose, pH 5.5, (medium II) were used. 10
gm of tissue (fresh weight) was incubated in 50 ml of medium in Erlenmeyer
flasks at 25ºC in dark or in continuous light (2000 ± 200 lux, osram-L fluora
tubes) on rotary shakers at 60 rpm. The time of incubation was 1 to 30 days. 2
mg of cardenolide dissolved in alcoholic solution was added to each flask.
Extraction procedure and analytical methods
For each experiment three flasks were used. The culture medium and the tissue
were separated by filtration. The medium as well as the tissue were freeze dried.
The cardenolides were extracted according to the Hammerstein and Kaiser
method (1972) modified by Boy (1975). The following solvent systems were
used for TLC on silica gel plates (Merck, Darmstadt):
a. Methyl-ethyl ketone, saturated with water;
b. Xylol, methyl-ethyl ketone, formamide (40:60:5 and 50:50:2 v/v). The silica
gel plates were impregnated by a solution of 20 vol % formamide in acetone;
c. Chloroform, methanol, water (80:18:2 v/v);
d. Isopropanol, acetic acid n-butylate, water (50:30:20 v/v).
The spray reagents of Raymond (1938), Jensen (1903), and Pesez (1952) were
used for the detection of cardenolides. Quantitative analysis of the cardenolides
was acomplished using TLC, Jensen’s reagent (1953) and measuring the
fluoresence intensity of the spots using a Zeiss PMQ II TLC spectral photometer.
Results and discussion
1. Biotransformation of digitoxin by the cell strains 72 L, 72 D and C3 of
Digitalis lanata: Strains 72 D and C3 are not able to produce lanatoside A and
C.
2. Digitoxin-disuccinate: Strain 72 D splits off the sucinoyl residues of the
substrate during the first 7 days of incubation. Afterwards, the same
biotransformation products are found as with digitoxin itself.
3. Biotransformation of digitoxigenin and digitoxose by cell cultures of
D.lanata, strain 72 L and according to Stohs and Rosenberg (1976):
4. Biotransformation of digitoxigenin-diethyl-amino-acetate, by cell cultures of
D. lanata, strain 72 L:
5. Biotransformation of digitoxigenin-succinate by cell cultures of D.lanata,
strain 72 L:
6. Biotransformation of gitoxin by cell cultures of D.lanata, strain 72 L:
7. a-acetyl digitoxin is glucosylated by cell cultures of D. lanata, strain 72 L to
lanatoside-A, which is hydroxylated to lanatoside-C. Simultaneously, the
substrate is deacetylated to digitoxin. Digitoxin is glucosylated to pupurea
glycoside A, which can be hydroxylated to deacetylanatoside C.
8. Biotransformation of peruvoside by cell cultures of D. lanata, strain 72 L, and
of Thevetia neriifolia:
9. Biotransformation of β-methyldigitoxin by cell cultures of D.lanata, strains
293 HL, 130, 228, 216, 447 and 508:
10. Biotransformation of β-methyldigitoxin by cell cultures of D.lanata, strains
347, B2, 287, 293, 72L, 364 and E:
11. Biotransformation of β-methyldigitoxin by the cell strains 72 D and 285
(D.lanata):
12. Biotransformation of β-methyldigitoxin by cell cultures of D. lutea:
 Along with β-methylgitoxin, traces of purpurea glycoside A and lanatoside-A
and B are present.
13. Biotransformation of 5β-pregnan derivatives by D. purpurea suspension
cultures: also,
14. Biotransformation of androgens by the suspension cultures of D. deltoidea.
15. Biotransformation of steroidal sapogenins by the cell cultures:
16. Biotransformation of phytosterols by cell cultures:
6.18.3 Clonal Propagation
Clonal propagation involves the propagation through cell, tissue or organ
culture. The advantages of this method are rapid multiplication of superior
clones, maintenance of genetic uniformity and multiplication of sexually derived
sterile hybrids. The shoot tips or axillary buds are utilised for propagation on
culture media without the intervention of the callus phase.
The main intent of clonal propagation is to establish plants that are uniform and
predictable with selected qualities. In view of the substantially superior rate of
plant increase, interest in the use of tissue culture as an alternative to traditional
asexual multiplication methods is spreading rapidly. The tissue culture approach
provides other equally significant benefits. Pathogen-free plants are recoverable
from clones that are systematically infected; international or regional exchange
of genetic stocks is expedited and rendered safer; and cryogenic preservation of
plant germ plasma is distinctly probable. Rapid clonal increase also signifies
faster and timely establishment of new cultivars. The tendency has been to
attempt plant multiplication by exploiting the phenomena of somatic cell
embryogenesis and adventitious shoot initiation. These phenomena may be
observed in freshly excised plant sections and re-cultured callus. Absence of
their manifestation should not be interpreted as precluding clonal propagation in
vitro of the plant in question. There are other alternatives.
Somatic cell embryogenesis
Somatic embryos are simply organised structures, the morphology of which is
fundamentally identical to that of embryos originating in zygotes but for the fact
that they originated from somatic cells. They will germinate in vitro and develop
into little plants. Clonal plant multiplication occurs fastest through somatic cell
embryogenesis. Yields of 500 carrot embryos/g callus/month have not been
unusual. Nevertheless, the method is severely limited in applicability. The
process remains restricted to a few genera; and even then, only to tissues excised
from embryos, seedling and other juvenile materials.
In practice, somatic embryogenesis is accomplished in two steps:
a. increase of callus;
b. development of embryos and plants.
A substantial stock of callus is established through monthly subcultures in a
nutrient enriched with ammonium nitrate and the auxin, 2,4-D. The number of
subcultures may require restriction, in as much as embryogenesis is often
progressively represented with each subculture. Transfer of the callus to auxin-
free or low auxin medium results in emergence of embryos and plants. Embryo
formation frequently occurs directly in the explants. In such instances the
intermediary callus may be avoided and plant multiplication can be achieved by
simply separating individual embryos and culturing them further in fresh media.
New embryos probably arise by budding from existing embryos.
Production of adventitious shoots and related organs
Adventitious shoots and related organs are stem and leaf structures that in tissues
are located in sites other than the normal leaf axil region. The structures in
question include the usual stems, bulbs, corms, tubers, rhizomes and others. The
development of these structures in vitro has been more probable than that of
somatic embryos, although plant multiplication is substantially slower.
The production of adventitious shoots and related organs might also proceed
through an intermediary callus. In experience, however, the emergence of organs
directly from explants has been observed more frequently and consistently. Thus,
the advisable method is to avoid the establishment of callus culture. Increase in
propagation is accomplished by separating and subculturing the emerged organs
at 4–6 week intervals. Organogenesis is stimulated by employing a medium
containing moderate amounts of auxin and cytokinin.
When the desired number of shoots is attained, they are rooted through Stage
III (described later under the section on sequential considerations). Bulbs, corms,
tubers, and similar storage organs require no rooting. They may however, have
dormancy requirements.
The problem of genetic variability may be especially pronounced among plants
arising through adventitious organogenesis. Repeated re-culturing generally
results in an increase of deviant plants. Associated with the increase, is often a
diminishing rate of organ multiplication.
Plants, the desirable qualities of which are attributable to a chimera, should not
be propagated through this method since the chimeral components may become
separated. Variegated plants will show modified patterns or lose the variegation
entirely.
Enhanced axillary branching
Axillary branches are simply shoots that emerge from their normal position, i.e.,
the leaf axil. Lateral buds and stem terminals, containing quiescent or active
shoot tips, are stimulated to axillary branching activity by furnishing a nutrient
medium containing extremely high concentrations (10–30 mg/l), of cytokinin. At
intervals of 4–6 weeks, the miniature branches are severed and subcultured
individually in fresh medium. After several repetitions of the subculturing
process, the tiny branches are transferred to stage III and rooted.
Although plant multiplication is slowest by this method, the method has
broadcast applicability among plant genera. A million-fold enhancement of
clonal plant increase in years time over traditional methods has been achieved
with some genera, e.g. Gerbera (Murashige et al. 1974). Furthermore, the plants
obtained are less variable genetically than those arising through the adventive
process.
Sequential considerations
Normally, the process of plant multiplication in vitro must proceed through a
series of steps, each with a distinct objective and sometimes with a specific set
of requirements. When the callus intermediary is excluded, as is the case in most
of the efforts, the common sequence involves three steps. They are currently
identified as stages I, II and III.
Stage I The concern in stage I is the freshly excised tissue. The aim is to achieve
prolonged survival in vitro of an infestation-free plant section. Frequently, stage
I can be fulfilled without too much peculiar problems.
Stage II The plant materials from stage I are now sub-cultured repeatedly until
the desired quantities of embryos, shoots or other organs are obtained. The bulk
of the culture activity and time will be spent in this stage.
Stage III Difficulties are usually encountered when propagules from Stage II are
transplanted directly into soil. The difficulties can be largely circumvented by a
preparatory step, just prior to the removal of propagates from the tissue culture.
In Stage III, unrooted shoots are reported. The plants are rendered capable of
autotropic development and made adaptable to the changes in moisture, stress
and light intensity. They are also conferred a degree of resistance to common
microbial infections. Bulbs, corms, tubers and other similar storage organs are
fulfilled of their dormancy requirements. Stage III is generally accomplished in
one passage and lasts 2–4 weeks.
6.18.4 Plant Improvement
The contributions of tissue culture in plant improvement may be broadly
discussed as below.
Propagation and production of virus-free material There is an unlimited
potential for the initiation and production of virus-free material in vitro,
especially with old clones and vegetatively propagated material.
Changes in methodology Numerous contributions can be envisaged. Most of the
current steps in plant breeding can be performed more easily in culture. This is
already the case with embryo culture, used also for recovery of haploids in
crosses with barley (Hordeum bulbosum). Another case where tissue culture aids
breeding is the accomplishment of fertilisation in vitro. Direct application of
pollen grains on excised ovules of Petunia axillaris overcomes self-
incompatibility in a homophoric, gametophytic system. Changes in ploidy and
their immediate propagation can be often achieved more easily in culture.
Significant progress in breeding was achieved by the combined use of tissue
culture and colchicin.
The major potential contribution in methodology is perhaps in the field of
screening. Screening has been done to check the resistance to disease, toxins and
stress conditions. While screenings at the tissue or cellular level might not
always render further field tests superfluous, they could lead to a considerable
saving in time and space especially with suitable material and subject to further
improvements in technique.
Disease resistance Tissue culture will probably provide considerable help in the
future to breeders in the the fields of disease resistance and host–parasite
relationships. A system for studying phytophthora parasitica on tobacco callus
has been described by Helgeson et al. Callus cultures derived from resistant
plants has been observed as not supporting the growth of the fungus.
Use of haploids from culture Some of the main prospective uses for haploids
include
a. To reach homozygosity in self-sterile plants.
b. As a basis for mutagenesis and selection in the vegetative phase.
c. For the development of isolated cells from individual plants for mutagenesis,
with the obvious advantage of easy recovery of recessives.
d. The raising of haploid embryos in culture and their use in breeding programs
requiring a combination of many dominant alleles.
Mutant induction and selection A main use of tissue culture for plant
improvement will be in the field of mutagenesis and selection in vitro. This
would also mean pre-selection on an unprecedented scale and a significant
contribution to new genetic variability.
Other approaches and prospects The subject of somatic hybridisation and
fusion has been reviewed recently. The possibility of bypassing sexual pathways
in hybridisation could be of basic interest in vegetatively propagating crops and
for the creation of entirely new plant types. Protoplast fusion has been effected
even in widely differing families and some interesting somatic hybrids have been
created in tobacco. But it is still uncertain how far the range of viable parasexual
hybrids can be extended beyond present sexual compatibilities.
Recent endeavours in producing chimeras in vitro are of special interest to
breeders of vegetatively propagated plants. Some of the best horticultural
varieties originated as somatic mutations and quite a few are periclinal chimeras.
In the past, chimeras have been produced experimentaly through compatible
graft combinations and wounding. The concept of a periclinal chimera with an
outer layer resistant to fungi has also been propounded.
Transfer of specific genetic information is a common technique in use for the
genetic modification of bacteria. Similar approaches have been made with
plants, including DNA-mediated transformation. Of particular interest are
experiments in which free-living nitrogen-fixing bacteria are to be associated
with easily cultured, non-leguminous species. A symbiosis was recently forced
between cells of the carrot and the free-living bacterium, Azotobacter vinelandi,
although no regeneration was achieved. Presumably, carrot cells made use of
reduced nitrogen from Azotobacter while the bacterium depend on carrot cells
for available adenine.
Present limitations In summing up, several limitations restricting the use of
tissue culture at present have to be mentioned.
a. The state of knowledge on how to induce shoot bud initiation is still
unsatisfactory. A better hormonal or combined control of bud differentiation
and a more complete understanding of de-differentiation in callus are required.
b. The need to produce variant or other breeding material in sufficient numbers
for further selection or trail is ultimately linked to improvements in
 regeneration and propagation to ensure maximum survival and use of material
produced in culture.
c. There is a lack of stability in many cultures.
d. There is a lack of methods for variant identification in many cases and a
growing need to relate cell selection to adult plant phenotype.
e. There is limited knowledge of important physiological and biochemical
processes in plants and of mechanisms (molecular and cellular) governing
important agronomical traits. There is a need for selection schemes with
relevance to agronomic traits and the intensification of detailed tissue culture
work on basic crop plants.
f. Juvenility in plants with a protracted cycle has to be overcome. There is, as
well, insufficient realisation of existing possibilities, especially in vegetatively
propagated crops.
g. Lastly, closer links between physiologists, biochemists, geneticists, breeders
and propagators is needed.

6.18.5 Immobilisation of Plant Cells

The immobilisation of plant cells has received much attention in the last
decades. There are many possible advantages of immobilisation. In general,
immobilisation can facilitate the use of continuous flow processes and the plant
cells can be reused. Apart from these general advantages, there are some
additional advantages as well. The cell or cell content that is induced by
immobilisation can be beneficial for secondary metabolite production. It has
been observed that in immobilisation, some differentiation occurs, which is
believed to be a prerequisite for the production of secondary metabolites.
Furthermore, immobilisation protects sensitive plant cells against shear forces,
while in some cases production and excretion can be stimulated.
The following methods are used for the immobilisation of plant cells: (a) gel
entrapment; (b) biofilms; (c) adsorption; (d) foam immobilisation.
Gel entrapment
The most common method used for plant cell immobilisation is gel entrapment
and the most common gel used is calcium alginate. A mixture of sodium alginate
and cell is extruded drop-wise into a calcium chloride solution, where bead
hardening starts to take place immediately. Alginate is the preferred gel because
of the ease with which it can be handled and because of its mildness. Moreover,
calcium alginate can have beneficial effects on the production of secondary
metabolites. Asada and Shuler (1989) immobilised Catharnthus roseus cells in
calcium alginate, which resulted in a three-fold increase in extra-cellular
ajmalicine. Kim and Chang (1990) obtained a 2.5 fold increase of shikonin
production when they immobilized Lithospermum erythrorhizon cells in calcium
alginate. Improved yields with cells immobilised in calcium alginate have also
been reported for the biotransformation of 2-(4-methoxybenzyl)-1-
cyclohexanone to its glucoside by Dioscorea deltoidea cells and for alkaloid
production by Solanum xanthocarpum cells.
In many cases, immobilised cells can be used for much longer periods than free
cells. Naoshima and Akakaba re-used immobilised Nicotiana tabacum cells
seven times for the biotransformation of various 3-oxobutanoates and the
conversion speed was increased with the number of uses. The immobilised cells
transformed cis-verbenol mainly into trans-verbenol, whereas free cells
transformed cis-verbenol to verbenone as the main product. This indicates that
even a mild immobilisation is not priori an inert operation, but can have a
pronounced effect on cell physiology.
Biofilms
Another application of gels for the immobilisation of plant cells is their use as a
support for biofilm formation. Krgi (1988) grew Catharanthus roseus cells on
the surface of calcium alginate beads within the interspatial volume of a packed
column. The cells showed some growth and the alkaloid content of the medium
were increased.
Adsorption
Several authors immobilised plant cells by adsorption to various support
materials, such as fibre glass, polystyrene, sulfonated polystyrene, fluorinated
ether etc (Plate 4). Mainly catharanthus roseus cells have been used and there is
ample evidence to show the determining role of the surface tension of the cells,
the surface and the culture liquid in the adhesion procedure. The culture age is
important in order to obtain good adsorption. Immobilisation is best, when the
cells have reached the age of 8–14 days, probably due to the higher secretion of
polysaccharides by the cells. Archambant et al. (1990) suggest that the adsorbed
biomass of Catharanthus roseus in their system contains a concentration of
indole alkaloids 3–12.5 times as high as the maximum quantity found in freely
suspended biomass in identical conditions.
Foam immobilisation
Another material that has found widespread use in plant cell immobilisation is
polyurethane foam. The immobilisation procedure is very simple; the cells
migrate into the foam without any force, so no changes in the temperature or
ionic strength influence the viability of the cells. The foam can be used in all
kinds of forms, as cubes, attached to stainless steel wire or wound sheets or
tubes. Usually, it takes about 10–15 days before all the cells are immobilised in
the foam, which is a disadvantage. The effects of the immobilisation in
polyurethane foam is not uniform for all cell cultures.
 7

Systematic Examination
of Powdered Drugs

Herbal drugs made from roots, stem and other parts of a plant are available in
different forms in the market. A common means of dispersal of a drug is in its
powdered form. But this form is prone to abuse. Description of the drug in
powdered form (so as to easily identify it and distinguish it from other drugs) is
necessary because of the following reasons: 1. Use of whole drugs, where
identification is more reliable, and preparation of the powder from these whole
drugs only prior to its incorporation in a formulation, is optimal, but
manufacturers are tempted to store or even buy drugs in the powdered form for
ease of handling and storage. When quality control officials come across
powdered drugs, samples are drawn for analyses, and are required to be tested as
per the pharmacopoeia, thus making the description of the drug necessary.
2. Chances for adulteration, spoilage, insect infestation or misidentification
increase for a powdered drug.
3. Knowledge of the diagnostic features of a powdered drug also help in the
identification of ingredients in a compound formulation when such powders
are added in situ to them.
Drugs can be classified as organised or unorganised depending upon whether
they have a definite structure or not. Organised drugs are those which have a
definite structure.

7.1 EXAMINATION OF ORGANISED DRUGS

About 500 mg of a representation sample is taken and
1. mixed thoroughly with water in a watch glass;
2. warmed, cleared in chloral hydrate and washed thoroughly in water; 3. stained
with phloroglucinol and concentrated hydrochloric acid. It is then washed
thoroughly in water; 4. stained with dilute iodine solution and again washed
thoroughly in water.
A drop from each of the above is mounted in glycerin. It is observed under a
microscope and the unique characters noted. The possible microscopical
characters of different organised drugs are given in Table 7.1.
Table 7.1 Microscopical characteristics of different organised drugs

7.2 EXAMINATION OF UNORGANISED DRUGS

Unorganised drugs, as the name suggests, are drugs that show no definite
structure. Unorganised drugs are fairly homogenous and may be solids, semi-
solids or liquids. For the unorganised drug, the following macroscopical data can
be recorded: 1. Form: Tears, lumps, semi-solid, masses etc
2. Size, colour, odour and taste
3. Surface appearance (as such and freshly broken): shiny, smooth, rough, dusty,
dull 4. Fracture: brittle, granular, conchoidal, clean, or hard to fracture, waxy,
etc 5. Any distinctive feature of fractured surface; any other peculiarity
6. Reaction towards water, acids and solvents
7. Fluorescence: as such and after preparing solutions, or after chemical reaction
with certain reagents.
Unorganised drugs may be sorted by observing the solubility towards alcohol
and, then applying other tests.

7.2.1 Preliminary Tests

Drugs insoluble in alcohol
1. Triturate the sample with water; if it gives the characteristic smell of
asafoetida or myrrh, it may contain these compounds.
2. Heat the sample with soda lime in a dry test tube; observation of ammonical
vapours may indicate the presence of gelatin or albumin.
3. One part of the representative sample is boiled with 100 parts of water; if a
formation of a stiff jelly-like mass is observed on cooling, it may be agar; if
there is coagulation of liquid, it may be albumin.
4. The sample is warmed in the presence of 5% caustic soda solution. Formation
of a canary yellow colour indicates the presence of agar or tragacanth.
5. A small quantity of the sample is mounted in alcohol and irrigated with water.
The behaviour of the particle of the sample is observed. If the particles of the
sample dissolve without swelling, it may be acacia. If the particles undergo
swelling and ultimately dissolve or become diffused, it may be tragacanth,
sterculia gum, gelatin or agar.
6. Mount the sample in ruthenium red. Formation of pink colour may indicate
the presence of agar or sterculia gum.
7. The sample is mounted in N/50 iodine solution; formation of olive green
colour with blue specks, may indicate the presence of tragacanth; formation of
crimson or brown colour, may indicate the presence of agar; if there is no
colour reaction, it may be acacia.
8. Presence of an acetous odour may indicate the presence of sterculia gum.
Drugs soluble in alcohol
1. Test for hydroxymethyl anthraquinone derivatives. Observation of rose pink
colour indicates the presence of aloes (exception: zanzibar aloes).
2. Treat the sample with potassium permanganate solution. Observation of
benzaldehyde odour (bitter almond smell), may indicate the presence of
benzoin.
3. Treat the sample with alcoholic ferric chloride solution. Observation of green
colour indicates the presence of gambier.
4. If an alcoholic solution of the drug is acidic to litmus, it indicates the presence
of colophony.
After the preliminary examinations, the unorganised drugs need to be identified
by specific chemical tests.

7.2.2 Chemical Tests for Different Unorganised Drugs

Indian gum (gum acacia) Prepare a solution of the sample by adding 10 ml
of water to 0.5 gm of the sample, stirring well. Use this solution for the
following tests:

Agar
The following tests can be performed on the sample to detect the presence of
agar.
Aloe Boil 0.5 gm of the sample with 50 ml of water until it is nearly
dissolved. Cool and add 0.5 g of Kieselguhr. Filter and use the filtrate for the
following tests.
Asafoetida/Devil’s dung Asafoetida can be detected by performing the
following tests.
Balsam of Tolu
The following tests can be used to detect the presence of tolu balsam

Bees wax
Bees wax is sometimes adulterated with other fatty substances or resins. The
purity of bees wax can be verified by performing the saponification cloud test.
Saponification cloud test Boil 0.5 gm of the suspect bees wax for 10 minutes in
10 ml of sodium hydroxide solution. Make up the original volume, filter through
glass wool and acidify with hydrochloric acid. If fatty substances, fatty acids or
resins are present, they will be precipitated. Fats may be saponified by boiling
with aqueous sodium hydroxide but waxes are unaffected by aqueous alkali
though they can be saponified by strong alcoholic potassium hydroxide.
Benzoin
The presence of benzoin can be detected by performing the following tests
Castor Oil
The following tests can be used to detect the presence of castor oil.

Black catechu
Black catechu is completely soluble in hot water and alcohol. It can be identified
by the following tests.

Gambier/pale catechu The following tests can be performed to confirm the

presence of pale catechu.
Chrysarobin
Chrysarobin can be identified by the following methods.

In addition to these tests, chrysarobin also answers the tests for anthracene
derivatives (given under aloes).
Colophony
The following tests can be performed on the drug to confirm the presence of
colophony.

Gelatin
The presence of gelatin can be confirmed if on heating the powder with soda
lime, ammonia gas is evolved. Gelatin can also be identified by performing a
number of tests on a test solution. The test solution is obtained by dissolving 0.5
gm of the substance in 100 ml of water and heating.

Honey
The presence of honey can be confirmed by adding 4 ml of alcohol to 1 ml of the
substance—a slight turbidity results. Honey is often adulterated with reducing
sugars and artificial invert sugar. Fiehe’s test is used to detect the presence of
artificial invert sugar.

Lanolin
The presence of lanolin can be detected by checking for the presence of
cholesterol.
Dissolve 0.5 gm. of the substance in 5 ml of chloroform. Add 1 ml of acetic acid
and 2 drops of concentrated sulphuric acid. A deep green colour confirms the
presence of lanolin.
Myrrh
The following tests can be used to detect the presence of myrrh.

Sesame oil
Oil can be identified as sesame oil by shaking 1 ml of the oil sample with a
solution of 0.5 gm sucrose in 10 ml of hydrochloric acid for 30 minutes. The
acid layer becomes bright red finally changing to dark red. This confirms the
presence of sesame oil.
Shark liver oil
The presence of shark liver oil can be confirmed by performing the following
tests.

Sterculia gum/Indian tragacanth

The same tests used to confirm the presence of gum tragacanth (explained later)
can be used to detect the presence of sterculia gum. Besides these, the following
tests can be used.
Storax
The following tests can be used to confirm the presence of storax.

Tragacanth
The presence of tragacanth can be confirmed by the following tests.
 8

Application of
Chromatography and
Spectroscopy in Plant
Drug Analysis

In the last chapter, we discussed general methods of distinguishing general

groups of drugs. For a systematic analysis of a drug, of course, those methods
are comparatively crude. New methods have been developed for sensitive
analysis of drugs. Two of these are chromatography and spectroscopy.

8.1 CHROMATOGRAPHY
Chromatography is a broad range of physical methods meant to separate and
analyse complex molecular mixtures. It depends on the differential affinities of
the solutes in two immiscible phases — a fixed bed with a large surface area and
a fluid which moves through, or over the surface of the fixed or stationary phase.

8.1.1 Classification
Chromatographic methods can be classified first according to the nature of the
stationary phase. The mobile phase may be a liquid or gas and the stationary
phase may be a solid or a liquid. There are four main sub-divisions of the
chromatographic process.

1. Liquid–solid: Adsorption chromatography; thin layer chromatography; ion

exchange chromatography
2. Gas–solid
 3. Liquid–liquid: Partition chromatography; paper chromatography
4. Gas–liquid

8.1.2 Adsorption Chromatography/Column Chromatography

The term column chromatography is used today to refer to those methods in
which the separation takes place within a packed column. The packing material
is the stationary phase and is solid with adsorptive or exclusion capabilities. A
liquid mobile phase is used as the eluent. In 1941, Martin and Synge developed a
liquid chromatographic process in which they used a packed column containing
water saturated with silica gel and a mobile phase of butanol chloroform.
To separate a mixture, a column is packed with an active solid such as alumina
(stationary phase) covered with a solvent (mobile phase).
A small sample of the mixture loaded at the top of the packed column forms a
band of adsorbed material. When a solvent is allowed to flow through the
column, it carries with it the components of the mixture. The rate of movement
of a given component depends on how much of it is retarded by adsorption on
the column packing. A weakly adsorbed substance travels more rapidly than a
strongly adsorbed one. The fractions which are obtained through running
different solvents in order of their increasing polarities are made from solvent
under vacuum to give the residue. On adsorbents like silica gel or alumina, the
order of the polarity of the solvents is as follows: Hexane → Benzene →
Chloroform → Ethyl acetate → Methanol→ Water.
The order is reversed if we use Sephadex as an adsorbent.
Column
The column is usually a glass or stainless steel tube tapered at the bottom and
often fitted with a top. A standard ratio of length to diameter ≥ 40:1 should be
taken as a standard. This tube is generally about 20–30 cm long with an internal
diameter of 2–3 cm, holds a capacity of 50–100 gms adsorbent and retains
several grams of adsorbate. In general, column chromatography is performed at
room temperature under the following conditions.

1. Column should be dried.

2. Silica gel should be moisture-free, otherwise it may get de-activated
affecting the desired separation.
3. The fraction should be made free of solvent, preferably using a thin
 evaporator.
4. Solvents to be used in the column should be completely dry.
5. It is advisable to collect small fractions when it is felt that the component
has started eluting.

The various adsorbents include silica gel, alumina, Kieselguhr, celite,

cellulose powder, ion exchange cellulose, starch, polyamide powder and
Sephadex.
Stationary phase
The general requirements of a stationary phase are

1. It should be spherical in shape and uniform in size.

2. Its mechanical stability must be enough to prevent the formation of dust,
which might be deposited in the channels of the packing.
3. It should not react chemically, either with the eluting solvents or with the
solvent components.

The stationary phase should be catalytically inactive and as a rule, have a

neutral surface (except ion exchangers). Adsorbents are classified based upon
the force with which they hold ions as

a. weak form eg sucrose and starch;

b. intermediate form eg CaCO3, Ca(OH)2, Mg(OH)2;
c. strong form eg silica gel, alumina, charcoal, and fuller’s earth.

Packing of the column

Dry packing
In this method, the adsorbent is packed in the column in dry form and the solvent
is allowed to flow until the equilibrium is reached. The disadvantage with this
technique is that air bubbles are entrapped between the solvent and the stationary
phase and hence the column may not be uniformly packed. Cracks may also
appear in the column. Hence a clear band of the separated component may not
be obtained.
Wet packing
In this method the adsorbent is mixed with the mobile phase in a beaker and
poured into the column. The stationary phase settles uniformly in the column.
There is no entrapment of air bubbles and cracking in the column. The flow of
characteristics and separation of bands is uniform. This is the ideal technique for
the separation. Figure 8.1 shows a diagrammatic representation of column
chromatography.

Fig. 8.1. Column chromatography

Sample introduction into the column

Mixtures of solutes are dissolved in a minimum volume of solvent, which is used
as an eluting agent. If the elution is for a quantitative purpose, then the solute
mixture is introduced by using a micropipette/ burette or syringe.
If the solute mixture is insoluble in the mobile phase, a large volume of
solvent should be introduced. The solute added is evaporated out and gets
impregnated on the adsorbent. This dry powder is placed in the column. The
flow rate depends upon the charge on the solute, the adsorbent and size of the
column. Generally, slow flow rates are preferred for better separation i.e., 5
ml/cm sq. cross-sectional area/ hour.
The solute–adsorbent proportion plays an important role in separation by
column chromatography.
Elution
There are three methods of elution, namely, isocratic, gradient and extrusion.
Isocratic elution technique
This technique involves the use of a fixed solvent or its combination throughout
the process of elution. The major disadvantage of this technique is that it takes a
long time to elute and there is poor resolution for complex mixtures.
Gradient elution technique
This method is the modified form of the isocratic elution technique. Here, two
eluting solvents, one weak and one strong are used. The weak solvent has a low
affinity for the solute, whereas the strong solvent has high affinity. The elution is
started with the low affinity solvent, later the concentration of the high affinity
solvent is increased gradually until the final mobile phase has a composition
approaching that of the strong solvent. The weak solvent elutes the weakly
retained solutes first, and the high affinity solvent elutes the strongly retained
solute, which results in short retention and good resolution.
Extrusion elution technique
In this method, the passage of irrigating solvent is stopped when a reasonable
resolution of bands has occurred. After the passage of solvent is stopped, the
column is drained and extruded carefully. It is then placed horizontally. The
bands are separated by a knife or a scalpel. It should be noted that the extracted
column is not to be cut perpendicular to the axis of the column at the boundaries
of the zones. The solutes from the different bands are then extracted with a
solvent and the resulting substance estimated by a suitable technique.
Applications

1. A tool of purification: The impurities get retained by the adsorbents and the
pure compound can be separated by using a suitable eluting agent.
2. Separation of complex mixtures of phytoconstituents can be done from
herbal extracts, for example, constituents like alkaloids, phenolic
compounds, aglycones, phytosterols can be separated.
3. Separation of components of interest, i.e., the desired constituent from the
extracts can be separated by using a proper solvent system, for example
quinine from cinchona extract.
4. The components can be separated in gram quantities, which are required to
 carry out suitable biological activity.

8.1.3 High Performance Liquid Chromatography (HPLC)

Introduction
In HPLC, a liquid mobile phase is pumped under pressure through a stainless
steel column containing particles of the stationary phase (Fig. 8.2).
The solute is loaded onto the head of the column via a loop valve and
separation of mixture occurs according to the relative length of the time spent by
the components in the stationary phase. It should be noted that all components in
a mixture spend more or less the same time in the mobile phase in order to exit
the column. Monitoring of the column effluents can be carried out with a variety
of detectors.
Advantages

1. It is easily controlled and precise sample introduction ensures quantitative

precision.
2. HPLC is a chromatographic technique which has gained extensive
development in recent years leading to improved column detectors and
software control.
3. The variety of columns and detectors available means that the selectivity of
the method can be readily adjusted.

Fig. 8.2 HPLC (Reproduced with permission from: Spinco Biotech Pvt
Ltd.)
 4. Compared to gas chromatography, there is less risk of sample degradation
because heating is not required in the chromatographic process.

Limitations

1. A reliable and inexpensive detector which can monitor compounds that lack
chromophores is required.
2. Drugs have to be extracted from their formulation prior to analysis.
3. Large amounts of organic solvent waste are generated which is expensive to
dispose.

Separation
Isocratic chromatography
A separation that employs a single solvent of constant composition is termed as
an isocratic elution.
Gradient chromatography
Depending upon the complexity of the sample, it may be required to change the
composition of the mobile phase during the analysis to achieve the desired
separation. This results in the separation of the later or slower eluting
components and completion of the analysis in a shorter period of time.
Mode of separation in HPLC
(i) Adsorption; (ii) partition; (iii) ion exchange; (iv) size exclusion; (v) affinity.
Adsorption
The solutes are retained as a result of the ability of the stationary phase to bind
them temporarily to its surface. It can be

1. Physical adsorption: In which the energy required to break the bonds

between the solute and the adsorbent is small and the mobile phase, through
its ability to dissolve and displace the solute effectively counteract these
attractive forces.
2. Chemisorption: In which strong chemical bonds form between the solutes
and the adsorbent, and the mobile phase is not able to provide sufficient
energy to adsorb the solutes.
3. Partition: The mixtures of solutes are separated according to the relative
 tendencies of their components to partition between the mobile phase and
the stationary phase. The stationary phase consists of a layer of liquid
coated or bonded onto the surface of the solid phase.
4. Ion exchange: This method provides a greater degree of selectivity due to
the larger number of combination of mobile and stationary phases, which
are employed.

It is especially useful in inorganic cations, amino acids or similar groups of

closely related compounds.
HPLC of natural products
The application of high performance liquid chromatography in the separation of
compounds from natural products has been summarised in Tables 8.1–8.10
(David G.I.Kingston, 1979).
Table 8.1 Separation of carboxylic acids and derivatives
Table 8.2 Separation of flavonoids and related compounds
Table 8.3 Separation of oxygen-containing heterocyclic compounds

Table 8.4 separation of terpenes and plant pigments

Table 8.5 Separation of steroids
Table 8.6 separation of steroid and other plant glycosides
Table 8.7 Separation of amines
Table 8.8 Separation of alkaloids
Table 8.9 Separation of antibiotics
Table 8.10 Separation of mycotoxins
8.1.4 Gas—Liquid Chromatography
In 1952, Martin and Synge developed a chromatographic technique in which the
moving phase is a gas. Gas chromatography (GC) makes use as the stationary
phase, a glass or metal column filled either, with a powdered adsorbent or a non-
volatile liquid coated with a non-adsorbent powder (Fig. 8.3). The mobile phase
consists of an inert gas loaded with the vaporised mixture of solutes flowing
through the stationary phase at a suitable temperature. The principle behind the
process is not known but it is generally held that separation of the components
occurs due to adsorption or partition effects.
Advantages

1. It has a very high frequency of separation and even complex mixtures may
be separated.
2. It has a very high degree of sensitivity in detection of components i.e., only
a few mg of sample is enough for complete analysis.
 Fig. 8.3 Gas chromatograph (Reproduced with permission from: Spinco
Biotech Pvt Ltd.)

3. Speed of analysis is quite rapid.

4. Gives reasonably good accuracy and precision.
5. The technique is fairly suitable for routine analysis because its operation
and related calculation do not require a highly skilled person.

Theory
As mentioned before, the principle behind the partition method is not fully
understood, though there are a number of theories to explain it. Three theories
have wide recognition and acceptance. They are

a. Plate theory
b. Rate theory
c. Random walk and non-equilibrium theory.

Plate theory
Martin and Synge first proposed the plate theory by GC method compared with
fractional distillation. The ‘theoretical’ plate is the partition of the column
wherein the solute is in complete equilibrium with the mobile and stationary
phase. The equilibrium can be expressed as:
Rate theory
The plate theory has certain limitations:

a. It does not speak of the separating power of a definite length of column.

b. It does not suggest means of improving the performance of the column.

The rate theory suggests that as the mobile phase flows continuously, solute
molecules are constantly being transformed and partitioned in the gas

chromatographic column. It is expressed as:

h = Plate height
u = Average linear gas velocity
λ = Measure of the packing irregularities
dp = Particle diameter
ɣ = Tortuosity factor
DG = Coefficient of gaseous diffusion of the solute in the carrier gas
K1 = Ratio of the amount of solute in the stationary phase to that in the gas
plate D L = Diffusion constant of solute in liquid phase
Df= Film thickness

Random walk and non-equilibrium theory

Giddings et al. described chromatographic separation in terms of a random walk.
Based on
a statistical concept, the virtual spreading of a solute band may be considered by
virtue of
molecular diffusion and mass transfer with the eddy diffusion being equated to
standard
deviation. Thus, plate height h employing the random walk approach may be

expressed as:
Cu = Resistance to mass transfer in a gas phase which should be treated
independently
A = Eddy diffusion
B = Longitudinal molecular diffusion in both mobile and stationary phase.
C = Kinetic or mass transfer term with origination in the stationary phase.
Application of GLC in natural drugs
Analysis of eucalyptiol (1,8-cineole) in eucalyptus oils by gas chromatography

Test sample : Eucalyptus oil

Column : 30 m fused silica capillary column walls coated with

FFAP

Carrier gas : Helium, 1.5 ml. min–1

Column : 90ºC for 5 minutes, then programmed at the rate of

temperature 4ºC min.–1 to 200ºC

InjectionPort : 220ºC
temPerature

Detector : 240ºC
temperature

Recorder : 2mV,chart speed 1 cm. min.–l, signal attenuation

1:100

For GC analyses, 0.1ml of pure test sample is injected with a 1.0 μl syringe
The identification of eucalyptol is done by comparing the retention time of
peaks by peak enrichment techniques with standard samples run under similar
operating conditions.
Analysis of trans-anethole infennel oil (Foeniculum vulgare Linn).

Test sample : Fennel oil

Gas : NUCON-5765
chromatograph
model

Column : Capillary 30 m long fused silica column

Stationary : FFAP
phase

Carrier gas : Helium

 Flow rate : 1.5 ml.min–l

Sample size : 0.01µl

Column : Isothermal 90°C for 5 minutes, then programmed from

temperature 90°C at the rate of 4°C min.–1

Injector : 240°C
temperature

Detector : 270°C
temperature

Recorder : 2 mV, signal attenuation 1. 100

Chart speed : 1 cm.min–1

Elution pattern : cis-anethole ≤ 0.20% (Retention time: 24.7 minutes)

transanethole (Retention time: 26.5 minutes)

Analysis of eugenol in clove oil

Test sample : Clove oil

Gas chromatography : NUCON-5765

model

Column : Capillary 30 m long fused silica column

Stationary phase : FFAP

Carrier gas : Helium

Flow rate : 1.5 ml. min.–1

Sample size : 0.20 µl

Column temperature : Isothermal 90°c for 5 minutes then

programmed from
90°C–202°C at the rate of 7°C min.–1
 Injector temperature : 240°C

Detector temPerature : 270°C

Recorder : 2mV, signal attenuation 1:100

Chart speed : 1 cm. min.–1

Elution pattern : Eugenol (Retention time: 30.7 minutes)

Analysis of coryone in dill oil

Test sample : Dill oil

Gas chromatography : NUCON-5765

model

Column : Capillary 30 m long fused silica column

Stationary phase : FFAP

Carrier gas : Helium

Flow rate : 1.5 ml. min.–1

Sample size : 0.20 µl

Column temperature : Isothermal 90°c for 2 minutes then

programmed from
90°C–210°C at the rate of 4°C min.–1

Injector temperature : 240°C

Detector temPerature : 270°C

Recorder : 2mV, signal attenuation 1:100

Chart speed : 1 cm. min.–1

Elution pattern : d-Limonene (Retention time: 6.2 minutes)

 Carvone (Retention time: 18.7 minutes)

Analysis of 1-menthol in mint oil

Test sample : Dill oil

Gas : NUCON-5765
chromatography
model

Column : Capillary 30 m long fused silica column

Stationary : FFAP
phase

Carrier gas : Helium

Flow rate : 1.5 ml. min.–1

Sample size : 0.20 µl

Column : Isothermal 100°c for 2 minutes then programmed from

temperature 100°C–210°C at the rate of 4°C min.–1,isothermal at
210°C for 5 minutes

Injector : 230°C
temperature

Detector : 260°C
temPerature

Recorder : 2mV, signal attenuation 1:100

Chart speed : 1 cm. min.–1

Elution pattern : l-Limonene (Retention time: 6.52 minutes)

Menthone (Retention time: 13.47 minutes)
Isomenthone (Retention time: 14.5 minutes)
Menthol (Retention time: 18.8 minutes)
GLC of fixed oils
The fatty acids obtained after acid hydrolysis are converted to methyl esters with
ethereal diazomethane and then analysed by GLC. Alternatively, the fatty acid
methyl esters can be obtained directly by trans-methylation of the parent lipids
by refluxing them for 90 minutes with methane-benzene-H2SO4 (20:10:1). GLC
is commonly carried out on columns of polyethylene glycol adipate (10% on
100–120 mesh celite) at 200° or of 3% SE 30 on 80–100 mesh Gas Chromosorb
Q at 204°. The peaks obtained are compared in retention times
with standard fatty acid methyl esters.
Determination of moisture content
The moisture content determination in several plant drugs can be analysed by the
GLC method.
For the identification of volatile terpenes in any plant material, it is essential
to combine the use of GLC with other procedures, especially with TLC. TLC is
useful, for example, for monitoring fractions separated by preparative GLC; on
the other hand, if a preparative GLC apparatus is not available, large-scale
separations can be carried out on TLC, with the TLC fractions subsequently
being monitored by GLC. At one time, IR spectra of separated oils were
determined routinely for confirming identity. It is now more usual to measure the
mass spectra, since most terpenes give characteristic fragmentation patterns. In
critical cases, both IR and mass spectra should be determined.

8.1.5 Thin Layer Chromatography

Thin layer chromatography is a method of analysis in which the stationary
phase, a finely divided solid, is spread as a thin layer on a rigid supporting plate
and the mobile phase, a liquid, is allowed to migrate across the surface of the
plate by capillary action.
In 1938 Izmailov and Schreiber introduced the TLC technique for analysing
plant material for alkaloids using thin layers of alumina on macroscopic slides.
Stahl in the 1950s published the method and developed a kit of basic equipment
for commercial use (Fig. 8.4).
Fig. 8.4 T LC kit (Reproduced with permission from: Toshiba Pvt Ltd.)
Adsorbents for TLC

Various types of tested adsorbents are available for TLC. They differ from the
usual material in that their structure is fine graded i.e., finally divided, with the
grain size of the adsorbent lying between 5 to 50 microgram and passing through
a #200 screen.
Silica gel
Silica gel is the most extensively used adsorbent. It is employed as such for
adsorbent TLC; and modified for reverse phase chromatography using
substances such as silicon oil, or by binding a non-polar functional group such as
octadecylsilyl (DS) to it. It is suitable for constituents like amino acids,
alkaloids, sugars, fatty acids, lipids, essential oils, steroids and terpenoids.
Alumina
Alumina has basic surfaces and is chosen over silica gel for separation of weakly
polar compounds. Similar to silica gel, alumina can be obtained in various forms
like alumina G, alumina H and alumina F 254 etc. It is suitable for alkaloids,
food dyes, phenols, steroids, vitamins, carotenes and amino acids.
Kieselguhr
Kieselguhr is a neutral adsorbent with low reactivity. It is not used to a very
large extent. Other inorganic adsorbents are CaSO4, magnesium silicate, MgO,
bentonite etc. Kieselguhr is suitable for sugars, dibasic acids, fatty acids,
triglycerides, amino acids and steroids.
Organic absorbents
Cellulose and its derivatives are used exclusively for separation of hydrophilic
compounds like amino acids, nucleic acids, carbohydrates and closely related
isomers. It is suitable for amino acids, food dyes and alkaloids.
Polyamides (nylon)
Nylon is a long-chain polymer which because of the presence of many free
amide and carboxylic groups on its surface is an adsorbent with strong hydrogen
bonding abilities. It is suitable for anthocyanins, antioxidants, flavonoids and
proteins.
Ion exchange in TLC
The presence of ion exchanging groups is the dominating feature of an ion
exchanging adsorbent. A fine grain ion exchanger is used in TLC. The ion
exchanger is usually a polyelectrolyte of high molecular weight, capable of
exchanging their bound ions with ions of the same charge that are in the
surrounding medium. Ion exchangers can be polyfunctional (i.e., they contain
various types of functional group). The acidity and basicity of the exchangers is
determined by the nature of the functional groups. The strongly acidic synthetic
resin exchangers may posses the SO3 group, the weakly acidic the COO–, the
strongly basic the N+ (CH3)2 and the weakly basic the amino group.
Cellulose Ion exchange cellulose adsorbent is suitable for the separation of
nucleotides and halide ions.
Celite It is suitable for steroids and inorganic cations.
Starch Suitable for amino acids.
Sephadex Suitable for amino acids and proteins.
Solvent system
The solvent system is chosen by the trial and error method. The rate of migration
of a compound depends on the solvent used. The simplest systems are mixtures
of organic solvents used to separate mono- and bifunctional molecules by
adsorptive chromatography on layers of activated silica gel or alumina. Solvents
at the bottom of the series are polar and move most of the compounds, whereas
those at the top are non-polar and move few compounds.
If the chemical nature of the solutes to be separated is known, a suitable
solvent can be selected using Stahl’s triangle.
Selection of supporting plate
The carrier or supporting plate is the backbone of the entire chromatographic
apparatus, and hence it must be stable in the presence of all types of solvents,
reactive spray reagents, and also at high temperatures. Glass plates fulfill these
requirements best. Their thickness is usually chosen between 1.8 mm to 5.5 mm.
Borosilicate glass is also used when a reaction is carried out at very high
temperatures.
Preparation of thin uniform layers
Adsorbent particle sizes of most TLC stationary phases lie between 5–50 micron
metres. The film thickness varies from 0–2 mm; generally 0.25 mm thickness is
used. The most important aspect in preparation of thin layers is the uniformity in
thickness and consistency throughout the layer. The adsorbent in finely divided
form is applied in the form of a suspension of slurry in a suitable solvent, which
evaporates leaving behind a thin layer of the adsorbent. Thin layers can be
prepared by the following methods: Dipping Plates are dipped into a slurry of
the stationary phase and suspended in a volatile solvent. The solvent is then
removed by air drying or by heating the plates in an oven. The development time
for this method is very short.
Spraying An aerosol spray is used to prepare this layer, using a slurry of suitable
consistency.
Pouring A known amount of slurry is poured onto the plate. The plate is then
tipped back and forth to spread the slurry.
Spreading An applicator or spreader is used and the layer thickness can be
varied as desired.
Sample preparation and application
The dried plates are conditioned, if necessary, in a controlled humidity chamber.
Samples ranging from a few micrograms are dissolved in 10–100 ml of a volatile
solvent. They are then applied as spots or as thin streaks with a capillary tube or
a micro litre syringe. The solvent must be evaporated between successive
applications. Area of sample application should be as small as possible.
Development
Chromatoplates are placed inclined at about 45 degree in a tank. The tank is
closed with a lid and the solvent allowed to move up the plate from the point of
application. When the mobile phase has travelled two-third of the plate, the plate
is removed from the chamber, the solvent front is marked and allowed to dry.
The various development techniques are: Linear development The TLC plates
are developed linearly in the ascending, descending or horizontal mode. In the
ascending mode, the sample is spotted at one end of the plate and then developed
in the ascending direction. It is also known as vertical development. In the
descending mode, the solvent is allowed to flow from the reservoir to the plate
through a strip of filter paper. It has no advantages in terms of efficiency of
separation and speed analysis. It also requires a more complicated apparatus. In
the horizontal mode, the test solution is placed in the middle of the plate and
developed either by slowly dripping solvent on to it from a micropipette or by
supplying the solvent from a reservoir through a wick. Like descending TLC, it
is used less often as it is a complicated process.
Radial development It is also known as circular TLC. It is performed on
horizontal chromatoplates.
Extended development The extended development consists of the following
techniques: continuous type, multiple development type and the two-
dimensional type. The continuous technique is used to achieve complete
separation of compounds, which resolve with small differences in Rf values.
Here the solvent is forced to run over the edges of the chromatoplates, where it
can be collected instead of being left to evaporate. In the multiple development
technique, repeated development is carried out with the same solvent in the same
direction, each time after drying. The two-dimensional technique is used to
separate complex mixtures. The plates are developed in one axis first and then,
after drying, they are developed in the other axis with the same solvent or
different solvents.
Gradient development In case of the gradient technique, two different
adsorbents are applied simultaneously. For example, silica gel and alumina can
be used to prepare a pH gradient across the width of the plate.
Temperature controlled development In this technique, development is carried
out at a definite temperature.
Detection
Before starting the procedure to detect the various solutes, the chromatogram is
allowed to dry. This is done either at room temperature or in an oven.
Application of visualisation agents to help in detection is done in a number of
ways:

Exposure of the chromatogram to vapours.

Dipping the plate in a reagent solution.
Spraying or impregnating the plate with reagent.
 Allowing the solution to develop as in the normal TLC.

The visualisation procedures may be classified, for convenience, into the

following:
Non-destructive method The non-destructive method consists of the following
techniques: UV method for fluorescent compounds under UV chamber at 254
and 365 nm; iodine vapour treatment also widely used, wherein some
phytoconstituents show brown, amber or yellow zones after exposure to the
iodine vapour.
Reagents causing irreversible reaction Charring is a widely used technique for
the detection of carbon containing compounds. This involves spraying the plates
with sulphuric acid and then heating in an oven at 110°C for 10–30 minutes. The
organic compounds are destroyed and a dark deposit of carbon remains.
Analysis
In thin layer chromatography, qualitative analysis of the unknown compound is
done by comparing the Rf values with standard values. As solutes never travel
the full length of the stationary phase in TLC all the Rf values are less than one.
The Rf value depends on the amount of the stationary phase, the humidity, layer
thickness, solvent quality, saturation of the chamber, development distance,
temperature, amount of substance added, and presence of impurities.

8.1.6 TLC of Plant Metabolites

TLC of volatile oils
Sample preparation for TLC

1. Extraction with methylene chloride (DCM) (Dichloromethane extract).

1 gm of the sample is subjected to extraction by shaking for 15 minutes
with 10 ml of methylene chloride. The suspension is filtered and the clear
filtrate evaporated to dryness. The residue is dissolved in 1 ml toluene; 5–
10 µ is applied for TLC.
2. Extraction with MeOH (methanolic extract)
 a. Crumcumae rhizome (cinnamoyl pigments): 1 gm of the powdered
drug is extracted by shaking for 5 minutes with 5 ml MeOH at about
60°C.
b. Gum resins (e.g: Myrrha): 0.5 gm of the powdered material is
extracted by shaking for 5 minutes with 5 ml of 96% ethanol. 20 µ of
the clear filtrate is applied for TLC.
c. Oleo-resins (Balsamum peruvianum, B. tolutanum): 0.5 gm of Peru
balsam is dissolved in 10 ml of ethyl acetate. 10 µl of this solution is
applied for TLC. For Tolu balsam: 10 µl of a 1:10 dilution in toluene is
applied for TLC.

Adsorbent: Silica gel 60 F254 pre-coated TLC plates.

Sample application: 5 ml of a 1:10 dilution of the essential oil in toluene is
applied to the TLC plate.
Solvent system: Toluene–ethyl acetate (93:7). This system is suitable for the
analysis and direct comparison of all important essential oils.
Detection

1. Without chemical treatment: either with UV 254 nm or UV 365 nm.

2. Spray reagents:
a. Anisaldehyde–sulphuric acid
b. Vanillin–sulphuric acid
c. Phosphomolybdic acid

TLC of alkaloids
Preparation of the drug extracts for TLC

1. Alkaloid drugs with high and medium alkaloid contents (>1%):

1 gm of the powdered drug is thoroughly mixed with 1 ml of the 10% NH3
solution or 10% Na2CO3 solution; it is then subjected to extraction by
shaking for about 5 minutes with 5 ml of methanol at 60°C (on a water
bath). The filtrate is cooled and concentrated so that 100 µl contains 50–100
µg alkaloids.
 2. Alkaloid drugs with low alkaloid contents (<1%):
a. Enrichment using an aluminium oxide column: 2 gm of the powdered
sample is mixed with 2 ml of 10% NH3 solution. It is then mixed with
7 gm of basic aluminium oxide. The whole mixture is packed loosely
into a glass column (1.5 cm diameter; 200 cm long). The bases are
eluted with about 10 ml CHC13. The first 5 ml of the elute is collected,
evaporated to 1 ml, then used for TLC.
This method is suitable for solanaceous drugs. Seed drugs must first be
de-fatted by using light petroleum. For drugs obtained from leaves, the
following official procedure is used as they contain chlorophyll which
interferes with the TLC separation.
b. 2 gm of the powdered drug is shaken for 5 minutes with 10 ml of 0.05
M H2SO4. It is then filtered. To the filtrate 1.0 ml of concentrated NH3
solution is added. The mixture is diluted to 10 ml with H2O and
extracted by shaking with 10 ml peroxide- free ether. The ether phase
is dried over anhydrous sodium sulphate, filtered and evaporated to
dryness on the water bath. The residue is dissolved in 0.25 ml
methanol and used for the TLC.

Adsorbent: Silica gel 60 F254 pre-coated TLC plates. Most of the alkaloids can
be separated on silicic acid.
Solvent system: The solvent systems used for some alkaloids are given in Table
8.11.
Table 8.11 Solvent sytems for certain alkaloidal drugs

Solvent system Alkaloidal drugs

Toluene:ethylacetate:diethylamine Suitable for the major alkaloids of most

(70:20:10) drugs.

CHCl3: diethylamine (90: 10) Cinchona alkaloids.

Toluene:acetone:ethanol:conc.NH3 Opium alkaloids.

(40:40:6:2)
 Ethylacetate: CH3OH:H2O (100: Rauwolfia alkaloids, colchicum
13.5 : 10) alkaloids, xanthine derivatives.

Acetone: H2O: conc. NH3 (90:7:3) Solanaceous dnrgs.

Toluene: CHCl3: ethanol. Secale alkaloids.

(28.5:57:14.5)

n-Heptane: ethyl methyl ketone: Rauwolfia alkaloids.

CH3OH (58:34:8)

CHCl3: CH3OH (85:15) Ipecacuanha alkaloids.

Toluene: CH3OH (86:14) Colchici semen (solvent run twice over

15 cm)

n-Propanol: formic acid:H3O (30: Berberidis cortex. Hydrastis rhizoma

1 :9)

Colombo radix.

Cyclohexane: CHCl3: glacial Berberine and protoberberine type

acetic acid (45:45:10) alkaloids.

Detection:

1. Without chemical treatment: A lot of alkaloids show a pronounced

quenching of fluorescence in UV 254 nm. Some alkaloids fluoresce blue or
yellow in UV 365 nm.
2. Spray reagents: Usually Dragendorff’s reagent is used. Brown or orange
zones appear immediately on spraying. Since the colours are not stable, the
alkaloidal zones are made more distinct by spraying first with
Dragendorff’s reagent and then with 5% sodium nitrite solution or 5%
ethanolic H2SO4.

TLC of glycosides
Anthraquinone glycosides
Preparation of sample for TLC : 0.5 gm of the powdered sample is extracted by
warming for 5–10 minutes on the water bath with 5 ml of CH3OH. The clear
filtrate is used directly for TLC.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent system: The solvent system suitable for the chromatography of all drug
extracts is ethyl acetate: CH3OH:H2O (100:17:13) (exception: senna extract).
Some other solvent systems for anthraquinone glycones include

a. Ethyl acetate: CH3OH:H2O (100:13.5:10)

b. n-propanol: ethyl acetate: H2O (40:40:30)
c. Toluene: ethyl formate: formic acid (50:40:10)
d. Light petroleum: ethyl acetate: formic acid (75:25:1)

Detection:

1. Without chemical treatment:

All anthraquinone derivatives quench fluorescence at UV 254 nm and give
yellow or red-brown fluorescence at UV 365 nm.
2. Spray reagents:
Borntrager’s reagent: After spraying with 5% or 10% ethanolic potassium
hydroxide, anthraquinones show red in visible, UV and red fluorescence in
UV 365 nm. Anthrones and anthranols show yellow in the visible, UV and
yellow fluorescence in UV 365 nm.

Bitter glycosides
Preparation of the sample for TLC: 1 gm of the powdered drug is extracted for
10 minutes with 10 ml CH3OH at 60°C on a water bath. The mixture is filtered
and the filtrate is evaporated to a volume of 2 ml, and used for TLC.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent system: Ethylacetate: CH3OH:H2O (77:15:8) Detection: By spraying
vanillin–sulphuric acid reagent or anisaldehyde–H2SO4, followed by heating at
1100°C for 5–10 minutes.
Coumarin drugs
Preparation of the sample for TLC: 1 gm of the powdered sample is extracted by
shaking with 10 ml CH3OH for 30 minutes on the water bath. The clear filtrate is
evaporated to about 1 ml and 20 µl is applied to TLC.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent systems:
Toluene: ether (1:1, saturated with 10% acetic acid)
Toluene (50 ml) and ether (50 ml) are shaken for 5 minutes with 50 ml of 10%
acetic acid in a separating funnel. The lower phase is discarded, and the toluene–
ether mixture is used for TLC. It must be freshly prepared.
Detection:

1. Without chemical treatment:

All coumarins show a distinct fluorescence quenching at UV 254 nm. They
also show intense blue or blue-green fluorescence. Furanocoumarins
sometimes show yellow, brown or blue fluorescence at UV 365 nm.
2. Spray reagents:
a. After spraying with 5% ethanolic KOH, blue fluorescent zones
appears. Ammonia vapour has the same effect.
b. Polyethyleneglycol reagent: This reagent intensifies and stabilises the
existing fluorescence of the native coumarins.
c. Antimony III chloride: Visnagin gives a lemon yellow fluorescence in
UV 365 nm after treatment with this reagent.

TLC of flavonoid drugs

Preparation of the sample for TLC: 1 gm of powdered sample is subjected to
extraction with 10 ml of methanol for 5 minutes on a water bath at 60°C. The
clear filtrate is used for TLC.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent system: Ethyl acetate–formic acid–glacial acetic acid–H2O
(100:11:11:27) Detection:
 1. Without chemical treatment:
All flavonoids cause fluorescence quenching, which is seen as dark blue
zones on the yellow background of the TLC plate at UV 254 nm.
Depending on the structural type, flavonoids fluoresce yellow, blue or green
at UV 365 nm.
2. Spray reagents:
Typical fluorescent colours in UV 365 nm after spraying with PEG.
Fluorescence behaviour is structure-dependent.
Flavonols: Glycosides of quercetin and myricetin → orange Glycosides of
kaemperfol and isorhamnetin → yellow-green
Flavones: Glycosides of luteolin → orange Glycosides of apigenin→
yellow-green

TLC of saponins
Preparation of the sample for TLC: 1 gm of the powdered sample is subjected to
extraction by heating for 10 minutes under reflux with 10 ml of 70% ethanol.
The extract is then heated under reflux for 1 hr with 30 ml of 0.5 M H2SO4and
cooled. The unfiltered mixture is shaken twice with 20 ml of CHC13 The
extracts are combined with dried over anhydrous sodium sulphate, filtered and
evaporated to dryness. The residue is dissolved in 2 ml of CHC13: CH3OH (1:1)
mixture, then used for chromatography.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent system: CHC13:CH3OH:H2O (64:5:10) Detection:

1. Without chemical treatment: No saponins are detected by exposure to UV

254 nm or UV 365 nm (exception: glycyrrhetic acid from liquorice)
2. Spray reagents:
a. Blood reagent: Haemolytic saponins are detected as white zones on a
reddish background. Haemolysis may occur immediately, or after
allowing the TLC plate to stand, or after drying the plate in warm air.
b. Vanillin–sulphuric acid reagent: saponins mainly form blue or blue-
violet and sometimes yellowish zones in the presence of vanillin–
sulphuric acid reagent.
c. Anisaldehyde–sulphuric acid reagent: similar responses with VS
 reagent.
d. Antimony (III) chloride reagent: Red-violet colours in visible; red-
violet, blue and green fluorescence in UV 365 nm.

TLC of cardiac glycosides

Preparation of the sample for TLC: 1 gm of the powdered sample is subjected to
extraction by heating for 15 minutes under reflux with 20 ml or 50% ethanol
with the addition of 10 ml of 10% lead acetate solution. Cool and filter. The
clear filtrate is treated with a small quantity of acetic acid; then extracted by
shaking gently with three 15 ml quantities of dichloromethane (gentle shaking is
required to avoid emulsion formation).The lower phases are combined, filtered
over anhydrous sodium sulphate and finally evaporated to dryness. The residue
is dissolved in 1 ml of dichloromethane–ethanol (1:1), and the solution is used
for TLC.
All cardiac glycoside drugs can be extracted by this method. Drugs containing
high cardenolide content (eg. Strophanthi semen), can be extracted by the
following simple procedure: 2 gm of the finely powdered seeds is de-fatted by
heating for 1 hr under reflux with light petroleum. 1 gm of the de-fatted and
dried seed powder is extracted for 5 minutes with 10 ml ethanol at 60°C. The
filtered solution is used for TLC.
Adsorbent: Silica gel 60 F254 pre-coated plates.
Solvent system: Ethyl acetate:CH3OH:H2O: 100:13.5:10.
Detection:

1. Without chemical treatment:

2. Cardenolides produce very weak fluorescence quenching in UV 265 nm,
but bufadienolides produce distinct zones of fluorescence quenching.
Cardiac glycosides do not fluoresce in UV 365 nm.
3. Spray reagents:
a. On spraying Kedde’s reagent cardenolides immediately form a pink or
blue-violet (visible) colour. Bufadienolides do not react. The colour
fades after a few minutes. Similarly cardenolides gives red-orange or
violet (visible) colour with Legal reagent (alkaline sodium
nitroprusside solution), Baljet reagent (alkaline picric acid solution)
 and Raymond reagent (alkaline m-dinitrobenzene solution)
b. Cardenolides and bufadienolides can be defeated by the following
general reagents. Antimony (III) chloride: The plate is sprayed with at
least 10 ml SbCl3 reagent, heated at 100°C for about 6 minutes and
then observed immediately in UV 365 nm. In visible light, the zones
appear mainly violet or brown.
Sulphuric acid reagent: The plate is sprayed with 5 ml of reagent, then
heated for 3–5 minutes at 100°C under observation. Blue, brown,
green and yellowish fluorescent zones are seen in UV 365 nm; the
same zones appear brown or blue in daylight.

NOTE: Liebermann–Burchard reagent can also be used for detection of cardiac

glycosides.

8.1.7 Preparative TLC

Preparative TLC is carried out using thick (up to 1 mm) instead of thin (0.10–
0.25 mm) layers of adsorbent. Manufactured plates are available for this.
Separated constituents are recovered by scraping off the adsorbent at the
appropriate places on the developed plate, eluting the powder with a solvent
such as ether and finally centrifuging to remove the adsorbent.

8.1.8 HPTLC
In high performance thin layer chromatography, a layer thickness of 100–150
microns is used to achieve separation. HPTLC uses open layers of adsorbents on
plates or foils to separate components of samples.
Retention factor: Rf is defined as the ratio of distance from the origin travelled
by the solute band to distance travelled by the mobile phase in a particular time.
Resolution: It is the distance between the band centres divided by average peak
width.
Number of plates: It gives the efficiency of the chromatography system.

where tr = Retention time

Wb = Width of base of chromatogram peak
Selectivity: It is defined as the difference between the moving distances of two
spots divided by the average moving distance as measured from the start R = E
×S
where R = Resolution
E = Efficiency
S = Selectivity
Application of HPTLC
HPTLC is used to detect andrographolide in Andrographis paniculata among
other substances.
Pre-coatedplates: Pre-coated TLC silica gel 60F, 0.25 mm, 10 x 20 cm (E.
Merck) Pre-washing: Methanol Sample preparation :
Test solution: Extract 5 g of powdered drug with methanol (50 ml) in a Soxhlet
apparatus (6.8 h). Evaporate the methanol extract under reduced pressure.
Dissolve the residue in 1 ml of methanol.
Reference solution: Prepare a solution containing 1 mg of andrographolide in 1.5
ml methanol.
Application of sample and standard:
Plate width : 100 mm
Band : 5 mm
sec/µl : 10 µl
Mobile phase: Chloroform: methanol (8:1)
Pre-conditioning: Chloroform: methanol (8:1)
Development and drying: Automatic development chamber
Detection:

1. UV at 254 nm
2. It is sensitive, accurate and serves as an essential tool for the
standardisation of herbal drugs.
3. It is a versatile separation technique for determining content uniformity,
purity profile and assay values.
8.1.9 Paper Chromatography
Chromatography on paper usually involves either partition or adsorption
chromatography. In partition chromatography, the compounds are partitioned
between a largely waterimmiscible alcoholic solvent (e.g. n-butanol) and water.
The classic solvent mixture, n-butanol–acetic acid–water 4:1:5, top layer)
(abbreviated as BAW) was indeed devised as a means of increasing the water
content of the n-butanol layer and thus improving the utility of the solvent
mixture. Indeed, BAW is still widely applicable in a general solvent for many
classes of plant constituents. By contrast, absorption forces are one of the main
features of paper chromatography (PC) in aqueous solvents. Pure water is a
remarkably versatile chromatographic solvent and can be used to separate
common purines and pyrimidines. It is also applicable to phenolic compounds
and to plant glycosides in minerals.
In PC, compounds are usually detected as coloured or UV-fluorescent spots,
after reaction with a chromogenic reagent, used either as a spray or a dip. For
large sheets, dipping is usually easier but the solvent content of the spray should
be modified in order to facilitate quick drying and thus avoid diffusion during
the dipping. The paper may then be heated in order to develop the colours.

8.2 INFRARED SPECTROSCOPY

8.2.1 Introduction
Infrared radiation refers broadly to that part of the electromagnetic spectrum
between the visible and microwave region i.e., the region in the electromagnetic
radiation spectrum from 0.8 to 200 µ. This region can be further sub-divided into
three parts namely:

a. 0.8 to 2.5 µ or 12500 to 4000 cm –1: Near IR

b. 2.5 to 15 µ or 4000 to 667 cm–1: Middle IR
c. 15 to 200 µ or 667 to 50 cm–1: Far IR.

The fundamental region (middle IR) between 2 and 15 µm is the region that
provides the greatest information for the elucidation of molecular structure and
most IR spectrophotometers are limited to measurements in this region.
8.2.2 Theory
The IR absorption spectra are due to changes in vibrational energy accompanied
by changes in the vibrational energy in the ground state of the molecule. The
transition from vibrational level 0 to vibrational level 1 gives rise to the
fundamental absorption of the molecule; overtones or harmonics are caused by
the transitions 0–2, 0–3 and so on, though the intensity of absorption for these
overtones is very much less than that for the fundamental frequencies. For
energy to be transferred from the light source to the molecule, the frequency of
vibration of each must coincide and moreover, must be accompanied by a
change in the dipole moment of the molecule.
Infrared radiation of frequencies less than about 100 cm–1 (wavelength longer
that 100 µm) is absorbed and converted by an organic molecule into energy of
molecular rotation. This absorption is quantised; thus, the molecular rotation
spectrum consists of discrete lines.
Infrared radiation in the range from 1000–100 cm–1 (1–100 µm) is also
absorbed and converted by an organic molecule. But the vibrational spectra
appear as bands rather than as lines because only a single vibrational energy
changes. It is these vibrational rotational bands, particularly those occurring
between 4000 cm–1 and 667 cm–1 (2.5 to 15 pm) which is the fundamental
region, that provide the greatest information for the elucidation of molecular
structure.
Band positions in infrared spectra are presented either as wave numbers or
wavelengths. The wave number unit (cm–1 reciprocal centimetre) is used most
often since it is directly proportional to the energy of the vibration and since
most modem instruments are linear in the cm–1 scale. Wavelength is reported in
micrometres (µm. 10–6 metres), although older literature refer to this quantity as
the micron (µ).
Band intensities are expressed either as transmittance (T) or absorbance (A).
Transmittance is the ratio of the radiant power transmitted by a sample to the
radiant power incident on the sample. Absorbance is the logarithm, to the base
10 of the reciprocal of the transmittance.
An IR spectrum is a plot of the absorption of IR light with variation of
wavelength (λ) or wave number. There are two kinds of molecular vibration–
stretching and bending. A stretching vibration is a rhythmical movement along
the bond axis such that inter-atomic distance increases or decreases. A bending
vibration may consist of a change in bond angle between bonds with a common
atom or the movement of a group of atoms with respect to the remainder of the
molecule without movement of the atoms in the group with respect to one
another.
Figure 8.5 shows an FT-IR spectrophotometer.
Tables 8.12–8.27 show the characteristic infrared frequencies of some
functional groups present in natural products.

Fig. 8.5 FT –IR spectrophotometer (Reproduced from: Anatak Pvt Ltd.) Table
8.12 Methyl groups
Table 8.13 Methylene groups
Table 8.14 Methane groups
Table 8.15 Groups with alkenes and double-bonded carbons
Table 8.16 Groups with phenyl nucleus
Table 8.17 Groups with triple bonds
Table 8.18 Carbonyl groups
Table 8.19 Hydroxyl groups
Table 8.20 NH groups
Table 8.21 Ethers

Table 8.22 C–N and N–N groups

Table 8.23 Nitro, nitroso and N–oxides

Table 8.24 Sulphur compounds

Table 8.25 Phosphorus compounds

Table 8.26 Silicon compounds

Table 8.27 Halides and inorganic ions

8.3 ULTRAVIOLET SPECTROSCOPY

The wavelength region of UV radiation encompasses the wavelength regions:
UV 200–300 nm and far UV 10–200 nm.
8.3.1 Principle and Theory Involved
Ultraviolet absorption spectra arise from transition of electrons within a
molecule or ion from a lower to a higher electronic energy level. For radiation to
cause electronic excitation, it must be in the UV region of the electromagnetic
spectrum.
When a molecule absorbs UV radiation of frequency v s–4 , the electron in that
molecule undergoes transition from a lower to a higher energy level or molecular
orbital. The energy difference is given by E = hv
The actual amount of energy required depends on the difference in energy
between the ground state E0 and the excited state E1 of the electrons.
So, E1 – E0 = hv
The total energy of the molecule = electronic energy + vibrational + rotational
energy.
Figure 8.6 shows an UV-visible spectrophotometer.

Fig. 8.6 UV-visible spectrophotometer (Reproduced with permission from:

Anatak Pvt Ltd.) 8.3.2 Beer–Lambert’s Law

Lambert’s law states that when a beam of light is allowed to pass through a
transparent medium, the rate of decrease of intensity with the thickness of
medium is directly proportional to the intensity of light.
Beer’s law states that the intensity of a beam of monochromatic light
decreases exponentially with the increase in concentration of the absorbing
substance.

8.3.3 Applications with Respect to Herbal Drugs

The UV spectrum provides a useful means of detecting conjugated unsaturated
chromophores within a molecule such as polyenes, α-, β-unsaturated ketones and
aromatic compounds. Within particular families of compounds, the position of
maximum absorption can reflect the degree of substitution of the chromophore.
UV absorption is characteristically broad. This form of absorption is found in
aromatic compounds.
It is also particularly helpful in detecting the presence of unstable
polyacetylene in culture broths of Basidiomycete fungi. The UV absorption of
the common fungal metabolite, ergosterol, is characteristic of its ring β diene.
But UV absorption is associated with the chromophore and not the whole
molecule. Thus, UV absorption would not distinguish between ergosterol and
ergosteryl esters, which often co-occur.
The presence of auxochromes, particularly phenolic and carbonyl groups,
modify aromatic chromophores. This can be particularly helpful in the
identification of chromones and flavones. The shifts in the absorption of
phenols, arising from the addition of a drop of alkali to produce the phenolate
anion, are quite helpful. Various complexing agents also produce useful
diagnostic changes in the UV spectrum. The UV spectrum may be caused by a
summation of chromophores from different parts of a polyfunctional molecule,
and this should be considered in the light of deductions drawn from other
spectroscopic methods and chemical degradation.
The UV spectral properties of different classes of plant pigments is given in
Table 8.28.

Table 8.28 UV spectral properties of different plant pigments

Pigment class UV range (nm)

Phenolic compounds 276, 282

a. Simple phenols: (ethanol λmax)
Orcinol

4-methyl resorcinol 282

2-methyl resorcinol 275, 280

Resorcinol 276, 283

Catechol 279

Hydroquinone 295
Pyrogallol 266

Phloroglucinol 269,273

b. Phenolic acids: (ethanol λmax )

Gallic acid 272

Protcatechuic 260, 295

Gentisic 237, 335

p-hydroxybenzoic 265

Syringic 271

Vanillic 285, 297

Salicyclic 225, 297

c. Hydroxy cinnamic acids

P. coumoric 227,310

Caffeic 243,326

Ferulic 235,324

Sinapic 239,325

p-methoxycinnamic 274,310

O-coumoric 227,275,325

Isoferulic 295,323

3, 4, 5 -trimethoxycinnamic 232,303

d. Hydroxy coumarins (aglycones)

Coumarin 212,274,282,312

Umbelliferone 210,240,325

Aesculetin 230,260,303,351

Daphnetin 215,263,325

Aseculin (glycoside) 224,252,293,338

Cichorilin 228,255,290,345

Scopolin 227 ,250,288,339

e. Flavonoid class: (ethanol λmax)

Anthocyanins 475–560(visible max)

Flavonols 254–235–386

Flavones 255–330–355

Chalcones and aurones 235–370–410

Glycoflavones (asflavonols) 255–350

Biflavonyls 269–33–350

Flavanones 224–275–290

Isoflavones 250–255–265

f. Quinone pigments

Emodin 223,254,276,290,440

Chrysophenol 225,258,279,288, 432

Physcion 226,255,267,288, 440

Aloe-emadin 225,258,279,287,430
Rhein 230,269,432

Emodio acid 227 ,252,274,290, 444

Organic acids, lipids and related compounds

a. polyacetylenes (λmax)

Falcarinol 229,240,254

Falcarinolone 259,274,290

Falcarinone 208, 246,260,274,291

Falcarindone 242,253,268,285,303

Aethusin 210, 248, 264,280,

294,313, 334

Nitrogen compounds

a. Alkaloids ( λmax in 0.1 M sulphuric acid)

Cystine 303

Nicotine 260

Morphine 284

Codeine 284

Berberine 228

Thebaine 254

Atropine 284

Quinine 250

Conine 268 .
b. Indoles ( λmax in methanol)

Indole-3-acetic acid 274,282,290

Indole-3-lactic acid 275,282,289

Indole-3 -acetaldehyde 244,269,292

Tryptamine 272, 279, 288

5-hydroxytryptamine 276,296

Tryptophan 275,281,290

c. Purines or pyrimidines (λmax in

0.1hydrochloric acid)

Adenine 262

Guanine 249

Uracil 260

Cystosine 276

Thymine 265

Adenosine 259

Guanosine 252

Terpenoids
a. Carotenoids (chloroform,λmax)

α-carotene 454, 485

β-carotene 466,497

γ-carotene 447,475,508
 ε-carotene 418,442,471

Lycopene 456,485,520

b. Xanthophylls Visible range (nm)

Lutein 428,456,487

Violaxanthin 424,452,482

Zeaxanthin 429,462,494

Neoxanthin 421, 447 , 477

Rubiaxanthin 439,474,509

Fuloxanthin 457,492

Cryptoxnathin 433,463,497

8.4 DIFFERENTIAL SCANNING CALORIMETRY

Differential scanning calorimetry (DSC) is an important technique for measuring
the energy necessary to establish a nearly zero temperature difference between a
sample and reference substances. The two specimens are subjected to identical
temperature regimes in an environment heated or cooled at a controlled rate and
the response of the sample compared with of the reference.
While calorimefiy is a simple measurement technique which measures the
heat of an isolated system, DSC is a heat based analytical method used to study
the thermal response of the specimen sample. In this method, the difference in
the amount of heat required to increase the ,temperature of the sample and the
reference are measured as a function of temperature. The differential scanning
calorimeter is an instrument which measures the rate of heat evolution or
absorption of substance, which is subjected to a programmed temperature
change. Besides comparing the heat response of the sample with that of the
reference, this method also gives information about the thermodynamics of any
reaction involved. The difference in temperature of the sample and reference is
due to the difference in mass, specific heat, heats of reaction or phase transitions.
The differential aspect of the measurement allows the amplification of small
differences, eliminating large signals and thereby enhancing its sensitivity. It
differs from the standard melting point determination apparatus — this apparatus
only determines a range of melting points for the compound whereas DSC gives
a sharp peak denoting the compound's melting point with a printout from the
computer attached to it (Fig. 8.7).
The basic principle behind this technique is, that, when the sample undergoes
physical transformation, such as phase transition, more heat (or less) will be
needed than the reference to maintain the uniform temperature. The extent of
heat flow depends upon the nature of the process—whether, it is exothermic or
endothermic.
In an endothermic process, the sample undergoes a phase transition from solid
to liquid, whereas in an exothermic process (such as crystallisation), less heat is
required to raise the sample temperature. Thus by recording the difference in
heat flow between the sample and the reference, the energy absorbed or released
during phase transitions can be detected.

Fig. 8.7 Differential scanning calorimeter

8.4.1 Parts of a Differential Scanning Calorimeter

A typical differential scanning calorimeter consists of two sealed pans—a
sample pan and a reference pan (which is generally an empty sample pan). These
pans have a very good thermal conductivity and are often covered by aluminium,
which acts as a radiation shield. The pans are heated and cooled uniformly and
usually at constant temperature (isothermally) in the experiment. It can also be
done by changing the temperature at a constant rate. This method is called
temperature scanning.
 The instrument detects the heat difference between the sample and reference
during the experiment. The information is then sent to a computer and the result
is a plot of the differential heat flow between the sample and the reference as a
function of temperature. When there is no thermodynamic chemical process, the
heat flow difference between the sample and the reference varies only slightly
with temperature. The result is a shallow or flat baseline (see the section on DSC
curve given in Section 8.4.2). But when an exothermic or endothermic process
takes place, within the sample, it results in a peak in the DSC curve. The
exothermic process will show a positive peak (above the baseline) but the
endothermic process will show a negative peak (below the baseline). The sample
in the form of powder, crystals or liquid is usually placed in the aluminium
sample pan. Similarly the reference pan is placed in the reference cell of the
instrument. The size of the sample varies from 0.1–5 mg. The sample cells and
the reference cells are often air-tight in order to shield the sample and reference
from external heat exposure. Thus experiments can be performed under variable
pressures and atmospheres.

8.4.2 Mode of Operation

The mode of operation of a typical DSC (DSC-60) is described below. The DSC-
60 is a heat flux differential scanning calorimeter which works on the same
principle as a differential thermal analysis. A schematic diagram of DSC-60 is
shown in Fig. 8.8.

Fig. 8.8 Schematic diagram of DSC-60

Starting the device

The power switches of the DSC-60, TA-60WS interface, the personal computer,
the display unit and the printer are turned on. When Windows© is activated,
double click the [TA-60WS acquisition: Status] icon. Then click the button of
the DSC-60 connected channel to open the module monitor window. Check the
indicated values (the sample section temperature and the differential heat flow)
and graphs (temperature and heat flow transitions with time) to make sure that
the instrument is operating normally.
Sample preparation
Weight out about 3–5 mg of the sample and put it in a standard aluminium
sample pan. Put an aluminium lid into the pan and crimp it with Shimadzu SSC-
30 sample sealer-crimper. For the reference material, use an empty aluminium
sample pan of the same type, covered with a lid and crimped in the same
manner.
Setting sample pans
The sample pans have to be set in the following manner. Remove the cell cover
by sliding it forward. Remove the furnace and the furnace lid with tweezers. Put
the pan containing the sample on the right stage of the detector plate and that
containing the reference material on the left stage. Return the furnace and
furnace cover and finally the cell cover with tweezers to their original positions,
respectively.
Entering measurement conditions
All the following procedures are performed on the personal computer. Activate
the [Ta-60WS acquisition: Status] window and execute the [Measure]-
[Measuring parameters] command to open the [Setting parameters] dialogue
box. Set the parameters as directed.
Starting and stopping measurement
After making sure that the temperature curve and the DSC curve (on the axis of
time) are stabilised (drawn horizontally), click the [Start] button. The [Start]
dialogue box will open to display the data file name. Change the data file name
as required. Click the [Start] button to start the measurement under the specified
conditions. When the sample section temperature reaches the target temperature,
the measurement stops automatically and a data file is created. For safety’s sake,
remove the pans after the sample section temperature becomes sufficiently low.
Data analyses
Activate the [Ta-60] window and execute the [File]-[Open] command to open
the dialogue box. Clicking the [Open] button opens the selected file and displays
the data curves (the temperature and DSC curves on the axis of time as default).
Next click the DSC curve (a marker will appear at each end of the curve).
Analysis of melting peak (Fig. 8.9)
Execute the [Analysis]-[Tangent] commands, click points A and B, and click the
[Analyze] button. The tangent lines AC’ and BB’ will be displayed. To correct
the gradient of a tangent line, drag at its end points (C’ and B’ respectively).
Clicking the [Analyze] button again displays the temperature at point P.
DSC curve (Figs. 8.10 and 8.11)
The result of a DSC experiment is a heating or cooling curve. This curve can be
used to calculate enthalpies of transitions. Integrating the peak corresponding to
a given transition gives this curve. The enthalpy of transition can be expressed
using the following equation: ΔH=KA
(where ΔH is the enthalpy of transition, K is the calorimetric constant, and A is
the area under the curve). The calorimetric constant will vary from instrument to
instrument, and can be determined by analysing a well-characterised sample
with known enthalpies of transition.

8.4.3 Types of Differential Scanning Calorimeters

There are two main types of differential scanning calorimeters—the heat flux
DSC and the power compensation DSC.
Heat flux calorimeter (Fig. 8.12)
In a heat flux calorimeter, heat is transmitted through silver or the copper–nickel
alloy, constantan to the sample and reference cells. The DSC monitors the
temperature difference between the sample and the reference. The disc of the
DSC functions as a temperaturesensing unit. The sample and reference resides
on raised platforms on the disc. A chromel wafer is seen beneath these platforms.
Chromel is an alloy of chromium, nickel and iron. The junction between these
two alloys forms a chromel–constantan thermocouple. The differential heat flow
is measured by analysing the signals from these sensors.
Power compensation DSC (Fig. 8.13)
In power compensated DSC, there are separate heaters for the sample and the
reference and they are maintained at the same temperature. The heating elements
are kept very small—weighing around 1gm. This is to aid speedy thermal
equilibrium, heating and cooling of the heating elements. The sample and
reference are seen above their respective heaters. The electronic sensors beneath
the samples monitor the temperature. Platinum resistance thermometers are
generally used due to its high melting point. The instrument consists of two
temperature control circuits. The purpose of this circuit is to control the
temperature scanning programme of the sample and reference. The output of the
differential temperature control circuit is used to generate the DSC curve.

Fig. 8.9 Analysis of melting peak

Fig. 8.10 A typical DSC curve

Fig. 8.11 Features of a DSC curve (From Wikipedia, the free encyclopedia)

Fig. 8.12 Heat flux differential scanning calorimeter (From Wikipedia, the free

encyclopedia)

Fig. 8.13 Power compensated differential scanning calorimeter (From

Wikipedia, the free encyclopedia) 8.4.4 Applications of Differential Scanning
Calorimeters

1. DSC is used to measure the melting temperature, heat of fusion, reaction

energy and temperature, glass transition and phase transition temperature
and energy.
2. It is widely used in quality control departments of industries and academic
institutions for evaluating the purity of samples and the curing of polymers.
3. It is used to measure the specific heat capacity (100–1200°C) and heats of
transition. Specific heat capacity can be used in conjunction with thermal
diffusivity to obtain thermal conductivity.
4. It is used to detect the temperature of phase changes and melting points in
the range of 20 to 1500°C. It is also used to determine the melting
behaviour of complex organic materials—both temperatures and enthalpies
 of melting.
5. To measure metal alloy melting temperatures and heat of fusion.
6. To measure structure transition temperatures and heat of transformation.
7. To measure oxidation temperature and oxidation energy.
8. To measure inter-metallic phase formation temperatures and exothermal
energies.
9. To study the exothermal energy of the polymer cure and also for the
determination of the degree and rate of cure.
10. To measure the glass transition temperature of glassy or plastic material or
softening temperatures.
11. To determine the transition temperatures of polymers and plastics from the
crystalline to the amorphous form and also to determine the energy
associated with the transition.
12. Glass transition may occur as the temperature of an amorphous solid is
increased. This is recorded as a baseline in the DSC curve. When
temperature is increased the amorphous form will be converted into the
crystalline form. This is an exothermic process and it results in a peak in the
DSC signal. When the temperature is further increased, it results in an
endothermic peak in the DSC curve. Hence DSC is an important tool in
producing the phase diagrams for various chemical systems.
13. To determine the thermal stability of a compound.
14. To determine the reaction kinetics of a material.
15. To determine the oxidation induction period of oil or fat.
16. To determine the crystallisation and melting temperature and phase
transition energies for inorganic compounds.
17. In food science research, DSC in conjunction with other thermal analytical
techniques is used to determine the water dynamics. The effects of curing
on confectionery products can also be analysed.
18. It is also used to study liquid crystals. Liquid crystals pass through a third
stage, from solid to liquid transitions, which exhibits the properties of both
phases. This stage is called the liquid crystalline or mesomorphous stage.
 DSC is used to study the small energy changes from solid to liquid crystal
and from liquid crystal to isotrophic liquid.
19. It is also used to evaluate dry and polymer purities. When a foreign solute is
added to a compound, it results in the depression of freezing point. Impure
compounds will exhibit a broadened melting peak, which begins at a lower
temperature than the pure compound.

8.5 NMR SPECTROSCOPY

The NMR spectra can be the most rewarding of the spectroscopic techniques in
terms of structure elucidation. At the preliminary characterisation stage, the
problem is often how to handle the wealth of data available and to distinguish
the significant structural features. In tabulating data from the proton NMR
spectrum, it is worth remembering that the chemical shift provides information
about the chemical environment of the proton (Table 8.29), the multiplicity of a
signal provides information about the relationship with neighbouring protons,
and the integral gives the relative number of protons contributing to a signal.
H1– NMR reflects the number and position of protons in a molecule. C13–NMR
gives an idea about the carbon skeleton. Table 8.30 gives the approximate range
of C13–NMR signals for some phytoconstituents.
Table 8.29 Some NMR chemical shifts of characteristic protons in
phytoconstituents
Table 8.30 Approximate ranges of l3C NMR signals

8.6 MASS SPECTROSCOPY

In the field of natural products, mass spectrometry is generally employed for
solving one or more of the following problems: establishment of molecular
weight and of empirical formula; detection of functional groups and other
substituents; determination of the over​all skeleton of the compound; and finally,
elucidation of precise structure and possibly of certain stereochemical features.
For all of these purposes, volatilisation of a given substance in the
undecomposed form into the electron beam is important; in fact, for the
determination of the molecular weight, it is essential. Although chemical
manipulation, in which a less volatile compound is transformed into a more
volatile derivative, sometimes offer a way out of the difficulties in volatisation, it
is generally most convenient to overcome this problem by instrumental
modifications. Indeed, it is largely these modifications, which have increased so
greatly and rapidly the scope of mass spectrometry applications in structural
organic chemistry.
The fragmentation behaviour of berberine alkaloids can be exemplified by the
mass spectrum of xylopinine (VIII). It can be seen that fission of the ring system
predominates. A Retro-Diels–Alder decomposition can again be involved to
explain the genesis of the most abundant ion (j, m/e 164) together with the
neutral 3,4-dihydro-6,7-dimethyoxyisoquinoline (IX).

If the charge remains with the nitrogenous fragment, further loss of a

hydrogen atom will provide the 6,7-dimethoxyisoquinolinium ion k (m/e 190).

This characteristic breakdown pattern may be employed for the localisation of

additional substituents. Thus tetrahydroberberine (X) gives the expected
fragments at m/e 164 (j) and m/e 149 (j-CH3), associated with the C-D moiety,
while rings A and B are responsible for the K (m/e 174) which immediately
points towards the structural difference between xylopinine (VIII) (k = m/e 190)
and tetrahydroberberine (X). Thalictricarvine (XI) on the other hand, shows its
most intense peak at m/e 178 (j), rather than m/e 164 (j), while the peak
corresponding to the A-B moiety remains at m/e 174 (k). It follows, therefore,
that thalictricarvine (XI) is a higher homolog of tetrahydroberberine (X) with the
additional methyl group in the C-D region of the molecule.

The only notable features in the high mass range of xylopinine (VIII) mass
spectrum (Figure 8.14) are the rather intense molecular ion and the appreciable
Ml fragment; the loss of a methyl radical from the molecular ion is also
discernible (m/e 340).
Fig. 8.14 Mass spectrum of xylopinine (VIII)

8.7 SPECTRAL PROPERTIES OF HERBAL PRODUCTS

Sterculic acid

Occurrence : Stinky sterculia (Sterculia foetida)

M.P : 19.3–19.9ºC

IRλ max μ : 5.35,9.92(cyclopropene),5.86(–

COOH),7.78,10.7s(characteristic in fattyacid spectra),7.27
(CH2)

NMR:δ : 6.83 (–COOH),3.50 (CH2),3.95 (CH3), 2.53 ( ),

Asperulosid(glycoside)

Occurrence : Karamu (Coprosma robusta) and Fern (Pyrola

monotropa)

M.P : 131.5–132ºC

UV λmaxEtOH : 234.5

IR : 1773 (γ–lactone), 1742 (CH3CO)

γKBrMaxcm–1
 NMR in : 7.20 (C3–H), 5.75 (C7–H), 5.68 (C1–H),5.49 d (J = 6)
CDCl3: δ (C6–H),
3.48 (C5–H),5.49–4.65 (four axial H on the sugar ring),
4.65 , 4.24 (C –H), 3.79 (C –H), 3.23 (C –H),
6 5 9
2.17s (CH3CO).

Ecucalyptin(Flavone)

Occurrence : Blue gum (Eucalyptus globulus), Eucalyptus E. cinerea

NMR
Flavones and isoflavones

Mass : m/z 270 (100), 269, 242, 241, 153, 133, 128, 129, 124, 123,
spectra l2l, 118

Ergochrysin (glyciside)

Occurrence : Ergot (Claviceps purpurea)

M.P : 285ºC

: 1802 (γ–lactone), 176l (

), l639(chelated
IR γmax
cm– 1
aromatic C=O)

NMRin : 8.80 d(J = 6c/s) (6H >CHMe),2.36 d,3.04 (J=8),2.51 d,2.88

CDCl3:τ d (J = 9) (two pairs of ortho aromatic protons)
Cucurbitacin A (triterpenoid)

Occurrence : Wild cucumber (Cucumis hookeri)

M.P : 207–208ºC

UV λ max : 229,290

IR γmaxcm–1 : 1730, 1258 (OAc), l715 (C=O), 1692, 1632 (CO–C=C)

NMRin : 2.98,3.46 (olefinic protons at H23and H24, J 14.0),

CDCl3:τ 4.20br. olefin (H6 ),7.95 (OAc),8.43 (2 ),
8.56(2 ), 8.68(2 ), 8.94( ,C–
13).

Panaxadiol (sapogenin of ginseng)

Occurrence : Ginseng (Panex ginseng)

 M.P : 244–250ºC

NMR in :
CDCl3: τ

Mass m/e:

Bands useful for characterising hydroxy steroids in CS2:

Diginin

Occurrence : Foxglove (Digitalis purpurea)

M.P : 155–l83ºc

UV λmax : 310

: 3585 (OH), 1735 (C=O), l7l2 (C=O), 1655 (C=C),

1095,1060,1032(C–O–C,891,872,853(–CO–C–O–C)

NMR: τ : 9.00s (18–Me), 8.67 d (J : 6.5 c/s),8.72 d (J = 6.5 c/s), 6.60s

(OCH3),
8.45 (19–Me).

Characteristic absorption bands of steroidal sapogenin acetates at 3100–2750

cm–1:
wherein a:CH–(3035); b:3000~2960; c:Asym.Me (2950); d:Asym.–CH2–(2930),
e:2920~2910; f: sym.Me (2870) g: sym. –CH2–(2860); h: 2850; i: 2830.
α–and β–4,8,13-Duvatriene-1,3-diol

Occurrence : Tobacco(Nicotiana tabacum)

M.P : 65–66ºC

: <220
 : 3300,1665,1345,1190,1160,1118,1024,995,974,954,818

NMR: τ : 4.81(3),5.07 (1),5.63 (1)(broad),8.36(3)(d, J = 1.2

c/s),8.50(3)(half peak width = 3.2 c/s),8.69(3),9.17(6)
(doublet with section splitting)
Nimbiol

Occurrence : Neem (Melia azadirachta)

M.P : 244ºC

: 231,286

: 30.5 (assoc. OH), 6.03 (Conj. C=0)

Andrographolide

Occurrence : Creat (Andrographis paniculata)

M.P : 230–231ºC

: 223

: 3448,3390~3279 (OH), 1828, 1647 , 907 (C

= CH2),
1727 (αβ-unsaturated γ-lactone), 1672 (conj.
C = C)

NMR of triacetate in : 0.75, l.02 ( ),2.03 (6) ,2.l0 (3)

CDCl3: δ (3 COCH3), 5.91 ( ), 6.97 t,J = 6.5 c/s
(exocyclic β-Protor, –CH2–CH = C<).
Quassin

Occurrence : Quassia wood (Quassia amara L., Picrasma quassioides)

M.P : 222ºC

: 255

: 1745(lactone), 1702,1688 (ketone), 1636(C = C).

NMR: τ : 4.17(J = 2) ( ),5.70 t(8–H)6.38,6.44(CH3O

8.15( ), 8.45, 8.79( ),8.86 d (J = 5.6)
( ).
Limonin

Occurrence : Valencia orange (citrus spp.)

M.P : 293–294ºC

: 207

: 3124,1760,1710,1608,1514,876.
Obacunoic acid

Occurrence : Phellodendron (Phellodendron amurense)

M.P : 205–208ºC

UV λmax : 205

IR γmaxcm– : 3220(OH),2800~3400(COOH),1745(δ-
1 lacton),1710(cyclohexanoneϕC=C–
COOH),1621(C=C)

NMR of : 8.88, 8.97, 9.01, 9.04 ( ), 8.62( ),

methylate: 4.24, 4.49 (α–H), 3.80, 4.08 (β–H), J = 13.4c/s (cis-β-
τ subst.acrylicester), 2.15, 2H (α–H), 3.66(β–H (furan
ring), 4.63( ).
Elemene

Occurrence : Rosewood (Dysoxylon fraserianum)

UV λmax : 250

: 1818, 1634, 1002, 912 (–CH=CH2), 1604(conj. C=C) , 877

( ),
809 ( ).

NMR τ : 3.65( ), 3.98 ~ 4.43( ), 4.92, 5.16( ),

820, 8.27 ( ) 8.97 d, J = 6.6(–CH(Me)2),
8.84( ).
Zederone

Occurrence : White turmeric (Curcuma zedoaria)

M.P : I 53.5~154ºC

: 1660 (α, β-unsatur. C = O), 1524, 898, 882 (β-furyl ketone),

1570~1560, 1520~1500 (c = c), 1155~1150, 875~870

NMRin : 2.12d, J = 1.3 (3) , 5.49 dd, (J = 3.7, 10.9)

CDCl3
(1)

Valeranone (Jatamanose)

Occurrence : Muskroot (Nardostachys jatamansi)

M.P : 206–207°C

: 220, 350

IR γmaxcm– : 1702, 1458~1442, 1385~1372, 1314, 1244, 1154, 1110,

1 1045, 935,
855~843, 828, 730
NMRin : 9.12s (3H)( ), 9.13 d, J=7.1(6H)( ) 9.02s (3H)
CCl 4: τ ( )
Methyl valerenate

Occurrence : Valerian (Valeriana officinalis)

B.P : 98–100°C

: 219

: 1710,1640

NMR in : 9.22 d, J = 8 c/s (3H),8.34s (3H) ,8.09 d,J = 1 c/s

CDCl3: τ: (3H),6.28s (3H),2.95d,J = 9c/s.
Pyrethrosin

Occurrence : Pyrethrum (Chrysanthemum cinerariifolium)

: 204,210

IR γmaxcm–1 : 1760 (γ-lactone), 1735,1242 (O.Ac), 1670, 1650 (C = C).

Vulegarin

Occurrence : Mugwort (Artemisia vulgaris L.)

M.P : 174~175°C

UV λmax : 215

IR γmaxcm– : 3250 (OH),1775 (γ-lactone), 1665(C=C –CO –)

1

NMRin : 3.42 d, 4.17 d, J = 10.2 c/s, 4.25 d , 8.46s,8.81s

CDCl 3: τ ( ), 8 .78 d, J = 6 c/s ( ).

γ-α-santonin

Occurrence : Wormwood (Artemisiacina)

M.P : 171–174°C

: 240,260

: 1780, 1665, 1635, 1615, 1135 ( ), 1030 (C–OCO)

NMR in CDCl3:τ : 3.13,3.38, 3.75,4.01,8.67.
Artilegin A

Occurrence : Tilesius
wormwood
(Artemisia tilesii)

M.P : 190–191°C

: 255

: 1780 (γ-lactone),
1740 (OAc),
1690
(cyclopentenone),
1645, 1622
(2 C = C)

NMR in CDCl3 : 40Mc, TMS, cps:

56.5m, J ≑1.5
(H3, H coupled
to non-adjacent -
H),
204, 208~209
(split) ( ), 215
(O–COCH3),
240.5–247
(split) (1l–Me).
Estafiatin

Occurrence : Mexican whitesagebrush (Artemisia mexicana)

M.P : 104~106°C

UV λmax : 214

IR γmaxcm–1 : 1755, 1668 (α,β -unsaturated lactone), 1640

NMR in CDCl3:δ : 5.48 d, 6.18 d, J = 3.3 c/s ( ),4.86 d, 4.96 d,

J :2( ),1.6s ( ).

UV-spectra of lignin model compounds

Name of the compound λmax

4-propylguaiacol 280

Eugenol 281

Dihydroconiferyl alcohol 281

Vanillyl alcohol 280

Vanillylethyl ether 280

4-propylveratrole 279

Veratryl alcohol 279

4-isopropylpyrocatechol 282
Isocresol 281

Pinoresinol 280

3 . 5-dimethoxy-4-hydroxy phenylpropane 273

4 -methyl- 6 -propylguaiacol 280

Dihydrodehydro-di-isoengenol 281

5-ethyl cresol 284

α-conidendrin 283

4-ethyl phenol 278

4 -4' - dipropyl -6, 6 -biguaiacol 290

Coniferyl alcohol 265

4, 4'- dihydrox y -3,3'-dimethoxystilbene 383

Coniferyl aldehyde 240,341

2 - ethoxy - 1 - (4 -hydroxy- 3 -methoxyphenyl) - 1 -propanone 280,305

Sesamin

Occurrence : Sesame (Sesamum indicum)

M.P : 123°C

: 237.5,287 .5

: 2860,1630,1505,1450,1370, 1255,1185, 1100, 1060, 1040,

930,
855 ,790 and 7 50
Picropodophyllin

Occurrence : Himalayan mayapple (Podophyllum emodi)

M.P : 232°C

: 3500, 1767, 1595, 1507 , 1480, 1422, 1330, 1250, 1130,

1040, 1005 , 926
Dendrobine
Occurrence : Orchid (Dendrobium spp.)

M.P : 134.5~136°C

: 1767 , 1763, 17 60

NMR in : 7.48 ( ), 5.15 ( ), 8.59 ( ), 8.94 d,

CDCl3:τ 9.05d ( ).

Mass M/e : 263, 235, 220, 210, 184, 96

Gentiamine

Occurrence : Qinjiao(Gentiona krilowi), Chitata (Sweritia chirata)

: 220,245

: 1728 (γ-lactone), 1626 (C = C),1592, 1574, 1475

(related to C = N vibration), 1129,1045 (pyridine ring)

NMR in : 5.33 t,(J = 5.9)(2–CH2–in lactone ring), 4.23 d,J =

CDCl3: τ 10.7,4.05 d, J = 17.9( ), 2.92, J = 10.7,
Vasicinone

Occurrence : Malabar nut (Adhatoda vasica)

M.P : 201~202°C

IR γmaxμ : 3.21, 3.42, 6.03, 6.875, 7.22, 7.516, 7.7 4, 8.275, 8.48, 9.06,
9.265, 9.73,
10.13, 10.31, 11.15, 11.33, 11.54, 11.71,12.92, 13.35, l3.89,
14.41
Ipecac alkaloids
Emetine :

M.P : 74°C

: 1610 (at 1550~1650)

O-methyl psychotrine

M.P : l2l–123°C

: 1618, 1610, 1576 (at 1550~1650)

Protoemetine anhydrous
M.P : 193 ~ 195°C

: 232,283,220

: 2810 (aldehyde C–H), l728(aldehyde C=O), 1622(aromatic

ring)

Morphine alkaloids
NMR spectra
Serpentinine
Occurrence : Rauwolfia (Rauwolfia serpentina)

M.P : 264~265°C

: 227,257

: 1712, 1623,3340 (NH or OH)

NMR in : 40 Mc, relative to H2O, CPS: +39.0,+52.0(2–

CHCl3, COOCH3),+113.9d,+142.7 d( ).
Ajmaline
Occurrence : Rauwolfia serpentina

M.P : l58~160°C

: 247,295

IR γmax μ : 3.00, 3.17 (OH), no absorption bands at 5.82,7.24 (= C–Me)

or
9.0 (ether bridge).

Alkaloids of aspidosperma (Aspidosperma obscurinervium)

Dihydro obscurinervine
M.P : 184~185°C

: 220,256,313

: 2780,1750,1610

NMR in : 6.32(1)(arom.H), 4.52,J = 5.2(1) (CH2CHOCO),4.28 d–

CDCl3: δ 4.12 d
(2) (CH2, of cyclic ether), 3.87,3.80 (6) (2-arom. OCH3)
2.62 d,
1.78 d, J = l 9 (2) (rigid C–CH2–),0.95 t, J = 7 (3) (C–
CH2–CH3).

Mass: m/e : 440 (M +), 425, 411, 244, 206 ((M–CO)2+)

Obscurinervine
M.P : 203~204°C

: 220,253,312

: 2780, 1750,1605

NMR δ : 6.32,5.71(2) (vinyl protons), 4.5.2,4.28 d~4.13 d, 3.87,3.80,

2.57,2.02 d, J = 18 (2) (rigid C–CH2–CO),0.97 t

Mass: m/e : 438 (M +), 423, 409,244,205.

Dihydro obscurinervidine.

M.P : 189~190°C

: 219,255,312

: 2780,1750,1610

NMR δ : 6.35 (l) (arom.), 4.53, J = 5.2 (1) (CH2–CHOCO), 4.18

(2) (CH2 of cyclic ether),3.88,3.82 (6) (2-OCH3) 2.62 d,
1.78 d,
J = 19 (2), (rigid C–CH2–CO), 1.07 d, J = 7 (3) (N–CH–
CH3)

Mass: m/e : 426 (M +), 411,246,244, 199.

Obscurinervidine
M.P : 206~207°C

: 219,253, 310

: 2780, 1760,1605

NMR δ : 6.35 (1) (arom.H), 5.73 (2) (vinyl protons), 4.55 (1)
(CH2CHOCO),
4.18 (2) (CH2 of cyclic ether), 3.88, 3.82 (6) (arom. OCH3),
2.57 d,
2.02 d,J = l8(2) (rigid C–CH2–CO),1.07d, J = 6 (3) (N–
CH–CH3)
Tuboxenine

Occurrence : Pleiocarpa (Pleiocarpa tubincina)

M.P : 139~140°C

: 206,244,295

: 3180 (>NH), 1605 (indoline), 739 (ortho

disubst.benzene)

NMRin : 6.5~7.5 (4.arom. protons), 3.9 (N–H),

CDCl,:δ

Mass: M : 280 (a), 171 (b), 156 (d), 136(e), 124(f), 123(g), 122(h),
110 (i)
Pleocarpine
Occurrence : Pleiocarpa mutica

M.P : 141~142°C

: 206,246, 282,295

: 5.74,5.87 (C = O), no absorption of OH & NH

NMR in : 60 Mc, CPS : 463 d, J = 7 , 430 (3) (arom. protons),229

CDCl3 (3), 223 (3),
(2 COOCH3), 180; no signals of C–CH3 olefinic protons
or C2–H.
Pleiocarpamine
Occurrence : Pleiocarpa mutica

M.P : l59°C

: 230,285

: 1736, 1770

NMR in deutero :
acetone: δ

Mass M + : 322, M–l5, M–59 (COOCH3) = 263,180.

Tuboflavine
Occurrence : Pleiocarpa tubicina

M.P : 207~208°C

: 401, 323, 289, 264, 215

: 1616 (C=O of pyridine), no absorption of >NH.

NMRin :
CDCl3
 8 (Part II)

Extraction, Isolation
and Analysis of
Phytopharmaceuticals

The process of separating active principle(s) from powdered crude drugs by

using suitable solvents is called extraction. The basic principle behind extraction
is the exceptional behaviour of the active principles towards the solvent system.
Following is a schematic representation of the procedure for obtaining
powdered drugs:
 NOTE1 For the extraction of volatile oil, freshly collected drug should be
taken.

NOTE2 Powder size can be coarse (10/44) or moderately coarse (22/60).

Coarse powder of size (10/44) is that powder of which all the
particles pass through sieve no. 10 and not more than 40% pass
through a no. 44 sieve. Moderately coarse powder of size (22/60) is
that powder of which all the particles pass through seive no 22 and
not more than 40% pass through a sieve no. 60. Hard drugs are
powdered while soft drugs like gentian are sliced or crushed.

The following methods are used for extraction: infusion; decoction; digestion;
maceration; percolation; successive solvent extraction; supercritical fluid
extraction; steam distillation; headspace techniques.

8.8INFUSION
This method is only applicable for soft drugs and drugs containing water-soluble
constituents.

1. The drug is mixed with water. It is allowed to stand for 15 to 20 minutes.

Shaking or stirring is done if required.
Drug + H20 Filtrate
2. Heat is not applied
3. It is then filtered

The filtrate is called infusion. The infusion should be used within 24 hrs.
Sometimes, 20–25% of ethanol is used as a preservative. Infusions containing
20–25% of ethanol can be stored for a long time eg. compound infusion of
chirata and compound infusion of gentian.

8.9DECOCTION
Decoction is applicable only for water-soluble and heat-stable drugs obtained
from hard and woody sources.
 1. The drug is mixed with water and heated for 15 to 20 minutes.
Drug + H20 Filtrate
2. It is filtered
3. Marc is pressed
4. Volume is adjusted

The filtrate is called decoction. It should be consumed within 24 hrs. At

present no decoction is officially recorded in IP or BP.

8.10 DIGESTION
Digestion is a modified form of maceration. The solvent action in the case of
digestion is enhanced by the application of heat. This process is suitable only for
heat-stable substances.
Processes like infusion, decoction and digestion are now obsolete and are
rarely used for extraction of drugs with a few exceptions.

8.11 MACERATION
During maceration the plant material is soaked in the solvent. The extraction
time can vary from several minutes up to weeks. Other variables include the
amount of agitation, plant–solvent ratio, moisture content, temperature and the
number of times the plant has been macerated. Usually, the solute–solvent
proportion is 1:10. Unorganised drugs like gums, resins, gum-resins and
oleogum resins are also extracted by the maceration process (eg. tincture of
benzoin, tolu and myrrh).
There are three types of maceration.

a. Simple (single) maceration;

b. double maceration;
c. triple maceration

In case of double and triple maceration, the menstruum is equally divided into
two and three parts respectively and used. The purpose is to get more extractive
yield.

8.12 PERCOLATION
In a percolation type extraction the plant material is continuously flushed with
fresh solvent. The extraction is continued until sufficient compound is extracted.
If necessary, the same material can be re-extracted with a second solvent.
There are three types of percolation i.e.,

a. Simple percolation (cold percolation)

b. Reserved percolation
c. Hot percolation

Simple percolation
Percolation (Fig. 8.15) involves the following steps:

1. Powdering of the drug.

2. Imbibition of the drug i.e. moisturing the drug with a little menstruum for 4
hrs.
3. Packing: The drug is packed up to 2/3rd capacity of the percolator.
4. Maceration: Sufficient menstruum is added and allowed to saturate the
drug. When the drops start to come down through the nozzle, the nozzle is
closed and the moistened drug allowed to stand for 24 hrs.
5. Percolation: The percolate is collected drop-wise. The process is continued
till complete extraction has taken place. Complete extraction can be
observed in the following ways:
 Fig. 8.15 Percolation process

a. Take the final drop. Allow to evaporate. If no residue remains,

complete extraction has taken place.
b. Test the specific gravity of the final percolate and menstruum.
c. Test the final percolate for absence of the phytoconstituents concerned.

This method is suitable for heat-sensitive, hard and woody drugs. It is not
suitable for drugs which may block the percolator.
Reserved percolation
Here, the first 3/4th of the percolate is collected and reserved. The final 1/4th of
percolate is then collected, evaporated to syrupy consistency and added to the
reserved 3/4th portion of the percolate. Usually, alcohol is used as a menstruum
for the reserved percolation. It has the following advantages:

a. Can be used to prepare concentrated liquid extracts.

b. The reserve 3/4th portion of the percolate contains maximum active
constituents. The final 3/4th percolate contains least concentration of the
principles. Thus subjecting this portion to evaporation tends to concentrate
 the extract, e.g. liquid extract of liquorice.

Hot percolation: Soxhlet extraction

In Soxhlet extraction, similar to percolation, the plant material is continuously
flushed with fresh solvent. But the fresh solvent is formed by boiling the solvent
containing the extracted analytes (Fig. 8.16). Thus, in contrast to percolation, the
total amount of solvent is limited. In spite of what is sometimes thought, a
Soxhlet extraction can be far from complete, due to channeling or the presence
of air in the semi-permeable thimble containing the plant material.

Fig. 8.16 Soxhlation

Suitability of the Soxhlet method: For heat-stable substances only, and also for
active constituents which are less soluble in the menstruum in the absence of
heat.
 As a general guideline, one can say that maceration is most used for the
extraction of compounds occurring in low concentrations which are strongly
bound and/or diffuse slowly. Percolation is more useful for extracting loosely
bound compounds which occur in high concentrations or have a low solubility in
the solvent. The Soxhlet extraction method is useful for certain quantitative
extractions of thermally-stable compounds.
A comparision of the advantages and disadvantages of maceration, percolation
and the Soxhlet type of extraction is given Table 8.31.
Table 8.31 Advantages and disadvantages of the different methods of
extraction

8.13 SUCCESSIVE SOLVENT EXTRACTION

By Soxhlet extraction
The air-dried powdered drug is extracted by the Soxhlet extractor with
successively different solvents, in increasing order of polarity Pet.ether
benzene solvent ether chloroform acetone
ethanol methanol Finally, the drug is macerated with chloroform–water.
Before extracting with a new solvent, the powdered material is dried in hot air in
an oven at temperatures below 50℃.
Each extract is concentrated by distilling off the solvent and then evaporating
the solvent to dryness on a water bath. The extract obtained with the solvent is
weighed. Its percentage yield is calculated in terms of the air-dried weight of the
plant material. The colour and consistency of the extract are noted.
By maceration (or cold maceration)
This is similar to the Soxhlet extraction method mentioned above, except that the
cold maceration principle is adopted.
Mother liquor method
Here, the air-dried powdered material is extracted by the Soxhlet method or by
cold macertion with the highest polar solvent; the semi-solid extract is prepared,
which is dispersed in 100 ml of distilled water and then fractionated with
different solvents in succession—200 ml each (thrice) time, by using a
separating funnel.

8.14 SUPERCRITICAL FLUID EXTRACTION

8.14.1 Supercritical Fluids
Supercritical fluid extraction is a form of liquid extraction where the usual liquid
solvent phase is replaced with a supercritical fluid.
A supercritical fluid is a substance that boils above its critical point. The
boiling point of a liquid is defined as either the temperature at which the
maximum vapour pressure of the liquid is equal to the external pressure, or the
temperature at which the liquid boils freely under that pressure. For water, the
boiling point at atmospheric pressure (1.01 325 bar) is 100℃.
However, if the pressure above a liquid is increased, the boiling point is
raised. The elevation of the boiling point of a substance with increase in pressure
is shown in the boiling point curve (Fig. 8.17). The curve continues up to a point
called the critical point. The temperature and pressure at this point are called the
critical temperature and pressure, respectively. Figure 8.18 shows the phase
diagram of carbon dioxide with the supercritical region indicated. A fluid in this
region is neither a true liquid nor a true gas and has some of the properties of
each. As indicated by the boundaries shown in the figure, the fluid will not
condense to a liquid irrespective of how much pressure is applied and equally
will not boil, if the temperature is increased.
Fig. 8.17 Generalised boiling point curve

Supercritical fluids have some very interesting and potentially useful

properties for extraction and chromatography. The solvation strengths of many
supercritical fluids approach those of liquid solvents; hence many substances
will dissolve in them. Supercritical fluids also have diffusion coefficients which
are close to those of gases, hence they are able to transport dissolved solutes
through materials very quickly. The low viscosity of supercritical fluids also
allows them to flow easily through small openings. Perhaps, the most dramatic
property of the supercritical fluid state is the absence of a surface meniscus. The
supercritical fluid exhibits no surface tension properties.
This combination of properties means that a supercritical fluid can penetrate a
material as though it was a gas under high pressure but with the very important
difference that it has solvation properties which approach those of a liquid.
Hence, these fluids may be very useful as phases for both extraction and
chromatography. The extractive properties of supercritical carbon dioxide have
been exploited for many years on an industrial scale for the decaffeination of
coffee and the removal of nicotine from tobacco. Their use in preparative
chromatography, however, is more recent and limited. The solvating power of a
supercritical fluid generally increases as its density increases. The mobile phase
elution strength can be increased by increasing the applied pressure on the fluid
to increase its density. The relationship between density and pressure of
supercritical carbon dioxide is shown in Fig. 8.5b. Although supercritical carbon
dioxide is a solvent of relatively low polarity, its solvation power varies
considerably with density. The curves are particularly steep close to the critical
point. For example, just above the critical temperature (31.1℃) a five-fold
increase in pressure changes the density from 0.4 to 0.9 g ml-1. If the pressure is
raised further, from 350 to 700 bar, the increase in density is only from 0.9 to
0.98 g ml-1. Pressure can be an effective way to control solvent elution strength.
In many cases, however, the range of solvation power available through density
variation is not great enough. For these cases, the addition of a small percentage
of a polar solvent such as methanol or acetonitrile to the supercritical carbon
dioxide can modify its properties. The modifying solvent may render the carbon
dioxide subcritical, but it now has the solvating power of the high-density carbon
dioxide combined with the polar nature of the added modifier.
Fig. 8.18 Phase diagram of carbon dioxide

8.14.2 Choice of Supercritical Fluids

Many fluids have been investigated for use in supercritical fluid extractions
(SFE). Table 8.32 lists the critical points of temperature and pressure for some of
them. The physical and chemical properties of a supercritical fluid depend upon
temperature and pressure which are of paramount importance when designing a
supercritical fluid chromatograph or extract. Excessively high pressure or
temperature requirements could make the apparatus too expensive to be
practical. Therefore, the choice of fluid is particularly important. The most
familiar solvent, water, is a poor choice for SFE. The critical parameters are
among the highest for common solvents. Ammonia is particularly unpleasant to
work with, so a fume cupboard, or other ventilation precaution, is needed to keep
it out of the laboratory atmosphere. It is also extremely corrosive and its use
requires that certain parts of the chromatograph be gold-plated to resist
dissolution. There is evidence from Sievers et al. of violent explosive reactions
between nitrous oxide and modifiers. The alkanes and alkenes pose fire hazards
and have low polarity.
The most popular and safe supercritical fluid for use in SFE is undoubtedly
carbon dioxide. It can be used with thermolabile substances as its critical
temperature is only slightly above room temperature. The critical pressure of
carbon dioxide (73.8 bar) is not difficult to maintain with modem high systems.
Carbon dioxide has the additional advantages of being non-flammable,
chemically inert, odour free, being available in a state of high purity at low cost
and not presenting a solvent disposal problem.
Table 8.32 Some critical points for a selection of fluids

Traditionally, SFE was used for preparative scale isolation of compounds from
plant matrices, for example coffee beans, hops, and spices. Supercritical fluid
extraction is now being increasingly used as a sample preparation method for the
analysis of solutes in solid or liquid matrices, eg. flavours and fragrances from
natural products. More recently, the trend has been towards the application of
SFE as an analytical extraction method for sample preparation prior to using
chromatographic systems. Using carbon dioxide as the supercritical fluid,
extractions can be performed under mild conditions, thus reducing both the risks
of thermal degradation and the poor collection efficiencies of volatile analytes
that can sometimes occur during the steam distillation or solvent extraction of
essential oils and fragrance components.
A hydraulic diagram of the preparative SFE is shown in Fig. 8.19.
Fig. 8.19 Hydraulic diagram of the preparative SFE system

8.14.3 SFE of Natural Products

Fats and lipids
The use of SFE both on an analytical and processing scale is quite widespread in
the food industry for the extraction of fats and oils from seeds, foodstuffs, and
other materials. In 1972, Vitzthum and Hubert demonstrated that lipids could be
extracted from copra (coconut kernel), sunflower seeds, shelled peanuts, and
soybeans with carbon dioxide at pressure ranging from 280 to 350 bar.
Gottscahu and Brunner examined rapeseed and soybean oils using carbon
dioxide, both in the pure state and in combination with either propane or nitrous
oxide. Stahl and co-workers described the extraction of vegetable oils from the
seeds of soya, sunflower, Helianthus annus and Brassica napus. Typically,
supercritical carbon dioxide at pressures of at least 250 bar and a temperature of
20 and 40℃ respectively were conditions suitable for the extraction of oil from
oil seeds. The yields of oil that can be extracted were found to be comparable to
those obtained by the conventional solvent extraction. Tilly et al. have also used
SFE to investigate the triglycerides present in vegetable oils. They used
supercritical carbon dioxide at pressures from 100 to 300 bar at temperatures
from 40 to 80℃ and reported that the solubility of triglycerides was dependent
upon the solvent density and the solute volatility.
Sakaki and co-workers studied the oil extracted from the fungus Mortierella
ramanniane var. using carbon dioxide, nitrous oxide, trifluoromethane and sulfur
hexafluoride under supercritical conditions. They found the oil solubility at 245
bar and 60℃ to be highest in supercritical nitrous oxide, followed by C02, while
both CHF3, and SF6 showed poorer solvent power. Over the range 157–295 bar
and 40–80℃, it was shown that free fatty acids and diglycerides were more
easily extracted than triglycerides and sterol esters. The fatty acid composition of
the fungal lipid fraction was reported to be myristic acid: 1.3 wt%; palmitic acid:
30.3 wt %; palmitoleic acid: 1.3 wt %; stearic acid: 5.5 wt%; oleic acid: 46.1 wt
%; linoleic acid: 8.5 wt %; linolenic acid: 6.3 wt %; and others: 0.7 wt%.
Poly-unsaturated fatty acids have been the subject of an investigation by
Higashidate et al. They examined the enrichment of eicosapentaenoic acid and
docosahexaenoic acid esters from esterified fish oil by programmed extraction–
elution with supercritical carbon dioxide. SFE was performed at 80 bar and
40℃, density 9 g minute1, on esterified fish oil derived from sardines. The
extract was directly introduced into a silica gel column coated with silver nitrate.
SFC was then performed and the eicosapnetaenoic acid and docosahexaenoic
acid methyl esters were fractionated by reducing the pressure of the column
eluent to atmospheric pressure, thereby enriching the methyl esters several folds.
Choi et al. employed supercritical carbon dioxide (with and without ethanol as
modifier) to remove lipids and pigments from protein concentrates of green
algae (Scenedesmus obliquus). The fatty acid content and composition of algal
lipids were determined using column chromatography, TLC, and GLC. The
neutral lipids and a part of the glycolipids were removed by single step
supercritical carbon dioxide, but phospholipids were not extracted. Addition of
ethanol increased the removal of glycolipids and phospholipids.
In the vegetable oil industry, Goncalves et al. investigated the use of SFE as a
de​acidification method for the removal of free fatty acids from olive and husk
oils. Conventional refining processes of olive oil can alter the triglyceride
composition, producing a decrease in the nutritional qualities of the oil.
Solubility of glycerol trioleate (the most abundant triglyceride in olive oil) and
free fatty acids in supercritical carbon dioxide under different conditions were
examined and SFE was proven as an alternative method for the removal of free
fatty acids from olive oil.
Steroids
Rossi et al. explored the possibility that SFE could be used to improve the
nutritional quality of goods by the elimination of potentially dangerous
ingredients from raw materials or derivatives formed during thermal treatment.
They used both carbon dioxide and methanol modified carbon dioxide SFE to
remove cholesterol and its oxidation products from powdered egg yolk Terpenes
One of the major uses of SFE is the extraction of natural raw materials for the
flavour and perfume industry. The flavour compounds isolated are terpenes,
mostly monoterpenes.
Hawthorne et al. have summarised SFE applications and the coupling of SFE
to gas chromatography. They reported the isolation of terpenes, oxyterpenes, and
sesquiterpenes along with many other natural product classes from a wide
variety of plant species such as basil, mint, oregano, parsley, sage, rosemary,
thyme, conifer trees, aromatic cedar wood, and the leaves of the eucalyptus tree.
Using SFE–GC and FID, Hawthorne et al. extracted and identified α-pinene,
camphene, cineole, camphor, borneol, bornyl acetate, and humulene from
rosemary; from thyme they were able to extract and identify borneol, thymol,
and carvacrol; from cinnamon, cinnamaldehyde and coumarin; α-pinene, β-
pinene, camphene, limonene, camphor, and bornyl acetate were extracted and
identified from spruce needles; cedrene and cedrol were extracted from cedar
wood. Hawthorne's group also isolated α- and β-pinene and limonene from
orange peel; α-pinene and 1,8-cineole from eucalyptus leaves, and 1,8-cineole,
estragole eugenol, and selinene from basil.
The citrus industry has found great use for SFE to remove limonene and other
terpenes from cold-pressed oils. The terpenes do not contribute much to the
flavour or fragrance of the oil. However, they are unstable at high temperatures
and in the presence of light, rapidly oxidising in air and often decomposing as
undesirable compounds. Several investigations into SFE of citms fruits have
been carried out. Sugiyama et al. have developed a simple micro-scale SFE
system, which they used at various pressures and temperatures to extract the
constituents of lemon peel oil. The extract obtained was analysed by GC–MS;
the compounds they extracted and identified were α-pinene, camphene, β-
pinene, myrcene, α-serpinene, ⍴-cymene, terpinolene, nonanal, limonene oxide,
tans-sabinene, citronellal, linalool, linalyl acetate, 4-terpineol, ceral, citral, neryl
acetate, geranyl acetate, nerol, and geraniol. Yamauchi et al. examined the
extraction of the oil from lemon peel and its fractionation by a coupled SFE-
prep-SFC system. The technique could be used for producing a new flavour by
remixing fractions in different proportions as well as for the simple deterpening
of the oil.
Huston and Ji used various densities of supercritical carbon dioxide to
selectively extract the components of ground clove buds (Syzygium aromaticum).
The extracts they obtained were recovered by using a direct on-column interface
of solvent recovery. An ion-trap GC–MS system was operated for solute
separation and identification. Some of the terpenoid components identified in
this work were eugenol acetate, eugenol, α-cubebene, carophyllene, humulene,
and isoeugenol. Optimum supercritical carbon dioxide extractions of cloves have
previously been reported to give oil yields of 16% and 19%. Neither study found
a distinct difference between steam distillation and SFE in terms of oil yield.
Pellerin describes the extraction of the volatile constituents from the flowers
of Lavandin grosso. Some of the extracted constituents identified were 1,8-
cineole, cis-ocimene, linalool, camphor, borneol, terpinene-4-ol, linalyl acetate,
lavanduyl acetate, coumarin, α- and β- caryophyllene, and hemiarin.
Supercritical carbon dioxide extraction was used by Nykanen et al. to isolate
flavour compounds from angelica root (Angelica archangelica L.). The
extractions were conducted at 40℃, at pressures ranging from 80 to 400 bars.
Capillary GC–MS was used to analyse the extracts, which were compared with
those obtained from angelica root by steam distillation. Seventy-six components
were extracted and identified; the major components were α-pinene, sabinene,
myrcene, α-phellandrene, δ-e-carene, ρ-cymene, β-phellandrene, chrsanthenyul
acetate, α-copaene, 15-pentadecanolide, 7-methoxy-8(3-methyl-2-butenyl)- 2H-
l-benzopyran-2-one, and oxypeucedanin.
Smith and Burford investigated the conditions required for quantitative
extraction of a range of test analytes (limonene, caryophyllene, carvone,
eugenol, and santonin) present at known concentrations in a model cellulose
matrix.
Stahl and Keller clearly demonstrated the advantage of SFE over traditional
methods of steam distillation in the extraction of thermolabile compounds.
Thermolabile sesquiterpenes were extracted with supercritical carbon dioxide
from β-asarone-free Calamus rhizomes (Acorus calamus L. var americamus).
During steam distillation this compound decomposes to shyobunone. With
carbon dioxide at 80–90 bar, 40℃, and with fractionated separation, higher
yields of the bitter principles of calamus, acorone and isoacorone, were obtained.
 Ma et al. have developed an analytical SFE system followed by GC–MS to
separate and determine the volatile components of three kinds of herbs used in
Chinese herbal medicines. The herbs examined were frankincense, myrrh and
Evodia rutaecarpa. The extractions were carried out using supercritical carbon
dioxide at 200 bar and 50℃. The results from the GC–MS profile showed that
the myrrh (gum resin) extract contains a large amount of sesquiterpenes, i.e., δ-
elemene, cis-α-bergamotene, β-cedrene, α-cubebene, α-ylangene, α-copaene, β-
bourbonene, β-elemene, trans-α-bergamotene, allo-aromadenendren, γ-
gurjunene, germacrene D, β-selinene, α-selinene, γ-selinene, γ-cardinene, δ-
cadinene, elemol, γ-elemene, cadinol, β-eudesmol, and α-eudesmol. However,
the most important components were the furano-sesquiterpenoids and
furanodienone.
Smith and Burford have described the extraction of the sesquiterpene lactone
parthenolide from the medicinal herb feverfew (Tanacetum parthenium). Dried
plant material was extracted with supercritical carbon dioxide at 250 bar and
45℃ at 0.85 ml per minute and analysed by GLC. In addition to parthenolide,
the extracts also contained significant amounts of camphor and chrysanthenol
acetate.
Alkaloids
Two important alkaloids—mainly because of the vast human consumption on a
daily basis—are caffeine and nicotine. The decaffeination of coffee by SFE has
been the subject of a large amount of research and development. A
comprehensive discussion on coffee decaffeination is given by McHugh and
Krukonis. The SFE process for the removal of nicotine from tobacco was
patented by Roselium et al. in 1979. Sharma et al. reported a fast reproducible,
single-step method for the isolation of organic compounds from chewing
tobacco (commercial snuff) using supercritical carbon dioxide as the mobile
phase at 551.5 bar and 60℃. Using this method, nicotine, cotinine, myosmine,
and other alkaloids have been quantitatively determined in commercial snuff.
Bicchi and co-workers investigated the applicability of off-line SFE-capillary-
GC for the analysis of pyrolizidine alkaloids in plants of the Senecio genus (S.
inaequidins and S. cordatus) in comparison with the classical Soxhlet extraction
method. The highest extraction recovery by SFE was achieved at 150 bar, 55℃,
with 5% methanol. The pyrrolizidine alkaloids extracted from S. inaequidins
were identified as senecivemine, senecionine, seneciphylline, integerrimine,
retrorsine, usaramine, desacetyldoronine, and doronine. The capillary GC-FID
pattern for S. cordatus identified senecivemine, senecionine, seneciphylline,
sparitoidine, integerrimine, jacobine, and jacozine. When compared to their
Soxhlet extraction with methanol, the SFE method produced a higher recovery
of pyrrolizidine alkaloids; a faster extraction time and used a smaller sample
size.
Janicot et al. used near-critical mixtures of carbon dioxide and polar modifiers
to extract the major alkaloids from poppy straw. The extraction of thebaine,
codeine, and morphine was achieved by percolating a mixture of carbon
dioxide–lmethanol–water (70:24:6 w/w, respectively) at 45℃ and 200 bar
through a column containing poppy straw (previously ground and sieved) for 20
minutes. Analysis was performed by SFC.
The extraction of pepper perfume using SFE has been investigated by Takagi
et al. SFE was performed with C02 at 40℃, 196 bar with 5% methanol for 60
minutes. The extracted berries produced yellow oil that was pepper perfume
rich. Analysis by SFC and HPLC showed that pipeline was the main component.
Oxygen-containing heterocyclic compounds
Saito et al. described the use of directly coupled SFE and semi-preparative-SFC
for the enrichment of tocopherols from wheatgerm powder. The extraction of α-
and β-tocopherol was performed at 250 bar and 40℃. Schneiderman and co-
workers used supercritical carbon dioxide at 551.7 bar and 65℃ to extract
anthraquinone from Kraft paper and pine plywood sawdust. Quantitative
extraction was reported to require only 20 minutes and did not lead to the
formation of emulsions or other phase separation problems encountered in
solvent extraction. Indeed, SFE was found to be far faster than Soxhlet
extraction. Schneiderman et al. also reported the use of SFE with carbon dioxide
at 551.7 bar and 60°C for the extraction of phylloquinone (vitamin K1) from
commercial soy-protein-based and milk-based powdered infant formulas.
Quantitative extraction required 15 minutes, and vitamin K1 was determined in
the extracts by HPLC with electrochemical detection.
Miyachi et al. examined the effects of pressure, temperature, time, and
modifiers on the SFE of Cnidium formosanum fruits. They found the optimum
temperature and pressure for the extraction of furanocoumarins (columbianadin,
bergapten, o-acetylcolumbianetin, edultin and columbianetin) using supercritical
carbon dioxide at 40°C and 400 bar. Miyachi and co​workers also applied SFE to
extract coumarins, lignans, and flavonoids from crude drugs and plants.
Scoparone and capillarisin, the main constituents of Artemisia capillaries were
easily extracted with supercritical carbon dioxide at 400 bar and 40°C. Ethanol
was used as the organic modifier to extract from Fraxinus japonica and Fraxinus
mandshurica var. japonica scopoletin, fraxidin, isofraxidin, and fraxinol, which
all possess a hydroxyl group on the coumarin skeleton. However, esculetin and
fraxetin, both of which possess two hydroxyl groups on the coumarin ring, could
not be extracted under the above conditions, even in the presence of ethanol or
water as a modifier.
A lignan, 1-hydroxypinoresinol was extracted from F. japonica and other
Fraxinus species using supercritical carbon dioxide with water as a modifier.
The extraction of lignans from the Forysthia species by supercritical carbon
dioxide compared well with the extracts produced by boiling n-hexane or boiling
methanol. Phyllygenin and (+)-pinoresinol were extracted from Forsythia
syspersa, and arctigenin and matairesinol were extracted from Forsythia
viridissima. The extraction of the prenylflavonoids, morusin and sanggenon A
(both having less than three hydroxyl groups), from the bark of Morus bombycis
was achieved using supercritical carbon dioxide without the need of modifiers.
However, those flavonoids with more than four hydroxyl groups could not be
extracted even with the use of modifiers. The trilogy of articles from Miyachi et
al. concludes with the extraction of pigments from Lithospermum root and
Glycyrrhiza (liquorice) root using supercritical carbon dioxide. Shikonin
derivatives, the red pigments of Lithospermum root (deoxyshikonin,β,β-
dimethylacrylshikonin, isovalerylshikonin, α-methyl-n-butylshikonin,
isobutylshikonin, acetylshikonin shikonin, and β-hydroxyisovalerylshikonin),
were effectively extracted with CO2 at 127.4 bar and 35°C. The addition of
ethanol or water as a modifier reduced the efficiency of this extraction.
Liquiritigenin could be extracted with CO2 from licorice root. However,
isoliquiritigenin, which has three phenolic hydroxyl groups, could not be
extracted. Similarly, the extraction of licochalcone A from licorice root required
only carbon dioxide, while the extraction of licochalcone B required the addition
of ethanol as modifier.
Aromatic and Phenolic Compounds
Chen et al. extracted the pungent principles of ginger (Zingiber officinale
Roscoe) using liquid carbon dioxide (41.3–48.2 bar). The identification of the
pungent compounds was performed by analytical HPLC and mass spectrometry.
The most abundant pungent compound identified was (6)-gingerol (11.88%
w/w). Other homologues of gingerol identified were (8)-gingerol (1.67% w/w)
and (10)-gingerol (2.38% w/w); trace amounts of (12)-gingerol and (14)-
gingerol, methyl-(6)-, methyl-(8)-, methyl-(10)-, and methyl-(12)-gingerols. (6)-
Shogoal, which is derived from (6)-gingerol during thermal process ageing, was
also identified (0.08% w/w).

8.14.4 Advantages of SFE

Mild technique.
Ideally no organic solvents are needed, i.e., environmentally friendly and
cheap.
Can give fast extractions.
Extraction can be automated.
With 100% CO2, there is no need for solvent removal.
Solvating power variable via pressure.

8.14.5 Disadvantages of SFE

Less suitable for very polar products.
Rather complex apparatus is needed.
Involves high pressure.
Less suitable for the extraction of leaves.
Trapping can be troublesome.
Scale-up to commercial levels is difficult.
Fresh plant materials are difficult to extract.

8.15 STEAM DISTILLATION

Steam distillation is used for the separation of volatile constituents from crude
drugs. It is done by distillation of the drug with a mixture of water and glycerin.
The distillate is collected in a graduated tube, in which the aqueous portion of
the distillate is automatically separated and returned to the distilling flask. The
volume of oil is then measured. The volume of volatile oil may be measured
directly or xylene may be used to take up the volatile oil. Clavenger’s apparatus
(Fig. 8.20) is used for the purpose.
PROCEDURE
50–10 g of the drug mixed with 30 ml/60 ml of glycerol and 300 ml of water,
and with a few small pieces of porous earthenware, is placed in the distillation
flask of the apparatus. The flask is connected to the still head, the stopper is
removed, and water is poured into the orifice, till it runs over. The flask is heated
with frequent agitation, until ebullition starts. The distillation is continued at a
rate which leaves the lower part of the condensor cold. The flask is rotated
occasionally to wash down any material adhering to the upper part of the walls.
After the distillation has been carried out for five hours, heating is discontinued
and the volume of oil in the graduated portion of the tube is read off after five
minutes. The distillation is again continued for a further period of one hour and
the volume of oil is read as before.

Fig. 8.20 Clavenger apparatus

If necessary, the distillation is continued again, till the volume of oil does not
differ in the two successive readings. This yield of volatile oil is taken as the
volatile oil content in the drug.

8.15.1 Advantages and Disadvantages of Steam Distillation

Advantages

Selective for volatile oils, eg. essential oils.

Very simple and cheap technique.
Only water is needed as solvent.
Well suitable for preparative scale extractions.
Very clean extracts.
High enrichment factor.

Disadvantages

Only suitable for polar volatile oils.

Possible decomposition due to the presence of water and high temperatures.
Unsuitable for mg scale operation.
Quantitative distillation is time-consuming.
Distillate varies during distillation.
Can loose more polar trace constituents.

8.16 HEADSPACE TECHNIQUES

The headspace of an object, eg, a flower, is the atmosphere directly surrounding
it. The headspace contains different volatile substances in varying amounts,
which originate either from the object or the environment. The headspace can be
collected and analysed. Two types of headspace analysis can be distinguished:

1. Equilibrium or static headspace: used for analytical purposes.

2. Dynamic headspace: used for analytical and preparative purposes.
8.16.1 Advantages and Disadvantages of the Headspace Technique
Advantages

Selective for volatile substances.

Very mild technique.
Can be fast, no clean-up is necessary.
Living plants can be sampled.

Disadvantages

Many variables in dynamic HS sampling.

Quantitatively dynamic HS is less suitable.
Impurities can be a problem in dynamic HS.
Static HS apparatus is complex.
Introduction of sample on GC is difficult.
Less suitable for preparative purposes.

8.17 SEPBOX
Access to an inexhaustible reservoir of natural compounds has become much
easier in recent years. But fractionation and separation of samples obtained from
nature remain time-consuming, tedious and extremely expensive, even though
the assays of testing these samples have become faster and cheaper due to the
invention of high throughput screening process.
A sepbox is a highly sophisticated completely automatic instrument for
isolation and separation of compounds from crude plant extracts. With this
technology a large number of samples can be efficiently processed. Up to 600
samples for high throughput screening can be obtained with a maximum purity
and more than 90% recovery rate using 2-dimensional separation, both for polar
and non-polar substances.
The sepbox instrument uses a combination of high performance liquid
chromatography (HPLC) and solid phase extraction (SPE) to separate complex
mixtures like natural product extracts. The use of sepbox will certainly enhance
the isolation of active principles of pharmacological interest from plant sources.

8.17.1 Types of Sepboxes

There are two main types of sepboxes—sepbox 2D-5000 and sepbox 2D-250.1.
Sepbox 2D-5000 (Fig. 8.21)
The sepbox 2D-5000 uses online coupled high performance liquid
chromatography with solid phase extraction with a second SPE-step to process
samples and gain fractions almost free from water and buffer (HPLC-SPE). In
this instrument 6 HPLCs and 18 SPE columns are placed in series. It also uses
UV and evaporative light scattering detection (ELSD) as detectors. The
minimum amount of extracts needed is 5 gm. It is a fully automated operation
and works on a user-friendly software so method development, process control,
data acquisition and data analysis is easy. This instrument allows a choice
between a liquid sample injection and an adsorbed (solid) sample injection. It
also allows the isolation of up to 600 compounds in 24 hours. The sepbox
fraction collector can contain up to 576 vials equipped with liquid sensors and
automatic shut off functions.

Fig. 8.21 Sepbox 2D-5000 (From www.Cm-corp.co.kr/products/products01_

03.htm)

Mode of operation
Initially a reverse phase separation takes place, and the obtained fractions are
trapped on 18 solid phase extraction columns. The second separation step takes
place on three alternative reverse phase columns, depending on the polarity of
the respective fraction. Figure 8.22 gives a flow diagram showing the different
stages of the whole process.

Fig. 8.22 Flow diagram of Sepbox 2D-5000 (From www.Cm-

corp.co.kr/products/products01_ 03.htm)

Sepbox 2D-250.1 (Fig. 8.23)

The sepbox 2D-250.1 uses online high performance liquid chromatography with
solid phase extraction (HPLC-SPE-HPLC). It is a fully automated operation
(Fig. 8.24) and is setup for a maximum of 100 or 250 mgs of extracts. It also
works on a user-friendly software so method development, process control, data
acquisition and data analysis is easy. It also allows a choice between liquid
sample injection and adsorbed (solid) sample injection. It consists of a bench-top
system. It can isolate up to 600 compounds in 24 hours and after isolation, the
various fractions can be collected on up to 12 micro titre plates. Figure 8.25
gives a flow diagram showing the different stages of the whole process.
Fig. 8.23 Sepbox 2D-250.1 (From www.Cm-
corp.co.kr/products/products01_03.htm)

Fig. 8.24 Sepbox automation (Referred from www.Cm-

corp.co.kr/products/products01_ 03.htm)

Fig. 8.25 Fig. 8.25 Flow diagram of Sepbox 2D-250.1 (From www.Cm-
corp.co.kr/ products/ products 01 _ 03.htm) 8.18 Selection of a Suitable
Extraction Process

The typical extraction process used for various types of drugs is tabulated in
Table 8.33.
Table 8.33 Methods of extraction for various types of drugs

Roots, rhizomes, stems, bark, seeds and fruits are the source of hard and
woody drugs. Similarly, soft drugs are obtained from flowers and leaves.
The solvent elution strength is listed with their increasing order of polarity:

NOTE: High polar drugs need a high active system and a higher solvent strength.
Low polar drugs require a low active system and a lower solvent strength.
The general protocol for extraction of different chemical constituents from the
plant material is described in Scheme 8.1.

8.19 CARBOHYDRATES
Carbohydrates or sugars occupy a central position in plant metabolism.
Therefore methods for their detection and estimation are very important to the
plant scientist. Not only are sugars the first complex organic compounds formed
in the plant as a result of photosynthesis, but they also provide a major source of
respiratory energy. They provide a means of storing energy (as starch), and
transport of energy (as sucrose) and are also the building blocks of the cell wall
(cellulose). In addition, many other classes of plant constituents, e.g., the nucleic
acids and the plant glycosides, contain sugars as essential features of their
structures. Finally, sugars play a number of ecological roles in plant–animal
interactions (flower nectars are mainly sugars), in protection from wounding and
infection and in the detoxification of foreign substances.
Sugars are conveniently classified into three groups, on the basis of molecular
size: the simple monosaccharides (e.g., glucose, fructose) and their derivatives;
the oligosaccharides, formed by condensation of two or more monosaccharide
units (e.g., sucrose); and the polysaccharides which consist of long chains of
monosaccharide units, joined head to tail, either as straight chains or with
branching.
Scheme 8.1 Protocol for extraction of different chemical constituents

8.19.1 Extraction of Monosaccharides

Method of isolation
Analysis: By paper chromatography
Adsorbent used: Whatmann filter paper no. 1
Solvent system:

The chromatogram should be run for 18 to 24 hours.

Spraying reagent: Dip the paper in aniline hydrogenpthalate and dry at 105°C
for 5–10 minutes.

8.19.2 Extraction of Oligosaccharides

Oligosaccharides resemble monosaccharides in their chemical properties, and
methods used for their separation are largely similar. The main difference is that
hot water (6–8 volumes) is used for their extraction. Heating at 90°C for 15
minutes is necessary. Filter the solution through celite while hot. Concentrate the
filtrate to 1/10th of its volume under vaccum; allow to crystallise in the
refrigerator.
Analysis
Similar to monosaccharides, but the chromatogram should be run for 48 to 96
hrs.
Examples
Disaccharides: sucrose (sugarcane), maltose (maltsugar), lactose (cow’s milk)
Trisaccharides: gentianose (gentian root), raffinose (beet and manna)
Tetrasaccharides: stachyose (manna)

8.19.3 Extraction of High Molecular Weight Carbohydrates

PROCEDURE
Isolation of starch from potato
Isolation of pectin from apple
Extraction of xylan from straw
Collection and purification of exudates
Examples of exudates are gums and resins.
Source: From the bark of trees and also from seeds and fruits.
PROCEDURE
Gums are pathological, abnormal plant products caused as a result of an injury to
the plant. Chemically, they are polymers of monosaccharides or mixed
monosaccharides and many of them are combined with uronic acids. Gums are
the mixtures of calcium, sodium or potassium salts of aldobionic acids.
Aldobionic acids on hydrolysis yield monosaccharides and uronic acids.
Analysis of high molecular weight carbohydrates: Same as that of
monosaccharides and oligosaccharides.

8.19.4 Tests for Carbohydrates

Test for reducing sugars
Heat the solution of substance and add a mixture of Fehling’s solution A and B
drop by drop. If reducing sugars are present the brick red precipitate of cuprous
oxide is formed.
NOTE: Reducing sugars include monosaccharides, many disaccharides (lactose,
maltose, cellobiose and gentiobiose); non-reducing sugars include some
disaccharides (sucrose) and polysaccharides. Non-reducing sugars can be
hydrolysed by boiling with acids, thereby giving reducing sugars. After
hydrolysis they should be neutralised with alkali, and only then can a brick red
precipitate be obtained, if heated with Fehling’s solutions A and B.
Molisch’s test
When carbohydrates are treated with α-naphthol and concentrated sulphuric acid,
purple colour is produced.
NOTE: If soluble carbohydrates are present and concentrated sulphuric acid is
gently poured in the test tube containing the carbohydrate, it will form a ring.
With insoluble carbohydrates like cotton wool (cellulose), the colour will not be
produced till it is shaken.
Osazone formation
Osazones are sugar derivatives formed by heating a sugar solution with phenyl-
hydrazine hydrochloride, sodium acetate and acetic acid. These derivatives can
be observed under the microscope in the form of crystals. These crystals are
characteristic of certain sugars.
Resorcinol test for ketones: Selivanoff’s test
Add a crystal of resorcinol to the solution and warm on a water bath with equal
volume of concentrated hydrochloric acid. A rose red colour is produced if
ketose sugar (e.g., fructose, honey) is present.
Test for pentoses
Heat the solution with an equal volume of hydrochloric acid containing little
phloroglucinol. Red colour is produced.
Keller-Kiliani’s test for deoxysugars
Deoxysugars are present in cardiac glycosides e.g., in Digitalis and Strophanthus
species. The sugar is dissolved in acetic acid containing trace amounts of ferric
chloride. It is then transferred to the surface of concentrated sulphuric acid. A
reddish brown ring will be formed at the junction and the colour slowly changes
to blue.
Benedict’s tests
To 5 ml of the reagent (Benedict’s reagent: 173 g sodium citrate + 100 g sodium
carbonate + 17.3 g copper sulphate) in a test tube, add 10 drops of test solution.
Mix well, boil the mixture vigorously for 2 minutes and allow to cool. A red,
yellow or green precipitate is formed, depending upon the amount of sugar.
Soluble starch test

a. Shows a deep blue colour with iodine.

b. Gives a canary yellow colour when heated with 5% KOH.

8.20 PROTEINS
Proteins contain carbon, hydrogen, oxygen, nitrogen and rarely sulphur. They are
very sensitive to temperature, pH, UV radiations and organic solvents. Proteins
are amphoteric in nature with high molecular weights, forming a colloidal
solution in water. They are soluble in 1 M NaCl and 0.1% NaOH, and are easily
extractable from plant sources. They are generally stored in the form of aleurone
grains in plants.

8.20.1 Isolation
Isolation of proteins from legumes
Legumin and vicilin are the main globular proteins of legume seeds. They may
be isolated as follows:
The above purification process should be repeated 2 to 3 times.
Isolation of proteins from seeds containing high oil content: e.g., Arachis
hypoge
NOTE: The globulin contains two components—arachin and conarachin—which

are fractionated as follows:

8.20.2 Analysis
Protien is generally analysed by thin layer chromatography. The particulars are
given in Table 8.34.
Table 8.34 Developing systems for various proteins
8.20.3 Estimation
The assay procedure was established by Lowry et al. (1951). It is based on the
reaction between the Folin–Ciocalteu reagent and the peptide bond.
About 0.1–0.2 ml of the protein sample (the protein content should be
between 10 and 100 mg) is mixed with 1ml of the reagent (made by adding 1 ml
of 0.5% CuSO4.5H2O in 1% sodium citrate to 50 ml 2% Na2CO3 in 0.1 M
NaOH). The solution is allowed to stand for 10 minutes at room temperature. 0.1
ml of the Folin–Ciocalteu reagent is added; mixed well, and the absorbance
measured at 750 nm after 0.5–2 hr. A standard curve is prepared at 750 nm by
using a pure protein sample. Then, the sample under test is graphically
estimated.

8.21 ALKALOIDS
The term “alkaloid” or alkali-like substances was proposed by the pharmacist W.
Meissner in 1819 for basic, nitrogen containing compounds of plant origin. They
occur only in certain genera and families and are rarely universally distributed in
large groups of plants. As more become known about the chemistry of the
various alkaloids, two more characteristics, viz., complex molecular structure
and significant pharmacological activity were added to the definition of
alkaloids. Furthermore, it was found that the basic property of alkaloids is due to
the presence of a nitrogen atom inside the ring. So, alkaloids are now generally
defined as physiologically active basic compounds of plant origin in which at
least one nitrogen atom forms part of a cyclic system.
There are exceptions to this definition. For example adrenaline, ephedrine,
muscarine and muscaline are alkaloids, though nitrogen atom is not present in
their cyclic ring system. Colchicine and ricinine, though defined as alkaloids, are
non-basic in nature. While thiamine and purins, even though they have a
nitrogen atom in their ring are not alkaloids.
There are some alkaloids which are not of plant origin but from
microorganisms. For example,
Batrachotoxin from Columbian arrow poison frog phyllobates aurotaenia.
Castoramine from the Canadian beaver.
Muscopylidine from the musk deer.
Gliotoxin from the fungus trichodermia viride.
From recent investigations, it has come to light that many alkaloids still
remain to be found in microorganisms, and that the traditional exclusive
relationship between alkaloids and higher plants may soon no longer be valid.

8.21.1 Sources of Alkaloids

Alkaloids in the plant world
About 5500 alkaloids are known at present, and new ones are being discovered
almost daily. But they are found only in 10–15% of all vascular plants. They are
also rarely found in lower plants like algae, fungi etc., (except ergot alkaloids
which occur in one or two families of fungi), gymnosperms or monocotyledons,
and thus chiefly occur in certain dicotyledons. Alkaloids occur in leaves, seeds,
roots and bark of nearly 40 plant families. The alkaloid content of plants varies
with its locality, the seasons and the age of the plant. Closely related alkaloids
generally occur together in the same plant e.g., nearly 20 alkaloids have been
isolated from opium. Similarly, it is also generally observed that a given genus or
related genera possesses the same or structurally related alkaloids, example,
seven different genera of the family solanaceae contain hyoscyamine. Further, it
is also true that simple alkaloids often occur in various different unrelated plants,
while the more complicated ones such as quinine, nicotine, colchicine etc.,
generally occur in one species or genus of a family.
Distribution of alkaloids in plant organs
Alkaloids are found in various parts of the mature plant as is illustrated below.
Seeds: Strychnos, Cola, Delphinium.
Roots: Aconitum, Baptisia,
Rhizomes: Sanguinaria.
Rhizomes and roots: Ipecac, Veratrum, Hydrastis.
Corm: Colchicum.
Leaves: Belladonna, Stromanium.
Fruits: Capsicum, Piper conium.
Barks: Cinchona, Xanthoxylum, Granatum.
The same plant can sometimes produce enough alkaloids in more than one
organ to warrant the production of more than one commercial entry, example
belladonna, (leaves and roots) or colchicum (seeds and corm).

8.21.2 Screening of Alkaloids

Many different procedures have been reported for detecting the presence/absence
of alkaloids in plant tissue. Such procedures are part of the economic
exploitation of higher plant species for their useful drugs. Indeed, more plants
have probably been surveyed for alkaloids than for any other class of secondary
constituents.
A typical procedure used by Hultin and Torssell (1965) for screening 200
Swedish plant species, involves a preliminary extraction of 4 g of dried tissue of
each sample with methanol. The aqueous solution of the acid-soluble portion of
this methanol fraction is basified with concentrated ammonium hydroxide and
then extracted with chloroform and ethanol. These concentrates are then tested
for alkaloids with six reagents and alkaloid is only recorded present if all six
reagents react positively.
In screening crude plant extracts, one has to be aware of false positive
reactions produed by some commonly used alkaloidal reagents such Mayers,
silicotungustic acid, Dragendorffs and Wagner’s reagent. For instance proteins
and other substances can cause precipitation with reagents containing heavy
metals.

8.21.3 Functions of Alkaloids

The purpose of the existence of alkaloids in plant i.e., their functions in plants, is
uncertain. But there are various views by different authorities such as:

They are of no importance and are only by-products of the plant

metabolism.
 They act as reservoirs for protein synthesis.
They may act as protective substances against animal or insect attacks.
Like hormones, they may have functions as stimulants or regulators in
activities like growth, metabolism and reproduction.
They may function as de-toxicating agents by methylating, condensing and
cyclising the compounds whose accumulation might otherwise cause
damage to the plant. But, it is also very important to note that as much as
85–95% of the plants manage very well without any alkaloid.

8.21.4 Nomenclature
Because of the complex structure of alkaloids and other historical reasons, no
attempt has been made for the systematic nomenclature for alkaloids. A large
number of alkaloids have been named according to the plants from which they
are obtained, viz., papaverine from Papaver soeniferum. A few alkaloids have
been named because of their physiological action viz. morphine (German:
morphin - God of dreams), emetine (Greek: emetekos – to vomit).
A single alkaloid group pelleteirine from pomegrenates has been named after
its discoverer PJ Pelletier. The names of minor alkaloids are derived by adding a
prefix or suffix to the name of the principal alkaloid.
All alkaloids end with the suffix ‘ine’. This is to discriminate alkaloids from
other classes of secondary metabolites.

8.21.5 General Characteristics and Properties of Alkaloids

Properties

1. Alkaloids are more or less complex compounds produced by plants.

2. They contain nitrogen in their molecular structure.
3. Many, probably most, alkaloids are derived, at least partly from various
amino acids as their direct precursors. The most common ones are
phenylalanine, tyrosine, lysine, ornithine, histidine, tryptophan and
anthranilic acid.
As an exception some alkaloids (eg., steroidal alkaloids) are in their
biogenesis, more directly derived from isoprenoid or other precursor
compounds of carbohydrates.
 4. They are basic in reaction, due to the availability of a lone pair of electrons
on nitrogen.
5. Solubility
The free bases of alkaloids are soluble in organic, non-polar immiscible
solvents. In contrast, the salts of most alkaloids are soluble in water. The
alkaloids containing quaternary bases are only water soluble. Some proto
alkaloids and pseudo alkaloids show higher solubility in water. For
example, colchicine is soluble in alkaline water, acid or water; and caffeine
is freely soluble in water.
6. Chemical tests
i. Precipitation by certain reagents: Table 8.35 lists the common
reagents used to test for the presence of alkaloids.
ii. Colour reaction with certain reagents
With 0.5 ml of selenium dioxide solution: Colouration
With 0.5 ml of ammonia molybdate solution: Colouration
With a saturated solution of ammonium vanadate: Colouration
Marquis reagent: concentrated sulphuric acid
+ 40% formaldehyde solution: Colouration
With a solution of 1 gm of ceric ammonium sulphate in 99 gm of
85% phosphoric acid (especially, useful for indole alkaloids):
Colouration
7. Sensitivity to heat and light (stability)
Most of them are susceptible to destruction by heat and most of them
undergo decomposition or degradation by exposure to air and/or light.
8. They are colourless, crystalline solids with a definite melting point or
decomposition range, and are non-volatile in nature.
Exceptions: Berberine: Yellow in colour.
Betanidine: Red in colour.
Nicotine and conine: Liquid and volatile in nature.
Some alkaloids are amorphous and gummy in nature.
9. Optical activity
They are usually laevorotatory in nature. A few exceptions are papaverine
which is optically inactive and conine which is dextrorotatory.
10. Pharmacological activity
 Most alkaloids exert some definite pharmacological action. In many cases,
a small quantity of alkaloid brings about a rather pronounced
pharmacological effect on various organs and tissues of animal and human
bodies.

Table 8.35 Test reagents used for alkaloids

8.21.6 Extraction
Method I

1. The powdered material is moistened with H2O, mixed with lime and treated
with ammonia solution.
2. The lime combines with acids, tannins and other phenolic substances and
sets free the alkaloids.
3. The liberated free alkaloidal bases are extracted with organic solvents such
as chloroform.
4. The above extract is concentrated; the concentrated organic liquid extract is
then shaken with aqueous acid and allowed to separate.
 5. Alkaloidal salts are now in the aqueous liquid phase while many impurities
remain behind in the organic liquid.
6. The aqueous liquid phase is collected and treated with diluted NH3; the
liberated free alkaloidal base is extracted with chloroform and finally
chloroform is evaporated to leave behind the pure alkaloid.

Method II

a. The powdered material is moistened with H2O or aqueous alcohol

containing dilute acid.
b. Add sufficient chloroform to the above moistened mass; shake well for
about 15 minutes. Filter and reject the filtrate. The process is repeated 2–3
times.
c. Add sufficient H2O to the residue, dissolve, and add excess sodium
bicarbonate or NH3, to liberate free alkaloidal bases.
d. Separate the free alkaloid either by filtration or by extraction with organic
solvents.

Method III: Isolation of alkaloids by overpressed thin layer

chromatography
Overpressured thin-layer chromatography (OPLC) is a planar chromatographic
method introduced by Tyihak et al. The technique makes use of a special
pressurised ultra microchamber, in which the sorbent layer is completely covered
with a flexible membrane under external pressure. Thus, the vapour phase above
the sorbent layer is virtually eliminated. A continuous development can be easily
performed by Chrompres 25 OPLC, the eluent can be pumped through the
sorbent layer and the eluates collected (J. Pothier et al., 1997).
The main purpose of OPLC is to obtain pure compounds from crude plant
extracts by coupling the chromatograph to a collector.
The advantages of this method are: efficiency, quickness, reproducibility and
small consumption of developing solvent. The disadvantages of the scratching
plates in classical TLC are also avoided by direct collection of eluting
compounds. Optimal experimental conditions are defined by preliminary
examinations of analytical OPLC conditions. The best results are obtained with
compounds having an Rf over 0.2, with a particular eluent system.
 Volatile liquid alkaloids like nicotine and conine can be isolated by the steam
distillation process.

8.21,7 Estimation of Weight of Total Alkaloids

PROCEDURE

1. Take an accurately weighed quantity of 10–20 gms of the drug, treated with
0.5 NH2SO4 and filter. Repeat the process till complete extraction of
alkaloids takes place.
2. Make the extract alkaline by adding excess of dilute NH4OH solution and
extract the alkaloid by shaking in a separating funnel, with a mixture of
chloroform–ether (66:34), till complete extraction of alkaloids takes place.
3. Pour out all the chloroform–ether fractions and shake with 20, 15, and 10
ml of 0.5 N H2SO4 for complete extraction.
4. Filter the combined acid extracts into a separating funnel, make alkaline to
litmus with dilute NH3 solution.
5. Extract the liberated alkaloids with successive portions of 20, 15 and 10 ml
of chloroform to bring complete extraction, and wash the combined organic
extract with 10 ml portions of H2O.
6. Again extract with two 10 ml portions of chloroform. Add the chloroform
washings to the main chloroform extract and filter the whole in a 100 ml
conical flask.
7. Evaporate chloroform on a water bath until a few ml are left and remove the
solvent completely by using a vaccum desicator.
8. Add 5 ml of alcohol (90%) to the residue and again evaporate the solvent.
Repeat the evaporation with alcohol, dry the residue to a constant weight in
the vaccum desiccator and weigh as total alkaloids.

8.21.8 Isolation of Solanine

Principle
Solanine is the major alkaloid of the potato plant. The sprout, berry and the
flower of the potato plant contain high concentrations of alkaloid (0.04% fresh
wt). Even the tubers contain a small concentration of alkaloids (0.001%). They
are extracted by macerating with 5% acetic acid and precipitating the alkaloid by
the drop-wise addition of 1 % NH4OH. The alkaloid is centrifuged, the
ppt.collected, purified and re-crystallised by dissolving in boiling methanol.

PROCEDURE

1. Extract the tissue by maceration with 5% acetic acid (15–20 parts) and filter
the extract to remove cellular debris. Warm at 70℃ and add conc. NH4OH
drop-wise till the pH is 10.
2. Centrifuge and discard the supernatant, wash the precipitate with 1%
NH4OH and re-centrifuge.
3. Collect, dry and weigh the crude solanine so obtained. This may be purified
by dissolving in boiling methanol (in which it is sparingly soluble); filter
and concentrate until the alklaloid starts crystallising out.
4. Check the purity of solanine by paper chromatography in ethylacetate–
pyridine, H2O (3:1:3) or TLC on silica gel G in acetic acid–ethanol (1:3).

Estimation

1. Dissolve a weighed amount of the crude alkaloid in 96% ethanol–20%

H2SO4 (1:1); so that the concentration of the alkaloid is between 0.2 and 3.0
mg/1.
2. The alkaloid solution (1 ml) is mixed with 5 ml 60% H2SO4and after 5
minutes, 5 ml of 0.5% solution of formaldehyde in 60% H2SO4 is added.
Allow to stand for 180 minutes and then measure the absorbance at 565–
570 nm.
 3. The actual amount of alkaloid in the crude isolate can then be determined
with reference to absorbance measurements of pure alkaloid solutions
treated in the same way with formaldehyde in 1 M H2SO4.

8.21.9 Isolation of Piperine

Principle
Powdered material is extracted with 95% ethanol, filtered and concentrated. 10
ml of alcoholic KOH is added and the solution is allowed to stand overnight.

PROCEDURE

1. Powdered black pepper (10 g) is extracted with 150 ml of 95% ethanol in a

Soxhlet apparatus for 2 hrs.
2. The extract is filtered and concentrated. Then, 10 ml of 10% alcoholic KOH
is added and the residue formed is discarded.
3. The solution is left overnight; yellow needles (m.p.: 125–126℃) of
piperine separates (yield: 0.3 gm). The purity of the product can be checked
by measuring its UV spectrum in ethanol ( λmax:245 nm).

Estimation
Method I: By HPLC
HPLC is precise, sensitive, reproducible and easy to perform. It has 0.1 μg
sensitivity and linearity in the range of 0.1–0.8 μg. The method can be used for
detection, monitoring and quantification of pipeline in different piper samples
(S.K. Chauhan et al., 1998).
PROCEDURE

1. Plant material: Piper nigrum fruits and roots of Piper longum are
purchased from a reliable source and authentified by the taxonomist.
 2. Extraction: About 50 gm of air-dried samples are ground to pass through a
20 mesh sieve. Batches of 10 gm of ground sample is weighed accurately
and extracted with alcohol (25 ml × 4 ml) separately over a steam water
bath. The respective extracts are filtered and volume made up to 100 ml
with alcohol. 1 ml of it is further diluted to 10 ml except for P. nigrum
where 1 ml extract is diluted to 25 ml with alcohol. The diluted test samples
can be used for HPLC analysis.
3. Standard preparation: A 0.2 mg/ml solution of piperine reference standard
is prepared in methanol. It is further diluted with methanol to yield the final
concentration of 5 μg/ ml, 20 μg/ml and 40 μg/ml. 20 μg from each of these
concentrations is injected into the HPLC column.
4. 20 μl each of different test solutions and the three different concentrations
of standard piperine are injected into an HPLC, using a Nova Pak Cl8
reversed phase column (3.9 ×150 mm), with a reversed phase guard
column. The mobile phase is methanol: water (75:25) and the flow rate is
0.6 ml/minute. The chromatogram is scanned up to 10 minutes and can be
detected at 340 nm. The amount of piperine in test samples can be
determined from the linear regression equation of the calibration graph
plotted between concentration and area.
5. Method validation and recovery: A known amount of piperine (around 10
mg) is added to about 1.0 g each of powdered test samples in which the
contents of piperine has been estimated previously by the proposed method.
The samples are extracted, diluted and analysed separately as per the
procedure mentioned above. The contents of piperine are quantified and
percentage recovery calculated.

Method II: By HPTLC

High performance thin layer chromatography is performed on silica gel 60 F254
HPTLC plates. The mobile phase is n-hexane: ethyl acetate (7:3 v/v). The
detection and quantification is performed at a wavelength of 340 nm. Linearity
of the detector response for piperine is 20–100 ng. The correlation coefficient
obtained for the linearity is 0.994. The standard error of the Y estimate is
133.2405 and standard error of the X coefficient is 0.67994.
The mean assay value of piperine is found to be 50.78 mg/gm of pepper. The
recovery value of piperine is 100.27%. Low values of standard deviation and %
coefficient of variation (COV) are indicative of the high precision of the method
(Dipal Kulkami et al., 2001).
PROCEDURE

1. Standard piperine solution: Accurately weigh pure piperine (10 mg) and
transfer into a 10 cm3 amber coloured volumetric flask. Cover with
aluminum foil since piperine in solution isomerises to isopiperine,
chavicine and isochavicine on exposure to light. Dissolve in methanol and
dilute to volume with methanol to give a 1 mg/cm3 solution (stock solution
A).
2. Sample preparation: Pepper is crushed and 1 gm of powder is extracted
using the Soxhlet apparatus in HPLC grade methanol for 6.0 hours. This
extract is filtered through a Whatmann filter paper no. 1 and the filtrate
collected in a 100 cm3 volumetric flask. The contents are diluted to volume
with methanol.
3. Chromatography: Chromatography is performed on aluminium backed
silica gel 60 F254 HPTLC plates (Merck). Before use, plates are pre-washed
with methanol. The mobile phase is n-hexane: ethyl acetate, (7:3 v/v).
Samples are applied to the plates as sharp bands by means of a Camag
Linomat IV sample applicator. After drying of the spots in a current of air,
the plate is placed in one trough of a Camag twin trough glass chamber. The
mobile phase (20 cm3) is poured into the chamber left to equilibrate for 10
minutes. The plate is then developed until the solvent front had travelled a
distance 7 cm above the position of sample application. The plate is
removed from the chamber and dried in a current of air. Detection and
quantification is performed with a Camag TLC scanner at a wavelength of
340 nm. Camag TLC software version 4.01 is used for detection and
evaluation of data.
4. Linearity of detector response: Linearity of detector response is performed
using a 10 μg/cm3 working solution, prepared from stock solution A. This
solution is applied to the HPTLC plate in the form of sharp 5 mm bands;
the distance between two adjacent bands must be 10 mm. The detector
response for piperine is measured for each band. The amount of piperine
(ng) is then plotted against the area under the curve. The same data can also
be subjected to statistical analysis using linear regression analysis and the
 correlation coefficient determined.
5. Assay: Standard and sample solution (5 μl) are spotted on an HPTLC plate.
The amount of piperine present per gram of pepper is calculated by
comparison of the areas measured for the sample with the calibration curve
constructed from the peak area obtained from a standard solution of
piperine (containing 20 ng to 100 ng) spotted with the sample.
6. Recovery study: To study the accuracy and precision of the method, a
recovery experiment can be performed by the method of standard addition.
The recovery of the added standard is studied at three different levels, each
being analysed in a manner similar to that described for the assay. Each set
of additions is repeated seven times and the recovery of the added standard
(%) is calculated using the formula:

where, X is the amount of standard added, Y is the amount of drug found by

the proposed method, N is the number of observations and E is the correlation
coefficient obtained for the linearity.

8.21.10 Tropane Alkaloids

Isolation
The powdered material is exhausted with absolute alcohol. The solvent is
removed by the distillation principle to get a syrupy residue. The residue is then
treated with 1% HCl. The acid solution is further purified by shaking with light
petroleum ether. Sufficient quantity of ammonia is added to make the solution
alkaline, then treated with an organic solvent like chloroform. The chloroform
solution is further shaken with dilute acid and the acid solution is made alkaline
with ammonia and again extracted with chloroform. The solvent is removed by
distillation and the residue comprises crude alkaloids. The crude alkaloidal
portion is neutralised with oxalic acid to get the oxalates of atropine and
hyoscyamine. Further, these may be separated by fractional crystallisation from
acetone and ether in which hyoscyamine oxalate is more soluble.
Hyoscine is also similarly isolated from the Datura species i.e., the finely
ground material is moistened with 10% sodium bicarbonate solution and
extracted with ether. After purification of the ether extract, hyoscine is
crystallised as hyosine hydrobromide from a mixture of ethanol and acetone.
For the isolation of cocaine from coca leaves, the powdered leaf drug is
treated with slaked lime to obtain a stiff paste. Then, the mass is subjected to
extraction with benzene at room temperature. The excess solvent is removed and
the residue is treated with 3% sulphuric acid. Then, sufficient quantity of
KMnO4 is added to saturate the solution at 5°C. The total solution is shaken with
dilute NH3 and cocaine is extracted by shaking with ether.

Identification
Vitali-Morin’s test

Rathenasinkam’s test
In this test, nitric acid is used to bring about the nitration of benzene ring in
atropine, hyoscyamine and hyosine. But, a mixture of HNO3 and H2SO4 is
required for the same reaction in case of cocaine.

To the residue, few drops of NH3 is added and extracted with CHC13. The
CHC13 is evaporated again and the residue is dissolved in acetone. A few drops
of 10% NaOH is added. A bluish-purple colour is formed.
Schaer’s test
Estimation of Belladonna alkaloids
The colorimetric method can be used for the quantitative estimation of total
alkaloids of Belladonna.
Method I: Colorimetric method
It is based on Vitali–Morin’s reaction and described by Allport et al.
PROCEDURE

1. 1 gm of accurately weighed powdered sample is taken in a 50 ml beaker.

About 1 ml of absolute alcohol and 0.1 ml of 10% w/w NH3 are added and
stirred well to impart uniform wetting.
2. The wetted mass is transferred to a miniature percolator and fitted with a
100 ml raduated measuring cylinder.
3. Chloroform is used as a percolating solvent. The percolation rate is
maintained at the rate of 1 drop per second. The percolation is continued
until the volume of the percolate is 30 ml. 6% acetic acid is added to the
percolate to make up the volume to about 80 ml. The contents are shaken
for 15–20 seconds, and then, allowed to stand for a thorough separation of
the two phases.
4. 5 ml of the upper layer is pipetted off, filtered through a Whatmann filter
paper. Exactly 1 ml of the filtrate is transferred to an evaporating dish, on a
water bath. The solution is evaporated to dryness and 0.2 ml of fuming
HNO3 is added immediately; the contents are mixed well for 3–4 minutes
and evaporated on a water bath. The alkaloidal residue is then dissolved in a
few ml of dry acetone.
5. The solution is transferred to a 10 ml volumetric flask; 0.1 ml of freshly
prepared 3% solution of KOH in methanol is added. The volume is made up
to 10 ml with dry acetone. The contents are mixed well and allowed to
stand for exactly 5 minutes. The colour intensity is then measured
 colorimetrically by using a green filter.
6. The concentration of the unknown solution is calculated with the help of a
calibration curve, which is prepared by using a known concentration of
lyosiamine sulphate.

Method II: By Spectrophotometry

A simple, rapid and sensitive spectrophotometric method can be used for the
determination of tropane alkaloids from plant extracts. Tropane alkaloids give a
yellow colour complex with bromophenol blue (BPB) in a buffer medium at pH
3.5. The absorbance is measured at 411 nm (S. Mandal et al., 1990).
PROCEDURE

1. Reagents: Bromophenol blue, 0.1 N NaOH, potassium hydrogen phthalate–

HCl buffer of pH 3.5. 10 mg of atropine free base dissolved in 100 ml of
methanol is used as standard.
2. The selection of proper pH for the formation and extraction of alkaloid–dye
colour complex is of prime importance.
3. Volumes of chloroform and BPB solution are varied keeping atropine at a
fixed concentration to get the maximum absorbance at 411 nm.
4. Different volumes of atropine free base solution are pipetted out one by one
in 10 ml volumetric flasks and distilled water used to make up the volume.
10 ml buffer, 5 ml dye solution and 10 ml standard are taken in a separating
funnel along with 20 ml CHCl3 The mixture is shaken for 2 minutes and
allowed to stand till the two phases are clearly separated. The chloroform
layer is separated out and its absorbance is measured at 411 nm against
similarly prepared reagent blank. The absorbance values are plotted against
the corresponding concentrations of a standard sample to give a calibration
curve. The linear is between 5μg/ml to 100μg/ml.
5. Assay of plant extract: Crude alkaloid extract is prepared from 100 gm
dried powder. The crude alkaloid is dissolved in 50 ml methanol and is used
as stock solution. From the stock solution, 0.25 ml is syringed out into a 10
ml volumetric flask and made up to 10 ml. The diluted solution is subjected
to further colour development as discussed earlier. The percentage of
tropane alkaloids as atropine, corresponding to the absorbance value is
found out from the calibration curve.
Estimation of hygrine in coca leaves
Method I: By GLC
PROCEDURE

1. Plant materials: Erythroxylum coca leaves are collected from the plants;
air-dried E. coca leaves are ground into a fine powder and stored in jars at
room temperature in a desiccator. The powdered leaf samples are extracted
within 2 hours after powdering to minimise the loss of volatile alkaloids.
2. GC analysis: The GC instrument equipped with a flame ionisation detector
and a HP 2934A integrator is used in the alkaloid analysis. A
dimethylsilicone capillary column (DB-5, 15 m × 0.25 mm (i.d.), 0.25 pm
film thickness) is used. Helium (99.95% purity) at approximately 60 cm/s
linear velocity is used as the carrier gas. The injection and detector
temperatures are 250 and 285℃, respectively. The temperature program
consists of an initial oven temperature of 70℃ followed by a ramp rate of
25℃/minute for 8 minutes and a hold at 280℃ for 1 minute. An external
standard is used for the quantitation of hygrine. The calibration curve for
hygrine is obtained by varying the concentrations (0.1–1.0 mg/ml) in
methanol.
3. Extraction: Powdered leaf tissue (~5 g) is combined with 100 ml of 95%
ethanol in a flask that is allowed to stand for 30 minutes. The extract is
stirred for 30 minutes at room temperature and allowed to stand for 40
minutes before passing it through a filter paper. The solvent is reduced to 5–
10 ml by rotary evaporation at 60℃ under vacuum (2–10 mm) to minimise
the loss of hygrine. The extract is re-dissolved in 75 ml of chloroform. The
flask is rinsed with 25 ml of an ethanol/water (3:1) mixture that is
combined with the extract in a separatory funnel. The extract is shaken with
a 75 ml volume of 1.5% citric acid in water (w/v) and then transferred to a
second separatory funnel. The extract is shaken again with a 25 ml volume
of citric acid. The larger citric acid volume is combined with the smaller
volume that is allowed to stand for about 15 minutes for phase separation.
After the chloroform layer is discarded, another 50 ml of chloroform is
combined with the citric acid layer. After the mixture is shaken and the
chloroform discarded, the aqueous fraction is poured into a beaker and
adjusted to pH 5.5 using NaCO3 powder. The aqueous fraction is shaken
 with two 40-ml volumes of chloroform to remove cocaine and other
interfering alkaloids. The aqueous layer is then adjusted to pH 8.8 with 10%
NH4OH in water. Hygrine is partitioned into chloroform by shaking the
aqueous layer with two 40 ml volumes of chloroform in a separatory
funnel. The chloroform extract is collected in a flask over anhydrous
Na2SO4. The extract is then transferred to a round-bottomed flask and is
subsequently reduced to a volume of 0.5–1.0 ml on a rotary evaporator. The
extract is diluted to 8 ml with methanol for GC analysis.
4. Fortified samples: Aliquots of a working standard solution (10.0 mg/ml) of
hygrine are added to 2.5–5.0 g of powdered E. coca leaf tissue to yield 10
and 20 mg of hygrine per sample. The alkaloid is extracted from fortified
samples using the same procedure as above. A minimum of two replicates
are made of each fortification level.

Method II: By HPLC

PROCEDURE

1. The procedure for extraction is the same as above.

2. Analysis: Detection is made with a model UV 2000 dual-wavelength
detector operated at 220 nm (0.05 AUFS). Hygrine is separated on a
Synchropak CM 100 weak cation exchange (WCX) column (10.0 cm × 4.6
mm; i.d.: 5 pm); the mobile phase consists of methanol/0.05 M KH2PO4,
pH 7.0 (75:25; v/v). The analytical column is used without a guard column.
The calibration curve is constructed by plotting the concentration of an
external standard (0.1–1.0 mg/ml) versus area counts of the corresponding
peaks.

8.21.11 Opium Alkaloids

Identification
Estimation
Method I: By Spectrophotometry
Moorhoff has described a method for estimating morphine in poppy capsules
using ferric chloride. It involves the following steps.
PROCEDURE
 1. A weak acid extract, containing 15–40 mg of morphine is made strongly
alkaline with sodium hydroxide solution.
2. Shake with 25 ml portions of benzene, thrice.
3. The benzene solutions are combined together and treated with 20 ml of 0.1
N NaOH solution.
4. Adjust pH to 9 by addition of NH4C1.
5. Then, extract with a mixture of chloroform—isopropanol (3:1). The
chloroform—isopropanol extract is filtered through anhydrous sodium
sulphate and made acidic with dilute sulphuric acid.
6. The acid extract is diluted to 100 ml and 15 ml of the extract is placed in
two flasks (1) and (2).
7. 15 ml of 0.1 N HCl is added to flask (1) and 17 ml to flask (2).
8. To flask (1), 2 ml of a 5% solution of iodic acid is added and after a period
of exactly 2 minutes, 17 ml of a 10% solution of ammonium carbonate is
added to both flasks.
9. Exactly 1 minute later, 1 ml of freshly prepared 0.3% solution of ferric
chloride is added to both flasks.
10. After 25–30 minutes, the colour intensity is measured at 510–520 mμ, by
using a spectrophotometer against a blank.

Method II: By HPLC (estimation of alkaloids in poppy straw)

A reversed-phase high-performance liquid chromatographic procedure can be
used for a quantitative analysis of the main alkaloids (morphine, codeine and
thebaine), together with oripavine, in poppy straw. The column consists of radial
compression cartridges packed with C18 as the stationary phase. The mobile
phase is a gradient of acetonitrile and an aqueous solution of octanesulfonic acid
sodium salt (M. P. Gomez-serramillos et al., 1995).
PROCEDURE

1. Column: RCM 25 × 10 radial compression model using a type 8 NV Radial

Pak cartridge (10 cm × 8 mm), containing C18 with a particle size of 4 pm
as the stationary phase.
 2. Standard alkaloid solution: Approximately 40 mg of oripavine and
thebaine, 70 mg of morphine and 150 mg of codeine are weighed precisely
and dissolved in 100 ml of 2.5% acetic acid solution (v/v). Injection
volume: 10 μl.
3. Extraction of the alkaloids from poppy straw: Poppy straw (1 g) is extracted
with 20 ml of 2.5% acetic acid (v/v), shaking mechanically for 30 minutes.
The mixture is then centrifuged and the supernatant decanted and filtered.
The extraction procedure is repeated thrice and each time the supernatant is
collected in a volumetric flask. The final volume is made up to 100 ml with
2.5% acetic acid (v/v).
4. Mobile phase: Solvent A: acetonitrile;
Solvent B: aqueous solution of 0.05 M sodium salt of octanesulfonic acid
(pH 3.5).
5. Elution gradient from 25 to 30% solvent A in 10 minutes; from 30 to 60%
solvent A in 7 minutes; return to initial conditions in 1 minute. Time of
equilibration between injections: 10 minutes. Flow rate: 1.5 ml/minutes.
6. Linear relationships are obtained by plotting the peak area against the
amount of each alkaloid injected (μg in 10 μl injection volume), with UV
detection at 248 nm, at least up to 20 μg of morphine, 5.0 μg of codeine, 4.0
μg of oripavine. The calibration ranges adequately cover the possible
weight per cent variations for these alkaloids in poppy straw samples.

8.21.12 Ipecacuanha Alkaloids

Isolation
The following method, for the isolation of ipecacuanha alkaloids, is described by
Paul and Cownley.
Identification

1. Solution of ipecauanha alkaloids + calcium hypochlorite an orange

colour.
2. Frohde’s test
 3. Emetine hydrochloride solution + Frohde’s reagent bright green
colour
4. Solution of psychotrine + conc. H2SO4 + conc. HNO3 cherry-red
colour
5. Psychotrine + Frohde’s reagent Pale-green colour

Estimation
By HPLC (estimation of emetine and cephaeline)
The emetine (EM) and cephaeline (CP), in ipecac syrup can be simultaneously
analysed quantitatively after direct dilution with distilled water, without any
other pre-treatment. The separation of EM and CP is accomplished by high
performance liquid chromatography (HPLC) with a reversed-phase column, and
peaks corresponding to EM and CP are examined by electron ionisation—mass
spectroscopy (EI—MS) (Daisuke Teghima et al., 1989).

PROCEDURE
1. Authentic samples: An authentic sample of emetine (EM) is purchased from
the Sigma Pharmaceuticals and cephaeline (CP) is isolated from ipecac
powder and purified. The purity of these alkaloids is confirmed by TLC.
2. Materials: Collect the ipecac syrup from a commercial source.
3. Preparation of samples: Two hundred microliters of ipecac syrup is diluted
to 4.2 ml with distilled water, and 200 μl of chloroquine (6 mg/ml) is added
as an internal standard. In order to examine the plant source of ipecac
syrup, two kinds of ipecac roots are powdered, and extracted with EtOH-
H2O (3:1), according to the method described in the USP. The diluted and
extracted samples are directly subjected to HPLC analysis.
4. HPLC: A Shimadzu liquid chromatography equipped with a variable-
wavelength Shimadzu ultraviolet (UV) spectrophotometer can be used. The
chromatogram of ipecac syrup is shown in Fig. 8.26.
Column: Pre-packed TSK gel ODS-80TM (5pm, 15 cm × 4.6 mm).
Injection volume: 20μl.
Mobile phase andflow rate: 10 mM sodium 1-heptanesulfonate solution
adjusted to pH 4 with glacial acetic acid—methanol (46:54) at 1 ml/minute.
Peaks are monitored at 285 nm. In the case of semi-preparative HPLC for
 EI-MS, a volume of 20 pi is injected (10 times). The combined eluates
corresponding to EM and CP each are concentrated and the residues of the
EM and CP fractions are extracted with ether and with chloroform,
respectively. Each extract is concentrated to dryness and the residues are
subjected to EI–MS. The calibration curves for EM and CP are shown in
Fig. 8.27.
5. EI–MS: Measurement conditions are ionising voltage: 30 eV; ionising
current: 100 A for EM and 300 A for CP; accelerating voltage: 3 kV; ion
multiplier: 1 kV; sample temperature: 300℃; chamber temperature: 200℃;
scan speed: 5 s. The EI–MS for emetine and cephaeline are shown in Fig.
8.28.
6. Constant weights of EM and CP are added to samples, and the recovery
rates are determined. This method gives good recovery: 99.5% and 105.2%
for EM and CP, respectively.

Fig. 8.26 HPLC chromatogram of ipecac syrup

Fig. 8.27 Calibration curves for emetine and cephaeline

Fig. 8.28a EI–MS for emetine

Fig. 8.28b EI–MS for cephaeline

8.21.13 Isolation of Cinchona Alkaloids

Isolation of cupreine
Cupreine is isolated from the bark of Remijia pedunculata. The extraction
procedure is similar to that of quinine.
PROCEDURE

1. The powdered bark is mixed thoroughly with Ca(OH)2 solution; a paste is

made with sufficient 5% NaOH solution, and extracted exhaustively with
CHC13 or benzene.
2. The organic extract is acidified with dil. H2S04 and neutralised exactly with
dilute NaOH. It is allowed to stand overnight so that the sulphate of quinine
and cupreine crystallises out.
3. The crystals of the compounds are collected and dissolved in dilute
sulphuric acid; it is made alkaline by adding excess sodium hydroxide
 solution and quinine is separated out by shaking with solvent ether.
4. The alkaline solution, which contains cupreine, is neutralised with dilute
H2S04. The crystals of cupreine sulphate separate out. They are further
subjected to purification by making them alkaline with NH3 and
crystallising the precipitate with ethanol.

Identification of cinchona alkaloids

1. Dilute acidic solution of quinine Strong blue fluorescence

2. Pthalleioquin test:

3. Erythroquinine test
Dilute acidic solution of quinine + few drops of bromine H20 + a drop of
10% solution of potassium ferrocyanide + a drop of strong NH3 solution →
Red colouration → shake with 1 ml of chloroform → the colour is taken up
by the chloroform layer.
4. Tests for distinguishing between quinine and quinindine

(b) The following solubility test can be used to distinguish quinine from
quinindine.
 5. Picric acid test
It is described by Summerford, to distinguish four cinchona alkaloids. 1 ml
of 1% solution of the respective alkaloid in glacial acetic acid + 1 ml
solition of picric acid in glacial acetic acid (1 in 70) → allowed to stand for
5 minutes. The following observations were noted:
a. A precipitate is formed with cinchonidine.
b. Upon scratching the sides of the tube with the remaining solution,
cinchonine picrate begins to crystallise and after a period of one hour,
quinine picrate is precipitated.
c. Quinidine picrate does not crystallise.
6. Tests to distinguish between cinchonine and cinchonidine

Estimation of cinchona alkaloids

Method I: by spectrophotometric method
This method is described by Loustalot and Pagan, for the determination of
quinine obtained from cinchona bark. It involves the following steps.
PROCEDURE
 1. 2 gm of finely powdered bark is weighed in a 100 ml beaker and mixed
with 0.5 gm of finely powdered CaO.
2. About 7–10 ml of water is added to make a smooth homogeneous paste,
and allowed to stand for 10 minutes.
3. The total pasty mass is transferred to a 200 ml volumetric flask with 150 ml
of ethanol; the suspension is shaken vigorously and allowed to stand for one
hour, with occasional shaking. The volume is made up with ethanol and
filtered through a Whatmann no. 5 paper. Care must be taken to avoid the
loss of ethanol during filtration.
4. 25 ml of ethanolic extract is pipetted into a 50 ml Erlenmeyer flask and 25
mg of Norite A is added.
5. The flask is shaken for 30 seconds and the solution filtered.
6. 8 ml of the clarified extract (representing 80 mg of bark powder) is pipetted
out into a 100 ml volumetric flask, 5 ml of 0.1 N HC1 is added and the
solution is made up to volume. Then, the percentage transmittance is
determined at 380 nm and the concentration is calculated using the standard
curve.
7. Standard curve: Prepare 5 ml aliquot of solutions containing 1.6, 3.2, 4.8,
6.4 and 8 mg of anhydrous quinine sulphate in 0.1N HC1. This series is
equivalent to 2, 4, 6, 8 and 10% respectively cf quinine sulphate. 5 ml of
each stock solution is transferred to 100 ml volumetric flasks; 8 ml of 95%
of ethanol is added to each flask and the volume is made up to the mark
with water. The percentage transmittance at 380 nm is determined and a
standard curve of percentage transmittance against concentration is plotted.
8. Blank solution: A blank solution containing the acid and ethanol is used as a
reference.

Method II: Spectrophometric estimation of total alkaloids (from cinchona

stem bark and marketed formulations containing cinchona)
The alkaloids of the stem bark of Cinchona officinalis and the marketed
formulations containing cinchona can be estimated by the spectrophotometric
method using tropaeolin ‘OO’ for the formation of the colour complex. The
method is based on the formation of a charge transfer complex between alkaloids
and tropaeolin ‘OO’ at pH 4.6 which can be extracted either in chloroform or
dichloromethane, followed by its reaction with acid reagent, to give a violet
coloured chromogen with λmax of 545 nm. Various parameters are involved in
colour development such as the effect of pH, the amount of acetate buffer, the
amount of tropaeolin ‘OO’ solution, the amount of acid reagent and the time
involved at various stages of reaction. Maximum colour intensity is obtained
with 3 ml of saturated solution of tropaeolin ‘OO’, 5 ml of acetate buffer and 3
ml of acid reagent. The violet coloured chromogen is stable up to 3 hrs. In
cinchona, alkaloids are present in combination with quinic acid and cinchotannic
acid. Tannins interfere during the extraction of alkaloids by the conventional
acid–base extraction method. Hence, during the extraction of alkaloids from the
stem bark and formulations, carboxy methyl cellulose (sodium salt) can be used
to adsorb the interfering substances. This facilitates the efficient extraction of
alkaloids in organic solvents (M. Rajani et al., 2001).
PROCEDURE

1. Chemicals and reagents: Acetate buffer of pH 4.6 (5.4 gm of sodium

acetate and 2.4 gm of glacial acetic acid in 100 ml of double-distilled
water), saturated solution of tropaeolin ‘OO’ and acid reagent (1 ml of
concentrated sulphuric acid + 99 ml of methanol).
2. Plant materials: Cinchona officinalis stem bark is purchased from a reliable
source and authenticated by a taxonomist. Homeopathic mother tincture
(China Q) and herbal tablet formulation (cinchona tablet) is purchased from
the market.
3. Linearity curve preparation: For the linearity curve preparation, a standard
solution of quinine (100 mg/ml) is prepared in methanol, aliquots of 1–3.5
ml are taken and colourimetric analysis is carried out according to the
following method. The solvent is removed at room temperature from the
above aliquots and 5 ml of acetate buffer and 3 ml of tropaeolin ‘00’
solution are added. The complex thus formed is extracted with chloroform
(3 ✕ 15 ml). The chloroform extract is transferred to a 50 ml volumetric
flask containing 3 ml of acid reagent and the volume is made up with
chloroform. The violet chromogen developed is measured at 545 nm
against a blank, using a double beam UV– VIS spectrophotometer. The
linearity curve of concentration vs absorbance is plotted.
4. Extraction of alkaloids: For the extraction of alkaloids from the samples (1
 g each of stem bark powder/1 ml of each homeopathic mother tinctures/1 g
tablet powder (from 20 tablets)), the method given in the European
Pharmacopoeia can be adopted, (20% sodium hydroxide solution can be
replaced with 25% ammonia solution and tragacanth can be replaced with
carboxy methyl cellulose (sodium salt)). A suitable quantity of sample
solutions is taken and the colour developed as described above. The
absorbance of coloured solution is recorded at 545 nm. The amount of total
alkaloids from the sample solution is calculated from the calibration curve
and represented as quinine.

Method III: By HPTLC

The following HPTLC method can be used for the estimation of alkaloids of
Cinchona officinalis stem bark and its marketed formulations (M. Rajani et al.,
2001).
PROCEDURE

1. Standard solutions of Qn (40–120 μg/ml), Qnd (1–5 μg/ml), Cn and Cnd

(5–25 μg/ml each) are prepared by suitably diluting the stock solutions of
Qn (400 μg/ml), Qnd (5 μg/ ml), Cn and Cnd (50 μg/ml) in methanol. 10 μl
each of Qn, Qnd, Cn and Cnd standard solutions are applied on pre-coated
HPTLC silica gel G 60F254 plates (E. Merck) using an automatic sample
spotter. The plates are developed with chloroform/diethylamine, (9.6:1.4
v/v) to a distance of 5 cm in a twin-trough chamber, previously saturated
with a solvent system for 30 minutes, dried in air for 15 minutes and
scanned and quantified at 226 nm. Data of peak area of each band is
recorded. 10 μi each of standard solutions of Qnd are applied on pre-coated
HPTLC silica gel G 60 plate (E. Merck) and developed as described above.
The plate is dried in air for 15 minutes, dipped for 3 seconds in 10% tartaric
acid solution (concentration optimised), and dried in air for 30 minutes. The
plate is scanned at 366 nm in the fluorescence/reflectance mode (with cutoff
filter K 400). Data of peak area is recorded.
2. For the extraction of the samples (1 g each of stem bark and tablet powder/1
ml each of homeopathic mother tinctures, n = 3), the method given in the
European Pharmacopoeia (1997) can be adopted (20% sodium hydroxide
solution can be replaced with 25% ammonia solution and tragacanth can be
replaced with carboxy methyl cellulose (sodium salt)). 10 μl of suitably
 diluted sample solutions are applied on pre-coated HPTLC silica gel G 60
F254 (silica gel G 60 for Qnd) plates. Peak area of Qn, Qnd, Cn and Cnd are
recorded. Amounts of Qn, Qnd, Cn and Cnd are calculated from peak area
using their respective calibration curves.

8.21.14 Isolation of Colchicine Principle

Cochicine is the chief constituent, obtained from the dried corm and seeds of
Colchicum autumnale (fam. Liliaceae).

Colchicine is soluble in water, aqueous ethanol, and in chloroform. It is less

soluble in absolute alcohol than in aqueous ethanol. It is practically insoluble in
solvent ether and light petroleum ether. Scheme 8.2 shows the procedure to be
followed to isolate Colchicine.

Uses
1. Used to relieve gout
2. Used to induce polyploidy

Identification

1. Colchicine solution + few drops of mineral acid Intense yellow

colour
2.

1.
2. Ethanoric solution of Colchicine + FeCl3 Garnet red colour

3.

4.
Scheme 8.2
4. Colchicine solution + Bromine H2O ppt

Estimation of colchicine
Method I
The following colorimetric method can be used for the estimation of colchicine
Colchicine, when treated with FeCl3 solution forms a green colour, which shows
maximum absorption at 470 nm. Satisfactory results can be obtained only if the
colchicine is present in a reasonably pure state. The method is suitable for the
estimation of amounts of colchicine ranging from 0.05–0.5 mg.
PROCEDURE

1. 5 gm of colchicine seed or corm is finely powdered and extracted

exhaustively with 100 ml of 70% alcohol.
2. The solvent is removed by evaporation on a water bath. The residue which
contains resinous matter must be removed by treating with 10 ml of H20
and little paraffin wax (the procedure is as explained above).
3. The resin-free aqueous extract is then shaken with light petroleum ether to
remove colouring matter and oil.
4. The aqueous solution is saturated with sodium chloride and extracted with
chloroform, three times. The chloroform fractions are combined together,
and dried with anhydrous sodium sulphate.
5. The chloroform extract is subjected to column chromatography as
explained above i.e., the alkaloid is eluted with 100 ml of chloroform, ,
which is removed by evaporation, leaving a yellow gummy residue. This
residue is taken up in a known volume of water and used for colorimetric
estimation.
6. Standard solution: A solution containing 2.5 mg of pure cochicine is
dissolved in 25 ml of 1 N HC1, in a 125 ml Erlenmeyer flask. The solution
is warmed on a water bath for an hour. It is then cooled to room temperature
and the volume made exactly 25 ml with 1 N HC1. Aliquotes containing
0.1–0.5 mg of colchicine are taken, the volume made up to 5 ml with 1 N
HC1 and treated with 0.1 ml of 5% ferric chloride solution. The absorbance
 is measured at 470 nm and the calibration curve prepared.
7. The aqueous solution of colchicine, collected after column chromatographic
purification, is treated as above and the concentration of colchicine
determined from the calibration curve.

Method II: By HPLC

The separation and determination of colchicine and its degradation products
formed in the presence of ultraviolet light can be carried out by HPLC. Reverse-
phase HPLC assay shows , good reproducibility, sensitivity and selectivity. It has
the advantage of being a relatively / simple, convenient and rapid method. This
method can provide an indication of colchicine concentration at ng level and in
as little as 10 minutes in biological fluids (Turhan Baykal et al., 1992).
PROCEDURE

1. Instrumental condition: The instrumentation consists of a Waters 6000 A

pump, a U6K universal injector, a M450 variable-wavelength UV detector
set at 245 mm, 0.2 aufs and a M730 data module. HPLC separations are
performed on a μ Bondapak C-18 column (3.9 mm x 300 mm). The mobile
phase used for the separation is MeOH: H20 (60:40) and the flow rate is
adjusted to 1.00 ml/ minutes.
2. Chemicals and solvents: Colchicine is purchased from Merck. HPLC grade
methanol and double-distilled water are used.
3. Sample preparation and HPLC determination: 8.5 mg of colchicine is
exactly weighed into a 100 ml volumetric flask and dissolved in MeOH. A
volume of 10 μl is injected for all samples. Colchicine is determined by
using a calibration graph. This is constructed by plotting the peak areas of
colchicine against the weights of colchicine.

8.21.15 Isolation of Protoveratrine

Scheme 8.3 shows the method used by Craig and Jacobs for the isolation of
protoveratrine from Veratrum species. The base is insoluble in water; sparingly
soluble in chloroform and hot ethanol, poorly soluble in solvent ether; and
soluble in benzene and light petroleum ether. Protoveratrine hydrochloride melts
at 234–236℃.
Isolation of Jervine
It is almost insoluble in water and light petroleum ether; soluble in ethanol,
ether, chloroform and benzene. It melts at 244–246℃.
PROCEDURE

1. The procedure to get a resinous residue, which contains a crude alkaloidal

mixture, is as in Scheme 8.3.
2. The residue is then treated with a mixture of 300 ml of 5% acetic acid and
40 ml of a saturated solution of ammonium sulphate. The precipitate is re-
suspended in fresh wash solution and centrifuged several times. The
sulphate is re-suspended in water and treated with excess of NaOH solution.
The mixture is shaken thoroughly with chloroform to re​dissolve the
liberated bases.
3. It is subjected to centrifugation, washed with H20, treated with sodium
sulphate, and concentrated to a small volume.
4. Sufficient absolute ethanol is added and all the residual chloroform is boiled
off.
5. To the above mixture, slightly excess of hydrochloric acid is added. When
allowed to cool, crystals of jervine hydrochloride are deposited.
6. Subjected to purification by dissolving in hot, diluted alcohol and
decomposed with NH3 solution, pure needles of alkaloidal base gets
separated out.

Identification

1. Sample of protoveratrine + Concentrated HC1 cherry-red colour.

2. Sample of jervine + Concentrated H2S04 yellow-bright green.
Scheme 8.3
8.21.16 Lobelia Alkaloids
Lobelia alkaloids are present in the dried aerial parts of Lobelia nicotianafolia,
belonging to the family lobeliaceae. It contains piperidine type of alkaloids
namely lobeline, isolobinine, lobelanine, norlobelanine, lobelanidine, etc.
Isolation of total alkaloids of lobelia

1. 10 gm of the finely powdered material is mixed with 10 gm of ignited sand

and added to 75 ml of a mixture of 4 volumes of ether and 1 volume of 95%
ethanol in a closed container. It is allowed to stand for 15 minutes with
shaking.
2. 5 ml of 10% ammonia solution is added; the container is shaken in a
mechanical shaker for one hour.
3. The total contents of the container are taken in a percolator, and allowed to
percolate. The percolation is continued with sufficient ether–ethanol
mixture, until the alkaloids are completely extracted.
4. The total percolate is treated with 30 ml of IN H2S04 in a separating funnel;
it is shaken well, and the phases are allowed to separate.
5. The lower layer is run off and the extraction is repeated with further
quantities of 20 ml each of the acid–ethanol mixture (IN H2S04and absolute
ethanol) until the alkaloids are completely extracted.
6. The mixed acid solutions are washed with different 15 ml portions of
chloroform; the chloroform solutions are rejected.
7. Sufficient ammonia solution is added until the acid solution is neutralised to
litmus.
8. The alkaloids are now extracted by shaking with successive quantities of 10
ml of CHC13. The chloroform solutions are combined, washed with 3 ml of
distilled water and the aqueous portion rejected.
9. Chloroform is removed by heating on a water bath; 5 ml of ethanol is added
to the residue and evaporation is continued. The process is repeated 2–3-
times. The residue comprises the total alkaloids of the lobelia.
Estimation
By colorimetry
A simple method is described for the selective estimation of lobeline in lobelia
herb and its liquid galenicals. The suggested method enables the determination
of the total lobelia alkaloids in the same run. Estimation of the total alkaloids is
carried out through complexation of the alkaloids with methyl orange at pH 5.
After extraction of the alkaloids- complex with chloroform and treatment with
0.1 N HC1, the dye released is measured colorimetrically at 510 nm. Due to the
selective solubility of lobeline hydrochloride in chloroform, the chloroform
phase is subjected to a second treatment with methyl orange solution to estimate
lobeline. The method is applied for the assay of lobelia powder, its tincture and
lobeline hydrochloride injections (Z. F. Mahamoud and S. EI-Masry, 1980).
PROCEDURE
Chemicals and reagents used

a. Lobeline HC1 reference solution: An aqueous solution (20 mg %) of

lobeline HC1 is used.
b. McIlvaine’s buffer solution (pH 5)
c. Dye solution: Solution (10 mg %) of methyl orange in a buffer of pH 5.0 is
used.
d. 5% sodium chloride in 0.1N HC1: The solution is prepared by dissolving 5
g of sodium chloride (analytical reagent grade) in 100 ml of 0.1 N HC1.
e. Lobelia powder and lobelia tincture, ethereal: Samples conforming to the
BPC are used.
f. Lobeline HC1 injections.
g. Acid methanol solution: The solution is prepared by mixing 50 ml of
methanol and 50 ml of 0.1 NHC1.

1. Assay of lobelia powder

One gram of fine powder is mixed with 20 ml of acid–methanol solution in a 50
ml glass toppered conical flask. After mechanical shaking at 37℃ for 30
minutes and cooling, the contents are centrifuged. The clear supernatant is
subjected to the following: (A) Determination of the total alkaloids: An aliquot
of the clear supernatant (2 ml) is transferred to a 50 ml separating funnel and 1.0
ml of 0.1 N NaOH is added for neutralisation. This is followed by 10 ml of the
dye solution and 10 ml of chloroform. After shaking for 1 minute, the contents
are left for complete separation. The chloroform layer is separated and extraction
of the aqueous phase is repeated using 2 x 10 ml chloroform. The combined
chloroform obtained after each extraction is shaken with 0.1 N HC1 (10 + 5 ml)
containing 5% w/v sodium chloride for 30 seconds. The combined acid solution
is transferred to a 50 ml volumetric flask and the volume completed with 0.1 N
HC1. The absorbance of the released dye is measured at 510 nm and compared
with a standard lobeline HC1 solution and is concomittantly assayed.
(B) Determination of lobeline content: The chloroform phase left in (A) and
containing lobeline hydrochloride is shaken with 10 ml of the dye solution. The
assay procedure is completed as before. After treatment of the combined
chloroform extracts with 10 + 5 ml of 0. 1 N HC1, the combined acid solution is
completed to volume in a 50 ml volumetric flask. The absorbance due to the
released dye is measured at 510 nm.
2. Assay of lobelia tincture, ethereal B. P. C.
An aliquot of the tincture (0.5 ml) is transferred to a 50 ml separating funnel
containing 10 ml of the dye solution and 10 ml chloroform. The assay is
completed as under lobelia powder.
3. Assay of lobeline injections
After dilution with water, an aliquot containing about 0.2 mg lobeline HC1 is
assayed as previously mentioned.
Use
Used in spasmodic asthma, chronic bronchitis, and for resuscitation.
8.21.17 Ergot Alkaloids
Ergot is the dried sclerotium of the fungus, Claviceps purpurea (family:
hypocreaceae), developed on rye plant, Secale cereale. It contains not less than
0.19% of the total alkaloids of ergot, calculated as ergotoxine, of which not less
than 15% consist of water-soluble alkaloids of ergot, calculated as ergometrine.
Basically, it contains indole type of alkaloids. Different alkaloids of ergot are
as follows:
1. Water-soluble alkaloids'.
Ergometrine group: Ergometrine and ergometrinine

2. Water-insoluble alkaloids:
Ergotamine group : Ergotamine, ergotaminine, ergosine and ergosinine
Ergotoxine group: Ergocristine, ergocristinine, ergocryptine, ergocryptinine,
ergocornine and ergocorninine

wherein:
Ergosine: R1 = R2 = H; R3 = CH2 CH (CH3)2
Ergocornine: R1 = R2= CH3, R3 = CH2 CH (CH3)2
Ergocristine: R1 = R2= CH3, R3= CH2 C6H5
Ergocryptine: R1 = R2 = CH3; R3= CH2 CH (CH3)2
Ergot alkaloids are more or less soluble in organic solvents, with the exception
of light petroleum. Ergots are practically insoluble in light petroleum. The
solubility in ether is much less than in benzene, chloroform, acetone or ethanol.
Isolation
The following method is used by the Barger and Carr, for the isolation of
different alkaloids of ergot.
NOTE: Separation of ergocryptine and ergocornine: Ergotoxine is subjected to
fractional crystallisation with di-(p-toluyl)-L-tartaric acid in MeOH. The salt of
ergocryptine crystallises out. Then, after the mother liquor is diluted with H20,
the salt of ergocornine crystallises out.
Isolation of ergosine

Isolation of ergotomine
Uses

1. Used as an oxytocic.
2. The drug has anti-epinephrine activity.
3. Used in the treatment of persistent migraine.

Identification

1. Van urk’s test

Solution of alkaloid + few drops of 1% solution of p-dimethylamino-
benzadelhyde solution in a mixture of equal parts of ethanol and HC1 + 0.1
ml of 5% FeCl3 + 65% v/v H2S04 (few drops) → Purple or blue colour
2. Keller’s test
Solution of alkaloid in glacial acetic acid + FeCl3 (anhydrous) + Conc.
H2S04 → An intense blue colour produced at the boundary of the two
layers.
3. Ergot alkaloids + Glyoxylic acid + Conc. H2S04 → Blue colour.
4. Identification by paper chromatography
The amino acids produced by the acid hydrolysis of water insoluble
alkaloids can be used for the identification of the members of the respective
 alkaloid.
5. Acid hydrolysis
Acid hydrolysis is done by heating the alkaloid with conc. HC1 in a sealed
tube at 100℃ for 16 hrs, evaporating the solution to dryness on a steam
bath, dissolving the residue in water and applying the aqueous solution to
paper.

The particular alkaloids of ergot, and the corresponding amino acid obtained
after hydrolysis, are as follows:
Upon acid hydrolysis

Ergocornine L-valine

Ergocristine L-phenylalanine

Ergocryptine L-leucine

Ergosine L-leucine

Ergotamine L-phenylalanine

Estimation
By colorimetry
The following method is described by Smith, for the quantitative estimation of
the total alkaloids of ergot (both water soluble and water insoluble).
PROCEDURE

1. Finely ground ergot powder is de-fatted by extraction with light petroleum

ether and allowed to dry in the air.
2. 15 gm of the de-fatted powder is placed in a 250 ml flask; add 147 ml of
acetone and 3 ml of 10% NH3 solution. Stopper the flask and shake on a
mechanical shaker for one hour. The solution is filtered.
3. About 100 ml of the filtrate (representing 10 gm of the powdered drug) is
transferred to an evaporating dish. The solution is concentrated to 20 ml
and transferred to a 125 ml separating funnel.
 4. About three volumes of pure ether is added and the solution made acidic
with 0.2 ml of 20% tartaric acid. The acetone–ether solution is extracted
with four 10 ml portions of 1% tartaric acid solution, each portion being
drawn off into a small round-bottomed flask. Traces of ether andacetone are
removed by evaporation at 40℃.
5. The solution is transferred to a 50 ml volumetric flask and washed with
small volumes of 1% tartaric acid solution to make up the volume. 5 ml of
the aliquot is removed (representing 1 gm of ergot), diluted to 50 ml and
used for the colorimetric estimation as follows:
4 ml of the ergot solution is mixed with 8 ml of the Van urk’s reagent (0.125
gm of p-dimethylamino benzaldehyde is added to 0.1 ml of 5% solution of
ferric chloride + 65% v/v of H2S04 to make 100 ml) in a 25 ml volumetric
flask. The mixture is allowed to stand for 3 minutes and absorbance is read
at 550 nm. A standard curve is prepared by using pure ergotoxine ethane
sulphate.

Isolation of water-soluble ergot alkaloids

1. 5 gm of the ergot powder is de-fatted with light petroleum, allowed to dry

in air, and mixed thoroughly with 0.3 gm of NaHCO3
2. The mass is made damp, by adding water drop by drop, with stirring.
3. The mixture is transferred to a percolator and extracted with peroxide free
ether containing 5% ethanol. Extraction of the alkaloids is carried out best
by drawing off 10 ml of the percolate at intervals of one hour until 70 ml is
collected.
4. The powder is left in contact with the solvent overnight, and percolation is
continued as above until another 100 ml percolate is collected. The
percolation is once more stopped and the powder is allowed to remain in
contact with the solvent overnight. The extraction is completed by drawing
off portions of percolate at 30 minute intervals until the last percolate shows
negative reaction for alkaloidal tests.
5. The ethereal extract is transferred to a separating funnel and the alkaloids
removed by shaking with six 10 ml portions of 5% lactic acid.
6. The combined extracts are collected and dried in a vaccum drier.
 The purity of the sample can be checked by paper chromatography in
butanol:acetic acid: H2O(4:1:5). The ergometrine and ergometrinine gets
separated as fluorescent spots at Rf 0.59 and 0.68 respectively.

8.21.18 Nux-vomica Alkaloids

Nux-vomica consists of the dried, ripe seeds of Strychnos nux-vomica, belonging
to the family loganiaceae. It contains indole type of alkaloids, namely
strychinine (not less than 1.2 %) and brucine.

Strychinine is practically insoluble in solvent ether and is very little slightly in

acetone and light petroleum. The base dissolves readily in chloroform (1 in 6)
and in a mixture of equal parts of ether and chloroform. It is soluble in benzene
(1 in 160) and in amyl alcohol (1 in 180). The base melts at 286–288℃.
Brucine is easily soluble in ethanol, chloroform and acetone. It is very slightly
soluble in solvent ether. It is practically insoluble in dilute solutions of NaOH
and KOH and is sparingly soluble in ammonia solutions. It melts at 178℃.
Isolation
Identification

1. Extract of the drug + 2 drops of sulphovanadic acid → purple-red colour

(presence of stryhinine).
2.

3. Sample of solution of the drug + 2 drops of Blood-red

colouration
4. Mandelin's test (for strychinine)
Sample solution + Mandelin’s reagent (H2S04 + ammonium vanadate) →
Violet-blue colour → cherry-red → orange colour.
 5. Malaquin’s test
Malaquin described the following test for strychnine. It is very sensitive and
can detect strychnine in a dilution of 1 in 100 ,000.

Method I: Estimation by colorimetry (strychinine)

The colorimetric method was used by Allen and Allport for the estimation of
strychinine in Nux-vomica seed. It is based on the colour formation with
Malaquin’s reagent.
PROCEDURE

1. 1 gm of finely powdered seed is accurately weighed, and placed in a 100 ml

conical flask; 3 ml of absolute alcohol is added and the mixture is heated on
a boiling water bath, until most of the alcohol has evaporated.
2. 10 ml of tetrachloroethylene and 0.6 gm of piperazine are added; it is
subjected to reflux condesation for 15 minutes. The contents are transferred
to a small percolator packed well; the percolation is continued at a speed of
1 drop/second, until the extraction is completed (solvent which is heated
extra is used for the percolation).
3. Sufficient 0.1 N H2SO4 is added to bring the volume of the percolate to 90
ml. The liquids are allowed to separate in a separating funnel.
4. After the immiscible liquids have separated, about 10 ml of the upper layer
is pipetted out and filtered through a dry filter paper.
5. Exactly 5 ml of the filtrate is transferred to a conical centrifuge tube of 15
ml capacity. 1.5 ml of 9% w/v aqueous solution of oxalic acid and 1 ml of
5% w/v solution of potassium ferrocyanide in 0.2% w/v aqueous solution of
sodium carbonate are added. The tube is allowed to stand at room
temperature for 15 minutes and is then immersed in a freezing mixture. The
tube is agitated until the contents are frozen, removed and gently warmed,
until melting takes place.
 6. Approximately 0.1 gm of acid washed Keiselguhr is added and the
precipitate is centrifuged down. The clear supernatent is poured off and
about 2 ml of a 0.1 % w/v aqueous solution of H2SO4 containing 1% of
oxalic acid is added.
7. The precipitate is washed by stirring with a glass rod. The washing is
repeated once more and the clear supernatant decanted, as completely as
possible.
8. 1 ml of conc. HC1 is added to the precipitate, the mixture is heated over a
flame and 10 ml of 10% w/v of HC1 is added. The mixture is transferred to
a 100 ml volumetric flask.The centrifuge tube is rinsed well with dilute
HC1 and the volumetric flask filled to the mark with the washings.
9. A portion of this solution is filtered and 5 ml of the filtrate is transferred to
a test tube containing 0.2 gm of zinc amalgam. The tube is immersed in a
boiling water bath for 7 minutes and cooled under a tap. 0.05 ml of freshly
prepared solution (0.1%) of sodium nitrite is added. The absorbance is
measured at 440 nm.
10. The calibration curve is prepared by using solutions of pure strychnine
ferrocyanide.

Method II: Estimation by colorimetry (brucine)

1. 0.1 gm of the alkaloidal sample (containing both brucine and strychinine) is

dissolved in 20 ml of 1% H2SO4with gentle heating.
2. The solution is transferred to a 100 ml graduated cylinder, rinsed and made
up to 30 ml with 1% H2SO4
3. If the alkaloids are present as salts, they must be converted to free bases by
treating with alkali and extracting with chloroform. After evaporation of the
chloroform, 0.1 gm of the dry residue is treated as above.
4. Different aliquots of standard brucine solutions are prepared from the stock
solution.
5. 10 ml of the mixture (1:1) of nitric acid and 20% H2SO4 is added to the
above test and standard samples. The solution is stirred and after one
minute, 2 ml of a saturated aqueous solution of potassium chlorate is added
 to the sample and standard.
6. The solution is well stirred and, if necessary, diluted to 50 or 100 ml,
according to colour intensity.
7. The absorbance is measured in a suitable absorption meter and the amount
of brucine is calculated from a calibration curve. cl

Method III: By HPLC

A reversed-phase high-performance liquid chromatographic procedure can be
used for the quantitative analysis of strychinine and brucine in S. nux-vomica
and S. ignatii (R. Gadi Biala and et al., 1996).
PROCEDURE

1. Chromatograph: LKB gradient pump 2249 equipped with a diode-array

detector, Hewlett-Packard model 1040 M, series 2, operating at 260 nm.
2. Column: Lichrospher 60 RP8 Select B Merck (250 ✕ 4 mm; with a particle
size of 5 pm) as the stationary phase.
3. Standard alkaloid solution: Approximately 10 mg each of strychnine and
brucine alkaloids are precisely weighed and dissolved in 100 ml of ethanol.
Injection volume: 10 μ l.
4. Plant material: Strychnos nux-vomica seeds and Strychnos ignatti seeds are
collected from a reliable source and authenticated by the taxonomist.
5. Extraction of the alkaloids: The dried powdered plant material (500 mg) is
macerated with a mixture of diethyl ether (1.5 ml), ammonia (0.25 ml) and
ethanol (0.5 ml) for 2 hr and then extracted with 50 ml of CHCl3 The
residue after evaporating the solvent is dissolved in ethanol and the final
volume is brought up to 50 ml with ethanol. Solutions are passed through a
0.45 pm filtration disc HVLP. Injection volume: 10 μ i.
6. Mobile phase: Solvent A: MeCN. Solvent B: aq solution of sodium salt of
heptanesulfonic acid (1 gm in 420 ml) and fitted at pH 3.2 with phosphoric
acid (0.5%). Elution is done with 25% of solvent A and 75% of solvent B.
Flow rate: 1.0 ml/minute.
7. Linear relationships are obtained by plotting the peak area against the
amount of each alkaloid injected (μ g in 10 μ l injection volume) with UV
 detection at 260 nm, at least up to 2.2 μ g of strychnine and 2.3 μ g of
brucine.

8.21.19 Ephedra Alkaloids

Ephedra is the dried stem and other aerial parts of Ephedra generdina, belonging
to the family gnetaceae. It consists of alkaloidal amines, namely, ephedrine,
pseudoephedrine, nor​ephedrine and methylephedrine.

Isolation of ephedrine and pseduoephedrine

Further, these compounds are purified by treating with alkaline solution,

extracted with a mixture of chloroform and ether (1:3) and converted to their
hydrochlorides.
Use
Used in the treatment of bronchial asthma and hay fever.
Identification

1. Solution of ephedrine in H2O + 0.1 ml of 10% solution of CuSO4+ 1 ml of

20% NaOH → Purple colour
2. Ephedrine + 2 ml of conc. H2S04 + 3 to 4 drops of 40% formaldehyde →
pink to red colour → warm the solution on a water bath wine-red

Estimation of ephedrine
Quantitative estimation of ephedrine can be done by the following method.
Spectroscopic method
The secondary amines (eg, ephedrine) in benzene in the presence of picryl
chloride produce a coloured complex, which shows maximum absorbance at 400
nm.

a. Test: Ephedrine is extracted as above and its solution is prepared in pure

benzene.
b. Standard: A standard curve is prepared by taking 1, 2, 3, 4, 5 and 6 ml of a
0.01% solution of pure ephedrine in benzene and transferred to the tubes.
The volume of the solution in each tube is adjusted to 90 ml with benzene.
c. Blank: It contains 9 ml of benzene.
d. 1 ml of 0.3% solution of picryl chloride is added to the test, standard and
blank.
e. All the tubes are closed tightly, allowed to stand for exactly 2 minutes and
placed in a water bath at 75–77℃ for 20 minutes.
f. Then, the tubes are removed from the water bath, allowed to stand for 3
minutes at room temperature and the absorbance measured at 400 nm. The
concentration of ephedrine in the test solution is measured from the
calibration curve.

NO T E : Amounts of ephedrine between 0.1 mg and 0.6 mg, shows a linear

relationship, when concentration is plotted against absorbance.
8.21.20 Isolation of Nicotine
Nicotine is obtained from Nicotiana tobacum, belonging to the family
solanaceae. It is a colourless liquid which boils at 246.1℃. Nicotine is easily
soluble in ethanol, ether, light petroleum and benzene.

Method I

Method II: By complexation

The active ingredients of medicinal plants are compounds containing donor
atoms such as N, O, S or P. As such, active ingredients of plants could act as
ligands for complexation with metal ions. The stability of such complexes
depends upon the nature of ions. If the plant extract is treated with aqueous
metal ions, its active compounds could preferentially coordinate with metals and
form precipitates. These precipitated complexes may further be purified and then
decomposed under mild conditions to recover the organic component in a fairly
pure state (Christy Munir et al., 1986).
PROCEDURE

1. The leaves of Nicotiana tobacum are dried in an oven at 50℃, and ground
to a fine powder to pass through a 0.3 mm mesh sieve. A dark coloured
extract is obtained by soaking 50 g of powdered tobacco in a toluene–
chloroform (9:1) mixture for an hour. The mixture is then filtered and the
filtrate subjected to removal of solvent using a rotary vaccum evaporator.
The dark coloured residue is then dissolved in about 25 ml of glacial acetic
acid.
2. The tobacco extract solution is cooled in ice and an excess of 5% solution
of zinc (II) chloride in acetic acid is slowly added with stirring. The yellow
green precipitate formed is removed after an hour and dried in vaccum. The
dried complex of dark brown colour is decomposed near 160℃. It is found
to contain 9.6% of Zn (determined by atomicabsorption spectrophotometric
method) as compared to 9.66% of Zn required for Zn (C10H14N2)3
C12.3H2O. The complex dissolved in NH4OH–water (1:10) solution,
displays absorption bands in UV region at 211, 254 (sh), 258 and 267 (sh)
nm.
3. The zinc nicotine complex is dissolved in dilute aqueous ammonia solution
and filtered. Excess of hydrogen sulphide is passed through it to precipitate
out ZnS. The filtrate after removal of ZnS is heated to 60℃ to expel excess
H2S. Nicotine is extracted with several portions of diethyl ether from the
filtrate. The combined ether extract is dried and evaporated to recover
nicotine as a yellow liquid. Several preparations are combined and vacuum
distilled to obtain a pure sample of nicotine.
It is evident that the complexation method can be successfully applied for
the extraction of compounds, especially Lewis bases from plants. The
method is relatively fast and isolation of a component takes 1–2 days at the
maximum, while conventional methods take several days and consume
large volumes of solvents and costly reagents. However, this method is
good only for isolation of those compounds which contain donor atoms and
are capable of forming insoluble and less stable complexes with metal ions.
Sometimes, if a number of closely related compounds are present in the
plant extract, a mixture of complexes may be formed, finally giving a
 mixture of a few compounds, which may be separated by other techniques
like TLC or column chromatography.

Identification

1. Nicotine + solution of vanillin in conc. HC1 → Rose-red colour.

2. Dilute solution of nicotine in ethanol + 2 ml of epichlorhydrin red
colour (The colour intensity varies depending upon the amount of nicotine
present).
3. Nicotine + cyanogen bromide + aniline → orange-red colour.

8.21.11 Isolation of Aconitine

Aconitine is a crystalline substance, isolated from the roots of Aconitum
napellus, family, ranunculaceae. It melts at 202–203℃. It is soluble in
chloroform or benzene; less soluble in ether, ethanol; and almost insoluble in
water or light petroleum.
8.21.22 Isolation of Pseudoaconitine
The following method can be used for the isolation of pseudoacontine from the
roots of Aconituan balfourii.
PROCEDURE

1. The roots of A. balfourii are air-dried, finely powdered and subjected to

percolation with 95% ethanol.
2. The ethanolic extract is concentrated to a small volume under reduced
pressure, and is treated with 0.5% HC1.
3. The acid solution contains impurities, like resin and oils which are removed
by agitating with solvent ether.
4. The solution is made alkaline and extracted with several portions of solvent
 ether.
5. Ether extracts are combined together and evaporated. Pseudoaconitine
crystallises out.
6. Crude pseudoaconitine is purified by converting it into its hydrobromide
and re-crystallising it with ethanol.

Identification

1. Dilute solution of aconitine + few drops of acetic acid + 2–3 drops of 0.2 N
potassium permagnate → Rosettes of red prismatic crystals.
2. A drop of 2% solution of aconitine nitrate in 10% acetic acid is mixed on a
slide with one drop of saturated solution of NaCl or KBr. The characteristic
microcrystalline salt of the alkaloid is obtained.
3. Very dilute solution of aconitine + ammonium reineckate in slightly acid
solution → ppt (observe under microscope) → Rosettes of fine crystals

Estimation
Method I: By HPLC
The method comprises extraction of aconite powder first with ammonical ether
and then with methanol (× 3 each); chromatography of the resultant extract over
neutral alumina and elution with ethyl acetate–methanol (7:3); prior to HPLC.
The method is found to be reproducible and suitable for routine analysis of
aconite and its pharmaceutical preparations, quantitatively (Hiroshi Hikino et al.,
1983).
PROCEDURE

1. Drug materials: Aconitine is purchased from Sigma Pharmaceuticals. The

raw aconite roots and processed aconites (prepared from raw aconite roots
by autoclaving at 120℃ for 40 minutes) are finely powdered and dried in a
desiccator.
2. Reagents: Acetonitrile (HPLC grade), tetrahydrofuran, sodium hydrogen
phosphate, phosphoric acid, sodium hexane sulphonate, alkaline alumina,
neutral alumina.
3. Instrumental conditions: The analysis can be carried out on an instrument
coupled to a stainless steel column (i.d.:30 cm × 4 mm), packed with ODS
 chemically bonded silica gel. The detector is a UV 8 variable wavelength
monitor with a flow cell of volume 8 μ l.
4. Extraction steps:
Ammoniacal chloroform/ ammoniacal ether/ ammoniacal methanol
method: The sample (5 g) is stirred with one of the above-named organic
solvents (60 ml), containing 5% aqueous NH3 (5 ml) at room temperature
for 30 minutes. The liquid phase is filtered and the residue is further
extracted 2 or 4 times in the same manner with the same solvent (60 ml
each). The combined solution is evaporated to dryness at a temperature not
exceeding 40℃.
Methanol or hydrochloric acid method: The sample (5 g) is extracted 3
times with either methanol or 1 N HC1 (60 ml each) at room temperature
for 24 hours each. The liquid phase is filtered, combined and evaporated
under diminished pressure at a temperature below 40℃.
Ammoniacal ether–methanol method: The sample (5 g) is extracted 3
times with ammoniacal ether (60 ml each) for 30 minutes each, and then
with methanol (100 ml each), once for 16 hours and twice for 3 hours each.
The organic phase is combined and evaporated to dryness under reduced
pressure at a temperature not exceeding 40℃.
5. Purification steps:
Chloroform or dichloromethane partition method: The methanol extract
prepared as above (575 mg), equivalent to 5 gm of crude drug, is suspended
in CHC13 or CH2C12 (20 ml) and extracted 3 times with 5 % HC1 (15 ml
each). The combined acidic layer is made alkaline (pH 10) with 25%
aqueous NH3 and extracted 4 times with CHCl3or CH2C12 (20 ml each).
The organic phase is washed once with water and evaporated to dryness.
Celite or alumina chromatography method: The methanol extract
equivalent to 5 gm of crude drug is mixed with celite (twice its weight),
placed on the top of a column of celite or alumina (10 g) and eluted
successively with benzene (100 ml), AcOEt-MeOH (7:3, 200 ml) and
MeOH (100 ml). Each eluate is evaporated to dryness.

Method II: By GLC/Selected ion monitoring (SIM)

SIM was used by Kitae et al. (1996) to determine aconitum alkaloids in the
tubers of Aconitum japonicum.
PROCEDURE

1. Materials: Aconitum tubers are purchased from a reliable source and

authentified by a taxonomist.
2. Reference alkaloids: Standard samples of hypaconitine, mesaconitine,
jesaconitine and aconitine are purchased from a commercial source.
3. Reagents: BSTFA [N,O-bis (trimethylsilyl)trifluoroacetamide].
4. Extraction: After the aconitum tubers are weighed and finely powdered,
alkaloids are extracted using 40 ml of methanol/2.0 gm of powder for 1 hr.
The extract is filtered and the residue is further extracted in the same
manner. The combined solution after removing undissolved matter is
evaporated to dryness. 30 ml of water is poured into the residue which is
then acidified to pH 3 by the addition of 10% aqueous tartaric acid. The
acidified solution is washed twice with 60 ml of diethyl ether. After
washing, the solution is made alkaline with 4% of aqueous NaOH and
extracted twice with CHC13(60 ml each). The organic phases are combined
and evaporated to dryness. Before converting to TMS (trimethylsilyl)
derivatives, the extract is diluted with CHC13 for analysis. Diluted sample
extracts are then evaporated to dryness and converted into TMS derivatives
by overnight treatment with 50 μl of BSTFA and used for GC/SIM analysis.
5. Apparatus: All analyses are carried out on a Jeol model DX-303 system
interfaced to a MS-GCG06 gas chromatograph. The column is a 15 m x
0.25 mm I.D. fused silica capillary cross-linked column with
methylsilicone. Temperature of the column oven is started at 250℃ and
increased to 320℃ at 16℃/minute. The carrier gas is helium with a linear
velocity of about 25 cm/s. The temperatures of the injection port and
transfer lines are kept at 320℃ and 250℃, respectively, and that of the ion
source at 250℃. The ionisation, the trap current, and the accelerating
voltage are 70 eV, 300 μ A, and 3 kV respectively. For GC/SIM analysis,
the ions at m/z = 596 for hypaconitine, at m/z = 684 for mesaconitine, at m/z
= 698 for aconitine, and at m/z = 728 for jesaconitine, are- monitored with a
dynamic resolution of 1,000 respectively.

8.21.23 Isolation of D-Tubocurarine

D-tubocurarine is isolated from the plant Chondrodendron tomentosum.
Use
D-tubocurarine is extensively used to promote muscular relaxation during
surgical operations.
Identification

1. Aqueous solution of D-tubocurarine chloride + FeCl3 solution → weak

green colour.
2. Aqueous solution of D-tubocurarine chloride + Na2CO3 solution → yellow-
brown ppt.
3. Aqueous solution of D-tubocurarine chloride + KBr or potassium
thiocyanate → ppt.

8.21.24 Isolation of Physostigmine

Physostigmine is a colourless crystalline substance, obtained from the seeds of
Physostigma venenosum, belonging to the family, leguminosae. The stable form
of physostigmine melts at 105–106℃; it is soluble in ethanol, ether, chloroform
and benzene, but insoluble in light petroleum.

The following method was used by Salway for the isolation of physostigmine
from Physostigma venenosum.

Uses

1. Used for contracting the pupil of the eye.

2. Used for reversing the effects of a number of sedatives.
 3. It has been shown to bring about slight improvement in the intellectual
capacity of humans and the cognitive performance of people with
Alzheimer’s disease.

Identification

1. Small quantity of physostigmine + 1 ml of strong NH3 solution →

yellowish-red colour.
2.

Estimation by Colorimetry
The estimation of physostigmine is based on the conversion of the compound to
rubreserine and measuring its absorbance at 300 nm.

a. Physostigmine is isolated from the finely powdered material of

Physostigma venenosum (1–5 gm) with ether according to the method
described by Salway.
b. The alkaloidal solution is mixed with 2–5 ml of 0.1 NH2SO4.
c. 1 ml of the acid extract is pipetted into a tube, neutralised with 10% NaOH
and one drop is added in excess.
d. The tube is shaken frequently for a period of 30 minutes, during which, the
full development of colour takes place. At the end of this period, one drop
of HC1 solution (conc. HC1 diluted with an equal volume of distilled
water) is added, and the mixture is again neutralised by the addition of
small amounts of solid sodium bicarbonate. The volume is made up exactly
to 5 ml with phosphate buffer, pH 7.
e. The absorbance of the coloured complex is measured at 300 nm and the
concentration of physostigmine is read from a calibration curve.

8.21.25 Isolation of Berberine

Berberine is found in a large number of plants belonging to the berberidaceae,
ranunculaceae, anonaceae families. It is a bright yellow crystalline substance
which melts at 145℃ and dissolves in cold water or ethanol. It is easily soluble
in hot water or hot ethanol, slightly soluble in benzene or chloroform and
insoluble in ether or light petroleum.

Uses

1. Cholagogue
2. Anti-emetic
3. Anti-catarrhal
4. Mild laxative
Identification

1. Berberine solution + Conc. HNO3 → reddish-brown colour

2. Berberine solution + Conc.H2SO4 → orange-yellow colour (upon warming)
→ olive-green colour.
3. Solution of alkaloid in sulphuric acid + a crystal of K2Cr2O7 → brownish-
violet colour.
4. Solution of berberine + Frohde’s reagent → Brown or green colour.
5. Solution of berberine + Mandelin’s reagent → Violet colour.
6. Solution of berberine + a drop of bromine water + few drops of HC1 → a
bright-red colour.

Estimation
Method I: by spectrophometry
This method was described by S.K. Chauhan et al. (2000)
PROCEDURE

1. Drug material: The roots of B. aristata D.C. are purchased from a reliable
source and authentified by a taxonomist.
2. Sample preparation: Around 20 gm of sample is ground to make fine
powder so that it passess through a 40 mesh seive. 1 gm of fine ground
powder is weighed accurately and extracted with methanol for about 6
hours using the Soxhlet apparatus. The extract is filtered using a Whatmann
filter paper No. 41 and the clear filtrate is transferred to a 100 ml
volumetric flask. The volume is made up to the mark with methanol. The
solution is used for spectrophotometric and HPTLC estimation.
3. Standard preparation: A 0.10 mg/ml solution of berberine in methanol is
used as a reference standard.
4. Assay: One ml of test sample which is further diluted to 25 ml with
methanol is taken. The dilution is made suitably so that the absorbance lies
in the range 0.5-1.0. The absorbance is recorded at 348 nm against
methanol as a blank. The standard solution is also serially diluted to yield a
final concentration of 0.02 mg/ml, 0.04 mg/ml, 0.08 mg/ml and 0.10 mg/ml.
 The absorbance of these different concentrations of standard berberine
preparations are read at 348 nm against methanol. The berberine content in
the test sample is calculated using a linear regression equation of the
calibration graph plotted between concentration and absorbance. The
equation for calculating berberine content is
y = 104.88 x - 0.0004
with a correlation coefficient of 0.999, where x is the concentration of the
compound analysed in mg/ml andy is the response in absorbance.

Method II: by HPTLC

This method was described by S.K. Chauhan et al. (2000)
PROCEDURE
The steps 1, 2 and 3 are the same as the above.

4. HPTLC assay: The test sample is further diluted (1:10) and 1, 2, 5 μ l of the
test sample alongwith 1, 2, 5 and 10 μ l of standard berberine (0.01 mg/ml)
are applied on a TLC aluminium plate pre-coated with silica gel 60 F254.
The chromatogram is developed in ethyl acetate: formic acid: acetic acid:
water (100:11:11:27) under chamber saturation conditions up to 80 mm.
The plate is air-dried and scanned at 348 nm in the absorbance mode. The
amount of berberine is calculated using the calibration curve plotted
between the concentration and the area of peak of standard berberine
preparations.
5. Varying known amounts of berberine (1, 3 and 5 mg) are added to about 1.0
gm of finely powdered test sample, in which the contents of berberine have
been estimated previously by the proposed methods. The samples are
extracted and analysed separately as per the procedure mentioned above.
The HPTLC chromatogram of the standard berberine and the test sample
taken from B.aristata is shown in Fig. 8.29. The contents of berberine are
quantified using the proposed method and percentage recovery calculated.
Fig. 8.29a HPTLC chromatogram of standard berberine

Fig. 8.29b HPTLC chromatogram of B. aristata

8.21.26 Isolation of Gelsemine, Gelsemicine and Sempervirine

These are the principal active constituents of roots and rhizomes of Gelsemium
sempervirens, belonging to the family loganaciae. These melt at 178℃, 170–
171℃ and 258– 260℃ respectively. The following method is described by
Forsyth, et al. for the isolation of alkaloids from the Gelsemium species.
Use
Analgesic, sedative and hypotensive.
Identification
 1. Solution of gelsemine in H2SO4 + cryst K2Cr2O7 red colour
violet green.
2. Solution of gelsemine in ethanol + p-dimethylaminobenzaldehyde in HCl
→ pink colour.
3. Ethanolic solution of gelsemine Intense fluorescence.

8.21.27 Isolation of Taxine

Taxine is a colourless granular powder, obtained from the leaves, shoots and
fruits of Taxus baccata, belonging to the family taxaceae. All the parts of the
plant are very poisonous; cattle and horses can die very rapidly after eating the
leaves and stems. Although poisonous, it produces a valuable wood. Taxine
melts at 121–124℃. The base is insoluble in water or light petroleum but
soluble in ethanol, ether, chloroform or benzene. Callow et al., have
recommended the following procedure for the isolation of taxine from the leaves
of Taxus baccata.
Identification

1. Pure sample of taxine + Cone. H2SO4 → deep-red colour.

2. Pure sample of taxine + Cone. H2SO4 + K2Cr2O7 → yellow colour →
(changes to) dull olive brown.

8.21.28 Isolation of Conessine and Holarrhenine

Conessine and holarrhenine are crystalline substances, .obtained from the bark
and seeds of Holarrhena antidysenterica, belonging to the family apocyanaceae.
Conessine melts at 125℃. It is sparingly soluble in water, but soluble in ethanol,
chloroform or light petroleum.
Holarrhenine melts at 197–198℃; is insoluble in water, easily soluble in
ethanol or chloroform, sparingly soluble in cold ethyl acetate, acetone or ether.
 Kanga et al., have described the following procedure for the isolation of
conessine from the seeds of H. antidysenterica.
Use
These constituents have anti-amoebic activity. Effective in dysentry,
haematuria, snake bites, bronchitis and as an anthelmintic.
Identification

1.

2. Solution of conessine + Marquis reagent → brownish-yellow colour →

(allowed stand) → a crimson colour with green fluorescence develops.
3.

Estimation
A turbidimetric method can be used for the quantitative estimation of the total
alkaloids of Kurchi bark (Holarrhena antidysenterica) in crude medicinal
preparations. The alkaloids are colloidally precipitated as complex salts of
potassium iodobismuthate in extremely dilute solutions with Dragendorff’s
reagent. The finely subdivided organic brown precipitate gives a coloured, clear,
homogeneous suspension in the presence of gum arabic. The optical density of
such suspensions changes linearly with change in alkaloid concentration, when
prepared within standardised experimental condtions, that include control of ion
concentration and temperature of the reaction mixture (R. K. Dwivedi and R. K.
Sharma, 1990).
PROCEDURE

1. Extraction of alkaloids: Bark powder (5 kg, 60 mesh) of authentic H.

antidysenterica is moistened for 15 minutes with 20 litres of 95% ethanol.
This is packed in a glass percolator and macerated for 4 hr to exhaustion.
The percolate is evaporated by distillation and then extracted with 7% w/v
citric acid. The acid extract is then made alkaline with dilute liquid
ammonia and the precipitated bases are extracted with chloroform. The
aqueous solution is further treated with 2 N sodium hydroxide solution and
the precipitated alkaloids are again extracted with chloroform. The
combined chloroform extract is washed free from alkali and the solvent
removed under reduced pressure. The residue, thus obtained, represents the
total tertiary alkaloids of H. antidysenterica (yield = 2.40 % w/w in terms
of starting material).
2. Reagents and solutions: Zinc sulphate (ZnSO4.7H2O AR) 5% w/v; barium
hydroxide (Ba(OH)2.8H2O, AR) 0.3 N; bismuth subnitrate (OBiNO3.AR)
8.5% w/v in glacial acetic acid and mixed in a 1:4 ratio with distilled water
(solution A); potassium iodide (KI, AR) 40% w/v (solution B); 75 mg of
egg albumin per litre of distilled water (protein solution).
3. Dragendorff’s reagent: Solution A, solution B, glacial acetic acid, and
distilled water are mixed in that order in proportions of 1, 1, 4 and 20,
respectively. The reagent is prepared fresh on the day of use.
Suspension: Gum arabic at a concentration of 2 mg/ml is used as the
suspending agent in Dragendorff’s reagent.
Diluent fluid: Distilled water is used as the diluent fluid for the dilution of
alkaloid–reagent mixture (reaction mixture).
4. Standard solutions: 50 mg amount of total alkaloids from H.
antidysenterica is weighed and dissolved in 0.20 ml of glacial acetic acid
with the volume made up to 5 ml with distilled water. Standard solutions
 contain 0.20–1.0 mg total alkaloids per ml and are processed as given
below. The supernatants obtained from the standard solutions, therefore,
contain 0.02–0.10 mg of total alkaloids per ml.
Kutjaristha alkaloidal mass solution (KAM-solution): The deep brown
alkaloidal mass extracted from kutjaristha is weighed and dissolved in a
suitable volume of acetic acid, diluted with distilled water and the final
volume recorded. An appropriate volume of KAM-solution is processed as
given below. The supernatant, thus obtained, is used for the estimation of
the alkaloid.
5. Pre-treatment of Kutjarishta: In a solution free from protein and sulphate,
the lowest concentration of total alkaloids possible to determine accurately
is 0.005 mg/ml. The dilution of the sample following removal of interfering
substances, therefore, is balanced accordingly. For a sample containing
0.020–1.0 mg of alkaloids per ml, the following procedure is applied. The
sample (1 ml) is pipetted into an Erlenmeyer flask and the following
reagents are added in the order given: 8 ml protein solution, 0.50 ml barium
hydroxide and 0.50 ml zinc sulphate. The mixture is shaken after each
addition and allowed to stand for 20 minutes at 10–15ºC and then
centrifuged at 3000 rpm for 30 minutes. A clear, colourless supernatant is
obtained. It is used for the estimation of the alkaloids as described below;
the precipitate is discarded.
Greater volumes of protein precipitants, barium hydroxide and zinc
sulphate, are required, however, to obtain a clear supernatant.
6. Determination of total alkaloids: The supernatant (1 ml) is pipetted into the
test tube and 1 ml of Dragendorff’s reagent added, followed by 3 ml of
distilled water. The contents are mixed after each addition. The mixture is
heated at 60ºC for 10 minutes in a water bath. After heating, the test tube
containing the reaction mixture is placed in a BOD incubator at 25º ± 1ºC
for 90 minutes. At the end of the incubation period, the contents are read at
490 µm using an Elico colorimeter (CL-20A). The reagent blank is
developed in an identical manner from the supernatant and is prepared from
a corresponding sample containing no alkaloid. It is also read at 490 µm
using the Elico colorimeter.

CALCULATIONS
Alkaloid content of kutjaristha per ml is calculated from the formula:
 where W = alkaloid in µg/ml of supernatant of KAM-solution, d = dilution factor (times
dilution of KAM-solution during the de-proteinisation procedure), Vka = total
volume of KAM-solution, Vkt = volume of kutjaristha used.

8.21.29 Isolation of Pilocarpine and Isopilocarpine

Pilocarpine and isopilocarpine are the principal active constituents of the leaves
of Pilocarpus microphyllus, belonging to the family rutaceae. Pilocarpine is a
colourless oil which boils at 260℃. The base is easily soluble in water, ethanol
and chloroform, but almost insoluble in ether and light petroleum. Isopilocarpine
is an isomer of pilocarpine. It is a colourless oil which boils at 261℃.

Use
Pilocarpine hydrochloride is used in ophthalmic practice, as it causes contraction
of the pupil of the eye. In early glaucoma treatment, it serves to increase the
irrigation of the eye and relieves pressure.
Identification

1. Solution of pilocarpine (or its salt) in dilute acid + hydrogen peroxide +

potassium dichromate → violet-coloured complex.
2.

Estimation of total alkaloids by colorimetry

PROCEDURE

1. 10 gm of the leaf powder is accurately weighed and shaken thoroughly with

50 ml of chloroform. The contents are allowed to stand for 10 minutes.
2. 5 ml of 10% NH3 solution is added and shaken continuously for an hour.
3. The contents are transferred to a percolator and eluted with sufficient
quantity of CHC13, until all the alkaloids are completely extracted. The
solution is concentrated to a small bulk.
4. About 150 ml of ether is added, followed by 20 ml of 0.5 N H2SO4and the
contents are shaken well in a separating funnel. The lower aqueous layer is
separated out. The ether solution is shaken with three portions of 10 ml of
0.1 N H2SO4.
5. The acid extracts are combined, made alkaline with NH3 and extracted with
four successive 20 ml portions of CHC13.
6. The chloroform is evaporated; the residue is washed with 10 ml portions of
water. The residue is dried for 5 minutes on a boiling water bath and
dissolved in 2 ml of ethanol. 15 ml of 0.05 N H2 SO4 is added.
7. An aliquot of the acid extract (containing approximately 1–5 mg alkaloids)
is placed in a 25 ml volumetric flask and neutralised with NaOH.
8. 1 ml of freshly prepared 2% sodium nitroprusside solution is added,
 followed by 1 ml of 1 N NaOH solution. The solution is mixed well and
allowed to stand for 5 minutes. 5 ml of 0.01N KMnO4 and 3 ml of 3 N
H2SO4 are added separately. The solution is made up to 25 ml with distilled
water.
9. Calibration curve is prepared by using pure pilocarpine nitrate (0.13%
solution of pilocarpine nitrate contains 1 mg of pilocarpine in 1 ml).

The absorbance of the coloured solutions should be measured colorimetrically

at 600 nm within 5–10 minutes. The concentration of the test is read from the
calibration curve.

8.21.30 Isolation of Coniine

Coniine is a colourless, strongly alkaline liquid, obtained from the seeds of
Conium maculatum, belonging to the family umbelliferae. It boils at 166–168℃.
It is soluble in ethanol and in most organic solvents. Scheme 8.4 is used to
isolate coniine.

Use
It is extremely toxic, causing paralysis of the motor nerve endings. Teratogenic
effects have been recorded after cows and swine have eaten hemlock during
pregnancy. Hemlock was used by the Greeks to execute criminals.
Identification

NOTE: With nickel chloride (instead of copper sulphate) thin, green rhomboid
crystals are found.
Scheme 8.4
8.21.31 Isolation of Pelletierine Alkaloids
Alkaloids are also obtained from both stem and root barks of Punica granatum
(promegranate), belonging to the family punicaceae. The plant contains about
0.5–0.9/o of volatile liquid alkaloids, the chief of which are pelletierine and
pseudopelletierine.
Pelletierine is a colourless alkaline oil which boils at 106℃. It is soluble in
organic solvents and water and readily forms crystalline salts with acids.
Pseudopelletierine with a b.p of 246℃ is easily soluble in water, ethanol,
ether and chloroform, but less soluble in light petroleum.
Isopelletierine has a b.p. of 102–107℃. It reacts with ethylchloroformate to
form a N- carbethoxy derivative, which on hydrolysis yields the unchanged base.
Methyl isopelletierine has a b.p. of 114–117℃. It is miscible with water.

The following procedure can be used for the isolation of pomegranate

alkaloids.
Identification
1. Solution of pelletierine + tannic acid → precipitate.
2. Solution of pseudopelletierine + few drops of H2So4 + K2Cr207 → intense
green colour.
8.21.32 Isolation of Yohimbine
Yohimbine is a yellow crystalline substance, isolated from the bark of
Pausinstalia yohimba, belonging to the family rubiaceae. It melts at 234℃. It is
soluble in ethanol, chloroform and hot benzene, but sparingly soluble in water or
ether.

The method described in Scheme 8.5 was used by Chemnititus for the
isolation of yohimbine from the bark of the plant. .
Use
Aphrodisiac; for impotence and weight loss.
Identification

1. Solution of yohimbine in conc. HC1 + few drops of 2% p-

dimethylammobenzaldehyde solution in conc. drops of 0.05%
sodium nitrite solution → a deep- violet blue ring appears after a short time,
and on shaking the solution, a blue colour develops.
2. Solution of yohimbine + few drops of a reagent consisting of 1 gm of
chloralhydrate dissolved in 5 ml of ethanol to which 10 ml of conc. H2So4is
added a deep blue colouration.
3. Solution of yohimbine + glyoxylic reagent + vanillin in rose-red
to violet colouration.
Scheme 8.5
8.21.33 Isolation of Arecoline
Arecoline is obtained from the seeds of Areca catechu, belonging to the family
palmaceae. It is a colourless, odourless, strong alkaline oil which boils at 209℃.
It is miscible with most organic solvents and water. The salts of arecoline are
crystalline but usually deliquescent.

The following method is described by Jahns for the isolation of arecoline from
the seeds of Areca catechu.
Use
It exhibits markedly toxic properties. It possesses para sympathetic stimulant
action, and anthelmintic activity.
Identification

1. Solution of arecoline + phospomolybdic acid → white ppt.

8.21.34 Isolation of Peroline
Peroline is a yellow crystalline substance, isolated from, the rye grass, Lolium
perenne. It is soluble in ethanol, chloroform or acetone; and very slightly soluble
in water, ether or benzene.
The following procedure can be used for the isolation of peroline from rye
grass.
Use
The plant is reported to be used in topical heat treatment of degenerative
rheumatic conditions.
Identification
1. Solution of peroline + gold chloride → precipitate
2. Solution of peroline + Mandeline’s reagent → brown colour
3. Solution of peroline + titanous oxide in H2SO4 → brick-red colour
8.21.35 Isolation of Sparteine
It is a colourless alkaline oil, soluble in ethanol, chloroform or ether; insoluble in
benzene or light petroleum. It is found in Cytisus scoparius, Lupinus arborens
and Chelidonium majus.

The alkaloid is also called lupinidine. The salts of the compound crystallise
well.

Alternatively, the sparteine can be isolated by the following procedure.

Use
Used in the treatment of functional disorders of the heart and circulation.
Identification
A chloroform solution of the base is allowed to evaporate on a filter paper and
the spot is exposed to bromine vapours for a few seconds. In the presence of
sparteine, a yellow stain develops which disappears on holding the paper in
ammonia fumes. If the spot is now warmed over a hot plate, a pink colour
appears.
8.21.36 Isolation of Lupinine
Lupinine is isolated from the seeds of Lupinus luteus (leguminosae). It is readily
soluble in cold water, less soluble in hot water and soluble in organic solvents.
The method described in Scheme 8.6 can be used to isolate lupinine.
Use
It is an insect anti-feedant, and a growth inhibitor for the grasshopper
Melanoplus bivittatus.

Scheme 8.6

8.21.37 Isolation of Harmine and Harmaline

These are obtained from the seeds and roots of Peganum harmala, belonging to
the family rutaceae. Harmine crystallises from methanol in colourless prisms
with an m.p. of 257– 259℃. It is sparingly soluble in water, ethanol and ether.
Harmaline occurs in colourless or pale yellow crystals with an m.p. 239–240℃.
It is very sparingly soluble in water but soluble in hot ethanol and chloroform.
The procedure described in Scheme 8.7 can be used to isolate harmine and
harmaline.
Use
Harmine is a CNS stimulant and is hallucinogenic at high doses.
Scheme 8.7
Identification
8.21.38 Isolation of Caffeine
Caffeine is a colourless, crystalline, silky substance; it has limited solubility in
water and alcohol. It is freely soluble in chloroform. It melts at 235–237℃. It
acts as a weak base and is able to form salts with strong acids which are
decomposed by water.

Caffeine can be extracted from tea leaves, by the following simple procedure.
PROCEDURE

1. Tea leaves are dried and 100 gm of the fine powder is subjected to Soxhlet
extraction with 400 ml of absolute alcohol for 3 hr.
2. The caffeine is then adsorbed onto 50 gm of magnesium oxide and acidifed
with 50 ml of 10% sulphuric acid.
3. It is then desorbed and extracted into chloroform. The soft silky needles of
caffeine are separated out.

Alternatively, the following method can also be used for the separation of
caffeine.
PROCEDURE

1. The leaves are dried, powdered and boiled with alkaline water and filtered.
To the filtrate, basic lead acetate is added to precipitate the tannins and
albuminoids. The excess of lead is removed from the filtrate as lead
sulphate by adding sulphuric acid.
2. To the filtrate, animal charcoal is added to de-colourise the solution. Then
chloroform is added to extract caffeine from the filtrate.
3. The chloroform layer is separated and then evaporated to yield caffeine
which can be purified by re-crystallisation from water.
Use
It is used as a diuretic, heart and CNS stimulant in the form of its citrate and
hydrochloride.
Identification
By the Murexide test: Dissolve the sample in a few drops of conc. HNO3;
evaporate to dryness and add two drops of conc. ammonium hydroxide; a purple
colour occurs in the presence of caffeine.
8.21.39 Isolation of Capsaicin
Capsaicin is obtained from the dried, ripe fruits of Capsicum frutescens or
Capsicum annum, belonging to the family solanaceae. It is soluble in methanol,
ethyl acetate, ethanol and acetone. Scheme 8.8 describes a method to isolate
capsaicin.
Further, pure capsaicin can be separated by preparative thin layer
chromatography of the elute.
Scheme 8.8
Use
It is used as a counter-irritant, rubefacient and for the relief of rheumatism.
Identification
By TLC: The plates are prepared according to the standard procedure with silica
gel. They are activated at 110℃, and Rf values in various solvents determined.
The standard Rf values of capsaicin in various solvents is given in Table 8.36.
Table 8.36 Rf values of capsaicin in different solvents

Estimation
By spectrophotometry
Capasicin forms a yellow-coloured complex with sodium nitrite and sodium
tungstate under acidic conditions and shows maximum absorbance at 430 nm.
The complex obeys Beer’s law for 2–35 μg capsaicin/ml.
PROCEDURE

1. Extraction: Dried sample of 30 mesh powder is ground. 0.5 gm powder is

exactly weighed, extracted with acetone for 4 hr in a Soxhlet apparatus, and
filtered after cooling to room temperature. Acetone (5 ✕ 2 ml) is repeatedly
added until the residue is free of pungency. Then, the solvent is evaporated
on a water bath (50℃) and the residue is transferred into a 25 ml standard
flask and diluted to volume with ethyl acetate and stored at 45℃.
The above extract can be directly used in the case of green chillies; but in
the case of red chillies, the extract is purified by column chromatography
on alumina. The capsaicin is eluted with an acetone: methanol: water
mixture (75:25:2) or chloroform or 50% aqueous ethanol.
2. Aliquot of standard capsaicin (0.05–0.5 ml) is pipetted out in a small
beaker. The solvent is evaporated below 65℃ with a current of hot air. To
each beaker, 1 ml of water and 2 ml of buffer solution is added, followed by
 1 ml of sodium nitrite and 1 ml of sodium tungstate. The contents are mixed
thoroughly and slightly warmed on a water bath. After 15 minutes, 2 ml of
1 N sodium hydroxide is added and made up to a definite volume.
Absorbance is read at 430 nm against a reagent blank.
3. Definite aliquots of extracted samples of capsicum and chillies are taken,
evaporated and the colour developed by following the above procedure.
The concentration of the test sample is determined from the calibration
curve.
8.21.40 Isolation of Rauwolfia Alkaloids
A total alkaloid extract may be obtained from the powdered root material of a
Rauwolfia species by extraction with a suitable solvent. Ethanol and ethylene
chloride are frequently used. The total alkaloidal extract is then usually
chromatographed on a column (acid- washed alumina is widely used). Benzene
is often used as the elution solvent for reserpine, deserpidine and rescinnamine.
In a chromatographic elution sequence, going from benzene to a degree of
solvent polarity, as represented by 10% methanol in chloroform, the sequence of
the principal Rauwolfia alkaloids elute is represented as follows:

10% methanol in chloroform Sarpagine

Serpentine

Ajmaline

Yohimbine and isomers

Rescinnamine

Reserpine

Tetrahydroserpentine

Reserpinine.

Isolation of reserpine and rescinnamine

In 1947, in connection with the estimation of the total alkaloids by the
conventional solvent extraction of Rauwolfia serpentina, Dutt et al. made an
extract of the powdered root material with alcohol. The solvent was then
distilled off, treated with several quantities of water, after which there was left
some insoluble matter (i.e., water-insoluble material from the alcohol-soluble
extract) and this was designated as oleoresin. A few years later, a number of
other investigators, found reserpine in this oleoresin fraction.
During that period, there was also commercially available, a fat-soluble
alkaloidal fraction extracted from R. serpentina root known as alseroxylon
(which is also in use at present in a number of medicinal preparations of
Rauwolfia). A number of investigators also isolated reserpine in the pure state
from this alseroxylon extract.
Reserpine was isolated in the pure state by Muller, Schlittler, and Bein in 1952
from the oleoresin fraction. Independently, Klohs et al., isolated reserpine from
the so-called oleoresin fraction, and obtained crystalline reserpine and crystalline
rescinnamine from the alseroxylon preparations.
8.21.41 Extraction of Vinca Alkaloids
Svoboda’s method
Svoboda et al. devised an extraction scheme by which numerous individual
Vinca alkaloids have been isolated. The plant material (ground whole plant,
Catharanthus roseus) after de-fatting with Skelly B (essentially n-hexane), is
extracted with 2% tartaric acid, before extraction with organic solvents under
acidic and alkaline conditions, thus, initially separating those alkaloids whose
tartrates are soluble in organic solvents and those which are not. Some alkaloids
are also removed from the plant material by the Skelly B together with fat. This
extraction scheme and the subsequent chromatography and gradient pH
extractions may serve to illustrate recent advances from the classical extraction
technique. The principles are applicable to the separation of certain other groups
of alkaloids.
In the following outline of the Svoboda extraction and separation schemes,
only the details pertaining to those steps and fractions which yield vinblastine,
vincristine, vinleurosine, and vinosidine are summarised. The details for those
other fractions yielding a number of Vinca alkaloids other than these four are
excluded.
The initial steps of extraction are summarised schematically in the flow sheet
given in Fig. 8.30. The different fractions are then chromatographed on alumina,
and eluted with various eluting solvents.
Some of these column fractions (i.e., eluted from the alumina column) are
further subjected to gradient pH extractions to achieve separations of certain
Vinca alkaloids.
In this scheme, vinblastine vinleurosine, vincristine, and vinrosidine are in
fraction A, together with a number of other alkaloids.
Chromatography
A benzene solution of 10 gm of the extracted fraction A is chromatographed on
400 gm of alumina Grade F-20 (de-activated by treatment with 12.5 ml of 10%
acetic acid). Eluting solvents used are benzene, benzene: chloroform,
chloroform, and chloroform: methanol in 500 ml fractions. Vinleurosine (0.234
gm) comes down in fractions 34–42 (benzene: chloroform 1:1) while vinblastine
(0.126 gm) is obtained as the sulphate from fractions 43–45 (benzene:
chloroform 1:1).
Post-vinblastine eluent fractions (3.6 kg of residue from evaporation of
combined chloroform fractions) are re-chromatographed on de-activated alumina
(120 kg) in a similar manner. The eluting solvents and fractions collected are:
benzene (fraction 1), benzene–chloroform 3:1 (fractions 2–15); benzene–
chloroform 1:1 (fractions 16–29); benzene–chloroform 1:3 (fractions 30–43);
chloroform (fractions 44–57); and chloroform–methanol 19:1 (fractions 58–67).
In these re-chromatographed fractions, vinrosidine is isolated from fractions
33–45, and vincristine is isolated from fractions 33–42. The isolation of these
alkaloids from these column fractions is achieved by subjecting each fraction to
gradient pH extractions.

Fig. 8.30 Flow chart for the Svoboda’s extraction

Gradient pH extractions
The eluted crude fraction (10 gm) is dissolved in 500 ml of benzene, and any
insoluble material is removed by filtration. The benzene solution is then
extracted into 500 ml of 0.1 M citric acid by steam distillation under reduced
pressure. This steam distillation procedure is used in order to avoid emulsion
formation. After removal of all the benzene, any insoluble material is filtered off.
The acidic solution (pH: 2.75–2.85) is extracted with 500 ml of benzene. The
aqueous phase is then adjusted with ammonia to pH 3.54, 3.9, 4.4, 4.9, 5.4, 5.9,
6.4 and 7.5 successively. At each pH level, the aqueous solution is extracted with
500 ml of benzene. The benzene extract in each case is dried over sodium
sulphate and evaporated to dryness. Both vinrosidine and vincristine are
extracted in this manner from the acidic solution, at pH levels 4.9–6.4.
Isolation of catharanthine, vindoline and vinblastine
Atta-ur-Rahma et al. (1983) described the following rapid procedure for isolating
catharanthine, vindoline and vinblastine.
PROCEDURE

1. 20 kg of air-dried leaves of Catharnathus roseus are finely crushed with 40

litres of ethanol. The crushed material is filtered and washed thoroughly
with 10 litres of ethanol. The ethanolic filtrates are combined and
evaporated first in a flash evaporator and then in a rotary evaporator until it
is concentrated to a gum. The gum is acidified with 5% HCl (5 litres) and
washed with chloroform (3 × 1 litre). The ice-cold solution is then basified
(1 litre, 33% NH3 solution), and extracted with 2 × 3 litres of chloroform.
Chloroform extracts are dried over anhydrous sodium sulphate and
concentrated to a crude alkaloidal gum (120 gm). The alkaloids (120 gm)
are dissolved in 400 ml of chloroform and extracted with pH 3 phosphate
buffer (one litre). The chloroform layer is dried over anhydrous Na2SO4,
filtered and concentrated under vacuum to afford 60 grams of the alkaloidal
mixture. This is dissolved in 240 ml of chloroform. 480 ml of petroleum
ether is added, which causes some alkaloids to precipitate out. Virtually no
cathranthine, vindoline and vinblastine are present in the precipitated
portion. The precipitates are filtered off and the filtrate is again
concentrated to a gum (36 gm). The gum is dissolved in 200 ml ethyl
acetate and extracted with pH 2 phosphate buffer (one litre). The aqueous
layer is separated and then extracted with chloroform (one litre) to afford a
 vindoline- and catharanthine-rich fraction (15 grams). The buffer layer is
then basified with ammonia solution to pH 10 and extracted with
chloroform (one litre) to afford the vinblastine-containing fraction (20
grams).
2. In order to obtain catharanthine and vindoline, a flash (dry) column
chromatographic procedure is applied. This consists of packing silica gel
(70–230 mesh) in a sintered column fitted with a ground glass bottom joint
and tap. This column is fitted onto a Buchner flask. The substance is loaded
on the column and the eluting solvent drawn down the column with the help
of vacuum applied on the collecting Buchner flask.
3. 15 grams of vindoline- and catharanthine-rich fraction is loaded on the top
of such a column, packed with 150 gms of silica gel (70–230 mesh). The
column is eluted first with 28% ethyl acetate in petroleumether (2 litres),
and then with 35% ethyl acetate in petroleum ether (5 litres) which affords
pure catharanthine (0.6 gm). Elution with 45% ethyl acetate in petroleum
ether (2 litres) removes the other alkaloids. Subsequent elution with 55%
ethyl acetate in petroleum ether (5 litres), affords pure vindoline (2.4
grams).
4. The vinblastine containing fraction (20 gms) is loaded on a flash
chromatography column packed with 200 grams of alumina (neutral
activity I). The column is eluted with 70% ethyl acetate in petroleum ether
(7 litres). The eluates are concentrated to a gum (10 gms), and again loaded
on the same type of column packed with 30 grams of TLC grade silica.
Elution with 50% ethyl acetate in petroleum ether removes the faster
moving substances. Subsequent elution with 0.5% ethanol in ethyl acetate
(2 litres), affords a vinblastine-rich fraction (0.6 gram).
5. The vinblastine-containing fraction (0.6 gm) is loaded on a preparative
HPLC (column diameter: 2.2 cm; column length: 50 cm; column packing:
Lichroprep 15–25 μm silica; solvent pressure: 1000–1200 lb/sq inch; rate of
flow: 13 ml/minute), and eluted successively with 50% ethyl acetate in
petroleum ether (2 litres), 60% ethyl acetate in petroleum ether (4 litres),
65% ethyl acetate in petroleum ether (2 litres) and finally with 70% ethyl
acetate in petroleum ether (2 litres). The last elution affords pure vinblastine
(60 mg) as the slowest moving material.

Estimation of serpentine in Vinca rosea

The method involves the quantitative separation of serpentine base by
preparative TLC and its estimation at 310 nm. It also involves conversion of
serpentine to ajmalicine and it estimation as ajmalicine at 283 nm (M. N. A. Rao
et al., 1981).
PROCEDURE

1. Extraction of serpentine with other strongly basic alkaloids:

An accurately weighed amount (5 gm in a 40 mesh) of V. rosea (roots,
stems, stem bark) is extracted with MeOH in a Soxhlet apparatus for 4 hr.
MeOH is completely removed in vaccum, the residue extracted with warm
0.5 N H2SO4 (20, 15, 10, 10, 10 ml) and adjusted to a pH of 7 with
NH4OH. The neutralised aqueous extract is shaken with CHCl3 (30, 20, 20,
15, 10 ml) and the CHCl3 extract washed twice with water (10 ml each).
Then the aqueous washings are mixed with the neutral aqueous extract and
the CHCl3 extract, dried, weighed and rejected. The neutral aqueous extract
is adjusted to a pH of 11.5 with aqueous KOH (20%) and the precipitated
alkaloids extracted with CHCl3 (30, 20, 20, 15 and 10 ml). The combined
CHCl3, extracts are washed twice with water (10 ml each), dried over
anhydrous Na2SO4 and filtered. The Na2SO4 is washed off with CHCl3.
The CHCl3, is removed and the residue weighed, dissolved in MeOH (50
ml) and labelled as solution A.
2. Isolation and estimation of serpentine in solution A:
A known volume of solution A is spotted on a preparative TLC plate and
developed with the solvent mixture (AcOH: CH3COCH3: MeOH:: 5: 70:25)
alongwith serpentine. The spot identical with serpentine is cut, eluted with
MeOH, made to a specified volume in a volumetric flask and labelled as
solution B. Graded dilutions from solution B are made and their absorbance
read out at 310 nm.
3. Preparation of standard curve for serpentine:
The standard curve for serpentine is obtained in the range 5–50 μg/ml at
310 nm.
4. Conversion of serpentine to ajmalicine:
An aliquot volume of solution A is reduced with NaBH4 in an ice bath
using a magnetic stirrer. After the completion of the reaction (1–2 hr,
 indicated by a colour change), the reaction mixture is adjusted to pH 7;
MeOH is removed and the residue treated with water (30 ml) and extracted
with CHCl3 (20, 15, 10, 10, 5 ml). The combined CHCl3 extracts are
washed twice with water (10 ml each) and dried over anhydrous Na2SO4.
They are filtered and the filtrate evaporated to obtain a residue which is
dissolved in MeOH to 50 ml in a volumetric flask. This solution is labelled
as solution C.
5. UV spectral analysis of solution C for ajmalicine:
Graded dilutions of solution C in MeOH are taken and their absorbance
read at 283 nm.
6. Standard curve for ajmalicine:
A standard curve for ajmalicine at 283 nm over a concentration range of 8–
40 μg/ml is obtained.

8.21.42 Isolation of Vasicine and Vasicinone

H. L. Bhalla et al. (1982) described the following procedure for the isolation of
vasicine and vasicinone.
PROCEDURE

1. Extraction and purification of the alkaloidal mixture:

5 kg of powdered vasaka IP leaves (60 #) are macerated with 20 litres of
alcohol (90%) for 48 hours with vigorous intermittent stirring. The extract
is filtered and the filtrate is concentrated to 1 litre at 60℃. The process is
repeated thrice. The concentrates are combined, the volume reduced to 500
ml and filtered. The filtrate is mixed with 500 ml of acetic acid (5%),
warmed at 60℃ for 30 minutes and the coagulated mass is filtered off. The
acidic filtrate is reduced to 200 ml under vacuum and shaken with 3 × 200
ml petroleum ether (60–80℃) and 2 × 100 ml chloroform respectively, to
remove non-basic red colouring matter. The acidic aqueous extract is
adjusted to pH 8.5 with dilute ammonia and extracted successively with 200
ml each of chloroform till the aqueous layer gives a negative test for
alkaloids when tested with Drangendorffs reagent. Chloroform is recovered
and the alkaloidal mixture dried. It is dissolved in a minimum quantity of
pure chloroform (free from water) and extracted with acetic acid (5%). The
aqueous acidic extract is shaken with activated charcoal (0.5% w/w) for 10
 minutes and filtered. The filtrate is adjusted to pH 8.5 with dilute ammonia
and the alkaloids back-extracted into chloroform. Chloroform is recovered
and the precipitate thus obtained, is dried over calcium chloride and
weighed.
2. Chromatographic separation of alkaloids:
i. Thin layer chromatography: The alkaloidal mixture (10 mg) is
dissolved in 1 ml of methanol and quantities approximating 0.01 ml of
the solution are applied to separate single spots on suitably prepared
and activated silica gel plates. The chromatogram is developed at
chamber saturation by the ascending technique, using chloroform:
methanol: ethyl acetate (8:2:1) as the solvent system. The plates are
dried and the spots corresponding to the alkaloids located by spraying
Drangendorff’s reagent. Their Rf values are then noted.
ii. Column chromatography: The alkaloidal mixture (0.5 g) is dissolved
in a minimum quantity of the solvent system, chloroform: methanol:
ethyl acetate (8:2.1) and the solution is carefully placed over a 12 inch
column (0.75 inch diameter) of silica gel. The column is then eluted
with the same solvent system, the eluates collected in 30 × 5 ml
fractions and subjected to TLC. Fractions corresponding to the same
spots are pooled together, and the alkaloidal residues are obtained by
evaporating the solvent at 60℃.

Estimation
Accurately weighed 10 mg amounts of each vasicinone and vasicine are
dissolved in 50 ml of chloroform. The solution (0.5 ml) is then applied in the
form of a band on an activated TLC plate and the chromatogram developed by
using chloroform: methanol: ethyl acetate (8:2:1) as the mobile phase. The upper
and lower bands corresponding to vasicinone and vasicine are scraped and
shaken with two successive quantities of about 76 ml of 0.5 N HC1. Silica gel is
separated by centrifugation, and the supernatant filtered. Adequate washing of
silica gel G is carried out with 0.5 N HC1 and the solution adjusted to volume. A
blank solution is simultaneously prepared. The absorbance of the samples is
noted against the blank at 233 nm and 285 nm for vascinone and vasicine
respectively (H. L. Bhalla et al., 1982).
8.22 GLYCOSIDES
Glycosides are compounds which yield one or more sugars upon hydrolysis i.e.,
which give sugar and non-sugar moieties. The sugar portion is called glycon and
the non-sugar portion is called aglycone. The most commonly occurring sugars
are β-D-glucose, ramnose, cymarose and digitoxose. Chemically, these are the
acetals in which the hydroxyl of the sugar is condensed with a hydroxyl group of
the non-sugar component. The secondary hydroxyl is condensed within the sugar
molecule itself to form an oxide ring. Some of the properties of glycosides are
listed below.
Properties

1. They are crystalline or amorphous substances.

2. They are soluble in H2O and dilute alcohol with the exception of resin
glycosides, but insoluble in organic solvents like benzene, ether, or CHCl3.
3. The aglycone moiety is soluble in organic solvents. Glycosides are easily
hydrolysed by water, mineral acids, and enzymes.

Isolation of glycosides by the Stats–Otto method

8.22.1 Isolation of Bitter Glycosides
Isolation of picrocrocin
We take for example the isolation of picrocrocin from Crocus sativus (saffron).
Picrocrocin is a colourless bitter glycoside obtained from the dried stigmas and
tops of the styles of Crocus sativus, belonging to the family iridaceae. It melts at
156℃.
Picrocrocin undergoes decomposition upon hydrolysis in the presence of
alkali to give safranal and glucose.

The dried powder is extracted with petroleum ether to remove lipids, then
with dry ether, which removes the picrocrocin. The crude picrocrocin is re-
extracted with dry ether and re-crystallised from methanol, ethanol and
methanol–chloroform–ether mixtures.
8.22.2 Isolation of Cyanogenetic Glycosides
Isolation of prunasin
Prunasin is obtained from the dried leaves of Prunus laurocerasus and dried
bark of Prunus serotiana, belonging to the family rosaceae. It is a D-
mandelonitrile glycoside which melts at 147–149℃.

Isolation of sinigrin
Sinigrin is isolated from the roots and leaves of Cochlearia armoracia
(cruciferae) and dried, ripe seeds of Brassica junica or B. Nigra (cruciferae). It is
a crystalline substance which melts at 127–128℃. It is readily soluble in water,
slighly souble in ethanol, and insoluble in ether and benzene.

During the isolation of sinigrin from Cochleria armoracia, the following

precautions need to be considered.

1. The fresh roots are cooled to –25℃ to inactivate the enzyme myrosin. The
roots are then extracted with methanol.
2. The sugars are removed by fermentation and the resulting solution is treated
with lead hydroxide to remove substances which interfere with
crystallisation.

Use
It stimulates digestion, increases gastric secretion and appetite. It is a good
diuretic and promotes perspiration. It is also an expectorant, and mildly
antibiotic. It can be used in both respiratory and urinary tract infections.
Tests for cyanogenetic glycosides
Cyanogenetic glycosides upon hydrolysis liberate hydrocyanic acid which
converts sodium picrate into sodium purpurate (colour changes from yellow to
brick red).
8.22.3 Isolation of Cardiac Glycosides
Cardiac glycosides can be isolated from the dried leaves of Digitalis purpurea
and Digitalis lanata, belonging to the family schrophulariaceae. The important
aglycones include digitoxigenin, gitoxigenin, gitaloxigenin and digoxigenin. The
extraction of cardiac glycosides basically involves the inactivation of the
glyosidase enzyme at a low temperature, and achieving neutrality by grinding
fresh leaves with ammonium sulphate. The total operation (Scheme 8.9) needs to
be carried out in a cold room.
Estimation
Method I: By enzymatic assay
Inhibition of the N+, K+ activated ATPase can be applied to estimate the total
cardenolides content in Digitalis lanata (Rodolfo Vingola and Macro Canella,
1982).
The enzymatic assay, based on inhibition of N+, K+ activated ATPase by
cardiac glycosides, is carried out on crude extracts of Digitalis lanata without
further treatments.
The enzymatic assay, based on inhibition of N+, K+ activated ATPase by
cardiac glycosides, is carried out on crude extracts of Digitalis lanata without
further treatments.
The enzymatic assay of digoxin cardenolides is compared to the Baljet
colorimetric procedure commonly used to determine the glycosidal content in
plant extracts. This assay proposed for the determination of cardiac glycosides in
plants provides a sensitive and highly specific method and can find useful
applications:

a. for screening new plants at a presumed cardiac activity;

b. as a complementary test in the biological assay in vivo for the
pharmacological research of natural products;
c. in plant cell culture experiments.
Scheme 8.9
PROCEDURE

1. Enzyme preparation: N+, K+ activated adenosine triphosphatase (E C 3. 6.

1. 3.) is purified from lamb kidney (outer medulla) as described by Lane et
al. (1979). The specific activity of these preparations corresponds to μmole
phosphorus liberated per mg of protein per hour. The enzyme is stored at –
20℃ in 0.5–1.0 ml stocks with a specific activity of 1,000 units/mg. The
loss of enzyme activity is up to 20% over a 6-month period of freezed
solution at –20℃. No substantial loss of activity is observed in the solution
at 5℃ over a 1-month period. 0.5 ml samples of stock solution of N+, K+-
activated ATPase are diluted with TRIS- EDTA buffer at pH 7.1 in order to
obtain 10–15 units in the assay. Protein concentration is determined by the
modified Lowry method with bovine serum albumin as the reference.
2. Glycoside solutions: Stock solutions of 40 mM digoxin are prepared in 80%
ethanol. The stock solutions are diluted to the required concentration with
the activity buffer constituted by 30 mM histidine–HCl, 100 mM NaCl, 5
mM MgCl2 at pH 7.1. The stock solutions and the dilutions of cardiac
glycosides are prepared immediately before use.
3. Extraction procedure: 1 gm sample of dried leaves of Digitalis lanata, after
grinding in a homogeniser to obtain a powder, is treated with 30 ml of 80%
aqueous ethanol for 1 hour at room temperature. The plant extract is diluted
with the activity buffer for the enzymatic assay.
4. Assay: To each tube containing 600 μl of activity buffer (30 mM histidine–
HCl, 100 mM NaCl, 5 mM MgCl2, pH 7.1) are added: 10 μl of plant
extract; 100 μl of ATP to a final concentration of 5 mM; 100 μl of a solution
containing about 10–15 units of N+, K+-activated ATPase. After pre-
incubation at 37℃ for 5 minutes, the reaction is started by adding 100 μl of
10 mM KC1. The reaction is interrupted after 5 min. at 37℃ by the
addition of 10% TCA to each tube. The protein precipitate is removed by
centrifugation (2,000 × g for 5 minutes). The supernatant solution is
decanted and analysed for inorganic phosphate (Pi), according to Le Bel et
al.’s method (1976). The amount of Pi liberated is quantitated from a
standard curve obtained by using a 1.0 mg/ml NaH2PO4 solution in distilled
 water. Control tubes include a blank for the assay of non-transport N+, K+-
activated ATPase activity, and a reference without inhibition. The test
requires 45 minutes of laboratory time.
5. Standard curve: In order to obtain a standard curve, which relates the
percentage activity of N+, K+-activated ATPase to the concentration of the
reference cardiac glycoside, 100 μl of digoxin solution (10, 20, 40, 60, 80,
100 mM) is added to the reaction mixture, following the assay procedure
described above.

Method II: By HPLC (determination of lanatoside C and digoxin in Digitalis

Lanata)
PROCEDURE

1. Drug material: D. lanata leaves are collected during the flowering stage.
They are immediately freeze-dried and then dried using P2O5 under reduced
pressure at room temperature. The dried leaves are pulverised and sifted
through a sieve of mesh width 500 μm. The leaf powder obtained is further
dried using P2O5 under reduced pressure for 5 days
2. HPLC analysis: Leaf powder (ca. 50 mg) is accurately weighed
anseparations of cardiac glycd extracted with 50% MeOH (25 ml),
containing desacetyllanatoside B (102.6 μg) as an internal standard. After
ultrasonication for 1 hr in an ultrasonic cleaning bath, the extract is filtered
and evaporated to dryness using a rotary evaporator. The residue is
dissolved m MeCN–H2O (2:8) (2 ml) and applied to a Sep-Pak C18 catridge
(Waters). Then, MeCN–H2O (2:8) (28 ml) and MeCN–H2O (3:7) (10 ml)
are successively passed through the cartridge. After evaporation of the latter
fraction using a rotary evaporator, the residue obtained is dissolved in
MeCN–CH3OH–H2O (20:1:50) (1 ml), and an aliquot (10 μl) of the sample
solution is submitted to HPLC. Lanatoside C and digoxin are determined by
the internal standard method. Calibration graphs are constructed by plotting
the ratio of the peak area of lanatoside C or digoxin to the peak area of the
internal standard against the weight of each compound. The average peak
areas from three chromatograms (Fig. 8.31) are used for the determination
(Yukari Ikeda et al., 1992).
Use
Cardiac glycosides are used in the treatment of congestive heart failure.
Tests for cardiac glycosides
1. Keller–Kiliani’s test (for deoxy sugars i.e., digitoxose): See tests for
carbohydrates
2. Baljet test: To the thick section of the drug, add a few drops of sodium
picrate solution and observe under a miscroscope. The yellow colour
changes to orange.
3. Legal's test: A few drops of pyridine and sodium nitroprusside are added to
the extract of the drug and made alkaline. A pink or red colour is obtained.
4. Liberman Burchard’s test: To a solution of the drug in glacial acetic acid,
one drop of concentrated sulphuric acid is added. A change in colour occurs
from rose, through red, violet, and blue to green. The reaction is due to the
steroid part of the molecule.

NOTE: Colours are slightly different from compound to compound.

Cardeinolides come out positive in the above four tests. Bufadeienolides test
positive in the LB test and negative in all the other tests.

Fig. 8.31 HPLC separations of cardiac glycosides. Peaks identified are: 1:

lanatoside C; 2: digoxin; 3: desacetyllanatoside B. Chromatographic conditions
are: column Chemcosorb 5 C8–U (4.6 mm × 150 mm); mobile phase MeCN–
MeOH–H2O (20:1:50); flow rate 0.5 ml/min; UV monitor 220 nm; sample
volume 10 μl. (a) Mixture of pure compounds. (b) Extract of Digitalis lanata
leaves without an internal standard, (c) Extract of D. lanata leaves with an
internal standard, (d) Extract of D. lanata leaves after addition of pure digoxin.
8.22.4 Isolation of Anthraquinone Glycosides
Isolation of rubiadin primveroside
Rubiadin primveroside is a glycoside containing rubiadin (aglycon) and
primeverose (glycon); it is obtained from the roots of Rubia tinctoria and
Galium verum, belonging to the family rubiaceae; the glycoside melts at 248–
250℃.
Estimation
By spectrophotometry (of sennosides and rhein glycosides in senna and its
preparations)
The method ensures complete elimination of other minor non-carboxylic
anthracene derivatives, as well as flavonoidal contaminants. The method
quantitates the actual total sennosides content, through the elimination of these
contaminates, and through correction for the interference due to the co-existence
of rhein with sennidins, in the final determinative step (Abdel-Azim M et al.
1980).
PROCEDURE

1. Preparation of reference sennidin A and sennidin B: In a boiling flask, 20

ml of water and 8 ml of 25% HC1 are added to 4.1 mg of sennoside A (or
sennoside B) and the mixture is refluxed in a boiling water bath for 20
minutes. After cooling, the mixture is quantitatively transferred into a
separator. The boiling flask is washed first with 5 ml of water, then with 20
ml of ethyl acetate. In each case, the washings are quantitatively transferred
to the separator. The mixture is shaken vigorously and, after phase
separation, the ethyl acetate phase is saved. The aqueous solution is re-
extracted with further fractions of 15 and 10 ml of ethyl acetate. The
combined ethyl acetate solution is washed with 10 ml of water and
transferred into a 50 ml volumetric flask. The original aqueous solution and
the aqueous washings are extracted with the same 5 ml of ethyl acetate and
the latter used in completing the volume of the solution in the volumetric
flask. As 4.1 mg of sennoside would give, by simple acid hydrolysis, 2.55
mg of sennidin, an additional 1 ml of ethyl acetate is added, so as to get 51
ml of a solution containing 5 μg of sennidin A (or sennidin B) per ml.
2. Standard solutions: For recording the UV spectra and determining
absorptivity, ethyl acetate solutions containing 20 μg per ml, are prepared
for each of sennidin A, sennidin B, aloe-emodin, and rhein.
3. Thin layer chromatography: TLC is performed on silica gel G
chromatoplates with benzene–acetic acid (4:1) as solvent system. Spots on
the developed chromatograms are visualised first under UV light, and then
by spraying with 5% methanolic potassium hydroxide solution.
4. Assay: The powdered drug (1 g) is extracted with 30 ml of water in a water
 bath at 80℃ for an hour with frequent agitation. After cooling, the aqueous
extract is decanted and filtered into a 100 ml volumetric flask; the marc is
saved in the original flask. It is then re-extracted with another portion of 30
ml of water for 30 minutes. The extract is decanted and filtered through the
same filter into the volumetric flask. The marc, the flask, and the filter are
washed with small successive volumes of warm water; the washings are
used to complete the volume of the solution in the volumetric flask.
A portion (10 ml) of the aqueous solution is transferred to a separatory
funnel, acidified with one drop of HC1, and extracted with two successive
portions of 40 and 20 ml of ether. The aqueous solution is saved in a boiling
flask. The combined ether extract is washed with 2 × 5 ml fractions of
water, the ether phase is discarded, and the aqueous washings are added to
the saved aqueous solution in the boiling flask.
8 ml of 25% HC1 is added and the mixture refluxed in a boiling water bath
for 20 minutes. The mixture, with any precipitate, is transferred
quantitatively to a separator; 5 ml of warm water is used to transfer any
retained precipitate. The mixture is then extracted with 3 × 30 ml fractions
of ethyl acetate. The brown deposit (at the interphase) is taken out each
time with the aqueous phase, and the ethyl acetate solution is filtered into a
100 ml volumetric flask. The filter is washed with small volumes of ethyl
acetate, and the filtered washings are used to complete the volume of the
ethyl acetate solution.

A series of three separators is prepared; 25 ml of the ethyl acetate solution is

transferred to the first, and 20 ml of pure ethyl acetate to each of the other two.
The three separators are shaken, in a consecutive manner, with the same two
portions of 25 and 20 ml of freshly prepared saturated sodium bicarbonate
solution. The ethyl acetate solutions are discarded. The combined bicarbonate
solution is acidified with 10 ml of HC1; and the mixture is shaken very gently to
help complete elimination of any latent effervescence; it is then extracted
successively with 25 and 20 ml of ethyl acetate. The combined ethyl acetate
solution is washed with 10 ml of water, transferred into a 50 ml volumetric flask
and completed to volume. The extinction of the solution is measured against
pure solvent at 377 and 430 nm, and the percentage of sennosides and total rhein
glycosides is calculated, by the application of formula A and formula B (below),
respectively.
 where A377 = absorbance of the test solution at 377 nm.
A430 = absorbance of the test solution at 430 nm.
arh;430 = absorptivity of rhein at 430 nm.
arh;377 = absorptivity of rhein at 377 nm
asd;377 = absorptivity of sennoside A: B (2:3) mixture at 377 nm.
asd;430 = absorptivity of sennoside A: B (2:3) mixture at 430 nm.
1.6074 = factor of transformation of sennidins to sennosides.
1.57 = factor of transformation of rhein to rhein monoglucoside.
47.5, 18.5, 35.4, and 8.2 = absorptivity values.
Assay of calcium sennoside tablets
The tablets (20 tablets) are weighed and reduced to a very fine powder. To a
weight equivalent to one tablet, 30 ml of water and 10 ml of 25% HC1 are
added; the assay is continued as in the case of reference sennidin A and B after
the 25% HC1 is added. The UV absorption spectra of sennidin A. sennidin B,
rhein and aloe-emodin is shown in Fig. 8.32.
Estimation
By micellar electrokinetic capillary chromatography (of rhubarb)
A micellar electrokinetic capillary chromatographic method for the separation
and quantitative determination of aloe-emodin, emodin, and rhein in rhubarb
sample can be used. The running electrolyte is a mixture of 0.025 M 3-
(cyclohexylamino)-l-propane sulphonic acid (CAPS) buffer (containing 0.025 M
sodium dodecyl sulfate (SDS)) and acetonitrile (100:10), pH 10.96, with an
applied voltage of 12 kV. This method resolves the three components as well as
chrysophanol and physicion from the other constituents present. The technique is
simple, rapid, and reproducible (Yu-Ying Zong and Chun-Tao Che, 1995).
Fig. 8.32 UV absorption spectra of sennidin A, sennidin B, rhein and aloe-
emodin in ethyl acetate

PROCEDURE

1. Analyses can be carried out on a Bio-Rad HPE 100 capillary

electrophoresis system equipped with a UV detector set at 500 nm and a
microsampler 100 cartridge (uncoated capillary, i.d: 50 cm × 50 pm.).
2. Plant material: The roots and rhizomes of Rheum plamatu, R. tanguticum,
and R. emodi are collected and authentified by a taxonomist. Plant materials
are air-dried, milled, and stored in enclosed containers until used.
3. Preparation of plant extracts: To prepare extracts of hydrolysed samples,
dried plant powder (100 mesh, 0.04 g) is added to 0.5 ml of 20% H2SO4
and stirred for 5 minutes. CHCl3 (50 ml) is then added and refluxed for 1 hr.
After cooling to room temperature, anhydrous Na2SO4 (2.5 g) is added, and
the solution is filtered through a No. 1 filter paper. The residue is further
washed by CHC13. The CHC13 extract and washings are then combined
and excess solvent removed in vaccum. The residue is dissolved in EtOAc
and diluted to 5 ml. An aliquot (0.1 ml) is then removed and after addition
of 6 μl of internal standard solution (1 mg of 1,8-dihydroxyanthraquinone
in 1 ml EtOAc), dried in vaccum. Finally, the residue is re-dissolved in 0.2
ml of running buffer and filtered through a 0.5 μm filter. The filtrate is
loaded directly (4 seconds) into the capillary electrophoresis system. To
prepare extracts of non-hydrolysed samples, dried plant powders (100
mesh, 0.1–0.3 g) are added to CHC13(50 ml) and refluxed for 20 minutes.
After cooling to room temperature, the extract is filtered. The extraction
procedure is repeated several times until the CHC13 extract become
colourless. The CHC13 solutions are combined, dried, and re-dissolved in 2
ml of EtOAc. An aliquot (0.1 ml) of the EtOAc solution is treated as
described for the hydrolysed samples.
4. Capillary electrophoretic conditions: Sampling time: 4 seconds; run time:
15 minutes; applied voltage: 12 kV (constant voltage, positive to negative
polarity). The electrolyte is a buffer solution consisting of 0.025 M CAPS
and 0.025 M SDS, mixed with MeCN (100:10). NaOH is added to a pH
value of 10.96.
 5. Recovery study: Known amounts of aloe-emodin, emodin, or rhein
standards are added to CHC13 extracts R. tanguticum having known
anthraquinone contents. The mixtures are processed and analysed as
described above. Figure 8.33 shows the electropherogram of reference
compounds and the internal standard, while Fig. 8.34 shows the
electropherogram of an unknown component compared to known aloe-
emodin, emodin and rhein standards.

Tests
Borntrager’s test
Boil the powdered drug with dilute sulphuric acid, filter and add chloroform to
the filtrate. Shake well and collect the organic layer. A few drops of strong
ammonia solution is added, shaken slightly and the test tube kept aside for a few
minutes. The lower ammonical layer takes on a pink or red colour. This test is
due to the presence of anthraquinones (O-glycosides) and comes out negative in
the case of reduced forms of anthraquinones i.e., anthranols.
Fig. 8.33 Capillary electropherogram of reference compounds and the internal
standard (peak 1, aloe-emodin; peak 2, 1, 8-dihydroxyanthraquinone; peak 3,
chrysophanol; peak 4, emodin; peak 5, physcion; peak 6, rhein) Modified
Borntrager’s test (for anthranols)

Boil the powdered drug with ferric chloride solution and a few drops of dilute
sulphuric acid. Filter. A few ml of chloroform is added to the filtrate. Shake well
and collect the organic layer. A few drops of strong ammonia solution is added,
shaken slightly and the test tube kept aside. The lower ammonical layer shows
rose pink to cherry red colour.
Fig. 8.34 Capillary electropherogram of a CHC13, extract of hydrolysed R.
tanguticum sample. (Unlabelled peak, unknown component; peak 1: aloe-
emodin; peak 2: 1, 8-dihydroxyanthraquinone; peak 3: chrysophanol; peak 4:
emodin; peak 5: physcion; peak 6: rhein) 8.22.5 Isolation of Flavanol Glycosides

Isolation of kampferol -3,7-dirhamnoside

Kampferol-3,7-dirhamnoside is a flavanol glycoside, found in leaves of
Celastrus orbiculate and other species. It melts at 190–195℃.
Isolation of kampferol-3-glucoside
Kampferol-3-glucoside is isolated from the flowers of Astraglus sinius and other
species. It melts at 178℃.
Isolation of quercitrin
Quercitrin is isolated from the bark of Quercus tinctoria.
Isolation of rutin
Rutin is a yellow crystalline powder obtained from the whole plant of Ruta
graveolens (rutaeceae) and flowers of Sambucus nigra (caprifoliaceae).
It is soluble in alkali and sparingly soluble in water. Rutin on hydrolysis yields
quercetin, rhamnose and glucose. It melts at 188–189℃.
Estimation
By spectrophometry (Gulnur Toker et al., 1997):
PROCEDURE

1. TLC: Methanolic extraction of the air-dried and powdered material and

rutin (Merck Pharmaceuticals) in methanol as a standard, are performed on
TLC. Kieselgel 60 PF254, pre-coated to 0.2 mm thickness plastic plates
(Merck), and ethylacetate: methanol: water (100:16.5:13.5) as a solvent
system are used in thin layer chromatography. Spots are visualised under
UV light (366 nm) after spraying with A1C13 in methanol.
2. Calibration curve of rutin standard solution: 10 mg of rutin (Merck) is
dissolved is 25 ml spectroscopic grade methanol (stock solution).
Absorption of the suitable rutin dilution is scanned between 220–500 nm
wavelengths. Maximum absorbance points are 257 and 355 nm
wavelengths. The A1C13 spectrum is measured immediately after the
addition of the six drops of the 5% A1C13 solution (in spectroscopic grade
methanol) to 2–3 ml of rutin solution. Maximum absorbance points are
273.5 and 436.5 nm respectively. Five dilutions in the range of 1.6 × 10–2
and 2.8 × 10–2 mg/ml are prepared from the stock solution. The calibration
curve is obtained at 273.5 nm, by using these solutions, after colouration
 with A1C13 solution.
3. Preparation of samples: Powdered and weighed plant material is extracted
with diethyl ether in a Soxhlet apparatus. After evaporation of the solvent,
the material is dried and extracted with methanol at room temperature, three
times. Combined methanol extract is evaporated to dryness in vaccum. The
crude extract is dissolved with spectroscopic grade methanol and a series of
dilutions prepared. The solutions in suitable concentration are scanned after
colouration with A1C13 solution, and the absorbance value determined at
273.5 nm wavelength. The amount of rutin is determined from the
regression equation
Y (absorbance) = 46.34 × (concentration mg/ml).

Isolation of andrographolide
It involves extraction of the leaf powder by cold maceration in a 1:1 mixture of
dichloromethane and methanol and direct isolation of andrographolide from the
resulting extract by re-crystallisation (M. Rajani et al., 2000).
PROCEDURE

1. The fresh leaves of A. paniculata is purchased from a reliable source and

authentified by a taxonomist. The material is air-dried under a shade for a
day and then in a hot air-oven below 60℃. It is powdered to 40 mesh and
stored in an air-tight container at 15–20℃ until further use.
2. Extraction and isolation: The sample of leaf powder (50 gm) is extracted
exhaustively with a 1:1 mixture of dichloromethane and methanol by cold
maceration. The extract is filtered and the solvent removed under vacuum.
3. The dark green crystalline mass obtained is washed separately with toluene
several times, until most of the colouring matter is removed from the
residue. The toluene is then completely removed from the residue. The
crystalline material left behind (yield:1.9–2.0 g) is dissolved in hot
methanol and cooled in a refrigerator for crystallisation. The process is
repeated several times until colourless plates of constant melting point 230–
231 ℃ are obtained.

Tests for flavonoids

1. To the test solution, add FeCl3 solution. A change of colour from green to
 black occurs.
2. Mg–HCl reduction test (Shinoda test):
To the plant extract, add a mixture containing a piece of magnesium ribbon
and concentrated hydrochloric acid – red colour indicates flavonoids,
flavonones, flavononols and xanthone
NOTE: Pink, purple or red magenta colour may also be observed in some
derivatives of flavonoids.
3. Sodium hydroxide solution test:
To the solution, add 10% sodium hydroxide – yellow colour is obtained.
4. Lead acetate solution test:
To the test solution, add 10% lead acetate solution – yellowish ppt. is
obtained.
5. Mineral acid test:
To the test solution, add concentrated sulphuric acid – yellow-orange colour
is obtained.
6. Flavonoids dissolve in alkalis giving yellow solutions, which become
colourless on addition of acid.

8.22.6 Isolation of Coumarins

Coumarins like cichoriin, frachinoside, fraxin, etc are obtained from the dried
leaves of Fraxinus chinensis, belonging to the family oleaceae.

The fractionation and isolation scheme for various coumarins is described

below.
8.22.7 Isolation of Saponin Glycosides
Isolation of gymnemic acids
The following-method was used by Joseph E. Sinsheimer, G. Subba Rao and
Hugh M. Mcllhenny to-isolate gymnemic acids from the leaves of Gymnema
sylvestre. The Rf values of the various gymnemic acids in different TLC solvent
systems is given in Table 8.37.
Table 8.37 Thin layer chromatography of gymnemic acids
a Listed in decreasing order of Rf values; b Solvent systems; I: chloroform–
formic acid–methanol (4:1:1); II: chloroform–acetic acid–methanol (5:1:1); III:
chloroform–formic acid–methanol–t-butanol (4:1:1:1); IV: isopropanol–
ammonium hydroxide–chloroform–t-butanol (5:2:1:1); V: isopropanol–
ammonium hydroxide–isoamyl alcohol (3:2:1); VI: isopropanol ammonium
hydroxide diethyl carbonate–isoamyl alcohol–t-butanol (3:2:2:1:1); VII: butyl
formate–methyl ethyl ketone–formic acid–water (5:3:1:1).
The results of the silicic acid chromatography done on acetone soluble acids
and ethyl acetate acids are presented in Tables 8.38 and 8.39.
Table 8.38 Silicic acid chromatography of acetone soluble acids
a Detection by TLC in solvent system III; b Purification procedures: (i):
adsorption chromatography on de-activated silica gel; (ii): reversed phase
partition chromatography on Teflon-6; (iii): preparative TLC; c Homogeneous by
TLC in solvent systems I–VII; d Stearic acid isolated from these fractions; e
Isolated as an amorphous solid.
Table 8.39 Silicic acid chromatography of ethyl acetate acids

a Detection by TLC in solvent system III; b Purification procedures; (i):

adsorption chromatography on deactivated silica gel; (ii): reverse phase partition
chromatography on Teflon-6; c Homogeneous by TLC in solvent systems I–VII.
Estimation of glycyrrhizin
By HPLC
A reversed phase high pressure liquid chromatography method to determine
glycyrrhizin in G. glabra and its extract can be used. The method involves
separation of the compound using the mobile phase acetonitrile: water:
phosphoric acid (32:67:1) and detection of chromatogram at 250 nm using a
photodiode array detector. The sensitivity of the method is observed to be 2.0 μg
and the linearity is observed in the range of 2.0–16.0 μg/μl. Being precise,
sensitive and reproducible, the method can be used to detect, monitor and
quantify glycyrrhizin in G. glabra and its extract (S.K. Chauhan et al., 1998).
PROCEDURE

1. Extraction: Around 25 gm of dried root is ground to pass through a 40 mesh

sieve. One gm from it is accurately weighed and extracted with water (15
ml × 6) over a steam water bath. The extracts are filtered, pooled and a
volume of 100 ml is made up with water. 1 ml of it is further diluted to 2 ml
with water. In case of G. glabra extract, around 250 mg of the extract as dry
powder is accurately weighed and dissolved in 100 ml of water. It is filtered
to remove the suspended, particles, if any, and the volume is made up to
100 ml. These solutions are used further for HPLC analysis as mentioned
below.
2. Standard preparation: A 1.0 mg/ml solution of glcyrrhizin reference
standard is prepared in methanol. It is further diluted with methanol to yield
the final concentration of 0.10, 0.20, 0.40, 0.60 and 0.80 mg/ml.
3. The test samples are passed through a 0.45 micron filter and 20 μl each of
test solutions and five different concentrations of standard glycyrrhizin (0.1,
0.2, 0.4, 0.6 and 0.8 mg/ ml) are injected to HPLC using Nova Pak C-18
reversed phase column (3.90 × 150 mm) with a RPC 18 guard column. The
mobile phase is acetonitrile: water: phosphoric acid (36:63:1), and the flow
rate is 1.4 ml/minute. The chromatogram is scanned up to 15 minutes which
is detected at 250 nm. The amount of glycyrrhizin is determined from the
linear regression equation of the calibration curve plotted between
concentrations, and area of standard glycyrrhizin. The equation for
 glycyrrhizin is
y = 215416 x + 56705
with a correlation coefficient of 0.9994 where y is the response in peak area
and x is the concentration of the compound analysed.
4. Method validation and recovery studies: A known amount of standard
glycyrrhizin is added to the powdered crude drug as well as to its extract, in
which the contents of glycyrrhizin has been estimated previously by the
proposed method. The samples are processed and analysed separately as per
the procedure mentioned above. The contents of glycyrrhizin are quantified
and percentage recovery calculated. The HPLC chromatograms of crude
and extracted glycyrrhizin are given in Fig. 8.35.

Fig. 8.35 HPLC chromatograms of glycyrrhizin: (a) crude G. glabra; (b)

extracted G. glabra

By second derivative UV spectrophotometry (extraction of total glycyrrhetic

acid)
Glycyrrhizin (G) obtained from Glycyrrhizae radix is hydrolysed into
glycyrrhetic acid in 2N HC1 and methanol (1:1), and extracted from the aqueous
phase in the form of an ion-pair complex with tetrapentylammonium bromide
(TPA) as a counter ion. Maximum D2 amplitude (Z value) is obtained when a
1000-fold or greater molar ratio of TPA is used at pH 11. Dichloromethane is an
effective extraction solvent of the ion-pair complex (Seung-BaeSong et al.,
1990).
PROCEDURE

1. Extraction and hydrolysis: Dried powder of Glyrrhizae radix (0.01 gm) is

weighed accurately, placed in 50 ml solution of 2 N HC1: methanol (1:1),
and refluxed on a heating mantle at 80℃ for 90 minutes. Methanol is
evaporated and a 2 N NaOH solution is added to adjust the pH to 11. This
solution is filtered (millipore size: 0.45 μm) and the residue is washed twice
with 10 ml distilled water. The extract and washings are put into a 100 ml
measuring flask and made up to 100 ml with distilled water for a sample
stock solution. For the standard, 4.07 mg of 18 (β-GA is weighed accurately
and 50 ml of 2 N HC1 and methanol mixture (1:1) is added. The rest of the
procedure is the same as for the sample.
2. Extraction of ion-pair complex: 10 ml of stock solution of GA is transferred
into 60 ml separating funnel and mixed with the same volume of TPA stock
solution (1 × 10–1 M). It is shaken for a minute at room temperature and
kept standing for 15 minutes to extract the ion-pair complex. 10 ml of
dichloromethane is added and shaken vigorously for two minutes and the
mixture is allowed to stand until the phase separation is completed. The
organic layer is transferred into a 50 ml volumetric flask and the same
procedure repeated twice. Dichloromethane is added up to the volume.
3. D2 UV spectrometry. For the determination of GA in a sample extract, the
standard addition method is used. 100, 200, 300 μl of standard stock
solution of GA are transferred into each separating funnel containing 10 ml
of sample extract. The total ion-pair complex with TPA is extracted with
dichloromethane following the procedure described. D2 UV spectra of the
samples are recorded. The contents of GA in the powder form and the
extract are calculated in comparison with D2 amplitudes (Z value at 255
nm). The D2 UV spectra is shown in Fig. 8.36.
4. HPLC determination: The hydrolysed sample stock solution (10.0 ml) is
 treated with 2 N HC1 for acidification, and then extracted with
dichloromethane. The organic phase is dried and re-dissolved in methanol
for HPLC injection. The mobile phase for HPLC is a mixture of methanol
and chloroform (1:1). The flow rate is 3.0 ml/minute at room temperature.
The eluent is monitored at 250 nm. Each 10 μl of the pre-treated stock
solutions of extract and powder is injected. The HPLC chromatograms of
GA (A) and extract of glycyrrhizae radix (B) is shown in Fig. 8.37. The
assayed contents of GA in powder and extract are calculated in comparison
with the peak height of the standard GA. A calibration curve is prepared
using GA as a standard solution (Fig. 8.38).

Estimation of diosgenin
ByGLC
B.S. Dixit et al. (1981) described a method to estimate the amount of diosgenin
obtained from Costus speciosus.
PROCEDURE

1. Dried rhizomes of C. speciosus are finely powdered in a mixer. 10 gms of

powdered sample is hydrolysed with 2.5 N hydrochloric acid for two hours.
The hydrolysed material is filtered and washed well with water, dried and
extracted with petroleum ether (60–80°) for eight hours in a Soxhlet
apparatus. The solvent is removed and the residue subjected to GLC.
2. GLC is carried out on a Varian Aerograph Model 1868-4 with a flame
ionisation detector. A stainless steel column 5' × 1/8" packed with
Chromosorb W (80–100 mesh) coated with 3% SE is employed. The
operating temperature of the oven is 250℃, the injector block 270℃ and
the detector 235℃. It is found that the column is quite stable. The flow rate
of nitrogen and the carrier gas, is 100 ml/minute. The retention time of into
stainless-steel tubings (i.d.: 500 × 20 mm and 500 × 50 mm). The system is
operated at room temperature.
3. Sample preparation from the crude drug: Roots of Panax ginseng are
pulverised and extracted with methanol. The extract is evaporated and
dissolved in water. The aqueous solution is passed through a solid phase, a
mega bond elute, pre-treated with water and methanol. After the solid phase
is washed with water and 30% methanol, the sample is eluted with
methanol and the eluate is evaporated to dryness under reduced pressure.
 The residue is dissolved in the eluent and injected into the preparative
HPLC system.

Estimation of ginsenosides
By HPLC (M. P. Gomez-Serranillos et al. 1997)
PROCEDURE

1. Standard reference solutions: About 2 mg of each ginsenoside (Rb1, Rb2,

Rc, Re, Rd and Rg1,) is accurately weighed and dissolved in 1 ml of the
mobile phase.
2. Chromatographic system: Variant 2510 pump and Varian Polychrom 9065
photodiode-array detector operating at 205 nm with a Varian DS 654 data
processor.
3. Column: Chromatographic runs are performed on a Hypersil ODS column
(20 cm × 4.6 mm; 5 μm) (Shandon Scientific Ltd., U.K.) at room
temperature.
4. Mobile phase: Water–MeCN (9:1 v/v) from pump A and MeCN from pump
B constitutes the mobile phase. The gradient elution is started with 75% A
and 25% B for 20 minutes and decreased up to 50% A in 20 minutes.
5. The equilibration time is 10 minutes, the flow rate is 2 ml/minute and
samples of 10 μl are injected.

The identification of the saponins is achieved by comparing both the retention

times (Rt) and the absorption spectra obtained for each eluted peak with those
obtained for the standards. To check the peak purity, the eluates are monitored
with a photodiode-array detector (200–400 nm). The three spectra corresponding
to the upslope, apex and downslope of each peak are normalised and
superimposed. Peaks are considered pure when there is an exact coincidence
between the three spectra.
Several aliquots of the standard solution of ginsenosides Rg1, Rb2, Rb1, Re,
Rd and Rc are diluted in the eluent to obtain reference solutions of decreasing
concentration. These solutions are analysed and the corresponding peak areas are
plotted against the concentration of ginsenosides injected. The HPLC profile of
the six ginsenosides is shown in Fig. 8.39.
The concentrations of the components are calculated from the chromatogram
peak areas, using the normalisation method.
Tests for saponin glycosides

1. Foam test: To 5 ml of aqueous extract, add a drop of sodium bicarbonate

solution, shake vigorously and allow to stand for 3 minutes. A honeycomb-
like mass is produced.
2. Haemolytic test: The haemolytic property of the sapogenins is used for their
identification.

Fig. 8.39 HPLC profile of six ginsenosides analysed. (1: Re; 2: Rg1; 3: Rb2;
4: Rc; 5: Rb1; 6: Rd)

3. LB's test: Used for steroidal saponin glycosides (see tests for cardiac
glycosides).

Estimation of picroside-l and kutkoside

By HPLC (from Picrorrhiza kurra by S. K. Chauhan et al. 1999)
PROCEDURE
About 25 gm of air-dried sample is ground to pass through a 40 mesh sieve. 1
gm of ground sample is weighed accurately and extracted with water (10 ml × 6
ml) or till the colour persists, over a steam water bath. The extract is filtered,
pooled and the final volume of 100 ml is made up with water. 1 ml of it is further
diluted to 4 ml with water and used for further HPLC analysis using a M/s
Waters HPLC system, consisting of a Rheodyne 7125 injector, a 996 photodiode
array detector, a 510 chromatographic pump and millennium software v. 2.10.
The test sample is passed through a 0.45 micron filter. 20 μl of test solution and
four different concentrations of standard picroside-I and kutkoside (0.0125,
0.0250, 0.050 and K. 075 mg/ml) are injected into a Nova Pak C-18 reversed
phase column (3.9 × 150 mm) with a reversed phase guard column. The mobile
phase is acetonitrile: water: phosphoric acid (16:83:1) and the flow rate is 1.0
ml/minute. The chromatogram is scanned up to 20 minutes which is detected at
270 nm. The amount of picroside-I and kutkoside in the test sample is
determined from the linear regression equation of the calibration graph, plotted
between concentration and area of respective standards. The equation for
picroside-I is Y= 1161528 x + 10900
with a correlation coefficient of 0.9995, while the equation for kutkoside is
Y= 1094118 x +73663
with a correlation coefficient of 0.9999, where x is compound analysed and Y
is the response in peak area. To validate the method, a known amount of
standard picroside-I and kutkoside are added to about one gram of powdered test
material in which the contents of picroside-I and kutkoside has been estimated
previously by the proposed method. The samples are extracted and analysed
separately as per the procedure mentioned above. The contents of picroside-I and
kutkoside are quantified and the percentage recovery calculated. Figure 8.40
depicts the HPLC chromatograph of P. kurroa.
Isolation of azadirachtin
Azadirachtin is the best natural anti-feedant. It occurs with many similar
tetranortriterpenes, in the seeds of Azadirachtin indica.
The schematic representation of an extraction procedure for azadirachtin from
the seeds of Azadirachta indica is given in Scheme 8.10 (Daniel R. Schroeder
and Koji Nakaishi 1987).
Fig. 8.40 HPLC chromatogram of P. kurroa
Scheme 8.10
Estimation
Method I: by HPLC
P. K. Gupta and V.V. Prabhu (1997) described the following procedure for the
estimation of asadirachtin.
PROCEDURE

1. The neem fruit sample is de-pulped and the seeds are shade dried. The seed
coats are removed manually to get NSK. 10 gm of NSK is powdered and
de-fatted with petroleum ether (40–60°) in a Soxhlet apparatus for twenty-
four hours.
2. The de-fatted marc is allowed to stand over 50 ml methanol at room
temperature and filtered. This process is repeated five times in forty-eight
hours. The extracts are pooled and concentrated to a volume of 50 ml using
a rotary evaporator, and to this, 50 ml of distilled water is added. The
aqueous methanolic extract is partitioned with 100 ml of petroleum ether
(60–80) five times, followed by extraction with 100 ml of dichloromethane,
five times. The dichloromethane extract is pooled together and the residue
obtained on distillation of the solvent is dissolved in 40 ml of HPLC grade
methanol. The solution is taken for determination of azadirachtin content
using an HPLC system. The conditions are as follows:

HPLC system : Waters

Pump : Model 501

Column : Reverse phase Novopack C18 stainless steel

column.

Column size : 300 × 3.9 mm2

Injector : U (6k)

Eluting : Acetonitrile: water (30:70)

solvents

Solvent flow : Isocractic

 Flow rate : 1ml/minute

UV wave : 214 nm
length

Retention time : 12.46 minute

Method II: by Colorimetry

A colorimetric method can be used for the determination of total azardirachtin-
related limonoids (AZRL) in neem seed kernel extracts. The method uses
acidified vanillin solution in methanol for the colourisation of the standard
azardirachtin or neem seed kernel extracts in dichloromethane (Jainming Dai et
al., 1999).
Extraction procedures
PROCEDURE 1: Microwave-assisted extraction (MAE) with dichloromethane
Blended fresh neem seeds kernel (2.00 g) is placed in a 250 ml quartz extraction
vessel of the Synthewave 402 microwave system. Petroleum ether (15 ml) is
then added. The vessel Is inserted inside the microwave cavity, fitted with a
condenser, and irradiated in the following sequence at 80% power (240 W): 1
minute on, 30s off, and 1 minute on. This process is repeated three times. The
petroleum ether extracts are combined and evaporated to yield 0.234 gm of fat.
The de-fatted sample is extracted three times with dichloromethane (15 ml)
using the following sequence: 30 s on, 30 s off, 30 s on, 30 s off, and 30 s on, at
50% power level (150 W). The dichloromethane extracts are filtered, and the
filter paper is washed with dichloromethane (5 ml). The combined
dichloromethane extracts are evaporated under vacuum on a rotary evaporator at
40℃ to give a yellow residue (0.047 gm) termed MAE-D. The extract is
dissolved in dichloromethane (0.40 mg/ml) for the vanillin assay.
PROCEDURE 2: MAE with methanol followed by dichloromethane partition
The extraction procedure is the same as that of PROCEDURE 1, except that the
solvent for extraction after the de-fatting step is replaced with methanol. The
petroleum ether extract yields 0.244 gm of fat and the methanol extract 0.214 gm
of residue. The methanol extract is re-dissolved in methanol (10 ml) and water
(10 ml), followed by the addition of 5% sodium chloride solution (1.0 ml). This
mixture is extracted with petroleum ether (6 × 20 ml) to further remove any
remaining fat. The residue is then extracted with dichloromethane (3 × 20 ml).
The combined dichloromethane extracts are dried over Na2SO4, and the solvent
is evaporated under vacuum to obtain 0.081 gm of an amorphous light yellow
solid, termed MAE-M. The product is dissolved in dichloromethane (0.33
mg/ml) and in methanol (0.029 mg/ml) for further analysis.
PROCEDURE 3: Room temperature extraction (RTE) with dichloromethane
A suspension of blended neem seeds kernel (2.00 gm) in petroleum ether (60 ml)
is stirred to room temperature for 12 hr. The petroleum ether extract yields 0.406
gm of fat. The de-fatted sample is extracted with dichloromethane (3 × 20 ml) by
stirring at room temperature for 12 hr. The extract is evaporated under vacuum to
give a yellow oil (0.337 gm) termed RTE-D. The product is dissolved in
dichloromethane (0.80 mg/ml) for the vanillin assay.
PROCEDURE 4: RTE with methanol followed by dichloromethane partition
The extraction procedure is the same as that of PROCEDURE 3, except that the
solvent for extraction after the de-fatting step is replaced with methanol (3 × 20
ml). The petroleum ether extract yields 0.393 gm of fat, and the initial methanol
extract yields 0.249 gm of residue. The partition procedure is the same as that in
PROCEDURE 2. The dichloromethane layer, after vacuum evaporation, gives an
amphorous solid (0.101 gm) termed RTE-M. The product is dissolved in
dichloromethane (0.20 mg/ ml) and in methanol (0.024 mg/ ml) for further
analysis.
PROCEDURE 5: Comparative study of different extraction methods
Blended neem seed (2.000 g) is stirred overnight in petroleum ether (60 ml) at
room temperature. After filtration, the de-fatted residue is used to study the
efficiency of the different extraction methods.

a. MAE: The de-fatted seeds are extracted with methanol (50 ml) using the
following irradiation sequence at 150 W: 30 s on and 30 s off for a total of
8.5 minutes of irradiation time. At the end of the irradiation sequence, the
solution is left for ~1 minute, before it is filtered and evaporated in vaccum
to yield an orange amorphous solid (0.25 gm). The extract is dissolved in
dichloromethane for vanillin assay.
b. RTE: The above procedure is followed; except for the fact that the
extraction step is performed with stirring at room temperature for 10
minutes. Total yield is 0.26 gm.
 c. RFX: The same procedure is followed, except that the extraction is carried
out in refluxing methanol for 10 minutes. Total yield is 0.26 gm.

Colorimetric determination of the total AZRL

To a dichloromethane solution (0.7 ml) of standard AZ or neem seed extract, is
added a methanol solution (0.2 ml) of vanillin (0.02 mg/ ml). After manually
shaking, the mixture is left at room temperature for 2 minutes. Concentrated
sulphuric acid (0.3 ml, 98%) is then added in three portions (0.1 ml each), and
the mixture is stirred for 10 s after each addition. After the addition of sulphuric
acid is completed, methanol (0.8 ml) is added to convert the two-layer mixture
into a homogeneous solution that instantly develops a blue-green colour. The
solution is left at room temperature for 4 minutes before the absorbance is
measured at 577 nm, using a Beckman DU-64 spectrophotometer, equipped with
a 10 mm quartz cell. The blank solution is obtained by substituting the test
solution with an equal volume of dichloromethane in the above procedure. The
total AZRL in the neem seed extracts (concentrations as stated under Extraction
procedures) are quantified using a calibration curve (R2 = 0.9995) generated
from standard AZ solutions in dichloromethane (0.01–0.10 mg/ml).

8.23 ISOLATION OF CAROTENOIDS

Carotenoids are C-40 tetraterpenoids (composed of isoprene units), lipid soluble,
unsaponifiable pigments, widely distributed in plant tissues. They are found in
all kinds of plants, from simple bacteria to yellow-flowered composites. All
green tissues of plants contain carotenoids, located mainly in the grana of the
chloroplasts. In plants, they perform two functions: they are the accessory
pigments in photosynthesis and are the colouring matters in flowers and fruits.
The following properties need to be considered for the isolation of
carotenoids.

a. Cartenoids are unstable pigments.

b. When exposed to air, they easily undergo oxidation.
c. They may also undergo trans-cis isomerism during handling.
d. They are soluble in organic solvents.
e. Solutions of carotenoids should be kept in the dark as much as possible, i.e.,
 they should be stored at low temperatures under nitrogen gas. Peroxide-free
solvents must be employed.
The schematic representation for the separation of plant carotenoids is
given in Scheme 8.11.

NOTE

1. The tissues must be fresh and undamaged.

2. The tissues must be first dehydrated with acetone or anhydrous NaSO4, and
then subjected to extraction.
3. The carotenoid extracts (especially xanthophyll fractions) should be
saponified as it removes the chlorophylls (the presence of which, often,
makes it difficult to follow the resolution of the xanthophylls) and neutral
fat (which interferes with chromatographic separation of the pigments and
also with the isolation and crystallisation of individual pigments).
4. The solvents used are light petroleum (sometimes, containing a little ether)
which form the epiphase and 90% methanol (90% v/v aqueous), the
hypophase.
5. If the light petroleum ether is shaken with 90% methanol, all the
xanthophylls containing two or more free hydroxy or keto groups are
extracted. The carotenes, their epoxides, monoketo- or monohydroxy-
derivatives, or xanthophylls with their hydroxy group esterified or
methylated, remain in the epiphase.
6. In general, the following adsorbents are used, for the separation of
carotenoids (arranged in order of increasing adsorptive power).
Starch < Sucrose < CaC03 < Calcium phosphate < Zinc carbonate < Bone
meal < Magnesium carbonate < Alumina (deactivated) < MgO (Merck) <
Calcium hydroxide < Calcium oxide <Alumina (Merck, activated)
7. The following solvents are used for the chromatographic separation of
carotenoids (arranged in order of increasing polarity).
light petroleum < solvent ether < acetone < benzene < chloroform < 1,2-
dihloroethane < ethanol < methanol < water + acetic acid < glacial acetic
acid
Tests

1. An ether or chloroform extract of crude drug when tested with 85%

sulphuric acid, gives a blue colour at the junction of two layers.
2. Most of the carotenoids give a blue colour with antimony trichloride in
chloroform.
3. Carotenoids give a dark blue colour with concentrated hydrochloric acid
containing little phenol.

Examples
Isolation of capsanthin
Capsanthin is a fruit carotenoid, isolated (Scheme 8.12) from the pods of red
pepper or paprika (capsium annum).
Identification

1. Solution of capsanthin in chloroform + concentrated sulphuric acid → deep

blue colour.
2. λmax 486 and 520 nm in benzene, 475 and 504 nm in n-hexane; 503 and 542
in carbon disulphide.
Scheme 8.12
Isolation of β-carotene and xanthophylls (lutein, violaxanthin and
neoxanthin)
β-carotene and xanthophylls are leaf carotenoids. The following simple
procedure can be used for the separation of these carotenoids.

Similarly, the xanthophylls band is re-run on a silica gel G plate in

dichloromethane: ethylaetate (4:1), and the three bands of decreasing Rf value,
namely lutein, violaxnathin and neoxanthin are eluted with ether. The separated
pigments can be identified by spectral measurements, β-carotene has λmax 466
and 497 nm in CHC13; lutein has λmax 428, 456 and 487 nm; violaxanthin,
424,452 and 482 nm; and neoxanthin 421,447,477 nm in CHCl3.
Alternatively, for the large-scale isolation of β-carotene, the following method
can be used.
 Further, the β-carotene from the elute is separated by preparative thin layer
chromatography on silica gel G, in the presence of benzene: light petroleum
ether (1.1). Under these conditions, the β-carotene has Rf 0.82. The compound is
further purified by crystallisation in the presence of mixture of benzene and
methanol.
Table 8.40 represents the necessary data for the separation of some important
carotenoids.
Table 8.40 Data for the separation of some important carotenoids
8.24 TANNIS
Isolation of embelin from Embelia ribes

Estimation of embelin
Method I: by spectrophotometry
An ultraviolet spectrophotometric method can be used to estimate embelin in the
berries of Embelia ribes and in different formulations. The maximum absorption
of embelin is found at 291 nm in chloroform and is found to obey the
photometric linearity (Saroj K. Pal et al., 1995).
PROCEDURE

1. Ten mg of pure embelin is accurately weighed and taken in a 100 ml

volumetic flask. The volume is made up with chloroform. The scanning is
performed at 240–400 nm in the ultraviolet region, using a 200–20 UV–Vis.
 spectrophotometer. The maximum absorbance is found at 291 nm (Fig.
8.41).

2. A standard solution of 0.1 mg/ml of embelin is prepared in chloroform.

From the stock solution, different concentrations of embelin solutions—
0.005 mg, 0.010 mg, 0.015 mg, 0.020 mg, 0.025 mg, 0.030 mg per ml—are
prepared. The absorbances of these solutions are measured at 291 nm.
3. For the estimation of embelin in berries of Embelia ribes, the embelin can
be isolated as per the above isolation procedure with each batch containing
10 gms of 40 mesh powdered berries.

For the estimation of embelin in market tinctures, three batches of market

tinctures are taken. In each case, 20 ml of the tincture is evaporated on a water
bath. The residue is extracted with ether. Embelin is isolated as per the procedure
adopted.
Each 500 mg of market tablet contains: Aconitum palmatum (100 mg),
Embelia ribes (100 mg), Piper nigrum (20 mg), Mentha arvensis (15 mg), and
base (q.s.).
A batch of 10 tablets are powdered and extracted with ether. The extract is
processed in the same way as the isolation of embelin
Method II
PROCEDURE
This method involves the complete extraction of the fruit powder in diethyl
ether, evaporation of the solvent and washing of the residue with petroleum ether
(40–60°). The residue left over is crude embelin. Three sets of 2.0 gm each of
the 60 # Embelia ribes powder is processed to isolate crude embelin. In another
experiment for studying percent recovery, 2.0 gm E. ribes fruit powder and 30
mg of pure embelin are taken. The residues are taken in 100 ml methanol. To 2
ml extract, 4 ml aniline (20% in methanol) and 6 ml methanol are mixed. The
resultant red colour is measured in a 1 cm cell at 445 nm on a Perkin–Elmer
Lambda 12/1.0 nm (1.31) UV/Vis. spectrophotometer.
Method III: By development of colour with 1N aqueous potassium hydroxide
PROCEDURE
Embelin forms a violet coloured complex with potassium hydroxide. Potassium
hydroxide (1 N) is used for the development of colour. Extraction of embelin is
done as before. Three sets of 2.0 gm of E. ribes powder and three sets of 2.0 gm
powder with 30 mg embelin are taken and extracted. The residues are re-
constituted in 100 ml methanol. 0.5 ml of this methanolic solution is mixed with
10 ml of IN KOH. The violet color complex is measured in a 1 cm cell at 515
nm on a Perkin–Elmer Lambda 12/1.0 nm (1.31) UV/Vis. spectrophotometer.
Tests for tannins

1. Tannin solutions can be precipitated by heavy metals

2. With FeCl3
i. a blue-black colour is produced, if gallitannins and ellagitannins are
present.
ii. a brownish green ppt. is obtained, if condensed tannins are present.
 3. If very dilute ferric chloride solution is gradually added to an aqueous
extract of Hamamelis leaves (containing both types of tannins), a blue
colour is initially produced which changes to olive green.
4. Gold beater’s skin test:
Soak a small piece of Gold beater’s skin in 2% hydrochloric acid, rinse with
distilled water and place in test solution for 5 minutes. Wash with distilled
water and transfer it to 1 % ferrous sulphate solution. Brown or black
colour on the skin indicates the presence of tannins.
5. Gelatin test:
0.5 to 1% solution of tannins can precipitate a 1% gelatin solution in 10%
sodium chloride.
6. Phenazone test:
To 5 ml of an aqueous extract of crude drug, add 0.5 of a sodium acid
phosphate, warm, cool and filter. To the filtrate, add 2% phenazone
solution. All tannins are precipitated.
7. Test for catechin:
When catechins are heated with acid, phloroglucinol is formed which can
be detected by a modification of the test for lignins. Dip a matchstick in the
plant extract, dry, moisten with concentrated hydrochloric acid and warm
on a flame. The phloroglucinol produced gives a pink or red colour to the
matchstick.
8. Test for chlorogenic acid:
An extract when treated with aqueous ammonia and gradually exposed to
air gives a green colour, if chlorogenic acid is present.

Isolation of artemisinin, artemisinic acid and arteannuin B

Hala N. Elsohly (1990) isolated artemisinin, artemisinic acid and arteannvin B,
terpenoidal drugs, (Scheme 8.13) from Artemisia annua.
Isolation of aristolochic acid and aristolic acid
Aristolochic acid and aristolic acid are extracted from dried ground roots of
Aritolochia indica (1 kg) in a Soxhlet extractor with 95% ethanol. The ethanolic
extracts are concentrated to a thick black-brown syrup under reduced pressure to
yield about 110 gm of residue. The residue is treated with chloroform (0.5 litre)
and 2% sodium bicarbonate solution (3 litres) and the suspension is subjected to
vigorous stirring for 24 hours. The wine-red alkaline solution obtained is
decanted and filtered. The combined filtered alkaline solution is then acidified
with 5% HC1 to yield a yellow-brown precipitate, which is filtered and dried to
yield 10–12 gm of total acid fraction. Extraction with the 2% sodium
bicarbonate is repeated several times until no further precipitation results upon
acidification of the bicarbonate extract.The crude total acid fraction (1.5 gm) is
extracted with chloroform (2 litres) in a Soxhlet apparatus for 10 hours (residue:
0.41 gm). The chloroform solution is added to a column of silicic acid–celite 545
(4:1, 500 gm). In 4–5 hours, a passage of 2 litres of chloroform through the
column results in resolution of 4 major coloured segments:

a. highly fluorescent reddish-yellow,

b. yellow-green non-fluorescent (the largest),
c. reddish-brown mixture of bands and
d. brown-black fluorescent mixture of bands.

Chloroform (5 litres) is used to elute band A (15 mg of a non-crystalline

substance) and band B (875 mg) which is aristolochic acid. The aristolochic acid
is subjected to purification by crystallisation of the crude acid from a mixture of
dimethyl formamide and ethanol; yellow-orange crystals with a melting point of
275–278℃ resulted (S. Morris Kupchan and John J. Merianos, 1968).
Scheme 8.13
Conversion of aristolochic acid to aristolic acid
To a solution of aristolochic acid in 1% aqueous ammonia hydroxide solution
(pH 12), is added sodium borohydride (for 100 mg of aristolochic acid, 76 mg of
sodium borohydride); the mixture is stirred at 25℃ for 4 hours. The reaction
mixture is poured onto crushed ice, acidified with hydrochloric acid to pH 4 and
extracted with ethyl acetate (5 ✕ 25 ml). The combined ethyl acetate phases are
washed with water, dried over sodium sulphate, filtered and concentrated in
vaccum to afford a homogeneous residue of artistolic acid (80 mg, 90% yield)
which is crystallised from dimethyl formamide and ethanol to afford fine needles
with a melting point 255–256℃ (Sibabrata Mukhopadhyay et al., 1983).

8.25 OLEORESINS
Extraction from black pepper (Piper nigrum) berries
Method A: Conventional cold percolation
Twenty-five grams of ground spice powder placed in a glass column is extracted
with 40, 30 and 30 ml each of 95% (v/v) ethyl alcohol. After a contact time of 30
minutes, 15 to 20 ml of the extract out of the 40 ml solvent used, is collected.
For subsequent extractions, 15 minutes each of contact time is given. The solids
in 10 ml of the combined extract are determined by evaporating on a tared Petri
dish using a steam bath. The Petri dish containing the solids is heated to 80℃
for 30 minutes in an oven, cooled and weighed.
Method B: Soxhlet extraction
Five grams of the powdered sample, in a thimble, is extracted by the Soxhlet
method for 8 hours with 150 ml of the solvent (95% ethyl alcohol). The extract
is evaporated on a steam bath and heated for 30 minutes in an oven at 80℃,
cooled and weighed.
Method C: Modified cold percolation
Five grams of powdered sample is weighed in an extraction tube and the
oleoresin is extracted using acetone (BDH) by cold percolation.
Three more extractions with 50 ml each of fresh solvent are done for 30
minutes. The combined extract is evaporated to dryness in a suitable tared
container over a steam bath and in an oven at 80℃ for 30 minutes, cooled and
weighed.
Extraction from ginger
PROCEDURE
Method A: Sequential extraction
Sequential extraction adopted in this study is as follows: Three 45 ✕ 4 cm
columns (A, B and C) are filled with 20 gm spice and 75 ml of the solvent. After
6 hours of the spice–solvent contact, the eluate from A is transferred to column,
B, from B to C and from C to A. This, is repeated after 6 hours without either
changing the solvent or spice. After operating this for 4 cycles, the eluates are
pooled up, the solvent stripped under vacuum and thick viscous oleoresin
separated out.
Method B: Counter-current extraction
A step-wise working pattern of a counter-current extraction is presented in Fig.
8.42. Three glass columns 45 x 4 cm are taken and filled with the solvent (125
ml). The sample under extraction is placed in the first column. After 6 hours
contact, soluble fraction from column I is taken to column II and from II to
column III, thereby, solvent from step I comes in contact with sample and eluate
from step II comes in contact with the spent sample of the successive step.
Method C: Soxhlet extraction
In a Soxhlet extractor, 30 gm of the spice powder is loaded with a cotton plug
onto the capillary of the extractor and extracted with 100 ml of the solvent for 3
hours at 90℃. Spice–solvent contact in this process is ensured for 10 cycles,
then the extract is removed, stripped of the solvent under vacuum and a thick
mass is separated.
Fig. 8.42 Schematic representation of counter-current extraction of ginger
oleoresin

Method D: Cold solvent percolation

10 gm of the spice powder is loaded in a glass column 45 x 1.5 cm and 125 ml of
the solvent is percolated after overnight contact. The percolate is stripped of the
solvent and viscous mass is separated for further analysis.

8.26 ESSENTIAL OILS

Steam distillation or, better, hydrodistillation is the procedure that is most
frequently used to isolate essential oils. In many cases a Clevenger-type
apparatus is used. However, long- duration distillation may influence the
composition of the oil isolated. This is because isomerisation, saponification and
other reactions may occur under distillation conditions. Other methods used to
isolate essential oils may also give rise to varying compositions. Therefore,
differences often found in the literature with regard to the composition of a
certain essential oil, will be partly due to the methods used to isolate the oil.
Hydrodistillation and solvent extraction
Isolation procedures, especially hydrodistillation, may influence the composition
of essential oils isolated from different kinds of plant material. When dill seed is
hydrodistilled, it takes more than 12 hr to obtain a fairly constant ratio of
carvone and limonene—the main components of the oil isolated. For
comparative purposes, dill seed oil is also isolated by solvent extraction. In
solvent-extracted oil, the ratio of the relative amounts of trans- dihydrocarvone
and cis-dihydrocarvone is found to be ca 1:5, but in distilled oils, the trans-
isomer is always more abundant than the cis-isomer. It is found that the
isomerisation observed is catalysed by metals present in the dill seeds, and that it
is not caused by heat alone (Koedam et al. 1979). Other oils, e.g., those from the
leaves of conifers, could be isolated in a relatively short period of time,
especially, when compared with the oils from the seeds of Umbelliferae.
Therefore, the chance of reactions is also smaller. However, a comparison of the
oils of the Leyland cypress isolated by hydrodistillation and by solvent
extraction, respectively, showed that the distilled oil contains 8.0% terpinen-4-ol,
whereas this compound amounted to 0.6% in the volatile fraction of the solvent
extract, and that the amount of sabinene is markedly lower in the distilled oil
(Koedam et al. 1981). The re-arrangements proved to be strongly dependent on
the pH of the distillation water.
Studies on the influence of distillation on the composition of oils from some
Labiatae showed that these oils are released much faster than the oils discussed
before. Of course, this is the result of the different localisation of these oils, but
it means that the chance of undesirable changes due to heat or due to the acidity
of the distillation water is strongly reduced. In view of this, it is interesting to
refer to a study of Schmaus and Kubeczka (1985), who reported that lavender oil
isolated by solvent extraction contained 43% linalyl acetate and 25% linalool,
whereas the oil isolated by hydrodistillation contained only 13% linalyl acetate
and 41% linalool.
be avoided. When a mixture of pentane and diethyl ether is used for the
extraction, the temperature will not exceed 40℃. However, a drawback in this
procedure is that the mixture of volatiles will be isolated along with non-volatile
compounds, which during subsequent GC analysis may give rise to problems.
Therefore, it will be necessary to apply some kind of isolated sample
preparation.
Simultaneous distillation–solvent extraction
An apparatus that is suited for the separation of volatile and non-volatile
compounds present in an extract, but which which can also be used for direct
isolation of volatiles from plant material, is the Likens–Nickerson-type
apparatus. If an extract obtained by solvent extraction is submitted to distillation
using this apparatus, the duration in which the volatile components are exposed
will be reduced, since the volatiles will be released much faster from the extract
than from the plant tissues containing them. Despite its advantages, there is still
a chance of artifact formation using this type of apparatus.
When the essential oil from flowers of Thymus lotocephalus was analysed
(Figueiredo et al. 1993), it was found that the oil isolated by simultaneous
distillation–solvent extraction consisted of large amounts of linalyl acetate and
linalool. In view of the study of Schmaus and Kubeczka (1985) mentioned, the
oil composition was compared with that of a corresponding solvent extract. The
composition showed that the ratio of linalyl acetate to linalol varied from 2:1 in
the distilled–extracted oil to 50:1 in the solvent extract, which confirmed that re-
arrangements of linalyl acetate occur easily.
In view of the possibility of a thermal generation of artifacts using a Likens–
Nickerson- type apparatus, a Swiss group described a modified devise for
simultaneous distillation– extraction; the isolation of volatiles is performed
under vacuum and at a temperature between 20℃ and 40℃ (Marginal et al.
1992). It is shown that the artifact formation from linalyl acetate does not occur.
Supercritical fluid extraction
Another method of isolating the volatile components of a plant material is
extraction by means of supercritical carbon dioxide, which is considered a
valuable alternative to the methods discussed before; it is superior to those
methods because of its speed of extraction, its selectivity and its suitability for
extraction of thermally labile compounds. Supercritical fluid extraction can be
applied under a wide range of conditions, such as various combinations of
pressure and temperature, and in this way it permits the extraction of a number
of different products from a certain plant material, just by changing the density
of the fluid.
The possibility of obtaining a certain composition by varying the process
condition is an advantage, if a product with a certain (desired) composition must
be obtained, but it is a drawback for other studies; for example, it is likely that
the composition of a mixture of volatiles extracted under certain conditions, is
hardly comparable to the composition of the corresponding oil obtained by
distillation. There are several examples which show that an extract and an oil
may be similar qualitatively, but that the percentage compositions are markedly
different. In addition, it is important to realise that the extracts obtained by
supercritical fluid extraction may also contain all kinds of non-volatile
compounds.
Microwave oven extraction
During recent years, a new procedure to isolate essential oils has been described,
i.e., using microwave ovens. Analysis of the oils obtained this way again showed
that the qualitative composition of the oils was the same as that of distilled oils,
but that the percentage composition varied significantly. But the procedure does
have its advantages, for example, because of the small amounts of plant material
needed and the speed of extraction, which is suitable when a number of samples
from the same plant must be analysed (Craveiro et al. 1989).
Thus, in summing up, the differences in essential oil composition can be
easily caused by the use of different apparatus for the isolation of the oil, and by
different conditions used during the isolation procedure. Each of the various
apparatus may have some typical advantages and some typical drawbacks.
In spite of the drawbacks of hydrodistillation, it is considered quite useful as
can be noted by the fact that the apparatus is described in the European
Pharmacopoeia. It is becoming more or less a standard apparatus. The results
obtained by various researchers using this standard apparatus will be more easily
comparable.
Tests for volatile oils (micro-chemical tests)
Presence of volatile oil in natural drugs can be detected by the following tests.

1. To a thin section of the drug, add an alcoholic solution of sudan III. A red
colour obtained by globules indicate the presence of volatile oil.
2. To a thin section of the drug, add a drop of tincture alkana; red colour
indicates the presence of volatile oil.
 9

Screening Methods Used

for Herbal Drugs

9.1 INTRODUCTION
Drug screening procedures are important and necessary in order to estimate the
harmful or therapeutic potential of useful drugs. Molecular pharmacological
procedures are used nowadays to screen herbal compounds/extracts.
The classical method of pharmacological screening involves sequential testing
of any new chemical entities (NCEs) or extracts from herbal sources by in vitro
and in vivo experiments conducted in lower animals like mice and rats and also
in higher animals, if necessary. Most drugs used in therapy nowadays have been
found and evaluated with these methods.
Both the pharmacologist and the chemist are highly concerned with drug
development, therefore they work jointly in evaluating NCEs and gaining a
better understanding of active ingredients. Today a large number of active
compounds are obtained from herbals, which are highly valuable in the treatment
of diseases like metabolic disorders, endocrinological disorders, cancers etc.
The continuing accumulation of fundamental knowledge about diseases and
the technological progress in pharmacology, herbal drug technology, molecular
biology and bio-technology has led to the development of much better animal
and laboratory models for many diseases. This makes the drug-screening process
more efficient and may also lead to a reduction in the use of animals for research
and pre-clinical development.
Research and development of new drugs are done under strict government
regulations (Drug Controller General of India (DCGI), Food and Drug
Administration (FDA) and Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA)), which have become very
systematic and stringent over the past couple of decades.
Drug development comprises two steps—pre-clinical and clinical
development.
The main aim of the pre-clinical development phase for a potential new
medicine is to explore the drug efficacy and safety before it is administered to
patients. In this pre-clinical phase varying drug doses are tested on animals
and/or in vitro systems.
With the introduction of advanced technologies such as SEPBOX it has now
become possible to isolate the active principle(s) from potent herbs. If active
compounds are found in a potential herb, they are extracted and their effect on
animals studied. These studies include pharmacodynamic, pharmacokinetic,
toxicology and special toxicological studies (carcinogenity and mutagenicity).
Most pre-clinical tests have to be conducted in accordance with the standards
prescribed. Following pre-clinical studies, the drug has to undergo a series of
different phases of clinical trials like phases I, II, III and IV. In this chapter, in
vitro and in vivo screening procedures mainly for anti-fertilty, anti-diabetics,
anti-cancer, anti-hypertensive, anti-anginal, anti-arrhythmic, cardiac glycosides,
diuretics, anti- asthmatics, anti-thyroid, anti-epileptics, neuropharmacological,
estimation of hormones using ELISA, transdermal delivery systems, clinical
trials and toxicological studies obtained from herbal sources are described.

9.2 SCREENING METHODS FOR ANTI-FERTILITY AGENTS

Anti-fertility agents are substances which prevent reproduction by interfering
with various normal reproductive mechanisms in both males and females.
An ideal contraceptive agent is one which possess 100% efficacy, reversibility
of action, which is free from side-effects and is easy to use.
Ancient literature has mentioned the use of a number of plants/preparations
for regulation of fertility in the form of emmenagogues, ecbolics, abortifacients
and local contraceptives. For centuries, virtually every indigenous culture has
been using plants and/or their various parts in one or the other form to restrict its
population. Women have used herbs since time immemorial to control their
fertility. The information was passed on from mother to daughter; midwives and
wise women all possessed this knowledge. But most of these plant’s activities
and their mechanism of action were not scientifically studied.
There are approximately 2,50,000 species growing on earth. It stands to
reason that not all of them can be used to regulate fertility; therefore some
criteria have to be laid down for selecting plants in order to evaluate their anti-
fertility potential. Three options are available:

1. Investigation of plants that have folkloric/traditional reputation as

contraceptives;
2. Evaluation of plants that are known to contain constituents which
theoretically affect the female cycle and thus produce anti-fertility effects
e.g. estrogenic sterols, isoflavones and coumestans or those, which have a
potential to contract the uterus;
3. Random collection of plants for mass screening.

During the last six decades, sporadic attempts have been made by Indian
investigators to evaluate anti-fertility plants. But there is variation in the reports
given by various investigators on the same plant part (from inactivity to 100%
activity). This appears to be due to inadequate attention given to proper botanical
identification, authentication and testing procedure. In spite of the detailed
description of plants found in ancient Ayurvedic and Unani literature,
documented experimental or clinical data on them are lacking. Furthermore the
efficacies of these plants have not yet been confirmed through repeated
investigations. In this section, we will describe some screening methods for anti-
fertility activity in females and males.

9.2.1 Screening Methods for Anti-fertility Activity in Females

Anti-fertility action of drugs acting in females may be due to

Inhibition of ovulation
Prevention of fertilisation
Interference with transport of ova from oviduct to endometrium of the
uterus
Implantation of fertilised ovum
Distraction of early implanted embryo
Screening methods for anti-ovulatory activity
Cupric acetate induced ovulation in rabbits
Rabbits are reflex ovulators. They ovulate within a few hours after mating or
after mechanical stimulation of vagina or sometimes even the mere presence of a
male or administration of certain chemicals like cupric acetate.
In this screening method, cupric acetate is used for the induction of ovulation.
The rabbit ovulates within a few hours after an i.v. injection of cupric acetate
(0.3 mg/kg using 1% cupric acetate in 0.9% saline). Injection of anti-ovulatory
drugs, 24 h before the induction procedure prevents ovulation.
Sexually mature female albino rabbits, weighing 3–4 kg, are used for the
study. Animals are kept in isolation for at least 21 days to ensure that they are
not pregnant and to prevent the induction of ovulation by mating. They are then
treated with the test drug and 24 h later an i.v. injection of cupric acetate is
given. The rabbits are sacrificed and the ovaries examined 18–24 h later. The
total number of ovulation points (Fig. 9.1) on both the ovaries is recorded for
each animal. Then the ovaries and uterus are excised and preserved in 10%
buffered formalin and subjected to histopathological evaluation.

Fig. 9.1 Ovulation point in rabbit ovary (HE × 40x)

HCG induced ovulation in rats

Immature female albino rats do not ovulate spontaneously and do not show
cyclic changes of the vaginal epithelium. Priming with human chorionic
gonadotropin (HGC) induces follicular maturation, followed by spontaneous
ovulation 2 days later. Injection of an anti-ovulatory drug prior to the induction
procedure will prevent ovulation. This principle is used for screening potential
anti-ovulatory agents.
Immature female albino rats (24–26 days old) are used for the experiment.
The animals are treated with various test drugs in different dose levels. After the
administration of the test drug, exogenous HCG is given to induce ovulation.
After 2 days, the animals are sacrificed. Their ovaries are preserved in a 10%
buffered formalin and subjected to histopathological evaluation. The results are
compared with the control group.
Screening methods for estrogenic compounds: In vivo methods
A primary therapeutic use of estrogen (both in vivo and in vitro) is in
contraception. The rationale for these preparations is that excess exogenous
estrogen inhibits FSH and LH and thus prevents ovulation.
Assay for water uptake
The principle of the assay is based on the observation that the uterus responds to
estrogens by increased uptake and retention of water. A peak in the uptake is
observed six hours after administration.
Ovariectomised adult animals may be used for this experiment. Although it is
simpler to use immature 18-day-old mice or 22-day-old rats obtained 2 days
prior to the beginning of the experiment. The animals are randomly grouped.
The control group is given 0.1 ml of cottonseed oil (vehicle for estradiol)
subcutaneously. The estrogen control group is given doses ranging from 0.01–
0.1 μg to establish a dose–response curve. In the initial test, the test compound is
given to groups at a high and low dose. In subsequent tests it is given over a
range of doses to provide the dose–response curve. All doses are given in 0.1 ml
of cottonseed oil.
Five hours after treatment, the animals are killed by cervical fracture and the
uteri are quickly excised. The operation is begun by a longitudinal slit through
the skin of the abdomen and through the body wall. The uterus is picked up with
the forceps and severed from the vagina. The uterine horns are separated from
the connective tissues and are then cut at their constriction point near the ovary.
The uteri are kept moist by placing them on damp (not wet) filter paper and by
covering them with damp filter paper. They are then rapidly weighed in a
sensitive balance. The uteri are dried in an oven at 60°C, for 24 h and are
reweighed. The percentage increase in water over control can be calculated, and
compared with the values of other groups.
Procedure for ovariectomy: The animals are anaesthetised with ether. A single
transverse incision is made in the skin of the back. This incision can be shifted
readily from one side to the other, so as to lie over each ovary in turn. A small
puncture is then made over the site of the ovary, which can be seen through the
abdominal wall, embedded in a pad of fat. The top of a pair of fine forceps is
introduced and the fat around the ovary is grasped, care being taken not to
rupture the capsule around the ovary itself. The tip of the uterine horn is then
crushed with a pair of artery forceps and the ovary together with the fallopian
tube removed with a single cut using a pair of fine scissors. Usually no bleeding
is observed. The muscular wound is closed by absorbable sutures and the outer
skin wound is closed by nylon suture.
Four-day uterine weight assay
This assay is based on the observation that estrogens cause an increase in protein
synthesis and thus bring about an increase in uterine weight. A peak is observed
after about 40 h.
Immature or ovarectomised albino mice or rats can be given the test drug
intramuscularly in cottonseed oil for three consecutive days. On the fourth day
animals are killed by cervical fracture, the uteri rapidly excised, and the uterine
contents gently squeezed out (results are unreliable if the uterine contents are not
removed). The uteri are weighed immediately in the wet state. They are then
dehydrated in an oven at 100°C for 24 h and re-weighed to obtain the dry weight
increase. The log dose is plotted against the wet weight to produce a sigoid
curve, and the ED50 can be determined for comparison of the test compound
with estradiol.
Vaginal opening
This assay is based on the principle that vaginal opening occurs in immature
female albino mice and rats when treated with estrogenic compounds. Complete
vaginal opening is considered a sign of estrogenic activity.
Immature female animals (18-day-old mice, 21-day-old rats) are used for the
study. The test and standard drugs are administered to the animals
intramuscularly in cottonseed oil. The vaginal opening is observed to determine
estrogenic activity.
Vaginal cornification
This assay is based on the fact that rats and mice exhibit a cyclical ovulation
with associated changes in the secretion of hormones. This leads to changes in
the vaginal epithelial cells. The estrus cycle is classified into the proestrus,
estrus, metestrus and diestrus stage. Drugs with estrogenic activity change the
animals from whatever stage they were into the estrus stage.
Adult female albino rats having a regular estrus cycle are used for the study.
Animals are treated with various test and standard drugs. Change in the vagina
can be observed by taking vaginal smears and examining these for cornified
cells, leucocytes and epithelial cells in the normal animals and treated animals
twice daily over a period of 4 days. Any drug which changes the animals into the
estrus stage skipping other stages is considered to have estrogenic activity.
Various stages of the estrus cycle in rats The estrus cycle is a cascade of
hormonal and behavioural events, which are highly synchronised and repetitive.
The short and precise estrus cycle of laboratory rats has been a useful model for
reproductive studies. The laboratory rat is a spontaneous ovulating, non-
seasonal, polyestrus animal. It ovulates every 4-5 days throughout the year
unless interrupted by pregnancy or pseudo–pregnancy.
A century ago, the English scientist Walter Heape described the progressive
stages of the estrus cycle. The cycle itself is divided into four stages, centered on
the period proceeding estrus, the “proestrus”, which signifies the period of
follicular growth in the ovary. He termed the period succeeding estrus as
“metestrus” which was a recovery period following ovulation and “diestrus” a
period when the ovarian secretions from the corpus luteum prepare the uterus for
implantation. The estrus cycle of a rat is usually completed in four to five days.
The cycle is roughly divided into four stages:
Proestrus: This is the beginning of a new cycle. The follicles of the ovary start to
mature under the influence of gonadotrophic hormones, and estrogen secretion
start increasing. The vaginal smear is characterised by nucleated epithelial cells;
the stage lasts for about 12 h.
Estrus: In this stage the uterus is enlarged and extended due to fluid
accumulation; estrogen secretion is at its peak. In the estrus stage, the smear
shows presence of squamous cornified cells (hexagonal or pentagonal cells). The
estrus stage is usually the period of heat and is characterised as a period of
sexual receptivity, when the female allows copulation. During this stage there is
increased running activity. It lasts for 12 h.
Metestrus: The ovary contains corpora lutea which secrete progesterone. This
stage is indicated by the presence of a mixture of cornified epithelial cells and
leucocytes indicating the post-ovulatory stage and desquamation of the epithelial
cells. The metestrus stage lasts for about 21 h.
Diestrus: The corpus lutea regress and the declining secretion of estrogen and
progesterone cause regression of the uterus. The vaginal smear shows only
leucocytes. This stage is the longest phase of the estrus cycle and has a duration
of about 57 h (Figs. 9.2–7).

Fig. 9.2 Rat vaginal smear at the proestrus stage showing nucleated epithelial
cells as seen in 40x
Fig. 9.3 Rat vaginal smear at the estrus stage showing cornified epithelial cells
as seen in 10x

Fig. 9.4 Rat vaginal smear at the metestrus stage showing both cornified
epithelial cells and leucocytes as seen in 10x
Fig. 9.5 Rat vaginal smear at the diestrus stage showing only leucocytes as seen
in 10x

Fig. 9.6 Rat vaginal smear of mated animal showing cornified epithelial cells
and sperms as seen in 20x
Fig. 9.7 Rat vaginal smear of mated animal showing sperms as seen in 40x

The experimental procedure for taking vaginal smears Holding the animal on
the ventral side up, a drop of normal saline is inserted into the vagina with a
Pasteur pipette. Care must be taken to avoid damage or injury to the vagina so as
to prevent pseudo-pregnancy. The drop of normal saline should be aspirated and
replaced several times. It is then transferred to a microscope slide and allowed to
dry. The smears are fixed by placing the slide in absolute alcohol for 5 s,
allowing it to dry, and staining it with a 5% aqueous methylene blue solution for
10 min. The excess stain is washed off with tap water, and the slide dried and
observed using a low power microscope.
Chick oviduct method
The weight of the oviduct of young chicken increases depending on the dose of
natural and synthetic estrogen. This principle is used for the screening of
estrogenic compounds.
Seven-day-old pullet chicks are injected subcutaneously twice daily with
solutions of the test compound in various doses for six days. Doses (0.02–0.5 pg)
of 17β-estradiol per animal serve as standard. Six to ten chicks are used for each
dosage group. On the day after the last injection, the animals are sacrificed and
the weight of the body and oviduct is determined.
Screening methods for estrogenic compounds: In vitro methods
Potency assay
This assay determines the affinity of the test compound for estrogen receptor
sites in the uterus.
The uptake of titrated estradiol by immature uteri must be established. The
inhibition of this uptake by pre-treatment with a test compound will then indicate
the estrogenic potency of the compound.
Four immature female mice (20 days old) are killed. The uteri are quickly
excised and are placed in a Krebs-Ringer phosphate buffer. Pieces of diaphragm
are taken from each animal to serve as control tissue for non-specific uptake of
estradiol. The uteri are divided at the cervix into two horns; this helps as one
horn can be used as the control and the other for testing the compound. The
tissues are placed in vials containing 5.0 ml of Krebs–Ringer phosphate buffer,
incubated, and shaken at 37°C with 95% oxygen. 5% carbon dioxide is bubbled
through. The radiochemical purity of the 3H-estradiol can be checked
chromatographically. Buffer solution of radioactive estradiol is made up so that
each 5 ml of buffer contains 0.0016 μg of radioactive estradiol (0.25 μci). A
stock solution can be made and kept refrigerated for up to 6 weeks.
The excised tissues are treated as follows:
Control: Four pieces of diaphragm are incubated and shaken with 5 ml of buffer
solution for 15 min. at 37°C and are then shaken for 1 h with 5 ml of buffer
containing the radioactive estradiol and 2% w/v bovine albumin.
Experimental: Four uterine homs are incubated and are shaken in 5 ml of buffer
at 37°C for 15 min. They are then incubated and shaken with 5 ml of buffer
containing 2% of albumin and radioactive estradiol at 37°C for 1 h. Both control
and experimental tissue are removed and washed with buffer at 37°C for 5 min.,
kept in damped filter paper and weighed. The tissues are then prepared for
counting. Samples of 100 μl of the incubation solution are also taken for
counting.
Treatment of tissues for counting: The tissues are dried to determine constant
weight and the dry weight recorded. Each piece of tissue is placed in a glass
counting vial and incubated at 60°C in a shaking water bath with 0.5 ml of
hyamine hydrochloride l0x until the tissue has completely dissolved. If the
solution is discoloured, 50 μl of 20% hydrogen peroxide may be added. 50 μl of
concentrated HC1 and 15ml of phosphor solution are added to each vial. The
vials are allowed to equilibrate in the packed liquid scintillation counter, and
counts are taken. Counting efficiency is determined by the addition of an internal
standard. The results are expressed as disintegrations per minutes per unit of wet
weight (dpm/mg). Test compounds can be incubated with the labelled estrogen.
This helps in assaying their effectiveness in competing for the receptors in the
uterus.
Estrogen receptor-binding assay
Estrogenic receptor-binding assay uses the principle of competitive binding of
labelled and unlabelled estrogen on the estrogenic receptors. Estrogenic
compounds displace the labelled estrogen in a concentration-dependent manner
from the estrogen receptor.
Cytosol preparation Uteri from 18-day-old female albino mice are removed and
homogenised at 0°C in 1:50 (w/v) of Tris-sucrose buffer in a conical
homogeniser. Human endometrium from menopausal women frozen within 2 h
of hysterectomy and stored in liquid nitrogen can also be used. The frozen
endometrium is pulverised and homogenised in 1:5 (w/v) of Tris-sucrose buffer.
Homogenates are centrifuged for 1 h at 105,000 g.
Determination of specific binding in mouse uterus cytosol as a function of
steroid concentration, incubation time and temperature Triplicate aliquots of
125 μl of cytosol are incubated with 5 or 25 nM labelled steroid either for 2 or 4
h at 0°C or for 2 or 5 h at 25°C in the absence (total binding) or presence (non-
specific binding) of a 100-fold excess of radio-inert steroid. Bound steroid is
measured by dextran coated charcoal (DCC) adsorption.
DCC adsorption technique A 100 μl aliquot of incubated cytosol is stirred for 10
min. at 0°C in a micro titre plate with 100 μl of DCC suspension (0.625%
dextran 80,000, 1.25% charcoal (Norit A)) and then centrifuged for 10 min. at
800 g. The concentration of bound steroid is determined by measuring the
radioactivity in a 100 μl aliquot of supernatant.
For calculation of the relative binding affinity, the percentage of radio-ligand
bound in the presence of a competitor compared to that bound in its absence is
plotted against the concentration of unlabelled competing steroid.
Screening methods for anti-estrogens: In vivo methods
Antagonism of physiological effects of estrogens
Anti-estrogenic compounds will inhibit some or all of the physiological effect of
estrogen such as water uptake of uterus, uterotrophy and vaginal cornification.
This principle is used for the screening of anti-estrogenic activity.
 The assay techniques used for anti-estrogens are modifications of the
estrogenic assays. The dose of estrogen used is that which is required to produce
50% of the maximum possible response. The test compound can be injected
simultaneously or at varying times before or after the estrogen. The procedure
for assays of water uptake, uterotrophy and vaginal cornification are followed as
described earlier except that the test compounds are given with the estrogen.
Scrrening methods for anti-estrogens: In vitro methods
Aromatase inhibition
This assay is based on the principle that some compounds which inhibit
aromatase (estrogen synthase) can produce anti-estrogenic activity. Anti-
estrogenic activity of compounds can be evaluated indirectly by evaluating
aromatase-inhibiting ability.
Ovarian tissue from adult golden hamsters is used. The estrus cycle is
monitored for at least 3 consecutive 4-day estrus cycles prior to the experiment.
The experiments for evaluating inhibitor effects are performed with ovaries
obtained from animals sacrificed on day 4 (proestrus) of the cycle. The ovaries
are excised free from adhering fat tissue and quartered. The quarters are
transferred into plastic incubation flasks with 2 ml of Krebs–Ringer bicarbonate
salt (KBR) solution (pH: 7.6) containing 8.4 mM glucose. The flasks are gassed
with O2:CO2 (95%:5%), tightly closed and placed in a shaker/water bath (37°C)
for incubation of the fragments. The incubation media are replaced with fresh
KBR after pre-incubation for 1 h. The ovaries are further incubated for 4 h in the
presence or absence of inhibitors. 4-OH androstendione is used as standard in
concentrations between 0.33 and 330 μM/1. At the end of the experiment, the
incubation media are removed and centrifuged. In the supernatant estrogen,
progesterone and testosterone are determined by radioimmunoassays. The data
of control and test group is compared with suitable statistical analysis.
Screening methods for progestins: In vivo methods
Proliferation of uterine endometrium in estrogen-primed rabbits: Clauberg–
McPhail test Female rabbits weighing 800–1000 g are primed with estradiol.
They are then administered with progestational compounds leading to the
proliferation of endometrium and converted into the secretary phase. This
principle is used for the screening of progestational compounds.
Female rabbits weighing 800–1000 g are primed with a daily injection of
oestradiol 0.5 mcg/ml in aqueous solution. On day 7, the drug treatment is
begun. The total dose is given in 5 equally divided fractions daily over 5 days.
Twenty-four hours after the last injection, the animals are killed. The uteri are
dissected out and frozen sections of the middle portion of one horn is prepared
and examined for histological interpretation. For interpretation of progestational
proliferation of endometrium, the beginning of glandular development may be
graded 1 and endometrium consisting only of glandular tissue may be graded 4.
Pregnancy maintenance test
Progesterone is responsible for the maintenance of pregnancy. This principle is
used for the screening of progestational compound.
Ovariectomy is done on day 5/10/15 of pregnancy in different groups of
pregnant rats. The animals are treated with different test and standard drugs.
Pregnant rats are killed 5/10/15 days later. An average of living foetuses at the
end of the experiment is compared with the standard and the control group
(without ovariectomy). The ED50 of progesterone is 5 mg/day in rat and less
than 0.5 mg/day in mouse.
Carbonic anhydrase activity in rabbit's endometrium
There is a linear dose-response relationship between dose of progestogens and
carbonic anhydrase activity in rabbit endometrium. This principle is used for the
screening of progestational compounds.
Immature female albino rabbits are used in this study. The animals are primed
with estradiol and administered test and standard drugs. After the drug treatment,
the animals are sacrificed and their uterus removed. The endometrial extract of
the uterus is evaluated for the carbonic anhydrase activity calorimetrically.
Prevention of abortion in oxytocin treated pregnant rabbits
Administration of oxytocin by IV to pregnant rabbits on the 30th day of
pregnancy causes abortion. Prior administration of progestational compounds
prevents the abortion. This principle is used for the detection and screening of
progestational compounds.
Ten units of oxytocin IV are administered to pregnant rabbits on day 30 of
pregnancy. Test and standard drugs in oil are injected twenty-four hours before.
The control animal not receiving any drugs aborts within 2–30 min. after
administration of oxytocin. Drugs which have progestational activity prevent
abortion.
Deciduoma reaction in rats
This study is based on the phenomenon of maternal/placental tumour formation
due to progestational drugs in traumatised uterus of ovariectomised rats. This
phenomenon is used for the screening of progestational compounds.
Ovariectomised adult female albino rats weighing between 150–200 g are
used for the study. The rats are primed with four injections of 1 μg oestrone. This
is followed by nine days of drug therapy. On day five, one uterine horn is
exposed and 1 mg of histamine dihydrochloride injected into the lumen. Twenty-
four hours after the last dose of drug, the animals are killed, the uterine horn cut
off, weighed and histologically examined.
Screening methods for progestins: In vitro methods
Progesterone receptor-binding assay
Progesterone receptor-binding assay uses the principle of competitive binding of
labelled and unlabelled progesterone on progesterone receptors. Progestational
compounds displace the labelled progesterone in a concentration-dependent
manner from the progesterone receptor.
Human uteri obtained after hysterectomy is frozen in liquid nitrogen and
stored at – 80°C until use. For cytosol preparation, uterine tissues are minced
and homogenised with a homogeniser at 0–4°C in ice-cold PENG buffer
composed of 10 mM KH2PO4, 10 mM K2HPO4 1.5 mM EDTA, 3 mM NaN3,
10% glycerol, pH 7.5. The homogenates are then centrifuged at 10, 5000 g at
4°C for 30 min. The supernatant is taken as cytosol.
The cytosol preparations are incubated with 3H-R5020 as radio-ligand at a
concentration of 8 nmol/1 and increased concentrations (1 × 10–10to 1 × 10–5
mol/1) of the competitor steroid overnight at 4°C. Then unbound steroids are
adsorbed by incubating with 0.5 ml of DCC (0.5% carbon (Norit A), 0.05%
dextran T400 in PENG buffer) for 10 min. at 4°C. After centrifugation (10 min.
at 1,500 g at 4°C), 0.5 ml of the supernatant is withdrawn and counted for
radioactivity. To calculate the relative binding affinity, the percentage of
radioligand bound in the presence of the competitor compared to that bound in
its absence is plotted against the concentration of unlabelled competing steroid.
Screening method for anti-progestational activity
Antagonisim of physiological effect of progesterone
The anti-progestational compound inhibits some or all the physiological effect of
progesterones. This principle is used to screen the anti-progestational activity of
drugs.
The procedures for assay of the Clauberg–McPhail test and deciduoma
formation are as described for progestational activity except that the test
compounds are given along with the progesterone.
Screening method for anti-implantation activity
Female albino rats of established fertility in the proestrous or estrous stage are
mated with mature male rats of established fertility (in the female: male ratio of
3:1). Each female is examined for the presence of spermatozoa in the early
morning vaginal smear. The day on which this sign of mating is seen is taken as
day 1 of pregnancy. The female is then separated and caged singly. The test drug
is administered orally to the animals once daily on specific days of pregnancy at
different concentrations. On day 10th of pregnancy, the animals are
laparotomised and the number of implants present in both the uterine horns as
well as the number of corpora lutea (CL) on each ovary is counted (Fig. 9.8).
The animals are allowed to complete the gestation period (usually 21–23 days)
and the number of litters delivered, if any are counted. Pre-implantation loss and
post-implantation loss are calculated using the following formula.
Pre-implantation loss = No. of CL on 10th day – No. of implants on 10th day
Post-implantation loss = No. of implants on 10th day – No. of litters delivered

Procedure for laparotomy The animal is anaesthetised with ether and the limbs
tied to a rat board (waxed) with the ventral side up. The hairs on the area around
the mid-line abdominal region are clipped with a curved scissor and the region
cleaned with 70% alcohol. An incision of 2 cm length is made along the mid-line
to expose the viscera. The superficially lying coils of ileum are lifted to expose
the two uterine horns. The horns are examined for implantation sites. Implants
are visible as clear swellings on the uterine horns giving the uterine tube a
beaded appearance. Embryos with a bright red dish aspect and a clear margin are
considered to be healthy. Those of a dull blue colour with no clear margin and
orientation with some exudates are considered resorbing. The number of
implants and resorption sites per horn are counted. The ovaries, which lie on the
upper end of the uterine horns, show corpora lutea as yellow spots over the
surface. The number of corpora lutea present on each ovary is also noted.
 After counting, the organs are replaced back. A small quantity of neosporin
powder is sprinkled over the organs to prevent any infection. The incision
through the muscular layer is closed with a continuous suture using absorbable
catguts. The skin layer is closed with continuous sutures using silk thread. An
antiseptic, povidone iodine solution, is applied on the sutured area after wiping
with 70% alcohol. The animal is maintained on light ether anaesthesia
throughout the experiment. After laparotomy, the rats are transferred to a warm
place till they recover from the anaesthesia.

Fig. 9.8 Rat uterus showing normal implants on the 10th day of pregnancy
Screening methods for abortifacient activity
Adult female albino rabbits are used for the study. The pregnancy date is counted
from the date of observed mating. The existence of pregnancy may be confirmed
by palpation after the 12th day of pregnancy. Intra-amniotic and intra-placental
injections are administered to the rabbits under ether anaesthesia on the 20th day
of pregnancy. The uterus is exposed through a mid-line incision, its various parts
are identified by transillumination from a strong source of light and a particular
site chosen for injection. Then the material is injected in 0.l of solvent into the
amniotic fluid or in 0.05ml of solvent into the placenta.
Alternatively, the drugs can be given through any route and duration from the
20th day of pregnancy. The effect of the drug is determined by looking for
vaginal bleeding, changes in weight, abdominal palpation, and by post-mortem
examination.

9.2.2 Screening Methods for Anti-fertility Activity in Males

Developing male anti-fertility agent involves interference with spermatogenesis
without loss of libido and varying sexual characteristics.
The general approaches include:

1. Emergent spermatozoa made non-functional

2. Production of oligospermia/aspermia

In vivo methods
Fertility test
Fertility test is based on the evaluation of the average litter size. Anti-fertility
agents negatively affect the average litter size.
Groups of 5–10 male rats of proven fertility are treated with the drug and are
paired with fertile females in the ratio of 1:3. Daily vaginal smears are examined
for the presence of sperms; normally within one week all females which have
passed through 1 estrus cycle would have mated. The mated animals are kept
separately till the gestational period. The litters are counted and using the
following formula, the average litter size is calculated.

If vaginal smear shows leucocytes in 10–14 days, pseudo-pregnancy is

confirmed. If insemination is not detected then inhibition of libido or aspermic
copulation might be the cause.
Fertility patterns can be obtained from changes in average litter size
Cohabitation test
This test determines the time interval for litter production after placing treated
males with 2 females each. The date of mating is calculated from the date of
parturition. This method is suitable for drugs known to cause sterility for several
weeks.
Adult female and male albino rats of proven fertility are used for the study.
They are kept for mating in the ratio of 2:1 till both females deliver litters. The
date of mating is calculated from the date of parturition. The time interval for
litter production after placing treated males with the two females is calculated.
Subsidiary test
This test determines the changes in spermatozoa count with time. The anti-
fertility drugs affect the spermatozoa count negatively.
Adult male albino rats weighing between 150–250 g are used for the study.
They are kept in a cage containing artificial or animal vagina. The vagina is
artificially simulated by a cylindrical plastic jacket with a rubber liner filled with
water at 5°C. 0.5 ml of ejaculate is diluted with saline containing traces of
formalin. The resulting suspension is counted on a haemocytometer.
In vitro methods
Spermicidal activity
Spermicidal drugs are diluted with normal saline and serial dilutions are made.
0.2 ml of human seminal fluid is mixed with 1 ml of spermicidal solution. Then
the mixture is incubated at 37°C for 30 min. A drop of the mixture is placed
immediately on a slide and at least five fields are microscopically observed (Fig.
9.9) under high power (× 400) for assessment of sperm morphological changes
and motility. Effective agents can immobilise and kill the sperms.
Immobilisation assay
The cauda portion of the epididymes of a ram is isolated and minced in 0.9 %
saline solution (pH: 7.5). It is filtered through a piece of cheese cloth to get a
sperm suspension. For human samples, ejaculates (n = 10) from normal subjects
after 72–96 h of sexual abstinence are subjected to routine semen analysis
following liquefaction at 37°C. Sperm count above 100 million/ml and viability
above 60% with normal morphology and rapid and progressive motility is
employed for the test.

Fig. 9.9 Inverted microscope

Ram epididymal sperm suspension (100–200 million/ml) or human ejaculate
(100–150 million/ml) are mixed thoroughly in a 1:1 ratio with different
concentration of drugs. A drop of the mixture is placed immediately on a slide
and at least five fields are microscopically observed under high power (× 400)
for assessment of sperm motility. The mixture is then incubated at 37°C for 30
min. and the above process is repeated.
Non-specific aggregation estimation
Different concentrations of drugs are treated with ram sperm suspension in a 1:1
ratio and kept at 37°C for 1 h. One drop of the sedimented sperm is then taken
from the bottom of the microcentrifuge tube, placed on a slide and the per cent
aggregation examined microscopically under 400x magnification. Since the non-
aggregated spermatozoa remain in the supernatant, the latter is collected and the
turbidity determined spectrophotometrically at 545 nm. The aggregation is
indirectly proportional to the sperm viability.
Sperm revival test
This assay determines the extent of spermicidal and immobilisation capability of
drugs by evaluating the revival of sperm motility.
To study the revival of sperm motility, after completion of the immobilisation
assay, the spermatozoa are washed twice in physiological saline. They are then
incubated once again in the same medium free of drug at 37°C for 30 min. to
observe the reversal of sperm motility.
Assessment of plasma membrane integrity
To assess the sperm plasma membrane, integrity ram sperm suspension (100–
200 million/ml) or human ejaculated sperm (100–150 million/ml) are mixed with
the drug at the minimum effective concentration, at a ratio of 1:1 and incubated
for 30 min. at 37°C. Sperm samples mixed with saline in a similar manner serve
as controls. For viability assessment, one drop each of 1% aqueous solution of
eosin Y and of 10% aqueous solution of nigrosin was placed in a
microcentrifuge tube. A drop of well-mixed sperm sample is added to it and
mixed thoroughly. The mixture is dropped onto a glass slide and observed under
400x magnification.
For the hypo-osmotic swelling test (HOS), 0.1 ml of aliquot is taken from
each of the treated and control sample, mixed thoroughly with 1ml of HOS
medium (1.47% fructose and 2.7% sodium citrate at a 1:1 ratio) and incubated
for 30 min. at 37°C. The curling tails are examined under phase contrast
microscope using l00x magnification.
5-nucleotidase is released possibly due to destabilisation of plasma membrane.
This can be estimated to determine the effect of the drug on the plasma
membrane integrity of the sperm. The activity of 5’-nucleotidase can be
determined by measuring the rate of release of inorganic phosphate from
adenosine 5’- monophosphate. After incubating the sperm suspension with the
drug, the sperm pellet is collected by centrifugation at 3,000 g at 37°C. It is then
washed twice in 0.9% saline and suspended in 0.1 mol/1 Tris-HCl buffer (pH:
8.5) with each reaction system containing (100–200) million spermatozoa. An
aliquot of 0.1 ml suspension of sperm is added to 0.9 ml of buffered substrate
containing 3 mmol/1 adenosine 5’- monophosphate and 50 mmol/1 MgCl2
dissolved in 0.1 mol/1 Tris-HCl buffer. The tubes are incubated at 37°C for 30
min. and 0.5 ml 20% TCA (0°C–4°C) is added to the mixture to stop the
reaction. The mixture is then centrifuged at 10,000 ×g at 4°C. The pellet is
discarded and the supernatant kept for phosphate estimation. The activity of 5’-
nucleotidase is expressed in terms of μg of phosphate released. The activity of
5’-nucleotidase is indirectly proportional to the plasma membrane integrity.
Evaluation of acrosomal status
This method evaluates the acrosomal status of sperm. The acrosome is the cap-
like structure on the head of the spermatozoa. It breaks down just before
fertilisation, releasing a number of enzymes that assist penetration between the
follicle cells that surround the ovum. The most widely studied acrosomal
enzyme is the acrosin that has been shown to be associated with acrosomes of all
mammalian spermatozoa. The highest substrate specificity was obtained with
BAEE (N-benzoyl-L-argine ethyl ester).
Different concentrations of drugs are mixed with ram sperm suspension in a
1:1 ratio and kept at 37°C for 1 h. The suspension is centrifuged and the pellets
collected. The pellets are extracted with 3 μmol/1 HC1 at pH 3 and the enzyme
activity is measured, following the hydrolysis of 0.5 μmol/1 BAEE dissolved in
0.05 mol/1 Tris-HCl buffer containing 0.05 mol/1 CaCl2 at pH 8. The activity of
acrosin is expressed in terms of mlU. One mlU activity means the amount of
enzyme, which causes the hydrolysis of one nano mole of BAEE in one minute
at 25°C. The activity of acrosin is directly proportional to the fertilising
capability of sperms.
Androgenic and anti-androgenic activities
Androgenic compounds increase the weight of the testes and seminal vesicles of
immature male mice. Anti-androgenic compounds suppress the increase in
weight responses of testosterone. This principle is used for the screening of
androgenic and anti-androgenic activity.
Immature male mice weighing around 20 g are used for the study. The drugs
are administered for seven days alone and along with testosterone. Twenty-four
hours after the last dose, the animals are weighed and sacrificed with an over-
dose of ether. The testes and seminal vesicles are removed and weighed rapidly
in a sensitive balance. The weights are compared with the control group.

9 .3 SCREENING METHODS FOR ANTERIOR PITUITARY

HORMONES
9.3.1 Gonadotropins
Follicle stimulating hormones (FSH)
Ovarian weight in HCG-primed rats
This method is based on the principle that administration of exogenous follicle
stimulating hormones (FSH) in immature rats increases the weight of ovaries by
virtue of promoting the follicular growth and maturation. This effect is greatly
enhanced by simultaneous administration of a constant use of human chorionic
gonadotropin (HCG).
Immature female albino rats weighing 40–45 g are used in the study. The
animals receive twice daily over a period of 3 days, subcutaneous injections of 3
different doses of the standard or the test. Subsequently human chorionic
gonadotrophin (HCG 25 IU) dissolved in 2% gelatin solution in saline is
administered to both the groups. Eight animals are used per group. Eighteen
hours after the last injections, the animals are sacrificed; the ovaries are removed
and freed from adherent fat and connective tissues. They are weighed on a
sensitive balance. The ovaries of two animals from each group is taken
separately and preserved in 10% buffered formalin and subjected to
histopathological evaluation.
[3H] thymidine uptake in cultured mouse ovaries
This method is based on the principle that a follicle-stimulating hormone dose
increases the [3H] thymidine uptake in cultured mouse ovaries.
Intact ovaries are obtained from 15-day-old mice. They are dissected carefully
with the aid of a binocular dissecting microscope and transferred to culture
dishes. Each ovary is placed on a strip of lens tissue supported on a stainless
steel mesh grid 4 mm above the floor of a plastic Petri dish and incubated in
Eagle’s medium supplemented with glucose and glutamine. The dishes are
gassed with 5% CO2 in air at 37°C in an incubator (Fig. 9.10). Three replicate
dishes are used for each concentration of the standard (0.1 and 0.4 IU/ml) and of
the test preparation. [3H] thymidine (0.02 μc) is added to each dish, the day after
the cultures are set up. Three days later, the tissue is prepared for counting. Each
grid is irrigated with about 5 ml saline solution. The ovary is then transferred to
a counting vial and dissolved in soluene. Scintillation solution is added to help in
counting in a liquid scintillation counter. After which dose–response curves for
uptake of [3H] thymidine are established for the standard and the test preparation
in order to calculate potency ratio with confidence limits.
Fig. 9.10 CO2 incubator

Granulosa cell aromatase assay

The granulosa membrane covers the ovum in the tertiary follicles. The granulosa
cells secrete estrogen while maturation of ovum. The following assay is based on
the stimulation of estrogen production by cultured granulosa cells due to FSH in
serum-free medium containing androstendione.
Albino rats (21–22 days old) are implanted with silastic capsules containing
diethyl stilbestrol to stimulate granulosa cell proliferation. Four days after
implantation, the animals are sacrificed and the ovaries dissected for granulosa
cell collection. The ovaries are decapsulated, follicles are punctured with a 27-
gauge hypodermic needle and granulosa cells are carefully expressed into
McCoy’s 5a medium. An aliquot is diluted with trypan blue stain and viable cells
are cultured in 24 × 16 mm well-cultured plates for 2-3 days at 37°C in a
humidified, 95% air and 5% CO2 incubator. Each well contains 5 × 104 viable
cells in 0.5 ml McCoy’s 5a medium supplemented with 2 mM L-glutamine, 100
U/ml penicillin, 100 μg/ml streptomycin sulphate, 10–7 M diethyl stilbesterol
(DES), 106 M androstenedione (as aromatase substrate), 0.125 mM 1- methyl-3-
isobuty 1-xanthine (MIX), 1 μg/ml insulin and 30 ng/ml HCG.
For the measurement of FSH bioactivity in serum samples, each test is
performed in triplicates at three dose levels (5, 10 and 20 μl). To ensure the
constant volume of say 20 μl serum in the total incubation volume of 500 μl, all
the samples are balanced with the addition of gonadotrophin-free serum. For the
FSH standard curve, 4% gonadotrophin-free serum is added to the cultured
medium. At the end of the cultured period of 3 days, estrogen contents in the
medium are measured by radioimmunoassay.
Luteinising hormone (LH)
Prostate weight in hypophystomised rats
This method is based on the principle that exogenous administration of LH into
hypophystomised immature male rats causes enlargement of the testes and varies
sexual organs. Hypophystomy is performed before administration of exogenous
LH, to avoid the interference of endogenous LH. The interstitial cells of the
testes are stimulated by LH and secrete androgen, which stimulate the accessory
organs, such as the ventral prostate of the rats.
Immature male albino rats at an age of 21 days are hypophystomised.
Hypophystomy is the surgical removal or destruction of the pituitary gland in the
brain. The operation may be performed by opening the skull or inserting special
needles that produce a very low temperature. From the second to the fifth day,
the rats receive daily subcutaneous injections of various doses of the test
preparation and the LH standard. They are sacrificed on the sixth day. Both the
testes and the ventral prostate are removed and weighed in a sensitive balance.
Super ovulation in immature rats
Super ovulation can be induced in immature female rats by priming them with
pregnant mare serum, followed by an intravenous injection of LH. This principle
is used for the detection and screening of LH.
Groups of five immature female albino rats are treated at an age of 21 days
with a priming dose of 30 IU of PMS (pregnant mare serum). Fifty-six hours
later they are given an intravenous injection of various doses of LH standard or
test preparation. Twenty-four hours after the final injection, the animals are
sacrificed. The autopsy is done and the fallopian tubes are removed and
examined under the dissecting microscope at a magnification of 40x (Fig. 9.11).
Ovulation is easily noted by the presence of an enlarged translucent segment
of the tube, through which the ova can be seen. The segment is then punctured
with a dissecting needle and the entire cumulus clot containing the ova is
expelled in a single mass. Due to the large number of eggs released at ovulation,
the egg mass has to be treated with hyaluronidase (a spreading substance). The
number of ova released is then counted.

Fig. 9.11 Dissecting microscope

Ascorbic acid depletion of ovaries in PMSG/HCG

Luteinising hormone induces dose-dependent depletion of ascorbic acid in the
ovaries of rats primed with pregnant mare serum gonadotropin (PMSG) and
human chorionic gonadotrophin (HCG). This principle is used for the detection
and screening of LH.
Female albino rats weighing 40±5 g are injected with 50 IU PMSG in 0.2 ml
saline subcutaneously on day 1 at 3:00 PM. On day 3, the rats receive HCG (25
IU) subcutaneously at 9:00 AM. On day 7, three different doses of the standard
and the test substance are injected subcutaneously. Eight animals are used per
group. Three hours later, the animals are sacrificed, both ovaries removed,
weighed and homogenised for determination of ascorbic acid content.

9.3.2 Prolactin
Pigeon crop method
The secretion of crop milk by pigeon and doves are initiated and maintained by
prolactin. This effect is used for the detection and screening of prolactin.
Two to three-month-old pigeons of both sex and uniform strain are injected
intra-muscularly with various doses of the test preparation or the standard, once
daily for 4 days. On the fifth day, the birds are sacrificed. A mid-ventral incision
is made through the skin and the crop and the adhering crop milk are removed.
The two lateral pouches are removed, the fat cleaned from the back of the gland
and the wet weight of the glands determined. Mean values of at least two doses
of test preparation and standard are plotted versus logarithms of doses. The
potency ratios with confidence limits are then calculated.
Lactation in rabbits
Exogenous administration of prolactin induces mammary growth and milk
secretion of pseudo-pregnant albino rabbits. This principle is used for the
detection and screening of prolactin.
Psuedo-pregnancy is induced in mature estrus rabbits by intravenous injection
of 50 IU of HCG. On the fourteenth day, the rabbits are examined for the
presence of well-developed mammary glands characteristic of pregnancy.
Various doses of the test preparation or the standard of lactogenic hormone are
injected subcutaneously once daily for 6 days. On the seventh day, the animals
are sacrificed and the abdominal skin is incised in the mid-line and separated
from the mammary gland underneath.
The degree of enlargement of the glands with secretion is rated as follows:

– : absence of response

+ : all ducts are filled with milk

++ : all ducts and most of the lobules are filled with milk through not
greatly thickened

+++ : entire gland is filled with milk.

 ++++ : mammary glands are greatly extended with milk throughout

The mean values of the test group are compared with the values of the
standard group.

9.3.3 Growth Hormone (GH)

Weight gain in growth plateaued rats
Female rats having reached maturity at six months, continue to gain weight but
at a very slow pace. The slowing-down of growth rate is described as plateauing.
Although such rats have reached a static weight level, they can readily be
induced to grow and gain weight by the administration of growth hormones.
This principle is used for the detection and screening of growth hormones.
Groups of ten adult female albino rats (6 months old) weighing between 220
and 280 g are used. A gain in weight of more than 10 g in a 20-day period is
considered a success. At least two doses of the hormone preparation and the
standard, dissolved in saline, are injected subcutaneously daily over a period of
20 days. During this time, weight gains between 10 and 40 g can be achieved. A
straight-line relationship exists between the logarithm of the daily dose level and
the response as measured in g of body weight increase.
Tibia test in hypophystomised rats
Hypophystomy is followed by cessation of epiphyseal growth (growth of long
bone). The width of the epiphyseal cartilage is markedly reduced after
hypophystomy. Administration of growth hormone to hypophystomised rats
induces a remarkable increase in the width of the epiphyseal cartilage plate. This
principle is used for the detection and screening of growth hormone.
Female albino rats are hypophystomised at an age of 26–28 days. They are
used for the bioassay 12–14 days after the operation. The increase in the body
weight during this period of time has to be less than 0.5 g per day indicating
complete hypophystomy. Six to ten animals are used for each group of two doses
of test preparation and standard. The solutions are administered intra-
peritoneally twice daily for four days.
On the fifth day, the animals are sacrificed, both tibiae are dissected free of
soft tissue, and the bones split in half with a sharp razor at the proximal end in
the mid- sagittal plane. The halves are washed in water for 10 min., immersed in
acetone for 6 min. and washed again with water for 3 min. They are then placed
in 2% silver nitrate solution for 2 min. and rinsed with water. During the water
rinse they are exposed to strong light, which turns the calcified portions of the
bone dark brown. The stained tibiae are then transferred to a microscope stage
and the width of the uncalcified cartilage plate, which does not stain and remains
white, is measured under low power with a calibrated micrometer eyepiece. Ten
individual readings are made across the epiphysis.
S35 uptake
This bioassay of growth hormone activity is based on the principle that cartilage
contains glucosaminoglycans, eg, chondroitin sulphate. The uptake of labelled
sulphate into the cartilage is greatly reduced after hypophystomy and restored
after growth hormone application.
Female albino rats are hypophystomised at 21 days of age and used for
experimentation three weeks later. The animals are given intra-peritoneal
injections of growth hormones together with radiolabelled sulphate once daily
for 4 days. Eight to ten animals are used for atleast two doses of test preparation
and standard. The rats are sacrificed 24 h after the last injection and the amount
of radiosulphate present in the seventh rib cartilage determined. A linear
relationship exists between the uptake of radiosulphate and the hormone given
over a range of 3–20 μg per day for 4 days.
Inhibition of glucose uptake in adipocytes in vitro
This bioassay is based on the principle of conversion of glucose to lipid in
murine adipocytes. It is dose-dependently inhibited by human growth hormone.
The medium, Dulbecco’s modified Eagle’s medium (DMEM) containing 5.5
mM glucose, 2% BSA, 25 mM dexamethasone, 37 mM estradiol, 10 μg/litre
insulin and 0.1 μci/ml uniformly labelled [14C] D-glucose is used. Cultures are
co-incubated with increasing concentrations of 22-kDa human growth hormone
(0.313–40 μg/litre) as standard or test substance or medium (controls) for 24 h.
For determination of HGH in patients 100 μl or 20 μl serum are added. After
incubation, the medium is removed and discarded. The cells are treated with
Doles reagent (one part heptane, 4% isopropyl alcohol, 0.1 part 1 N H2SO4), the
plates scraped and the contents transferred to a glass tube. Lipids are extracted
and radioactivity of the lipid is determined by scintillation counting in a liquid
scintillation spectrophotometer (Fig. 9.12). The results are expressed as C14
counts per min./dish. Lipid accumulation in controls without HGH is taken as
100%. Activity ratios are calculated from dose–response curves.
Fig. 9.12 Liquid scintillation counter

9.4 SCREENING METHODS FOR ANTI-DIABETIC DRUGS

Diabetes mellitus is a metabolic disorder characterised by increased blood
glucose level associated with discharge of glucose in urine. There are two major
types of diabetes mellitus i.e. insulin dependent diabetes mellitus (IDDM) and
non-insulin dependent diabetes mellitus (NIDDM). Insulin dependent diabetes
mellitus, also called type 1 diabetes, occurs due to complete loss of pancreatic β-
islet cells and hence there is insulin deficiency. Non-insulin dependent diabetes
mellitus, also called as type 2 diabetes, is due to insulin resistance. Insulin
resistance is developed due to defects at the receptor level or insulin signalling at
the post-receptor level. This defect may be in the effector cells such as the
skeletal muscle, the adipose tissue etc or in the β-islet cells. A large number of
drugs including herbs and minerals with suspected anti-diabetic activity have
been successfully tested in the laboratory. The various animal models to screen
anti-diabetic activity are listed in this section.

9.4.1 Models for Insulin Dependent Diabetes Mellitus (IDDM)

Alloxan induced diabetes
Alloxan is a cyclic urea compound which induces permanent diabetes. It is a
highly reactive molecule which produces free radical damage to β-islet cells and
causes cell death. Alloxan at a dose level of 100 mg/kg in rats produces diabetes.
In rabbits, a dose level of 150 mg/kg infused through a marginal ear vein
produces diabetes in 70% of the animals.
Albino rats of either sex weighing 150–200 g are injected with a single dose
of alloxan monohydrate (100 mg/kg body weight) dissolved in normal saline
(0.9%) by i.p route. The animals are kept for 48 h during which food and water
is allowed ad libitum. The blood glucose level shows the triphasic response with
hyperglycemia for 1 h followed by hypoglycemia that lasts for 6 h and stable
hyperglycemia after 48 h. The animals showing fasting blood glucose level
above 140 mg/dl after 48 h of alloxan administration are considered diabetic.
Drug samples to be screened are administered orally for a period of six weeks.
After six weeks of treatment, blood samples are collected from 8 h fasting
animals through a caudal vein. Serum is separated by cooling centrifuge (2°C–
4°C) at 3000 rpm for 10 min. The serum glucose level is estimated by glucose
oxidase–peroxidase method (GOD–POD kit) using an autoanalyser.
Streptozotocin induced diabetes
Streptozotocin is a broad-spectrum antibiotic which causes β-islet cells damage
by free radical generation. Streptozotocin induces diabetes in almost all species
of animals excluding rabbits and guinea pigs. The diabetogenic dose of
streptozotocin varies with species. In mice, the dose level is 200 mg/kg through
i.p and in beagle dogs 15 mg/kg through i.v. for 3 days.
Adult albino rats of either sex weighing 150–200 g are injected with
streptozotocin (60 mg/kg body weight) prepared in citrated buffer (pH: 4.5)
solution by i.p. The citrated buffer is prepared by mixing 53.9 parts of 0.1 M
citric acid and 46.1 parts of 0.2 M disodium hydrogen orthophosphate and
finally adjusted to a pH of 4.5. The blood glucose level shows the same triphasic
response as seen in alloxan treated animals. Animals showing fasting blood
glucose level above 140 mg/dl after 48 h of streptozotocin administration are
considered diabetic. Drug samples to be screened are administered orally for a
period of six weeks. After six weeks of treatment, blood samples are collected
from 8 h fasted animals through a caudal vein. Serum is separated by cooling
centrifuge (2°C–4°C) at 3000 rpm for 10 min. The serum glucose level is
estimated by glucose oxidase–peroxidase method (GOD–POD kit) using an
autoanalyser.
Virus induced diabetes
Viruses are one of the etiological agents for IDDM. They produce diabetes
mellitus by infecting and destroying β-islet cells of pancreas. Various human
viruses used for inducing diabetes include RNA picornovirus, coxsackie B4
(CB-4) and encephalomyocarditis (EMC-D).
Six to eight-week-old mice are inoculated by 0.1 ml of 1:50 dilution of D-
variant encephalomyocarditis (EMC) through i.p. The 0.1 ml of the above
dilution contains 50 PFU (plaque forming units) of EMC virus. Mortality due to
this concentration of virus is approximately 10–20%. A less infecting variant
produces a comparable damage by eliciting autoimmune reactivity to the β-islet
cells. Infected animals are considered hyperglycemic if their non-fasting levels
exceed by 250 mg/dl the levels of uninfected animals of the same strain. Drug
sampies to be screened are administered orally for a period of six weeks. After
six weeks of drug treatment, blood glucose estimation is done to determine the
anti-diabetic activity.
Insulin antibodies induced diabetes
A transient diabetic syndrome can be induced by injecting guinea pigs with anti-
insulin serum. It neutralises the endogenous insulin with insulin antibodies.
Diabetes persists as long as the antibodies are capable of reacting with the
insulin remaining in circulation.
Preparation of antibody
Bovine insulin, dissolved in acidified water (pH: 3.0) at a dose of 1 mg is
injected to guinea pigs weighing 300–400 g. Anti-insulin sera is collected after
two weeks of antigenic challenge.
Adult albino rats are injected with 0.25–1.0 ml of guinea pig anti-insulin
serum. Insulin antibodies induce a dose-dependent increase of blood glucose
level up to 300 mg/dl. Slow rate intravenous infusion or an intra-peritoneal
injection prolongs the effect for more than a few hours. However, large doses
and prolonged administration are accompanied by ketonemia. The drug sample
to be screened is administered by a suitable route and blood glucose level is
analysed to determine the activity.
Hormone induced diabetes
Dexamethasone
Dexamethasone is a steroid possessing immunosupression action which causes
an autoimmune reaction in the islets and produces type 1 diabetes.
Adult rats weighing 150–200 g are injected with dexamethasone at a dose
level of 2–5 mg/kg body weight by i.p. twice a day. The repeated injection of the
same dose level is carried out for a period of 20–30 days resulting in IDDM. The
sample to be screened is administered through a suitable route and blood glucose
analysed to determine the activity.
Genetic models
Non-obese diabetic mouse (NOD)
NOD mouse is a model of IDDM. Hypoinsulinemia is developed which is
caused by autoimmune destruction of pancreatic β-islet cells in association with
auto-antibody production.
Mice are breed at the laboratory by sib-mating over 20 generations. After 20
generations of sib-mating, spontaneous development of IDDM in mice is
obtained. Diabetes develops abruptly between 100–200 days of age. Weight loss,
poly urea and severe glucosuria are common. The animals are treated with the
drug sample to be screened. Blood sample is analysed for glucose level to
determine activity.
Bio-breeding (BB) rat
Diabetes is inherited as an autosomal recessive trait and develops with equal
frequency and severity among males and females. Insulin deficiency and
insulitis are due to autoimmune destruction of pancreatic β-islet cells.
Spontaneous diabetes is diagnosed in a non-inbreed but closed out-breed colony
of rats at bio-breeding laboratories.
Rats are breed at the laboratory by sib-mating over 20 generations. After 20
generations of sib-mating, spontaneous development of IDDM in rats is
obtained. The onset of clinical diabetes is sudden and occurs at about 60–20 days
of age. The clinical presentation of diabetes in the BB rat is similar to that of its
human counterpart. Marked hyperglycemia, glycosuria and weight loss occur
within a day of onset and are associated with decreased plasma insulin, that if
untreated will result in ketoacidosis. The animals are treated with the drug
sample to be screened for a required period of time. The blood sample is
analysed for glucose level to determine activity.

9.4.2 Models for NIDDM

Streptozotocin induced neonatal model for NIDDM
Streptozotocin causes severe pancreatic β-cells destruction, accompanied by a
decrease in pancreatic insulin stores and a rise in plasma glucose level. In
contrast to adult rats, the treated neonates partially regenerate and become
normoglycemic by three weeks of age. In the next few weeks, the β-cell number
increase, mainly from the proliferation of cells derived from ducts, leads to
hyperinsulinemia and shows symptoms similar to insulin resistance.
Neonatal rats are treated with streptozotocin (90 mg/kg body weight) prepared
in citrated buffer (pH: 4.5) by i.p at birth or within the first 5 days following
birth. After six weeks, the rats develop symptoms similar to NIDDM. Rats
showing fasting blood glucose level above 140 mg/dl are considered diabetic.
Further steps are similar to that of the alloxan induced model. The drug sample
to be screened is administered by a suitable route and blood glucose level is
analysed to determine the activity.
Other chemically induced NIDDM models
Adrenaline induced acute hyperglycemia
Adrenaline is a counter-regulatory hormone to insulin. It increases the rate of
glycogenolysis and glucose level in blood causing acute hyperglycemia.
Adult albino rats are injected at a dose level of 0.1 mg/kg through s.c route.
The dose produces peak hyperglycemic effect at 1 h and lasts up to 4 h. The drug
sample to be analysed is administered through a suitable route and blood glucose
determined. Oral hypoglycemic agents can be screened by this method.
Chelating agents
Dithizone induced diabetes
Organic agents react with zinc in the islets of Langerhans causing the destruction
of the islet cells and producing diabetes. Severe necrosis and disintegration of β-
cells (insulin producing cells) were observed while ⍺-cells (cells which produce
glucoagon which maintains the glucose level in the blood) remain unaltered.
Compounds such as dithizone, EDTA, 8-hydroxy quinoline are used to induce
spontaneous type 2 diabetes in experimental animals. Dithizone at a dose level
of 40–100 mg/kg (i.v.) produces type 2 diabetes in mice, cats, rabbits, and
golden hamsters.
Adult rabbits weighing 1.8–2 kg are divided into two groups of six animals
each. An exactly weighed amount of dithizone is dissolved in dilute ammonical
solution (0.2 to 0.5%). The solution is warmed to 60°C–70°C for 10 min. to aid
solubility of dithizone. Dithizone injection at a dose level of 50–200 mg/kg will
produce triphasic glycemic reaction. Initial hyperglycemia will be observed after
2 h and normoglycemia after 8 h, which persists for up to 24 h. Permanent
hyperglycemia is observed after 24–72 h. The drug sample to be analysed is
administered through a suitable route and the blood glucose determined.
Models for insulin sensitivity and insulin-like activity
Euglycemic clamp technique
This method has proved to be a useful technique of quantifying in vivo insulin
sensitivity. A variable glucose infusion is delivered to maintain euglycemia
during insulin infusion. The net glucose uptake is quantified and the sensitivity
of the body tissue to insulin determined.
Adult albino rats weighing 150–200 g are fasted overnight and anaesthetised
with pentobarbital (40 mg/kg i.p.). Catheters are inserted into a jugular vein and
a femoral vein for blood collection and insulin and glucose infusion,
respectively. To evaluate the insulin action under physiological hyperinsulinemia
(steady state plasma insulin concentration during the clamp test is around 100
μU/dl) and maximal hyperinsulinemia, two insulin infusion rates e. 6 and 30
mU/kg/min. are used. The blood glucose concentrations are determined from
samples collected at 5 min. intervals during the 90 min. clamp test. The glucose
infusion rate is adjusted so as to maintain basal level. The glucose metabolic
clearance rate is calculated by dividing the glucose infusion rate by the steady
state blood glucose concentration. The drug sample to be analysed is
administered through a suitable route and the blood glucose determined.
Assay for insulin and insulin-like activity
This assay involves comparing two standard solutions of insulin with the test
drug for its insulin-like activity.
Four groups of six rabbits weighing at least 1.8 kg are used. Two standard
solutions of insulin containing one unit and two units respectively and two
dilutions of sample whose potency is being examined are prepared. As diluent, a
solution of 0.1 to 0.25% w/v of either m-cresol or phenol and 1.4 to 1.8 w/v of
glycerol acidified with hydrochloric acid to a pH between 2.5 to 3.5 is used.
Each of the prepared solution (0.5 ml) is injected subcutaneously. After one hour
and 2.5 h of each injection, a suitable blood sample is taken from the ear vein of
each rabbit and the blood sugar determined preferably by glucose oxidase
method.

9 .5 SCREENING METHODS FOR THYROID AND ANTI-THYROID

DRUGS
The thyroid gland consists of follicular cells which secrete thyroxin (T4) and
triidothyronine (T3) and para follicular cells which secrete calcitonin
respectively. The main biological effects of T3 and T4 are on growth and
development, the calorigenic effect, cardiovascular function and metabolic
functions. The primary feedback effect is inhibition of TSH-secretion. These
effects can be used for testing thyroid hormone analogues and metabolites. Anti-
thyroid drugs can inhibit the iodine uptake and utilisation in the thyroid.

9.5.1 Changes in Metabolic Rate

Oxygen consumption rate (Smith et al. 1947)
This method is most commonly used for diagnostic procedures in man as well as
for evaluation of thyroid hormones and their derivatives in animals (indirect
calorimetry). It works on the principle that thyroid hormones increase basal
metabolic rate, oxygen consumption and carbon dioxide production.
The main requirements include 200 ml wide neck bottles, stop watches and
albino mice.
Mice are placed individually into 200 ml wide-necked bottles. Groups of 10
mice are used for each dose of test preparation or standard. The bottom of the
bottles is covered with filter paper to soak up urine. The bottles are sealed, tilted
to a 60° angle and rotated 5 times per min. in a special apparatus. The time when
the mice start to undergo an asphyctic seizure is noted. Immediately after
observation of seizures, the mice are released for recovery. Due to the defined
muscle work, the test drug increases the time it takes for the mice to start
seizure. Average time to seizures is calculated for each dosage and dose-
response curves are established. The main disadvantage of this method is that the
mice may sometimes die due to convulsions.
Mouse anoxia method
Resistance of rats to oxygen deficiency was decreased when they were treated
with thyroid extracts. This increased sensitivity to anoxia forms the basis of an
assay method in which mice were dosed with a graded amount of iodinated
casein.
The main requirements for the experiment include a closed jar, thermometers,
thyroxin and healthy adult albino mice.
20 healthy adult albino mice are used. They are divided into three groups:
control, standard and test compound. The standard compound is thyroxine at a
dose of 5, 10 and 20 μg per animal given in the form of a subcutaneous injection
once daily on 3 alternate days; 2 or 3 days after the last injection they are placed
in a closed jar of 1 litre capacity at 23°C and the survival time determined.The
end point is taken as the last viable respiration just before convulsions start.
Carbon dioxide output
This assay is based on the principle that thyroid extracts alter the carbon dioxide
output of mice. Mice are kept for 24 h in a glass respiratory chamber through
which a steady stream of carbon dioxide free air is passed. The carbon dioxide
output of the mice is measured. The carbon dioxide in the atmosphere is sucked
in KOH solution and the amount absorbed determined by titration. This causes
the chamber to cool, so care should be taken that the temperatue is maintained at
23°C.
The main requirements for this assay include closed chambers and healthy
adult albino mice (16–22 g).
Healthy adult albino mice weighing (16–22 g) are taken and kept at a standard
diet. After 8 days, the normal carbon dioxide outputs are determined for 5
consecutive days by placing the mice along with food in closed chambers at
23°C. After 3 weeks, animals are dosed with test substances. Daily doses are
adjusted according to body weight. The carbon dioxide output is also
determined.
If there is loss of weight of mice, carbon dioxide output is expressed in terms
of body weight and if there is gain of weight it is expressed as body surface area.
Anti-goitrogenic activity
Increased secretion of TSH induces thyroid enlargement and weight increase
within a few days. In normal animals, the secretion of TSH by the pituitary is
regulated by feedback from thyroid hormones. The administration of goitrogenic
compounds which block thyroid hormone synthesis and/or secretion reduces the
concentrations of circulating T4/T3 and their pituitary effect, thus releasing TSH
from its feedback inhibition. The TSH rise induces hyperplasia of the thyroid
follicles as indicated by an increase in thyroid weight. Hyperplasia is prevented
by injection of thyroxine or thyroid hormone analogs.
The main requirements include propyl thiouracil (0.1%), thyroxine (10–40
μg/kg) and healthy albino rats (150–180 g).
Healthy albino rats weighing 150–180 g are used in groups of 8–10 animals.
0.1% propyl thiouracil (PTU) is added to the food or to the drinking water or
equivalent doses are given by gavage for 2 weeks. The rats are treated
(subcutaneously or by gavage) with various doses of the test compound or the
thyroxine standard (10–40 μg/kg). The PTU control group receives only PTU
and saline injections. The rats are sacrificed after 14 days; their thyroid glands
are dissected out cleanly and weighed rapidly to avoid evaporation loss. The
two- to three-fold increase of thyroid weight by PTU is reversed dose-
dependently to normal values by thyroid-active substance. Dose–response curves
are plotted for test compounds and the standard and activity ratios calculated.
Inhibition of iodine release (Perry 1951)
In rats, the release of iodine-131 from the thyroid is inhibited by treatment with
thyroxine and the degree of inhibition is related to the dose administered. This
phenomenon was used to compare thyroid hormone derivatives with the standard
thyroxine.
The main requirements include propylthiouracil, ether, iodine-131 and healthy
albino rats.
Healthy albino rats weighing 180–240 g are fed a commercial laboratory
chow with or without addition of 0.03% propyl thiouracil (reference compound
for thyroid peroxidase inhibition). Food is withheld 8 h before and for 24 h after
iodine-131 or iodine-125 is injected intraperitoneally at 25°C. Under light ether
anaesthesia, radioactivity over the thyroid region of the neck is determined 40 h
later. This value is taken as zero time and all further counts made at 24 h
intervals, are expressed as a percentage of zero time-counts after correction for
physical decay of the isotope. After the reading at zero time, the diet is changed
to a diet containing 0.03% propyl thiouracil and various doses of the test
preparation or the standard are injected subcutaneously at 24 h intervals for a
total of four doses. The daily loss of iodine-131 is inversely proportional to the
dose of thyroid hormone. The percentage of zero time-counts after 96 h of
iodine-131 remaining in the thyroid after the last of 4 doses is plotted against
logarithm of the dose. From these dose –response curves, potency ratios are
calculated.
Thyroidectomy (Bomskov 1937)
Endocrinological experiments for pharmacological evaluation of thyroid
hormones and analogues may be performed in thyroidectomised rats. The
thyroid in rats consists of three lobes (left, median and right).
The main requirements include nembutal anaesthesia, surgical equipments and
albino rats.
The rat is anaesthetised with nembutal and the legs fixed on a surgical table.
The fur of the neck is removed with electric clippers and the area disinfected. A
median skin incision of 2.0 cm length is made. On both sides of the neck, large
salivary glands and maxillary lymph nodes are found. They are pushed aside,
making visible the musculus hyoideus covering the trachea. This muscle is split
in the mid-line. The isthmus of the thyroid connecting both lobes is located.

9.5.2 Anti-thyroid Drugs

Anti-thyroid drugs are characterised by their ability to interfere with synthesis,
release and/or peripheral action of the thyroid hormone, thereby lowering the
basal metabolic rate. Decrease of T4/T3 reduces thyroidal inhibition of the
pituitary gland; TSH secretion increases and induces a goitrogenic response.
This response is used for detecting anti-thyroid drugs.
Inhibition of iodine uptake into the thyroid of rats (McGinty and Bywater
1945)
Propyl thiouracil (PTU) and a wide spectrum of drugs may inhibit thyroid
hormone synthesis. Some of these drugs can be used for the treatment of
thyrotoxicosis. As a consequence of thyroid peroxidase inhibition, the iodine
uptake and content of the thyroid is decreased. This phenomenon is dose-
dependent.
The main requirements include potassium iodide, thiouracil and immature
albino rats.
Groups of immature albino rats of age 26–28 days, weighing 40–45 g, are
placed in metabolism cages. They are fed a normal diet, and potassium iodide is
added to the drinking water. The test compounds or the reference standard are
added in various concentrations to the diet over a period of ten days. The amount
of compound that each rat receives is calculated from the total food consumption
over 10 days and expressed as milligram daily per kilogram of body weight.
After 10 days of treatment, the rats are sacrificed and the thyroids dissected free
from the adjacent tissues. The thyroid is weighed and iodine content determined.
In daily doses between 0.1 and 10.0 mg/kg, thiouracil decreases the iodine
content of the thyroid depending on the dose. Dose–response curves of test
compounds are compared with those of the reference standard for calculation of
potency ratios with confidence limits.

9 .6 ESTIMATION OF VARIOUS HORMONES INVOLVED IN

REPRODUCTION BY ELISA
Various drugs can affect the levels of reproductive hormones such as estrogen,
progesterone, LH and FSH. Plants like ginseng (Ginseng radix), liquorice root
(Glycyrrhiza radix) can affect the levels of FSH; neem (Azadirachta indica) and
mango (Mangnifera indica) can affect the progesterone; soyabean (Glycine max)
and alfalfa (Medicago sativa) can affect estrogen levels. These changes in
hormone levels can be estimated by various ELISA techniques.
For nearly three decades, enzyme-linked immunosorbent assays (ELISA) have
been used to detect diseases, toxin food, for environmental monitoring and
related fields. ELISA is a useful and powerful method in estimating ng/ml to
pg/ml ordered materials in the solution, such as serum, urine and culture
supernatant. This assay is based on the principle of antibody-antibody
interaction. It allows for easy visualisation of results and can be completed
without the additional concern involved in using radioactive materials. The plate
used to fix the sample for the assay is shown in Fig. 9.13.

Fig. 9.13 A 96-well microtitre plate used in the ELISA reader

9.6.1 Luteinising Hormone (Vermes et al. 1991)

The LH quantitative test is based on a solid phase enzyme-linked
immunosorbent assay (ELISA). The assay system utilises a mouse monoclonal
anti-alpha LH antibody for the solid phase (microtitre wells) in the antibody
enzyme (horse radish peroxidase, HP) conjugate solution. The test sample is
allowed to react simultaneously with the antibodies, resulting in LH molecules
being sandwiched between the solid phase and the enzyme-linked antibodies.
After a 45-min. incubation at room temperature, the wells are washed with water
to remove unbound labelled antibodies. A solution of TMB (tetra methylene
benzidine) reagent is added and incubated at room temperature for 20 min.,
resulting in the development of a blue colour. The colour development is stopped
by addition of 1 N HC1, and the absorbance is measured spectrophotometrically
at 450 nm. The intensity of the colour formed is proportional to the unlabelled
FSH in the sample.
Add 50 μl of calibrators to some wells to prepare standard wells and 50 μl of
sample to prepare sample wells. Both the types of wells are then labelled as
standard or sample. Add 50 μl of anti-LH-HP conjugate. Incubate for 30 min. at
room temperature with continous shaking at 700 rpm. Aspirate the contents of
each well. Wash 3 times with 400 μl of wash solution and aspirate. Add 200 μl
of revealing solution to each of them. Incubate for 15 min. at room temperature
with continuous shaking at 700 rpm and then add 50 μl of the stop solution.
Record the absorbance of each well at 450 nm. LH concentrations in each of the
urine/plasma sample can be calculated by extrapolation of absorbance observed
in the ELISA reader (Fig. 9.14) through standard curve of LH.

Fig. 9.14 ELISA reader

9.6.2 Estradiol (McNasty et al. 1976)

This assay is based on the principle of competitive binding between E2 in the test
specimen and E2–HP conjugate for a constant amount of rabbit anti-estradiol. In
the incubation, goat anti-rabbit IgG-coated wells are incubated with 25 μl E2
standards, control, patient samples, 100 μl estradiol–HP (horse radish
peroxidase) conjugate reagent and 50 μl rabbit anti-estradiol reagent at room
temperature (18–25°C) for 90 min. During the incubation, a fixed amount of HP-
labelled E2 competes with the endogenous E2 in the standard, the sample or
quality control serum for a fixed number of binding sites of the specific E2
antibody. Thus the amount of E2 perioxidase conjugate immunologically bound
to the well progressively decreases as the concentration of E2 in the specimen
increases. Unbound E2 peroxidase conjugate is then removed and the wells
washed. Next, a solution of TMB (tetra methylene benzidine) reagent was added
and incubated at room temperature for 20 min., resulting in the development of a
blue colour. The colour development is stopped by addition of 1N HC1. The
absorbance is measured spectrophotometrically at 450 nm.The intensity of the
colour formed is proportional to the unlabelled E2 in the sample.
The desired numbers of coated wells are placed in the holder. About 25 μl of
standards, specimens and controls is dispensed into appropriate wells. 100 μl of
working estrogen–HP conjugate reagent will be added into each well. Then 50 μl
of rabbit anti-estrogen reagent is added into each well. It is thoroughly mixed for
30 s and incubated at room temperature (18–25°C) for 90 min. It is then rinsed 5
times with distilled or de-ionised water. 100 μl of TMB reagent is then added
into each well. It is then gently mixed for 5 s and incubated at room temperature
(18–25°C) for 20 min. The reaction is stopped by adding 100 μl of stop solution
to each well and mixed for 30 s. It is important to make sure that all the blue
colour changes to yellow completely. Absorbance is read at 450 nm with a
microtitre well reader within 15 min. Estradiol concentrations in each of the
urine/plasma sample can be calculated by extrapolation of absorbance observed
in the ELISA reader through the standard curve of estradiol.

9.6.3 Progesterone (Johansson et al. 1971)

The progesterone enzyme immunoassay is based on the principle of competitive
binding between progesterone in the test specimen and progesterone–HP
conjugate for a constant amount of rabbit anti-progesterone. In the incubation,
goat anti-rabbit IgG-coated wells are incubated with 25 μl progesterone
standards, control, patient samples and 100 μl progesterone-HP conjugate
reagent and 50 μl rabbit anti-progesterone reagent at room temperature (18–
25°C) for 90 min. During the incubation, a fixed amount of HP-labelled
progesterone competes with the endogenous progesterone in the standard,
sample or quality control serum for a fixed number of binding sites of the
specific progesterone antibody.
Thus the amount of progesterone perioxidase conjugate immunologically
bound to the well progressively decreases as the concentration of progesterone in
the specimen increases. Unbound progesterone peroxidase conjugate is then
removed and the wells washed. Next, a solution of TMB reagent is added and
incubated at room temperature for 20 min., resulting in the development of a
blue colour. The colour development is stopped by addition of 1 N HC1, and the
absorbance is measured spectrophotometrically at 450 nm.The intensity of the
colour formed is proportional to the unlabelled progesterone in the sample.
 The desired number of coated wells are attached to the holder. About 25 μl of
standards, specimens and controls is dispensed into appropriate wells. 100 μl of
working progesterone–HP conjugate reagent is added into each well. Then 50 μl
of rabbit anti-progesterone reagent is added into each well. It is thoroughly
mixed for 30 s and incubated at room temperature (18–25°C) for 90 min. It is
rinsed 5 times with distilled or de-ionised water. 100 μl of TMB reagent is then
added into each well. It is gently mixed for 5 s and incubated at room
temperature (18–25°C) for 20 min. To stop the reaction 100 μl of stop solution is
added to each well and mixed for 30 s. It is important to make sure that all the
blue colour changes to yellow completely. Absorbance can be read at 450 nm
with a microtitre well reader within 15 min. The progesterone concentration of
the specimens and controls can be calculated from the standard curve.

9.6.4 Follicle Stimulating Hormone

The FSH quantitative test is based on a solid phase enzyme-linked
immunosorbent assay (ELISA). The assay system utilises a mouse monoclonal
anti-alpha FSH antibody for the solid phase (microtitre wells) in the antibody
enzyme (horse radish peroxidase) conjugate solution. The test sample is allowed
to react simultaneously with the antibodies, resulting in FSH molecules being
sandwiched between the solid phase and the enzyme-linked antibodies. After 45
min. incubation at room temperature, the wells are washed with water to remove
unbound labelled antibodies. A solution of TMB reagent is added and incubated
at room temperature for 20 min., resulting in the development of blue colour.
The colour development is stopped by addition of 1 N HC1, and the absorbance
is measured spectrophotometrically at 450 nm.The intensity of the colour formed
is proportional to the unlabelled FSH in the sample.
The desired number of coated wells is placed in the holder and 50 μl of
standards, specimens and controls is dispensed into appropriate wells. 100 μl of
working enzyme conjugate reagent is added into each well and thoroughly
mixed for 30 s. They are then incubated at room temperature (18–25°C) for 45
min. and rinsed 5 times with distilled or de-ionised water. 100 μl of TMB
reagents is added into each well, gently mixed for 10 s and kept for incubation at
room temperature (18–25°C) in the dark for 20 min. The reaction is then stopped
by adding 100 μl of stop solution to each well and gently mixing for 30 s. It is
important to make sure that all the blue colour changes to yellow completely.
Absorbance can be read at 450 nm with a microtitre well reader within 15 min.
9.7 CARDIOVASCULAR SCREENING METHODS
An animal model is a non-human animal that has a disease or injury that is
similar to a human condition. Animal models are the backbone of any research
project. The choice of animal models in cardiovascular research is dictated by
size consideration alone. When dealing with hemodynamics, the need is for
larger vessels, whereas for histopathological examination, it is wise to use mice
where the structure is to be serially sectioned into small pieces. In other words,
the animal model must match the design of the experiment.

9.8 SELECTION OF AN ANIMAL MODEL FOR CARDIOVASCULAR

SCREENING
9.8.1 Primates
A wide variety of animal species and strains have been used as models of CVD,
with variable degrees of success. As seen in humans, an ideal model – small and
economical, yet large enough to permit desired experimental procedures – will
precisely mimic CVD. In many respects, nonhuman primates such as
chimpanzees and rhesus monkeys are ideal models. They are phylogenetically
close to humans, eat a similar omnivorous diet, have similar metabolism, and
develop both metabolic syndrome and CVD as they age. On the negative side,
nonhuman primates live for long periods of time (requiring lengthy
experiments), are expensive to maintain, and often carry viral zoonoses that are
dangerous to humans. The use of primates also raises significant ethical issues,
such that it is only indicated in limited circumstances.

9.8.2 Dogs
The dog can appear to be an attractive species for the study of CVD and has, in
the past, been widely used for studies of circulatory physiology. However, while
dogs can be of an ideal size and are easy to work with, there are problems
associated with the status of dogs in the Western culture and with
anthropomorphic attitudes toward them. In addition, as canines, they are
carnivores and are, in turn, resistant to high-lipid diets and atherosclerosis. Their
cardiovascular system also has important physiological characteristics that differ
from those of humans, such as a contractile spleen that compensates for blood
loss and hypotension. In addition, dogs have an extensive myocardial collateral
circulatory system, much greater than that seen in pigs, primates and rats. This
confers a degree of protection against myocardial infarction not seen in other
species, except cats. While most mammalian species respond to chronic
ischemia with angiogenesis and/or growth of collateral vessels, these processes
are much more efficient in dogs than in pigs or humans. Thus, while the dog is a
useful model for the study of processes underlying revascularization, the results
can only be extrapolated to the human condition with great care.

9.8.3 Swine
The domestic pig shares many of the advantages of the nonhuman primate,
including susceptibility to atherosclerosis, similar dietary preferences, and
similar gastrointestinal system and metabolism. A major drawback is the large
size of adult pigs with resultant management difficulties and, for drug
assessment, a need for large amounts of experimental agents. Experimental use
of swine, including miniature pigs, to study CVD has generally depended on
feeding of high-cholesterol and/or high-sugar diets and/or chemical induction of
quasi type 1 diabetes with alloxan or streptozotocin. Spontaneous development
of metabolic syndrome and insulin resistance, however, is not common in this
species.

9.8.4 Rabbits
The rabbit has been widely used in studies of atherosclerosis and has the merits
of being small and relatively inexpensive, but large enough to permit
physiological experiments. An herbivore, the rabbit is not inherently
atherosclerosis-prone, and induction of vascular lesions necessitates a high-
cholesterol diet. Even mild cholesterol supplementation leads to
hypercholesterolemia and aortic lesions in these sensitive animals. Lesions start
as fatty streaks, primarily on the aorta, and can develop into advanced lipid-
laden intimal lesions. There are reservations about the nature of the lesions and
the mechanisms of induction, given the highly abnormal diet and the major
differences in lipid metabolism between rabbits and humans. In addition, there is
evidence that even modest dietary manipulation, such as the substitution of
casein for soy protein in the diet is atherogenic in the rabbit, in the absence of
dietary cholesterol. On the other hand, familial hypercholesterolemia (FH) has
long been recognized as a definitive risk factor for atherosclerotic disease in
humans. Kondo and Watanabe isolated a mutation in the rabbit and developed
the Watanabe heritable hyperlipidemic strain, which has extreme
hypercholesterolemia and associated advanced atherosclerosis. The mutation
was shown to cause a defect in the low-density lipoprotein receptor (LDLR),
providing an animal model of the previously recognized genetic defect
underlying FH in humans. This facilitated the characterization of the effects of
genetic defects on LDL metabolism. These studies, based on a genetic animal
model, confirmed the importance of elevated LDL-C levels in CVD in humans
and fostered the development of hypocholesterolemic agents, particularly
inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase or statins.

9.8.5 Hamster
The Syrian hamster is a small rodent of Middle Eastern origin that is also
sensitive to high-fat, cholesterol-supplemented diets. The hamster that is also
sensitive to high-fat, cholesterol-supplemented diets. The hamster carries a
significant portion of its plasma cholesterol in the LDL lipoprotein fraction, and
is thus, closer to humans than to rodents and this has led to numerous studies of
lipid metabolism in the hamster, largely involving high-fat, cholesterol-
supplemented diets. Cholesterol-fed hamsters develop mild atherosclerosis that
is exacerbated by the inhibition of NO synthesis. The aortic lesions seen are
confined to fatty streaks, and the cardiovascular effects reported do not include
advanced intimal lesions or myocardial infarcts. Hamsters fed a very high (60%)
fructose diet become insulin-resistant and have been used to study the effects on
insulin signaling and lipoprotein metabolism, but evidence of CVD has not been
reported in this model. Ablation of pancreatic h cells with streptozotocin, to
create quasi type 1 diabetes, results in significant atherosclerosis and glomerular
sclerosis in cholesterol-fed hamsters that can develop into advanced aortic
lesions. Some of the reservations regarding mouse models, as detailed above,
also apply to the hamster as a model of human CVD, especially because the
creation of CVD requires highly abnormal diets and/or treatment with a
cytotoxic chemical agent, such as streptozotocin.

9.8.6 Rat
Rats are generally considered to be resistant to atherogenesis, although lesions
have been produced by heroic measures. Naturally-occurring lesions in rats are
not very similar to that seen in man. Although lipid-containing lesions can be
produced in rats, they are generally considered to be residual lesions following
acute arteritis.
 The development of genetically-engineered mice with disorders of lipid
metabolism, such as apolipoprotein E (apoE) and LDL receptor knockout mice,
was therefore a major step forward in animal models of atherosclerosis. These
mice develop atherosclerosis spontaneously. The plaques that develop are
widespread and reproducible and have some architectural features reminiscent of
human lesions. These mice have formed the basis for a plethora of studies
identifying specific molecules critical to atherosclerosis, in particular, those
regulating monocyte adherence/chemotaxis and macrophage differentiation/foam
cell development. The major dissent has been that lesions occur at sites very
different from human lesions – the aortic root and thoracic aorta, for instance.
Lesions in the aortic root are also foam cell-rich, rather than smooth muscle cell-
rich, and may not have a single definable fibrous cap, and represent xanthomata
rather than clinically important advanced lesions. Most important of all, these
mice are models of atherogenesis, not advanced atherosclerosis, and they do not
exhibit the single most important event in human atherosclerosis, that of plaque
rupture leading to vessel occlusion.

9.8.7 Guinea Pig

The most striking similarity between guinea pigs and humans is that the majority
of circulating cholesterol is transported in LDL19 .Guinea pigs carry the
majority of cholesterol in LDL and possess cholesterol ester transfer pfotein and
lipoprotein lipase activities, which results in reverse cholesterol transport and
delipidation cascades equivalent to the human situation. Guinea pigs develop
atherosclerosis, and gender and hormonal status affect the extent of the
atherosclerotic plaques. The Guinea pig is a model to study the inflammatory
component of diet-induced atherosclerosis.

9.8.8 Advantages and Disadvantages of Animal Models

Due to their short life span they react and age faster. They can be sacrificed after
the entire disease process is studied and sufficient numbers are available, sp we
can study the interactions of various factors, as the physiology and anatomy of
the animals may be already known. The disadvantages of animal models are that
we may not be able to reproduce the same history of pathogenesis in animals as
in man. To reproduce some models may not be feasible. Some may be difficult
to reproduce or not possible to reproduce uniformly. Greater variability can
occur in the experimental results and there is difficulty in reducing the number of
animals used (one experimental result is obtained per animal).
9.8.9 Types of Animal Models
1. Experimental Model
This type is surgically induced, should mimic the disease being studied, and be
easily manipulated and readily reproducible. If this model does not reproduce the
disease exactly, the correlation between animal and man must be significant,
verifiable and predictable.
2. Negative Model
This type of model does not develop the disease and is usually avoided. It can be
helpful in determining why some species are resistant.
3. Orphan Model
This type of model includes diseases of animals, which do not have human
counterparts, or diseases similar to those in man with dissimilar etiologies or
pathogenesis.
4. Spontaneous Model
These are naturally-occurring diseases of animals, which mimic those occurring
in man.

9.9 SCREENING METHODS FOR ANTI-ANGINAL DRUGS

Blood is circulated into and from the heart through veins and arteries. The heart
is perfused in the retrograde direction, from the aorta at constant pressure or at
constant flow with oxygenated saline solutions. Retrograde perfusion closes the
aortic valves, just as in the in situ heart during diastole. The perfusate is
displaced through the coronary arteries flowing off the coronary sinus and opens
the right atrium. Parameters usually used to determine whether the heart is
functioning properly are contractile force, coronary flow and cardiac rhythm.
Drugs may influence the rate (chronotrophy) and force of contraction
(inotrophy). An increase in the heart rate is called a positive chronotrophic
response while a decrease in response is called a negative chronotrophic
response. Similarly an increase in force of contraction is called a positive
inotrophic response and a decrease in force of contraction is called a negative
inotrophic response. Adrenergic drugs like epinephine and norepinephine
produce positive inotrophic and chronotrophic effects while cholinergic drugs
like acetylcholine produce negative inotrophic and chronotrophic effects.

9.9.1 Langendroff’s Experiment Using Guinea Pig Isolated Heart

(Broadley 1979)
The main requirements for this assay involve polygraphs, pentobarbital sodium
(30–40 mg/kg), ringer solution, sodium thiopental, propanolol (0.1 mg), a drop
counter and male/female guinea pigs.
Guinea pig of either sex weighing 300–500 g is selected. The heart is
surgically removed from barbiturate-anaesthetised pigs. The pigs are placed
under artificial respiration, while the heart is placed in a dish containing ringer
solution at 37°C. Pericardial and lung tissues are removed. The aorta is located
and cut just below its division. A cannula is inserted into the aorta and tied with
2 threads. The perfusion apparatus is started and kept at 37°C by water from a
thermostat (Fig. 9.15). Oxygenated ringer is perfused at a constant pressure of 40
mm of Hg at a temperature of 37°C from a reservoir. A small steel hook with a
string is attached to the apex of the heart. The contractile force is measured using
a force transducer and the coronary flow is measured using a drop counter. The
heart rate is measured using a chronometer coupled to a polygraph. Test drugs
are injected just above the aortic cannula.

9.9.2 Positive Inotrophic Effects

Positive inotrophic effects of a test compound can be evaluated by reducing the
cardiac force. Injecting an overdose of sodium thiopental or calcium antagonists
induces experimental heart failure. This effect can be reversed by β-adrenergic
antagonists which can again be reversed by β-adrenergic drugs, cardiac
glycosides. The β-sympathomimetic activity of a new drug can be assessed by
using isoprotemol as standard. After administering thiopental sodium, the left
ventricular pressure (LVP) and (dp/dt)max decreases considerably, but coronary
flow is slightly increased. The (dp/dt)max and LVP are restored by β-adrenergic
drugs. Cardiac glycosides increases LVP and (dp/dt)max and the coronary flow is
unchanged.

Fig. 9.15 Assembly showing thermostatic organ bath and students kymograph

9.9.3 Negative Inotrophic Effects

β-adrenergic drugs such as isoproterenol at doses of 0.05 to 0.2 μg increase the
contractile force of the heart. After injection of β-adrenergic blockers, the
contractile force of isoproterenol will be attenuated. Comparing isoproterenol
inhibition versus the standard propanolol (0.1 mg) will assess negative inotropic
effects of isoproterenol.

9.9.4 Coronary Vessel Dilating Effects

Coronary vessel dilating effects are assessed using Langendroff’s heart
experiment. This experiment is also used to test the influence of test compounds
on cardiac rhythm; anti-fibrillatory effects, anti-arrhythmogenic effects etc.

9.9.5 Experimental Model for Myocardial Infarction in Cats (Harris

1950)
An infarct is an area of tissue that has died because of lack of oxygenated blood.
The myocardium is affected when a branch of coronary artery is occluded. The
extent of myocardial damage depends on the size of the blood vessel and the site
of the infarct. The damage is permanent, because cardiac muscle cannot
regenerate and the dead tissue is replaced with non-functional fibrous tissue.
When the left ventricle is involved, the effects and complications are great,
because the left ventricle has to withstand greater pressure as it receives oxygen-
rich blood from the veins.
The main requirements for this assay are pressure transducers, polygraphs,
thermo dilution catheters, multichannel polygraphs, pentobarbital sodium (19
mg/kg/i.p) and adult healthy cats of either sex.
Adult mongrel cats of either sex are selected. They are anaesthetised using
sodium pentobarbitone (19 mg/kg/i.p). Ventilation is maintained by
tracheostomy. A left lateral thorcotomy and a wide pericardiostomy are
performed. A metallic cannula is connected to the pressure transducer and
introduced into the left ventricle for registration of left ventricular pressure. The
systolic and diastolic blood pressures are measured by placing a catheter in the
right femoral artery. The left femoral vein will be cannulated for infusion
(drug/vehicle/saline) purposes. The heart rate can be assessed by using a scalar
electrocardiogram. The left anterior descending coronary artery is dissected. A
descending coronary artery is occluded for 40 min. and released gradually for re-
perfusion.
Re-perfusion is instituted for 3 h. The animal is sacrificed and the heart
prepared for determination of infarct size. Cardiac output and related
haemodynamic parameters are measured. The thermo dilution catheter that
senses the blood temperature is inserted in the right carotid artery. The external
jugular vein is cannulated for injection of reference solution. The following
haemodynamic variables are measured throughout the experiment period using a
cardiac output monitor and a multichannel polygraph: heart rate; systolic arterial
blood pressure; diastolic arterial blood pressure; left ventricular end diastolic
pressure; cardiac output; total systemic vascular resistance; stroke volume.

9.9.6 In vivo Model for Regional Myocardial Ischemia

The myocardium is composed of specialised cardiac muscle tissue found only in
the heart. When an artery is completely blocked, the tissues it supplies rapidly
undergo degeneration and die from ischaemia, which leads to infarction. When a
coronary artery is occluded, myocardial infarction occurs. In the present
experiment the occlusion of the left anterior descending coronary artery leads to
regional left ventricular ischaemia.
The main requirements are polyethylene catheters, polygraphs, pentobarbital
sodium (65 mg/kg/i.p), 10% formaldehyde and adult rabbits.
Albino rabbits weighing between 1.5–2.5 kg of either sex are selected. They
are anaesthetised with sodium pentobarbitone (65 mg/kg/i.p). After induction of
anaesthesia, tracheostomy is conducted. Ventilation is done using a rodent
ventilator. A polyethylene catheter is placed on the right femoral vein and one on
the right femoral artery. In the fifth intercostal space, a left lateral thoracotomy is
conducted. The heart is exposed. The left anterior descending coronary artery is
isolated just proximal to the first branch and a silk thread is passed around this
part of the artery. After 30 min. of post-surgical recovery period, the left anterior
descending coronary artery is occluded for 15 min. to produce regional left
ventricular ischemia. Re-perfusion is instituted by releasing it. This is continued
for 60 min. The following haemodynamic parameters are studied: heart rate;
mean arterial pressure; LV peak (+) and (–) dp/dt; electrocardiogram; left
ventricular end diastolic pressure; rat pressure product.
Computerised haemodynamic monitors and polygraphs are used to measure
the different parameters. The experimental animal is sacrificed. The affected left
ventricle is excised and stored in liquid nitrogen for determining the biochemical
parameters. Part of the tissue is fixed in 10% formaldehyde for histopathological
examination.
9.9.7 Myocardial Infarct Studies: Macroscopic Enzyme Mapping of
Ischemic Myocardium
The principle behind these studies is based on the fact that when an artery is
completely blocked, the tissues it supplies rapidly undergo degeneration and die
from ischaemia, which leads to infarction. When a coronary artery is occluded,
myocardial infarction occurs. The dye monostral blue and triphenyl tetrazolium
chloride stain myocardial ischemic sites.
The main requirements are incubators, triphenyl tetrazolium chloride (TTC),
phosphate buffer and monostral blue dye.
Triphenyl tetrazolium chloride (TTC) is used for macroscopic enzyme
mapping of the ischemic myocardium. 150–200 ml of TTC is freshly made up in
phosphate buffer. It is pre-warmed at 37–40°C for 15–30 min. Excess of blood
can be removed by washing the heart with cold water. The excess epicardial fat
is trimmed off and the left ventricle separated. The heart is cut transversely
across the left ventricles to obtain slices not more than 1 cm in thickness. Heart
slices are kept in the glass dish containing the pre-warmed TTC solution. The
dish is put in the incubator and heated between 37–40°C for 30–45 min. The
heart slices are turned over once or twice and kept immersed and covered by 1
cm of the TTC solution at all times. Finally the heart slices are placed in the
fixing solution. Normal myocardium will turn bright red, ischemic (enzyme
deficient) myocardium turns pale gray or grayish yellow and fibrous scars,
white. Differential staining with monostral blue dye and triphenyl tetrazolium
chloride can be done to the following zones of the myocardium:

1. Area of risk (AR)

2. Area of necrosis (AN)
3. Viable tissue within the area at risk
4. Area at risk destined to necrosis can be calculated using the formula,

where ‘t’ and ‘c’ represents test and control groups respectively.
9.9.8 Isoproterenol Induced Myocardial Necrosis in Rats (Rona et al.
1959)
Cardiac necrosis can be produced by injection of natural or synthetic
sympathomimetics in high doses. In rats infarct-like myocardial lesions can be
produced by isoproterenol. These lesions can be totally or partially prevented by
several drugs such as sympatholytics or calcium antagonists.
The main requirements for this assay are isoproterenol, nitroglycerin, calcium
channel blockers and adult albino rats.
Myocardial necrosis can be induced by the injection of natural or synthetic
sympathomimetics in high doses. Groups of adult albino rats are selected. They
are treated with the test drug or standard drug either subcutaneously or orally for
1 week. After one week, isoproterenol is given subcutaneously at a dose of 5.25
and 8.5 mg/kg for 2 consecutive days. The mortality rate and its symptoms in
each group are recorded and compared with rats given isoproterenol alone. Rats
are sacrificed after 48 h of the first isoproterenol administration. The hearts are
removed and weighed. The frontal sections are subjected to histological
examination. Microscopic examination allows the following grading.

Grade 0: No change
Grade 1: Focal interstitial response
Grade 2: Focal lesions in many sections
Grade 3: Confluent retrogressive lesions with necrosis
Grade 4: Massive infarct with occasionally acute aneurysm and mural
thrombi.

This test is used for evaluation of coronary-active drugs such as nitroglycerin

and calcium channel blockers.

9.9.9 Coronary Thrombosis Induced by Stenosis in Dogs (Folts et al.

1982)
Stenosis is the narrowing of a valve opening thus impeding blood flow through
the valve. It occurs when inflammation and excrustations roughen the edges, so
that they stick together, narrowing the valve opening. When healing occurs,
fibrous tissue is formed, which shrinks as it ages, increasing the stenosis and
leading to incompetence. When a coronary artery is occluded, not only does
myocardial infarction occur. If the epithelium lying over a plaque breaks down,
platelets are stimulated by the damaged cells and a blood clot (thrombus) is
formed, blocking the artery and causing ischaemia and infarction. The pieces of
the clot may break off, travel in the blood stream and lodge in small arteries
distal to the clot, causing small infarcts (areas of dead tissues).
The main requirements for this assay are pressure transducers, micro tip
catheters, rectal thermometers, pentobarbital sodium (45 mg/kg/i.v) and healthy
dogs.
Dogs of either sex weighing between 15–40 kg are selected. They are
anaesthetised with pentobarbital sodium (45 mg/kg/i.v). Respiration is
maintained through a tracheal tube using a positive pressure respirator. The heart
is exposed through a left thoracotomy at the fourth or fifth intercostal space. The
left coronary arterial circumflexes (LCX) are opened and exposed. The coronary
blood flow is measured by placing an electromagnet on the proximal part of the
LCX. A haemostatic clamp is placed for 5 s iistal to the flow probe. A small
plastic cylindrical constrictor is placed around the artery at the site of damage. In
case of occlusion of the artery, re-flow is induced by lifting the vessel with
forceps. The test substances are given by i.v bolous injection or continuous
infusion. Continuous flow variations (CFVs) are registered for 2 to 5 × 60 min.
and compared before treatment. The peripheral arterial blood pressure is
measured by cannulating the right femoral artery and connected to a pressure
transducer. Left ventricular pressure (LVP) is measured by inserting a micro tip
catheter via the carotid artery. By amplifying the LVP, the left ventricular end
diastolic pressure (LVEDP) is evaluated. From the initial slope of the LVP curve,
the contractility is determined. From the blood pressure curve, the heart rate is
determined. The body temperature is monitored using a rectal thermometer (Fig.
9.16) probe. The ECG is recorded. At the end of the test, an overdose of
pentobarbital sodium is injected to kill the animal. Stenosis induced coronary
thrombosis are quantified by using the following parameters:
Frequency of cyclic flow variations = cycle number per time
Magnitude of cyclic flow variations = cycle area (mm2)
Change in cycle number and cycle area after drug treatment is calculated and
compared to pre-treatment controls. Finally the statistical significance is
assessed using student’s t/-test.
Fig. 9.16 Rectal thermometer

9.10 SCREENING METHODS FOR ANTI-HYPERTENSIVE DRUGS

Hypertension is the most common cardiovascular disease. It is conventionally
defined as a condition when the blood pressure ≥140/90. Blood pressure may be
defined as the force or pressure, which the blood exerts on the walls of the blood
vessels. When the left ventricle contracts and pushes blood into the aorta, the
pressure produced is called the systolic blood pressure. In adults it is about 120
mm of Hg. When complete cardiac diastole occurs, and the heart is resting
following the ejection of blood, the pressure within the arteries is called the
diastolic pressure. In adults it is about 80 mm of Hg.

9.10.1 Methods to Induce Experimental Hypertension

The animal models of hypertension share many features which are common to
human hypertension. Many of these models have been developed by utilizing the
etiological factors that are presumed to be responsible for human hypertension
such as excessive salt intake, hyperactivity of reninangiotensin–aldosterone
system (RAAS) and genetic factors. Since regulation of blood pressure (BP) is
multifactorial, the effectiveness of an antihypertensive agent in one model does
not necessarily mean that the mechanism of action of a given agent in a given
model is related to the pathogenesis of elevated blood pressure.
Animals used: Whereas in the past, most studies on experimental
hypertension were carried out on dogs, currently rats are the preferred animal
species. The spontaneous hypertensive rat (SHR), a genetic strain of the
hypertensive rat, is the animal of choice for screening antihypertensive agents.
 Rabbits, monkeys, pigs and mice are also used to produce experimental
hypertension.
The various types of animal models of hypertension being used are:

1. Renovascular hypertension
2. Dietary hypertension
3. Endocrine hypertension
4. Neurogenic hypertension
5. Psychogenic hypertension
6. Genetic hypertension
7. Other models

Two kidney-one clip (2K1C) hypertension

The renal artery is constricted on one side only, with the other artery (or kidney)
left untouched. This results in a sustained increase in BP due to increased plasma
rennin activity (PRA), which in turn increases circulating angiotensin-II, a potent
vasoconstrictor. However, there is no salt and water retention because the other
normal kidney is intact. Thus, the resultant hypertension at this stage is renin–
angiotensin dependent. After about 6 weeks, the increased angiotensin-II
releases aldosterone from the adrenal cortex leading to a gradual retention of salt
and water. Retention of salt and water leads to decreased rennin production.
From this stage onwards, hypertension is volume dependent. Hence, salt and
water balance is critically involved in the pathogenesis of renovascular
hypertension. Increased BP and increased renin activity returns to normal by the
unclipping or removal of the affected kidney.
Hypertension induced by cholinomimetic agents
Physostigmine (10–80 μg/kg, i.v.), a cholinesterase inhibitor, and oxotremorine
(20–40 μg/kg, i.v.), a direct muscarinic cholinergic agonist, cause a dose-
dependent increase in BP. Cholinomimetic-induced hypertension has been
shown to be elicited through the activation of the central cholinergic mechanism
and mediated peripherally through the sympathetic nervous system. Pretreatment
with methyl scopolamine (1 mg/kg), 5–10 minutes before giving oxotremorine,
prevents initial hypotension in this model.
Transgenic rat (TGR) models
Transgenic models of hypertension have revolutionized the experimental work
on hypertension. Several transgenic rat lines expressing candidate genes for
hypertension have been produced. Introducing an additional renin gene, the
murine Ren-2 gene, into the germ line of rats results in a transgenic hypertensive
rat strain, TGR (mREN2) 27, with an overexpression of renin. Genetic linkage
studies have shown that the components of RAAS are associated with this type
of hypertension. Linkage has been described for the angiotensinogen gene in
human hypertension. The TGR model may be useful for studying the role of the
local RAAS system in hypertension.

9.10.2 Acute and Chronic Renal Hypertension in Rats/Dogs

(Goldblatt et al. 1934)
This method is based on the fact that clamping of the renal artery in rats leads to
renal arteriosclerosis, which leads to ischemia. The reduced blood flow to the
kidney causes the elevation of the renin–angiotensin–aldosterone mechanism.
Aldosterone is the hormone secreted by the cortex of the adrenal gland, which
regulates the secretion of sodium ions. Renin is an enzyme which converts the
plasma protein angiotensinogen to angiotensin-I, and finally to angiotensin-II
with the help of angiotensin-converting enzymes. It is responsible for the
hypertension.
The left renal artery is partially clamped for 4 h. After 4 h, the clamp is re-
opened. The accumulated amount of renin is released into the renal circulation.
This leads to the formation of angiotensin-II leading to acute renal hypertension.
The main requirements are a surgical table, Dieffenbach clip/small bulldog
clamp, hexobarbital sodium (100 mg/kg)/urethane (175 mg/kg), pentolinium
(100 mg/kg/i.v)/ hexamethonium bromide (10–20 mg/kg/i.v), pentobarbital
sodium (30–40 mg/kg), adult healthy albino rats.
Adult male Wistar/Sprague-Dawley rats weighing 200–250 g are used. They
are anaesthetised by injecting them with hexobarbital sodium (100 mg/kg) or
urethane (450 mg/kg) intraperitoneally for 3–4 h. An incision of 1 cm length is
made on the lower abdomen. The rat is secured on the operating table and the
renal artery exposed. The renal artery is occluded using either a Dieffenbach clip
or a small bulldog clamp (Fig. 9.17) for 3.5–4 h. The animals are again
anaesthetised by injecting pentobarbitone sodium (30–40 mg/kg/i.p.). The
trachea is exposed to allow free breathing and cannulated in order to clear any
secretions. A respiratory pump is also used if artificial respiration is required.
Carotid arteries which lie close to the trachea on either side are recognised by
their elastic and pulsating nature and one of them is exposed and cannulated
using an arterial cannula. The cannula in the carotid artery is connected to the
Condon’s mercury manometer or pressure transducer for measuring systolic and
diastolic blood pressure. Test compounds are injected through the cannula placed
on the jugular or femoral vein. The femoral vein is seen on the medial surface of
the upper part of the thigh; whereas the jugular vein is seen under the skin at the
side of the neck. Test compounds can be injected by i.v at different doses.
Ganglionic blockers like pentolinium (100 mg/kg/i.v) or hexamethonium
bromide (10–20 mg/kg/i.v) can be injected in order to provide a stable reduced
pressure. The bulldog clamp/renal arterial clip is then removed. A stable
hypertension is achieved within 15 min. Blood pressure is closely monitored in
the manometer. The hike in blood pressure after re-opening the renal artery is
measured. Similarly the reduction in blood pressure after the test drug
administration is also measured. The percentage of inhibition of hypertension is
calculated. The results are analysed by student’s t-test. The duration of action is
determined in minutes.
Fig. 9.17 Bulldog clamps

9.10.3 Chronic Renal Hypertension in Rats using 1-kidney-1-clip

Method
The 1-kidney-1-clip method is a modified version of the chronic renal
hypertension model. The principle behind the method is the same i.e. the
ischemia of the kidneys. It takes around 4–5 weeks to develop hypertension.
The main requirements are wound clips/surgical sutures, U-shaped silver
clips/small bull dog clamps, surgical table, pentobarbital sodium (50 mg/kg/i.p),
adult healthy albino rats.
Adult healthy albino rats weighing 200–250 g are selected. They are
anaesthetised by intraperitoneal injection of pentobarbital sodium (50 mg/kg).
The animal is put on the surgical table. The backside of the rat is properly
shaved and the skin disinfected using a disinfectant. The renal artery is exposed.
A U-shaped silver clip is put around it and it is partially blocked near the aorta.
The size of the clip can be adjusted using special forceps. The right kidney is
removed through an incison. The renal pedicle is tied. Skin incisions are closed
using wound clips or surgical sutures. The blood pressure is measured using
Condon’s manometer (Fig. 9.18) or a pressure transducer using polygraphs or
physiographs after 4–5 weeks of clipping. Values above 150 mm of Hg are
chosen for the experiment. Blood pressure readings are taken once every 3 days
prior to the experiment on each rat. The test drug is administrated orally for 3
days. The pre- and post-drug blood pressure readings are taken. The pre-test
drug blood pressure will give the control values. The changes in systolic blood
pressure are expressed in mm of Hg. The results are compared using the
student’s t-test.

9.10.4 Chronic Renal Hypertension in Dogs using the Wrapping

Technique (Abram and Sobin 1947)
The main requirements for this assay are a tail-cuff instrument, surgical table,
polygraph, surgical sutures/wound clips, thiopental sodium (15 mg/kg/i.v) and
healthy dogs weighing 10–12 kg.
Healthy dogs weighing 10–12 kg are selected for the experiment. They are
anaesthetised with an intravenous injection of thiopental sodium (15 mg/kg).
Under anaesthesia, a midline incision is made on the abdomen. One of the
kidneys is exposed. The exposed kidney is wrapped with cellophane and then
replaced. The contralateral kidney is then exposed. The artery, vein and the
ureter are ligated, and the non-wrapped kidney is surgically removed. The
wound is closed using surgical sutures or wound clips. Antibiotics are given on
the day of the surgery and then for the next 3 days in order to avoid any post-
surgical infection. Body temperature is measured twice daily for the next 4 days
after surgery. Blood pressure is measured using a tail-cuff instrument, which is
connected to a polygraph (Fig. 9.19). Those animals with a systolic blood
pressure more than 150 mm of Hg are considered to be hypertensive. The test
compounds can be given orally or intravenously. The blood pressure is recorded
every 2 h on day 1, 2 h before, 2 and 4 h after oral administration of test
compounds. Test drugs are repeated for 5 days. The blood pressure readings are
taken before and 2 and 4 hours after treatment on the 3rd and 5th day. The fall in
blood pressure at various recording times are calculated.

Fig. 9.18 U-tube manometer

Fig. 9.19 Multi-channel polygraph

9.10.5 Neurogenic Hypertension in Rats

Neurogenic hypertension is useful for acute experiments. The method is based
on the principle that the removal of the buffer nerve leads to elevation of blood
pressure. The vasodilator and depressor reflexes, originating in the baroreceptor
areas of the carotid sinus and aortic arch, play an important role in the regulation
of blood pressure. The stimulation of the efferent buffer fibres exerts an
inhibitory influence on the vasomotor centre. The removal of the buffer nerve
leads to a persistent rise in blood pressure.
The main requirements are a micro tip pressure transducer, surgical table,
polygraph, thiopental sodium (15 mg/kg), sodium barbital (200 mg/kg), sodium
pentobarbital (60 mg/kg) and adult healthy dogs weighing 10–15 kg.
Adult healthy dogs of either sex, weighing between 10–15 kg are selected.
They are anaesthetised using an anaesthetic mixture of sodium thiopental (15
mg/kg/i.v.), sodium barbital (200 mg/kg/i.v.) and sodium pentobarbital (60
mg/kg/i.v.). The dogs are placed on the dissection table. An incision is made on
the lower abdomen. The femoral vein and artery are exposed and cannulated in
order to administer the test compounds intravenously. The arterial pressure and
heart rate are recorded. The left ventricular pressure is measured using a micro
tip pressure transducer (Fig. 9.20). A catheter is inserted into the pulmonary
artery via the jugular vein and also to the heart. 5 ml of cold 5% dextrose is
injected into the right atrium. The temperature change in the pulmonary artery is
recorded. The cardiac output is calculated using a cardiac output computer
connected to it. A polygraph is used to record the readings. The carotid arteries
are cleared up to the bifurcation of both internal and external carotid arteries.
The carotid sinus nerves are isolated, ligated and sectioned. Neurogenic
hypertension is induced by bilateral vagotomy. The animal is observed for 30
min. to see that it is stabilised. The test compound is injected intravenously.
Cardiac parameters like heart rate, arterial pressure and left ventricular pressure
are monitored for 90 min. Changes of the cardiovascular parameters are
expressed as percentage values.

Fig. 9.20 Pressure transducer

9.10.6 Desoxycorticosterone Acetate Induced Hypertension in Rats

Desoxycorticosterone acetate is a mineralocorticoid. Mineralocorticoids cause
sodium and water retention, edema and progressive rise in blood pressure.
Desoxycorticosterone acetate induced hypertension is due to the sodium-
retaining properties of the steroid, causing increase in plasma and extra-cellular
volume.
In this assay, the hypertensive effects are due to salt loading and unilateral
nephrectomy in rats.
The main requirements are adult healthy albino rats, anaesthetic ether, desoxy-
corticosterone acetate (20 mg/kg), surgical table, 1% NaCl solution, olive oil.
Adult healthy albino rats weighing 200–250 g are selected. They are
anaesthetised with ether and placed on the surgical table. An incision is made on
the lower abdomen. The left kidney is removed through a flank incision. The rats
are injected (s.c) twice weekly with desoxycorticosterone acetate in olive oil at a
dose of 20 mg/kg for 4 weeks. Drinking water is replaced with 1% sodium
chloride solution. The blood pressure increases after 1 week and the systolic
values reaches between 160 and 180 mm of Hg after 4 weeks. The test
compound is given orally. The changes in systolic b.p are expressed in mm of
Hg. The results can be analysed statistically by the student’s t-test.

9.10.7 Fructose Induced Hypertension in Rats

Fructose is the monosaccharide form of carbohydrate. It is the simplest form in
which a carbohydrate can exist. Increase in carbohydrate intake can raise blood
pressure in experimental animals.
The main requirements for this assay are a tail–cuff instrument, an auto
analyser, adult healthy albino rats 10% fructose solution,
Healthy albino rats weighing 210–250 g are used. They are divided into 8
groups and housed 2 per cage on a 12 h light and 12 h dark cycle. They are also
allowed free access to standard laboratory diet and drinking fluid. The drinking
fluid consists of 10% fructose solution. Parameters like body weight and food
intake of each rat are measured every week during the treatment. The systolic
blood pressure (Fig. 9.21) and the pulse rate are measured using the tail-cuff
method before and every week during treatment. Blood samples are also
collected before and every week during treatment. Plasma glucose, insulin and
triglycerides are determined from the blood samples (Fig. 9.22). Statistical
analysis is performed using one-way or two- way ANOVA (analysis of
variance).

9.10.8 Monocrotaline Induced Pulmonary Hypertension in Rats

Monocrotaline is a toxic substance. A single injection of monocrotaline leads to
progressive pulmonary hypertension resulting in right ventricular hypertrophy
and cardiac failure.
The main requirements for this assay are monocrotaline, Krebs–Hanseleit
buffer, normal saline solution, potassium chloride and adult healthy albino rats.
Fig. 9.21 Rat blood pressure measurement assembly showing student
physiograph, Condon’s manometer

Fig. 9.22 Auto analyser

Adult healthy albino rats weighing 200–250 g are selected. They are treated
with the test drug. After one week of test drug administration, a single
subcutaneous injection (100 mg/kg) of monocrotaline is given. They are
sacrificed on 4, 7 and 14 days after treatment using pentobarbital anaesthesia.
The heart and lungs are excised from the thoracic cavity. The atria is removed,
the right ventricle and the left ventricle separated from the septum. They are
blotted and weighed separately. The left lung is blotted, weighed, minced and re-
weighed after drying at room temperature for 14 days. Three pulmonary artery
segments—the main pulmonary artery, the right pulmonary artery and an
intrapulmonary artery from the right lower lobe—are isolated. The tissue
segments are suspended between stainless steel hooks in 10 ml isolated tissue
baths containing modified Krebs-Hanseleit buffer. They are maintained at 37°C
and oxygenated using 95% O2/5% CO2. At the end of each experiment, the
vessel segments are blotted, weighed and the dimensions measured. The cross-
sectional area of each artery is determined from tissue weight and diameter. The
arteries are equilibrated for 1 g of passive applied load and are made to contract
using potassium chloride. The procedure is repeated after washing and the
applied loads increased by 1g increments. The responses are normalised to the
maximum active force development generated by an artery in each experiment
and the data are plotted. Changes in isometric force are recorded on a polygraph
using force displacement transducer. The responsiveness to contractile and
relaxant agonists is assessed in pulmonary arteries from saline and
monocrotaline treated rats. The concentration–response curves to hypertonic
KC1, angiotensin-II, nor-epinephine are generated in vessels at resting phase.
The contractile and relaxation responses are plotted as a function of negative
logarithm of agonist concentration. The differences in mean responses are
compared by student’s t- test.

9.11 INDIRECT BLOOD PRESSURE OR NON-INVASIVE BLOOD

PRESSURE (NIBP)
NIBP is widely used for routine BP measurement in animals that are awake or
have been anesthetized (Fig. 9.23). The basic technique is straightforward: a cuff
is placed at the base of the tail and inflated to occlude blood flow, then the cuff is
slowly deflated while monitoring for the return of pulsatile flow (Fig. 9.24). The
cuff pressure at the time of the first appearance of the pulse indicates systolic BP.
Rats and other rodents control blood flow to the tail as a means of
thermoregulation. When the ambient temperature exceeds approximately 28°C–
30°C, blood flow to the tail increases, making NIBP measurement easier.
Fig. 9.23 Non Invasive Blood Pressure Instrument

Fig. 9.24 Animal restrainer showing cuff placement

Principle
The cuff is quickly inflated to well above the suspected systolic blood pressure;
the pulse will then be obliterated. Thereafter, pressure in the cuff is slowly
released and, as the pressure falls below systolic blood pressure, the pulse will
reappear. The method is analogous to sphygmomanometry in humans and can be
applied not only at the tail of rats that are awake, but also in dogs and small
primates. The indirect tail cuff method is widely used to evaluate the influence
of antihypertensive drugs in spontaneously and experimentally hypertensive rats
(Fig. 9.25)

Fig. 9.25 Software screenshot

Test Procedure
Male spontaneous hypertensive rats weighing 300–350 gm, or rats or mice
with experimentally induced hypertension are used.
To measure blood pressure, a tubular inflatable cuff is placed around the
base of the tail and a piezo-electric pulse detector is positioned distal to the
cuff.
The cuff is inflated to approximately 300 mm Hg. As the pressure in the
cuff is slowly released, the systolic pressure is detected and subsequently
recorded on a polygraph.
The test substance is administered intraperitoneally or by gavage once per
day over a period of 5 days.
The usual screening dose of a new compound is 25 mg/kg.

Standard compounds:

1. endralazine (3 mg/kg p.o.)

2. nifedipine (3 mg/kg p.o.)
3. urapidil (5 mg/kg p.o.)

9.12 SCREENING METHODS FOR ANTI-ARRHYTHMIC DRUGS

Changes in the rhythm of the heart also affect health. A cardiac arrhythmia is any
disorder of the heart rate or rhythm, and is the result of abnormal generation or
conduction of impulses. The arrhythmia could be a sinus bradycardia, a sinus
tachycardia, fibrillation, or a flutter. The normal cardiac cycle gives rise to a
normal sinus rhythm, which has a rate between 60 and 100 beats per minute.
Sinus bradycardia is a sinus rhythm below 60 beats per minute. It is an
abnormality if it follows myocardial infarction or accompanies raised
intracranial pressure. Sinus tacycardia is a sinus rhythm above 100 beats per
minute when the individual is at rest. It accompanies anxiety. Fibrillation is the
contraction of the cardiac muscle fibres in a disorderly sequence. The chambers
do not contract as a whole and the pumping action is disrupted. Flutter is the
disturbance of the normal heart rhythm that like fibrillation may affect atria or
ventricles; although the arrhythmia is less rapid and chaotic.

9.12.1 Chemically Induced Arrhythmias

The principle behind this group of assays is based on the fact that a large number
of chemical agents alone or in combination is capable of inducing arrhythmias.
Administration of anaesthetics like chloroform, ether and halothane followed by
a precipitatory stimulus such as intravenous adrenaline or cardiac glycosides,
aconitine and veratrum alkaloids cause arrhythmias. The sensitivity to these
arrhythmogenic substances differs from species to species due to inherent
biological variations.
Aconitine induced arrhythmias in rats (Yamamoto et al. 1993)
The infusion of aconitine in the anaesthetised rat causes ventricular
arrhythmiasis.
The main requirements for this assay include an ECG instrument, aconitine (5
μg/kg/ i.v), urethane (1.25 g/kg/i.p), 0.1 N nitric acid and healthy albino rats.
Healthy albino rats weighing 330–400 g are used and anaesthetised by an i.p
injection of 1.25 g/kg of urethane. 5 μg/kg aconitine is dissolved in 0.1 N HNO3.
It is administered by continuous infusion into the saphenous vein at the rate of
0.1 ml/min. The ECG is recorded every 30 s. The test drugs are administered
orally or intravenously at a dose of 3 mg/kg, 5 min. before the start of aconitine
infusion. Each group should contain 6–8 animals. The anti-arrhythmic effect of
the test drug is measured by the amount of aconitine/100 g animal which induces
ventricular extra systoles, ventricular tachycardia, ventricular fibrillation and
finally death.
A higher dose of aconitine in the treated group as compared to an untreated
control group is an indication of anti-arrhythmic activity. Statistically it is
assessed by the student’s t-test.
Digoxin induced ventricular arrhythmias in anaesthetised guinea pigs
An overdose of cardiac glycoside, such as digoxin induces ventricular extra
systoles, ventricular fibrillation and finally death in guinea pigs.
The main requirements for this test include a respiratory pump, an ECG
instrument, pentobarbital sodium (35 mg/kg/i.v), lidocaine (93 mg/kg/i.v),
ramipril (1 mg/kg/p.o), digoxin (85 μg/kg) and adult guinea pigs.
Adult healthy guinea pigs weighing 350–550 g are selected. They are
anaesthetised with pentobarbital sodium (35 mg/kg/i.p). Catheters are inserted
into the trachea, one in the jugular vein for administration of the drug and one in
the carotid artery for recording the blood pressure. Positive pressure ventilation
is applied with a respiratory pump at 45 breaths/min. The systolic blood pressure
is monitored in the carotid artery using a pressure transducer with a polygraph or
a physiograph. Digoxin is infused into the jugular vein with a perfusion pump at
the rate of 85 μg/kg; 0.266 ml/min. until cardiac arrest. The ECG is recorded
with steel- needle electrodes placed subcutaneously. The control animals receive
only the digoxin infusion and test animals receive the test drug either orally or
intravenously .for 1 min. prior to infusion. The onset of ventricular extra
systoles, ventricular fibrillation and cardiac arrest are recorded. The total amount
of digoxin infused to induce ventricular fibrillation is measured. The standard
drugs could be a local anaesthetic having anti- arrhythmic activity like lidocaine
(93 mg/kg/i.v) or ACE inhibitors like ramipril (1 mg/kg/p.o). The test values are
compared with standard values and statistically analysed by student’s t-test.
Strophanthin induced arrhythmias
Acute intoxication with cardiac glycoside strophanthin-K induces ventricular
tachycardia and multifocal ventricular arrhythmias in dogs.
The main requirements for this assay include an ECG instrument,
strophanthin-K (3 μg/ kg/min.), quinidine (3 mg/kg/i.v and 10 mg/kg/i.d),
lidocaine (3 mg/kg/i.v and 10 mg/kg/ i.d), pentobarbitone sodium (45 mg/kg/i.v)
and healthy dogs.
Male or female dogs weighing approximately 20 kg are used. They are
anaesthetised by injection of pentobarbital sodium (35–45 mg/kg/i.v). A cannula
is inserted to the femoral or jugular vein for the administration of drugs. The
duodenum is also cannulated for intraduodenal administration. ECG is registered
with needle electrodes. The heart frequency can be assessed from the R-peaks of
ECG. Strophanthin-K is administered by continuous i.v infusion at the rate of 3
μg/kg/min. The signs of cardiac toxicity like ventricular tachycardia or
multifocal ventricular arrhythmias appear about 30–40 min. later. The
strophanthin infusion is terminated at this stage. When the arrhythmias are
stable, the test compounds are administrated intravenously in doses between 1.0,
5.0 mg/kg for 10 min. or intraduodenally in doses between 10 and 30 mg/kg.
ECG recordings are obtained at times 0.5, 1, 2, 5, 10 min. following the
administration of the test drug. The standard drugs are quinidine and lidocaine
which re-establishes normal sinus rhythm at doses of 1 and 3 mg/kg/i.v and 10
mg/kg/i.d respectively.

9.12.2 Electrically Induced Arrhythmias: Ventricular Fibrillation

Electrical Threshold
A serial electrical stimulation result in flutter and fibrillation. It is possible to use
this stimulation to reproduce some of the main types of arrhythmias which are of
clinical importance. The flutter threshold or the ventricular multiple response
thresholds may be determined in anaesthetised dogs before and after the
administration of the test drug.
Several electrical techniques are used to measure ventricular fibrillation
threshold such as single pulse stimulation, continuous 50 Hz stimulation and
sequential pulse stimulation.
The main requirements include respiratory pump, pentobarbital sodium (45
mg/kg/i.v) and adult healthy dogs.
Adult healthy dogs weighing 8–12 kg are selected. They are anaesthetised
with sodium pentobarbital (45 mg/kg) and also ventilated using a respiratory
pump. The systolic blood pressure is monitored. A thermal blanket is used to
maintain the body temperature. A mid-line sternotomy is performed, and the
heart suspended in a pericardial cradle. The sinus node is crushed. The heart is
then driven by 3-ms square anodal constant current pulses for 400 ms of the
basic cycle and is prematurely stimulated by 3-ms test stimulus through the
driving electrode. A recording electrode is placed on the surface of each
ventricle. A silver plate is implanted under the skin in the right femoral region as
the electrode. The ventricular fibrillation threshold is determined by delivering
0.2–1.8 s train of 50-Hz pulses for 100 ms after every eighteenth basic driving
stimulus. The intensity of the current is increased, from the diastolic threshold in
increments of 10 μgA to 100 μgA until ventricular fibrillation occurs. When
ventricular fibrillation occurs, the heart is immediately defibrillated and allowed
to recover to control conditions for 15–20 min. Anti-arrhythmic drugs are
administered through the femoral vein. Ventricular fibrillation threshold (VFT) is
determined before and after administration of the test drug at given time
intervals. The mean values are compared by the student’s t- test.
Action potential and refractory period in isolated left ventricular papillary
muscle of the guinea pig
The intracellular action potential in the left ventricular papillary muscle of a
guinea pig is recorded after electrical stimulation. The stimulation frequency is
varied in order to determine the refractory period.
The main requirements for this experiment include glass micro electrodes, a
stimulator, potassium chloride solution (3 M), ringer solution and adult guinea
pigs.
Adult guinea pigs of either sex weighing 250–260 g are selected. They are
sacrificed by stunning. The carotid arteries are separated and the thoracic cage
opened. The heart is removed and placed in a container containing pre-
oxygenated, pre-warmed ringer solution. The pericardium and the atria are
trimmed away. The left ventricle is opened and the two strongest papillary
muscles are removed. They are then fixed in a suction electrode for electrical
stimulation and a force transducer for recording contractions. A standard micro
electrode technique is applied to measure the action potential via a glass micro
electrode containing 3 M KC1 solution. The papillary muscle is stimulated with
rectangular pulses of IV and 1 min. duration at an interval of 500 ms. The
intracellular action potential is amplified and differentiated for recording
upstroke velocity. The effects on sodium channels as well as on calcium
channels can be studied. The relative refractory periods are estimated and are
defined as the minimum time interval between two stimuli at which each of the
stimuli is answered by a contraction. The stimulation threshold is also measured.
After 30 min. of equilibrium time, the test compounds are added. The following
parameters are compared with pre-drug values after 15 and 30 min.: resting
potential; upstroke velocity; duration of action potential; stimulation threshold;
refractory period and contraction force.

9.12.3 Mechanically Induced Arrhythmias

This method is based on the principle that arrhythmias can be induced directly
by ischaemia or by re-perfusion. After ischaemia either by infarction or by
coronary ligation, several phases of arrhythmias are found. The two-stage
coronary artery ligation technique focuses on late arrhythmias. The influence of
re- perfusion arrhythmias can be tested in various species.
Ventricular arrhythmias after coronary occlusion
Coronary artery occlusion in anaesthetised dogs is characterised by increase in
heart rate, heart contractility, left end diastolic pressure as well as ventricular
arrhythmias.
The main requirements include a respiratory pump, an ECG instrument,
thiobutobarbital sodium (30 mg/kg/i.v), chloralose (20 mg/kg/i.v), pentobarbital
sodium, urethane (250 mg/ kg), morphine (2 mg/kg) and adult healthy dogs.
Dogs of either sex weighing 20–25 kg are used. They are anaesthetised by
injection of thiobutobarbital sodium 30 mg/kg/i.v. and maintained in that state by
20 mg/kg chloralose and 250 mg/kg urethane, followed by 2 mg/kg morphine.
The respiration is maintained through a tracheal tube using a positive pressure
respirator (Fig. 9.26). The test compounds are administrated through a cannula
inserted into the peripheral vein. The ECG is recorded. The cannula of the
femoral artery is connected to a pressure transducer for recording theperipheral
systolic and diastolic blood pressure. A micro tip catheter is inserted via the
carotid artery for determining the left ventricular pressure (LVP). The heart rate
is measured from the LVP waveform. Myocardial contractility is measured as the
rate of rise of LVP. The heart is exposed by left thoracotomy between the fourth
and fifth intercostal space and the pericardium opened. A silk suture is placed
around the left anterior descending artery. After a period of 45 min., the test
substance or the vehicle are administered as i.v bolous. After 20 min., the
ligature at the coronary artery is partially closed for 90 min. During the
occlusion period, test drugs are given by continuous infusion. After release of
coronary obstruction, the animal is monitored for 30 min. re- perfusion period.
All parameters are recorded. Finally the animal is killed by injecting it with an
overdose of pentobarbital sodium
Fig. 9.26 Artificial respirator

Harry’s dog model of ventricular tachycardia by double ligation of the

coronary artery
The main requirements include an ECG instrument, methohexitone sodium (10
mg/kg), halothane and healthy dogs.
Dogs of either sex are selected and anaesthetised by i.v injection of
methohexitone sodium (10 mg/kg). They are maintained in this state with
halothane. The heart is exposed through incision in the fourth and fifth
intercostal space. The descending branch of the left coronary artery is dissected
and ligated in two stages. Ligatures are placed around the artery and a 21-gauge
needle. The first ligature is partially tied around the artery and the needle. The
second ligature is partially tightly tied around the artery after 30 min. The dog is
allowed to recover. Observations are made 22–24 h after ligation of the coronary
artery. The mean blood pressure is recorded from the catheter placed in the
femoral artery. The ECG and blood pressure are recorded for a period of 30 min.
before and during drug administration. Drugs are administered either by
injection or by continuous infusion via a hind leg vein. The number of sinus and
ectopic beats are counted for each successive 5-min period. Beats with P-wave
and a mean frontal QRS vector of normal duration are counted as sinus in origin,
all others are ectopic.

9.13 SCREENING METHODS FOR CARDIAC GLYCOSIDES

9.13.1 Binding Kinetics on Ouabain Receptor
Cardiac glycosides can be characterised by their binding kinetics i.e. association
process, equilibrium binding and dissociation process on the ouabain receptor.
Cardiac glycosides have cardiotonic activity. They increase myocardial
contractility and output in a hypodynamic heart, without a corresponding
increase in oxygen consumption. Thus myocardial efficiency is increased. They
are used for treatment of congestive heart failure.
In this experiment, cardiac glycosides are screened for their binding kinetics
i.e. association and dissociation process on the ouabain receptor.
The main requirements include HAWP millipore filters, ouabain, Tris-HCl
solution and healthy male/ female rats.
Healthy adult rats are killed and the heart isolated. The sarcolemma is
obtained from the rat heart. Coronary perfusions myoctes are obtained from rat
heart by collaginase digestion from this membrane. Fractions of myocyte
sarcolemma are thus obtained. Ouabain with a specific radioactivity of 20
Ci/mMol is incubated with ligands to be tested in 10 ml of binding medium. The
binding medium consists of 1 mm of MgCl2, ImM of inorganic phosphate and
50 mM of Tris-HCl at pH 7.4 at 37°C for 10 min.
Association process
200 μg of membrane preparation and ouabain (either 10 or 100 nM) are added to
initiate the reaction after temperate equilibration. 4.5 ml samples are removed at
various time intervals.
Equilibrium binding
At the end of temperature equilibration, ouabain at concentrations of 10 nM to
40 μg of membrane is added. After 30 min., duplicate aliquots of 4.5 ml are
removed and filtered.
Dissociation process
After attaining equilibrium, 10 ml of pre-warmed Mg2+ plus Tris-HCl solution
supplemented with 0.2 mM of unlabelled ouabain are added to initiate the
dissociation of ouabain. At various times, aliquots of 0.9 ml are removed and
rapidly filtered. Filtration is done under vacuum on HAWP millipore filters and
3 times rinsed with 4 ml of ice-cold buffer. The radioactivity bound to the filters
and specific binding measurements are assessed. The kinetic parameters for the
association and dissociation process are calculated. Scatchard plots help in
analysing the results.
Isolated cat papillary muscle (Cattell and Gold 1938)
During congestive heart failure, the right ventricle fails when pressure developed
within it by the contracting myocardium is less than the force needed to push
blood through the lungs. When compensation has reached its limit and the
ventricle is not emptying completely, the right atrium and venacavae become
congested with blood and this is followed by congestion throughout the venous
system. It is characterised by edema of the limbs and ascites. Isolated cardiac
tissues are chosen to study the decrease of performance after prolonged electrical
stimulation and during the restoration of force under the influence of cardiac
glycosides. Papillary muscles are the projections of myocardium covered with
endothelium. These muscles are used for the present study.
The main requirements include a polygraph, anaesthetic ether, ouabain (300
ng/ml), and adult healthy cats of either sex.
Cats of either sex weighing 2.5 to 3 kg are used. The animal is anaesthetised
with anaesthetic ether and the heart removed. The papillary muscles (Fig. 9.27)
are isolated from the right ventricle. It is fixed in an organ bath containing
oxygenated ringer solution at 36°C. One end of the muscle is tied to a tissue
holder and the other end to a strain gauge. Electrical stimulation of the muscle is
conducted with 4–6 V for 2 min. duration at the rate of 30 min. The contraction
is recorded using a polygraph. The muscle begins to fail after 1 h with the force
of contraction also diminishing to a fraction of the control. Cardiac glycosides
are added at this point to the bath restoring the contractile force to a level
approaching the control. The standard dose is 300 ng/ml ouabain. The potency of
natural and semi- synthetic glycosides can be determined.
Fig. 9.27 Papillary muscle of cat

Isolated hamster cardiomyopathic heart (Jasmin et al. 1979)

Special strains of Syrian hamsters are used for evaluating cardiotonic drugs. Bio
14/6 strains of Syrian hamsters develop cardiomyopathy. These animals can be
used for evaluation of cardiotonic drugs. Cardiomyopathy is a chronic disorder
affecting the muscles of the heart. It results in enlargement of the heart, heart
failure, arrhythmiasis and embolism. In this experiment the transgenic Syrian
hamsters Bio 14/6, which exhibits cardiac myopathy is used.
The main requirements include Langendroff’s apparatus, chronometer,
heparin (5 mg/kg/ i.p), heart ringer solution and adult Syrian hamsters (Bio
14/6).
Hamsters with cardiomyopathy (Bio 14/6) at the age of 50 weeks are used.
Controls are normal Syrian hamsters (FIB hybrids) at the same age. The animals
are pre-treated with heparin (5 mg/kg/i.p) and 20 min. later the heart is prepared
for the LangendorfFs apparatus. It is then perfused with heart ringer solution.
The preparation is allowed to equilibrate for 60 min. at 32°C with a diastolic pre-
load of 1.5 g. The force of contraction is recorded isometrically using a
polygraph strain gauge transducer. A chronometer is used for measuring the
heart rate. The coronary flow is measured using an electromagnetic flow metre.
The contractile force and the coronary flow from diseased and normal animals
are noted before and after application of the test drugs. The results are compared
using the student’s /-test.
Cardiac toxicity in cats using Hatcher’s method (Hatcher and Brody 1910)
The purpose of this method is to establish a “cat unit” for cardiac glycoside
preparations. “Cat unit” is the amount of crystalline ouabain which is fatal
(within about 90 min. to a kg) to a cat when the drug is injected slowly and
almost continuously into the femoral vein. Time to cardiac arrest after
intravenous infusion of a solution with defined concentration of the standard is
used as a reference. The unknown solution of the test preparation is compared
with the standard. The method can be used to test natural and semi-synthetic
glycosides.
The main requirements include an ECG instrument, anaesthetic ether,
chloralose (70 mg/ kg/ i.v) and healthy male/ female cats.
Cats of either sex weighing 2–3.5 kg are selected. They are anaesthetised with
ether anaesthesia and maintained in this state using chloralose at a dose of 70
mg/kg/i.v. The animal is fixed on its legs on a heated operating table.
Tracheastomy is performed and a tracheal cannula is inserted. ECG is recorded.
The test solutions are administered by i.v infusion. Cardiac arrest is the end point
and it is generally reached within 30–60 min. of the infused solution.
Decay rate and enteral absorption rate of cardiac glycosides (Knaffl-Lenz
1926)
The basic aim of this method is to determine decay rates of cardiac glycosides.
The decay of efficacy can be due to excretion or metabolic degradation of the
glycoside.
The main requirements include operation table, an ECG instrument,
pentobarbital sodium (35 mg/ kg /i.v) and healthy dogs of either sex.
Healthy dogs of either sex weighing 8–20 kg are selected. They are
anaesthetised with pentobarbital sodium (35 mg/kg/i.v.). The animal is fixed on
its legs on a heated operating table. Tracheostomy is performed and a tracheal
cannula inserted. Test compounds are given through the cannula inserted into the
vena formalis. The ECG is recorded. The symptoms of initial cardiac toxicity
(extra systoles, A-V block) are recorded. Infusion is stopped. After 4, 8, 12 or 24
h the infusion procedure is repeated. The glycosides are partially metabolised or
excreted and hence the dose needed for observation of ECG changes during the
second infusion is lesser than the changes during first. The decay rate of the
glycosides is determined and the half-life times calculated. The dose required in
the second experiment for introduction of ECG changes is equal to the amount
of metabolised or excreted glycoside. This value is expressed as a percentage of
the amount required in the first experiment and indicates the decay rate of the
glycoside.
Cardiac insuffiency in guinea pigs
The principle behind this assay is based on the fact that cardiac insuffiency is
created by placing a ring-shaped clamp around the base of the heart. The
interruption of the flow of blood towards the heart leads to the development of
symptoms of congestive heart failure (CHF). This method was developed to
induce CHF in guinea pigs with symptoms very close to human pathology—
cardiac hypertrophy, peripheral edema, lung and liver congestion, dyspnea,
hydrothorax and ascites. Effective treatment is achieved by cardiac glycosides.
The main requirements include operation table, a ring-shaped clamp/bulldog
clamp, anaesthetic ether and healthy guinea pigs.
Male guinea pigs weighing 250–400 g are used. The animal is anaesthetised
with ether. The fur of the ventral thorax is shaved and the skin disinfected. The
skin is cut with a pair of scissors and the pig opened at the fourth intercostal
space with two forceps. The heart is pressed against the opening with the left
hand. The pericardia are opened with a fine forceps. The beating heart is
extruded from the thorax wound by pressure with the left hand on the right
thorax wall. A ring-shaped clamp is placed around the basis of the heart. A
thread soaked with diluted disinfectant is placed as a loop around the apex of the
heart. It is then tightened. Necrosis due to complete interruption of the blood has
to be avoided. After removal of the clamp, the heart is placed back. The incision
between the fourth and fifth costal rib is closed and the musculus pectoralis
placed over the wound. Antibiotic emulsion is applied on the wound and the skin
wound closed. The animal develops symptoms of severe congestive heart failure
with a death rate of 80% within 14 days. Lung weight and relative heart weight
increases. Lung edema and liver congestion are found histologically. Peripheral
edema and pre-terminal dyspnea and tachypnea are also observed. Various doses
of cardiac glycosides (0.1 to 100 μg/kg/s.c or i.m) over a 14-day period can
diminish the symptoms of cardiac insuffiency and death rate.
Cardiac hypertrophy in mice
The principle behind this assay is that blood flow restriction of the aorta in
rats/mice induces not only hypertension but also cardiac hypertrophy within
several weeks. In this method, the occlusion of aorta using nylon suture ligature
leads to myocardial hypertrophy and finally CHF.
The main requirements include a ventilator, ketamine (100 mg/kg/i.p),
xylazine (5 mg/ kg), morphine (2 mg/ kg) and adult mice (18–22 g).
Adult mice weighing between 18–22 g are selected. They are anaesthetised
with an injection (i.p.) of a mixture of ketamine (100 mg/kg), xylazine (5 mg/kg)
and morphine (2 mg/kg). Animals are placed under a dissecting microscope. A
mid-line cervical incision is made to expose the trachea and carotid arteries.
After endotracheal intubation is done, the cannula is connected to a rodent
ventilator. Both left and right carotid arteries are cannulated with PE50 tubing
catheters and connected to modified P50 transducers. The aorta is isolated
between the carotid arteries and constricted by a 7.0 nylon suture ligature using
27-gauge needles. The latter is removed to yield a constriction of 0.4 mm
diameter with a reproducible aortic constriction of 65–75%.
The systolic and mean arterial pressure at baseline and during total occlusion
when the ligature is tied and after transverse constriction (early and late) is
recorded. The rise in systolic pressure provides a mechanical stimulus for
cardiac hypertrophy. The myocardial hypertrophy can be confirmed by
examining the aortic-constricted hearts and sham-operated hearts, 7 days after
operation. Finally the mice are killed, the heart excised and weighed. The
ventricles and atria are frozen in liquid nitrogen for northern blot analysis. The
heart is examined for cell size and is fixed using 10% buffered neutral formalin
solution. Tissue blocks are sectioned at a thickness of 1 μm, mounted on slides
and stained with toluidine blue. The sections are analysed by histopathological
studies. Cell areas are measured Statistical significance between sham-operated
and aortic-constricted rats are assessed by student’s t-test.

9.14 LANGENDORFF HEART SYSTEM

In 1895, Langendorff described studies on isolated surviving mammalian hearts,
using mainly cats as donors. Since then, the method has been improved and is
now used for studies on guinea-pig, rabbit or rat hearts.
9.14.1 Principle behind the method
The principle behind the method involves the cannulation of the aorta above the
sinus of Valsalva, and subjecting the heart to retrograde perfusion (Fig. 9.28).
The aortic valve is forced shut by the pressure of the perfusate, which then enters
the coronary system, eventually leaving the heart via the cut ends of the
pulmonary arteries. Throughout this procedure, the chamber of the heart remains
essentially empty. By adjusting the perfusion equipment, the investigator can
choose to perfuse either in a constant flow or in a constant pressure mode.

9.14.2 Langendorff System–Technical Information

Both, constant flow and constant pressure systems, need a reservoir in which the
buffer is aerated with carbogen (95% C02 and 5% O2), and the pH can be
checked and adjusted to 7.45–7.50, by modulating the gas output thermostated
heat exchanger in which the buffer is warmed up to 37°C, and a thermostated
heart chamber in which the buffer is delivered to the heart. The exchanger and
the chamber are double jacket glassware connected to a thermostated water
pump. A roller pump is also required for both systems. In the constant pressure
system, it keeps the level of the buffer in the reservoir at 108 mm water column
(about 80 mmHg); in the constant flow system, it drives the buffer from the
reservoir into the heat exchanger at a speed sufficient to give the desired constant
flow.

Fig. 9.28 (a) Normal circulation (Opie, 1997). (b) Circulation in a Langendorff-
perfused heart (Dhein, 2005). In the Langendorff- perfused heart, the perfusate
fluid enters the heart via the aorta (RA, right atrium; RV, right ventricle; PA,
pulmonary artery; LA, left atrium; LV, left ventricle; Ao, aorta; 02, oxygen).

Electronic transducers are necessary for monitoring heart function and

controlling and regulating the functionality of the Langendorff system. The most
commonly used transducer is the pressure transducer connected to an isovolumic
balloon inserted into the left ventricle. The balloon is inflated to a maximum
pressure of 10 mmHg and gives data about the heart rate and the developed
pressure. From these raw data, other functional parameters can be calculated
(e.g. rate pressure product, dp/dt).
Usually Tyrode, Locke or Krebs–Henseleit buffers are used. They have to be
filtered, because even the purest chemicals may contain insoluble particles
which will obstruct the small coronary vessels. We use a modified Krebs–
Henseleit buffer bubbled with carbogen, with a pH between 7.4 and 7.5.
Modified Krebs-Henseleit Buffer

Compound Concentration (mM)

NaCl 118.0

NaHCO3 25.0

KC1 4.7

KH2PO4 1.21

MgS04_7H2O 1.22

Glucose 11.0

CaCl2 H2O 1.84

The perfusion buffer we normally use is the modified Krebs–Henseleit buffer.

9.14.3 Preparation of Hearts for Perfusion

Anesthesia and Anticoagulation
All animals are weighed prior to experimentation. Heparin 100 IU, is
administered via intraperitoneal (i.p.) injection for prophylaxis against the
formation of thrombus within the coronary vasculature or ventricular chambers.
After 5 min (time allowed for activated partial thromboplastin time (APTT), to
increase), the animals should be killed, either by an anesthetic overdose (e.g.
sodium pentobarbital, 60 mg/kg i.p.) or by rapid cervical dislocation. The animal
is then transferred to a dissection block and the limbs secured with adhesive
tape.
Dissection
A skin incision is performed at the xyphoid sternum and continued to the lateral
ends of the left and right costal margins. The incision is then continued through
the ribs at the left and right anterior axillary lines to create a clam-shell
thoracotomy. The anterior chest wall is deflected upwards providing an optimal
operating field. The heart is removed by transecting the descending aorta and
inferior vena cava, followed by the ascending aorta and superior vena cava and
transferred to a dissection dish containing ice cold Krebs–Henseleit buffer. At
this stage of the experiment, the priority is to transfer the heart into the cold
buffer as quickly as possible, to avoid any detrimental effects of hypoxia.
Cannulation of the aorta and transfer of the heart to the Langendorff
apparatus
The excess of extra cardiac tissues such as lungs, vessels and thymus is trimmed
off and the ascending aorta is exposed and cannulated with a primed 21G
stainless steel murine cannula under Krebs solution, to avoid air embolisation of
the coronaries. The cannula is inserted into the aorta by manoeuvring the heart
with as little trauma as possible. This is achieved using two small forceps, which
hold the atrial appendages. It should be noted that care should be taken not to
insert the cannula too deep into the aorta to avoid forcing the aortic valve open
and reaching into the left ventricle. This situation is not redeemable by rapid
withdrawal of the cannula as the aortic valve is invariable irreversibly damaged,
resulting in a reduced coronary perfusion (buffer will be lost via left ventricle)
and the heart will not perform as expected. The opposite situation, i.e. not
inserting the cannula deep enough, may have the same result; if the cannula is
tightly secured above the aortic branches (e.g. brachiocephalic trunk), some
buffer will be lost through them. Consequently, the perfusion flow entering the
coronary system will be diminished. Therefore, it is essential that insertion of the
cannula deeper than the point where the aorta leaves the base of the heart should
be avoided. The aorta is secured to the cannula using a 5-0 suture, and
transferred to the Langendorff perfusion apparatus (Fig. 9.29). The buffer should
run slowly through the system prior to mounting the cannula, so that the heart
will receive nutrients and oxygen as soon as it is placed in the system. Once
securely attached to the perfusion apparatus, retrograde perfusion at a constant
pressure or constant flow must be started. The time taken from the moment of
the opening of the thorax until the heart is mounted and perfused on the
Langendorff system has to be under 3 min, in order to avoid the potential effect
of ischemic preconditioning due to delayed perfusion.

9.14.4 Applications
Positive inotropic effects
While negative inotropic substances can be tested in a heart beating with normal
force, the evaluation of a positive inotropic compound usually requires that
cardiac force is first reduced. Acute experimental heart failure can be induced by
an overdose of barbiturates, such as sodium thiopental, or calcium antagonists.
This kind of cardiac failure can be reversed by alpha-sympathomimetic drugs,
cardiac glycosides, or increased Ca+2 concentration. In this way, the potential
alpha-sympathomimetic activity of a new drug can be measured, using
isoproterenol as standard. After thiopental-Na treatment, left ventricular pressure
(LVP) and dp/dt max decreases considerably, whereas coronary flow is slightly
enhanced. Alpha-sympathomimetic drugs restore LVP and dp/dt max and keep
coronary blood flow elevated. Cardiac glycosides increase LVP and dp/dt max
and leave coronary flow unchanged.

Fig. 9.29 (a) Heart mounted on the cannula (AA, atrial appendage), (b) Heart
mounted in the LangendorfF system (a ventricular balloon (VB) is inserted in the
left ventricle; a thermoprobe (TP) is inserted via the pulmonary artery; C,
cannula).
Negative inotropic effects
The effects of a sympathomimetic drug such as isoproterenol at doses of 0.05 to
0.2 pg that increases contractile force as well as heart frequency are registered.
After the injection of a beta-blocker, the effects of isoproterenol are attenuated.
The effects of a potential beta- blocking agent can be tested by comparing the
isoproterenol inhibition versus a standard, such as propranolol (0.1 mg).
Coronary vessel dilating effect
The LangendorfF heart has been extensively used for assessing the coronary
dilating activity of drugs.
Calcium-antagonism
In order to demonstrate the effect of calcium-antagonists, 1 to 5 mg BaCl2 are
injected, which induce a pronounced spasm of the coronary arteries, thereby
reducing the coronary flow. Five minutes later, the test drug is injected. Active
compounds have a relaxing effect on the coronary arteries indicated by an
increase of coronary flow. After this effect has weaned, BaCl2 is injected again
and the test drug or a standard drug, e.g. nifedipine, is tested. The increase of
coronary flow is expressed as a percentage of flow during the BaCl2 spasm and
compared with the effect of the standard. Using various doses, dose-response
curves can be established.
Effect on potassium outflow induced by cardiac glycosides
In this method, by using the Langendorff heart contractile force, coronary flow,
and the potassium content in the coronary outflow can be determined by flame
photometry. Increase in potassium outflow correlates well with the positive
inotropic effect.
Gradual determination of hypoxic damage
The enzymes, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), a-
hydroxy- butyrate dehydrogenase (a-HBDH), and glutamic-oxalacetic
transaminase (GOT), in the effluent of a Guinea pig heart preparation under
varying degrees of hypoxia, can be measured. Potassium content and oxygen
tension in the inflowing and out-flowing solution can be determined.
Arrhythmogenic, anti-arrhythmic and antifibrillatory effects
The Langendorff heart preparation is also used to test the influence of
compounds on cardiac rhythm. For recording monophasic action potential,
suction electrodes are applied on the heart. Ventricular fibrillation can be
induced by simultaneous injection of digitoxin (12.5–25.0 μg) and aconitine
(12.5– 25.0 μg) into the perfusion fluid. Cardiac glycosides shorten the
refractory period, decrease the conduction velocity and increase heterotopic
stimulus generation. Aconitine increases markedly heterotopic stimulus
generation. Both compounds together, invariably induce ventricular fibrillation.
Anti-arrhythmic compounds can be tested in this way. Fibrillation is inhibited, at
least partially, by 10–20 μg quinidine.
Electrical stimulation and antifibrillatory effect
Ventricular fibrillation can be induced in the Langendorff preparation by
reducing the glucose content of the perfusion medium to 0.25 g/1000 ml and the
KC1 content to 0.12 g/1000 ml. After a perfusion period of 20 min, 10 μg
epinephrine is injected into the perfusion cannula. Immediately afterwards, the
heart is stimulated with a current of 40 Hz and 5 mA for 2 min. This procedure is
repeated every 10 min. Standard conditions are achieved when the fibrillation
continues without further electrical stimulation. Hearts treated in this way serve
as controls. Other hearts stimulated in the same way are treated with continuous
infusion of the test drug or the standard via the perfusion medium. Differences in
the incidence of fibrillations are calculated using the chi square test (Fig. 9.30).

Fig. 9.30 (a), (b), (c) represent working heart and normal ECG recording of a
working heart by SPEL Haemosys recording software, respectively.
9.15 SCREENING METHODS FOR DIURETICS
Drugs that induce diuresis (enhances urine out-flow) are known as diuretics.
Many herbal plants like cantaloupe (Cucumis melo), Dolichos biflorus (virus),
radish (Raphanus sativus), kanguni (Satania italica), Oriental sweet gum
(Liquidamber orientalis) and kapok tree (Ceibia pentandra) possess diuretic
activity. Extracts from these drugs are used in various diseases like hypertension,
congestive heart failure, edema, nephrolithiasis and urolithiasis. The diuretic
activity of these drugs can be evaluated by the following methods.

9.15.1 Diuretic Activity in Rats (Lipschitz Test)

This test is based on the principle that water and sodium excretion in test animals
is different as compared to rats treated with a high dose of urea. The “Lipschitz
value” is the quotient between excretion by test animals and excretion by the
urea control.
Adult albino rats weighing 100–200 g are used for the study. Six animals per
group are placed in metabolic cages (Fig. 9.31) individually provided with a wire
mesh bottom and a funnel to collect the urine. Stainless steel sieves are placed in
the funnel to retain faeces and allow the urine to pass. The rats are fed with
standard diet and water ad libitum. 17 to 24 h prior to the experiment, food and
water are withdrawn. The test compound is administered orally. The other group
is treated with urea (lg/kg) orally. Additionally 5 ml of 0.9% NaCl solution per
100 g of body weight are given by gavage. Urine excretion is recorded after 5
and 24 h. The sodium content of the urine is determined by flame photometer.
Fig. 9.31 Metabolic cage in which the rats are placed

Urine volume excreted per 100 g body weight is calculated for each animal in
the group. Results are expressed as the “Lipschitz value” i.e. the ratio T/U in
which T is the response of the test compound and U that of urea treatment. The
value of 1.0 and more are regarded as a positive effect. Potent diuretics having a
Lipschitz value of 2.0 and more have been found.

9.15.2 Chronic Renal Failure in Rats

Chronic renal failure is a frequent pathological condition in man. The following
assay is used for special pharmacological studies as well as evaluation of renal
toxicity of new chemicals.
Albino rats weighing between 150–200 g are used for the study. Rats are
anaesthetised by i. m injection of ketamine (40 mg/kg) and droperidol (0.25
mg/kg). An incision is made in the abdominal wall and the small bowel and
caecum are lifted and placed on saline-soaked sponges. The right kidney is
exposed and dissected from the retroperitoneal area and the vascular and ureteric
pedicles are ligated with silk sutures, and the kidney removed. The renal artery
of the left kidney is dissected into the hilum to expose the three main segmental
renal arteries. The kidney is not dissected out of the peritoneum. The anterior
caudal branch of the artery is then temporarily ligated to establish the volume of
renal tissue supplied. The area of ischemia becomes demarcated within 10–15 s.
If this approximates ¼ to ⅓ of the kidney, a permanent ligature is placed. The
viscera are then carefully replaced in the abdomen and peritoneum and linea alba
is closed with a continuous suture. The skin is closed with stainless steel clips.
Blood for serum creatinine is collected by retro orbital puncture under
anaesthesia at various time intervals up to 12 months. In association with this,
urine is collected every 24 h for the measurement of creatinine, protein and
specific gravity.
Compounds which posses diuretic activity will increase the volume,
accompanied by decrease in urine specific gravity which indicates the decrease
in concentrating ability of the kidney. Proteinuria is also significantly increased.
Terminal uremia occurs after 14–15 months.

9.15.3 Diuretic and Saluretic Activity in Dogs/Cat

Renal physiology of the dog is claimed to be closer to man than that of rats. So
dogs have been extensively used to study renal physiology and the action of
diuretics. Using catheters, periodic collection of urine can be made with more
reliability than in rats.
Dog or cats are anaesthetised using sodium pentobarbitone 35–50 mg/kg. The
lower abdomen is opened. The femoral vein is exposed and cannulated with a
suitable venous cannula. The venous cannula is used for the administration of
test drugs and saline. The control group receives only water. The standard group
receives 1 g/kg urea or 5 mg/kg furosemide per day. The test groups receive
different test drugs. The urinary bladder is catheter- ised through the urethra and
connected to a measuring cylinder. The urine is collected, the volume measured
and analysed for Na–/K– ions using flame photometry. Chloride ions are
estimated using argentometry. The urine volume and electrolyte concentration of
test compounds are compared with the control group to determine the diuretic
activity.

9.16 NEUROPHARMACOLOGY
Neuropharmacology is that branch of health sciences which is concerned with
the study of drugs affecting the nervous system. Neuropsychopharmacology is
the study of drugs used for the treatment of neurological as well as psychiatric
diseases. Measurement of alterations in the behavioural pattern is the central
component in the field of neuropsychopharmacology. Scientists assume that the
emotions experienced by animals are similar to those experienced by humans
because of similar physiological changes that animals and human undergo.
Based on this assumption it is believed that we can learn more about humans by
observing animal behaviour and physiology. The entire concept of an animal
model is based on this assumption.
Selection of animals for neupropharmacological studies
Selection of animals for evaluation of neuropharmacological studies is of
paramount importance. In general the animals selected for the animal model in
neuropharmacological studies should exhibit normal behaviour. These animals
should not show signs of abnormal behaviour such as excessive jumping, biting,
sniffing, licking, lacrimation and salivation. The animals showing such
behaviour should be excluded from the study. The selected animals should be
randomly distributed in age and sex.
Neuropharmacological agents can exhibit either stimulant or depressant
actions. A battery of tests is needed to screen these agents. The screening should
always be initiated with the effects on gross behaviour.

9.16.1 Gross Behavioural Effects in Mice or Rats

The test drug is administered orally or parentally as intended in therapeutics to
different animal groups in graded doses. The dose selected should be the
minimum expected therapeutic dose, intermediate dose (3 times higher than the
therapeutic dose) and maximum expected therapeutic dose (10 times higher than
the therapeutic dose). After the drug administration to different groups of
animals they are observed by the naked eye for:

1. CNS stimulant effects as evidenced by excessive jumping, biting, sniffing

and scratching etc. CNS depressant effects as evidenced by excessive
reduced spontaneous motor activity, sleepiness or passiveness.
2. Other effects can also be observed such as autonomic effects as evidenced
by changes in papillary size, lacrimation, salivation, defecation, and
urination.

At the end of 24 h all groups should be checked for mortality if any. LD50 can
also be determined if possible.
9.16.2 Behavioural Test in Golden Hamsters/Mice
This test is used to assess efficacy of drug on gross behavioural patterns. Golden
hamsters of average weight of 60 g or albino mice (35–45 g) are crowded in
cages for at least 2 weeks. During this time animals develop a characteristic
fighting behaviour. From these animals, one animal is placed in a glass jar of 2
litres. In this situation the animal assumes a squatting position while resting
during the day. If the animal is touched with a stick or forceps, it will arouse
immediately from its resting position. If one tries to hold the hamster with a
blunt forceps, a characteristic behaviour—the hamster throws itself on its back
and tries to bite or push the forceps away with its legs and utters angry shrieks—
is elicited. Stimulus can be applied every 20 min. for 3 h. Only animals
responding with all three defense reactions (turning, vocalising and biting) are
included in the test. Suppression of defense reactions in a test group can be
compared with that in the control group.

9.16.3 Spontaneous Motor Activity in Rats

Spontaneous motor activity is normal in rodents—it is very high especially in the
dark. Any drug that produces sedation will reduce this spontaneous motor
activity. Rats, which are randomly divided in control and study groups, are pre-
treated with either vehicle or drug and are placed in a digital photoactometer
(Fig. 9.32). A digital photoactometer is made like a box and is provided with 12–
16 photocells, which can detect the spontaneous activity of rodents. After one
min. acclimatisation of the activity of the animals i.e. the number of rounds the
animal completes within the chamber are recorded and compared among groups.
Alterations in other gross behavioural pattern such as jumping, biting, blinking,
licking, lacrimation and salivation can also be compared among groups. Rotarod
apparatus (Fig. 9.33) can also be used for neuro-skeletal muscular and motor
activities of drugs.
Fig. 9.32 Digital photoactometer

Fig. 9.33 Rota-rod apparatus

9.16.4 Open Field Behaviour

This test is also performed to assess the effect of drugs on gross behavioural
patterns. The rats or mice are "placed in a large circular enclosure 85 cm in
diameter with white sun mica floor. The enclosure is divided into 25 segments
by the intersection of 3 concentric circles with the lines radiating from the
centre. Two types of stimuli are presented to the animals: a bright light of 165
foot candles produced by 5 photographic lamps and a noise of 78 dB emanating
from an oscillator and a loudspeaker. The behavioural response is noted by using
scores of ambulation (number of floor segments entered by the animal) and
rearing (number of times the animal stood on its hind limbs) frequencies,
immobility time (times when there is lack of movement), defecation and
urination. The test is based on the assumption that effects dissipate with
experience. Therefore, two types of animals are employed: one group consisting
of animals with no prior experience to the open field and another group with
prior experience. The animals of the latter group are given 10 trials in open field,
2 per day at least 6 h apart for 5 days before being tested on day 6 with drugs.
Plus maze (Fig. 9.34) and Y maze can be used for evaluating the gross behaviour
as well as anxidytic activities of drugs.

Fig. 9.34 Plus maze

9.16.5 Sodium Barbitone Induced Hypnosis

This test is done to evaluate the sedative and hypnotic effect of drugs. Albino
mice are randomly divided into control and test groups and are pre- treated with
the test vehicle or drug respectively. 1 h later pentobarbitone sodium (35
mg/kg/i.p.) is administered. The mice start to fall asleep in 15 min. As soon as
they are asleep they are gently put on their backs. Recovery from sleeps allows
them to correct their posture. The time is noted when they fall asleep and noted
again when they spontaneously correct their posture. The difference between the
two is taken as sleeping time. Sleeping time can be compared among groups and
with control.

9.16.6 Effect on Body Temperature

Normal rectal temperature of selected animals is recorded. Animals are then
divided into groups and the desired dose of the drug is administered to one of the
group while the other group is treated as for control. The rectal temperature is
then recorded at hourly intervals for 4 h and then 24 h after the drug
administration. The change in rectal temperature can be compared among
groups. Drugs like chlorpromazine and reserpine are known to cause
hypothermia because of their central depressant effects.
9.16.7 Inhibition of Amphetamine Stereotypy in Rats
Amphetamine, which is a sympathomimetic drug and releases catecholamines
from neuronal storage pools induces a characteristic stereotypic behaviour (lip
smacking, grooming, catalepsy, gnawing) in rats. This behaviour can be
prevented by neuroleptic drugs. This test demonstrates efficacy of anti-psychotic
drugs with dopamine D2 receptor antagonism.
Wistar/Sprague–Dawley rats (120–200 g) are injected with amphetamine (10
mg/kg/s.c or 5 mg/kg/i.p) 30 min. after pre-treatment with vehicle or test drug.
Animals are then placed individually in stainless steel cages (40 cm x 20 cm x
18 cm). The stereotypic behaviour is evaluated at 30 min. interval for 3 h and
alterations in test group are compared with control group.

9.16.8 Conditioned Avoidance Reflex in Rats (the pole climbing test)

This test was developed by Pavlov. It evaluates effects on learning and memory.
Rodents are trained to avoid a noxious stimulus (electric stimuli) in response to a
cue (light or sound buzzer). The anti-psychotic drugs decrease the number of
conditioned avoidance responses (CAR) and prolong the total waiting time
(TWT). CNS depressants affect both avoidance and escape i.e. the animal does
not try to avoid the noxious stimuli and does not escape when stimulus is
applied. Anti-psychotics in low doses affect only avoidance tactics with no effect
on the ability to escape whereas in high doses both are affected due to ataxia and
hypnosis.
Adult healthy rats (250–300 g) are housed in a controlled environment with 22
± 2°C temperature, 12 h light/dark cycle and free access to food and water. Rats
are then trained for a period of 8 days with 10 trials each day (inter-trial period:
30 s), which lasts for 5 min. By these trials, rats learn to climb the pole during 3
s of light conditional stimulus before an electric stimulus (2 mA) of 3 s duration
is applied in the pole climbing apparatus (Fig. 9.35). At the end of the training
period, the animals which fail to learn pole climbing in response to light stimulus
are excluded from the study.
The test is done after pre-treatment on the 9th day by giving 3 s light stimulus
followed by 3 s electric stimulus (2.5 mA). Parameters evaluated include CAR
and TWT. Haloperidol (0.2 mg/kg/i.p) and clozapine (10 mg/kg/i.p) are used as
standard drugs (Agrawal 1982).
Fig. 9.35 Pole climbing apparatus

9.16.9 Forced Swim Test (Porsolt et al. 1978)

This test is designed for testing the behavioural despair in animals. Animals are
forced to swim individually for 15 min. in a glass cylinder containing fresh water
at a temperature 25 ± 2°C to avoid variations and to maintain consistency in the
immobility time between different groups. This constitutes the pre-test session.
24 h later, the animals are treated with the drug or the vehicle and each animal is
again forced to swim in a similar environment for a period of 6 min. The
duration of immobility is recorded. In this experimental paradigm, after a phase
of vigorous swimming, the animals show an immobile floating posture. This
immobility is postulated to represent a depressant-like state and is reduced or
eliminated by clinically effective anti-depressant drugs. Animals are judged to be
immobile if they seize struggling and remain floating motionless in water
making only the necessary movements to keep their head above water.

9.17 SCREENING OF ANTI-EPILEPTIC AGENTS FROM HERBAL

SOURCES
9:17.1 Epilepsy: General Considerations
Epileptic seizures are caused by sudden, excessive, rapid, synchronised and focal
or general neuronal discharge. The generation and spread of seizures involves
the following mechanisms:

1. Abnormalities in the membrane properties of neurons, which can make the

neuronal membranes hyper-excitable thus reducing the seizure threshold
and leading to easy spread of seizures.
2. Changes in the ionic micro-environment surrounding neurons can also
 reduce the threshold for depolarisation.
3. Decreased inhibitory neurotransmission within the CNS often leads to
enhanced and uninhibited excitatory transmission thus favouring the
generation of seizure activity.
4. Enhanced excitatory neurotransmission can easily lead to rapid neuronal
firing, which can spread to generate epileptic seizure.

Epileptic seizures can be classified into two types—generalised and partial.

Generalised seizures
In these types of seizures there is no evidence of a localised onset.
Generalised tonic–clonic (Grand mal) seizures: These seizures are characterised
by tonic rigidity of extremities for 15–30 s followed by clonic jerks (alternate
contractions and relaxations of groups of muscles) This stage lasts for 60–120 s
and is followed by deep sleep.
Absence (Petit mal) seizures: The attacks are characterised by sudden onset of
loss of consciousness and ongoing motor activity. The attacks last for less than
10 s.
Myoclonic seizures: The attacks are characterised by sudden shock-like
contraction of group of muscles
Partial seizures
Partial seizures are localised in activity.
Simple partial: Consciousness is not lost. Sensory or motor symptoms are
experienced depending upon the part of the brain involved.
Complex partial: Consciousness is lost. Temporal lobes are commonly involved.
The person may keep staring or may fall down. Automatism characterised by lip
smacking, swallowing, fumbling, scratching is often observed.
Partial with generalised tonic–clonic seizures: Partial seizure immediately
precedes a generalised tonic–clonic seizure.
Mechanisms of anti-epileptic drugs
Anti-epileptic drugs act by one of the following mechanisms:
Inhibit GABA (inhibitory neurotransmitter) mediated inhibition Gamma amino
butyric acid (GABA) is the predominant inhibitory neurotransmitter in the brain.
The receptors for this neurotransmitter are divided into two classes: GABAA and
GABAB. GABAA receptors incorporate a chloride ion channel within it while
GABAB are G protein-coupled receptors. Majority of anti-epileptic drugs act by
affecting GABAA transmission. These anti-epileptics can act by:
Increasing the permeability of chloride channels (GABAA receptor): This
potentiates the action of GABA. Examples are Barbiturates, valproate and
benzodiazepines.
Increasing GABA release: Thus increasing the amount of GABA available at
neural synapses. Example: Gabapentine.
Inhibiting GABA transaminases: Thus inhibiting the breakdown of GABA and
making a large amount available at neural synapses. Examples: Vigabatrin,
Valproic acid.
Inhibiting GABA re-uptake: Thus increasing the amount of GABA available at
neural synapses. Examples: Tiagabine.
Prolongation of sodium channel inactivation Thus making the neuronal
membrane refractory for the generation of the next action potential. Examples:
Phenytoin, carbamazepine, valproic acid and lamatrigine.
Inhibit low threshold ('T' type) calcium ion current in thalamic neurons This
increases the refractory period of the neuronal membrane. Examples:
Ethosuximide, trimethadone and valproic acid.
More recent drugs act using a variety of mechanisms, which can be a
combination of the above described mechanisms, or by other mechanisms such
as inhibition of NMDA receptors (excitatory receptors). Examples:
Levetiracetam, topiramate, zonisamide, stiripentol, 2-en- VPA, denzimol,
oxacarbazepine, eterobarb and milacemide.

9.17.2 Screening
Anti-epileptics are screened primarily by using animal models, which can be
divided into two categories:

1. Electrically induced seizures

2. Chemically induced seizures
Electrically induced seizures
MES induced seizures
Efficacy of drugs against primary and secondary generalised tonic–clonic
seizures is screened by this method. It was developed by Merritt and Putnam
(1938).
Adult rats (150–200 g) or mice (18–30 g) are used. Animals are pre-treated
intramuscularly/orally (the intraperitoneal route can also be used) with the test
compound or the vehicle. Electric stimulus is applied via the corneal or ear
electrodes, which are moistened with saline solution before application. The
strength of stimulus used is 150 mA in rats and 50 mA in mice at a frequency of
50–60/s for a 0.2 s duration (Swinyard 1972; Loscher and Schmidt 1988).
Rectangular pulses or sinusoidal pulses can be used. All control mice are
expected to exhibit a tonic limb flexion phase (1.5 s), an extensor tonus (10 s)
followed by a variable short clonic phase. Disappearance of the hind limb
extensor tonic convulsion is considered the measure of efficacy. The percentage
of animals showing inhibition of seizures in the control group can be compared
with that of the test group. Using varied dose levels ED50 can be calculated.
This model represents the grand mal type of epilepsy and therefore, drugs like
phenytoin, carbamazepine, phenobarbitone and promidone are effective while
ethosuximide is inactive. The drugs, which are effective, can be used as positive
control.
The electroconvulsiometer used to stimulate the seizures is shown in Fig.
9.36.
Threshold model
It is also known as the tonic extension model and is used to determine the ability
of a drug to modify the seizure threshold for tonic limb extension. It is simple
and highly reproducible. The behavioural and EEG changes generated are
consistent with the human disorder.
Fig. 9.36 Electroconvulsiometer

Healthy albino mice (18–30 g) are used. An electric stimulus is applied as

described in the MES induced seizures method and threshold is determined as
the current or voltage inducing hind limb extension in 50% of the animals (CC50
and CV50 or EV50 respectively). Elevation of threshold by the drug is taken as a
measure of its efficacy. Calculation of the dose that elevates the threshold by
20% helps in comparing the effect of the drug. Threshold determination of
control animals should be undertaken on each day parallel to the threshold
determination in drug- treated animals.
Kindled rat seizure model (Goddard et al 1969)
Kindling (although it generally means setting on fire) is the name given to the
procedure by which administration of sub-convulsive electric stimulus results in
progressive intensification of stimulus-induced epileptic activity finally leading
to generalised seizures. The phenomenon shows that epilepsy (even mild)
induces epilepsy.
This method can differentiate between drugs which raise the seizure threshold
and which limit seizure threshold (Goddard et al. 1969).
Wistar/Sprague–Dawley rats (270–400 g) are implanted with an electrode in
the right amygdala for electrical stimulation. Other regions of the fore brain can
also be used. After 1 week of surgery, an electrical stimulus is applied daily
either as fixed current strength (400– 500 microampere, 1 ms monophasic square
wave pulse for 1 s with 50–60/s frequency) or as individual threshold current.
Seizures developing during stimulation of the amygdala are classified into 5
stages:

1. Immobility, eye closure, twitching of vibrissae, stereotypic sniffing

 2. Facial clonus and head nodding
3. Facial clonus, head nodding and forelimb clonus
4. Rearing, often accompanied by bilateral forelimb clonus
5. Rearing with loss of balance and falling accompanied by generalisd tonic-
clonic seizures.

When stage 5 seizures develop, rats are said to be fully kindled. Continuous
stimulation for a few weeks results in the development of spontaneous seizures
persisting for 7 months even after stopping the stimulation.
Test animals are administered with the test compound i.p/orally/i.m and the
drug efficacy is measured by observing the following four parameters on the day
before and after treatment:

1. Seizure latency (time from stimulation to the first sign of seizure activity)
2. Seizure severity
3. Seizure duration
4. After discharge duration

ED50 can be calculated for total suppression of generalised seizures, focal

seizures and amygdala after seizures.
The above-described method can help in measuring the efficacy of a drug
against the process of epileptogenesis as well as the fully kindled state.
Chemically induced seizures
Pentylene tetrazole (PTZ) induced seizures
PTZ (cardiazole/metrazole), which is a tetrazole derivative can produce
convulsion in a variety of animals such as rats, mice cats etc. It acts by
antagonising the GABA neurotransmission. This model represents absence
seizures in man and therefore can be used for the screening of drugs effective in
petit mal epilepsy.
For calculating the threshold for clonic seizures, 1% solution of PTZ is given
by i.v infusion at the rate of 0.3 ml/min. (Loscher and Schmidt 1988). The
animal at first develops isolated jerks followed by generalised tonic–clonic
seizures with loss of the righting reflex and then maximal tonic–clonic seizures
after a time lag. The mean dose required for inducing generalised tonic–clonic
seizures with loss of righting reflex is calculated. The mean dose is about 50
mg/kg for clonic seizures and 85–90 mg/kg for maximal seizures in mice.
Subcutaneous PTZ test is used to assess drug efficacy. CD97 (convulsive dose
in 97% animals) is first calculated. It is usually 70 mg/kg in rats and 80–100
mg/kg in mice (Loscher and Schmidt 1988). Within 30 min. after subcutaneous
administration of the above- mentioned dose, mice develop a sequence of
excitement, myoclonic jerks, clonic seizures, maximal tonic seizures and death.
The first occurrence of clonic jerks for 5 s or the first clonic seizure with loss of
righting reflex is taken as end point. ED50 for suppression of clonic seizure is the
measure of efficacy of the drug. Drugs effective in absence seizures show
efficacy in this test.
Systemic penicillin test
This model is used for screening of drugs effective in treating petit mal epilepsy.
Cats are used. Recent research reveals that rats can also be used in this method
(Fariello 1976).
Penicillin (300000 units) is injected intramuscularly. The seizures begin about
one hour after injection and continue intermittently for 6–8 h. The seizures are
characterised by recurrent episodes of arrested activity, staring, myoclonus,
facial oral twitching and occasional progression to generalised tonic–clonic
seizures (Taylor and Gloor 1984). The model represents the absence seizures in
man and therefore ethosuximide and valproate are effective in this model
(Pellegrini et al. 1978; Guberman et al. 1975).
Strychnine induced seizures
Strychnine is a CNS stimulant. It can act as an antagonist of glycine receptor and
produce convulsions in mice. The control and study animals are administered
with 2 mg/kg strychnine nitrate 1 h after vehicle or drug treatment. The number
of protected animals in the treated group is calculated as a percentage of the
affected animals in the control group.
Picrotoxin induced convulsions
Picrotoxin, a GABAA antagonist is used in doses of 3.5 mg/kg/s.c in mice (18–
22 g) to induce seizures within 30 min. Diazepam (10 mg/kg/ip) can be used as
control. Protection is expressed as a percentage of the inhibition relative to
control.
Isoniazid (INH) induced convulsions
INH (300 mg/kg/sc), a GABA synthesis inhibitor can be given 30 min. after
treatment in mice (18–22 g) and seizure activity can be observed over the next
120 min. Protection is expressed as a percentage of the inhibition relative to
control.
Bicuculline induced convulsions
Bicuculline, a GABAA inhibitor can be used in Wistar/Sprague–Dawley rats 1–2
h after treatment in a dose of 1 mg/kg/i.v. The seizures develop within 30 s of
injection. Effect of drug can be evaluated as in the case of INH.
4-Aminopyridine model
4-Aminopyridine, a potassium channel blocker can be given 15 min. after
treatment in a dose of 13.3 mg/kg/s.c. to Swiss mice (25–30 g) and inhibition of
seizure activity evaluated. The mean latency to death at LD97 is about 10 min.

Seizures induced by focal lesions

Simple partial seizures can be produced in animals by topical or intracerebral
application of certain metals or chemicals. These models are useful for
evaluation of drugs effective in partial seizures.
Metals which can be used to produce focal lesions in the cerebral cortex are
aluminium hydroxide, cobalt, tungstic acid and iron. Aluminium hydroxide is
the most commonly used, although other chemicals have also been used. These
include intra hippocampal injections of kainic acid, tetanus toxin, topical
application of penicillin, anticholinergics, cholinergics, picrotoxin, strychnine
etc. Cryogenic injury to the brain can also produce focal lesions.
Bicuculline methiodide (2 mg/kg) administered to rats 3–6 months after a
radiation dose to a volume of cerebrum equivalent of 0.25 ml produces seizure
focus. These seizures last for several weeks after single injection (Remler and
Marcussen 1986). Drugs can be evaluated for efficacy using this model.

9.18 SCREENING METHODS FOR ANALGESIC ACTIVITY

9.18.1 Centrally Acting Analgesics
Hot plate method
The paws of mice and rats are very sensitive to heat even at temperatures which
do not damage the skin. They respond by jumping, withdrawal of paws and
licking of paws. The time until these responses occur can be prolonged after
administration of centrally acting analgesics, whereas peripheral analgesics of
the acetyl salicylic acid or phenyl acetic acid type do not generally affect these
responses.
The hot plate consists of an electrically heated surface (Fig. 9.37). The
temperature is controlled for 55°–56°C. Adult albino rats are used for the test.
The animals are placed on the hot plate and the time until either licking or
jumping occurs is recorded by a stopwatch. The delay in response is recorded
after administration of the standard or the test compound.
Fig. 9.37 Eddy’s hot plate
Haffner’s tail clip method
In this method the raised tail phenomena in mice is observed. 6 mice per group
are used. A clip is applied to the base of the tail of mice and the reaction time is
noted. The test compounds are administered orally to fasted animals. The animal
quickly responds to the stimuli by biting the clip or the tail near the location of
the clip. The time between stimulation onset and response is measured by a
stopwatch.
Tail immersion test
This method is based on the observation that morphine-like drugs are selectively
capable of prolonging the reaction time of the typical tail- withdrawal reflex in
rats induced by immersing the end of the tail in warm water of 55°C.
Adult albino rats are used for the test. They are placed in individual cages
leaving the tail hanging out freely. The animals are allowed to adapt to the cages
for 30 min. before testing. The lower 5 cm portion of the tail is marked. This part
of the tail is immersed in a cup of freshly filled water of exactly 55°C. Within a
few seconds, the rat reacts by withdrawing the tail. A stopwatch records the
reaction time. The reaction time is determined before and periodically after
either oral or subcutaneous administration of the test substance. A withdrawal
time of more than 6 s is regarded as a positive response.
Radiant heat method
This test is useful for quantitative measurements of pain threshold against
thermal radiation in man and for evaluation of analgesic activity. It is very useful
for discriminating between centrally acting morphine-like analgesics and non-
opiate analgesics.
The animal is put into a small cage with an opening for the tail at the rear
wall. The investigator holds the tail gently. By the opening of a shutter, a light
beam exerting radiant heat is directed at the end of the tail. For about 6 s, the
reaction of the animal is observed. The mouse tries to pull the tail away and
turns the head. The shutter is closed with a switch as soon as the investigator
notices this reaction. Mice with a reaction time of more than 6 s are not used in
the test. The escape reaction is the end point of this test. Before administration of
the test compound or the standard, the normal reaction time is determined. The
test compounds and the standard are administered either orally or
subcutaneously. The analgesiometer can also be used to measure analgesic
activities (Fig. 9.38).
Formalin test in rats
Rats weighing 180–300 g are administered 0.05 ml of 10% formalin into the
lower surface of the front paw. The test drug is administered simultaneously
either s.c. or orally. Each individual rat is placed in a clear plastic cage for
observation. Readings are taken and scored according to a pain scale. Pain
responses are indicated by elevation of the paw or excessive licking and biting of
the paw. Analgesic response or protection is indicated if both paws are resting on
the floor with no elevation of the injected paw.

Fig.9.38 Analgesiometer
Tooth pulp stimulation
This test is based on the fact that stimulation of the tooth pulp induces
characteristic reactions, such as licking, biting, chewing and head flick which
can be observed easily.
Adult healthy rabbits are used for the test. Rabbits are anaesthetised and their
pulp chambers exposed with a high-speed dental drill. On the day of the
experiment, clamping electrodes are placed into the drilled holes. Aftei an
accommodation period of 30 min., stimulation is started to determine the
threshold value. The stimulus is applied with a frequency of 50 Hz and a
duration of 1 s. The electrical current is started at 0.2 mA and increased until the
phenomenon of licking occurs. The test substance is either injected
intravenously or given orally. The animals serve as their own controls.
Grid shock test
This test measures the analgesic properties by the “flinch– jump” procedure in
rats.
The floor of the box used is wired with stainless steel wire, spaced about 1
mm apart. The stimulus is given in the form of an electric current, 30 cycles per
s with a duration of 2 ms per pulse. With increasing shock intensities, the mice
flinch, exhibit a startling reaction, increase locomotion or attempt to jump. The
behaviour is accurately reflected on the oscilloscope by marked fluctuations of
the pulse and defined as the pain threshold response. The current as measured in
milliamperes is recorded for each animal before and after administration of the
drug.
Electrical stimulation of the tail
This method is based on the fact that since the tail of mice is known to be
sensitive to any stimulus, the stimulus can be varied either by the duration of the
electric shock or by an increase in the electric current to check the efficacy of the
analgesic agent.
Male mice weighing 20 g are placed in special cages. A pair of clips is
attached to the tail and the positive electrode is placed at the end of the tail.
Electric current at an intensity of 40–50 V is applied. The frequency of the
stimulation is 1 shock/s, and the pulse duration 2.5 ms. The normal response
time range of the stimuli is 3–4 s. Following administration of the drug, the
response time is registered at 15 min. intervals until the reaction time returns to
control levels.

9.18.2 Peripherally Acting Analgesics

Pain in inflamed tissue (Randall–Selitto test)
This method is based on the principle that inflammation increases the peripheral
analgesic sensitivity to pain. Inflammation decreases the pain reaction threshold
but the threshold is readily elevated by non-narcotic analgesics of the salicylate–
amidopyrine type as well as by the narcotic analgesics.
Groups of healthy albino rats (130–175 g) are used. The animals are starved
18 to 24 h prior to administration. To induce inflammation, 0.1 ml of a 20%
suspension of Brewer’s yeast in distilled water is injected subcutaneously into
the plantar surface of the left hind paw of the rat. Three hours later, pressure is
applied through a tip to the plantar surface of the rat’s foot at a constant rate to
the point when the animal struggles, squeals or attempts to bite. Each animal is
tested for its control pain threshold. Any animal with a control pain threshold
greater than 80 g is eliminated and replaced. The mean applied force is
determined for each time interval.
Writhing tests
Pain is induced by injection of irritants into the peritoneal cavity of mice. The
animals react with a characteristic stretching behaviour which is called writhing.
The test is suitable to detect analgesic activity. An irritating agent such as phenyl
quinone or acetic acid is injected intraperitoneally to mice and the stretching
reaction evaluated.
Mice of either sex of weight 20–25 g are used. Phenyl quinone in a
concentration of 0.02% is suspended in a 1% suspension of carboxy methyl
cellulose. 0.25 ml of this suspension is injected intraperitoneally. The mice are
placed individually into glass beakers and observed for a period of ten min. The
number of writhes is recorded for each animal. A writhe is indicated by a
stretching of the abdomen with simultaneous stretching of at least one hind limb.

9 .1 9 SCREENING OF HERBAL DRUGS FOR ANTIPYRETIC

ACTIVITY
Pyresis induced by lipopolysaccharides from E.coli in rabbits
Lipopolysaccharides from gram-negative bacteria, e.g. E. coli, induce fever in
rabbits after intravenous injection. An increase in body temperature of 1ºC or
more at a dose between 0.1 and 0.2 μg/kg is observed.
Rabbits of both sexes with a body weight between 3 and 5 kg are used. The
animals are placed in suitable cages and thermocouples connected with an
automatic recorder are introduced into the rectum. Then 0.2 ml/kg of solution
containing 0.2 μg lipopolysaccharide are injected intravenously into the rabbit’s
ear. Sixty min. later, the test compound is administered either subcutaneously or
orally. Body temperature is monitored for at least 3 h. A decrease of body
temperature by at least 0.5ºC for more than 30 min. as compared with the
temperature value before administration of the test compound is regarded as a
positive effect.
Pyresis induced by Brewer’s yeast suspensions in rats
A subcutaneous injection of Brewer’s yeast suspension is known to produce
fever in rats. A decrease in temperature can be achieved by administration of
compounds with antipyretic activity.
A 15% suspension of Brewer’s yeast in 0.9% saline is prepared. Rats with a
body weight of 150 g are used. Initial rectal temperatures are recorded by
insertion of a thermocouple to a depth of 2 cm into the rectum. The animals are
subcutaneously injected with 10 ml/kg of Brewer’s yeast suspension in the back
below the neck. Immediately after yeast administration, food is withdrawn. 18 h
post-administration, the rise in rectal temperature is recorded. The measurement
is repeated after 30 min. Only animals with a body temperature of at least 38ºC
are taken into the test. The animals receive the test compound or the standard
drug by oral administration. Rectal temperatures are recorded again after dosing.
The differences between the actual values and the starting values are registered
for each time interval.

9.20 SCREENING METHODS FOR ANTI-CATARACT AGENTS

Cataract, the opacification of the lens of the eye, is the leading cause of visual
impairment, responsible for blindness in 40 million people around the world. In
India, cataract is responsible for almost 80% of the blind population. The
recently launched Vision 2020, a global initiative for the elimination of
avoidable blindness introduced by the World Health Organization and other
organisations, singled out cataract as a priority disease. The situation can be
remedied surgically by extirpation of the cataractous lens. However, the
disability remains prevalent in a substantial number of the population. Effective
surgical procedures are available for treatment, but besides not being free from
complications, they require highly trained personnel and pose a substantial
economic burden. This is the reason why biochemical solutions or
pharmacological interventions that maintain the transparency of the lens are
urgently required. A variety of natural and synthetic compounds of different
chemical structures have been reported to render a protective shield against
cataract. Herbal drugs like tulsi, green tea and amla have been reported to
prevent the process of cataractogenesis in various experimental models.

9.20.1 Mechanism
During the process of cataractogenesis, the eye lens loses its transparency
because of a variety of complex metabolic and physiological mechanisms. These
complex mechanisms act together to change the refractive index. The result is
that the lens is not able to focus light on to the retina. Post- translational
modifications occur in the lens protein during cataract formation and are the
result of chemical actions that include oxidation, glycation, proteolysis, Schiff’s
base formation, transmutation and carbamylation.
The etiology of sugar cataract is well established. When hyperglycemia
occurs, the increased amount of glucose is converted into sorbitol by the enzyme
aldose reductase (AR) within the lens. Opacity is caused by the electrolyte
imbalance and accumulation of polyols. The main mechanisms involved in
cataractogenisis are: non-enzymatic glycation; oxidative stress; and polyol
pathway.

9.20.2 Experimental Cataract

Several in vitro and in vivo experimental models for cataract have been
developed which have indicated targets for lens damage and which resembles the
human cataract. There is often considerable overlap in mechanism between one
etiology and another.
In vitro models
Enucleated animal lenses are individually maintained in 2 ml of physiologically
competent tissue culture medium (TC-199/MEM) with 10% foetal calf serum at
37ºC and 5% CO2 atmosphere in a 24-well Falcon plate. The lenses are
incubated for 2–16 h before beginning the experiment. If any of the lenses
develop opacities during this period, they are eliminated from the experiment.
Incubation helps in detecting lenses which are damaged during the extraction
procedure. Substituting the medium with different agents generating oxidative
stress conditions directly or indirectly induces cataractogenic injuries to these
lenses. Incubation of lenses in the presence of high sugar concentration mimics
sugar cataract.
To see the effect of the anti-cataract agent on the biochemical status of the
lens, the following parameters are evaluated:

1. Glutathione and malondialdehyde

2. Sugar alcohol (polyols)
3. ATP
4. Soluble and insoluble proteins
5. Anti-oxidative enzymes.
Oxidative stress induced experimental cataracts
Oxidative free-radical damage is an initiating or very early event in the overall
sequence that leads to cataract. Oxidative stress may cause direct modification of
the inner lens proteins such as cross-linking, aggregation and precipitation.
Photo-chemically induced cataract
Culture of rat lenses in the presence of fluorescent light in a medium containing
micro molar (10–100 µM) quantities of riboflavin acting as a photosensitiser,
leads to severe physiological damage and opacification in 24 h. The initial
membrane damage in this system is evidenced by a disturbed cation ratio
between lens water and the medium of incubation. Riboflavin on becoming
photosensitised generates free radicals in a sequence of reactions.
Enzymatically induced cataract
Culture of rat lenses in the presence of 1 mM xanthine and 0.1 unit of xanthine
oxidase acting as substrate and enzyme respectively, generate superoxide radical
and lead to severe oxidative damage and opacification of lens within 24 h.
Selenite induced cataract
This model is useful for experimental studies on biochemical mechanisms and to
study the role of calcium induced proteolysis in cataract formation.
Incubation of rat lenses in a tissue culture medium with 100 µM concentration
of sodium selenite results in membrane damage and faint cortical opacities
within 24 h.
Hydrogen peroxide induced cataract
Hydrogen peroxide causes oxidative damage and converts the reduced form of
lens protein to the oxidised form.
Hydrogen peroxide stress produces cataract in cultured lenses. The loss of
transparency begins in the equatorial region within 24 h and the entire superficial
cortex becomes opaque by 96 h. After an additional 48 h, the nuclear region also
becomes opaque. For the hydrogen peroxide stress model, 200 µM of hydrogen
peroxide is added in the culture medium and the medium changed every two
hours during the first eight hours.
Steroid induced cataract
Incubating the isolated lenses in a tissue culture medium with 1.5 mg/ml of
methyl prednisolone for 24 h induces in vitro steroid cataract.
Naphthalene induced cataract
Isolated rat lenses are exposed to naphthalene metabolites containing medium
Tc-199 for 48 h. Under this condition, lenses develop opacities. They become
swollen and thus heavier than the normal lenses. The opacities are seen in the
outer layer of the cortex with regions of obvious liquification.
Sugar induced cataracts
Diabetes is one of the most important risk factors of cataract. The enzyme aldose
reductase is responsible for initiating the cataractous process via polyol pathway.
Galactose is a better substrate than glucose for AR which results in more polyol
being formed. The thus formed polyols accumulate in the eye lens (Kinoshita J
H.1974)
 Sugar cataracts in rat lenses are produced in vitro by incubating the lenses in
the presence of 30 mM xylose or 30 mM galactose in Tc-199 with foetal calf
serum. The lenses maintained under such conditions show raised water and
polyol levels. Lenses develop opacity in the subcapsular equatorial region on day
1 and in the central region on day 2.
In vivo models of cataract
Various rat/rabbit models of cataract are established for the screening of anti-
cataract agents. These models use the following mechanisms: sugar induced
cataract, oxidative stress, radiation induced cataract etc.
Sugar (diabetic) cataract
Streptozotocin induced cataract The chemical structure of streptozotocin (STZ)
has a glucose molecule with a highly reactive nitrosourea side chain, which
supposedly initiates its cytotoxic action. The glucose moiety directs this agent to
the pancreatic 2-cells. There it binds to the membrane receptor to generate
structural damage.
The requirements for this test include: albino rats weighing 150–200 g,
streptozotocin and slit lamp.
Albino rats (Wistar/Sprague–Dawley) of 150–200 g body weight are used.
Diabetes is induced by injection of streptozotocin (50–70 mg/kg/i.p). Care
should be taken not to puncture the intestine. Streptozotocin is dissolved in 0.02
M sodium citrate buffers. The solution is filtered through a 0.22 µm-millipore
filter into a sterilised container kept on ice and used within 10 min. of
preparation. The control group rats are injected with sterilised buffer alone. After
3 days, the blood glucose level in each rat is estimated. STZ injected rats having
blood glucose level less than 150 mg/dl are re-injected with a fresh solution of
STZ and tested again for blood glucose (Blakytny and Harding 1992).
Progression of cataract stages is observed through a slit lamp (Fig. 9.33). The
initial stages of cataract appear 15 days after STZ injection. The sutures become
prominent and the fully mature cataract appears in nearly 110 days depending
upon the age of the rat at the time of injection. The cataractogenic changes can
be compared with the control rats (injected with buffer).
The stages of streptozotocin induced cataract are as follows:

1. Lenses similar to normal lens

2. Small vacuoles appearing in the periphery or central suture or cloudiness
 3. a. Large vacuoles spreading to the centre
b. Increased radial opacities, fused vacuoles and cloudiness
4. Disappearance of vacuoles and the radical opacities become more
prominent
5. Full opacity.

Galactose induced cataract Elevated sugar levels in the aqueous humor induces
lens fibre swelling by osmosis and increases the lens fibre permeability resulting
in disruption of the fibre. Cataractogenic sugars entering the lens are converted
into their respective sugar alcohols in the presence of the enzyme aldose
reductase. These alcohols accumulate in the fibres creating hypertonicity, which
is corrected by an influx of water. Initially the activity of a cation pump
compensates for the increased water content of the fibre and leads to an
electrolyte imbalance.

Fig. 9.39 Slit lamp

The requirements for this test include: galactose, albino rats of either sex
weighing 50–70 g, slit lamp and 1% tropicamide. Galactose feeding can induce
cataract more rapidly in comparison to streptozotocin/alloxan induced cataract.
The galactose-fed rat model has became a popular cataract model, as it is
reproducible compared to the diabetic model.
Wistar/Sprague–Dawley rats weighing 50–70 g are fed with a diet containing
30% galactose. Diet and water are given ad libitum. The eyes of the rats are
examined weekly by a slit lamp to see the cataractogenic changes. 1%
Tropicamide is used to dilate the pupil size. Vacuoles which may be attributed to
globular degeneration of the fibre lens start appearing in the periphery of the lens
within a period of one week. The vacuoles gradually increase in number and size
as they move towards the centre. This takes about 14 days. All these changes are
not visible through the naked eye. During the third week, the lens shows
opalescence, visible without any aid and becomes totally opaque in 30 days time.
The different stages of cataract are graded in the following way (Sippel
TO.1966) (Fig. 9.40).

Stage 0: Lenses similar to normal lenses

Stage I: Lenses showing faint peripheral opacity

StageII : Irregular peripheral opacity with slight involvement of the

lens in the centre.

Stage III: Faint opalescence visible with the naked eye

Stage IV: Mature nuclear cataract.

Fig. 9.40 Stages of galactose cataract

Selenite (oxidative stress) induced cataract

Selenium, an essential nutrient and a hazardous element, plays a critical role in
maintaining normal physiological conditions. Selenite induced cataract models
have been used to quantify and characterise events that occur in the lens prior to
the formation of a nuclear cataract. Selenium oxidises the sulph- hydryl (SH)
group or opens ion channels in the lens epithelial membrane. This allows influx
of calcium from the aqueous humor. Elevated calcium levels activate calpain, a
cytosolic calcium activated protease, by causing autolysis (Shearer and David
1982; Thomas et al. 1992). This model has been used extensively as a model for
nuclear cataract; a transient cataract also forms 15–30 days after injection.
The requirements includes sodium selenite, 9–10 day old albino rat pups along
with their mothers and slit lamp.
Albino young rats (Wistar/Sprague–Dawley>) are housed together with their
mother. The mother is fed a normal diet and water ad libitum and she suckles the
pups. The young rats are grouped into three. One group is left as a normal group.
The other two groups—control and treated—are given subcutaneous injections
of sodium selenite (25–30 µ moles/kg) in the scruff of the neck. The treatment
group is given the anti-cataract agent intraperitoneally. On the 16–18th day, the
eyes of the young rats open. 100% of the eyes show incidence of cataract in the
control group. The eyes of the treated group are compared with the control group
to evaluate the efficacy of the drug.
Naphthalene induced cataract
The naphthalene cataract model is a suitable model for human age related
cataract. The injected naphthalene is first oxidised in the liver to an epoxide and
is then converted into naphthalene dihydrodiol. This stable compound on
reaching the eye gets converted enzymatically to dihydroxy naphthalene. Being
unstable at physiological pH, 1,2-dihydronaphthalene spontaneously auto-
oxidises to 1,2 naphthoquinone and hydrogen peroxide. 1,2-dihydronaphthalene
is also a highly reactive compound which can alkylate proteins, glutathione,
amino acids. It can also generate free radicals.
The requirements for this test include: albino male rats weighing 125–150 g,
10% naphthalene solution, 1% tropicamide and slit lamp
125–150 g albino male rats are used for this model. The rats are dosed with
10% naphthalene solution using an 18-gauge needle at 0.5g/kg/day for three
days and 1 g/kg/day thereafter. A batch of rats is dosed with a same amount of
mineral oil to serve as controls. The drug treatment is given orally by gavages
one hour prior to naphthalene administration. Morphological changes in the eyes
of the rats are observed through slit lamp examination after dilating the pupil
with 1% tropicamide. The lenses are examined twice a week during the first two
weeks and thereafter at weekly intervals. One week after the administration,
spoke-like opacities in the cortex are seen. By the third week, an opaque shell is
visible in the deep cortex region which becomes denser and slightly deeper with
time.
Grading of naphthalene cataract is as follows:

Stage 0: Clear lens

Stage 1: Water clefts and spoke-like opacities

Stage 2: More water clefts and spoke-like opacities

Stage 3: Opacities merge to form a shell

Stage 4: Shell partly thick and white

Stage 5: Shell fully thick and white

Radiation (UV) induced cataract

The relationship between cataract and UV exposure has been well established
epidemiologically and experimentally (Marcelo et al. 2004; Linda et al. 2005).
Ultraviolet radiations of wavelengths that can pass through the cornea to reach
the lens and be absorbed can cause intensive damage to the lens.
The requirements for this test include: albino rats, a UV radiation source and a
photodetector.
Albino rats are anaesthetised and exposed to UVB (290 to 320 nm) radiation.
The UV radiation source is a mercury lamp with a water filter and a
monochomator (set at: 300 nm and 9 nm full bandwidth at half maximum). The
dose range should be 0.1 and 20 kJ/m² and the exposure time is 15 min. Pupil
dilating eye drops is applied before irradiation and one eye in each rat is
irradiated. The animals are kept between 6 h and 32 weeks after exposure.
Photo-documentation is done after the extraction of the lenses. One or eight
weeks after exposure, the forward light scattering in the lenses is determined.
The lens is placed in a cuvette filled with salt solution. The probing light from
the dark-field illumination will, in the case of a perfectly transparent lens, pass
through the lens and not reach the photodetector.
9.21 SCREENING METHODS FOR ANTI-GLAUCOMA AGENTS
Glaucoma is another major cause of blindness. Many synthetic drugs are
available for the treatment of glaucoma. There are also several nutrients and
botanicals that hold promise in improving circulation to the optic nerve and even
lowering IOP (intraocular pressure) including vitamins B12 and C, melatonin,
lipoic acid, ginkgo (Ginkgo biloba), bilberry (Vaccinium myrtillus), topical
forskolin, and red sage (Salvia miltiorrhiza).
The method for raising IOP should be simple, produce significant rise in
intraocular pressure, allow tonometric monitoring in the experimental animals
and minimise secondary ocular changes like inflammation. IOP can be measured
by tonometry, using a Schiotz tonometer, a non-contact tonometer (Fig. 9.35) or
a pneumotonometer.
Water-loaded rabbit model of glaucoma (acute model)
This model was established by Sugiyama et al.(1989). Water loading in rabbits
reduces the serum osmolarity and a temporary change in IOP is observed. This is
an acute model and is widely used as a provocating test in diagnosing primary
open angle glaucoma (Thorpe and Kolker 1967) and for detecting the anti-
glaucoma potential of several drugs (Santafe et al. 2000).
The requirements for the model are: New Zealand white albino rabbits, infant
feeding tube and a tonometer.

Fig. 9.41 A non-contact tonometer

 New Zealand white albino rabbits of either sex weighing 2–2.5 kg are used for
the study. The animals are fasted overnight and basal IOP is measured using a
tonometer. Ocular hypertension is induced in conscious rabbits by inserting an
infant gastric tube into the stomach and loading 70 ml/kg. The IOP is measured
at 15, 30, 45, 60, 90, 105 and 120 min. For evaluating the anti-glaucoma activity
of an agent, it is administered in the form of eye drops to one of the eye prior to
water loading of the animal, while the vehicle is instilled in the contra-lateral
control eye. The difference in the IOP is measured in the two eyes at various
time intervals after the water loading till the IOP reaches the baseline value. Any
significant change in the IOP shows the potential of the drug as an
oculohypotensive.
⍺ chymotrypsin-induced experimental glaucoma (chronic model)
Sears and Sears (1974) established this chronic model of glaucoma.
Intra vitreal injection of α chymotrypsin induces a sustained rise in IOP for
several weeks due to predominant changes in the trabecular meshwork.
The requirements for this test include: New Zealand white albino rabbits, α
chymotrypsin, 2% xylocaine, a 30-guage sterile needle and a tonometer.
New Zealand white albino rabbits of either sex weighing 2–2.5 under
nembutal (sodium pentobarbitone) anaesthesia are used. One of the eyes is
anaesthetised with local anaesthetic, 2% xylocaine. The eyeball is fixed with a
forceps and a 30- guage sterile needle is inserted carefully for an intravitreal
injection (viathe limbus). Antibiotic (2–3 drops) is applied to the eye to prevent
infection. Freshly prepared (150 units) α chymotrypsin dissolved in sterile saline
(0.5 ml) is injected into the posterior chamber. Initially and after two days, the
IOP is measured on regular intervals using a tonometer. Within 15 days, a stable
increase in IOP is observed.
To evaluate anti-glaucoma activity, the test agent is administered topically or
orally in a suitable formulation once the glaucoma is established (stable IOP).
Steroid induced glaucoma
Experimental glaucoma induced by steroids is reported in young rabbits and cats
(Knepper et al. 1978).
The relationship between long-term use of corticosteroids and the rise in IOP
has already been established. Suppression of lysosomal activity by
corticosteroids is assumed to result in reduced elimination of
mucopolysaccharides within the trabecular meshwork with subsequent
augmentation of the extracellular matrix contributing to steroid-induced
glaucoma (Kasavina and Chesnokova 1973).
The requirements for this test include: New Zealand white young rabbits, 1%
dexamethasone/prednisolone eye drops and a tonometer
A subset of the general population experiences a significant increase of IOP in
response to glucocorticoid administration. Young rabbits (1.25–1.5 kg) are
trained to accept tonometry and then IOP is measured daily for 15 days so as to
make a record of baseline IOP. These rabbits are then instilled with
dexamethasone/prednisolone 1% eye drops (10 µl) in the test eye and normal
saline (10 µl) in the control eye, twice a day for a period of 40 days. IOP is
measured twice a week during steroid treatment period. At the end of 40 days,
rabbits are subjected to evaluation of anti-glaucoma activity.
Methyl cellulose (MC) induced glaucoma
Injection of MC causes a blockade in the trabecular meshwork and thus aqueous
outflow from the anterior chamber of the eye is inhibited.
The requirements for this test are: methylcellulose, 0.9% saline and a three-
way stopcock.
Injection of 0.5–4% MC (a polymer) in the anterior chamber of rabbits
induces glaucoma (Lorenzetti and Sancilio 1967). Methylcellulose solution
(0.5%) made in 0.9% saline, free of air bubbles is injected into the anterior
chamber (just above the aperture of the iris) of the rabbit’s eyes after removing a
volume of aqueous humor equal to the volume of methylcellulose (150–190 µl)
added using a three-way stopcock for evacuation and refilling. The IOP increases
considerably in the next 2 h. 8 weeks of intraocular hypertension can be induced
by a series of four intra-anterior chamber injections of 1% or 2% methylcellulose
in rabbits (Zhu and Cai 1992).
9 .2 2 SCREENING OF HERBAL DRUGS FOR ANTI-CANCER
ACTIVITY
Cancer is characterised by uncontrolled division of cells and the ability of
cancerous cells to invade other tissues, either by direct growth into adjacent
tissue, through invasion or by implantation into distant sites by metastasis.
Metastasis is defined as the stage in which cancer cells are transported through
the bloodstream to sites far removed from the original site. Cancer is the leading
cause of death in many countries today, exceeded only by heart diseases. It is
one of the principal causes of death in developed countries.
It is usually treated with a combination of surgery, chemotherapy and
radiotherapy. While chemotherapy forms an important modality for treating
cancer, there are several hurdles to its efficient usage such as multi-drug
resistance, excessive toxicity to normal cells, insufficient amount of drug
reaching the target cells, angiogenesis (formation of new blood vessels from pre-
existing vessels) and metastasis. Thus there is an urgent global need to develop
non-toxic anti-cancer agents for various kinds of carcinomas. Many non-toxic
herbs have been found to possess anti-cancer activity, thus reducing the toxic
side-effects of chemotherapy and radiotherapy and playing a vital role in the
prevention and treatment of cancer.
Interest in the use of herbal products has grown considerably. With advanced
knowledge of molecular science and refinement in isolation and structure
elucidation techniques, it is easier to identify various anti-cancer herbs and
develop a remedy that might cure cancer. The continuous search for new anti-
cancer drugs from plants will be a fruitful frontier in cancer treatment and
chemo-prevention as plants are safer than synthetic moieties.

9.22.1 Herbal Drugs as Anti-cancer Agents

Common or Madagascar periwinkle (Catharanthus roseus or Vinca rosea)
contains vinca alkaloids, which were the first phytoconstituents ever used to treat
cancer. Common yew (Taxus baccata) contain taxanes which arrest
multiplication of cancerous cells by cross-linking the microtubules, whereas
vinca alkaloids and podophyllin which is present in Himalayan or Indian
mayapple (Podophyllum hexandrum) act by breaking down the cytoskeletal
organelle (microtubule) into smaller subunits. Pharmacological studies have
demonstrated that curcumin, a constituent of the spice turmeric, has been shown
to reduce the adenoma burden in rodent models of colorectal cancer. The anti-
cancer effect of curcumin has been demonstrated in all the stages of cancer
development, i.e. initiation, promotion and progression of cancer. It inhibits the
growth of cancer by preventing production of harmful eicosanoids such as PGE-
2 and increases the level of glutathione. Studies have shown that green drinks
which contain a combination of various herbs including common barley
(Hordeum vulgare), alfalfa (Medicago sativa) and Spirulina enhance the activity
of the immune cells against cancer.
Garlic(Allium sativum) bioflavonoids, quercetin and cyanidin are responsible
for its anti-oxidant properties. Bromelain, a mixture of proteases isolated from
the stem and fruit of pineapple (Ananas comosus), stimulates the defence
mechanism of the body against cancer by enhancing the cytotoxic activity of
monocytes and macrophages, thus inhibiting the growth of cancer. Green tea
(Camellia sinensis) contains epigallocatechin gallate, which protects against
cancer by preventing the covalent bonding of carcinogens to the DNA.
Aloe (Aloe vera) contains aloe-emodin, which activates the macrophages and
enhances the activity of the immune cells against cancer. Milk thistle (Silybum
marianum) protects the body against liver cancer by accelerating regeneration of
the liver cells. Studies have revealed that ashwagandha (Withania somnifera)
enhances the therapeutic effect of radiotherapy. The glycosides withaferin A and
withanolide D found in ashwagandha are known to inhibit growth of cancer.
Camptothecin, a quinoline alkaloid isolated from seeds of camptotheca
(Camptotheca acuminate) is a well-known anti-cancer agent. Recent studies
have revealed that Creole cotton (Gossypium barbadense) which contains
gossypol possesses selective toxicity towards cancerous cells. Cape bushwillow
(Combretum caffrum) contains combretastatin, which has been isolated recently.
It executes its therapeutic action against cancer by inhibiting the blood supply to
the tumour. Derivatives of camptothecin such as 18-OH-camptothecin, 11-OH-
camptothecin and 10-OH-camptothecin have been found to possess a strong anti-
leukaemic activity.
Other important anti-cancer herbs include ash gourd (Benincasa hispida),
cabbage (Brassica oleracea), calendula (Calendula officinalis), Western
leatherwood (Dirca occidentalis), creosote bush (Larrea divaricata or Larrea
tridentate), white mulberry (Morus alba), paniculate ostodes (Ostodes
paniculata), gonnabos (Passerina vulgaris), kadsura stem (Piper futokadsura),
bitterwood (Soulamea soulameoides), creeping red thyme (Thymus serpyllum),
snakegourd (Trichosanthes kirilowii) and stinging nettle (Urtica dioica).
 The initial step in identifying anti-cancer activity of herbs is pre-clinical
evaluation by means of either in vitro or in vivo models. After this the drug
undergoes standard toxicology testing in animals before being put through
clinical trials.

9.22.2 Advantages of the in vitro Method Over the in vivo Method

In vitro and in vivo screening by a human cell line panel is a new and useful
system for evaluation of anti-cancer drugs. To date 85,000 compounds have been
screened against this in vitro cell line panel in a short-term assay.
In vitro approaches have the potential to provide information about the
function of specific organs and how a toxic agent can adversely affect them. The
in vitro screening is thus more specific, accurate and less time-consuming than
the in vivo method.

9.22.3 In Vitro Methods for Screening Cytotoxicity (anti-cancer

activity)
Protocols that can be used to indicate cytotoxicity in vitro are included in this
chapter. They fall into three categories:
1. To indicate that the drug has altered cell viability: These tests are valuable
for initial general screening. They can also be used in combination with direct
endpoint assays, which are more specific to the toxicant or target tissue, to
indicate if the more specific changes that occur in cells at concentrations that are
below the levels of concentration needed to decrease viability. This group
includes:

a. Trypan blue dye uptake

b. Lactate dehydrogenase (LDH) leakage
c. Neutral red dye retention
d. Propidium iodide or Fluorescent marker attachment to double-stranded
nucleic acids.

2. To assess if the drug has an effect on cell growth and cell proliferation:
These tests are generally more sensitive to lower concentrations of toxicants than
tests affecting cell viability. These tests can also be used at concentrations of the
drug that do not necessarily cause irreversible cell damage. Therefore, they
could be used to determine if recovery occurs when the cells are no longer
exposed to the toxicant. These tests are used to assess and indicate whether the
toxicant has affected cellular metabolic processes or not. This group includes:

a. [3H]thymidine uptake assay

b. Total DNA synthesis measured by [1251] Udr uptake
c. cell cycle analysis with propidium iodide

3. To assess whether the toxicant has affected cellular metabolic processes:

These tests may be useful to indicate if the toxicant is likely to target the cell
membrane, an intracellular organelle, or a biochemical process. They can be
used along with viability tests to determine concentration and time sensitivities.
This group includes:

a. MTT dye conversion by mitochondria

b. Alamar blue reduction by respiring cells
c. Fluorescent dye loss

Determination of cell viability by trypan blue exclusion method

Trypan blue dye exclusion assay is the simplest and cheapest method to check
cell viability but it is qualitative and indicates only if a cell is alive. It is a
common test for estimating cell viability, and is based on the ability of a cell
with an intact plasma membrane to exclude trypan blue dye which is
impermeable to healthy cells but can permeate through compromised
membranes of dying cells. Thus trypan blue stains dead cells which can then be
counted manually using a haemocytometer (Fig. 9.42). It detects loss of
membrane integrity during the later stages of cell death. The cell suspension is
prepared directly from culture (depending on the cell density) and 20 μi of cells
is mixed with 20 μi of trypan blue dye in an Eppendroff tube. The mixture is
allowed to stand for 5 min. The surface of the haemocytometer slide is cleaned
with 70 per cent alcohol and the cover slip placed by wetting the edges over the
grooves and semi-silvered counting area. A small amount of trypan blue cell
suspension is transferred to both chambers of the haemocytometer by carefully
touching the edge of the cover slip with a pipette tip. Each chamber is allowed to
fill by capillary action.
The total number of stained cells and the total number of cells in the four
1mm comer squares (Fig. 9.43) are counted. Cells which touch the top and left
middle line are included and those which touch the lower or right boundary are
excluded. The cells in the four comer squares in both chambers are counted. The
whole process is depicted in Fig. 9.44.
CALCULATIONS
Cells/ml = average cell count per square x dilution factor x 104

Fig. 9.42 Haemocytometer

Fig. 9.43 One of tyhe corner squares of a haemocytometer

Fig. 9.44 Counting the number of cells using a haemocytometer

Lactate Dehydrogenase (LDH) Leakage to Assess Viability

This procedure is used as an indicator of increased membrane permeability and
subsequent cell death. The increase in permeability associated with cell death
causes the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH),
which is present in mammalian cells. LDH is membrane- impermeable and
remains within live cells.
Procedure: IX 105 cells are taken and treated with a drug or vehicle in
suspension. Wash the cells one to three times with PBS, preparing a final re-
suspension in an exact volume of PBS. Add 250 pi of NADH solution (0.194
nmol/liter NADH; 54 mmol/liter phosphate buffer, pH 7.5). For suspension cells,
add 10 pi of drug-treated cells (>104 cells/ml) or control cells to these wells. For
attached cells, add 10 μi of the supernatant of toxicant- treated or control cells to
thess wells. The reaction is initiated by adding and mixing 25 μi of a solution of
6.48 mmol/liter pyruvate. The decrease in absorbance at 340 nm at 3 to 5 min
intervals over a period of time (up to 1 hour) is recorded. Rate of decrease of
drug (change in absorbance/min) to that of the rate of decrease of positive
control is compared and the result is expressed as the change in absorbance
units/min.
Neutral red toxicity assay
This cytotoxicity study is performed using neutral red (NR) uptake assay while
cell proliferation study is done by MTT. The assay is based on the principle that
cells which possess an intact membrane and are healthy, will take in the vital
stain, neutral red, in lysosomes. The amount of dye taken up by the cells is a
function of the cell number. Failure to take up NR therefore indicates that the
cells have suffered damage.
In this method, cells are placed in 24-well plates and treated with 2 ml of
various concentrations of test agents overnight at 37°C/5% C02 in a humidified
incubator. The media is aspirated and replaced with 1 ml per well of DMEM
containing 50 pg/ml neutral red (NR). The plates are incubated for 3 h at
37°C/5% C02 after which NR is aspirated and the discs washed twice with 1 ml
PBS containing calcium and magnesium for 3–5 min. This is followed by
another wash for 1–2 minutes with 1 ml of an aqueous solution containing 0.5%
formaldehyde and 1% calcium chloride. The formaldehyde is aspirated and the
NR absorbed by the cells is solvent-extracted with 2 ml of 1% acetic acid in 50%
aqueous ethanol on a shaker platform for 1 h. Two hundred micro litre aliquots
of each of the extracted NR solutions are transferred to a 96-well plate. The
optical density of each well at 540 nm (OD540) is determined
spectrophotometrically by a Vmax micro plate reader (Molecular Devices Corp.).
Before placing the plates with the cells, a blank correction is made to the pre-
treated nylon mesh (without cells) which has been similarly incubated with NR.
The mean OD540 of the untreated control wells is set to represent 100% viability.
Results for each concentration are plotted as a per cent of untreated controls vs.
concentration of test agent, and an NR 50 value (concentration of test agent that
reduces the NR uptake by 50% compared to untreated controls) is determined
directly from the graph. The lower the NR- 50 value, the more toxic is the
compound because less is required to achieve 50% mortality of the cells on the
mesh. The 1 ml of the NR medium is replaced using a a sterile pipette or sterile
pipette tips and incubating for 1 h at 37°C. Plates are then observed under the
inverted microscope. The medium is removed from the cells by gentle aspiration
and cells are washed with PBS. 1 ml of de-staining solution is added. Finally the
plates are read on the Bio-Tek micro plate reader.
Propidium Iodide Attachment to Double-stranded Nucleic Acids to Assess
Viability
The fluorescent dye propidium iodide (excitation 530 nm, emission 645 nm) is
excluded from living cells, but enters and combines with the double-stranded
nucleic acids (i.e., DNA) of dead cells. The assay for cell viability using
propidium iodide attachment to double​stranded nucleic acids can be done
simultaneously with the determination of membrane integrity, by using a variety
of membrane-permeant fluorescent probes (e.g., CFDA-AM, which has an
excitation 485 nm and emission of 530 nm), because the fluorescent spectra do
not overlap (Nieminen et al., 1992).
Procedure: 1 X 105 cells are placed in wells of a flat-bottom 96-well
microtiter plate and are allowed to adhere to the plate for 24 hours. The cells
were taken and treated with a drug or vehicle control. Then, 100 μi propidium
iodide (30 μM) was added to all the wells and incubated for 30 min at 37°C. The
plate was read in a microtiter plate fluorescence spectrophotometer with
excitation set at 530 nm and emission at 645 nm. The cells exposed to test
compounds and the wells exposed only to vehicle (negative control) were
compared, and the percentage of viable cells was calculated.
|3H] Thymidine Uptake to Assess Cell Growth
This procedure can be used as an indication of cytotoxicity because toxicants can
decrease the rate of cell division. The assay can also be used as an indicator of
cell proliferation, as [3H] thymidine incorporates into the nucleic acids of
proliferating cells. This is a relatively complex, yet sensitive procedure that
requires the use of radio-labelled chemicals.
Procedure: 1 X 105 cells are placed in wells of a flat- bottomed 24 well
microtiter plate and are allowed to adhere to the plate for 24 hours. The medium
from the wells is replaced with 1 ml serum-free, chemically defined medium, 24
to 48 hours before treating cells with test compounds. Then, 50 μi drug or
vehicle control is added to the wells. Thereafter, 50 μl/well [3H] thymidine
solution is added for the last 2 hours of incubation with the test substance and
incubated at 37°C. The reaction is stopped by placing plates on ice and the cells
are washed 3 times with 1 ml of ice-cold serum-free medium, and then carefully
removed from the medium by vacuum aspiration. 1 ml of ice-cold trichloroacetic
acid (10%TCA) is added to each well. The plates are left on ice for 10 min to
allow precipitation of DNA and protein. 1 ml of 10% TCA is added to the plates
and left on ice for an additional 5 min. It is washed again for the third time with
TCA and left on ice for an additional 5 min to complete the precipitation, and
finally the TCA is removed. Precipitated protein is solubilized with 0.5 ml of 0.3
N sodium hydroxide by incubating for 30 to 60 min at room temperature. 250 μi
is transfered to a mini- scintillation vial, with 5 ml scintillation fluid and count.
DNA fragmentation assay
Apoptosis, or physiological cell death, can be distinguished from necrosis on the
basis of a series of morphological and biochemical parameters. In general, in
order to describe a death event as apoptotic, more than one parameter should be
evaluated. Among these parameters, DNA fragmentation is very typical of the
apoptotic process. Measurement of DNA fragmentation is preferentially used to
evaluate apoptosis in resting cells or in other cell populations where DNA
labelling is impossible or difficult.
1.0 ml of cell suspension (not less than 5 × 105 and no more than 5 × 106, in
order to obtain for the DNA, an OD600 of 0.04 < DNA < 1.200) is placed in
tubes labelled B. The cells are centrifuged at 200 × g at 4ºC for 10 min. The
supernatants are transferred carefully into new tubes labelled S (supernatant). To
the pellet in tubes B, 1.0 ml TTE solution is added and vortexed vigorously. This
procedure allows the release of fragmented chromatin from nuclei, after cell
lysis (due to the presence of Triton X-100 in the TTE solution) and disruption of
the nuclear structure (following Mg++ chelation by EDTA in the TTE solution).
To separate the fragmented DNA from the intact chromatin, tubes B are
centrifuged at 20,000 × g for 10 min. at 4ºC and the supernatants transferred
carefully into new tubes labelled T. To the small pellet in tubes B, 1.0 ml of TTE
solution is added, followed by the addition of 1.0 ml of 25% TCA to tubes T, B
and S. They are vortexed vigorously and precipitation allowed to proceed
overnight at 4ºC. After incubation, precipitated DNA is covered by pelleting for
10 min. at 20,000 × g at 4ºC and the supernatants discarded by aspiration.
DNA is hydrolysed by adding 5% TCA to each pellet and heating for 15 min.
at 90ºC in a heating block. A blank is prepared with 5% TCA alone. To each
tube, freshly prepared DPA solution is added, then vortexed. The colour is
allowed to develop for about 4 h at 37ºC or overnight at room temperature. The
aliquots of coloured solution (ignoring dark particles) are transferred from each
tube to a well of a 96-well microtitre plate. OD600 is read with a multi-well
spectrophotometer reader, setting blank to 0. The excitation wavelength of 600
nm is the optimal one, but wavelengths from 560 to 620 can be used as well. The
percentage of fragmented DNA can be calculated using the formula:

where S, T and B are the OD600 of fragmented DNA in the S, T and B

fractions, respectively.
Cell Cycle Analysis with Propidium Iodide to Assess Cell Growth
This procedure can be used as an indicator of cytotoxicity for toxicants that
affect DNA synthesis and, therefore, cause cell arrest mostly in the G1/G0 phase
of the cell cycle. Some of these cells may undergo apoptosis later, whereas
others may have decreased rates of cell growth and proliferation. Propidium
iodide (PI), a DNA-binding fluorochrome, is the most commonly used DNA dye
for cell cycle analysis. Because it can be excited at 488 nm, it can be used on
most common flow cytometers. Its stoichiometric binding to double- stranded
nucleic acids allows fluorescent intensity (>620 nm) to be used as an indicator of
cellular DNA content. Using propidium iodide is now a common procedure for
DNA analysis (Krishan, 1975).
Procedure: 1 X 105 cells are placed in wells of a flat-bottom 24-well
microtiter plate and are allowed to adhere to the plate for 24 hours. Cells are
harvested and centrifuged for 5 min at 10000 rpm. Supernatant is discarded and
the pellet is vortexed. Cells are re-suspended in 1 ml standard azide buffer,
counted and recentrifuged for 5 min at 10000 rpm. 0.5 ml of Vindelov’s PI is
added. And let cells sit at 4°C for 5 min to 1 h, then filter through a 30-nm mesh,
if necessary, to remove noncellular particulate material. Cells are analyzed by
flow cytometry using 488 nm excitation, gating out doublets and clumps using
pulse processing, and measuring fluorescent emission at >620 nm.
Determination of cell viability by MTT assay
MTT assay is essentially a standard colorimetric assay (an assay which measures
changes in colour in accordance with Beer-Lambert’s law) for measuring cellular
proliferation (cell growth).
The requirement for this assay includes: a microtitre plate reader with 650–
and 570– nm filters, an inverted microscope (Fig. 9.45), a multi-channel pipette,
a C02 incubator (Fig. 9.46), a laminar flow hood, a microtitre plate (flat-
bottomed), sterile tubes (5 ml), serological pipettes and sterile pipette tips.
The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)
assay, first described by Mosmann (1983), is based on the ability of a
mitochondrial dehydrogenase enzyme present in viable cells to cleave the
tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals
which are largely impermeable to cell membranes, thus resulting in its
accumulation within healthy cells. The MTT system is a quantitative and very
sensitive test, because there is a linear relationship between cell activity and
absorbance. Thus the growth or death rate of cells can be measured easily. It
detects the mitochondrial function of dying cells during the early stages of cell
death.

Fig. 9.45 Inverted microscope (magnified view)

Fig. 9.46 C02 incubator

The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)

assay, first described by Mosmann (1983), is based on the ability of a
mitochondrial dehydrogenase enzyme present in viable cells to cleave the
tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals
which are largely impermeable to cell membranes, thus resulting in its
accumulation within healthy cells. The MTT system is a quantitative and very
sensitive test, because there is a linear relationship between cell activity and
absorbance. Thus the growth or death rate of cells can be measured easily. It
detects the mitochondrial function of dying cells during the early stages of cell
death.
 500–10,000 cells is plated in 200 μi media per well in a 96-well plate. 8 wells
are left empty for blank controls and incubated (37°C, 5% C02) overnight to
allow cells to attach to the wells. 2 μi of the drug of interest dissolved in DMSO
is added to each well. They are placed on a shaking table at 150 rpm for 5 min.,
to thoroughly mix the samples into the media. 20 μi MTT solution is added to
each well and incubated for a further 2 h at 37°C to allow for intracellular
reduction of the soluble yellow MTT to the insoluble purple foimazan crystals.
Then, 100 μi of the lyses buffer is added into each well and allowed to incubate
overnight at 37°C in order to solubilise the formazan crystals. Quantification of
cell viability is done on a micro plate reader at a wavelength of 600 nm.
The percentage cell viability is calculated using the following formula:

Alamar Blue Dye Reduction to Assess Metabolic Capability

This bioassay uses a water-soluble indicator of cytotoxicity, eliminating the need
for the washing and fixation steps needed in other assays using vital dyes (e.g.,
neutral red, MTT). As dye extraction is not done, the time needed for data
collection is decreased. Data may be collected as either fluorescence (excitation
at 530 to 560 nm; emission at 590 nm) or absorbance at 570 or 600 nm. Either
cells adhering to the plastic of microtiter plates or cells in suspension culture can
be used for this assay. Since the dye is nontoxic to the cells, the same cultures
can be monitored over a series of time points. This assay can be used for cell
proliferation determination as well as for cytotoxicity assays. Cells are harvested
in log- phase growth.
Procedure: IX 105 cells/ml cell were allowed to grow at 37°C, 5% C02. Cells
are treated with drug or vehicle control that is added to the wells. A quantity of
alamar blue equal to 10% of the culture volume (e.g., 20 pi in a well that
contains 200 μi total or 22 μi to a well containing 200 μi medium) is added, and
incubated. Fluorescence (excitation 530 to 560 nm; emission 590 nm) or
absorbance (at 570 and 600 nm) is measured in a microtiter plate reader at
desired time points. Cytotoxicity as absorbance (or fluorescence) in treated cells,
and over absorbance or fluorescence in control cells is calculated. A graph that
plots log concentration of test compound on the x axis is generated and percent
decrease in absorbance (or fluorescence) is calculated from the control on the y
axis. The dye reduction over time is compared in the wells containing test
compound and control wells.
Measuring Fluorescent Dye Loss to Assess Metabolic Activity
The acetoxymethyl ester derivative of 5-carboxyfluorescein diacetate (CFDA-
AM) is one of several membrane-permeant dyes available from Molecular
Probes that can be used to indicate membrane integrity (Haugland, 1996). For
CFDA-AM and other such dyes, membrane integrity is indicated because the
product formed upon esterase-induced cleavage will not leave the cells, if the
cell membrane is intact. The ability to leak CFDA is considered an early
indicator of cell damage. 1 X 106 cells are added to each well of a 96-well plate
and allowed to attach overnight. Cells are incubated with test compounds,
according to the experimental protocol. 100 μi of a 50 μg/ml solution of CFDA-
AM is added and incubated for 30 min at 37°C in an incubator. Fluorescence is
read using excitation at 485 nm and emission at 530 nm and wells treated with
toxicant are compared with wells containing cells without test compound or
vehicle.
Estimation of ATP levels
Cell death (especially apoptosis) is an energy-dependent process that requires
ATP. If ATP levels fall to a point where the cell can no longer perform basic
metabolic functions, the cell dies. A typical apoptotic cell exhibits a significant
decrease in ATP level. Therefore, loss of ATP level in cells has been used as an
indicator of cell death. In contrast, cell proliferation has been recognised by
increased levels of ATP.
The assay utilises bioluminescent detection of the ATP levels to rapidly screen
both apoptosis and cell proliferation in mammalian cells. Luciferase is used to
catalyse the formation of light from ATP and luciferin. The light can be
measured using a luminometer or a beta counter.
The assay can be fully automatic for high throughput (10 s/sample) and is
extremely sensitive (detects 10–100 mammalian cells). The high sensitivity of
this assay has led to many other applications for detecting ATP production in
various enzymatic reactions, as well as for detecting low level bacterial
contamination in samples such as blood, milk, urine, soil, and sludge.

9.22.4 Methodology of in vivo Cancer Screening

Agents found to have reproducible activity in the in vitro anti-cancer drug
screening are then tested in vivo. The most common models used are rats.
Models of spontaneous and chemically induced colorectal cancer (CRC)
Mouse models of spontaneous and carcinogen-induced CRC are the earliest
models described. Spontaneous formation of colorectal cancer in mice occurs in
<1% of the mice population. In order to increase the incidence of CRC, new
models were developed in which mice were exposed to carcinogens. Most
carcinogens cause malignancies in multiple organs, but there are some which
predominantly induce CRC. Frequently used carcinogens are dimethylhydrazine
(DMH) and its metabolite azoxymethane (AOM), N-methyl-N-nitro-N-
nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). The incidence
of CRC development depends on the carcinogen used, the dosage, the duration
and frequency of administration, as well as on the routing and timing of
administration. DMH and AOM can be administered via four routes: oral,
subcutaneous, intrarectal and intramuscular. The effectiveness of various
carcinogens in inducing colorectal cancer has been compared. Oral dosing of
DMH in rats results in a low tumour incidence (14–30%). Subcutaneous
administration of DMH or AOM results in a colon tumour incidence varying
from 0 to 100%. Intrarectal administration of DMH induces mild hyperplasia
and preneoplastic lesions in the colon after 34 weeks. Repeated intramuscular
injections of AOM result in 80% incidence of CRC after 12 weeks. A major
disadvantage of carcinogen-induced CRC development is the low incidences of
tumour formation, making it necessary to use a large number of animals.
Moreover, these models are not suitable for studying metastasis formation as this
occurs very slowly and infrequently. However, carcinogen-induced CRC
development in mice is ideal for studying the influence of diet on tumour
development.
Chemically induced colon cancer has been used to test the sensitivity of
tumours to chemotherapeutic agents. Seventy-one Sprague–Dawley/Wistar rats
were injected with dimethylhydrazine (20 mg/kg/s.c) once weekly for 20 weeks
to induce colon cancer. A barium enema was then performed to see the size of
the colon tumours. The animals were divided into three groups that are subjected
to the following treatments: 5-fluorouracil (5-FU); 1-(2-tetrahydrofuryl)-5 FU
(FT); and a mixture of FT and uracil (UFT). After 5 weeks of treatment, the
barium enema was repeated. “Response” was assessed on the basis of tumour
doubling time. Response rates in the 5-FU, FT, and UFT groups were 25%, 33%
and 36%, respectively. This reflects the clinical data of these drugs. The
described system may be a predictive model for screening anti-cancer drugs for
human colorectal cancer.
Models of chemically induced breast cancer
Chemical carcinogen-induced rat mammary tumorigenesis has been the model of
choice for many tests. 1-methyl-1-nitrosourea (MNU), a well- characterised
carcinogen, is used to induce adenocarcinomas in the rat’s mammary glands.
Mammary carcinogenesis is induced in female Sprague–Dawley rats by the
i.p. administration of MNU (50 mg MNU/kg body wt) at 21 days of age. Rats
are randomised to one of four dietary treatment groups:ad libitum fed or
restriction of calorie intake to 90, 80 or 60% of ad libitum intake. Energy
restriction (ER) reduces the ductal extension of the mammary gland into the fat
pad in proportion to its effect on the growth measured as body weight. However,
the reduction in ductal branching, breast density and carcinoma volume by ER is
greater than its effect on body weight. An animal’s breast density is predictive of
its carcinogenic response, irrespective of the level of ER imposed. While ER
inhibits cell proliferation and induces apoptosis in pre-malignant and malignant
mammary gland lesions, the magnitude of these effects makes it unlikely that
they fully account for the protective effects of ER against mammary
carcinogenesis.
Hollow fibre assay
This assay has the ability to provide quantitative indices of drug efficacy in
heterogeneous tumours with minimal expenditure of time and materials. It is
currently being used as the initial in vivo experience for agents found to have
reproducible activity in the in vitro anti-cancer drug screening.
Standard panels of 12 tumour cell lines are used for the routine hollow fibre
screening of the in vitro actives. These include NCI-H23, NCI-H522, MDA-MB-
231, MDA-MB-435, SW-620, COLO 205, LOX, UACC-62, OVCAR-3,
OVCAR-5, U251, SF-295 and HepG2 (Fig. 9.41). In addition, alternate lines can
be used for specialised testing of compounds on a non-routine basis. The cell
lines are cultivated in RPMI-1640 containing 10% FBS and 2 mM glutamine.
On the day preceding the hollow fibre preparation, the cells are given a
supplementation of fresh medium to maintain the log phase growth. For fibre
preparation, the cells are harvested by standard trypsinisation technique and re-
suspended at the desired cell density ((2–10 × 10 6 cells/ml). The cell suspension
is flushed into 1 mm (internal diameter) polyvinylidene fluoride hollow fibres
with a molecular weight exclusion of 500,000 Da. The hollow fibres are heat-
sealed at 2 cm intervals and the samples generated from these seals are placed
into a tissue culture medium and incubated at 37º C in 5% CO2 for 24 to 48 h
prior to implantation.

Fig. 9.47 HepG2 cell line

A total of 3 different tumour lines are prepared for each experiment so that
each mouse receives 3 intraperitoneal implants (1 of each tumour line) and 3
subcutaneous implants (1 of each tumour line). On the day of implantation,
samples of each tumour cell line preparation are quantitated for viable cell mass
by a stable end point MTT assay so that the time zero cells mass is known. Mice
are treated with experimental agents starting on day 3 or 4 following fibre
implantation and continued daily for 4 days. Each agent is administered by
intraperitoneal injection at 2 dose levels. The fibres are collected from the mice
on the day following the fourth compound treatment and subjected to the stable
end point MTT assay. The optical density of each sample is determined
spectrophotometrically at 540 nm and the mean of each treatment group is
calculated.
The per cent net growth for each cell line in each treatment group is calculated
and compared to the per cent net growth in the vehicle-treated controls. A 50%
or greater reduction in per cent net growth in the treated samples compared to
the vehicle control samples is considered a positive result. Each positive result is
given a score of 2 and all the scores are added for a given compound. The
maximum possible score for an agent is 96 (12 cell lines × 2 sites × 2 dose levels
× 2 [score]). A compound is referred for xenograft testing if it has a combined i.p
+ s.c score of 20 or greater, an s.c score of 8 or greater, or produces cell mortality
of any cell line at either dose level evaluated.
Xenograft Tumour Models
Xenograft tumour models are generated in immunodeficient mice following the
implantation of tumour cells or tumour tissue into ectopic (e.g. subcutis, renal
capsule) or orthotopic sites. The commonly stated advantages of tumour
xenografts are the ease of model generation and the fact that therapeutic
assessment occurs in human cancer tissue as opposed to tissues of another
species. Further, patient-specific xenografts have recently been described as a
means to develop personalized therapies for some malignancies.
Xenograft tumours are generally established by the subcutaneous (s.c.)
inoculation of tumour cells into nude mice (1.0 * 107 cells per mouse). Growth
of solid tumours is monitored using in situ caliper measurements and models
may be at an advanced stage or an early stage. Generally, activity is defined by
tumour growth delay, optimal % T/C (T/C – median- treated tumour
mass/median control tumour mass) or net log cell kill. Drug-related deaths and
body weight loss are used as parameters of toxicity. Many compounds have
shown promising activity in s.c. xenograft models, and have progressed to
clinical studies. It is anticipated that the s.c. xenograft model will still be of
value in this modem era of target- driven anticancer dmg discovery, if used
appropriately. There is increasing appreciation of the fact that xenograft models
should be characterized to ensure that the molecular drug target is expressed and
that xenograft studies should integrate both pharmacokinetic and
pharmacodynamic investigation.
Orthotopic and Metastasis Tumour Models
Orthotopic means “the transfer of living organs or tissue from one part of the
body to another”. Compounds are usually screened against a panel of poorly
characterized human tumour xenografts implanted s.c. in nude mice. These s.c.
tumour models do not represent the primary site of common human cancers or
sites of metastasis. It has been suggested that the disparity between preclinical
and clinical activity is related to the treatment of advanced metastatic disease in
the clinic, whereas conventional s.c. xenograft models do not represent advanced
metastatic disease nor is the orthotopic site represented. Orthotopic
transplantation models attempt to mimic the morphology and growth
characteristics of clinical disease and are thought to represent a more clinically
relevant tumour model with respect to tumour site and metastasis. One of the
most obvious advantages of orthotopic systems is that attempts to target
processes involved in local invasion (e.g., angiogenesis) can be carried out in a
more clinically relevant site. Several other models are also used to assess the
antiangiogenic properties of novel agents (e.g., corneal micropocket assay,
sponge implant assay). Since the early studies showing orthotopic
transplantation of colon tumours and metastasis to the liver, tumour material has
been grown orthotopically in mice at most common sites of human cancer.
Whether preclinical models representative of clinical disease (e.g., orthotopic/
metastatic models) should be employed as a replacement for traditional s.c.
nonmetastatic xenografts is an interesting question.
Autochthonous Models
Autochthonous tumours include spontaneously occurring tumours and induced
tumour growth (e.g. by chemical, viral, or physical carcinogens). It is thought
that autochthonous tumours may mimic human tumours more closely than
transplanted tumours (i.e., s.c./ orthotopic). Advantageous properties include
orthotopical growth, tumour histology devoid of changes introduced by
transplantation, and a route of metastasis through the lymph and blood vessels
that surround early tumour growth. Despite such properties, the use of
autochthonous tumour models has not been widespread due to several
limitations. A large variability in take rate and growth exists; as apart from the
large number of animals needed, time frames of several months to years exist for
a single experiment due to long tumour latencies as opposed to weeks in
transplanted xenograft models, and there is lack of spontaneous metastasis.
Autochthonous models are used occasionally as tools for advanced or phase II
screening, but more recently, in this “postgenome era”, autochthonous models
have largely been replaced by genetically engineered mouse (GEM) models.
Genetically Engineered Cancer Models (GEM)
GEM models that accurately model human cancer, at both the molecular and
phenotypic levels, are the new tools that are available for experimental
therapeutic studies. Genetically engineered mouse (GEM) models are a
promising alterative to xenograft models for biological and therapeutic
investigations. GEM models are generated through the introduction of genetic
mutations associated with particular human malignancies. Such mutant genes
may be gain-of-function oncogenes or loss-of-function tumour suppressor alleles
that are either constitutively or conditionally expressed in mouse models. Over
the past 20 years, GEM models have made a significant contribution to the field
of cancer research. GEM models have increased our understanding of the
molecular pathways responsible for the initiation and progression of human
cancer, and have highlighted the importance of specific oncogenes and tumour
suppressor genes (TSG) in particular types of cancer. GEM models possess well-
validated molecular/genetic characteristics (e.g., gene mutations), which
ultimately facilitate the rational design of small molecule therapeutics. The main
aim of GEM models is to recapitulate genetic/molecular changes in human
cancer and use these to test novel anticancer therapeutics in an attempt to
accurately predict the clinical response. The first strains of genetically
engineered mice predisposed to cancer were transgenic mouse models whereby
cellular/viral oncogenes were introduced to the mouse germ line. One of the first
transgenic cancer models involved the constitutive expression of the c-myc
oncogene under the control of the mouse mammary tumour virus promoter
leading to the development of mammary tumours. Many transgenic experiments
have followed and clearly shown that the manipulation of the mouse germ line
could predispose the mice to cancer. The most common human cancer, basal cell
carcinoma, has also been modelled by the exposure of Ptch heterozygous mice to
UV light. Etiologic factors such as diet are also being modified and have been
shown to affect tumour development in GEM models. Transgenic and knockout
mouse models involving manipulation of the mouse germ line are often limited
by embryonic ethality. In an attempt to overcome this, several new approaches to
create GEM strains have been introduced, including transient conditional gene
targeting, latent oncogenes, inducible oncogene expression and the use of avian
sarcoma leucosis viruses, and chromosome engineering. These novel methods
have been facilitated by recent technological advances [e.g., cytogenetic
(spectral karyotyping)], genomic comparative genomic hybridization,
comparative genomic hybridization microarrays and gene expression profiling.

9 .2 3 SCREENING METHODS FOR EVALUATION OF

HEPATOPROTECTIVE AGENTS
A toxic or repeated dose of a known hepatotoxin is administered to induce liver
damage in experimental animals. The test substance is administered prior to or
after the toxin treatment. If the hepatotoxicity is prevented or reduced, the test
substance is effective. There are various models of inducing hepatotoxicity in
rodents (rats and mice).
Hepatitis in Long–Evans Cinnamon rats
The Long–Evans Cinnamon strain of rats has been recommended as a useful
model to study genetically transmitted hepatitis and chronic liver disease. It has
been speculated that this strain of rats is prone to liver diseases due to excessive
copper accumulation in the liver.
Long–Evans Cinnamon rats are housed in temperature- and humidity-
controlled rooms at a 12: 12 light/dark cycle. Groups of 6–10 rats are given
different diets based on a 15% purified egg protein diet and supplemented with
vitamins or drugs. Drugs are applied via mini pumps intraperitoneally implanted
under ether anaesthesia. The occurrence of jaundice is easily observable as the
time when the ears and tail turn yellow and the urine becomes bright orange,
staining the fur in the lower abdominal region. Usually, the jaundice
progressively worsens, ending in death of the animal within about a week.
Incidence of jaundice and mortality vs. time are used as parameters to measure
the extent of hepatoprotective activity.
Allyl alcohol induced liver necrosis in rats
In this method allyl alcohol is used as a liver necrosis inducing agent in rats.
Albino rats weighing 120–150 g are used. On the first day, food but not water
is withdrawn. After 6 h, the compounds to be tested for protective activity are
administered i.p or orally. One hour later, the animals are dosed orally with 0.4
ml/kg of a 1.25% solution of allyl alcohol in water. Next morning, the treatment
with the potentially protective drugs is repeated. Food but not water is withheld
until the third day. Next morning, the animals are sacrificed and the liver is
removed. The parietal sides of the liver are checked using a stereomicroscope
with 25 times magnification. Focal necrosis is observed as white-green or
yellowish haemorrhagic areas clearly separated from unaffected tissue. The
diameter of the necrotic areas is determined using an ocularmicrometer. These
values are added for each animal to obtain an index for necrosis.
Carbon tetrachloride induced liver fibrosis in rats
Chronic administration of carbon tetrachloride to rats induces severe
disturbances of hepatic function together with histologically observable liver
fibrosis. This model is used for the screening of hepatoprotective agents.
Albino rats are treated orally twice a week with 1 mg/kg carbon tetrachloride,
dissolved in olive oil 1:1, over a period of 8 weeks. The animals are kept under
standard conditions (day/ night rhythm: 8:00 AM to 8:00 PM; 22°C room
temperature; standard diet; water ad libitum). Twenty animals serve as controls
receiving only olive oil; 40–60 animals receive only the carbon tetrachloride.
Groups of 20 rats receive in addition to carbon tetrachloride, the compound
under investigation in various doses by gavage twice daily (with the exception of
the weekends, when only one dose is given) on the basis of the actual body
weight. The animals are weighed weekly. At the end of the experiment (8
weeks), the animals are anaesthetised and exsanguinated through the caval vein.
The serum is analysed for parameters like total bilirubin, total bile acids, 7 S
fragment of type IV collagen, procollagen III N-peptide. The liver, kidney, aortic
wall and tail tendons are prepared for determination of hydroxyproline. They are
weighed and completely hydrolysed in 6 N HC1. Hydroxyproline is measured by
HPLC and expressed as mg/mg wet weight of the organs.
For histological analysis, 3–5 pieces of the liver weighing about 1 g are fixed
in formalin and Camoy solution. Three to five sections of each liver are
embedded, cut and stained with azocarmine aniline blue (AZAN) and evaluated
for the development of fibrosis using a score of 0–IV.

Grade Normal liver histology

0:

Grade Tiny and short septa of connective tissue without influence on the
I: structure of the hepatic lobules

Grade Large septa of connective tissue, flowing together and penetrating

II: into the parenchyma; tendency to develop nodules

Grade Nodular transformation of the liver architecture with loss of the

III: structure of the hepatic lobules

Grade Excessive formation and deposition of connective tissue with

IV: subdivision of the regenerating lobules and development of scars

The values of all the parameters of the test group are compared with the
control group using suitable statistical methods.
Bile duct ligation induced liver fibrosis in rats
Ligation of the bile duct in rats induces liver fibrosis, which can be evaluated
histologically and by determination of serum collagen parameters. This model is
used for the screening of hepatoprotective agents.
Albino rats are anaesthetised and laparotomy is performed under aseptic
conditions. A mid-line incision in the abdomen is made from the xiphostemum
to the pubis, exposing the muscle layers and the linea alba, which is then incised
over a length corresponding to the skin incision. The edge of the liver is then
raised and the duodenum pulled down to expose the common bile duct, which
pursues an almost straight course of about 3 cm from the hilum of the liver to its
opening into the duodenum. There is no gall bladder, and the duct is embedded
for the greater part of its length in the pancreas, which opens into it by numerous
small ducts. A blunt aneurysm needle is passed under the part of the duct
selected, the pancreas is stripped away with care, and the duct is double ligatured
with cotton thread.
The peritoneum and the muscle layers as well as the skin wound are closed
with cotton stitches. The animals receive normal diet and water ad libitum
throughout the experiment. Groups of 5–10 animals receive the test compound in
various doses or the vehicle twice daily for 6 weeks. They are then sacrificed
and the blood harvested for determination of bile acids, 7 S fragment of type IV
collagen, and procollagen III N-peptide. The liver is used for histological studies
and for hydroxyproline determinations. Control animals show excessive bile
duct proliferation as well as formation of fibrous septa. This is consistent with
complete biliary cirrhosis. The value of the test is compared with the control
using suitable statistical analysis.
Galactosamine induced liver necrosis
A single dose or a few repeated doses of D-galactosamine causes acute hepatic
necrosis in rats. Prolonged administration leads to cirrhosis. This model is used
for the screening of hepatoprotective agents.
Adult albino rats weighing 110–180 g are injected intraperitoneally three
times weekly with 500 mg/kg D-galactosamine over a period of one to 3 months.
The test substances are administered orally with food or by gavage. The control
group receives only vehicle or food without drugs. The rats are sacrificed at
various time intervals and the livers excised and evaluated by light microscopy
and immunohistology using antibodies against macrophages, lymphocytes and
the extracellular matrix component.
Country made liquor (CML) model
Country made liquor (containing 28.5% alcohol) is used to produce
hepatotoxicity in this model. CML is administered orally at a dose of 3 ml/100
g/day for 30 days, which results in severe fatty changes in liver.
Rats are divided into groups of 8 each. The control group receives 1% gum
acacia as vehicle, corn oil (1 ml/100 g/day) and glucose isocaloric to the amount
of alcohol. The positive control group receives CML (3 ml/100 mg/day) in two
divided doses and corn oil (1 ml/100 g/day) in a single dose. Other test groups
receive drugs in respective doses along with CML (3 ml/100 g/day) and corn oil.
After 21 days, the blood is withdrawn for analysis of SGOT, SGPT, alkaline
phosphatase, serum cholesterol, albumin, total proteins, bilimbin, glucose, and
creatinine. The rats are sacrificed and the livers dissected out for
histopathological analysis. The value of the test is compared with the control
using suitable statistical analysis.
Paracetamol model
This model is used to produce experimental liver damage only in mice, since rats
are resistant to paracetamol-induced hepatotoxicitiy. Paracetamol administered
orally as a single dose of 500 mg/kg in mice produces hepatotoxicity.
Adult albino mice are used for the study. Paracetamol is administered as a
single dose of 500 mg/kg. After 48 h, they are treated with the test drugs for 5
days. At the end of the experiment, blood is withdrawn for biochemical analysis
of SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total
proteins, bilirubin, glucose, and creatinine. The liver is subjected to
histopathological studies. The value of the test is compared with the control
using suitable statistical analysis.
Partial hepatectomy model
In this method partial hepatectomy (removal of 70% of liver mass) is done and
the action of drugs on the re-generation of liver cells studied. Hepatoprotective
agents improve the re​generation capability of liver.
Rats were used for this study as they can withstand surgical infections better
than mice. They are anaesthetised using light ether anaesthesia. A median line
incision reaching 3–4 mm posteriorly from the xiphoid process of the sternum is
done and the large median lobe of the liver with the left lateral lobe taken out.
These lobes are ligated by coarse linen and excised. Around 68 ± 2% of the total
hepatic parenchyma is also removed. The peritoneum is closed using absorbable
suture and the integument closed using non​absorbable surgical suture. Various
hepatoprotective drugs can be screened for their activity using these
hepatectomised rats.
At the end of the screening experiments, the blood is collected for analysis of
the serum. The following parameters are determined: SGOT, SGPT, alkaline
phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and
creatinine. The animals are sacrificed. The liver is excised out, weighed and
subjected to histopathological evaluation. The value of the test is compared with
the control using suitable statistical analysis.

9.24 SCREENING METHODS FOR ANTI-INFLAMMATORY DRUGS

WHO has identified 2000–2010 as the decade for musculoskeletal disorders.
Herbal drugs like holy basil (tulsi; Ocimum sanctum), turmeric (Curcuma longa),
Indian olibanum tree (Boswellia serrata), ginger etc are widely used for the
treatment of various inflammatory disorders. They are not only found to be safer
and have fewer side-effects, but they also cover a large domain of mechanisms
involved in inflammation thus proving to be more beneficial than synthetic
drugs. Inflammation expresses the response to damage of cells and vascular
tissues. The five basic symptoms of inflammation— redness, swelling, heat, pain
and deranged function—have been known since the ancient Greek and Roman
era.
The major events occurring during this response are an increased blood supply
to the affected tissue by vasodilation, increased capillary permeability caused by
retraction of the endothelial cells which allows the soluble mediators of
immunity to reach the site of inflammation and leukocytes migration out of the
capillaries into the surrounding tissues. Neutrophils, monocytes and lymphocytes
also migrate towards the site of infection.The development of inflammatory
reactions is controlled by the following systems: cytokines, complement, kinin
and fibrinolytic pathways; by lipid mediators (prostaglandins and leukotrienes)
released from different cells; and by vasoactive mediators released from mast
cells, basophils and platelets.
The response is accompanied by the clinical signs of erythema, edema,
hyperalgesia and pain. Inflammatory responses occur in three distinct phases,
each apparently mediated by different mechanisms:
Acute, transient phase: Characterised by local vasodilatation and increased
capillary permeability
Sub-acute phase: Characterised by infiltration of leukocytes and phagocytic cells
Chronic proliferative phase: Tissue degeneration and fibrosis occur
Drugs preventing acute and sub-acute inflammation can be tested using the
following models: paw edema in rats, croton oil ear edema, pleurisy tests, UV-
erythema in guinea pigs, oxazolone-induced ear edema in mice, granuloma
pouch technique and vascular permeability. The effectiveness of drugs which
work at the proliferative phase can be measured by methods for testing
granuloma formation, such as the cotton pellet granuloma, adjuvant induced
arthritis, glass rod granuloma and PVC sponge granuloma.

9.24.1 Testing of Drugs Preventing Acute and Sub-acute

Inflammation
Paw edema
This technique is based upon the ability of anti-inflammatory agents to inhibit
the edema produced in the hind paw of the rat after injection of a phlogistic
agent (irritant). Rats with a body weight between 100–150 g are required. Many
irritants have been used, such as brewer’s yeast, formaldehyde, dextran, egg
albumin, kaolin, Aerosil® and sulphated polysaccharides like carrageenan. The
animals are fasted overnight. The control rats receive distilled water while the
test animals receive drug suspension orally. Thirty minutes later, the rats are
subcutaneously injected with 0.1 ml of 1% solution of carrageenan in the foot
pad of the left hind paw. The paw is marked with ink and immersed in the water
cell of a plethysmometer (Fig. 9.48) up to this mark. The paw volume is
measured plethysmographically immediately after injection, 3 and 6 h after
injection, and eventually 24 h after injection. The paw volumes for the control
group are then compared with those of the test group.
Croton oil ear edema in rats and mice
This method mainly evaluates the anti-phlogistic activity of topically applied
steroids.

Fig. 9.48 Digital plethysmograph

Mice (22 g) or rats (70 g) are required. For tests in mice, the irritant is
composed of (v/ v): 1 part croton oil, 10 parts ethanol, 20 parts pyridine and 69
parts ethyl ether; for rats the irritant is composed of (v/v): 4 parts croton oil, 10
parts ethanol, 20 parts pyridine and 66 parts ethyl ether. The standard and the test
compound are dissolved in this solution. Irritants are applied on both sides of the
right ear (0.01 ml in mice or 0.02 ml in rats under ether anaesthesia). Controls
receive only the irritant solvent. The left ear remains untreated. Four hours after
application, the animals are sacrificed under anaesthesia. Both ears are removed
and discs of 8 mm diameter are cut. The discs are weighed immediately and the
weight difference between the treated and untreated ear is recorded indicating
the degree of inflammatory edema.
Pleurisy test
Pleurisy is the phenomenon of exudative inflammation in man. In experimental
animals, pleurisy can be induced by several irritants, such as carrageenan,
histamine, bradykinin, prostaglandins, mast cell degranulators and dextran.
Leukocyte migration and various biochemical parameters involved in the
inflammatory response can be measured easily in the exudate.
Male rats weighing 220–260 g are required. The animal is lightly
anaesthetised with ether and placed on its back. The hair from the skin over the
ribs on the right side is removed and the region cleaned with alcohol. A small
incision is made into the skin under the right arm. The wound is opened and 0.1
ml of 2% carrageenan solution is injected into the pleural cavity through this
incision. The wound is closed with a clip. One hour before this injection and 24
and 48 h thereafter, rats are treated (subcutaneously or orally) with the standard
or the test compound. A control group receives only the vehicle. The animals are
sacrificed 72 h after carrageenan injection and pinned on a dissection board with
the forelimbs fully extended. One ml of heparinised Hank’s solution is injected
into the pleural cavity through an incision. The cavity is gently massaged to mix
its contents. The fluid is aspirated out of the cavity using a pipette. The aspirated
exudates are collected in a graduated plastic tube. One ml (the added Hank’s
solution) is subtracted from the measured volume. The values of each
experimental group are averaged and compared with the control group. The
white blood cell number in the exudate is measured using a Coulter counter or a
haematocytometer.
Ultraviolet erythema in guinea pigs
Anti-inflammatory agents delay the development of ultraviolet erythema on
albino guinea pigs. They are shaved on the back 18 h before testing. The test
compound is suspended in the vehicle and half the dose of the test compound is
administered orally 30 min. before ultraviolet exposure. Control animals are
treated with the vehicle alone. The guinea pigs are placed in a leather cuff with a
hole of 1.5–2.5 cm size punched in it, allowing the ultraviolet radiation to reach
only this area. An ultraviolet burner is warmed up for about 30 min. prior to use
and placed at a constant distance (20 cm) above the animal. Following a 2 min.
ultraviolet exposure, the remaining half of the test compound is administered.
The erythema is scored 2 h and 4 h after exposure.
Oxazolone-induced ear edema in mice
The oxazolone-induced ear edema in mice is a model of delayed contact
hypersensitivity that permits the quantitative evaluation of the topical and
systemic anti-inflammatory activity of a compound following topical
administration.
Mice of either sex (25 g) are required. A fresh 2% solution of oxazolone in
acetone is prepared. This solution (0.01 ml) is injected on the inside of both ears
under anaesthesia. The mice are injected 8 days later, again under anaesthesia,
with 0.01 ml of 2% oxazolone solu​tion (control) or 0.01 ml of oxazolone
solution in which the test compound or the standard is dissolved, on the inside of
the right ear. The left ear remains untreated. The maximum of inflammation
occurs 24 h later. At this time the animals are sacrificed under anaesthesia and a
disc of 8 mm diameter is punched from both ears. The discs are immediately
weighed on a balance. The weight difference is an indicator of the inflammatory
edema.
Granuloma pouch technique
Irritants such as croton oil or carrageenan produce aseptic inflammation resulting
in large volumes of exudate, which resembles the sub-acute type of
inflammation. Rats (150–200 g) are selected for the study; the back of the
animals is shaved and disinfected. With a very thin needle, an air pouch is made
by injection of 20 ml of air under ether anaesthesia. Into the resulting air pouch
0.5 ml of a 1% solution of croton oil in sesame oil is injected. 48 h later the air is
withdrawn from the pouch and 72 h later any resulting adhesions are broken.
Instead of croton oil, 1 ml of a 20% suspension of carrageenan in sesame oil can
be used as irritant. Starting with the formation of the pouch, the animals are
treated every day either orally or subcutaneously with the test compound or the
standard. On the 5th day, the animals are sacrificed under anaesthesia. The pouch
is opened and the exudate collected in glass cylinders. The average value of the
exudate of the controls and the test groups is calculated.
Vascular permeability
This test is used to evaluate the inhibitory activity of drugs against increased
vascular permeability, which is induced by a phlogistic substance. Mediators of
inflammation, such as histamine, prostaglandins and leucotrienes are released
following stimulation e.g. of mast cells. This leads to a dilation of arterioles and
venules and to an increased vascular permeability. As a consequence, fluid and
plasma proteins are released and edemas are formed. Vascular permeability is
increased by subcutaneous injection of the mast cell- degranulating compound
48/80. The increase of permeability can be recognised by the infiltration of the
injected sites of the skin with the dye Evan’s blue.
Male rats (160 and 200 g) are used. 5 ml/kg of 1% solution of Evan’s blue are
injected intravenously. One hour later the animals are dosed with the test
compound orally or intraperitoneally. 30 min. later, the animals are lightly
anaesthetised with ether and 0.05 ml of 0.01% solution of compound 48/80 is
injected subcutaneously at 3 sites. 90 min. after the injection of compound
48/80, the animals are sacrificed by ether anaesthesia. The abdominal skin is
removed and the dye-infiltrated areas of the skin measured. The per cent
inhibition in the treated animals as compared to the control group is calculated.

9.24.2 Testing of Drugs Preventing the Proliferative Phase

(Granuloma formation) of Inflammation
Cotton pellet granuloma
Foreign body granulomas are induced in rats by the subcutaneous implantation
of pellets of compressed cotton. After several days, histologically giant cells and
undifferentiated connective tissue can be observed besides fluid infiltration. The
amount of newly formed connective tissue can be measured by weighing the
dried pellets after removal. More intensive granuloma formation has been
observed if the cotton pellets are impregnated with carrageenan.
Male and female rats with an average weight of 200 g are used. The back skin
is shaved and disinfected with 70% ethanol. An incision is made in the lumbar or
neck region. Subcutaneous tunnels are formed and a sterilised cotton pellet is
placed with the help of a blunted forceps. The animals are treated for 7 days
subcutaneously or orally. They are then sacrificed, the pellets taken out and
dried. The net dry weight, i.e. after subtracting the weight of the cotton pellet is
determined. The average weight of the pellets of the control group as well as that
of the test group is calculated. The per cent change of granuloma weight relative
to the vehicle control group is determined.
Adjuvant arthritis in rats
Adjuvant induced arthritis in rats exhibit many similarities to human rheumatoid
arthritis. An injection of complete Freund’s adjuvant into the rat’s paw induces
inflammation as a primary lesion with a maximum inflammation after 3 to 5
days. Secondary lesions occur after a delay of approximately 11 to 12 days and
are characterised by inflammation of non-injected sites (hind legs, forepaws,
ears, nose and tail), a decrease in weight and immune responses.
Male rats with an initial body weight of 130 to 200 g are used. On day 1, rats
are injected in the sub-plantar region of the left hind paw with 0.1 ml of
complete Freund’s adjuvant. The adjuvant consists of 6 mg mycobacterium
butyricum thoroughly ground with a mortar and pestle and suspended in heavy
paraffin oil (Merck) to give a concentration of 6 mg/ml. Dosing with the test
compounds or the standard is started on the same day and continued for 12 days.
Both paw volumes and body weight are recorded on the day of injection. The
paw volume is measured plethysmographically with equipment as described in
the paw edema tests. On day 5, the volume of the injected paw is measured
again, indicating the primary lesion and the influence of therapeutic agents on
this phase. The severity of the induced adjuvant disease is determined by
measuring the non-injected paw (secondary lesions) with a plethysmometer. The
animals are not dosed with the test compound or the standard from day 13 to 21.
On day 21, the body weight is determined again and the severity of the
secondary lesions evaluated visually and graded according to the following
scheme:
Score

ears: absence of nodules and redness................................ 0

presence of nodules and redness.............................. 1

nose: no swelling of connective tissue............................... 0

intensive swelling of connective tissue ............................... 1

tail: absence of nodules...................................................... 0

presence of nodules .................................................. 1

forepaws: absence of inflammation............................................ 0

inflammation of at least 1 joint................................. 1
hind paws: absence of inflammation............................................ 0
slight inflammation................................................... 1
moderate inflammation.............................................. 2
marked inflammation................................................. 3
Sponge implantation technique
Foreign body granulomas are induced in rats by subcutaneous implantation of a
sponge. Sponges used for implantation are prepared from polyvinyl foam sheets
(thickness: 5 mm). Discs are punched out to a standard size and weight (10.0 ±
0.02 mg). The sponges are then soaked in 70% v/v ethanol for 30 min., rinsed
four times with distilled water and heated at 80°C for 2 h. Prior to implantation
in the animal, the sponges are soaked in sterile 0.9% saline in which either
drugs, antigens or irritants have been suspended. Typical examples include 1%
carrageenan, 1% yeast, 1% zymosan A, 6% dextran, heat- killed Bordetella
pertussis or 0.5% heat-killed Mycobacterium tuberculosis.
Sponges are implanted in rats weighing 150–200 g under ether anaesthesia.
An incision is made and separate cavities are formed into which sponges are
inserted. Up to 8 sponges may be implanted per rat. The incision is closed with
Michel clips (Fig. 9.49) and the animals maintained at a constant temperature of
24°C. For short-term experiments, the animals are treated with the test drug or
standard once before implantation orally or subcutaneously. For long-term
experiments, the rats are treated daily up to 3 weeks.
Glass rod granuloma
Glass rod induced granulomas reflect the chronic proliferative phase of
inflammation. Of the newly formed connective tissue, not only can the wet and
dry weight be measured, but also the chemical composition and mechanical
properties. Glass rods with a diameter of 6 mm are cut to a length of 40 mm and
the ends rounded off. They are sterilised before implantation. Rats are
anaesthetised with ether, the back skin shaved and disinfected. From an incision
in the back region, a subcutaneous tunnel is formed with a blunted forceps. A
glass rod is introduced into this tunnel. The incision wound is closed by sutures.
The animals are kept in separate cages. The rods remain in situ for 20 or 40 days.
Animals are treated orally. At the end of 20 days the animals are sacrificed. The
glass rods are removed together with the surrounding connective tissue, which
forms a tube around the glass rod. By incision at one end, the glass rod is
extracted and the granuloma sac inverted forming a plain piece of pure
connective tissue. Wet weight of the granuloma tissue is recorded. The
specimens are kept in a humid chamber until further analysis. Biochemical
analyses, such as determination of collagen and glycosaminoglycans, can also be
performed.

Fig. 9.49 Wound clips

9.25 SCREENING METHODS FOR ANTI-ULCER DRUGS

An ulcer is a local defect, or excavation of the surface of an organ or tissue,
which is produced by the sloughing of inflammatory necrotic tissue. The term
“peptic ulcer” refers to a group of ulcerative disorders of the upper GIT, which
appears to have in common, the participation of acid pepsin in their
pathogenesis. A peptic ulcer probably results due to an imbalance between
aggressive (acid, pepsin and H. pylori) and defensive (gastric mucous, and
bicarbonate secretion, prostaglandins, innate resistance of the mucosal cells)
factors. In gastric ulcers acid secretion is normal or low.
There are various methods for screening anti-ulcer drugs:

1. Pylorus ligation in rats

2. Stress ulcer through immobilisation stress
3. Stress ulcers by cold water immersion
4. Indomethacin induced ulcers in rats
5. Ethanol induced mucosal damage in rats (cytoprotective activity)

Pylorus ligation in rats (Shay et al. 1945)

The principle is based on ulceration induced by accumulation of acidic gastric
juice in the stomach.
The requirements include: stereo microscope, adult albino rats (150–170 g),
anaesthetic ether, plastic cylinder, 0.1 N sodium hydroxide.
Adult albino rats weighing 150–170 g are starved for 48 h although they have
access to drinking water. Normally ten animals are used per dose and as control.
After they are ether anaesthetised, a mid-line abdominal incision is made and the
pylorus ligated. The abdominal wall is closed by sutures and test compounds are
given either orally by gavage or injected subcutaneously. The animals are placed
for 19 h in plastic cylinders with an inner diameter of 45 mm being closed on
both ends by a wire mesh. The animals are then sacrificed using carbon dioxide
anaesthesia. The abdomen is opened and a ligature is placed around the
aesophagus close to the diaphragm. The stomach is removed and the contents
drained into a centrifuge tube. Along the greater curvature, the stomach is
opened and pinned onto a cork plate. The mucosa is then examined with a stereo
microscope.
The evaluation is done by counting the numbers of ulcers and the severity
graded according to the following scores:
0: no ulcers; 1: superficial ulcers; 2: deep ulcers; 3: perforations
The volume of gastric content is measured after centrifugation. Acidity is
determined by titration with 0.1 N NaOH. Ulcer index U1 is calculated using the
following formula:
U1=Un+Us + Up × 10-1
where Un = the average of the number of ulcers per animal.
Us = average of severity score
Up= percentage of animals with ulcers.
Ulcers through immobilisation stress
The principle behind this method involves psychogenic factors, such as stress,
which play a major role in the pathogenesis of gastric ulcers in man.
The requirements include: adult albino rats, anaesthetic ether, C02 anaesthesia,
and a stereo microscope.
A group of 10 adult albino rats (150–170 g) per dose of the test drug and for
controls are used. Food and water are withdrawn 24 h before the experiment.
After oral and subcutaneous administration of the test compound or a placebo
solution, the animals are slightly anaesthetised with ether. Both the upper and
lower extremities are fixed together and the animals wrapped in wire gauge.
They are horizontally suspended in the dark at 20°C for 24 h and finally
sacrificed using carbon dioxide anaesthesia. The stomach is removed, fixed on a
cork plate and the number and severity of the ulcers registered with a stereo
microscope.
Stress ulcer by cold water immersions
The principle behind this assay is that cooling the rats in water when they are
restrained according to the previous model accelerates the occurrence of gastric
ulcers and shortens the time of necessary immobilisation. In this model, the
gastric ulcer formation is mainly due to gastric hypermotility, which could lead
to mucosal over-friction.
The requirements include: Wistar rats, cages, Evans blue dye, 2% formol
saline, C02 anaesthesia, and a magnifier.
Groups of 8–10 Wistar rats weighing 150–200 g are used. After oral
administration of the test compounds, the rats are placed vertically in individual
restraint cages in water at 22°C for 1 h. They are then removed, dried and
injected intravenously through a tail vein with 30 mg/kg Evans blue. 10 min later
they are sacrificed using C02 anaesthesia and their stomachs removed. Formol
saline (2% v/v) is then injected into the totally ligated stomachs for storage
overnight. The next day, the stomachs are opened along the greatest curvature,
washed in warm water and examined under a three-fold magnifier.
The evaluation is done by measuring the lengths of the longest diameter of the
lesions. This is summated to give a total lesion score (in mm) for each animal;
the mean count in control rats should be about 25 (range 20–28). Inhibition of
the lesion production is expressed as a percentage value.
Indomethacin induced ulcers in rats
This assay is based on the fact that use of non-steroidal anti- inflammatory
agents like indomethacin and acetyl salicylic acid induces gastric lesions in man
and in experimental animals by inhibition of gastric cyclo- oxygenase.
The requirements include: Wistar rats, 0.1% tween 80, 2% formol saline and
C02 anaesthesia.
Groups of 8–10 Wistar rats weighing 150–200 g are used. The test drugs are
administered orally in 0.1% tween 80 solutions, 10 min. prior to an oral
administration of indomethacin (20 mg/kg). 6 h later, the rats are sacrificed in
C02 anaesthesia and their stomachs removed. Formol saline (2% v/v) is then
injected into the totally ligated stomachs for storage overnight. The next day, the
stomach are opened along the greater curvature, washed with warm water and
examined.
The mean score is calculated in control rats. It should be about 25. Inhibition
of the lesion production is expressed as a percentage value.
Ethanol induced mucosal damage in rats (cytoprotective activity)
The principle behind this method is that intragastric application of absolute
ethanol, which is a reproducible method, produces gastric lesions. The lesions
can be inhibited by drugs with cytoprotective activity.
The requirements include: adult albino rats, absolute ethanol, C02 anaesthesia,
Plexi glass template, densitometer, camera, an Aristo model T-16 cold cathode
transilluminator.
Adult albino rats weighing 250–300 g are deprived of food 18 h prior to the
test although water is given. The rats are administered the cytoprotective drugs
intragastrally 30 min. prior to administration of 1 ml absolute ethanol. Untreated
animals are taken as control. 1 h after administration of ethanol, the animals are
euthanised with C02 and the stomachs excised, cut along the greater curvature
and rinsed with tap water. The stomachs are stretched on a piece of foam core
mat. The subjective scores of the treated tissue are recorded; the response is
graded from the least (0) to the most (3) damaged. Using a cork borer, a circular
area about 13 mm in diameter is cut from each lobe of the fundus just below the
ridge dividing the glandular from the non-glandular portion of the stomach. The
excised pairs of tissue from each stomach are placed into the holes of the
template. Pairs of tissue from each stomach are examined to minimise sampling
errors. The template is positioned on a rectangular central open area of an Aristo
model T-16 cold cathode transilluminator (38 x 38 cm) containing a W-45 blue-
white lamp. A camera is mounted on a copy stand directly above the template.
Photographs are taken. The film is pressed in a standard manner and the contact
sheet made from the negatives. A light transmission densitometer is used to
evaluate the negatives and the optical density determined.
The evaluation is done by observing haemorrhagic or damaged areas that
appears bright on the negatives, whereas undamaged tissues appear dark
showing greater optical density.
The significance of the differences in optical density between ethanol-treated
tissue and control is evaluated by non-paired single tail student’s t-test.

9.26 SCREENING OF ANTI-ASTHMATIC DRUGS

Asthma is primarily an inflammatory condition characterised by hyper-
responsiveness of the bronchial smooth muscle to a variety of stimuli, resulting
in the narrowing of air tubes which leads to reversible airway obstruction. It is
usually accompanied by increased secretion, mucosal edema and mucous
plugging. The mucous membrane and muscle layers of the bronchi thicken and
the mucous glands enlarge reducing airflow in the lower respiratory tract. During
the asthmatic attack, the spasmodic contraction of the bronchial muscle
constricts the artery and there is excessive secretion of thick sticky mucous
which further reduces the airways. The lungs become hyper inflated and there is
severe dyspnoea and wheezing. The duration of the attack varies from a few
min. to h.
Anti-asthmatic drugs can be evaluated by different techniques.
Autocoid induced bronchoconstriction
This assay is based on the fact that autocoids like histamine and leukotrienes
induce bronchoconstriction. Histamine is an important mediator of immediate
allergic and inflammatory reactions. It causes bronchoconstriction by activating
H receptors. Calcium ionophores induce the release of leukotrienes via the 5-
lipoxygenease pathway. Leukotrienes are powerful bronchoconstrictors that
appear to act on smooth muscles via specific receptors. This is used to detect the
H and leukotriene receptor blocker properties of test compounds.
In vivo models using isolated lungs of guinea pigs
In this model, the drugs samples are evaluated for their capacity of inhibiting
bronchospasm induced by histamine or calcium ionophore.
Albino guinea pigs of either sex weighing 300 to 450 g are used for the
experiment. The animals are sacrificed with an overdose of ether. The lungs are
removed by opening the chest. They are cut into strips of 5 cm each and
transferred into a physiological solution. The isolated tissues are mounted in an
organ bath containing a nutritive solution (the nutritive solution contains
anhydrous salts in the following percentages: NaCl: 0.0659; NaHC03: 0. 0252;
KC1: 0.046; CaCl2:0.005; MgCl2: 0.0135; NaH2P04: 0.01; Na2HP04:0.008;
glucose: 5 with pH: 8). The bath is maintained at 37°C and bubbled with carbon.
The tissue is allowed to equilibrate for 30 to 60 min. under a load of 0.5 to 3 g.
Carbachol is added to the bath to test the lung strip for its ability to contract prior
to adding the spasmogen. 20 min later, the spasmogen is added to the bath and
the contractile force at its maximal level is recorded to obtain the two pre-valves.
The spasmogen is administered again by maintaining a 20 min. equilibrium
period. After 5 min., the test compound is administered. Isometrically, the
contractile dose is determined. The percentage inhibition of spasmogen-induced
contraction by the test sample is also determined.
Bronchospasmolytic activity in anaesthetised guinea pigs (Konzett-Rossler
1940)
The principle of the method is based on the fact that air volume in the bronchi
changes during bronchospasm. The air volume changes of a living animal are
measured by placing it in a closed system consisting of a respiration pump, the
trachea and the bronchi as well as a reservoir permitting measurement of volume
or pressure of excess air. Bronchospasm decreases volume of excess air. Thus
the degree of bronchospasm can be quantified by re​ cording the excess air. This
method permits the evaluation of a drug’s bronchospasmolytic effect by
measuring the volume of air, which is not taken up by the lungs after
bronchospasm.
Adult guinea pigs of either sex weighing 250–500 g are used. The animal is
anaesthetised with urethane and the trachea cannulated using a two-way cannula.
One arm is connected to a respiratory pump and the other to a Statham
transducer. The animal is artificially inspired using a Starling pump (inspiratory
pressure: > 90–120 mm of water; tidal volume: > 3ml/100 g body weight;
frequency: > 60 strokes/min.). The excess air not taken up by the lungs is
measured and recorded on a polygraph. The internal jugular vein is cannulated
for administration of spasmogens and test compounds. Spasmogens like
histamine dihydrochloride or acetylcholine hydrochloride may be given
intravenously. After obtaining two bronchospasms of equal intensity, test
compounds are administered i.v., s.c., or intraduadenally (i.d.). Spasmogen is
again given at time intervals 5, 15 and 30 min. after i.v. administration of the
drug or at time intervals 15, 30 and 60 min. after i.d. administration of the drug.
The results are expressed as a percentage of inhibition of induced bronchospasm
over control agonistic responses.
Pre-convulsive dyspnoea model using histamine or acetyl choline aerosol
method
Healthy guinea pigs of either sex weighing between 250–500 g are selected. The
animals are housed in clean cages and kept in laboratory conditions. Food and
water are supplied ad libitum. The animals are placed in control, test and
standard groups. All the animals are placed in an anaesthetic box and histamine
(15 ml of 0.5%) or acetylcholine (15 ml of 10%) is sprayed or introduced by
means of a compressor. As soon as pre-convulsive dyspnoea commences, the
animals are placed in fresh air. The percentage protection is calculated by the
formula:

where
T1 = The mean of the control pre-convulsion time 2 days before and 2 days
after the administration of drugs.
T2 = The pre-convulsion time determined with the administration of drugs.
After two days of pre-exposure to bronchoconstrictor agents, the test, standard
and vehicle samples are administered orally one hour before the aerosol spray.
Pre-treatment with an effective agent should delay the pre-convulsion dyspnoea.
In vivo model using tracheal chain of guinea pigs
Guinea pigs are very susceptible to the action of histamines. The trachea of a
guinea pig is removed and placed on a Petri dish with De Jalon solution and
carbogen gas (95% oxygen and 5% C02). Tracheal chain is made and mounted
on an organ bath using De Jalon solution and carbogen gas is passed through it.
The contraction of histamine is recorded on a moving drum or on a physiograph
using transducers. The test drug and histamine contraction are separately
recorded. The anti-asthmatic agent action is estimated by analysing how much it
will reduce the amplitude of contractions.

9.27 SCREENING METHODS FOR ANTI-LITHIOTIC DRUGS

Kidney stones are solid crystals of dissolved minerals found inside the kidneys
or ureters. Clinically the presence of stones in the kidneys is referred to as
nephrolithiasis, urolithiasis or renal calculi. The drugs used for the treatment of
this condition are called anti-lithiotic drugs. Stones can develop in the kidney,
the ureter or the urinary bladder. Kidney stones (renal calculi) originate as
microscopic particles and develop into stones over time. The stones typically
leave the body in the urine stream. If they grow large before passing, they
acquire a crystalline shape and can cause severe pain in the ureter and urethra.
Complications of stone disease may result in severe infection, renal failure or in
rare cases death.
About 75–80% of kidney stones contain calcium compounds, mostly calcium
oxalate and calcium phosphate; they are called calcium stones. Magnesium
ammonium phosphate (struvite) stones form 15% of urinary stones. Uric acid
stones account for 5–10 % of urinary stones. Cystine stones account for only
approximately 1% of urinary stones.
Calcium stones have numerous causes and 85% of these stones are idiopathic
or primary. 10% of cases of primary hypercalciuria are renal in origin. The
remaining 15% of calcium stones are secondary which may result from
hyperparathyroidism or with sarcoidosis. Renal tubular acidosis and
hyperoxaluria are other secondary causes of calcium stones.
Struvite stones forms a complex with calcium phosphate. Uric acid stones can
be removed by the alkalisation and dilution of urine. Surgical intervention for the
treatment of stones is available. Laser techniques can also be used for removing
stones. Besides the above- mentioned methods, kidney stones can also be treated
using the pharmacological approach. Screening of these products can be done
using the following methods.
Ethylene glycol induced nephrolithiasis (Yamagushi et al. 2005)
Nephrolithiasis occurs due to salts, minerals and other substances normally
found in the urine sticking together and building up on the inner surfaces of the
urinary system. In this experiment, ethylene glycol is used as an inducing agent
for nephrolithiasis. The feeding of 1% ethylene glycolated water for 35 days
results in hyperoxaluria. This is due to the conversion of ethylene glycol to
glyceraldehydes, glyoxalic acid, glyoxal and oxalate by the enzyme alcohol
dehydrogenase.
The requirements include: adult albino rats, 1% ethylene glycosylated water,
metabolic cages, haematoxylin-eosin, Von Kossa stain, Pizzolato’s stain, X-ray
powder diffraction, polarising microscope and a flame photometer.
Adult albino rats weighing 180–200 g are used for the study. The animals are
fed with 1% ethylene glycolated water for 35 days. This results in hyperoxaluria.
A test drug’s curative effect is evaluated by treating the rats with it after the
ethylene glycol treatment. A drug’s prophylactic effect is evaluated by treating
simultaneously with ethylene. The animals are kept in the metabolic cages for 24
h in order to record the secretion of urine for 24 h. The urine is subjected to
evaluation of pH and also for various minerals like calcium, potassium, sodium,
oxalates and total proteins by flame photometer. The animals are sacrificed, the
kidneys excised and stained with haematoxylin-eosin, Von Kossa stain and
Pizzolato’s stain. Crystal morphology is determined using polarising microscopy,
while composition is determined using a high-resolution X-ray powder
diffraction.
Porcine model of calcium oxalate kidney stones: Urolithiasis induced by
trans-4-hydroxy-l-proline (Mandel et al. 2004)
Pigs have been extensively used in biomedical research because of their
similarities in origin, structure and function to humans. The present animal
model is an excellent model of oxaluria and urolithiasis with physiological,
anatomical and nutritional characteristics that closely resemble human beings. In
this experiment the pigs fed with trans-4-hydroxy- 1-proline have hyperoxaluria,
calcium oxalate crystalluria and calcium oxalate papillary deposits that may be
the precursors of kidney stones.
 The requirements include: adult healthy pigs, 10% trans-4-hydroxy-l-proline
(weight per weight HP/food).
Adult healthy pigs are selected and fed with 10% trans-4-hydroxy-l-proline
(HP) mixed with food for up to 20 days. Hyperoxaluria is caused, which will
reach a maximum by day 6 of the treatment. Urine is collected and analysed for
oxalate and urate levels using a flame photometer. After 20 days of feeding with
HP, the pigs are killed, the kidneys are removed and examined grossly and
microscopically for indications of injury, crystal deposition and urinary stone
formation. Histopathological examination of the papillary tissue is also done in
order to assess the tissue injury and crystal deposition.
Urolithiasis in rat model using calcium oxalate/zinc disc implants (Choong
et al. 2000)
In this experiment, a calcium oxalate crystal or zinc disc is inserted into the
lumen of the urinary bladder through an incision made in the supra-pubic region.
The implants lead to kidney stones.
The requirements for this experiment include adult healthy rats, anaesthetic
ether, calcium oxalate implant and zinc implant.
Healthy adult albino rats weighing between 200–250 g are selected and
divided into 3 groups i.e. control, positive control and test groups. They are
anaesthetised using ether anaesthesia and an incision is made in the lower
abdomen. The urinary bladder is exposed. Through the incision in the supra-
pubic region, a calcium oxalate crystal or zinc disc is inserted into the lumen of
urinary bladder. These animals are given the test drugs for weeks. After 4 weeks
of treatment, the animals are killed, the urinary bladder is incised and weighed in
a balance.The characteristics of the formed urinary bladder stones are
determined. Histopathological examination of the urinary bladder is performed
to reveal the injury if any and graded. In case of zinc implants, the treatment has
to be started after 4 weeks of surgery and the animals are killed 8 weeks later.

9 .2 8 DEVELOPMENT AND EVALUATION OF TRANSDERMAL

DRUG DELIVERY SYSTEMS (TDDS) OF HERBAL DRUGS
Transdermal therapeutic systems are defined as self-contained, discrete dosage
forms which, when applied to the intact skin, deliver the drug(s), through the
skin, to the systemic circulation at a controlled rate.
 Recently, there has been an explosion in research on drug delivery via the
skin, due primarily to the success of several transdermal devices in sustaining
drug delivery to the systemic circulation. Some transdermal patches containing
herbal drugs are nicoderm patches containing nicotine for smoking cessation
programmes, transdermal patches for motion sickness containing scopolamine
etc. With the development of new technologies such as the SEP box containing
18 HPLC and solid phase extractions connected parallely, it has become possible
to isolate many more pure compounds from the crude source. With this
explosion of new compounds from herbal sources, it has become easier to
prepare their TDDS.
While it is true that product approvals for new TDD products have not
exploded as some predicted following the rapid success of TDD nicotine
products in the early and mid-90s, an increasing number of TDD products
continue to deliver real therapeutic benefits to patients around the world. More
than 35 TDD products have now been approved for sale in the US, and
approximately 16 active ingredients are approved for use in TDD products
globally.
The advantage of delivering drugs across the skin for systemic therapy is well
documented. These advantages are however, counter-balanced by a number of
limitations. Table 9.1 shows the advantages and disadvantages of TDDS.
Table 9.1 An analysis of TDDS
Stratum corneum as the skin permeation barrier
The stratum corneum forms the outer-most layer of the epidermis and consists of
many layers of compacted, flattened, dehydrated and keratinised cells in
stratified layers. These cells are physiologically inactive and are continuously
shed with constant replacement from the underlying viable epidermis. The
stratum corneum (10 mm thick) has a water content of only 20% as compared to
the normal physiologic level of 70% in the physiologically active stratum
germinativum. The stratum corneum is responsible for the barrier function of the
skin as proven by studies using isotopic tracers. It also behaves as a primary
barrier to percutaneous absorption. Figure 9.50 shows the structure of the human
skin.
Fig. 9.50 Structure of human skin

9.28.1 Basic Components of Transdermal Drug Delivery System

The basic components of a TDDS include a polymer matrix or matrices, the
drug, permeation enhancers and other excipients like backing membrane and
adhesive layer.
Polymer matrix
The polymer controls the release of the drug from the device. It should have the
following properties:

1. Molecular weight, glass transition temperature and chemical functionality

should be such that the specific drugs diffuse properly and get released
through it.
2. It should be stable, non-reactive with the drug, easy to manufacture and
fabrication into the desired product should be cheap.
3. The degradation product of polymer should be non-toxic and non-
antagonistic to the host.
4. It should sustain its mechanical properties and not deteriorate excessively
when large amounts of active drug are incorporated into it.

Sources of polymers
Natural source Cellulose derivatives, chitosan, zein, gelatin, shellac, waxes,
proteins, gums, and their derivatives, natural rubber, starch etc.
Synthetic elastomers Polybutadiene, hydrin rubber, silicone rubber, nitrile,
acrylonitrile, butyl rubber, styrene butadiene rubber etc.
Synthetic polymers Polyvinyl alcohol, polyvinyl chloride, polyethylene,
polypropylene, polyacrylate, polyamides, polymer, polymethyl etharylate etc.
Drugs
For successfully developing a transdermal drug delivery system, the drug should
be chosen with great care. Usually those drugs which are degraded in the GI
tract or undergo hepatic first pass metabolism are good candidates for this. The
drug should have various physico​chemical and biological properties.
Physico-chemical properties

1. The drug should have a molecular weight less than 500 Dalton.
2. The drug should have affinity for both lipophilic and hydrophilic phases.
3. The drug should have low melting point.

Biological properties

1. The drug should be potent with a daily dose of the order of a few mg/day.
The half-life (t1/2) of the drug should be short.
2. The drug must not induce a cutaneous irritation or allergic response.

Compounds from herbal sources which meet the above criteria are suitable
candidates for transdermal drug delivery systems.
Permeation enhancers
Permeation enhancers are chemicals which increase the permeability of the
stratum corneum by altering the skin’s barrier properties. These chemicals
adsorb into the stratum corneum and interact with tissue components to reduce
the barrier properties of the membrane without causing damage to the underlying
skin cells. These should act reversibly; that is the reduction in the stratum
corneum’s barrier properties should be temporary.
Desirable properties for penetration enhancers

1. It should have no pharmacological activity within the body.

2. It should be non-toxic, non-irritant and non-allergic.
3. It should work rapidly and the extent of action should be predictable and
reproducible.
4. It should work unidirectionally, i.e. it should allow entry of drug into the
body whilst preventing the loss of endogenous material from the body.
5. It should be compatible with the drug and excipients.
6. It should be cosmetically acceptable i.e., it should not feel bad, and should
be ideally tasteless, odourless and colourless.

A large number of compounds have been investigated for their ability to

enhance the stratum corneum permeability. They may be classified as solvents
and surfactants.
Solvents
These compounds increase penetration possibly by swelling the polar pathway
and/or by fluidising lipids e.g. water and alcohols like methanol and ethanol.
Alkyl methyl sulphoxides Dimethylsulphoxide, dimethylformamide,
dimethylacetamide
Pyrrolidones 2-pyrrolidone, N-methyl, 2- pyrrolidone, azone
Tulsi (Ocimum sanctum) oil Tulsi oil has been used as a penetration enhancer
by the Delhi Institute of Pharmaceutical Sciences in transdermal drug delivery
and has been patented (Application no: 673/Del/2001; Inventors/Applicants: Dr.
S.S. Agrawal; Prof. Suraj Prakash Aggarwal; Alka Goel). It has been used in
transdermal conception control systems.
Miscellaneous solvents Propylene glycol, glycerol, silicone fluids, isopropyl
palmitate
Surfactants
These compounds enhance the polar pathway transport, especially, of
hydrophilic drugs. Surfactants have the ability to alter the penetration of the
drug, which is a function of the polar head group and the hydrocarbon chain
length. These compounds are however, skin irritants, therefore, a balance should
be struck between the intensity of penetration enhancement and degree of
irritation. Anionic surfactants like dioctyl sulphosuccinate, sodium lauryl
sulphate, can penetrate and interact strongly with the skin. Once they have
penetrated the skin, the surfactants can induce large alterations.
Cationic surfactants cause a higher degree of irritation than anionic surfactants
and hence are not widely studied. Non-ionic surfactants like pluronic F-127,
pluronic F-68, have long been recognised as those with least potential for
irritation. Bile salts like sodium taurocholate, sodium deoxycholate, and sodium
tauroglycocholate are also used. Other miscellaneous chemicals used in
transdermal drug delivery systems are terpenes, urea, calcium thioglycollate, etc.
Other excipients
Adhesives
The fastening of all transdermal devices to the skin has so far been done by
using a pressure-sensitive adhesive. It can be positioned on the face of the device
or in the back of the device extending peripherally. It should not irritate the skin
or stick aggressively to the skin. It should be easily removed and not leave
unwashed residue on the skin. The face adhesive system should also be
physically and chemically compatible with the drug and excipients and not affect
the permeability of the drug. The widely used pressure-sensitive adhesives
include polyisobutylene, acrylics, and silicones.
Backing membrane
Backing membranes are flexible and provide good bonding to the drug reservoir.
They prevent the drug from leaving the dosage form through the top and accept
printing. It is an impermeable substance that protects the product during use on
the skin e.g. metallic plastic laminates, aluminum foil, flexible polyurethane etc.

9.28.2 Design Features of Transdermal Drug Delivery Systems

Technically TDDS may be categorised into two types, monolithic (matrix type)
and membrane-controlled systems.
Monolithic systems incorporate a drug matrix layer between the backing and
frontal layer. The drug–matrix layer is composed of a polymeric material in
which the drug is dispersed. As mentioned before, the polymer matrix controls
the rate at which the drug is released for percutaneous absorption.
Membrane-controlled transdermal systems are designed to contain a drug
reservoir or “pouch”, usually in liquid or gel form, a rate controlling membrane,
and a backing, adhesive layer.
Fabrication of polymer matrices
For preparing transdermal matrices, the polymer is dissolved or dispersed in a
suitable solvent (acetone, methanol, water etc.) by stirring on a magnetic stirrer.
After the polymer dissolves or disperses completely, an accurately weighed
quantity of the drug is added and stirred further. For increasing the permeation of
the drugs, a penetration enhancer may also be added prior to the addition of the
drug in the matrices.
Matrix casting
The above solution so prepared is poured into Teflon moulds and dried in hot air
oven.

9.28.3 In vitro Evaluation of Transdermal Matrices through Rat Skin

A Keshary–Chien diffusion cell (Fig. 9.51) is used in the diffusion study of
transdermal matrices across rat skin. It consists of an upper (donor) compartment
and a lower (receptor) compartment. The temperature of the receptor solution is
maintained at 32 ± 1°C, by circulating water in the outer jacket. It is maintained
at a constant hydrodynamic condition by a small magnetic bar rotating at 600
rpm (approximately). The volume of the receptor phase is maintained at 60 ml.
The skin surface area is taken as 3.14 cm2 (internal diameter of the cell: 2 cm).
The receptor compartment is provided with a sampling port for sampling the
receptor solution at scheduled time intervals for assay.

Fig. 9.51 Keshary–Chien diffusion cell used for in vitro permeation studies

The matrices are subjected to in vitro evaluation across rat dorsal skin. The
skin is clamped between two portions of the Keshary–Chien diffusion cell with
the dermal side facing the receptor solution. The transdermal matrix is placed
over the epidermal side of the skin and covered with parafilm to provide
occlusive covering. The donor compartment is then clamped over it. Samples of
5 m volume are withdrawn at some definite time intervals and analysed for drug
content.

9.28.4 Primary Skin Irritation Studies

An optimal transdermal patch has to be evaluated for skin irritation before it can
be put to use. Primary skin irritation studies have been done on rabbits (Draize et
al. 1944).
The hair on the back of the rabbit was removed using scissors and the skin
washed properly one day prior to study. One side of the back of each rabbit was
taken as control and the other side as test.
A patch containing the drug was secured on the experimental side and one
without any drugs was attached to the control side of the rabbits. Both the
patches were covered with occlusive covering. The medicated patches were
changed after 72 h and fresh patches secured at the same site (Modified Draize’s
irritation test). The patches were placed on the back for one week.
When the patches were removed, each of the areas under the patches was
observed for signs of erythema or edema for 7 days. Signs for recording degrees
of patch test reactions were as follows:

No reaction : -

Erythema : +

Erythema and papules : ++

Erythema, papules and vesicles : +++

Marked edema and vesicles : ++++

Throughout the past 2 decades, the transdermal patch has become a proven
technology that offers a variety of significant clinical benefits over other dosage
forms. Because transdermal drug delivery offers a controlled release of the drug
into the patient, it enables a steady blood-level profile, resulting in reduced
systemic side-effects and, sometimes, improved efficacy over other dosage
forms. In addition, because transdermal patches are user-friendly, convenient,
painless, and offer multi-day dosing, it is generally accepted that they offer
improved patient compliance.
Although transdermal drug delivery patches have a relatively short regulatory
history compared to other, more traditional dosage forms, the technology has a
proven record of FDA approval. Since the first transdermal patch was approved
in 1981 to prevent nausea and vomiting associated with motion sickness, the
FDA has approved, throughout the past 22 years, more than 35 transdermal patch
products, spanning 13 molecules. The US transdermal market approached $1.2
billion in 2001 and was based on 11 drug molecules: fentanyl, nitroglycerin,
estradiol, ethinyl estradiol, norethindrone acetate, testosterone, clonidine,
nicotine, lidocaine, prilocaine, and scopolamine. Two new, recently approved
transdermal patch products (a contraceptive patch containing ethinyl estradiol
and norelgestromin, and a patch to treat over-active bladder, containing
oxybutynin) should help to expand the US transdermal market.
Clearly, the clinical benefits, industry interest, strong market, and regulatory
precedence show why transdermal drug delivery has become a successful and
viable dosage form. Yet, the pharmaceutical industry tends to view TDD as a
limited technology that serves only a niche set of drugs. Certainly, transdermal
drug delivery is not suited nor clinically justified for all drugs. The skin barrier
also limits the number of drugs that can be delivered by passive diffusion from
an adhesive patch. Yet, its many advantages cannot be ignored.
The market for transdermal products has been in a significant upward trend
and is likely to continue so for the foreseeable future.

9 .2 9 DEVELOPMENT AND EVALUATION OF INTRA-UTERINE

CONTRACEPTIVE DEVICES (IUCDS) FOR HERBAL DRUGS
Numerous herbs have been used historically to reduce fertility, and modern
scientific research has confirmed the anti-fertility effects in at least some of the
herbs tested. Herbal contraception offers alternatives for women who have
difficulty with modern contraceptive options or who just want to try a less toxic
way of preventing conception. The various herbal sources having anti-fertility
activity are as follows: Wild yam, neem oil, wild carrot or Queen Anne’s lace
seeds (Daucus carota), pomegranate (Punica granatum), rutin, smartweed leaves
{Polygonum hydropiper), apricot kernels, cotton, rue and vitamic C (ascorbic
acid).
Besides these, phytoestrogens are increasingly becoming popular.
Phytoestrogens are chemicals produced by plants that act like estrogens in
animal cells and bodies. They are often found in trace amounts in food. These
chemicals mimic and supplement the action of the body’s own hormone,
estrogen. Phytoestrogens mainly fall into the class of flavonoids. The
coumestans, the prenylated flavonoids and isoflavones are three of the most
potent in this class. Phytoestrogens when taken orally is not as effective as when
applied directly onto the uterus.
Vaginal creams and suppositories made with neem oil are quickly becoming
the birth control method of choice in India. This is because they are non-
irritating and easy to use while being almost 100% effective. They have the
added benefit of preventing vaginal and sexually transmitted diseases. Neem has
a proven ability to prevent pregnancy.
The vast potential of these herbal drugs as a contraceptive option and as an
intra-uterine contraceptive device (IUCD) which has the added advantages of
low dose as well as increased activity due to direct application onto the uterus
have not yet been tapped. But at the Delhi Institute of Pharmaceutical Sciences
& Research (DIPSAR), for the first time in the history of IUCD, a novel frame-
less medicated IUCD containing herbal drugs such as neem oil has been
fabricated using a variety of polymers (biodegradable as well as non-
biodegradable).

9.29.1 Intra-Uterine Contraceptive Devices (IUCD)

An intra-uterine contraceptive device (intra: within; uterine: uterus) is a birth
control device. It is also known as an IUCD or a coil (this colloquialism is based
on the coil-shaped design of early IUCDs). It is a device placed in the uterus and
is the world’s most widely used and inexpensive method of reversible birth
control. The device has to be fitted inside or removed from the uterus by a doctor
or qualified medical practitioner. It remains in place the entire time contraception
is desired. Depending on the type IUCDs (Fig. 9.52), can usually remain
effective for 2, 5 or 10 years.

Fig. 9.52 Different types of IUCDs

Types of IUDs
There are mainly two types of IUDs: Copper containing intra-uterine devices
(IUD) and medicated IUDs (hormone containing intra-uterine device e.g. the
Levonorgestrel Intra​uterine system (LNG IUS)).
Fabrication of IUDs
Medicated IUCDs can be fabricated in two ways—the reservoir system and the
matrix system.
Reservoir system
It is a sandwich-type of system containing a polymer layer with the drug
dispersed in it and sandwiched between two polymer layers without the drug
(Fig. 9.53). It is released through the polymer by dissolution into the polymer
and then diffusion through the polymer wall.
Polymers A variety of polymers are employed in the fabrication of IUCDs. Table
9.2 lists out just a few of them.

Fig. 9.53 Reservoir system

Table 9.2 Examples of polymers used in polymeric drug delivery

Preparation of polymer solution (top and bottom layer) The polymer is made to
dissolve in a suitable solvent under constant stirring on a magnetic stirrer. The
solution is then poured into a mould or a glass Petri plate and made to dry.
Preparation of polymer layer containing drug (middle layer) The polymer
solution is made in a suitable solvent so as to be of pourable consistency. When
the polymer has been dissolved completely, an accurately weighed amount of
drug is added in small fractions under constant stirring on a magnetic stirrer. The
polymer solution containing the dispersed drug is poured into the Petri plate and
kept in glass desiccators, overnight. The solvent is allowed to evaporate slowly.
Fabrication of sandwich The film containing the dispersed drug is stripped off
and the dried film placed over the dried film (bottom layer) in the Petri plate.
The film is pressed gently with the help of fingertips so as to make it stick to the
bottom layer. Over these films, another portion of the polymer solution (top
layer) is poured and kept overnight for drying.
The resulting sandwiched film is stripped off from the Petri plate and kept in
the oven at 40°C, for 3 to 4 h.
Matrix system
In this system (Fig. 9.54), the drug is dispersed in a matrix (polymer). In
comparison to reservoir systems, these devices are simpler (since they are
homogeneous and, hence, easier to produce) and potentially safer (since a
mechanical defect in a reservoir device, but not a matrix, can lead to dose
dumping).

Fig. 9.54 Matrix system

Preparation of matrix The selected polymer is made to dissolve in a suitable

solvent so as to be of pourable consistency. When the polymer has been
dissolved completely, an accurately weighed amount of drug is added in small
fractions under constant stirring on a magnetic stirrer.
Pouring of mixture The polymer containing drugs is then poured into a mould
or a glass Petri plate taking care that no air bubble is entrapped in the layer while
pouring. The matrix is then made to air-dry for a while. It is then dried in an
oven at 35°C. The film so prepared is then stripped carefully and stored in
aluminium foil.
Preparation of the intra-uterine system
The films are cut into size of 1.5 cm x 1.5 cm and packaged in an aluminium
foil.
In vitro evaluation
In vitro drug release studies provide valuable information about the in vivo
behaviour of the dosage form. It tells us about the amount of drug available to
the biological system. Different methods to study the in vitro diffusion of the
drug from intra-uterine devices are given in literature (USP 24 NF 19, 2000).
Dissolution apparatus
A modified form of USP type II, paddle-type dissolution apparatus is used for
carrying out the release profile studies.
Composition of IUDs
Copper IUDs consist of a plastic (polyethylene) carrier T- shaped frame wound
with copper wire/bands (Fig. 9.55). A silver core is sometimes used to prevent
fragmentation of the copper. A frameless copper-based IUCD has been
introduced recently, the GyneFix®. It is a knotted polypropylene cord sheathed
with 6 copper sleeves. It is secured in the uterus by embedding the knot into the
fundal myometrium using a special applicator. Some are impregnated with
barium sulphate to make them radio-opaque.

Fig. 9.55 Copper releasing IUCD

The LNG IUS, marketed by Berlex Laboratories as Mirena, consists of a T-

shaped polyethylene frame with a reservoir around the vertical stem that
contains levonorgestrel. It was approved for use by the FDA in 2000. Initially the
LNG IUS releases levonorgestrel at a rate of 20 μg per day. This rate decreases
to approximately half that rate by five years. It is indicated effective for up to
five years of use, although limited data support its effectiveness for at least seven
years.
Mode of action of IUCDs
IUDs mainly act by inducing a sterile inflammatory response, changing the
composition of the uterine and tubal fluids, reducing the viability of both the
sperm and the ova, thus making fertilisation unlikely. This is the main mode of
action for copper-bearing devices. Post-fertilisation mechanisms of action may
also play a role, particularly in non-copper-bearing devices.
The levonorgestrel-bearing device (Mirena) is an effective contraceptive. It
works by causing endometrial suppression and preventing implantation. It
thickens the cervical mucus to prevent sperm penetration, impairs sperm
migration and, in some women, and some cycles, suppresses ovulation. Its actual
presence in the uterus may also add to its contraceptive effect.
Effectiveness of IUCDs
Depending upon the type of device used, IUCDs have a <1% failure rate per
year. The hormonal IUD is as effective as the contraceptive pill at preventing
pregnancy; and the copper IUDs effectiveness ranges from 98% to over 99%,
depending on the brand. Generally devices with a large surface area of copper
(>250 mm2) are more effective, as are those containing levonorgestrel.
Pregnancy rates appear similar for those using the Gynefix® system.
Analysis of IUCDs
Table 9.3 provides an analysis of IUCDs. The advantages of using IUCDs
outnumber its disadvantages.

Table 9.3 Advantages and disadvantages of IUCDs

Advantages Disadvantages

1. Highly effective, even 1. Inter-menstrual spotting and bleeding.

immediately after fitting into the
uterus.

2. It does not interfere with sexual 2. Heavier, longer and more painful
intercourse. periods.

3. Periods become shorter, lighter 3. Expulsion, displacement or migration

and less painful after three of IUCD.
months.

4. Reversible with no adverse 4. Increased risk of pelvic inflammatory

effect on fertility. Fertility returns disease, uterine perforation, ectopic
quickly once it is removed. pregnancy, pelvic actinomycosis.

5. One does not have to think

about contraception for as long as
it works.

6. In order to terminate the

therapy, it can be easily removed
as and when desired.

7. No adverse “hormonal” effects

are seen with IUCDs.

Currently available IUCDs

Copper containing IUCD

GyneFix® : Frameless (polypropylene cord)

 Flexi-T® 300*: Copper wire on a fundus-seeking modified T- shaped plastic
carrier
Multiload® Cu 375: Copper wire on a polyethylene stem with flexible U-
shaped side arms
Nova-T® 380: Copper wire with a silver core on a modified plastic T-
shaped carrier
T-Safe® CU 380 A : Copper wire wound on a T-shaped carrier with a copper
collar on the distal portion of each arm
ParaGard T 380A by Barr Laboratories

Numbers indicate surface area of copper in mm2

Medicated IUCD
Levonorgestrel-bearing IUCD: Mirena-T-shaped intra-uterine system
(containing 52 mg levonorgestrel)
Fibroplant: Frameless intra-uterine device.

9.30 TOXICITY STUDIES

Toxicokinetic studies (generation of pharmacokinetic data either as an integral
component of non-clinical toxicity studies or in specially designed studies)
should be conducted to assess the systemic exposure achieved in animals and its
relationship to dose levels and duration of treatment.
Toxicity testing is of paramount importance while screening drugs. From the
1950s to the 1960s thalidomide was sold in Great Britain to pregnant women to
combat morning sickness. But inadequate testing was performed to assess the
drug’s safety. This led to catastrophic results for the children of the women who
had taken thalidomide during their pregnancies. Children were born malformed
or with phocomelia (seal-like limbs, congenital absence of upper limbs). After
this thalidomide tragedy, many countries decided to go for toxicity testing and
teratogenicity in both the sexes, so as to prevent further tragedies. Although
herbal drugs are less toxic as compared to synthetic drugs, drugs like black
nightshade (Solanum nigrum) have shown cardiac toxicity in rabbits after 21
days of administration.
Toxicity studies are conducted with the assumption that man will behave in
the same manner as the animals. Toxicity studies are of the following types:
acute toxicity studies, sub-acute toxicity studies and chronic toxicity studies.

9.30.1 Acute Toxicity Studies

Acute toxicity studies are conducted to determine the median lethal dose (LD50
or LD90, the dose required to kill 50% or 90% respectively) of laboratory
animals. In addition, such studies may also indicate the probable target organ of
the chemical and its specific toxic effect. It provides guidance on the doses to be
used in more prolonged studies. Acute toxicity tests form part of a complete
programme of toxicity testing that provide the basis on which to design further
testing programmes.
Experimental design
Selection of species of animals
These studies should be carried out in at least 2 rodent (mice or rats) species. Rat
and mice are preferred because they are economical, readily available and easy
to handle. When LD50 values in rats and mice are markedly different or when the
rate of bio-transformation in humans is known to be significantly different from
rats and mice, use of a non-rodent species is desirable.
LD50 determination is done in animals of both sexes; also in both adult and
young animals because of their difference in susceptibility. The age of the
animals used is of considerable importance. In general, young immature animals
are preferable because of the rapidity of their growth, which enables even a
slight depression in growth rate to be measurable. On the other hand, the
detection of many actions, particularly on the endocrine and reproductive
systems, requires the use of sexually mature animals.
Number of animals
The above doses can be estimated using 6–9 animals i.e., a small group of
animals (3–5 rodents /sex/dose).
Dose
Toxicity studies are performed in each species at the same dose level as intended
to be used in treatment. Three other doses are also to be administered: that
amount that would kill half of the animals (LD50), another that would kill more
than half (preferably less than 90%), and a third dose that would kill less than
half (preferably more than 10%) of the animals.
Rloute of administration
Toxicity studies are performed in each species using the route by which it is
going to be used in humans. Oral route is the most commonly used method.
Parentral routes are used in accessing the acute toxicity of parentral drugs. In
addition, unless the intended route of administration in humans is only
intravenous, at least one more route should be used in one of the species to
ensure systemic absorption of the drug. This route should depend on the nature
of the drug A limit of 2 g/kg (or 10 times the normal dose that is intended in
humans, whichever is higher) is recommended for oral dosing.
Duration of study
If the drug is intended to be used only once in the lifetime of an individual such
as a general anaesthetic, only 24 h of acute toxicity studies are performed.
Otherwise, they are done for 48 h or so.
Evaluation
The number and time of death should be examined in order to estimate the LD50.
The observation period is usually 7–14 days but may be much longer. Signs of
toxicity should also be recorded.
Some methods for acute toxicity evaluation are described in this section.
Graphical method of Miller and Tainter (1944)
This method is most commonly used for calculating any ED50 value. A special
co-ordinate paper—a logarithm-probit paper—is used. It has two decades of
logarithmic scale as the abscissa and a scale of probits as the left ordinate. The
right ordinate is marked in a scale of per cent corresponding to the probit scale.
The probit corresponding to a per cent value must be found in a table of probits.
Arithmetical method of Reed and Muench (1938)
This method employs cumulative values. It is assumed that an animal killed by a
certain drug would have been killed by a large dose, and that a surviving animal
would have survived a smaller dose. The cumulative dead and survivors are
recorded. The per cent of survival is calculated and the LD50 is computed. This
computation is less reliable than the Miller and Tainter method.
Arithmetical method of Karber (1931)
The interval mean of the number of dead in each group of animals is used in this
method, as well as the difference between doses for the same interval.
Observations and examinations
Daily water and food intake of the animals have to be monitored. The initial
weight and weight after the study has to be recorded.
Laboratory tests
Tables 9.4–9.7 list some of the biochemical parameters, body organs and
systems that might be affected by the drug and which have to be accessed during
the study.
Table 9.4 Hematological parameters

Haemoglobin
Total RBC count
Haematocrit
Reticulocyte
Total WBC count
Differential WBC count
Platelet count
Terminal bone marrow examination
ESR (non-rodents only)
General blood picture: A special mention of abnormal and immature cells
should be made
Coagulation parameters (non-rodents only): Bleeding time, coagulation
time, prothombin time, activated partial thromboplastin time

Table 9.5 Urinalysis parameters

Colour
Appearance
Specific gravity
24-hour urinary output
 Reaction (pH)
Albumin
Sugar
Acetone
Bile pigents
Urobilinogen
Occult blood
Microscopic examination of urinary sediment.

Table 9.6 Blood biochemical parameters

Glucose
Cholesterol
Triglycerides
HDL cholesterol (non-rodents only)
LDL cholesterol (non-rodents only)
Bilirubin
SGPT (ALT)
SGOT (AST)
Alkaline phosphatase (ALP)
GGT (non-rodents only)
Blood urea nitrogen
Creatinine
Total proteins
Albumin
Globulin (calculated values)
Sodium
Potassium
 Phosphorus
Calcium

Table 9.7 Gross and microscopic pathology

Brain*: Cerebrum, cerebellum, mid-brain

Spinal cord**
Eye
MiddLe ear
Thyroid
Parathyroid**
Spleen*
Thymus
Adrenal*
Pancreas**
Trachea**
Lung*
Heart*
Aorta
Oesophagus
Stomach
Duodenum
Jejunum
Terminal ileum
Colon
Rectum**
Liver*
Kidney*
 Urinary bladder
Epididymis
Testis*
Ovary
Uterus*
Skin
Mammary gland
Mesenteric lymph node
Skeletal muscle

*Organs to be weighed.
**Organs should be examined as the drug might have an external effect on it.
OECD guidelines for acute oral toxicity studies
The Organization for Economic Co-operation and Development has set down
various guidelines for the testing of chemicals. These guidelines are based on the
procedure adopted by the American Society of Testing and Materials (ASTM) in
1987 and revised in 1990. They help in minimising the number of animals
required to estimate the acute oral toxicity of a chemical.
Definitions as per OECD guidelines
Acute oral toxicity Refers to those adverse effects occurring following oral
administration of a single dose of a substance or multiple doses given within 24
h.
Dose Refers to the amount of test substance administered. Dose is expressed as
weight (g or mg) or as weight of test substance per unit weight of test animal
(eg. mg/kg).
The preferred rodent species, as per this guideline, is the rat, although other
rodent species may be used. Normally female rats are used since females are
generally slightly more sensitive than males. Females should be nulliparous and
non-pregnant. When the test is conducted on males, adequate justification should
be provided.
Each animal should be between 8 and 12 weeks old and its weight should fall
in an interval within ± 20% of the mean initial weight of any previously dosed
animals.
The temperature in the experimental animal room should be 22°C (±3°C). The
relative humidity should be at least 30% and preferably not exceed 70%.
Lighting should be artificial—the sequence being 12 h light and 12 h dark. The
animals should be randomly selected, marked to permit individual identification,
and kept in their cages for at least 5 days prior to dosing to allow for
acclimatisation to laboratory conditions. In general test substances should be
administered in a constant volume over the range of doses to be tested by
varying the concentration of the dosing preparation. Maximum volume of liquid
that can be administered at one time depends on the size of the test animal. In
rodents, the volume should not normally exceed 1 ml/100 g of body weight;
however, in the case of aqueous solutions, 2 ml/100 g body weights can be
considered.
The test substance should be administered in a single dose by gavages using a
stomach tube or a suitable incubation cannula. Animals should be fasted prior to
dosing (e.g., for the rat, food but not water should be withheld overnight; for the
mouse, food but not water should be withheld for 3-4 h). Following the period of
fasting, the animals should be weighed and the test substance administered. The
fasted body weight of each animal is determined and the dose calculated
according to the body weight. After the substance has been administered, food
may be withheld for a further 3–4 h in rats or 1–2 h in mice.
Limit test: Guideline 425 (acute oral toxicity: updated guidelines adopted,
December 20, 2001)
A limit test is generally performed before the main test. As per the 425
guidelines, one animal is dosed at the test dose i.e. 2000 mg/kg. If the animal
dies, the main test is conducted to determine the LD50. If the animal survives,
four additional animals are dosed sequentially so that a total of five animals are
tested. If three animals die, the limit test is terminated and the main test is
performed.
The LD50is less than the test dose (2000 mg/kg) when three or more animals
die. If a third animal dies, the main test is conducted. The LD50 is greater than
the test dose (2000 mg/kg) when three or more animals survive.
If an animal unexpectedly dies late in the study, and there are other survivors,
it is appropriate to stop dosing and observe all animals to see if other animals
will also die during a similar observation period. Late deaths should be counted
the same as other deaths.
Main Test: Guideline 425
Single animals are dosed in sequence usually at 48 h intervals. However, the
time interval between dosing is determined by the onset, duration and severity of
toxic signs. Treatment of an animal at the next dose should be delayed until one
is confident of survival of the previously dosed animal.
The first animal is dosed a step below the best preliminary estimate of the
LD50. If the animal survives, the second animal receives a higher dose. If the
first animal dies or appears sick, the second animal receives a lower dose.
Dosing continues depending on the fixed-time interval (e.g., 48 h) outcomes
of all the animals up to that time. The testing stops when one of the following
stopping criteria is met:

a. 3 consecutive animals survive at the upper bound;

b. 5 reversals occur in any 6 consecutive animals tested;
c. At least 4 animals have followed the first reversal and the specified
likelihood ratios exceed the critical value.

Animals are observed individually at least once during the first 30 min. after
dosing, periodically during the first 24 h (with special attention given during the
first 4 h), and daily thereafter, for a total of 14 days,
The times at which signs of toxicity appear and disappear are important,
especially, if there is a tendency for toxic signs to be delayed. All observations
are systematically recorded with individual records being maintained for each
animal.
Additional observations will be necessary if the animals continue to display
signs of toxicity. Observations should include changes in skin and fur, eyes and
mucous membranes and also respiratory, circulatory, autonomic and central
nervous systems, somatomotor activity and behaviour patterns. Attention should
be directed to observations of tremors, convulsions, salivation, diarrhoea,
lethargy, sleep and coma.
Individual weights of animals should be determined shortly before the test
substance is administered and at least weekly thereafter. Weight changes should
be calculated and recorded. At the end of the test, surviving animals are weighed
and then humanely killed.
All animals (including those which die during the test are removed from the
study for animal welfare reasons) should be subjected to gross necropsy. All
gross pathological changes should be recorded for each animal.

9.30.2 Sub-acute Toxicity Studies

Sub-acute toxicity studies are conducted to determine the organs affected by
different dose levels. These studies access the nature of toxic affects under more
realistic situations than the acute toxicity studies.
Experimental design
Selection of species of animals
These studies should be carried out in at least 2 phylogenetically different
species, of which one should be a non-rodent and the other a rodent (mice, rats)
species. Ideally, the animals chosen should bio-transform the chemical in a
manner essentially identical to humans.
Number of animals
Equal number of male and female animals should be used. Generally 10–50 rats
are used in each dose group as well as in the control group so as to provide the
data, which is statistically analysable.
Dose
It is advisable to select three doses: a dose that is high enough to elicit definite
signs of toxicity but not high enough to kill many of the animals, a low dose that
is expected to induce no toxic effect, and an intermediate dose. A control group
should also be included.
Doses are generally selected on the basis of information obtained in acute
toxicity studies, using both the LD50 and the slope of the dose–response curve.

Route of administration
Toxicity studies are performed in each species using the same route as that
intended in humans.
Duration of study
The duration of the study in rats is generally 90 days. In dogs, the duration is
often extended to 6 months or even 1 year. The duration of sub-acute toxicity
studies depends on the intended duration of the test drugs in therapeutics (Table
9.8).
Table 9.8 Duration of the sub-acute toxicity studies in relation to intended use in
therapeutics.
 Intended use in therapeutics Duration of sub-acute toxicity
studies

Once in a life-time, say, general 7 days

anaesthetics

Up to 5 or 7 days 21 days

More than 7 days 30 days

More than 21 days 180 days

Evaluation
They provide an indication of the effect on the target organs, dose–effect, and
dose–response relationships. Autopsy of the animals and gross pathological
examination provides useful information on the toxicity of the chemical under
test.
Observations and examinations
Body weight and food consumption: This should be determined weekly.
Decreased body weight is an index of toxic effects. Food consumption is also a
useful indicator.
General observations: These include monitoring of general appearance, gross
behaviour and any other abnormalities.
Laboratory test: Examinations should include the tests listed in Tables 9.4–9.7.
Postmortem examination: Lists of organs, which are to be histopathologically
examined, are enlisted in Table 9.5. The organs isolated should be weighed; the
number of organs and all the gross lesions has to be determined as they serve
useful indicators of toxicity.

9.30.3 Long-term Toxicity Studies (Chronic Toxicity Studies)

In these studies, the animals are exposed over a long period of time to the toxic
effects of the drug in order to mimic more realistic situations. On the basis of
information obtained in sub-acute toxicity studies, the main aim of these long-
term studies is to determine the organs affected and determine whether the drug
is potentially carcinogenic or not. These tests may be conducted concurrently
with the initial studies in humans (phase 1 clinical trials). The procedures
involved in both sub-acute and chronic studies are similar except for their
duration. The duration of long-term studies in rats is generally 1–2 years, and it
can extend to 7 years in case of dogs.

9 .3 1 COMMITTEE FOR THE PURPOSE OF CONTROL AND

SUPERVISION ON EXPERIMENTS ON ANIMALS (CPCSEA)
All animal experimentation and breeding is controlled by the Committee for the
Purpose of Control and Supervision on Experiments on Animals (CPCSEA).This
was decided by the Act of the Parliament of India in December 1998. The
guideline enforced by this committee is the amended form of the Prevention of
Cruelty to Experimental Animals Act 1960. By this act, it is mandatory to obtain
separate licenses to conduct breeding and maintenance for experimental animals.
A licence is also required for conducting research experimentation in animals.
As per the CPCSEA, it is mandatory to form an Institutional Animal Ethics
Committee (IAEC). The committee should consist of 8 members—2 nominees
from the ICMR, 1 scientist from the concerned field, 1 pharmacologist, 1
veterinarian, 1 social activist, 1 member secretary (IAS officer) and 1 chairman
within the institution. The goal of these guidelines is to promote the human care
of animals used in biomedical and behavioural research and testing with the
basic objective of providing specifications that will enhance animal well-being
and the quality of biological knowledge that is relevant to humans and animals.
All animals must be acquired lawfully as per the CPCSEA guidelines. A
health surveillance program for screening incoming animals should be carried
out to assess animal quality.
Quarantine—the separation of newly received animals from those already in
the facility until the health and possibly the microbial status of the newly
received animals have been determined—must be observed. Regardless of the
duration of quarantine, newly received animals should be given a period for
physiological, psychological and nutritional stabilisation before their use.
Physical separation of animals by species is recommended to prevent
interspecies disease transmission and to eliminate anxiety and possible
physiological and behavioural changes due to interspecies conflict. All animals
should be observed for signs of illness, injury, or abnormal behaviour.
Unexpected deaths and signs of illness, distress, or other deviations from normal
health condition in animals should be reported promptly to ensure appropriate
and timely delivery of veterinary medical care. Animals that show signs of a
contagious disease should be isolated from healthy animals in the colony. No
animal should be used for experimentation for more than 3 years unless adequate
justification is provided.
Transportation of experimental animals
The transport of animals from one place to another is very important and must be
undertaken with care. The main considerations for transport of animals are the
mode of transport, the containers and the animal density in cages, food and water
during transit, protection from transit infections, injuries and stress.
Animal experimentation
Scientists should ensure that the procedures which are considered painful be
conducted under appropriate anaesthesia as recommended for each species of
animals. In the event of a decision to sacrifice an animal on termination of an
experiment or otherwise, an approved method of euthanasia should be adopted.
Before using actual anaesthetics, the animal is prepared for anaesthesia by
overnight fasting and using pre-anaesthetics, which block parasympathetic
stimulation of the cardio-pulmonary system and reduce salivary secretion. Local
or general anaesthesia may be used, depending on the type of surgical procedure.
General anaesthetics are also used in the form of intravenous or intramuscular
injections such as barbiturates.
Euthanasia
Euthanasia is resorted to when an animal is required to be sacrificed on
termination of an experiment or for other ethical reasons. The procedure should
be carried out quickly and painlessly in an atmosphere free from fear and
anxiety. To make the euthanasia as humane as possible, the method applied
should have an initial depressive action on the central nervous system for
immediate insensitivity to pain. The choice of the method will depend on the
nature of study and the species of animal to be killed.
All scientists working with laboratory animals must have a deep ethical
consideration for the animals they are dealing with. From the ethical point of
view it is important that such considerations are taken care at the individual
level, at the institutional level and finally at the national level.

9.32 CLINICAL TRIALS

Clinical trials include any investigation done on human subjects to determine:

1. The clinical, pharmacological, pharmacokinetic of an investigational agent;

2. and/or other pharmacodynamic effects of an investigational agent;
3. and/or to identify any adverse reactions to an investigational agent;
4. and to assess the agent’s safety and efficacy.

In fact, clinical trials may also be considered as bioassays where actions and
interaction(s) of a molecule (likely to be used as drug) are observed in human
volunteers/patients. Clinical trials are conducted to evaluate the efficacy and
safety of the drug molecule/molecules in human subjects before
commercialisation.
A clinical trial (also clinical research) is a research study in human volunteers
to answer specific health questions. Carefully conducted clinical trials are the
fastest and safest way to find treatments that work in people and ways to
improve health. Interventional trials determine whether experimental treatments
or new ways of using known therapies are safe and effective under controlled
environmental conditions. Observational trials address health issues in large
groups of people or populations in natural settings. Participants also receive up-
to-date care from the scientists/ experts/ investigators.
Participants in clinical trials can play a more active role in their own health
care, gain access to new research treatments before they are widely available,
and help others by contributing to medical research.
Research in clinical trials involving human subjects is conducted according to
strict scientific and ethical principles. Based on these principles and guidelines,
an action plan i.e. protocol is prepared which acts like a recipe for conducting
the trial. The plan is carefully designed to safeguard the health of the participants
as well as answer specific research questions. It describes what would be done in
the study, how it would be conducted, and why each part of the study is
necessary. The same protocol in used in every study under multi-centre trials in
the same project.
The clinical trial process depends on the kind of trial being conducted. The
clinical trial team includes pharmacologists, pharmaceutical scientists,
physicians, clinical pharmacologists, statisticians and nurses as well as social
workers and other health care professionals. They check the health of the
participant at the beginning of the trial. They gives specific instructions for
participating in the trial, observe various parameters of the study in the
participant as indicated in the protocol and continue to stay with the participants
even after the trial is completed.
Protection of participants
All federally funded clinical trials and trials to evaluate a new drug or medical
device are subject to the Food and Drug Administration (FDA) regulation or the
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) guidelines. In India such
statutory requirement is outlined in Schedule Y of the Drugs and Cosmetics Act
and Rules where it is necessary to get the protocol approved or reviewed by the
Institutional Review Board (IRB) or the Institutional Ethical Committee (IEC).
Many institutions require that all clinical trials, regardless of funding, be
reviewed and approved by a local IRB/IEC. The Board or Committee, which
includes physicians, researchers, community leaders, medical professionals, non-
medical members and other members of the community, reviews the protocol to
make sure the study is conducted fairly and that the participants are not exposed
to risk, endangering the health. The legal status, composition, function,
operations and regulatory requirements pertaining to the IRB/IEC may differ
among countries. The IRB/IEC also decides how often to review the trial once it
has begun. Based on this information, the IRB/IEC decides whether the clinical
trial should continue as initially planned and, if not, what changes should be
made. An IRB/ IEC can stop a clinical trial if the study centre is not following
the protocol or if the trial appears to be causing unexpected harm to the
participants.
Eligibility criteria
Each study protocol has guidelines for who can or cannot participate in the
study. These guidelines, called eligibility criteria, describe characteristics that
must be shared by all participants. The criteria differ from study to study. Using
inclusion/exclusion criteria is an important principle of medical research that
helps to produce reliable results. The factors that allow someone to participate in
a clinical trial are called “inclusion criteria” and those that disallow one from
participating are called “exclusion criteria”. These criteria are based on such
factors as age, gender, the type and stage of a disease, previous treatment history,
current health status and other medical conditions. Before joining a clinical trial,
a participant must qualify for the study. Some research studies seek participants
with illnesses or conditions to be studied in the clinical trial, while others need
healthy participants. It is important to note that inclusion and exclusion criterias
are based on scientific norms. Thus, the criterias are used to identify appropriate
participants and keep them safe. The criterias are so chosen and grouped so that
at the end of the study, researchers would be able to answer the questions they
plan to study.
Enrolling participants with similar characteristics ensures that the results
would be due to what is under study and not because of other factors. In this
way, eligibility criterias help researchers achieve accurate and meaningful
results. These criteria also make certain that people who are seriously ill but
participating in the study, are not exposed to the risk.
Informed consent
Informed consent is the process by which participants learn the key facts about a
clinical trial before deciding whether or not to participate. It is also a continuing
process throughout the study so as to provide information to the participants. To
help someone decide whether or not to participate, the physicians and nurses
involved in the trial explain the details of the study. The informed consent form
should be bilingual. If the participant’s native language is not English,
translation assistance should be provided. The research team should provide an
informed consent document that includes details about the study, such as its
purpose, duration, required procedures, and key contacts. Risks and potential
benefits should be explained in the informed consent document. The participant
then decides whether or not to sign the document. Informed consent is not a
contract, and the participant may withdraw from the trial at any time. People
who agree to take part in the study are asked to sign the informed consent form.
However, signing the form does not mean that the person must stay in the study.
People can leave the study at any time either before the study starts or at any
time during the study or the follow-up period. If new benefits, risks, or side-
effects are discovered during the study, the researchers must inform the
participants. They may also be asked to sign new consent forms if they want to
stay in the study.
Place
Clinical trials usually take place in hospitals, medical or pharmaceutical
institutions, cancer centres, other medical centres, clinics and military hospitals
in cities and towns across India and in other countries. Clinical trials may
include participants at one or two highly specialised centres, or they may involve
hundreds of locations at the same time.
Benefits
Clinical trials that are well-designed and well-executed are the best approach for
eligible participants to:

1. Play an active role in their own health care.

2. Gain access to new research treatments before they are widely available.
3. Receive regular and careful medical attention from a research team that
includes doctors and other health professionals.
4. Help others by contributing to medical research.
5. Be the first to benefit from the new method under study. The approach
being closely studied and properly monitored may be more effective than
the standard approach.

Risks
There are also risks to clinical trials.

1. There may be unpleasant, serious or even life-threatening side-effects to

experimental treatment.
2. The experimental treatment may not be effective for the participant.
3. The protocol may require more of their time and attention than would a
non-protocol treatment, including trips to the study site, more pathological
tests, more treatments, hospital stays or complex dosage requirements.
4. New drugs or procedures under study are not always better than the
standard care to which they are being compared.
5. Participants in randomised trials will not be able to choose the approach
they receive.
6. Health insurance and managed care providers may not cover all patient care
costs in a study.

Sponsors
Clinical trials are sponsored or funded by a variety of organisations or
individuals such as physicians, medical and pharmaceutical institutions,
foundations, voluntary groups, and pharmaceutical companies, in addition to
federal agencies such as the National Institutes of Health (NIH), the Department
of Defense (DOD), and the Department of Veteran’s Affairs (VA). Trials can
take place in a variety of locations, such as hospitals, universities, doctors’
offices, or community clinics.

9.32.1 Types of Clinical Trials Treatment trials

Treatment trials test experimental treatments, new combinations of drugs, or new
approaches to surgery or radiation therapy to evaluate the efficacy of the
treatment. They are designed to answer specific questions about, and evaluate
the effectiveness of, a new treatment or a new way of using a standard treatment.
Prevention trials
Prevention trials look for better ways to prevent diseases in people who have
never had the disease or to prevent a disease from returning. These approaches
may include medicines, vitamins, vaccines, minerals, or lifestyle changes.
Diagnostic trials
Diagnostic trials are conducted to find better tests or procedures for diagnosing a
particular disease or condition. Diagnostic trials usually include people who
have signs or symptoms of disease.
Screening trials
Screening trials test the best way to detect certain diseases or health conditions.
They involve people who do not have any symptoms of disease.
Quality of life trials (or supportive care trials)
These trials explore ways to improve comfort and the quality of life for
individuals with a chronic illness.

9.32.2 Phases of Clinical Trials

The trials at each phase have a different purpose and help scientists answer
different questions.
Phase I trials (clinical pharmacological evaluation)
Phase I trials are the first step in testing a new approach in humans. In Phase I
trials, researchers test an experimental drug or treatment in a small group of
healthy human volunteers (20–80) for the first time to evaluate its safety,
minimum effective dose, determine a safe dosage range, and identify side-
effects. Drugs with significant potential toxicity, for example, cytotoxic drugs
are usually studied in patients instead of healthy volunteers. Diseases, which are
incurable including AIDS, are also studied in patients. Phase I trials usually take
place at a few locations only. The volunteers are divided into smaller groups,
called cohorts. Each cohort is treated with an increased dose of the new therapy
or technique. The highest dose with an acceptable level of side-effects is
determined to be appropriate for further testing. Early phase I clinical trials are
always of “open” design. Scientists start by giving a single dose, which is one-
twentieth to one-tenth of the predicted therapeutic dose based on previous
experiments to a smaller group of people, a slightly larger dose to the next
group, and so on, while closely monitoring the patients for side-effects.
Generally, the trial is only intended to determine the maximum tolerated dose
and the toxicity of the drug. This stage is also used to determine the best method
of administration of the drug. Some phase I trials test new combinations or new
dose schedules of drugs that are already shown to be effective. Cross-over
designs are also followed in this phase.
Pharmacokinetic studies conducted at this stage provide basic data for
subsequent phase II and phase III trials.
Phase II trials (controlled clinical evaluation)
Phase II trials study the safety and effectiveness of an investigating molecule or
agent or intervention, and evaluate how it affects the human body. In phase II
trials, the experimental study drug or treatment is given to a large group of
patients (50–300). Phase II trials are done to determine if a drug is actually
effective or not.
Most phase II studies are well-controlled, randomised trials following “cross-
over” design. Here one group of patients (subjects) receives the experimental
drug, while a second “control” group receives a standard treatment or placebo.
Placement of the subject into the drug treatment or placebo group is by random
chance (as if by the flip of a coin). Many investigators feel that the double blind
protocol—neither the patient nor the researchers (investigator, coordinator, etc.)
know who is getting the experimental drug—which is used in phase III trials
should also be used in phase II studies. Additionally, phase II studies are often
designed to determine the correct dosage, that is, the dosage with the least
number of side-effects that is most effective. These are often referred to as dose-
ranging studies. In general, the purpose of phase II studies is to provide the
pharmaceutical company and the FDA comparative information about the
relative safety of the experimental drug, the proper dosage, and the drug’s
effectiveness. The main targets of a “late” phase II clinical trial are to find out
the appropriate clinical dosage schedule and to establish the incidence of
beneficial and undesirable effects in the patient population.
In certain cases, phase II clinical studies may not be randomised and patients
in an advanced stage of a disease may get a promising treatment at the best dose
as can happen in a phase III study. Only about one-third of experimental drugs
successfully complete both phase I and phase II studies.
Phase III trials (extended clinical evaluation)
In phase III trials, the experimental study drug or treatment is given to large
groups of people (250–1000) to confirm its effectiveness, monitor side-effects,
compare it to commonly used treatments, and collect information that will allow
the experimental drug or treatment to be used safely. Comparison of the new
drug with the standard current therapy is carried out using randomly assigned
groups. This method, called randomisation, helps to avoid bias and ensures that
human choices or other factors do not affect the study results. Randomisation is
important to the scientific validity of the research. “Placebo controlled double
blind cross-over design” procedures are preferred. Undesirable interactions of
the new drug with other drugs are also studied. Further studies may have to be
conducted to find out the mechanism of action of the drug in humans.
In most cases, studies move into phase III testing only after they have shown
promise in phases II and I.
Phase IV trials (Post-marketing surveillance)
Phase IV trials are conducted to further evaluate the long-term safety and
effectiveness of a treatment. They usually take place after the treatment has been
approved for standard use. Several hundred to several thousand people may take
part in a phase IV study. These studies are less common than phase I, II, or III
trials. They may also monitor the use of the drug or treatment in different
populations, or look at quality-of-life issues. The main aim is to monitor efficacy
and safety under prescription use. Adverse drug reaction(s) and toxicity are also
revealed in this trial.
Schedule Y which lays down guidelines for undertaking clinical trials was
amended in December 2000 and published in January 2005.
Bioavailability and bioequivalence
Bioavailability is defined as the rate and extent to which a substance reaches the
systemic circulation. Bioavailability testing is a means of predicting the clinical
efficacy of a drug. They are designed to determine either an absolute
bioavailability or relative bioavailability i.e. bioequivalence (with a reference
dosage form which has good absorption characteristics). It is required by law to
undertake bioequivalence studies whenever a new formulation of an existing
drug product is introduced into the market.
Besides being legally necessary, bioequivalence studies are required for a
variety of reasons.

1. Results from clinical studies indicate that different drug products produce
different therapeutics results.
2. Results from bioavailability studies indicate that different products are not
bioequivalent.
3. Drug has a narrow therapeutic range.
4. Some drugs have a low solubility and/or a large dose is required.
5. Absorption is considerably less than 100%
6. High excipient/active drug ratio present in the drug product.

Bioequivalence criteria
The FDA (Food and Drug Administration) criteria for establishing
bioequivalence between two formulations is that the 90% confidence interval for
the ratio of the mean AUC and mean Cmax for the test product must be between
80–120% of the respective mean values for the reference or the innovator
product.
Methods of assessing bioavailability
There are several methods, which determine the bioavailability or
bioequivalence of a drug product. The vast majority of bioavailability studies
involve the administration of the test dosage form to a group of healthy human
subjects, followed by the collection and assay of the drug concentration in blood
(plasma or serum) samples. The second most frequent method utilises urinary
excretion measurements. Occasionally, other types of biological material, such
as saliva, cerebro-spinal fluid, bile, or faeces are also collected. For those drugs,
for which assay methods are not available for the determination of drug
concentrations in biological fluids, a pharmacological response may be
measured. Finally, some bioavailability assessments are made on the basis of a
determination of the therapeutic response of patients to a given dosage form.
FDA (US) recognises pharmacokinetic, pharmacodynamic, clinical, and in vitro
studies as acceptable approaches to document the bioavailability or
bioequivalence of a drug product.
Good clinical practice
It is vital to have a standard for the design, conduct, performance, monitoring,
auditing, recording, analyses, and reporting of clinical trials that provides
assurance that the data and reported results are credible and accurate, and that
the rights, integrity, and confidentiality of trial subjects are protected.
Guidelines for good clinical practices (GCP) are given in ICH topic E6. These
were adopted by FDA (US) as “Guideline for good clinical practice ICH
harmonised tripartite” in May 1996 and became operational in January 17, 1997.
The objective of this ICH GCP guideline is to provide a unified standard for the
European Union (EU), Japan and the United States to facilitate the mutual
acceptance of clinical data by the regulatory authorities in these jurisdictions.
GCP is an international ethical and scientific quality standard for designing,
conducting, recording and reporting trials that involve the participation of human
subjects. Compliance with this standard provides public assurance that the rights,
safety and well being of trial subjects are protected consistent with the principles
that have their origin in the Declaration Helsinki, and that the clinical trial data
are credible. “Ethical principles for medical research involving human subjects”
was adopted by the 18th WMC (World Medical Association) General Assembly
Helsinki, Finland in June 1964 and amended many times. The latest amendment
was by the 52nd WMA Gereral Assembly, Edinburgh, Scotland in October 2000.
The guideline was developed with consideration of the current good clinical
practices of the European Union, Japan, and the United States, as well as those
of Australia, Canada, the Nordic countries and the World Health Organization
(WHO).
This guideline should be followed when generating clinical trial data that are
intended to be submitted to regulatory authorities. The principles established in
this guideline may also be applied to other clinical investigations that may have
an impact on the safety and well being of human subjects.
Principles of ICH GCP

Clinical trials should be conducted in accordance with the ethical principles

that have their origin in the Declaration of Helsinki, and that are consistent
with GCP and the applicable regulatory requirement(s).
Before a trial is initiated, foreseeable risks and inconveniences should be
weighed against the anticipated benefit for the individual trial subject and
society. A trial should be initiated and continued only if the anticipated
benefits justify the risks.
The rights, safety, and well being of the trial subjects are the most important
considerations and should prevail over interests of science and society.
The available non-clinical and clinical information on an investigational
product should be adequate to support the proposed clinical trial.
Clinical trials should be scientifically sound, and described in a clear,
detailed protocol.
A trial should be conducted in compliance with the protocol that has
received prior inst tutional review board (IRB)/independent ethics
committee (IEC) approval or favourable opinion.
The medical care given to, and medical decisions made on behalf of,
subjects should always be the responsibility of a qualified physician or,
when appropriate, of a qualified dentist.
Each individual involved in conducting a trial should be qualified by
education, training, and experience to perform his or her respective task(s).
Freely given informed consent should be obtained from every subject prior
to clinical trial participation.
All clinical trial information should be recorded, handled, and stored in a
way that allows its accurate reporting, interpretation and verification.
The confidentiality of records that could identify subjects should be
protected, respecting the privacy and confidentiality rules in accordance
with the applicable regulatory requirement(s).
Investigational products should be manufactured, handled, and stored in
accordance with applicable good manufacturing practice (GP). They should
be used in accordance with the approved protocol.
Systems with procedures that assure the quality of every aspect of the trial
should be implemented.
 10

Standardisation of
Herbal Drugs

The process of evaluating the quality and purity of crude drugs by means of
various parameters like morphological, microscopical, physical, chemical and
biological observations is called standardisation.
Standardisation of herbal drugs is a painstaking problem as one prescription
might involve the mixture of many drugs in proper proportions.

1 0 .1 IMPORTANCE OF STANDARDISATION AND PROBLEMS

INVOLVED IN THE STANDARDISATION OF HERBS
10.1.1 Role in Identitification of Botanical Source
The botanical identity of a majority of the plants mentioned in the
pharmacopoeia of various indigenous systems of medicine has been established
since the introduction of the modem system of plant classification in India.
There are however a number of crude drugs where the plant source has not yet
been scientifically identified. In other cases it has been seen that, more than one
plant species, sometimes with widely different morphological and taxonomic
characters are considered the source of a particular drug. For example, Bacopa
monnieri, and Centella asiatica both described as Brahmi, have different
ingredients and have hardly any relationship with the properties ascribed to the
drug Brahmi. The Department of Indian Systems of Medicine and Homeopathy
(ISM & H) have established that Bacopa monnieri is Brahmi (Nira-Brahmi) and
Centella asiatica is Mandukapami (Brahmamanduki).
The true source of the crude drug in such cases can be located only after
detailed chemical and pharmacological studies. There are also various drugs
whose isolated chemical constituents demand a further investigation so as to
ascertain their usefulness.
Among the crude drugs which can be more than one plant or which have more
than one constituent, those belonging to the species of the same genera do not
pose much of a problem, as in most cases, the chemical composition is similar,
though, there may be a difference in the concentrations of the various chemical
compounds present. This happens when Ayurvedic doctors use substitutes.
While dealing with a substitute, we must keep in mind that Ayurvedic and
Unani drugs came into being long before the lineal concept of binomial
nomenclature was adopted for naming the plants. Thus a number of plants
belonging to the same genera were considered to be the source of a same
particular drug.
Sometimes plants were found having therapeutic properties similar to those
attributed to a particular one (a plant of different genera or even family) in
classical literature – they were then given the same name. This perhaps became
necessary due to the non-availability of the original classical plant source, in all
regions of a country having a large geographical expanse and markedly different
agro-climatic conditions.

10.1.2 Useful in Identification of Common Adulterants

The use of other plants than that mentioned in classical texts can cause
degradation of the quality of the drug. Adulteration of the genuine drug is the
other cause of degradation. Adulteration is practiced either knowingly or through
ignorance on the part of the collector. Since Ayurvedic doctors are generally
knowledgeable and conscientious in their field, it is only when a drug is
commercially produced that adulterants are noticed.
Pharmacognostic studies have been done on many important drugs, and the
resulting observations have been incorporated in various pharmacopoeias. It is,
however essential, that these should be defined properly in terms of chemical
and physico-chemical standards since pharmacognostic standards alone may not
always be adequate to ensure their quality.
In recent years, chromatography, a highly useful tool, has been used to find
out the quality and genuineness of a drug. Among the chromatography
techniques, TLC has been widely employed for the analysis of extractives
obtained from crude drugs. On account of its extreme rapidity and ease of visual
evaluation, TLC has become the most ideal analytical method for the systematic
identification and control of purity in plant drugs systematically.

10.1.3 Significant Role in the Quality Evaluation of Crude Drugs

Originating from Different Localities
It is well known that agro-climatic conditions and locality factors effect the
chemical composition and therapeutic properties of a drug. Ayurveda also
recognises these properties.
Sometimes, various varieties of the same plant, though originating from
different parts of India have been attributed with different therapeutic properties.
Determination of the major chemical constituents responsible for
pharmacological actions will help in formulating an appropriate yardstick to
evaluate the quality, as it is the whole crude drug which is used in drug
formulations based on indigenous systems of medicine.

10.1.4 Determination of Suitable Time of Harvest

The concentrations of the required chemical constituents in a plant are closely
influenced by the stage of growth and season.
Classical Ayurvedic literature also gives great importance to the period and the
stage of the plant’s growth when a required plant should be collected. In majority
of the cases, stress is laid on the collection of mature ripe parts. A lot of
importance is also attached to the period during which the crude drug is
collected.
For example, Holarrhena antidysenterica revealed a maximum concentration
of the total required alkaloids (3.2 to 3.9%) during the two flowering seasons,
i.e. March–April–May, and September–October.
Also, a study of the total alkaloidal contents in the leaves of Adhatoda vasica
during different periods of the year and geographical conditions revealed that the
highest yield (2.5%) is obtained during the months of July–October.
The stage of maturation of the part of the plant to be collected is also an
important factor.
For example, the fruits of Terminalia chebula are collected when they attain an
optimum size but are still green.
Roots of Withania somnifera are dug out after just 8–10 months of planting
while the stem of Tinospora cordifolia is collected only when fully mature.
 Literature pertaining to different systems of indigenous medicine provide this
information for a large number of crude drugs.
This however, has to be substantiated with scientific proof based On detailed
chemical examination of samples collected under different conditions of growth
and maturity.

10.1.5 Role in Specification of the Conditions for Drying, Storage,

and Shelf-life
Post-harvest processing, which requires a reduction in bulk of the material and
removal of moisture from it, is a very important exercise in maintaining the
quality of the material. Quick drying is necessary under controlled temperature
with the exposure conditions differing from material to material. Standards must
be laid down on the basis of minimum active principle contents in dried and
stored material. Crude drugs containing essential oils, glycosides, or flavonoid
compounds are dried under controlled temperature within a short time. For
example, Valeriana wallichi, Jatamansi roots, if dried at temperatures above
25°C lose much of its main pharmacologically active chemical compound.
Some drugs have to be used fresh to get the maximum concentration of active
principle(s) for a therapeutic action while there are plants which have to be bone
dry e.g. Rubia cordifolia.

10.2 STANDARDISATION OF SINGLE DRUGS AND COMPOUND

FORMULATIONS
The following schematic diagram explains the standardisation procedure
followed for single drugs and compound formulations (ISM &H).
Scheme 8.1 Work on the component ingredients

Scheme 8.2 Work on the formulation

1 0 .3 WHO GUIDELINES FOR QUALITY STANDARDISED
HERBAL FORMULATIONS
WHO has set certain standards for herbal drugs. It involves the following
parameters.
Botanical parameters
Sensory evaluation: Includes visual macroscopy/touch/odour/taste
Foreign matter: Includes foreign plants, foreign animals, foreign
minerals, etc.
Microscopy: Includes histological observation and measurements

Physico-chemical parameters
TLC/HPTLC finger print
Ash values: Total, acid-insoluble, water-soluble
Extractive values: In hot water, cold water and ethanol
Moisture content and volatile matter: Loss on drying (LOD),
azeotropic distillation
Volatile oils: By steam distillation
Pharmacological parameters
Bitterness value: Unit equivalent bitterness of standard solution of
quinine hydrochloride
Haemolytic property: On ox blood by comparison with standard
reference solution of saponin
Astringent property: Tannins that bind to std. Frieberg Hide
powder
Swelling index: In water
Foaming index: Foam height produced by 1 gm material under
specified conditions
Toxicological parameters
Arsenic: Stain produced on HgBr2 paper in comparison to standard
stain
Pesticide residues: Includes total organic chloride and total
organic phosphorous
Heavy metals: Like cadmium and lead.
Microbial contamination: Total viable aerobic count of pathogens:
Enterobacteriaceae, E. coli, Salmonella, P. aeruginosa, S. aureous.
Aflatoxins: By TLC using standard aflatoxins (B1 B2, G1 and G2)
Radioactive contamination

1 0 .4 ESTIMATION OF THE PARAMETER LIMITS USED FOR

STANDARDISATION
10.4.1 Determination of Foreign Matter
Drugs should be free from moulds, insects, animal faecal matter, and other
contaminants, like dust, soil, stones and extraneous matter. Foreign matter
sometimes also consists of parts of the organ(s) of the plant other than that
required for the drug by definition or beyond limits set by the WHO guidelines.
The amount of foreign matter should not be more than prescribed limit.
100–500 g of the drug sample should be weighed, or a quantity prescribed by
the WHO guidelines may be used. Foreign matter may be detected by inspection
with the unaided eye or by the use of a lens of 6X power. Separate the foreign
matter and calculate the percentage present.

10.4.2 Thin Layer Chromotagraphy

Preparation of plates
Silica Gel-G chromato plates of 0.2 to 0.25 mm thickness are used for the
purpose.
The plates can be prepared on a glass plate of 20 cm length. Prepared plates on
an aluminium base are also available (Sethi PD et al., 1997)
The coated plates should be activated for at least 1 hr at 100–105°C.
It is important to note that in case cellulose plates are being used, ten minutes
heating is sufficient.
Chromato plates should be stored in a place free from moisture and the plates
should be re-dried if necessary. The use of chromato plates prepared more than
2–3 days before should be avoided.

10.4.3 Determination of Ash Values

The quality of a drug can also be determined by the ash left after ignition. There
are four different methods which measure the ash: (i) total ash; (ii) acid-
insoluble ash; (iii) water soluble ash; (iv) sulphated ash.
Total ash
The method of total ash is designed to determine the amount of material that
remains after ignition. Ash can be classified as physiological ash which is
derived from the plant tissue itself and non-physiological ash which is the
residue after ignition of extraneous matter (e.g. sand and soil)
It is usually carried out at low temperatures possibly because alkali chlorides,
which are volatile at low temperatures, may be lost. The total ash consists of
carbonates, phosphates, silicates and silica.
PROCEDURE
About 2–4 gm of grounded material, should be accurately weighed into a
previously ignited and tared crucible (platinum or silica).
The material should be spread in an even layer in the crucible and ignited by
gradually increasing the heat to 500–600°C until it is white indicating that it is
free from carbon. The crucible should be cooled in a desiccator and weighed.
If carbon-free ash is not obtained in this manner, the crucible should be cooled
and the residue moistened with about 2 ml of water or saturated solution of
ammonium nitrate. After which, it should be dried on a water bath, then on a hot
plate, and ignited to a constant weight. The total ash should be calculated as the
percentage of ash with reference to the air- dried plant material.
Acid-insoluble ash
Acid-insoluble ash is the residue obtained on boiling the total ash with dilute
hydrochloric acid, and igniting the washed insoluble matter left on the filter. This
determination measures the presence of silica, especially sand and siliceous
earth.
PROCEDURE
25 ml of hydrochloric acid is added to the crucible containing the total ash, and
covered with a watch glass. It is boiled gently for 5 minutes. The watch glass is
rinsed with 5 ml of hot water and the liquid is added to the crucible. The
insoluble matter is collected on an ash-less filter paper and washed with hot
water until the filtrate is neutral. The filter paper containing the insoluble matter
is transferred to the crucible, dried on a hot plate, and ignited to a constant
weight.
 The content of acid-insoluble ash is calculated as a percentage of ash with
reference to the air-dried plant material.
Water-soluble ash
Water-soluble ash is the difference in weight between the total ash and the
residue obtained after boiling the total ash in water.
PROCEDURE
The total ash obtained, should be boiled for 5 minutes with 25 ml of water. The
insoluble matter may be collected on a Gooch crucible or an ash-less filter paper.
It should be washed with hot water, and ignited for 15 minutes at a temperature
not exceeding 450°C. The total weight of insoluble matter should be subtracted
from the weight of the ash. This difference in weights represents the water-
soluble ash. The percentage of water-soluble ash should be calculated with
reference to the air-dried drug.
Sulphated ash
Sulphated ash is the residue obtained when total ash is boiled with sulphuric acid
and washed and the insoluble matter left on the filter paper is ignited.
PROCEDURE
The residue obtained in total ash determination is cooled and moistened with 1
ml of sulphuric acid. Gentle heating is done until white fumes are no longer
evolved. Ignition is carried out at a temperature of 800 ± 25°C until all the black
particles disappear and is done in a place protected from air currents.
The crucible is cooled and a few drops of sulphuric acid are added and heated.
Ignition is carried out as before. Cooling is allowed and crucible weighed. The
operation is repeated until two successive weighings do not differ by more than
0.5 gm.

10.4.4 Determination of Extractable Matter

This method determines the amount of active constituents extracted with
different solvents from a given amount Of plant drug.
Alcohol-soluble extractive value
5 gms of the air-dried drug should be coarsely powdered, and macerated with
100 ml of alcohol of the specified strength in a closed flask for 24 hrs, shaking
frequently for the first six hours and allowing to stand for 18 hours. The
macerate is filtered rapidly taking precautions against loss of solvent.
25 ml of the filtrate is evaporated to dryness in a tared flat bottom shallow dish
and dried at 105°C, to constant weight. The weight is recorded. The percentage
of alcohol-soluble extractive should be calculated with reference to the air-dried
drug.
Determination of water-soluble extractive value
5 gm of the air-dried drug should be coarsely powdered and macerated with 100
ml of distilled water in a closed flask for 24 hrs, shaking frequently for the first
six hours, and allowing to stand for the next 18 hours. The macerate should be
filtered rapidly, taking precautions against loss of solvent.
25 ml of this filtrate is evaporated to dryness in a tared flat bottom shallow
dish, and dried at 105°C, to constant weight and weighed. The percentage of
water-soluble extractive should be calculated with reference to the air-dried
drug.

10.4.5 Determination of Moisture Content

Determination of moisture content helps in the estimation of the amount of
volatile mater i. e. the water that dries off from the drug. This procedure is more
appropriate for substances which appear to contain water as the only volatile
constituent.
About 10 gm of drug, (without preliminary drying), is weighed accurately
(within 0.01 gm) and placed in a tared evaporating dish.
For drugs made from underground tubers, the sample should be prepared by
cutting and shredding so that the parts are about 3 mm in thickness.
Seeds and fruits which are smaller than 3 mm should be cracked.
The use of high speed mills should be avoided and care must be taken that no
appreciable amount of moisture is lost during the sample preparation. After
placing the drug in the tared evaporating dish, drying should be carried out at
105°C for 5 hours. The sample is then weighed.
The drying and weighing is continued at one-hour intervals till the difference
between two successive weighing corresponds to not more than 0.25 per cent.
Constant weight is reached when two consecutive weighing after drying for 30
minutes, and cooling for 30 minutes in a desiccator, shows not more than 0.01
gm difference.

10.4.6 Determination of Volatile Oil in Drugs

Separation and determination of volatile oil is discussed under Section 8.15 and
8.16.

10.4.7 Determination of Minerals

The powdered drug should be cleaned visually to ensure that there are no dust
particles. The drug should be dried at 150°C to a constant weight.
The dried drug should be used for determination of dry ash. Pre-cleaned silica
crucible should be heated at 600°C until the weight of the cmcible is constant.
About 5 gm of drug should be taken in a silica crucible and heated in a muffle
furnace till there is no evolution of smoke. The crucible should be taken out,
cooled at room temperature by keeping it in a dessicator and the ash moistened
with concentrated sulphuric acid. It should then be heated on the heating mantle
till the fumes of sulphuric acid ceases. The crucible with sulphated ash should
then be heated in a muffle furnace at 600°C till the weight of the contents is
constant (2 to 3 hours).
The sulphated ash obtained above should then be dissolved in 100 ml of 5%
hydrochloric acid solution.
The solution should be stored in tightly-capped plastic bottles and directly used
for the determination of various mineral elements by using an atomic absorption
spectrophotometer.
NOTE: The standard solutions of all the mineral elements should be prepared.
The instruments should be caliberated and the concentration of minerals in ppm
should be measured directly in the test solution.

10.4.8 Qualitative Detection of Aflatoxins

Sample preparation
About 50 gm of powdered drug is weighed into a mixing jar. 25 ml of saturated
sodium chloride solution and 250 ml of methylene chloride should be added and
blended for 3 minutes at high speed. The resulting mixture should be filtered
through a high porosity folded paper into a 50 ml graduated cylinder.
50 ml of the filtrate is transferred to a 250 ml glass stoppered Erlenmeyer flask.
The extract is evaporated to near dryness on a steam bath and methanol–5%
sodium chloride hexane (50:50:50) added. Shaking should be done for 10
minutes on a wrist action shaker, and contents transferred to a 250 ml separating
funnel. The contents are allowed to stand for 5–10 minutes, and the lower
aqueous layer drained into another 250 ml separating funnel. 50 ml of carbon
tetrachloride should be added to the aqueous layer, and it should be shaken for 1
minute. The methylene chloride layer is drained into a 250 ml Erlenmeyer flask,
and the aqueous layer extracted with additional 25 ml methylene chloride. The
methylene chloride extracts should be combined and the combined extract
evaporated to near dryness on a steam bath.
Column chromatography
A bed of glass wool is kept in the bottom of the chromatographic column, and 1
cm high anhydrous sodium sulphate is added to give a base for the silica gel.
Methylene chloride is added to settle silica—it may be as high as 3 cms. Slowly
a 2 cm bed of anhydrous sodium sulphate is added.
The extract is dissolved in 5 ml methylene chloride and the extract solution
charged to the column. Elution should be done sequentially at maximum flow
rate with 40 ml methylene chloride; 40 ml benzene–acetic acid (9:1), 40 ml
hexane and 40 ml anhydrous ether. The elutes should be discarded.
Chloroform–acetone (80:20) is used as an eluting system for aflatoxins. The
elute is collected and evaporated to dryness on a steam bath under nitrogen. This
extract should be exercised for thin layer chromatography.
Aflatoxins reference sample
Different aflatoxin reference samples can be prepared in benzene–acetonitrite
(98:2).
Particulars for TLC
Thin layer plates: Pre-coated silica gel G F254 plates (10 x 20 cm) of uniform
thickness (0.2 mm) may be used.
Chromatographic chamber:Glass tank with lid
Solvent system: Chloroform–acetone–isopropanol (85:10:85)
Detection: Under a UV chamber fitted with a 15 watt long wave ultraviolet
lamp.
NOTE:For more information on extraction of volatile oil, please refer to Chapter
8, Part II.

10.4.9 Determination of Microbial Contaminants

Test for E. coli
A drug is tested for the presence of E. coli by taking 10 gms of powdered
material and making the volume up to 100 ml with lactose broth. This mixture
should be incubated for 4 hrs at 35–37°C. 1 ml sample from this is taken and
serial dilutions are made with 9 ml of MacConkey broth of concentrations 100
mg/ml, 10 mg/ml, 0.1 mg/ml and 0.01 mg/ml. These samples are incubated at
37°C for 18 hours. 1 ml of each is inoculated on MacConkey agar media, and
further incubated for 24 hrs at 35–37°C.
Colony characteristics
Growth of red, generally non-mucoid colonies of gram –ve rods indicating the
presence of E, coli.
Biochemical test
The colonies obtained on the MacConkey agar media are inoculated on peptone
water (5 ml) and the culture incubated for 24 hours at a temperature of 35–37°C.
After the incubation period, 3 ml of peptone culture is to be pipetted into a test
tube and equal volume of Kovac’s reagent is added. Appearance of a pink ring is
the indication of presence of E.coli. Similarly parallel tests are conducted with
pure strain.
Test for Salmonella
10 gm of powdered material is taken and the volume made up to 100 ml with
lactose broth. The mixture is incubated at 35–37°C for 4 hrs. A further 10 ml of
this sample is taken. 100 ml of tetra methylate brilliant green bile broth is added
to it and incubated at 37–40°C for 24 hrs. 1 ml sample is taken from it and
planted on a xylose lysin deoxycholate agar media. Deoxycholate citrate agar or
brilliant green agar can also be used. This is incubated at 35°C for 50 hours.
Colony characteristics
Small transparent and colourless with an opaque, pink (surrounded by a pink to
red zone) zone indicates the presence of S.typhi.
Biochemical test
The colonies obtained from the above culture are treated with polyvalent
salmonella antisera. This is known as serotyping or slide agglutination test.
Appearance of clumping of the cells confirms the presence of salmonella
species.
Test for Pseudomonas aeruginosa
10 gm of powdered material is taken and the volume made up to 100 ml with
nutrient agar broth. This mixture is incubated at 35°C for 4 hrs. 1 ml of this
sample is taken and 100 ml of soybean–casein digest broth is added to it. This
mixture is incubated at 35°C for 48 hours. 1 ml of this sample is plated on a
centrimide agar media, and incubated at 35°C for 48 hours.
Colony characteristics
Gram –ve rods with a usually greenish fluorescence indicates the presence of
P.aeruginosa.
Biochemical test
Two or three drops of freshly prepared 0.01 g/ml solution of N" N" N" N"
tetramethyl-P- phenylenediamine dihydrochloride R (known as oxidase reagent)
is applied on filter paper (Whatmann no. 1) and a smear of the suspected colony
is applied. Appearance of deep purple colour within ten seconds, indicates the
presence of Pseudomonas aeruginosa.
Test for Staphylococcus aureus
10 gm of the powdered material is taken and the volume made up to 100 ml with
NA broth. This mixture is incubated at 35–37°C for 4 hrs.
1 ml of this sample is plated on Baird-Parker agar media F, and incubated at
35–37°C for 48 hours.
Colony characteristics
Black colonies of gram +ve cocci, surrounded by clear zones indicate the
presence of S.aureus.
Biochemical test
Colonies obtained from Baird–Parker agar media are subjected to the
coagulase/DNAse test. In the coagulase test, formation of coagulum after adding
the Staphylococcus aureus culture and plasma (5–10 times diluted sample) and
after subsequent incubation for 2–4 hours, is the indication of the presence of
coagulase (i.e., Staphylococcus aureus positive). With the DNAse test, clearing
is seen on the agar medium.
Test for aerobic bacteria, yeasts and mould
10 gm of powdered material is taken and the volume made up to 100 ml with NA
broth. This mixture is incubated separately at two different temperatures i.e. at
35–37°C for 4 hrs and at 40–42°C for 4 hours.
From the mixture incubated at 35–37°C for 4 hours, 1 ml of sample is plated on
caseinsoyabean digest agar, and incubated at 35–37°C for 4 days.
Streptomycin is added to the mixture incubated at 40–42°C for hours. From
this mixture 1 ml of sample is plated on com meal agar media, and 1 ml of
sample is plated on SDA (Sabouraud’s dextrose agar media) media. Both the
com meal agar media sample and the SDA media sample are incubated at 35–
37°C for 4 days.
Limits of microbial contamination in medicinal plant materials
The limits vary according to the use of the material.
Plant materials harvested under acceptable hygienic conditions:

Per gram maximum 104 cells E.coli

maximum 105 Mould propagules

Plant materials which are pre-treated or plant materials to be usedfor topical
dosage forms:

Per gram maximum 107 Aerobic bacteria

Maximum 103 Saccaromeycetes and hyphomycees

Maximum 102 E.coli

Maximum 104 other enterobacteria

no salmonella at all

Other plant material for internal use:

Per gram maximum 105 Aerobic bacteria

maximum 103 Saccaromeycetes and hyphomycees

maximum 101 E.coli

maximum 103 other enterobacteria

no salmonella at all

10.4.10 Determination of Pesticide Residues

Pesticide residues produce toxic effects like irritation of the eye, lacrimation,
salivation, sweating, blurring of vision, breathlessness, colic; the systemic effects
includes hypotension, tachycardia, cardiac arrhythmias, vascular collapse,
respiratory paralysis, excitement, ataxia, convulsions. Hence the presence of
these contaminants in medicinal plants must be avoided.
Preparation of test sample
20–50 gm of powder is taken. Acetonitrile–water mixture (650:350) is added and
blended for 5 minutes at high speed and filtered. The filtrate is transferred to a
litre separating funnel and 100 ml of light petroleum ether is added. The contents
are then shaken for one or two minutes and 10 ml of sodium chloride (400g/4
litres) and 600 ml of distilled water added.
The separating funnel is shaken vigorously for 30–45 seconds, and the solvent
layer allowed to separate. The petroleum ether layer should be collected, and
washed with water thrice. This is then treated with anhydrous sodium sulphate.
The extract is then subjected to column chromatography. The column is packed
with activated fluorosil and eluted with petroleum ether. Three fractions of 200
ml each are collected. The first elute contains chlorinated pesticides like aldrein,
benzene hexachloride, DDT etc, while the second elute contains dieldrein and
the third elute contains malathion. The elutes are to be concentrated to 10 ml and
then used for thin layer chromatography.
Particulars for TLC
Standard samples: All the reference samples are prepared in petroleum ether.
Adsorbent: Pre-coated silica gel G F254 plate (10 x 20 cm) of uniform thickness
(0.2 mm).
Solvent system: N-hexane: acetone (7:3).
Detection: Under iodine treatment or UV chamber.

10.5 HERBAL EXTRACTS

The quality of the plant determines the quality of herbal extracts which can be
obtained from it. This necessitates that the medicinal herbs be looked after and
taken care of
For extraction, quality plants are first selected and from these, only the active
part or organ is used. These are dried and the drug obtained is checked for
pesticides. After clearance, they are sent to companies for extraction. These
companies after making sure that the drug is from the right species, first perform
a lab-scale extraction to determine/check the quality of the drug i.e. to ascertain
the concentration of active constituents in it.

10.5.1 Preparation of the Extracts

Extracts are produced as a result of a solvent’s action on the raw material i.e. the
drug/ crude plant. Various types of solvents can be used for this purpose, such as
water, alcohol, propylene glycol, butylene glycol and vegetable oils. The
concentration of active matter found in the final extract is dependent on the
concentration of the drug in the solvent. However, the actual quality of
extraction is more specifically determined by the extraction conditions, as they
affect the delivery of the extract. With alcohol, propylene glycol, butylene
glycol, water or a blend of them, we can extract water-soluble components such
as biophenolic fractions, vitamins, terpenes and amino acids.
Carotenoids and essential fatty acids can be extracted with oil.
Extraction can be carried until the point where the solvent is saturated by all
dissolved matters. Usually, 200 gms of dried herb per kg solvent is the limit in
the case of glycolic extracts and therefore, all commercial claims exceeding this
limit have to be taken very seriously.
 The consumer has also to keep in mind the shelf-life of the drug. An extract
made with 5 kilos of plant per kilo of solvent is useless if the initial plant has lost
most of its active matters because it has been stored for 3 years. It is also
worthless to buy an extract whose active matters have been destroyed by an
improper extraction process.

10.5.2 Evaluation of Herbal Extract

A herbal extract has to be clear, free of foreign particles, with no sludge or
precipitate and should stay like this, even if it is stored for a long time.
Colour of the extract
The colour of the extract depends upon the nature of plant. For example,
carotenoids from carrot give a red extract, and copper derivatives of spirulina
give a blue colour extract. However, if the colour of all the extracts is the same
brownish colour, it indicates unsatisfactory extraction techniques like oxidation,
beginning of degradation or beginning of the polymerisation of sugar
derivatives. So, the dark colour of extracts, especially, if it found in all the
extracts of the range, is not always a guarantee of quality, as it is widely thought.
Smell of the extract
Similarly, the smell of the extract is related to the nature of the plant and if the
extract does not smell like the plant, it indicates that the extract has been ill-
treated. For e.g. chamomile extract should smell like chamomile flowers.
However, glycolic extracts and even most hydroglycolic extracts are never
fragranced to the point that they replace perfumes in the final formulation.
From the above discussion, eyes and nose can be considered as the first
laboratory equipment to evaluate extracts.
Freedom from microbial contaminants
The extracts should be free from microbial contaminants. Though, we cannot
speak of absolute sterility, yet an extract should not have more than 100 non-
pathogenic germs per gram.
The extracts can be kept safe by using the right preservatives at the light
dosage level. This can be done either by the direct injection into a specific gelose
media or a filtration on a 0. 2 µ. membrane and then inoculation to the media.
Dry residue
Another indicator of the quality of an extract is dry residue. If a large amount of
drug was used to make the extract, there should be a high percentage of dry
residue. This dry residue comes from the extractible soluble part of the plant in
that solvent. Dry residue varies from plant to plant depending upon the chemical
composition.
The maximum theoretically extractible part of a dried plant is generally 10 to
20%, which means that herbal extracts made with 20% of the dried plant cannot
show a dry residue above 4%.
All other figures (lower or higher) have to be checked often by a
complimentary analytical method. The dry residue percentage has been
correlated by an UV spectrum to check whether there is indeed some active
constituents in the extracts or whether this dry residue is only obtained by adding
preservatives, salts or sugars.
The amount of dry residue can be estimated by placing 1 gram sample in an
oven at 118°C for 2 hours. It is then checked that a constant mass is obtained
even after two hours in the oven.
Refractometry
Reffactometry is also employed in the evaluation of a herbal extract.
Refractometry is linked to the results obtained on the dry residue test. The more
residue in solution, the higher is the refractive index. However, the final
results/figure also depends on the nature of the solvent i.e. the refractive index of
propylene glycol is higher than that of water.
PH of the extract
PH of the extract is also an indicator of the quality of an extract. Generally the
pH of cosmetic extracts range from 5 to 7. Excessive pH values whether acidic
or alkaline may lead to bad conservation.
UV and visible spectrophotometry
UV and visible spectrophotometry offer very fast and reproductive ways to assay
the presence of preservatives and also the amount of preservative, if present.
Further, it also helps to characterise the extract and evaluate its strength. This is
made possible as many active matters of plant absorb UV rays at a set
wavelength which is invariable and characteristic of the plant. The more active
the matter is, the stronger is the peak.
The procedure followed is given below: the product which is to be analysed is
diluted into a solvent, generally water in order to obtain an absorbance value
between 0, 1, and 1 for the characteristic wavelength. This measure is made
against a blank which is the dilution solvent used. In order to obtain values
which may be compared, the theoretical absorbance for the product is calculated
with a standard dilution at 1%. This characteristic value is generally called A%
or E%.
This has a wide appreciation at different dilutions. The spectra of the extracts
are made after dilution (using a volumetric flask) of each extract in order to
obtain an optical density between 0.2 to 1.0 absorption maxima. The absorbance
at the absorption maxima are measured in 1 cm thick quarter cells. In order to
make a quick comparison between, two extracts, the absorbance is brought to a
value, taking into account the dilution of the extract according to the formula:

where, E = Absorbance at an nm wavelength for a solution at 1%.

DO = measured optical density
D = Dilution in times number.
The higher the E% value, the stronger the substance concentration absorbing it
at this wavelength is. Thus, the general aspects of the curve is used as an
evaluation method during production as it helps to spot foreign matter or check
the presence of all the active matter for that extract. In other words, it helps to
check whether the extraction has been successfully achieved.
However, when compared to other analytical methods, UV spectrophotometry
is not very accurate. Still it is fast and does not necessitate experienced
laboratory personnel. The UV curve, therefore, may be established once all the
other tests have been completed. It will reflect the quality of the extract and
gives a rough finger print which may be a reference for quick quality control
approval.
Colorimetry assays
Colorimetry assays can also be used for evaluation of drug extracts. The general
principle of colorimetry assay is to allow contact between the components or the
family of components to be evaluated with a specific reagent to obtain a
coloured complex, which should follow Beer–Lambert’s law.
The intensity of the colouration is measured by spectrophotometry and is
directly proportional to the analysed substance. One may calculate this
concentration in relation with the standard range of the pure product.
This technique is simple although not very precise or accurate. However, it
may be performed to make a comparative analysis between two competitive
extracts of the same plant. A more accurate computation may be effected by
densitometry or by HPLC.
Thin layer chromatography
Thin layer chromatography offers a good method for evaluation of drug extract.
Thin layer chromatography is a first step towards the identification of the
chemicals present in the herbal extracts. It is a physio-chemical separation
method. The thin layer which is also called the stationary phase, is made of a
substance finely dispersed on a rigid plate (glass, aluminium, plastic). It is very
often made of silica. The solution of the unknown mixture is spotted on the plate
as a drop or as a lime-sized ball.
The loaded plate will then be introduced onto a well-closed tank containing the
appropriate element. The separation is obtained by the migration by of the eluant
using capillarity on the stationary phase.
All the chemicals in the mixture to be analysed migrate from the initial
injection point on the plate towards a final point. Each component will migrate
until it reaches this specific point according to its chemical structure. Once this
set distance has been reached, one can measure the relative position of each spot
of ingredient as compared to the front (Rf). A spot is said to have an Rf of 0.5 if it
reaches half the distance between the injection point and the front (of the
solvent).
The detection of the various spots may be effected either by a simple visual
observation if the components are naturally coloured or by the observation of
fluorescence emitted under UV. Sometimes it becomes possible to detect the
ingredient only after pulverisation of the plate by a reagent. The pulverised spot
gives, through a more or less specific reaction, a colouration visible under
fluorescence.
A chemical will always migrate to the same distance. Depending on the
chemical components, the plate and solvent used, the migrating phase and the
front will be different. If we have data on the standard Rf values of various
compounds, we can identify the chemicals in the extract by comparing the value
of the R f of the spot and the Rf value of the standard.
These TLC methods have wide applications. They are one-dimensional, easy
and fast. They are very often sufficient to characterise the extract of the plant.
However, separating these chemicals from the solvent is quite a problem as
most herbal extracts for the cosmetic industry contain glycols.
Densitometry
Densitometry is a process by which optic density in light-sensitive materials can
be measured. Lately it has become a standard in evaluation of extracts. It helps
to check the concentration of active matters which are spotted on the thin layer
chromatography plate.
A UV light beam is generated in the shape of a thin line and moved in curves
over the spots to be measured. A relationship between the quantities of each
substance on the plate may be measured by the difference of intensity of the
optical signal reflected by the plate and the intensity of the optical signal
reflected by the component. This difference in optical signals is transformed into
an electrical signal, the variation of which can be used to sketch a densitogram.
The densitogram can also be compared to the reading given by a standard on
the same plate.
Qualitative results can also be obtained as the respective surfaces of the peaks
in the densitogram (which denote the quantity of the chemical) can be calculated
by integration. Thus, densitometry is both a qualitative and a quantitative
method. This technique is very accurate, precise and valuable provided we use
good chromatographic plates. The calibration of the method is generally faster
than that made on HPLC, although the direction level is not as accurate.
HPLC technique
HPLC technique is also employed in the evaluation of herbal extracts. HPLC is a
kind of chromatography performed on columns, which separates and identifies
the various components of the herbal extracts and computes their relative
concentration. This type of analysis helps in the detection of very low
concentrations of ingredients to the level of ppm. New diode-based photo
detectors enable the analytical chemist to get a chromatogram where each peak
represents an isolated component. This makes it possible to draw the UV
spectrum of each peak at the same time. It is also possible to make sure that the
component being observed is the one required and that it is the only one to be
detected by comparing the spectrum with a spectrum of the standard.
This is used mainly to check the quality of raw materials or of their
pharmaceutical spray- dried herbal extracts. However, HPLC takes more time in
the preparation of solvents and columns. This has limited the use of HPLC in
standard quality control procedures. It is used more in research.
Infrared spectroscopy
Infrared spectroscopy can also be used to evaluate herbal extracts. Although it
can only confirm the nature of the solvent, it gives us some idea about the
concentration of active matters. This method enables suppliers to meet
specifications with a mere dilution of a so called ‘active’ into the chosen solvent.
It also makes an impure solvent to be proportionally very expensive.

10.5.3 Validation of Herbal Products

Validation is a tool for total quality management and ensures products of good
quality. Hence validation needs to be introduced into herbal science.
Herbal medicines are manufactured under different pharmaceutical processes to
result in various dosage forms such as extracts, tinctures, decoctions, pills,
powders, tablets, capsules, semi-solid pastes and jellies.
The concept of validation has expanded to encompass a wide range of activities
from analytical methods used for the quality control of drug substances and drug
products to checking equipment, facilities and the process for the manufacturing
of drug substances and drug products.
The purpose of validation of any material, equipment or process is achieved by
means of a validation protocol, which details the tests to be carried out, the
frequency of testing and results expected i.e. the acceptance criteria (K P R
Chowdary et al., 1997).
Process validation is done in two stages. In the first stage, critical process
parameters are identified and a protocol is designed defining the acceptance
criteria for each parameter. The critical process of the process parameters should
relate to various aspects of the process and give data to ensure that the product is
manufactured to consistently meet the specifications.
Raw material validation include identification of critical properties/characters
and designing a protocol laying specifications for each. Some critical properties
for raw material validation include (i) impurity profile and residual solvents; (ii)
colour; (iii) polymorphism; (iv) particle size distribution; (v) bulk density; (vi)
satisfactory microbiological profile.
In brief, validation of herbal products includes the following:
Raw material validation
Identification of the crude drug or drugs by

i. Pharmacognostical identification
ii. Botanical taxonomy
iii. Chemical identity/chemical tests
iv. Analytical data such as ash values, solubility, extractive values, LOD (%),
acid values, saponification values.
v. Identification of adulterants and substitutes
vi. Determination of active constituents/chemical composition
vii. Quality and purity of the herb
viii. Efficacy and toxicity testing.
Finished product validation

i. Organoleptic properties: colour, taste, odour, touch etc.

ii. Physical characteristics: viscosity, particle size, specific gravity, refractive
index.
iii. Chemical characteristics: pH, chemical tests/chemical identity/assay.
iv. Biological characteristics: efficacy and toxicity tests.
v. Microbiological characteristics: total count, tests for the absence of
pathogenic organisms.
vi. Stability testing to define shelf-life
vii. Storage conditions
viii. Packing system/unit.
Process validation
Some of the critical parameters for liquid/extractive formulations in general are
as follows:
1. Ratio of crude drug to solvent
2. Temperature
3. Length of time of extraction
4. Method of collection of extract
5. Method of concentration
6. Light sensitivity during process
7. Storage conditions and precautions during process.
For solid dosage formulation (powders, pills, capsules, tables)
1. Particle size distribution of drugs
2. Blending order and time of blending
3. Granulating fluid, binder concentration, granulating time.
4. Drying temperature and time
5. Moisture content
6. Tablet hardness
7. Tablet characteristics such as disintegration, friability
8. Tablet weight and thickness control
9. Spray rate of film coating solution
10. Core: coat (polymer) ratio
Semi-solid formulations
1. Solvent blend composition
2. Extraction process parameters such as amount of solvent, temperature,
length of time, method of collection of extractives etc.
3. Method of concentration
 4. Semi-solid blending time
5. Blend-homogenecity
6. Viscosity/rheological character
7. Light sensitivity, storage and other precautions during processing.
Validation consumes significant resources. However there are substantial
benefits. Materials and processes consistently under control require less process
support, will have less down-time, fewer batch failures and operate more
efficiently with greater output.
 11

Herbal Formulations:
A Comparative Study of
Ayurvedic and Modern
Dosage Forms

Pharmaceutical dosage forms prepared from parts like leaves, roots, rhizomes,
wood, bark, fruits, seeds, tubers, rhizomes, corms, flowers and flowering buds
used to combat diseases, are termed as herbal formulations.

11.1 CLASSIFICATION
Conventionally, herbal dosage forms can be categorised into two types, as
follows.
11.2 GENERAL CONSIDERATIONS
11.2.1 Selection of Herb or Herbal Part
Depending upon the pharmacological action required, the herb/herbal part can be
selected by reference to traditional literature, by phytochemical investigation of
each part of the plant and/or by clinical trials of extracts of different plant parts.
11.2.2 Purchase of Herbs
It is mandatory to buy the herbs from reliable botanical sources and get it
authenticated by a taxonomist. Dried herbs can be generally obtained from
herbal suppliers, some of who offer mail order service. Buying from shops may
seem more convenient and reliable as the herbs can be examined before
purchasing, but mail order companies often have a much higher rate of turnover
and may supply better quality and fresher herbs as a result. To gain the best
medicinal effect, a good quality product is essential. The following points should
be kept in mind, before buying the herb:

a. The herb should not have been stored in clear glass jars or in direct
sunlight, as this causes oxidation which affects their efficacy.
b. Good quality aromatic herbs should have a distinct scent and taste.
c. Signs of infestation due to poor drying techniques or adulteration should be
checked.
d. Herbs lose their colour as they age. Herbs of bright hue that have been well
dried and stored, and that are not too old should be bought, for example,
marigold flowers that are a vivid yellow/orange colour are likely to make
good medicine. If they have been stored on a shelf for 18 months, they will
probably look drab and pale and not be as effective as those of vivid colour.
11.2.3 Storage of Herbs
It is necessary to preserve the herbs properly, to prevent deterioration. Leaves,
flowers, roots and other parts should be stored in sterilised, dark glass containers
with air-tight lids. They may also be stored in new brown paper bags, which are
kept dry and away from light. Metal and plastic containers are not advisable, as
they may contaminate the herb. If stored in a cool and dark place, herbs can be
kept for about 12 months after harvesting. Herbs frozen in plastic freezer bags
can be used for up to six months. The container must always be marked with the
name of the herb, the source, the strength of preparation if appropriate, and date
of harvesting. The material must also be regularly checked for insect infestation.
If this occurs, put all affected material in a sealed plastic bag and discard.
11.2.4 Collection of Herbs
Aromatic herbs: Collect the leaves or flowers in clean dry weather, in the
morning after the dew has disappeared.
Flowers: Collect before or immediately after flowers open completely. The
collection must be done in clear, dry weather in the morning after the dew has
disappeared.
Leaves: Collect leaves after full development just before the leaf fades. Biennial
leaves should be collected during the second year.
Bark: It is usually collected in the spring or early summer by making suitable
longitudinal and transverse incisions on the stem or root of the plant.
Seeds: Collect seeds at the time of full maturity.
Bulbs: Bulbs should be dug out from the soil, just before the leaves decay.
Stalks: Collect stalks in the autumn.
Twigs: Collect twigs in autumn.
Root and rhizomes: Annuals are to be collected just before flowering. Biennials
are collected after vegetation of first year has ended while perennials are
collected either in the spring before vegetation begins or in the fall after
vegetation ends.
11.2.5 Drying of Herbs
Leaves: Leaves containing aromatic principles must be dried in the shade. But
they must be placed in sunlight for a short period of time to prevent fungus
attack. Unscented leaves may be dried in the sun, although it is best to dry them
in an airy, dry room.
Flowers: It is necessary to dry all the flowers carefully and rapidly in the shade
to preserve the colour and aroma (if the flowers contain volatile oils).
Roots: Fibrous roots are dried in the sun or artificially at temperatures from 65°F
to 80°F. Fleshy roots and rhizomes are cut into small transverse slices of about a
half inch in length and dried in the sun.
Bulbs: The outer membranes are peeled and the bulbs cut into transverse slices
about a half inch in length and dried in sunlight.
Barks, woods and twigs: May be dried in the sun.'
Apart from simple air-drying, there are other ways to preserve the herb’s
medicinal properties.
De-humidifying
De-humidifying is an effective but expensive way to dry herbs. It sucks the
water out of the plant. The dehumidifier should be placed in a more or less
sealed small room in which the herbs are hung in loose bunches or placed on
mesh trays. Herbs dry quickly with this method.
Freeze-drying
Freeze-drying retains the colour and flavour of the herb but is more suited to
culinary than to medicinal herbs. Whole herbs such as sage (Salvia officinalis)
can be frozen in plastic freezer bags. There is no need to de-frost before use as
the leaves crumble easily when still frozen.
Microwaving
It is possible to dry the herbs in a microwave oven. The cut parts should be
spread out on a sheet of paper and dried in the microwave according to the
temperature guidelines provided by the manufacturer. It takes about 2–3 minutes,
but the progress should be checked every 30 seconds. Re-arrange the parts to
ensure even drying.
11.2.6 Selection of Dosage Forms
Once final selection of herb or herbal part is made depending upon the medicinal
properties, the next decision the formulator has to make is about the dosage
form. He can select any dosage form from the list of traditional or modem
dosage forms as mentioned earlier or he can make a modified dosage form by a
combination of the traditional and modem dosage forms.

11.3 DIFFERENT STAGES OF HERBAL FORMULATION

The plant material is subjected to the following process steps.
11.3.1 Grinding
The selected plant material is dried and subjected to powdering with the help of
a hammermill or disc pulveriser, which has built-in sieves. The size reduction of
plant material is kept between 30–40 mesh. The purpose of powdering the
material is to disintegrate the organ, tissue and cell structure of the plant
material, so that the medicinal ingredients present therein are exposed to the
solvent with which it is to be extracted. Further, the size reduction provides
maximum surface area of the particle which in turn gives better mass transfer
between the material in contact with solvent. Size reduction between 30–40
mesh is optimal; a reduction beyond this may make the material slimy with
solvent which creates difficulty during filtration.
11.3.2 Extraction
The process of separation of medicinally active principles from the plant
material, with the help of suitable solvent, is called extraction. Generally, there
are three methods of extraction:
1. Hot aqueous extraction (decoction);
2. Cold percolation;
3. Solvent extraction
Hot aqueous extraction (decoction)
An open type of extractor is used for this purpose. The body of the extractor
contains a cy​lindrical vessel with a steam jacket. The bottom of the vessel is
provided with a false bottom having a filter cloth. One part of powdered drug
and sixteen parts of water are fed into the extractor. The heating is done by
injecting steam into the jacket. The material is allowed to boil till the volume of
the water is reduced to one-fourth of its original volume. By this time, the
medicinal ingredients present in the plant material are extracted out.
Cold percolation
The extraction of plant material is sometimes carried out in a percolator which is
a tall, cy​lindrical vessel with a conical bottom fitted with a built-in false bottom.
The percolator is connected to a condenser and a separator for stripping out
solvent from the marc.
The powdered plant material is fed into the percolator along with the suitable
solvent. The contact between the material and solvent is allowed to remain till
equilibrium of solute between the material and the solvent is achieved. The
solvent extract known as miscella is taken out; Fresh solvent is added into the
percolator and the miscella is drained out after the system acquires equilibrium.
In this way four to five washes of solvent are fed to the plant material till it gets
completely extracted. All washes from the percolator are clubbed and subjected
to concentration. Decoctions are explained later in Section 11.4.2 with a graphic
representation of the whole process.
Solvent extraction
The Soxhlet extractor is used for the solvent extraction process. It consists of an
extractor, a distillation still and a tubular condenser. The plant material is fed
into the extractor. Solvent is added till it reaches the siphon point of the extractor
and then the extract gets siphoned out into the distillation still. The distillation
still is heated with the help of steam. The vapours of the solvent get condensed
and the condensed solvent goes back to the extractor. The level of the solvent in
the extractor again rises to the siphon point and the extract is siphoned out into
the distillation still. In this way a number of cycles in which fresh solvent comes
in contact with plant material are carried out till the plant material is completely
extracted. The final extract in the distillation still is concentrated for recovery of
the solvent and the rich con​centrate is fed into a vacuum chamber drier where
the last traces of solvent are removed and a dry cake of the product is obtained
which is used as such or further purified as the need might be.
11.3.3 Filtration
The extract so obtained is separated out from the marc (exhausted plant material)
by allow​ing it to trickle into a hold tank through the built-in false bottom of the
extractor, which is covered with a filter cloth. The marc is retained on the false
bottom and the extract is re​ceived in the hold tank. From the hold tank, the
extract is fed into a sparker filter with the help of a pump to remove any fine or
colloidal particles present in the extract.
11.3.4 Concentration
The liquid extract so obtained is fed into a wiped film evaporator and
concentration is carried out under vacuum to get concentrated extract. The
concentrated extract is further fed to a vacuum drier to get a solid mass free from
solvent. The solid mass so obtained is either used directly in the desired
pharmaceutical formulation or further purified depending on the purity of the
product desired.
11.3.5 Spray Drying
The concentrated extract is subjected to a spray drier with the help of a high
pressure pump at a controlled feed rate to get dry powder.
11.3.6 Pharmaceutical Dosage Form
The dried powdered material so obtained is subjected to various procedures to
obtain the suitable dosage forms like capsule, tablet, pill or ointment.
The schematic representation of the various stages involved in the herbal
formulation process is shown in Fig. 11.1.

11.4 DOSAGE FORMS

11.4.1 Infusions
The dilute solutions of the readily soluble constituents of crude drugs are called
infusions. There are two types of infusions, namely, cold infusions and hot
infusions.

Fig. 11.1 Schematic representation of the various stages involved in the herbal
formulation process: 1: Open extractor, 2: Pump, 3: Fine filter, 4: Hold tank, 5:
Rotameter, 6: Spray drier, 7: Cyclone separator, 8: Tablet machine, 9: Percolator,
10: Hold-tank, 11: Wiped-film evaporator, 12: Condensor, 13: Separator, 14:
Solvent extractor, 15: Distillation-still, 16: Receiver, 17: Vacuum pump, 18:
Disintegrator
Cold infusions
When the active principles of the drug are volatile in nature or sensitive to heat
treatment, cold infusions are preferred. These are prepared by soaking the drug
in cold water or milk for several hours, and then straining the herb.
Hot infusions
These are prepared by pouring boiling water over the herb or herbal part and
keeping the in​fusion in a covered container for 5–10 minutes. Herbal teas are
usually hot infusions.
An infusion is the simplest way to prepare the more delicate aerial parts of
plants, espe​cially leaves and flowers, for use as a medicine or as a revitalising or
relaxing drink. It is made in a similar way to tea, using either a single herb or a
combination of herbs and may be drunk hot or cold.
METHOD OF PREPARATION
1. Place 2–3 gm of dried or 4–6 gm of fresh herb in the strainer of the tisane
cup.
2. Fill the cup with freshly boiled water
3. Fill the cup with the lid and infuse for 5–10 minutes before removing the
tisane strainer. Add a teaspoon of the sweetener, if desired.
The preparation is diagrammatically represented in Fig. 11.2.
NOTE: If the herb contains, volatile oils, proper precautions are needed during the
prepara​tion to prevent the loss of same by evaporation. Loss can be prevented by
covering the cup with a lid.

Fig. 11.2 Diagrammatic representation of the process of hot infusion

Pot infusion
Pot infusions are prepared by warming the pot, then placing 20 gm of dried herb
or 30 gm of fresh herb in it. Water that has just been boiled is then poured into
the pot. The lid is replaced and the mixture allowed to infuse for 10 minutes.
Some of the infusion is strained into a cup. A teaspoon of honey may be added,
if desired.
Dosage: 3–4 doses (500 ml) each day.
Storage: The infusion can be stored in a covered jug in a refrigerator or cool
place for up to 24 hours.
The following herbs can be used in infusion preparations.
Flowers: Chamomile, elderflower, clove, yarrow
Leaves: Peppermint, spearmint, pennyroyal, alfalfa, blackberry, blueberry,
catnip, costmary, lemon balm, red raspberry, rosemary, sage, thyme Berries:
Blueberry, juniper, raspberry, rose hips
Seeds: Anise, caraway, coriander, cumin, dill, fennel
Bark: Cherry, cinnamon
Root: Comfrey, ginger, ginseng
Rind: Organic rind from peeled oranges, lemons, grapefruit
Some infusion preparations
Infusion for cold and digestion
½ cup dried peppermint leaves; 1 cup dried alfalfa leaves; 3 tablespoons dried
lemon balm leaves; 3 tablespoons dried, grated organic lemon rind PROCEDURE
Add boiling water and steep for 5–10 minutes.
Infusion for de-toxification
2 cups clover blossoms; 2 thin sticks cinnamon, slightly bruised; 1 teaspoon
grated, dried organic orange rind PROCEDURE
Add boiling water and steep for 5–10 minutes.
Infusion containing vitamins
Mint; alfalfa; filaree; dandelion; romaine leaves; parsley; celery tops; carrot tops;
2 cups pineapple PROCEDURE
Place handfuls of these weeds and home greens in the juice extractor and use 1
cup to each 2 cups of pineapple. The juice obtained may be made into ice cubes
and can be used.
Infusion containing vitamin C
1 cup dried rose hips; 1 cinnamon stick; ¼ cup dried lemon balm leaves; 1
teaspoon dried, grated organic lemon rind.
PROCEDURE
Add boiling water and steep for 5–10 minutes.
Infusion for stress
Indian mallow (Abutilon indicum): 30%; country mallow (Sida cordifolia): 20%;
liquorice (Glycyrrhiza glabra): 20%; holy basil (Ocimum sanctum): 20%;
ashwagandha (Withania somnifera): 40%
PROCEDURE
Steep the mixture in boiling water. Add honey and lemon to taste: q.s.
Antitussive infusion
Cinammon (Cinnamomum zeylancicum) bark: 25%; liquorice (Glycyrrhiza
glabra): 30%; sweet violet ( Viola odorata) flowers: 45%
11.4.2 Decoctions
When plant constituents are not soluble in standing cold or boiling hot water, it
will often yield its soluble ingredients if simmered in almost boiling water for 30
minutes or more.In this process, the crude drug is boiled in a specified volume of
water for a defined time, cooled and strained.
A decoction is generally made using roots, rhizomes, bark and berries but
sometimes leaves and flowers may also be included. For decoctions, fresh herbs
should be sliced while dried herbs should either be powdered or well bruised.
METHOD OF PREPARATION
1. lace 20 gm of dried or 40 gm of fresh herb in a saucepan.
2. Cover with 750 ml of cold water and subject to boiling. Simmer for about
20-30 minutes, until the liquid is reduced to about 500 ml.
3. Strain the liquid through a sieve into a jug. Pour the required amount into a
cup, cover the jug and store in a cool place.
The schematic representation of the preparation is shown in Fig. 11.3.

Fig. 11.3 Schematic representation of the preparation of a decoction

NOTE:
1. Decoctions should always be strained while hot, so that the matter which
separates on cooling may be mixed again with the fluid by shaking when
the remedy is used.
2. Use glass, ceramic or earthenware pots or clean unbroken enameled cast
iron. Do not use plain cast iron with astringent plants.
Dosage: 3–4 doses (500 ml) each day.
Storage: It can be stored in a covered jug in a refrigerator or cool place for up to
48 hours.
Some decoction preparations
Decoction for indigestion
Calumba (Jateorhizapalmata): 5 gm; sweet flag (Acosus calamus): 10 gm;
water: 750 ml Dose: ½ cup twice a day.
Contra-indication: It should not be taken during pregnancy.
Decoction for relieving menstrual pain
Corydalis (Corydalis yanhusuo):10 gm; cinnamom: 3 gm; water: 500 ml
Dose: 100 ml twice a day.
Contra-indication: Not to be taken during pregnancy
Decotion for constipation
Senna pods : 1–2 gm; fresh ginger: 1 gm
PROCEDURE
Steep in 1 cup of freshly boiled water for 6–12 hours, strain and drink
Decotion for anaemia
Astragalus (Astragalus membranaceus): 12 gm; angelica (Angelica ginensis): 12
gm; water: 500 ml Dose: Two cups daily.
Decoction for bronchial asthma
Coleus (Coleus forskohlii) root: 30 gm; water: 1000 ml
Dose: Should be drunk in small doses over two days.
Decoction for coughs and shortness of breath
Schisandra (Schisandra chinensis, crushed berries): 10 gm; water: 200 ml Dose:
Should be divided into 3 doses and drunk during a 24-hour period.
Contra-indication: Large doses can cause heartburn.
Decoction for poor circulation
Ginger : 3 teaspoonful; prickly ash berries (Zanthoxylum americanum): 3
teaspoonful; water: 750 ml Dose: 1 cup twice a day.
Contra-indication: It is not to be used during pregnancy nor taken internally, if
suffering from inflammatory stomach conditions.
Decoction for stress
Ashwagandha root ( Withania somnifera): 10 gm; water: 200 ml.
Dose: Drink in small doses over two days.
11.4.3 Tinctures
Tinctures are solutions of medicinal substances in alcohol or diluted alcohol.
They are made by soaking a herb in alcohol. This encourages the active plant
constituents to dissolve, giving tinctures a relatively stronger action than
infusions or decoctions. They are convenient to use and last up to two years.
Tinctures can be made using a jug and a jelly bag, instead of a wine press.
METHOD OF PREPARATION
1. Place the herb (200 gm dried or 300 gm of fresh, chopped herb) in a large,
clean glass jar and pour the alcohol (35–40%) on it ensuring that the herb is
covered. Close and label the jar. Shake well for 1–2 minutes, then store in a
cool dark place for 10-14 days, shaking the jar every 1–2 days.
2. Set up the wine press, placing a muslin or nylon mesh bag securely inside.
Pour in the mixture and collect the liquid in a jug.
3. Slowly close the wine press, extracting the remaining liquid from the herbs
until no more drips appear. Discard the left-over herbs.
4. Pour the tincture into clean, dark glass bottles using a funnel. When full,
stopper with a cork or screw top and label the bottles.
Figure 11.4 is a diagrammatic representation of the process.
Storage: Can be stored in sterilised, dark glass bottles in a cool dark place for up
to 2 years.
Dosage: 5 ml (1 teaspoonful) to be taken 2–3 times a day diluted in 25 ml of
water or fruit juice.
Tinctures are strong preparations and it is essential to check that the
recommended dosage is used. Industrial alcohol, methyl alcohol or rubbing
alcohol (isopropyl alcohol) should never be used in tinctures.
Fig. 11.4 Schematic representation of the preparation of tinctures
Alcohol-reduced tinctures
Alcoholic tinctures should sometimes be avoided, for example, during
pregnancy or a gastric inflammation. Adding 5 ml of tincture to a small glass of
almost boiling water and leaving it for 5 minutes allows the alcohol to evaporate.
To make non-alcoholic tinctures, replace the alcohol with vinegar or glycerol.
Some tinctures preparations
Tincture for gastric uneasiness, nausea and flatulence: Compound tincture of
lavender ¼ fluid ounce oil of lavender; ¼ fluid ounce oil of rosemary; ½
ounce cinnamon powder; ¼ ounce fine nutmeg powder; 1 handful
moderately fine red saunders (sandalwood) powder; 1 ½ pints alcohol; 2
cups water.
PROCEDURE
Dissolve the oils in the alcohol. Add the water. Mix the powders. Moisten the
mixture with a fluid ounce of the alcoholic solution of the oils. Pack it into a
conical percolator such as the chemex, and gradually pour over it the remainder
of the alcoholic solution.
Strong tincture of ginger for digestive problems
Ginger (moderately coarse powder); alcohol (90%)
PROCEDURE
Moisten the drug with a portion of the menstrum and set aside for 4 hours. Pack
in the per​colator. Pour on sufficient menstrum to saturate the column of drug
throughout its length and also form a layer above it. Set aside for 24 hours.
Commence percolation and continue until the volume of percolate collected is
75% of the volume of the finished product. Press the marc. Mix the liquids and
add menstrum to produce the required volume and filter.
Tincture of strophanthus (a heart tonic)
PROCEDURE
Moderately coarse powder of strophanthus is taken and dried at 45°C. Pack in a
percolator. Macerate with light petroleum for 24 hours. Commence percolation
and continue until the percolate is colourless. Dry the de-fatted mass in a current
of warm air. Re-pack in percola​tor and macerate for 48 hours with the menstrum
(70% alcohol). Commence percolation and continue until 500 ml has been
collected.
Tincture for arithritis
Pepper (Capsium frutescens); willow bark
PROCEDURE
Make the tinctures of the above herbs separately; combine 20 drops of capsicum
tincture with 100 ml of willow bark tincture.
Dose: 1 teaspoonful with water twice a day.
Tincture for poor appetite
Cardamom (Elettaria cardamomum); yellow gentian {Gentian lutea)
PROCEDURE
Make the tinctures of the above herbs separately; combine 5 drops of cardamom
tincture and 15 drops of gentian tincture.
Dose: One teaspoonful, three times a day.
Tincture for bronchitis
Wild sunflower (.Inula helenium); thyme
PROCEDURE
Make the tinctures of the above herbs separately; mix 50 ml of Inula helenium
tincture with 50 ml of thyme tincture for use.
Dose One teaspoonful, 3 times a day
11.4.4 Capsules
Powdered herbs are most easily taken as capsules but can be sprinkled on food
or taken with water. Externally, they can be applied as dusting powder to the skin
or mixed with tinctures as a poultice.
Reputable herbal suppliers are the best source of powdered herbs and in
general, the finer the powder the better the grade and quality. Gelatin or
vegetarian capsule cases are also available from specialist outlets.
METHOD OF PREPARATION
1. Pour the powder into a saucer and slide the capsule halves towards one
another, scoop​ing up the powder (or use a capsule-making tray).
2. When the halves of the capsule are full of powder, slide them together
without spilling the powder and store.
Storage: Store in air-tight, dark glass containers in a cool place for up to 3–4
months.
Dosage: Take 2–3 capsules twice a day or as directed by the physician.
A schematic representation of the preparation of capsules is given in Fig. 11.5.
Some capsule preparations
Capsule for cystitis
Dried fine powder of Buchu (Barosma betulina)
PROCEDURE
Fill 500 mg of powder in an empty capsule.
Dose: 500 mg capsule, twice a day

Fig. 11.5 Schematic representation of the preparation of capsules

Capsule for bronchial catarrh
Dried fine powder of myrrh (Commiphora molmol)
PROCEDURE
Fill 300 mg of powder in each capsule.
Dose: 300 mg capsule twice a day
Capsule for cold
Dried fine powder of coneflower (Echinaceae angustifolia)
PROCEDURE
Fill 500 mg of powder in each capsule.
Dose: 500 mg capsule three times a day
Capsule for hyperacidity
Adulsa (Adhatoda Vasica) leaves; holy basil (Ocimum sanctum) herb; ginger
(Zingiber of​ficinale) rhizome PROCEDURE
Mix the dried powder of the above herbs in equal quantities and fill 250 mg in
each capsule.
Dose: 250 mg capsule three times a day.
Capsule for hypertension
Garlic (Allium sativum) bulbs; arjuna (Terminalia arjuna) bark
PROCEDURE
Mix the dried powder of the above herbs in equal quantities and fill 250 mg in
each capsule.
Dose: 250 mg capsule twice a day
11.4.5 Medicated Wines
Medicated wines are also called tonic wines. These are an agreeable way to take
strengthen​ing and tonic herbs to increase vitality and improve digestion. These
are made by steeping tonic herbs, such as Chinese angelica or bitter herbs such
as southernwood, in red or white wine for several weeks.
The wine is rather inexpensive and has some long-range advantages in plant
medicine. Because of its alcohol content, it immediately dissolves any plant
substances which are normally insoluble in water. Alcohol helps to resist the
plant’s tendency to spontaneously change. The grape acid also helps to increase
the solvent power of the wine.
Wine is less stimulating for the body than high proof spirits and can be used for
sipping in small quantities, while, tinctures may only be used by the teaspoon.
METHOD OF PREPARATION
1. Place 100 gm dried or 200 gm fresh tonic herbs or 25 gm dried bitter herbs
in a clean jar or vat. Pour in enough wine (usually one litre of red or white
wine) to cover the herb completely. Close the jar securely, shake carefully
and allow to stand.
2. Allow the wine to mature over 2 (or preferably 6) weeks, then take a dose
from the tap or jar. Regularly top up the mixture with wine.
Figure 11.6 gives a diagrammatic representation of the preparation of
medicated wines.
NOTE: During the preparation, the jar needs to be covered well. If exposed to air,
the herbs may go mouldy, making the remedy not only ineffective but unsafe to
take.
Storage: Use a ceramic vat with a tap at its base or a sterilised glass jar. It can be
stored for 3–4 months, ensuring that the wine covers the herbs. If the herbs go
mouldy, discard the medicine.
Dosage: One sherry glass (about 70 ml) each day.
Fig. 11.6 Schematic representation of the preparation of medicated wine
Herbal wines
Herbal wines are made by fermenting the herb in the same way that wine is
fermented from grapes. With the correct equipment, this is a simple process, but
fermentation alters the activity of the herbs and reduces their medicinal values.
Some medicated wine preparations
Medicated wine for digestive problems
1 bottle white wine or Madeira red wine; 1 handful chamomile flowers.
PROCEDURE
Add the flowers to the wine. Steep the mixture for 7–10 days and strain. Use in
tablespoon doses for stomach problems.
Medicated wine as digestive aid
2 pints claret wine; 2 tablespoons sage leaves; 2 tablespoons thyme leaves; 2
tablespoons hyssop leaves; 2 tablespoons spearmint leaves; 2 tablespoons
wormwood leaves; 2 table​spoons Maijoram herb.
PROCEDURE
Chop the herbs into a coarse powder. Moisten the powders with some of the
claret. Prepare the wine as for digestive problems.
Medicated wine for nervous problems (like epilepsy and vertigo)
1 bottle white wine; 1 handful fresh rosemary leaves; 2 tablespoons dried bonage
leaves.
PROCEDURE
Steep leaves in white wine for about a week or ten days. Strain out the herbs.
11.4.6 Syrups
Preparations formulated by the incorporation of sugar with vegetable infusions,
decoctions, expressed juices, fermented liquors or simple water solutions are
called syrups. Honey and unrefined sugar are effective preservatives and can be
combined with infusions or decoctions to make syrups and cordials. They have
the additional benefit of having a soothing action and therefore make a perfect
vehicle for cough mixtures as well as relieving sore throats. With their sweet
taste, syrups can disguise the taste of unpalatable herbs and are therefore greatly
appreciated by children.
A syrup is made with equal proportions of a herbal infusion or decoction and
honey or unrefined sugar. When making an infusion or decoction for a syrup, it
needs to be infused or simmered for a long time in order to optimise its
medicinal action. Infusions should be sim​mered for 15 minutes and decoctions
should be simmered for 30 minutes. Press the soaked herb through the strainer or
sieve to remove as much liquid as possible. A small amount of neat tincture can
be added to the cooled syrup to increase its effectiveness.
Syrups may also be made with tinctures instead of infusions or decoctions. 500
gm of honey or unrefined sugar is combined with 250 ml of water. It is gently
heated until all the sugar or honey has dissolved and the mixture has thickened.
It is removed from the heat. Once cooled, one part of the tincture, or mixture of
tinctures is stirred, into three parts of the syrup and stored as directed above.
Some examples of syrup include simple syrup, orange syrup, tolu syrup,
raspberry syrup, wild cherry syrup.
METHOD OF PREPARATION
1. Pour the 500 ml of infusion or decoction into a pan. Add sufficient honey or
sugar. Heat for a sufficient period of time with constant stirring until all the
honey or sugar has dissolved and the mixture has a syrupy consistency.
Remove from heat and cool.
2. Pour the cooled syrup into sterilised glass jars using a funnel and store in a
cool dark place. Seal the jars with cork stoppers.
Preservation and storage: Any simple syrup can be preserved by substituting
glycerine for a certain portion of the syrup. They can be stored in dark glass
bottles with cork tops in a cool place for up to 6 months. They should be stored
at a temperature not exceeding 30°C.
 A schematic representation of the preparation of syrup is given in Fig. 11.7.

Fig. 11.7 Schematic representation of the preparation of syrup

NOTE:Always make syrups in small quantities.
Dosage: 1–2 teaspoonful, three times a day

PREPARATION
Sucrose: 667 gm; purified water: q.s. to 1,000 ml
Dissolve the sugar in the water while heating. Raise the temperature to the
boiling point, and strain the solution while hot. Then add enough extra distilled
water through the strainer to make required weight.
Some syrup preparations
Raspberry syrup
Raspberry syrup is prepared from the fresh juice of ripened raspberries and
possesses a very pleasant taste that is enjoyed by children. It is particularly
useful for masking the taste of bromides, citrates and other saline drugs, as well
as for antipyrine and substances having an acid taste.
Cherry syrup
Cherry syrup is prepared from the juice of fresh ripe fruit. It is used for
antibiotics, cough preparations and sulfa-antibiotics. It has a pH of 3.8 and a
buffer action, which makes it especially valuable as a vehicle for acids.
Compound sarsaparilla syrup
Sarsaparilla syrup is a suitable vehicle for iodides. Many pharmacists have
considered the syrup to be an inert vehicle. However, it has been suggested that
the saponin present in sarsaparilla may increase the absorption of medicaments
administered in preparations contain​ing sarsaparilla.
Glycyrrhiza syrup
Glycyrrhiza syrup is an excellent masking agent for bitter substances. It is not
suitable for disguising the sour taste of acidic drugs as the glycyrrhizin is
decomposed by the acid with resulting precipitation. It has a pH of 6.
Mulberry syrup
1 pint mulberry juice; 2 pounds refined sugar; 2 ½ fluid ounces rectified spirit
(or gin) PROCEDURE
Heat the mulberry juice to boiling point and when it is cooled, filter it. Dissolve
the filtered sugar in the filtered liquid with gentle heat, and add rectified spirit.
Let the strained juice stand for eight to fifteen hours, and ferment. The juice
separates into two portions. The upper layer is clear. Separate this section by
straining and make into syrup by adding water.
Ginger syrup
Strong ginger tincture: 50 ml; syrup sufficient to produce 1000 ml. Mix
Tolu balsam syrup
Tolu balsam: 12.5 gm; sucrose: 660 gm; purified water, q.s. to produce 1000 gm
PROCEDURE
Add boiling purified water to the Tolu balsam contained in a tared vessel. Cover
the vessel lightly and boil the contents gently for 30 minutes, stirring frequently.
Add purified water to adjust the specified weight. Cool, filter the solution and
add sucrose. Heat the solution on a water bath to dissolve the sucrose. Finally,
add sufficient purified water to produce the required volume.
11.4.7 Tablets
Tablets are the solid dosage forms of powdered herbs, herbal extracts or their
constituents prepared by moulding or compression. Certain additives are also
added to the medica​ments in the formulation of tablets. Tablets are usually
circular in shape and may be flat or biconvex.
METHOD OF PREPARATION
The following steps are involved in the preparation of tables:
1. Weighing the ingredients;
2. Mixing the powdered ingredients and excipients;
3. Converting the mixed ingredients into granules;
4. Compression of granules into tablets;
5. Coating of tablets;
6. Evaluation of tablets;
Weighing of ingredients
If crude drugs are used they must first be ground into fine powder and passed
through a no. 100 mesh sieve. The fine powder (or other medicaments) and other
ingredients must be weighed accurately using a balance of good quality.
Mixing
All the medicaments and excipients are mixed uniformly, to prepare a
homogeneous mass, so that uniform tablets can be manufactured. The mixing of
ingredients should be done in an ascending order of their weight.
Converting the mixed ingredients into granules
Granules offer the following advantages over fine powder.
Flow property. Granules flow evenly through the hopper of the tablet machine.
Hence, tablets of uniform weight can be prepared.
Uniform composition: Granules may be bigger or smaller but are uniform in
composition. The ingredients are bound together and cannot separate. Therefore,
separation of smaller granules at the bottom is not going to change the
composition of the tablets.
Knitting power: Granules have more knitting power. Hence, on compression, the
granules form a sound tablet.
Sticking: Granules being heavier do not blow out of the die and therefore do not
stick to the machine.
The mixed ingredients can be converted into granules by the following methods.
1. Moist granulation method
2. Dry granulation method
3. Granulation by preliminary compression
Moist granulation method
This is the most widely used method. Here, the uniformly mixed ingredients are
moistened with a sufficient quantity of granulating agent to make a coherent
mass. Then, the mass is passed through a sieve no. 8 or 10. If the mass sticks to
the wire of the sieve it indicates over-moistening. The wet granules are spread in
trays and dried at 60°C in a hot air oven. The dried granules are passed through
sieve no. 20 to collect granules of uniform size. A schematic representation of
this process is shown in Fig. 11.8.
Dry granulation
The crystalline medicaments or granular medicaments are passed through sieve
no. 20 or any other specified sieve and then mixed with any additional excipient
(Fig. 11.9).
Granulation by preliminary compression
This is also called the slugging method, preferred in those cases where the
medicament is unstable in the presence of moisture. Here, the dry powder is
compressed into large tablets or slugs. These slugs are subjected to breaking into
small pieces which are passed through a specified sieve to collect the granules of
suitable size (Fig. 11.10). The lubricating agent and disintegrating agent are
mixed with these granules before compression into the tablet machine.
Fig. 11.8 Schematic representation of the wet granulation method

Fig. 11.9 Schematic representation of the dry granulation method

Fig. 11.10 Schematic representation of the direct compression method
Compression of granules into tablets
The dried granules obtained above are compressed into tablets by using a ‘tablet
making machine’. The compression is achieved by filling the required quantity
of granules into the die and then compressing them in between the lower punch
and the upper punch. The various tablet making machines in use includes single
punch tablet machine, multi-punch tablet machine, rotary tablet machine (Fig.
11.11) and dry cota tablet machine.
The single punch tablet machine is used for small-scale preparation (Fig.
11.12). It may be hand operated or electrically operated. The other machines are
used for large-scale manufacturing.
Coating of tablets
Coating of a tablet is required to mask the unpleasant taste and odour, to improve
the appear​ance of the tablet, to protect the medicament from atmospheric effects,
to control the site of action of drugs and to produce sustained release of the
product. The coating is generally carried out either by using pan coating or press
coating.
Pan coating is done in a pan (Fig. 11.13) made up of copper or stainless steel.
The pan is rotated with the help of an electric device. At first, the tablets to be
coated are placed in the pan. Hot air is blown at a particular speed or the pan
adjusted so that the tablets remain sepa​rated from each other. After coating,
polishing is done in a polishing pan. The pan coating technique is used for sugar
coating, film coating and enteric coating.
Sugar coating involves various stages which include sieving, sealing, sub-
coating, syrup coating and polishing.
In film coating, the tablets are coated by a single or mixture of film-forming
polymers. The polymer is dissolved in some volatile organic solvent and is
sprayed over the tablets in a rotating pan. The coating process is continued till a
uniform good film is formed over the tablets. Film coating is also used to make
the tablets water-proof before the sugar coating.
Enteric coating of the tables is carried out in a rotating pan. The solution of
enteric coating material is prepared in a volatile organic solvent and sprayed
over the tablets which are rotated in the coating pan. The hot air which is blown
into the pan helps in the evapora​tion of the organic solvent.

Fig. 11.11 Rotary tablet machine

Fig. 11.12a Single punch tablet making machine (hand operated)
Press coating is a completely automatic process wherein granules of coating
material are prepared and a layer of coating material is placed below and above
the tablet to be coated.
Evaluation of tablets
The following parameters should be checked while evaluating the quality of a
tablet: shape; appearance; content of the medicament in the tablets; uniformity of
weight; disintegration; dissolution; mechanical strength; friability.
Shape
Tablets are usually circular with either flat or biconvex faces. The diameter size
and shape of the tablets depends upon the die and punches used for preparing the
tablets.
Fig. 11.12b Single punch tablet making machine (hand operated): expanded
Fig. 11.13 Tablet coating pan
Appearance
Overall, the tablet must be aesthetic in appearance. It involves the measurement
of many factors such as size, shape, surface texture, odour and taste.
Content of the medicament in the tablets
It is necessary to ensure that every tablet (either coated or uncoated) contain the
stated amount of medicaments within the prescribed limits. The required
amounts of medicaments are given in the pharmacopeias. The tablet’s contents
must be within the limits.
Uniformity of weight
All the tablets of a particular batch must be uniform in weight, although a small
variation in the weight of individual tablets may occur. A little variation is
allowed by pharmacopoeias. Table 11.1 gives the percentage deviation in weight
variation allowed for uncoated tablets.
Table 11.1 Permitted percentage duration in weight of tablet

SI. No. Average wt.of the tablets (mg) Maximum % difference allowed

1 130 or less 10

2 130-324 7.5
 3 More than 304 5

PROCEDURE
Weigh 20 tablets randomly. Calculate their average weight. Compare the
individual tablet weights to the average weight. Not more than two of the
individual weights must deviate from the average weight by more than the limits
given in Table 11.1 and none should deviate by more than twice the percentage.
Disintegration test
While swallowing, the process of breaking a tablet into smaller particles or
granules is called disintegration. The time required to disintegrate the tablet is
known as disintegration time. The disintegration test is very important and
essential for all the tablets (coated or uncoated). This is because the dissolution
rate depends on the disintegration time which in turn affects the absorption rate
of drugs.
The apparatus used for the disintegration test is called the disintegration
apparatus (Fig. 11.14).
Usually, the rate of disintegration depends upon the nature of the tablet. The
disintegration test is not required for those tablets which are supposed to be
dissolved by slow digestion in the mouth or chewed or are to be dissolved in
water before administration. Generally, the disintegration time for uncoated
tablets is 30 minutes and for coated tablets one hour.
Dissolution test
The disintegration test, simply determines the time required for the tablet to
break up under the conditions of the test and for all particles to pass through a 10
mesh screen. It offers no assurance that the resultant particles will release the
drug in solution at an appropriate rate.
Fig. 11.14 Tablet disintegration test apparatus
Hence, the dissolution tests have been developed for nearly all tablet products.
The disso​lution test (Fig. 11.15) is carried out to determine the amount of time
required for a given percentage of the drug substance in a tablet to go into
solution under specified conditions in vitro.
The rate of dissolution of a solid drug plays a vital role in the absorption and
physiologi​cal availability of the drug in the blood stream. Hence, the
determination of the dissolution rate of any solid drug substance is very
important.
Mechanical strength (hardness)
The hardness of a tablet depends upon the nature of the excipients used in the
formulation, weight of the material used, pressure applied during compression
and the space maintained between the upper and lower punches at the time of
compression.
Fig. 11.15 Dissolution test apparatus
The pharmacopoeia has not fixed any standards for the hardness of the tablets.
The tablet hardness is defined as the force required to break a tablet in a
diametric compression test. Some of the devices, which can be used to determine
the hardness of the tablets, are the Monsanto hardness tester (Fig. 11.16) and the
Pfizer tablet hardness tester (Fig. 11.17).
Friability
The friability test is used to evaluate the ability of the tablet to withstand wear
and tear during packing, handling and transporting. The apparatus used for this
purpose is called a friabilator (Fig. 11.18).

Fig. 11.16 Monsanto tablet hardness tester

Fig. 11.17 Pfizer tablet hardness tester

Fig. 11.18 Friabilator

Some tablets preparations
Tablet for bronchitis
Gum traganth: 180 gm; wheatstarch: 180 gm; glycyrrhiza: 180 gm; kantakari:
180 gm; cu​cumber (Cucumis sativalinn): 180 gm; orchid (Orchis latifolia): 180
gm Dose: Two tablets (250–500 mg) daily
Tablet for acute fever
Tabasheer (Bambumanna): 60 gm; quinine bark (Cinchona officinalis): 30 gm;
tinospara (Tinospora cordiofolia): 30 gm; gum acacia: q.s.
Dose: Two tablets (250–500 mg) daily
Tablet for constipation
Croton (Croton tiglium): 30 gm; aloe (Aloe barbadensis): 30 gm; gum
tragalanth: 30 gm Dose: 125–250 mg
Tablet for jaundice
Aloe (Aloe barbadensis): 150 gm; cocklebur (Agrimonia eupatoria): 150 gm;
myrobalan (Terminalia chebula): 300 gm; wild celery (Apium graveolens): q.s.
Tablet for strengthening the uterus (uterine tonic)
Betel nut palm (Areca catechu linn): 90 gm; nutmeg (Myristica fragrans): 30
gm; jaggery: 15 gm; marijuana (Cannabis sativa): 6 gm; saffron: 3 gm; clove:
1.5 gm; musk: 750 mg; opium: 375 mg Tablet for digestive disorders
Asafoetida: 3 parts; ginger: 3 parts; common salt: 3 parts; borax: 3 parts
Dose: One tablet a day
Tablet for bronchitis
Pomegranate (Punica granatum): 180 gm; long pepper (Piper longum): 90 gm;
black pepper (Piper nigrum): 45 gm; jaggery: 720 gm Dose: Two tablets daily.
Tablet for epilepsy
Castorium: 300 gm; summer savory (Satureja hortensis): 300 gm; caraway
(Carum carvi): 300 gm Tablet for malarial fever
Assam ginseng (Myrica nagi thumb): 150 gm; nicker bean (Caesalpinia crista):
300 gm
11.4.8 Ointments
Ointments are a class of semi-solid dosage forms meant for external application
to the skin or mucous membrane. Ointments contain medicament or
medicaments, suspended, emulsi​fied or dissolved in a base. They perform
emollient and protective action.
An ointment can also be considered as a soothing, healing, slightly oily or fatty
substance into which the essence of a healing plant has been dissolved.
Basically, this is accomplished by heating the fat or oil with the plant until it
loses its normal colour and the oil or fat has absorbed the healing chemical
principles. The plant is then strained out, and beeswax is added to harden the
ointment. Preservatives, such as tincture of benzoin or glycerin can also be
added.
The ointment should be stable, smooth, free from grittiness, melt or soften at
body tem​perature and be easily applied. The medicament used must be finely
divided and uniformly distributed throughout the bases. The ointment base
should be non-irritating and should have no therapeutic action.
METHOD OF PREPARATION
1. Melt petroleum jelly (500 gm) or wax in a glass bowl set in a pan of boiling
water.
2. Add the finely cut herb (60 gm dried or 150 gm flesh herb) and simmer for
fifteen minutes with continuous stirring.
3. Pour the mixture (herb mixture) into a jelly bag secured to the rim of a jug
with a string, and allow the liquid to filter through.
4. Squeeze as much as of the hot herb mixture (by wearing rubber gloves) as
possible through the bag into the jug.
5. Quickly pour the molten ointment into jars before it sets in the jug. Place
the lid on each jar without securing it firmly. When cool, tighten the lids
and label.
Figure 11.19 gives a schematic representation of the preparation of ointments.
Storage: Can be stored in sterilised, dark glass jars with lids for up to three
months.
Dosage: Apply a little 3 times a day.
Fig. 11.19 Schematic representation of the preparation of ointments
Ointment bases
Bases can be conveniently categorised into the following types.
Hydrocarbon bases: Soft paraffin; hard paraffin; liquid paraffin
Absorption bases: Wool fat (anhydrous lanolin); hydrous wool fat; wood
alcohol; beeswax Neutral oil bases: Almond oil; coconut oil; olive oil; vitamin
E; wheat germ Usually, more than one ointment base is used in the preparation
of ointments.
Some ointment preparations
Ointment for sore legs
1 handful chickweed; 1 handful red rose leaves; olive oil (required quantity)
PROCEDURE
Cook the chickweed and the red rose leaves together in the oil on top of a stove
or in the oven. After one to three hours, strain out the herbs.
Juniper berry ointment
2 cups of juniper berries (ripe); 2 cups of olive oil; 2–3 tablespoons of beeswax
The ointment is useful for wounds, itching, scratches, scars from bums, and
festering sores.
PROCEDURE
Soak the berries overnight. Strain out the water. Simmer the berries in the oil and
take care that they do not bum. Heat the wax in a separate container. Strain out
the berries. Add the melted wax while the preparation is hot. Required amount of
wax may be added after the hardening process starts. If necessary, re-heat and
add additional melted wax.
Ointment for burns and scalds
Calcium carbonate: 90 gm; yellow beeswax: 45 gm; almond oil: 675 ml
PROCEDURE
The oil is heated and wax dissolved thoroughly and mixed in it. The fine powder
of calcium carbonate is added, stirred well and allowed to cool till it forms a soft
and semi-solid mass.
Ointment for wounds
Almond oil: 1800 ml; yellow beeswax: 900 gm; pine (Pinus longisima): 180 gm;
Indian copal tree (Vateria indica): 120 gm; pistachio (Pistacis lentiseus): 90 gm
PROCEDURE
The almond oil is heated and the stated quantity of yellow beeswax is dissolved
thoroughly and mixed in it. The fine powders of other ingredients are added,
stirred well and allowed to cool till it forms a soft and semi-solid mass.
Ointment for scabies
Camphor tree (Cinnamomum camphora): 90 gm; zinc oxide: 90 gm; cardamom
(Elettoria cardamomum): 45 gm; white barked acacia (Acalia leucophloea): 45
gm; beeswax: 27 gm PROCEDURE
The method of preparation is similar to the above.
11.4.9 Creams
Creams are viscous semi-solid ointment-like preparations. They may be oil in
water type (aqueous) creams or water in oil type (oily) creams. Due to the
presence of water soluble bases in oil in water type creams, they can easily be
removed from skin and clothing. Aqueous creams are susceptible to bacterial
and mould growth, therefore a preservative must be added.
METHOD OF PREPARATION
1. Melt the emulsifying wax (150 gm) in a glass bowl set in a pan of boiling
water (80 ml) or a double boiler. Add glycerine (70 gm), water and herb
while stirring and simmer for 3 hours.
2. Strain the mixture through a wine press or a jelly bag. Stir slowly but
continuously until it cools and sets.
3. With a small knife or spatula, place the set cream into dark glass jars.
Tighten the lids and label. Store in a refrigerator as soon as possible.
Figure 11.20 gives a schematic representation of the preparation of creams.
Storage: It can be stored in sterilised, air-tight, dark glass jars in a refrigerator
for up to 3 months.
Dosage: Rub a little into the affected area 2–3 times a day.

Fig. 11.20 Schematic representation of the preparation of creams

11.4.10 Ayurvedic Dosage Forms
Ayuh means life and veda means knowledge. Ayurveda may be defined as the
science of life or the true knowledge of life. Solid, liquid and semi-solid dosage
forms meant both for inter​nal and external use are available. The ayurvedic
dosage forms are given below.
Solid dosage forms
Some common solid dosage forms are chuma (powders) and vatika (pills).
Churnas
Churnas are powdered preparations of drugs used for oral administration. There
are two types—simple and compound churnas. Simple churnas contain only one
medicament, and compound churnas contain more than one medicament.
METHOD OF PREPARATION
The herb is thoroughly cleaned, properly dried and reduced to fine powder. The
powder is sieved through a 80 mesh sieve. If the churna contains more than one
drug, each one is sepa​rately powdered, sieved, weighed accurately and mixed
together. The churnas must be stored in air-tight containers. They are usually
taken as such or mixed with sugar, honey, milk, water. Examples of churnas are
Maha sudarshan churna and Sitopaladi churna.
Bhasma
Bhasmas are preparations containing ash which are obtained through the process
of incinera​tion. They are meant for oral administration. They are obtained by
treating metallic and non- metallic minerals like gold, silver, zinc and animal
derived substances like shells and homs, with plant juices by special process in
closed crucibles, in pits and with cow dung cakes.
METHOD OF PREPARATION
1. The material is subjected to the sodhana (de-toxification) process, which
involves purification.
2. The purified material is then reduced to a fine powder.
3. The powdered material is then subjected to react with suitable herbal
extracts (or miner​als) and kneaded to form a wet mass.
4. The wet mass is converted into pellets or slugs, which are incinerated in a
 furnace.
Examples of bhasmas are Swam bhasma and Abhraka bhasma.
Vati and gutika
These are solid unit dosage forms, prepared in the form of tablets or pills. They
are prepared by compression with the help of a tablet making machine or by
hand.
METHOD OF PREPARATION
1. The plant drugs are dried, cleaned and finely powdered, separately. The
minerals are made into bhasmas.
2. Finely powdered medicaments and additives are mixed to a uniform mass
and moistened.
3. The mass is kneaded with syrup or extract of herbs and converted into a
uniform moist mass.
4. The moistened mass is rolled into uniform cylindrical pencils, which are
then divided into a number of pieces of the same size. Each piece is then
converted into a pill, by rolling in between the fingers. The final pill mass
should not stick to the fingers when rolled. The resultant pills are dried in
shade or sun and stored in air-tight containers. They can be used up to two
years. Pills containing minerals can be used for an indefinite period.
Semi-solid dosage forms
Common semi-solid dosage forma are avaleha (soft extract—jam, jelly, syrup)
and kalka (ointment and pastes).
Avaleha
Avalehas are basically sugar based semi-solid preparations for oral use. The
preparation of avaleha involves, evaporating the kwatha (decoction; explained
under liquid dosage forms) to a semi-solid consistency and then adding
medicaments, sweetening and flavouring agents to it. The medicaments can also
be mixed in syrup and evaporated to get a product of highly viscous or semi-
solid consistency. Chyawanprash is an example of an avaleha.
Lepa
Lepas are external preparations used in the form of a paste. The method of
preparation in​volves powdering the drugs and mixing with a liquid medium like
water, ghee, cow’s urine etc to make a soft paste. An example of a lepa is the
Dasaga lepa.
Liquid dosage forms
Common liquid dosage forms are kasaya (extracts), kwatha (decoction) and vasti
(enemas).
Asava and arishta
These are liquid ayurvedic preparations prepared by the process of fermentation.
METHOD OF PREPARATION(arishta)
1. At first, the ingredients of the formula are powdered coarsely and the
decoction prepared.
2. The decoction is filtered and placed in the fermentation vessel.
3. Required quantity of sugar (or jaggery or honey) is added.
4. The vessel is covered tightly with an earthen lid, taking care to maintain
constant tem​perature throughout the process of fermentation.
5. After completion of the fermentation, the fluid is decanted and then strained
after three days. When the fine suspended particles settle down, it is
strained and bottled.
The preparation of asava is similar except that the sugar (or jaggery) is
dissolved in water, boiled, cooled, and poured into the fermentation vessel. Only
then is the fine powder of the drug added to the vessel.
Arka
Arka is the liquid formulation obtained by distillation of fresh flowers and soft
parts of plants containing volatile constituents. If the arka is to be prepared from
hard parts of a plant, the drug is first powdered and moistened with water (or
other suitable liquid) for a sufficient amount of time until it softens. A boiling
chamber made of copper or brass is used for the purpose.
METHOD OF PREPARATION
1. The coarsely powdered drug is soaked in water over-night.
 2. The total mass is placed in the distillation chamber. Water, usually 8 to 10
times the weight of the drug is added and the condenser attached.
3. Heat is applied and the distillate collected in the receiver.
4. The initial distillate should be rejected as it may contain impurities. The
subsequent dis​tillate is collected and mixed together.
Examples of arkas are arka of mint and arka of fennel.
Kwatha
Liquid preparations, prepared by the method of decoction, meant either for oral
administra​tion or external use are called kwathas.
METHOD OF PREPARATION
1. The drug material is reduced to coarse powder and moistened with a solvent
for 12 hours.
2. The softened drug is boiled with solvent for about 30 minutes. The mixture
is stirred well.
3. It is then allowed to cool and the preparation is filtered through a piece of
muslin cloth.
Taila
These are preparations consisting of lipophilic liquid in the form of medicated
oil. They are meant for external use. Taila is prepared by dissolving or extracting
the medicated substanc​es with an oil like mustard oil.
Ghrita
Ghirta preparations are used as tonics. The preparation involves extracting or
dissolving the medicinal substances with ghee.
 12

Herbal
Cosmetics

1 2 .1 S O U R C E , HISTORICAL BACKGROUND AND PRESENT

STATUS
All human beings want to look beautiful, so they have been using different types
of materials, from time immemorial, to improve their looks. Originally,
cosmetics were associated with religious practices. It is true especially in the
case of old civilizations like the Indian, Chinese, Egyptian and Greek
civilizations. The practice of ubtan – the application of a paste of flour, turmeric
and vegetable oil before marriage – is still practiced in India. Kumkum is applied
by Hindu married women.
In the past, all cosmetics were made at home. Natural materials like aromatic
materials, spices, herbs, resins, dyes, fats, oils, perfumes were used. In Egypt,
high priests were often recognized as medical practitioners, and over time,
everything related to health and body care came to be associated with medicine.
Egyptian physicians were highly specialized in various branches of their
medicine; however, astrology, magic, mysticism and religion had an influence on
medical practices. The Egyptians were famous as teachers throughout the
Middle East. Their knowledge and practices were passed on, in succession, to
the Assyrians, Babylonians, Chaldeans, Hebrews, Persians and Greeks.
Cosmetics have been unearthed from tombs, temples and other religious places.
In 4 B.C.E., after the conquest of Persia by Alexander the Great, a new culture
developed in which significant advances were made in the fields of medicine and
cosmetology. Medical schools were established in various Greek states, but it
was not until Hippocrates, that all inherited knowledge was systematized into
some semblance of scientific principles. It was Hippocrates, who dissociated
medicine from magic, superstitious and religion. He advocated correct diet,
exercise, sunlight, special baths and massages for good health and beauty.
In 7 C.E., the Prophet Mohammed started a new religion – Islam. Under the
Prophet and his successors, countries like Syria, Persia, Judea, Mesopotamia and
Egypt were brought under Muslim control. The works of Galen, Dioscorides,
Paul, Hippocrates, Oribasios, Aristotle, Pliny and others were translated, mostly
by the Arabs. The study of physiology and hygiene began to gain prominence,
and attention was paid to the therapeutic effects of bathing, proper diet,
gymnastics and massage. But early in the eleventh century, the Mohammedan
Empire began to crumble. Toledo was lost to the Christians; Spain and Sicily to
the French.
The Arabs continued to work on herbs and plants and their use in medicine,
perfumery, hygiene and skin care. Abdekar, the court physician of Mohammed
II, the Ottoman Sultan, in daily conferences instructed his favourites among the
beauties of the Sultan's seraglio, in the care of skin and hair and the virtues of
various types of baths.
In lndia, perfumery had developed centuries ago, and several essential oils
known as attar were used in the country and also exported. Henna has been
traditionally for colouring the palm and hair. Except for a few multinational
companies, the cosmetic industry has been a cottage industry in India. Even
today, the cosmetic industry is a largely in the small scale sector. However, all
types of cosmetics are been manufactured by this industry.
Between 1900 and 1920, many books on cosmetics, perfumery, cosmetology
and related subjects were written. Most of these were either in German or in
French. "Perfumes, Cosmetics and Soaps" by W.A. Poucher was the first book of
its kind in English. Published in 1923, it has been revised and updated from time
to time. "Harry's Cosmeticology", another good book on cosmetics, was first
published in the mid-50s.

12.2 RAW MATERIALS: OILS, WAXES, GUMS, HYDROPHILLIC

COLLOIDS, COLOURS, PERFUMES, PROTECTIVE AGENTS,
B LEACHING AGENTS, PRESERVATIVES, ANTIOXIDANTS AND
OTHER AUXILLARY AGENTS
Oils: Oils are derived from vegetable and mineral sources, and are used in
cosmetics. Examples of vegetable oils are almond oil, arachis oil, castor oil,
olive oil and coconut oil. Examples of mineral oils are light and heavy paraffin.
Almond Oil: It is a fixed oil obtained by expressing the seeds of Prunus
amygdalus, Family Rosaceae. The oil is pale yellow in colour, with a
characteristic odour. The active principles are mainly the mixture of glycoside
with oleic acid, linoleic acid, myristic and palmitic acid. It has an emollient
action, so it is used in the preparation of creams and lotions.
Arachis Oil: This is also a fixed oil obtained from the seeds of the Arachis
hypogea belonging to the family Leguminoseae. The oil is pale yellow in colour,
with a faint nutty odour. Refined groundnut oil is colourless, with active
principles like oleic, linoleic acid and a small amount of other acids. At 3° C, it
is cloudy, at a lower temperature, it solidifies. It is used in the preparation of hair
oils and brilliantines.
Castor Oil: Oil is obtained from the seeds of Ricinus communis belonging to
the family, Euphorbiaceae. It has a slight odour; the oil is either yellow in colour
or colourless. It consists of a mixture of glycosides, in which 80% of ricinoleic
acid is the major constituent. At 0° C it forms a clear liquid. It is used as an
emollient, in the preparation of lipsticks, hair oils, creams and lotions.
Olive Oil: This oil is obtained from the fruit of the Olea europea, belonging to
the family, Oleaceae. The oil is either pale yellow or greenish yellow in colour, it
has a slight odour. It consists of the glycerides of oleic acid, palmitic, linoleic,
stearic and myristic acids. At a lower temperature, it is solid or partly solid. It
has emollient, soothing properties. It is used in the manufacturing of creams,
lotions and bath oils.
Coconut Oil: This oil is obtained from the dried solid part of the endosperm of
the coconut – Cocos nucifera, family Palmea. It is a white or pearl-white
unctuous mass in winter and colourless in summer.
Light liquid paraffin: It consists of a mixture of hydrocarbons in the form of
an oily liquid which has no colour or odour. Viscosity and weight per ml (0.83–
0.87g) are both low in light liquid paraffin. It is used in the manufacture of bath
oils, hair oils, brilliantines, lotions and creams, due to its better spreadibility.
Heavy liquid paraffin: It is composed of a mixture of hydrocarbons in the
form of a colourless and odourless oily liquid. Due to its soothing effect on the
skin, it is used in creams, lotions, brilliantines, hair oils and bath oils. Heavy
liquid paraffin is obtained from petroleum.
 Waxes: Waxes are the esters resulting from the condensation of high molecular
straight chain fatty acids with high molecular straight chain monohydric alcohol
of the methanol series. They are used in cosmetics as a base, along with oils and
fats. Example: lipsticks.
Commonly used waxes are briefly discussed below.
Beeswax: It is a purified wax separated from the honeycomb of bees, Apis
mellifera which belong to the Family, Apidae. Beeswax is composed of 70%
ester myricyl palmitate. It is yellowish brown in colour, solid, with a honey-like
odour. Under cold conditions it becomes brittle; when bleached, it becomes
yellowish-white solid with a faint characteristic odour. The melting point of
beeswax is 62°C–65°C. Beeswax helps in the incorporation of water to form an
emulsion.
Carnauba Wax: This is obtained from the leaves of the Brazilian wax palm,
Copernica cerifera, which belongs to the Palmae family. Camauba wax is
available in various grades. The highest grade is light-brown to pale-yellow in
colour. It is in the form of moderately coarse powder or flakes, with a
characteristic bland odour. The melting range of this wax is 81°C–86°C. It is a
hard wax and is used in the manufacture of candles, wax varnishes, leather and
furniture polishes.
Paraffin Wax: It is derived by the distillation of petroleum. It is a mixture of
solid hydrocarbons consisting mainly of n-paraffins and, to some extent, their
isomers. So, it also called hard paraffin wax. Physically, the paraffin wax is
colourless, odourless or a white, translucent, wax-like solid, which is slightly
greasy to touch. Paraffin wax melts at 50°C–57°C.
Spermaceti: It is a solid wax obtained from the head, blubber and ear case of
the sperm whole, Physester colodon, which belongs to the Physeteridae family.
It consists mainly of cetyl palmitate and cetyl myristate spermaceti in a solid
wax, which is a translucent crystalline, pearly-white, unctuous mass with little
odour and taste. It melts at a specific gravity of about 0.94.
Spermaceti is also available synthetically and is composed of a mixture of
esters of saturated fatty alcohols and saturated fatty acids. Synthetic spermaceti
is available as white to off-white translucent flakes with a crystalline structure
and a pearly lustre. The melting range of synthetic spermaceti is 43°C–47°C.

12.2.1 Colours
Colours have been used in cosmetics, since time immemorial, by human beings.
Basically, the desire to buy a cosmetic product is controlled by three senses,
namely, sight, touch and smell. So colour is one of the most important
ingredients of cosmetic formulations. Colour is a visual sensation which can be
caused by a definite wavelength or a group of wavelengths by an object through
one or more of the following phenomena – emission, refection, refraction or
transmission.
Natural colours such as cochineal, saffron and chlorophyll are discussed in
brief here.
Cochineal: Cochineal is a red dyestuff derived from the dried female insect,
Dactilopius coccus, which belongs to the Coccidae family. Carminic acid is the
main colouring constituent in cochineal. On crystallization, carminic acid forms
red needles and at 130°C, the needles darken and also carbonize at 250°C.
For the preparation of caramine, the cochineal is extracted with water. Alum is
added to this solution to precipitate the red aluminium salt called carmine lake.
Saffron: It consists of the stigmas and tops of the styles of the plant, Crocus
sativa, which belongs to the Iridaceae family. It is a perennial plant grown in
Jammu and Kashmir in India. Saffron powder is yellowish and is easily soluble
in water, so it is used as a flavouring and colouring agent in food preparations.
Saffron contains a number of carotenoids – crocin is an important natural
saffron carotenoid. Picrocrocin is a colorless bitter glycoside responsible for
saffron’s characteristic odour.
Chlorophyll: It is the natural green pigment, found abundantly in nature. It is
the component that is responsible for photosynthesis.
Rose: It is obtained by the steam distillation process from the flower petals of
Rosmarinas officinalis which belongs to the Labiatae family. For obtaining rose
oil, the blossoms are collected before they open, a little before sunrise.
Jasmine Essential Oil: Obtained from the flowers of Jasminum grandiflorum
which belongs to the Oleaceae family, the oil is obtained by the solvent
extraction method and its essence is used in the perfumery industry.
Lavender: It is obtained from the flowers and stalk of lavandula officinals
which belongs to the Labiatae family.
Tuberose: The nickname of the tuberose is “mistress of the night”. The oil is a
brown, viscous liquid with a sweet, heavy and sensuous scent.
 Geranium: This oil is obtained from the flowers, leaves and stalks of the
Pelargonium graveolens, which belongs to the Geranigceae family. Its essence is
obtained by the distillation process, from the flowers and stems of the plant. The
geranium is known as geranium bourbon.
Champa: It is obtained from the flowers of the Michelia champaka. The
colour of the flower is yellow to deep orange.
Cinnamon: Cinnamon oil is obtained from the different parts of the cinnamon
tree – its leaves, bark and roots. Cinnamon zeylanicum belongs to the family,
Lauraceae. The oil obtained from the bark is most valuable. The oil has a warm,
spicy and sweet character.
Neroli: It is an essential oil obtained through the distillation process from the
flower of the bitter orange tree. It can be stored in amber-coloured bottles in the
refrigerator.

12.2.2 Hydrophilic Colloids [Surfactants]

Surfactants or active agents reduce the boundary tension in one or more than one
interface in the system. They also provide stability to one or more than one
interface by the formation of absorbed layers. These properties are taken
advantage of in the preparation of cosmetics.
In the preparation of cosmetics, there are mainly five uses for which surfactants
are used.

i. Detergency
ii. Wetting
iii. Foaming
iv. Emulsification
v. Solubilization

Every surfactant has a common feature – all are amphipathic molecules which
have two distinct parts:
1. a hydrophobic part
2. a hydrophilic part
 The hydrophobic part of the molecule basically consists of hydrocarbons, chain
rings or a mixture of both. On the other hand, the hydrophilic part usually
consists of a polar group, such as a carboxylic, sulphate or sulphonated group. In
the case of non-ionic surfactants, they contain a certain number of hydroxyl or
ether groups; due to this reason, they absorb at interfaces.
Anionic Surfactants: This kind of surfactant contains anionic groups which
are connected directly to the hydrophobic unit. For example, fatty acid soaps [R
– C00- mts].
Cationic Surfactant: The cationic group is separated from the hydrophobic
group in case of cationic surfactants; for example, quatemized amides of
ethylene diamine.
RCONH-CH2-CH2 N+ [CH3]3 x-
Non-ionic Surfactant: In non-ionic surfactants, the molecule is made up of
multiple unchanged polar groups; for example, fatty acid alkanolamide.
RCONH-CH2-CH2-OH.
Clove: It contains essential oils, obtained from the buds of the Euginca
carryophylus, which belongs to the family, Myrataceae. In small doses, it can be
used in the perfume industry.
Ambrette: Ambrette seeds contain oil; it can be obtained by using the
expression method. The oil is rich; it is sweet, floral and musky in nature. The
oil can be used as an anti-aging agent.
Sandalwood: It is obtained by the steam distillation process from the hard
wood of Santalum album belonging the family, Sundalaceae. In most perfumes,
it is used as a fixative agent.

12.2.3 Protective Agents

In the formulation of creams, silicones act as protective agents; a combination of
silicones with other barrier agents like petroleum jelly beeswax, paraffin etc can
produce excellent barrier creams.

12.2.4 Bleaching Agents

The most commonly used bleaching agents are given below:
Mercury Compounds: Mercuric chloride (Hgcl), red mercuric oxide (HgO)
and ammoniated mercury are examples of mercury compounds that can be used,
for their skin bleaching effects. Currently, the use of mercury compounds is
prohibited in cosmetics.
Hydroquinones: They are mostly used as bleaching agents for temporarily
lighting skin at a concentration of 1.5%–2%. In the case of 5% concentration,
redness and burning may be produced. Reverse action of hydroquinones takes
place on exposure to sunlight. If the cosmetics containing hydroquinone are
discontinued, then too, a similar effect can be observed.
Catechol and its derivatives: Catechol exhibits skin lighting effect to an
extent. 4- Isopropy catechol has been found to be among the most potent de-
pigmenting agents. They can produce irritation and a sensitization reaction at
concentrations of 3% or more.
Ascorbic Acid and its derivatives: Ascorbic acid does not seem to be very
effective as a de-pigmenting agent, but its use has been found to be safe. It is
mostly used in skin bleaching creams, which contain hydroquinone as a
stabilizer (antioxidant). Ascorbyl oleate used in skin bleaching cream for
bleaching freckles in human skin is used at a concentration of 3% and 5%.

12.2.5 Oxidising Agents

Hydrogen peroxide has been used as an oxidizing agent in skin bleach creams.
Sometimes, zinc peroxide is also used in anhydrous ointments such as bleaching
agents, although the properties of zinc peroxide have been not proved.

12.2.6 Opaque Covering Agents

Various cosmetic products which contain white or pale pigments like titanium
dioxide, zinc oxide, talc, kaolin, bismuth etc. can provide a temporary but
remarkable change in the colour of the skin.

12.2.7 Preservatives
These are the agents which are used to prevent spoilage of cosmetic products.
They are products of the oxidation of oils and fats and also the growth of
microorganisms. Most cosmetic preparations, especially those containing water
are likely to deteriorate if preservatives are not added.
Properties of preservatives
An ideal preservative must possess the following attributes;
 1. It should be compatible with the formulation.
2. Soluble to the extent needed to achieve an effective concentration.
3. Stable enough to provide a sustained antimicrobial effect.
4. Colourless and odourless or nearly so.
5. Non-irritant and non allergic in the concentrations used.
Organic acids: Benzoic acid, Formic acid
Alcohols: Ethyl alcohol, Isopropyl alcohol
Aldehydes: Formaldehyde, Cinnamic aldehyde
Phenolics: Cresol, Phenol
Esters: Methyl p-hydroxy benzoate, Ethyl p-hydroxy benzoate
Mercury: Thiomersol, Nitromersol
Surface active agents: Benzalkonium chloride, Cetyl pyridinium chloride
Miscellaneous compounds: Ethyl Vanillin and Vanillin

12.2.8 Antioxidants
Natural antioxidants like tocopherols present in fats and oils are destroyed
during the refining process. Hence, the addition of antioxidants is essential to
avoid the rancidity of fats and oils in cosmetics due to oxidative deterioration.
Some of the common antioxidants used in cosmetic preparation are:
Amines: Purins and lecithin
Phenols: Gallic acid, Methyl gallate
Quinones: Tocopherols, Hydroxy chromans
Alcohols: Sorbitol and Mannitol
Esters: Di-lauryl thiopropionate
Organic acids: Ascorbic acid

1 2 .3 FORMULATION ASPECTS OF INCORPORATING HERBAL

EXTRACTS IN VARIOUS PREPARATIONS
1. Almond Complexion Lotion:
Almond Oil - 1 tbsp

Cucumber/Carrot - 1 tsp
juice

Glycerin - 2 tsp

Liquid Paraffin - 1 tsp

Extract of cornflower - 1 tsp

Heat the almond oil and paraffin together and add all the ingredients to it. Shake
it well, apply it and let it remain till it dries. Wash with lukewarm water and then
rinse off with cold water.
2. Lavender Complexion Lotion:
Borax Powder - 1 tsp

Rose water - 1 cup

Olive oil - 2 tbsp

Lavender extract - ½ cup

Mix borax powder in rose water and add boiling oil to the mixture. Keep
stirring; when cool, add lavender extract too. It can be kept under refrigeration
for more than two months.
3. Cucumber Cleansing Cream:
Beeswax - 30 gm

Spermaceti - 30 gm

Olive Oil - 500 ml

Cucumber Juice - ½ cup

Sodium benzoate - 1 tsp

Put beeswax and spermaceti in a bowl; place the bowl in an open pot filled with
boiling water. After these two substances have melted, remove the bowl from the
pot. Pour olive oil and cucumber juice. Keep stirring; while the mixture is still
warm, add sodium benzoate.
4. Nail cream:
Avocado pulp - 1
teaspoon

Egg yolk - 1
teaspoon

Honey - 1
teaspoon

Salt - a pinch

Blend avocado to a smooth consistency. Combine all the ingredients with

avocado. Massage gently but well, onto nails and rinse off after half an hour.

12.3.1 Face Scrub

Face packs which enhance the quality of rubbing or scrubbing of the skin are
called face scrubs. By scrubbing, they bring the blood to the surface of the skin,
remove dead cells, smooth and clean the skin. Scrubs eliminate the buildup of
dirt and oil on the skin surface and improve the lustre of the skin, lending it a
deep glow. The recipes for some face packs are given below.
1. Orange Face Scrub:
Dried orange peel : 2 tbsp
[powder]

Oatmeal : 2 tbsp

Cold cream : 2 tbsp

Mix all the ingredients to get a paste and apply it on the face. When it dries up
slightly, remove the pack by scrubbing. Finally, wash off with cold water. This
preparation can be used for normal skin.
2. Pea Face Scrub:
Pea powder : 4–6 tbsp

Lemon juice : ¼ tbsp

Rose water : 2 tbsp

Soak the powder in rose water and lemon juice for 30 minutes. Stir it well and
the preparation is ready for use.
3. Carrot Face Scrub for Oily Skin:

Yeast : 1 tbsp
powder/brewer’s
yeast

Yoghurt : 1½ tsp

Lemon juice : 1 tsp

Carrot juice : 1 tsp

Olive/Almond oil : 1 tsp

Mix all the ingredients and blend well together. Apply this on the face for 15
minutes. Then wash off with lukewarm water. If the skin is very oily, exclude the
oil; if dry, add more oil to it. Do not use this on the skin if you have pimples, as
it may cause an infection.

12.3.2 Face Packs or Face Masks

These are required to supplement the primary phase of skin care – cleansing.
Masks stimulate blood circulation, tone the muscles and maintain the elasticity
of the skin. Also they draw out the impurities from the pores. Some recipes for
face packs are given below.
1. Bael fruit face pack:

Bael fruit powder - 2 tsp

 Date extract - 2 tbsp

Honey - 1 tbsp

All the ingredients are mixed into a paste and applied on the face for 10–15
minutes and then washed off.
2. Lemon Astringent Lotion:
Lemon juice - 2 large
lemons

Distilled water - 16 tbsp

Tincture of benzoin - 1 tbsp

Mix all the ingredients together and apply using a cotton pad.
3. Nutmeg Astringent Lotion:
Honey - 1 tbsp

Nutmeg powder - 1 tbsp

Clove powder - ½ tbsp

Grated lemon peels - 2 tbsp

Rose water - 2 tbsp

Orange flower extract - 2 tbsp

Tincture of benzoin - 1/8 tsp

Mix all the ingredients thoroughly, allow to stand for seven days. The
preparation can be used for more than two months if kept under refrigeration.

12.3.3 Herbal Shampoos

1. Lime Shampoo:
Amla - 100 gm

Shikakai - 200 gm

Char - 100 gm

Chorillo - 100 gm

Khus - 100 gm

Reetha - 200 gm

Water - 2½ liter

Glycerin - 8 tsp

Lime juice - 4 tsp

Sodium benzoate - l½ tsp

Boil the first five ingredients in the water till the mixture is reduced to half the
quantity. Strain it and add remaining ingredients.
2. Neem Shampoo:
Gram flour - 1 kg

Sandal wood powder - 250 gm

Neem leaves powder - 4–5 cups

Shikakai powder - 1 kg

Mix these thoroughly by sieving. Keep it in an airtight bottle. When needed for
washing, soak 2 tbsp of it in a cup of water, then apply.
3. Clove hair-setting preparation:
Almond oil - 1 cup

Palm oil - 2 tsp

Benzoated lard - 500 gm

Lemon juice - 2 tsp

Oil of cloves - 1 tsp

Heat the benzoated lard and mix it with the remaining ingredients. It will make a
pleasantly scented cream to set the hair.
4. Gum tragacanth hair setting preparation:
Gum tragacanth - 15 gm

Alcohol - 20 ml

Castor oil - 2 tsp

Glycerin - 1 tsp

Water - 250 ml

Perfume - as
required

Dissolve the gum tragacanth in the alcohol. Stir it over a very low flame and add
castor oil and glycerin to it. Keep stirring and add water. Cool and bottle. If
desired, a few drops of fragrance can be added before it cools down.
5. Saffron hair dye:
Saffron - 1 pinch

Boiling water - 500 ml

Soak saffron in water for 10 minutes. Strain and use it on the hair. Grey hair will
acquire a rich golden hue.
6. Walnut hair dye:
Walnut husk - 500 gm

Water - 500 ml

Boil husk in the water for 15 minutes, then strain and use this liquid to dye your
hair. It will change the colour of brown hair to a dark shade.
7. Anti-dandruff lemon preparation:
Lemon peel - 1 handful

Spatula - 1 handful

Water - 500 ml

Soak lemon peel and spatula in boiling water. Let it stand for 5–6 hrs then strain
it. Use this preparation for rinsing the hair after shampooing.

12.3.4 Deodorants and Powders

1. Deodorant powder:
Orris root powder - 100 gm

Orange peel powder - 100 gm

Lemon peel powder - 100 gm

All the ingredients are mixed together and a final quantity of calamus root
powder is added, then it is sieved, using a fine sieve.
Note: Orris root can cause allergy in some persons, hence, before using this
powder a small quantity should be tried first.
2. Baby powder:
Talc - 200 gm

Powder of - 100 gm
chamomile flowers

Powder of calendula - 100 gm

flowers

All the ingredients are mixed and sifted, using a very fine sieve. This preparation
is effective for skin rash and irritation.
3. Body powder:
Talcum powder - 100 gm

Lavender powder - 100 gm

The ingredients are mixed thoroughly and sieved, through a fine sieve. A small
quantity of lemon peel powder can also be added to this powder.

12.3.5 Bath Oils

These oils help to improve circulation, soothe tiredness and help fight infection.
Rose, lavender and chamomile are the best oils for relaxing.
1. A rich bath oil:
Olive oil - ¼ cup

Shampoo - 1
tablespoon

Vegetable oil - 1 cup

Lemon juice - 1
tablespoon

All the ingredients must be blended together. Store in a glass bottle. Use 1–2
tablespoon for your bath.
 13

Nutraceuticals:
A Modern Approach.

13.1 INTRODUCTION AND CLASSIFICATION

Major scientific and engineering advances in recent decades have led to an
increase in the development of expensive, high-technology medical and surgical
procedures and drug therapies. At the same time, however, there has been an
increase in the number of people turning to alternative medical therapies, which
emphasize the importance of a ‘good diet’ in maintaining and restoring health.
The term “nutraceutical” was coined from “nutrition” and “pharmaceutical” in
1989, by Stephen DeFelice, MD, founder and chairman of the Foundation for
Innovation in Medicine. A nutraceutical can be defined as “any substance that
may be considered a food or part of a food and provides medical or health
benefits, including the prevention and treatment of disease. Such products may
range from isolated nutrients, dietary supplements and diets to genetically
engineered ‘designer’ foods, herbal products and processed foods such as
cereals, soups and beverages.” Such foods are commonly referred to as
functional foods, signifying that these foods and/or their components may
provide a health benefit that goes beyond basic nutrition.
At present, there are no universally accepted definitions for nutraceuticals and
functional foods, although commonality clearly exists between the definitions
offered by different health-oriented professional organizations. According to the
American Dietetics Association, the term “functional” implies that the food has
some identified value leading to health benefits, including reduced risk of
disease, for the person consuming it. Functional foods include everything from
natural foods, such as fruits and vegetables endowed with antioxidants and fibre,
to fortified and enriched foods, such as orange juice with added calcium or
additional carotenoids, to formulated ready-to-drink beverages containing
antioxidants and immune-supporting factors. The Nutrition Business Journal
states that it uses the term, nutraceutical, for anything that is consumed primarily
or particularly for health reasons. Based on that definition, a functional food
would be a kind of nutraceutical. On the other hand, Health Canada states that a
nutraceutical is a product that is “prepared from foods, but sold in the form of
pills or powders (potions), or in other medicinal forms not usually associated
with foods. A nutraceutical is demonstrated to have a physiological benefit or
provide protection against chronic disease.”
Nutraceuticals can be classified on the basis of food source (Table 13.1),
mechanism of action (Table 13.2), and chemical nature (Table 13.3).
Table 13.1 Classification of nutraceuticals based upon food source

Table 13.2 Classification of nutraceuticals based upon mechanism of action

Table 13.3. Classification of nutraceuticals based upon chemical nature
13.2 NUTRACEUTICALS AND DISEASES
13.2.1 Cardiovascular diseases
Cardiovascular diseases (CVD), is the name for the group of disorders of the
heart and blood vessels; it includes hypertension, coronary heart disease,
cerebrovascular disease, heart failure, peripheral vascular disease, etc. In 1999,
CVD alone contributed to a third of global deaths, and by 2010, it was expected
to be the leading cause of death in developing countries. Many research studies
have identified a protective role for a diet rich in fruits and vegetables against
CVD. Nutraceuticals in the form of antioxidants, dietary fibres, omega-3
polyunsaturated fatty acids (n-3 PUFAs), vitamins and minerals are
recommended, together with physical exercise for the prevention and treatment
of CVD.
Polyphenols range from simple phenolic molecules to highly polymerized
compounds with molecular weights of greater than 30,000 Da. Stilbenes,
anthocyanins, condensed tannins (proanthocyanidins), in grape and wine,
tetrahydro-β-carbolines, dietary indoleamines, melatonin and serotonin, in
different plant foods are hypothesized to impart health benefits.
Flavonoids are widely distributed in onion, cruciferous vegetables, black
grapes, red wine, grapefruits, apples, cherries and berries. Flavanoids in plants
are available as flavones (containing the flavonoid apigenin found in
chamomile); flavanones (hesperidin – citrus fruits; silybin – milk thistle) and
flavonols (tea: quercetin, kaempferol and rutin grapefruit; rutin buckwheat;
ginkgo flavonglycosides - ginkgo), play a major role in curing cardiovascular
diseases. Flavonoids block the angiotensin-converting enzyme (ACE) that raises
blood pressure; by blocking the “suicide” enzyme cyclooxygenase that breaks
down prostaglandins; they prevent platelet stickiness, and hence, platelet
aggregation. Flavonoids also protect the vascular system and strengthen the tiny
capillaries that carry oxygen and essential nutrients to all cells. Flavonoids block
the enzymes that produce estrogen, thus reducing the risk of estrogen-induced
cancers.
Cholesterol has long been implicated as a significant risk factor in
cardiovascular disease. Phytosterols compete with dietary cholesterol by
blocking the uptake as well as facilitating its excretion from the body.
Phytosterols in diet have the potential to reduce morbidity and mortality from
cardiovascular disease. Fagopyrum esculentum Moench (common buckwheat or
sweet buckwheat) seeds possess proteins, flavonoids, flavones, phytosterols,
thiamin- binding proteins etc. Buckwheat proteins are beneficial in dealing with
constipation and obesity, and more important, lower cholesterol and high blood
pressure.
Dietary fibre preparations from defatted rice bran have laxative and
cholesterol-lowering ability with attendant benefits, such as the prevention or
alleviation of cardiovascular disease, diabetes and colon cancer.
Milk and eggs are important animal sources of nutraceuticals, like proteins and
polyunsaturated fats or essential fatty acids (EFAs). EFAs are required for the
production and rebuilding of cells, to reduce blood pressure, lower cholesterol
and triglycerides, reduce the risk of blood clots, help prevent many diseases
including arthritis, arrhythmias and other cardiovascular diseases. The nutritional
value of egg is increased because of gamma linolenic acid (GLA) which has
many benefits, including the prevention and management of CVDs, like
hypertension. Fatty acids of the omega-3 series (n-3 fatty acids) present in fish
are well established dietary components affecting plasma lipids and preventing
major cardiovascular disorders, such as arrhythmias.

13.2.2 Obesity
Obesity, defined as an unhealthy amount of body fat, is a well-established risk
factor for many disorders like angina pectoris, congestive heart failure,
hypertension, hyperlipidemia, respiratory disorders, renal vein thrombosis,
osteoarthritis, cancer, reduced fertility etc. Obesity is now a global public health
problem; about 315 million people are estimated to fall into the WHO-defined
obesity categories.
Excessive consumption of energy-rich foods (snacks, processed foods and
drinks) can encourage weight gain, which calls for a limit in the consumption of
saturated and trans-fats, apart from sugars and salt in the diet. A tolerable and
effective nutraceutical that can increase energy expenditure and/or decrease
caloric intake is desirable for body weight reduction. Herbal stimulants, such as
ephedrine, caffeine and green tea have proved effective in facilitating body
weight loss. Nutraceuticals like conjugated linoleic acid (CLA), capsaicin,
Momordica Charantia (MC) and Psyllium fibre possess potential anti-obese
properties.

13.2.3 Diabetes
Diabetes mellitus is characterized by abnormally high levels of blood glucose,
either due to insufficient insulin production, or due to its ineffectiveness. The
most common forms of diabetes are type I diabetes (5%), an autoimmune
disorder, and type II diabetes (95%), which is associated with obesity. Globally,
the total number of people with diabetes was projected to rise from 171 million
in 2000 to 366 million in 2003.
Although there is widespread use of herbal dietary supplements that are
believed to benefit type II diabetes mellitus, few have been proven to do so in
properly designed randomized trials. Isoflavones are phytoestrogens. A high soy
isoflavone intake (20–100 mg/ day) is associated with lower incidence and
mortality rate of type II diabetes, heart disease, osteoporosis and certain cancers.
For the synthesis of the long chain n-3 fatty acids, insulin is required; the heart
may thus be particularly susceptible to their depletion in diabetes. Ethyl esters of
n-3 fatty acids may be beneficial in diabetic patients. Docosahexaenoic acid
modulates insulin resistance and is also vital for neuro-visual development. This
is especially important in women with gestational diabetes mellitus for which
essential fatty acids during pregnancy is recommended.
Dietary fibres from Psyllium have been used extensively in processed food to
aid weight reduction, for glucose control in diabetic patients and to reduce lipid
levels in hyperlipidemia. Good magnesium status reduces diabetes risk and
improves insulin sensitivity. Chromium picolinate, calcium and vitamin D
appear to promote insulin sensitivity and improve glycemic control in some
diabetics.
13.2.4 Cancer
Cancer has emerged as a major public health problem in developing countries, to
match its occurrence in industrialized nations. A healthy lifestyle and diet can
help in preventing cancer. People who consume large amount of lutein-rich
foods such as chicken, eggs, spinach, tomatoes, oranges and leafy greens
experience the lowest incidence of colon cancer. A broad range of phyto-
pharmaceuticals with a claimed hormonal activity, called phyto​estrogens, is
recommended for the prevention of prostate/breast cancer.
Flavonoids found in citrus fruit appear to offer protection against cancer by
acting as antioxidants. Soy foods are a unique dietary source of isoflavones
(genistein, daidzein and biochanin), which have been shown to inhibit prostate
cancer cell growth.
Lycopene is a major carotenoid and is found almost exclusively in tomatoes,
watermelon, guava, pink grapefruit and papaya. Because of its unsaturated
nature, lycopene is considered to be a potent antioxidant and a singlet oxygen
quencher. Lycopene prevents cancer, cardiovascular disease and diseases of the
gastrointestinal tract. Recently, it was reported that lycopene-containing fruits
and vegetables exert a cancer-protective effect via a decrease in oxidative and
other damages to the DNA in humans. Beta-carotene, the important precursor of
vitamin A has antioxidant properties and helps in preventing cancer and other
diseases. It can be found in yellow, orange and green leafy fruits, and vegetables
like carrots, spinach, lettuce, tomatoes, sweet potatoes, broccoli, cantaloupe,
oranges and winter squash.
Tannins, also called proanthocyanidins, detoxify carcinogens and scavenge
harmful free radicals. Tannins are found in blackberries, blueberries, cranberries,
grapes, lentils, tea and wine. Ellagic acid is a proven anti-carcinogen used in
alternative medicine to prevent cancer. It is present in strawberries, cranberries,
walnuts, pecans, pomegranates; the best source is red raspberry seeds.
Pectin is a soluble fibre found in apples. A new form of citrus pectin called
modified citrus pectin (MCP) has been shown to prevent prostate cancer
metastasis by inhibiting the cancer cells from adhering to other cells in the body.
Several studies have also shown pectin to have a positive influence in decreasing
serum cholesterol levels without affecting serum triglyceride levels. Pectin also
has the ability to reduce the rise of blood sugar when combined with a meal.
 Phenolics such as ferulic, caffeic, gallic acids and curcumin are reported to
possess anticancer activity. Glucosinolates, closely-related sulfur compounds,
are found in cruciferous vegetables including the Brassica crops – Brussels
sprouts, broccoli, cauliflower, cabbage, watercress, oilseeds like rape and
mustard. They are powerful activators of liver detoxification enzymes and a high
intake of cruciferous vegetables has been associated with a lower risk of lung
and colorectal cancer. Broccoli, rich in sulforaphane (a glucosinolate), is a potent
phase 2 enzyme inducer. It produces D-glucarolactone, a significant inhibitor of
breast cancer.

13.3 IMMUNE BOOSTERS AND ANTI-INFLAMMATORY AGENTS

Various nutrients in the diet play a crucial role in maintaining an optimal
immune response, on the organism’s immune status and susceptibility to a
variety of disease conditions. Probiotics –,living micro-organisms – when
ingested in certain amounts, have a positive impact on host health, which goes
beyond conventional nutritional effects. The bacteria most often used as
probiotics are Lactobacilli and Bifidobacteria. They can be given with fermented
foods such as yoghurt, fermented vegetables or meats and they may briefly
establish in the gut. Prebiotics – ingredients or compounds that have a beneficial
effect on the microflora in the host itself, such as fibre, fructooligosaccharides,
inulin, lactulose and sugar alcohols. They are short-chain carbohydrates that may
be fermented in the large bowel and stimulate the growth of potentially
beneficial bifidobacteria. An improvement of the non​specific immune
phagocytic activity of granulocytes has been shown in the blood of human
volunteers after consumption of Lactobacillus acidophilus and Bifidobacterium
bifidum.
Many studies have pointed out that micronutrients such as selenium, vitamins
A, C and E can influence several components of the immune system. Studies
suggest that deficiency of Se is accompanied by a loss of immuno-competence.
On the other hand, supplementation with Se, even in ‘selenium-replete’
individuals, has marked immuno-stimulant effects, including an enhancement of
the proliferation of activated T-cells and an improvement of NK-cell activity.
Oxidant-mediated tissue injury is a particular hazard to the immune system,
since phagocyte cells produce reactive oxygen species as part of the body’s
defense against infection. Many antioxidants can be obtained directly from the
diet. Vitamin A deficiency can affect the function of different cells of the
immune system. Different studies have reported defects in phagocytic activity
and impairment of T and B cell function. In general, improvement of immune
function and increased resistance to infection is observed in vitamin A-deficient
hosts after supplementation. The decrease in cellular immunity with ageing or
during the development of degenerative diseases is markedly improved by the
intake of a high vitamin E diet. In addition, vitamin E plays a role in the
differentiation of immature T cells in the thymus.

13.4 INFLAMMATORY DISORDERS

Inflammation is the response of body tissues to injury or irritation, characterized
by pain, swelling, redness and heat. Arthritis is a general term that describes
inflammation in the joints. Micronutrients for which preliminary evidence of
benefit exists include vitamin C and vitamin D. In addition, numerous
nutraceuticals that may influence osteoarthritis pathophysiology, including
glucosamine, chondroitin, sadenosylmethionine, ginger and avocado/soybean
unsaponifiables, have been tested in clinical trials. These products are safe and
well tolerated, but interpretation of the collective results is hampered by the
heterogeneity of the studies and inconsistent results.
Resveratrol is present in the fruits of bilberry (Vaccinium myrtillus), the low-
bush “wild” blueberry (Vaccinium angustifolium), the rabbit-eye blueberry
(Vaccinium ashei Reade), and the high-bush blueberry (Vaccinium corymbosum).
Although blueberries and bilberries were found to contain resveratrol, the level
of this chemoprotective compound in these fruits was <10% than reported for
grapes. Resveratrol shows the strongest sirtuin-like deacetylase action of any
known phytochemical. Sirtuins have been shown to extend the lifespan of yeast
and fruit flies. It acts as an anti-inflammatory agent, antifungal and inhibits
cyclooxygenase- 1 enzyme. Other beneficial health effects include anti-cancer,
antiviral, neuro-protective, anti-aging and life-prolonging effects.
The omega-3 and omega-6 series play a significant role in health and disease
by generating potent modulatory molecules for inflammatory responses,
including eicosanoids (prostaglandins and leukotrienes), and cytokines
(interleukins) and affecting the gene expression of various bioactive molecules.
Gamma linolenic acid (GLA) is produced in the body from linoleic acid, an
essential fatty acid of the omega-6 series by the enzyme delta- 6-desaturase.
Preformed GLA is present in trace amounts in green leafy vegetables, nuts,
vegetable oils such as evening primrose oil, blackcurrant seed oil, borage oil and
hemp seed oil, and from spirulina and cyanobacteria. It is a nutraceutical used
for treating auto-immune diseases and problems of inflammation. The most
significant source of GLA for infants is breast milk. GLA is further metabolized
to dihomogamma linlenic acid (DGLA), which undergoes oxidative metabolism
by cyclooxygenases and lipoxygenases to produce anti​inflammatory eicosanoids.
Phytoconstituent gentianine present in Gentian root is an effective anti-
inflammatory agent. Anti-inflammatory herbal nutraceuticals and anti-
inflammatory nutraceutical compounds derived from plants or herbs may also be
used as anti-inflammatory agents. These include bromolein, a proteolytic
enzyme found in pineapple, teas and extracts of stinging nettle; turmeric,
extracts of turmeric, or curcumin, a yellow pigment isolated from turmeric.
Glucosamine (GLN) and chondroitin sulfate (CS) are widely used to alleviate
symptoms of osteoarthritis. These nutraceuticals seem to regulate gene
expression and synthesis of NO and PGE2, providing a plausible explanation for
their anti-inflammatory activities.

13.5 DEGENERATIVE DISEASES

13.5.1 Macular degeneration
The prevalence and effects of age-related macular degeneration (AMD) are
increasing dramatically as the proportion of elderly in our population continues
to rise. A combination of vitamin C, vitamin E, beta-carotene and zinc (with
cupric oxide) is recommended for AMD. Healthy lifestyle with a diet containing
foods rich in antioxidants, like lutein and zeaxanthin, n-3 fatty acids, appears
beneficial for AMD. Herbs or herbal extracts, such as garlic (which contains
allicin), and green tea (containing catechins and bioflavonoids such as QR,
hesperidin and rutin), are effective antioxidants. Bioactive components of food,
which are of special interest, include vitamins E and C, polyphenols, carotenoids
– mainly lycopene and β-carotene – and coenzyme Q10 which possess
antioxidant properties. The high content of polyphenolic flavonoids in
nutraceuticals and functional foods is attributed as the reason for their
antioxidant/radical scavenging ability. Antioxidant therapy is supposed to be
effective in the early stages of atherosclerosis by preventing LDL oxidation and
oxidative lesions of the endothelium.
 Astaxanthin is an important naturally-occurring molecule and the most
abundant carotenoid in the marine world. It can be found in fish such as salmon,
trout, sea bream and in shrimp. Natural astaxanthin is produced from
Haematococcus pluvialis microalgae. Unlike β-carotene, astaxanthin has no pro-
vitamin A activity. It has a number of essential biological functions in aquatic
animals, such as protecting them against the oxidation process, protecting
against UV light effects, immune response and pigmentation. It is also a very
potent antioxidant with ten times more powerful antioxidant activity than other
carotenoids. For more than ten years, astaxanthin’s role in enhancing the
immune system and preventing oxidative stress has been the subject of
international research. It offers powerful protection for the eyes and prevents
macular degeneration. It prevents heart disease due to oxidative damage, boosts
immune system function and protects the nervous system from degenerative
diseases like Alzheimer’s disease. It is used in drug delivery for medicines that
are insoluble in water. In vivo antioxidant activity of carotenoids from green
microalgae (Dunaliella salina) has been reported.
Vision-improving agents
Lutein is a carotenoid found in many fruits and vegetables including mangoes,
corn, sweet potatoes, carrots, squash, tomatoes and dark, leafy greens such as
kale, collards and bokchoy. Lutein dipalmitate is found in the plant Helenium
autumnale. Lutein, also known as helenien, is used for the treatment of visual
disorders. Zeaxanthin is used in traditional Chinese medicine mainly for the
treatment of visual disorders. Food sources of zeaxanthin include corn, egg yolk
and green vegetables and fruits, such as broccoli, green beans, green peas,
Brussels sprouts, cabbage, kale, collard greens, spinach, lettuce, kiwi and
honeydew. Lutein and zeaxanthin are also found in nettles, algae and the petals
of many yellow flowers. In green vegetables, fruits and egg yolk, lutein and
zeaxanthin exist in non-esterified forms. They also occur in plants in the form of
mono- or di-esters of fatty acids. A new source of these carotenoids, a crystalline
lutein product, is an extract from the marigold flower (Tagetes erecta) that
contains approximately 86% by weight of the carotenoids, lutein and zeaxanthin.

13.5.3 Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by progressive dementia, with
memory loss as the major clinical manifestation. In 1996, approximately 4
million people in the United States were clinically diagnosed with AD; this is
expected to triple in the next 50 years. Several lines of evidence strongly suggest
that oxidative stress is etiologically related to a number of neurodegenerative
disorders including Alzheimer’s disease. Nutraceutical antioxidants like β-
carotene, curcumin, lutein, lycopene, turmerin etc may exert positive effects on
specific diseases by neutralizing the negative effects of oxidative stress,
mitochondrial dysfunction and various forms of neural degeneration.

13.5.4 Parkinson’s disease

Parkinson’s disease is a brain disorder that results from nerve damage in certain
regions of the brain causing muscle rigidity, shaking and difficulty in walking,
usually occurring in mid to late adult life. Canadian researchers have indicated
that vitamin E in food may be a protection against Parkinson’s disease. Creatine
appeared to modify Parkinson’s disease features as measured by a decline in the
clinical signs. Nutritional supplements have shown some promising results in
preliminary studies; however, it is important to remember that there is not
sufficient scientific data to recommend them for Parkinson’s disease at present.

13.5.5 Miscellaneous
Angiogenesis is a physiological process involving the growth of new blood
vessels from pre-existing vessels. Angiogenesis is a normal process in growth
and development, as well as in wound healing. However, this is also a
fundamental step in the transition of tumours from a dormant state to a
malignant state. Antiangiogenic compounds that are selective against newly-
formed blood vessels while sparing existing ones, may not lead to side effects
even after prolonged exposure. Available indirect evidence suggests that
anti​angiogenic compounds may prevent diseases involving a degenerative
process like arthritis, multiple sclerosis, Alzheimer’s, Parkinson’s, osteoporosis,
diabetes and cancer. Many inhibitors of angiogenesis are being isolated from
functional foods. Naturally-occurring bioactive compounds are speculated to be
potentially effective and safe anti-angiogenic compounds. Such compounds
include catechins, flavins, curcumin, isoflavones, Resveratrol,
proanthocyanidins, flavonoids, saponins, terpenes, chitin, chitosan, vitamin B3,
vitamin D3, fatty acids, peptides and amino acids (alpha 2-macroglobulin,
arginine, phenylalanine etc.).
Psyllium, a dietary fibre, is valuable in the management of irritable bowel
syndrome, inflammatory bowel disease, ulcerative colitis, colon cancer and
constipation. Moringa oleifera Lam (Moringaceae) has an impressive range of
medicinal uses, with high nutritional value. Different parts of this plant contain a
profile of important minerals, and are a good source of protein, vitamins, beta-
carotene, amino acids and various phenolics. It provides a rich and rare
combination of zeatin, QR, beta-sitosterol, caffeoylquinic acid and kaempferol.
Various parts of this plant such as the leaves, roots, seed, bark, fruit, flowers and
immature pods act as cardiac and circulatory stimulants, possess anti-tumour,
antipyretic, antiepileptic, anti-inflammatory, anti-ulcer, anti-spasmodic, diuretic,
antihypertensive, cholesterol lowering, antioxidant, anti-diabetic,
hepatoprotective, antibacterial and antifungal activities, and are being employed
for the treatment of different ailments in the indigenous system of medicine,
particularly in Asia.

13.6 CURRENT STATUS AND LEGAL ASPECTS

The nutraceutical industry has emerged as an important part of the food industry.
With high economic growth, increasing income and changing lifestyles, the
market is growing enormously. Globally, the nutraceutical market was placed at
65 billion dollars in 2002 and was expected to grow to 250 billion dollars by
2005. In India, this market is at about Rs 1, 600 crore at present, with an annual
growth rate of 25%.
In developed countries, predictable factors have been largely responsible for
encouraging the growth of the nutraceutical industry. High disposable incomes,
changing lifestyles with unhealthy eating habits, increasing incidence of health
problems, an increasingly larger aging populations with unique dietary needs to
maintain health, etc. have all prompted the development of new nutritional
solutions, especially the use of nutraceuticals. Therefore, there is a significant
correlation between the growth of nutritional ingredients and demographic issues
and confidence in the growth of nutraceutical products over the next 20 years.
Although this industry is also expanding in developing countries, it is difficult
to predict its growth rate. Some of the reasons for this are – high population,
disparity in levels of disposable income, spectrum of malnutrition including
over- and under-nutrition etc. Notwithstanding the above, the Indian
nutraceutical industry also has great prospects.
Over the last decade, a wide range of products have been available, giving an
insight into the potential for tremendous growth. On the one hand, a booming
economy has resulted in an overall increase in disposable incomes. Added to
this, unhealthy eating habits coupled with a sedentary lifestyle have led to
increased incidence of diet and related health issues. On the other hand, there is
growing awareness on the importance of nutrition and diet for long-term good
health. These have contributed to favourable market conditions for the
nutraceutical industry in India. Apart from this, India has other advantages like
well-qualified and intelligent human resources for setting up R&D facilities of
international standards. The country is also a cost-effective source of
sophisticated raw materials, due to technological advances in areas like
fermentation processes, plant extraction and chemical synthesis. These
converging economic and demographic trends in India have laid the groundwork
for opportunities in the nutraceutical industry.
While prospects are high for this industry, India faces certain challenges too.
The supply chain is a long one and is further affected by poor infrastructure in
terms of roads, cold chain facilities and storage conditions. The wastage of fresh
food is as high as 50% due to lack of infrastructure facilities. Despite surplus
food, the productivity of agricultural/horticultural crops is very low and the land-
holding pattern is fragmented. In addition to this, the taxes levied on packaged
and branded foods are very high – about 30% in India. In comparison, the taxes
in EU countries are below 10% and even in other Asian countries like China, it
is only 13%. All these disadvantages hinder India from gaining a competitive
edge in the global market.
The regulatory framework in India also needs the attention of the relevant
authorities. Globally, the regulatory authorities are aware of the changing needs
of consumers and proactively protect consumers by amending existing laws to
accommodate changes. NLEA (Nutrition Labelling and Education Act) of 1990
and DSHEA (Dietary Supplement and Health Education Act of 1994 are fine
examples of this in the USA. Similarly, the FOSHU Act (Foods of special Health
Uses) was introduced in Japan much earlier. But the scenario in India is very
different. Old laws such as the Prevention of Food Adulteration Act, 1954,
which regulates packaged foods, still exist. In addition, manufacturers need to
abide by many other cumbersome laws (Fruit Products Order, 1955 (FPO);
Vegetable Oils Products (Regulation) Order, 1998 (VOP); Agricultural Produce
(Grading and Marking) Act, 1937 (as amended up to 1986) and General Grading
and Marking Rules, 1986 and 1988 (AGMARK)).
 In India, there is lack of clarity in classifying items into functional foods and
nutraceuticals. This causes confusion among the regulators. At times, the drug
regulators are tempted to classify these products as drugs. This has resulted in
trouble for genuine manufacturers. Proper legislation is the need of the hour. The
Government of India has taken certain welcome steps like the amendment of the
PFA (Prevention of Food Adulteration Act) which defines “Food for special
dietary uses”. Another revolutionary step that is being planned is to introduce a
Food Safety and Standards Act. This will replace the old PFA with new
legislation. The new Act will take India on to the path of a new regulatory
framework to make it capable of taking on global competition.
 14

Chemotaxonomy

Three centuries ago, James Petiver demonstrated that plants bearing a close
resemblance to each other, often exhibit similar physiological activities. As
medicinal properties are influenced by the nature of the constituents present in
them, it is inferred that related plants contain similar constituents, and thus, exert
similar medicinal properties. For example, all the species of cinchona contain
quinine, and therefore, all are useful in the treatment of malaria.
The phytochemical screening of many plants has revealed that there is a close
relationship between the chemical constituents of the plants and their
taxonomical status. In many instances, parallel trends emerge between chemical
taxonomic groupings and chemical constituents. The earlier observations and the
recent phytochemical findings together form the ‘essence’ or concept of a new
branch of science called chemotaxonomy.

14.1 CHEMOTAXONOMY
Chemotaxonomy is defined as ‘the classification of organisms based on the
differences and similarities in their biochemistry’. Also known as
chemosystematics, it is an attempt to classify and identify organisms (originally
plants), according to demonstrable differences and similarities in their
biochemical compositions.
The term ‘chemotaxonomy’ is self explanatory, as it deals with the
classification of the plant kingdom on the basis of the chemical information
obtained from the plants. In this classification, equal importance is given to
taxonomy and the biogenesis of the chemical constituents. Chemotaxonomy
aims at investigating the distribution of chemical compounds or groups of
biosynthetically-related compounds in related species or supposedly related
plants.
14.1.1 Chemical Constituents as Taxonomic Markers
Certain characters are considered for classifying the taxonomy (systematic
classification) of the plant kingdom. Such characters are known as ‘taxonomic
markers’ or ‘features’. They are usually morphological, anatomical, cytological,
ecological or genetic characters. Chemical constituents can also be utilized as
markers in taxonomy, along with others, particularly morphological features.
Such a system of classification is aptly called ‘chemotaxonomy’, as chemical
information is used for this purpose.
Advantages of Utilizing Chemical Constituents as Markers
Many are of the opinion that chemical characteristics are closer in genetic terms
than some of the more visible characters like morphological features. This makes
them less likely to come under the influence of modifying factors. Therefore,
chemical principles are more precisely defined and thus, assume greater
taxonomical significance than other features. Certain kinds of chemical evidence
may also act as a reliable guide to understand the phylogenetic relationships of
living organisms i.e. their origin and evolution.

1 4 .2 ORIGIN AND CHRONOLOGICAL DEVELOPMENT OF

CHEMOTAXONOMY (HISTORY)
Chemotaxonomy, though it seems to be a recent approach, actually has its roots
in antiquity. It has witnessed a gradual development over the years and its
chronological development can be distinguished into two distinct phases – the
primitive phase and the modem phase.

14.2.1 Primitive Chemotaxonomy

The early classification of herbs was based on their medicinal and edible
properties. On this basis, they were classified into medicinally useful and non-
useful varieties and also as poisonous and non-poisonous plants. This was the
origin of primitive chemotaxonomy. Primitive chemotaxonomy, thus, was an
applied science with extremely practical aims and benefits.
As early as in the 15th century, grouping of medicinal plants was done
according to their active constituents, which is evidenced in the writings of
herbalists. John Griffith Vaugh was among the pioneers of chemotaxonomy. The
importance of chemotaxonomy was anticipated in the 17th century by Nehemiah
Grew and James Petiver. Nehemiah Grew (1673) quoted some chemical
examples like umbelliferous fruits as carminatives, while commenting on the
predictive values of taxonomic characters. James Petiver (1699) demonstrated
that “plants of the same likeness have for the generality much the same virtues
and use”. He showed that natural groups of plants have chemical as well as
morphological and anatomical similarities. Further, in the 18th century, several
significant attempts were made to correlate the chemistry of plants with their
phylogenetic development.

14.2.2 Modern Chemotaxonomy

Helen Abbott (1886) noted that all saponin-containing groups are closely allied
with some structural similarities. She also presumed that the chemical content of
the plants could be a valuable source of evidence not only for classification, but
also in understanding their evolution. In her opinion, the theory of evolution in
the plant kingdom is best illustrated by the chemical constituents present in
them. She concluded that chlorophyll would be of little value in distinguishing
the categories of green plants because of its wide occurrence. This is
appropriately acknowledged as the beginning of the modem phase of
chemotaxonomy.
By the 19th century, emphasis had been directed towards the precise
characterization of chemical constituents and also towards investigating their
occurrence in different species. These attempts could not provide much by way
of conclusive results, but they paved the way for the enormous development of
chemotaxonomy in the years to come.
A. P. De Candolle (1804) had related natural plant classification to the
medicinal properties of plant species. For example, all species of cinchona have
similar constituents and are therefore, used as febrifuge. Greshoff (1909) coined
the term ‘Comparative Phytochemistry’, which he defined as “the knowledge
of connection between natural relationship of plants and their chemical
composition”.
It was recognized quite early that not all the diverse chemical constituents of
plants would be equally useful as sources of taxonomic evidence or as guides to
evolutionary relationships.
Kowarski and Nuttall were among the first to utilize serological methods to
study classification and phylogeny. During this period, the evidences in
chemotaxonomy were culminated into a publication ‘Stammebaum' (a family
tree of plant relatives), which is now only a matter of historical importance.
McNair (1935) noted that more closely-related taxas produce more similar
chemical products. He also mentioned that more highly evolved members form
larger molecules like alkaloids, volatile oils and glycerides. He highlighted the
taxonomic usefulness of variations in the alkaloidal content as well as in the fat
or oil content, in his research publications. According to McNair, chemical
classification may be compared with or used as a supplement to morphological
classification. It is not a replacement for the existing systems of plant
classification, but contributes to their improvement. Thus, he predicted that
comparative chemistry would be an aid to taxonomy. He had foreseen the
importance of chemotaxonomy in the development of the true natural system of
angiosperm phylogeny.
Baker and Smith (1920) studied the essential oils of eucalyptus and concluded
that primitive species were also chemically primitive. Babcock (1947)
chemically investigated the genus Crépis L. and made a phylogenetic grouping
within the genus. Dedio et al (1969) analyzed the phenolic constituents of Secale
(Tourn.) L. and proposed a definite relationship between the species. The
chemotaxonomy of the family Compositae was studied by many researchers
with a reference to sesqui-terpene lactones, flavonoids and some other
compounds.
The tremendous progress which has been witnessed in recent years has been
made possible by:

the screening of a large number of plants for isolation and the

characterization of many new natural products
the elucidation of biosynthetic pathways

Such a rapid expansion of chemotaxonomy as witnessed in recent times is

largely due to the advent of separation techniques like column chromatography,
HPLC, TLC, paper chromatography, gas chromatography, electrophoresis etc.
and also the determination of structures by Mass and NMR spectra.
In the 20th century, several researchers have contributed immensely to the
development of chemotaxonomy. Among them, the excellent work done by
Hegnauer should be recognized.
14.3 PHYTOCONSTITUENTS USEFUL IN CHEMOTAXONOMY
With the enormous advances made in the field of phytochemistry, a vast number
of compounds have been isolated and characterized. Some of them have been
recognized as being chemotaxonomically important.
The phytoconstituents that are useful in chemotaxonomy are classified as:
1. Primary metabolites
2. Secondary metabolites
3. Semantides and macromolecule-like enzymes

14.3.1 Primary Metabolites

These are the products of vital metabolic pathways i.e. products of the
respiratory chain, TCA cycle, fatty acids and essential amino acids etc. They are
of universal occurrence, and thus, lack systemic significance in chemotaxonomy.
However, in certain organs of a few plants, they are stored in excess. Apart from
their accumulation, the nature of accumulation is important.
For example,

Sac-shaped starch grains in ginger and turmeric

Sucrose, though of widespread occurrence, is present in large quantities in
sugarcane and beetroot.

Quantitative variations often serve as a ‘chemotaxonomic substance’. For

instance, the presence of relatively high amounts of the amino acid, arginine, in
the family Rosaceae, irrespective of its common occurrence in the plant
kingdom, is chemotaxonomically significant.

14.3.2 Secondary Metabolites

Secondary metabolites are less widespread in the plant kingdom, as they perform
only non vital functions. They are usually large molecules with many side
chains. The secondary metabolites that have received attention in
chemotaxonomy are alkaloids, glycosides, terpenoids, volatile oils, flavonoids
etc. Their role in plant classification has been increasingly recognized.
14.3.3 Biosynthetic Complexity and the Occurrence of Secondary
Metabolites
The relationship between the biosynthetic complexity of a secondary metabolite
and its taxonomic significance is discussed here.
The occurrence and distribution of a phytoconstituent in the plant kingdom
depends on its biosynthetic complexity. The more difficult the reaction for the
formation, the less likely it is to occur independently in unrelated plants.
Therefore, the biosynthetic complexity of a compound determines its occurrence
i.e. whether it occurs only in a limited number of species, or in an entire family
or order of the plant etc. For example, the biosynthetically complex alkaloid,
morphine, is found only in two species of Genus Papaver (P. somniferum and P.
setigerum). On the other hand, the relatively simpler alkaloid protopine, occurs
in all plants of the poppy family.
Thus, those compounds, which are biosynthetically simple, are widely
distributed, hence, lack systemic significance. Similarly, those compounds which
are present in only a few species are also of little interest, unless biosynthetically
similar compounds occur in other plants.
Chemical compounds, though diverse in their nature, occurring in different
plants, appear to be biosynthetically analogous. Such plants probably contain
similar enzyme systems in them for their biosynthesis. The formation of these
compounds reflects a relationship (for example, having similar enzyme systems)
between them.
Apart from formation, their accumulation is another important aspect. Some
secondary metabolites like nicotine and related alkaloids are produced in trace
quantities by many plants, but accumulated in large quantities only in few genera
such as Nicotoiana and Duboisia.

14.3.4 Secondary Metabolites in the Perspective of Chemotaxonomy

Phenolic Compounds
Phenoloic compounds, so far, have provided the maximum taxonomic data for
chemotaxonomy. Among them, flavonoids have been extensively studied. They
have a common nucleus, with a great variety of types and patterns of side chains
(Figure 14.1).
 Two classes of water-soluble pigments – anthocyanins and betacyanins – are
responsible for the red, purple and blue colours in many plants. The
anthocyanins are flavonoids,whereas betalains (including the betacyanins) are
nitrogen-containing heterocycles, while performing the same functions as the
former. The anthocyanins are biosynthesized via the shikimic acid pathway,
while betalains are formed by an altogether different metabolic route.

Fig. 14.1 Structure of a Flavanoid

The distribution of anthocyanins and betalains in the plant kingdom is
chemotaxonomically important. The occurrence of anthocyanins is widespread
in the plant kingdom, ranging from mosses to gymnosperms, as well as in the
angiosperms. These are absent in a few families, which alternatively contain
betalains – this is an interesting point As it reveals the presence of one and the
absence of the other metabolic pathway.
Betalains as Chemotaxonomic Markers
A total of 9 families contain betalains, out of which 7 are placed in the order
‘Centrospermae’. The other 2 families containing betalain come under the orders
‘Cactales’ (Cactaceae) and ‘Sapindales’ (Didicraceae). The other families
namely, Gyrostemonaceae, Caryophyllaceae and Mollginaceae, of the order
‘Centrospermae’, are free from betalains, but contain anthocyanins.
Hence, from the perspective of chemotaxonomy, the inclusion of these 9
families containing betalains in ‘Centrospermae’, and excluding the remaining 3
families from it, is ideal. Similarly, the species of the genera ‘Primula’ can be
distinguished on the basis of its petal flavonoids
Alkaloids
Alkaloids are nitrogen-containing bases, usually with a heterocyclic ring.
Though they generally lack significance in chemotaxonomy, alkaloids assume
importance in a few cases due to their specific distribution.
True alkaloids are of rare occurrence in the lower plants, such as ephedrine in
ephedra (a gymnosperm), annotine type of alkaloids and traces of nicotine in
lycopodium (a pteridophyte). The distribution of alkaloids in angiosperms is
uneven. Certain groups of alkaloids have been associated with particular families
or genera. Some of the significant chemotaxonomical findings are discussed
below.

The inclusion of families Solanaceae and Convolvulaceae in the same order

is justified as they contain tropane alkaloids.
Similarly, due to the presence of benzyl isoquinoline and the absence of
glucosinolates, it may be concluded that the Papaveraceae family is nearer
to Ranunculaceae than Cruciferae and Capparaceae.
Papaveraceae and Fumariaceae are closely related, as both contain the
alkaloid protopine.

Glycosides
Glycosides, occurring in plants and animals, are acetals chemically, which on
hydrolysis yield one or more sugars along with a non-sugar moiety. They are
classified on the basis of the linkage between the sugar and non-sugar moiety.
The distribution of the various types of glycosides is discussed below.
’O’-glycosides: Among glycosides, ’O’-glycosides are widespread, and are
therefore, less important in terms of chemotaxonomy. But, in a few cases, they
may attain some importance. For example, the nature of sugar moiety, like deoxy
sugars in cardiac glycosides or the unusual position of attachment of sugars to
aglycone may be characteristic.
‘C’-glycosides: In these glycosides, sugars are attached to the aglycone by
carbon–carbon linkage. These are relatively fewer in occurrence. Aloin (aloes),
cascarosides (cascara bark) are important anthraquinone derivatives which are
’C’-glycosides.
About 15 ‘C’ glycosides of the flavonoid group are present in 8 unrelated
families, ranging from ferns to monocotyledons, but in greater amounts in
Papilionaceae.
‘S’-glycosides: These glycosides give isothiocyanate on hydrolysis. The
occurrence of thioglycosides in all species of the families of Cruciferae,
Capparaceae, Papaveraceae and Fumariaceae unites them into a natural group,
under the order ‘Rhoedales’. Based on chemical and other evidence, Cruciferae
and Capparaceae are now included in the order Capparales (containing
glucosinolates), whereas Papaveraceae and Fumariaceae are placed in the order
‘Papaverales’ (not containing them). Some ’S’-glycosides occur in 8 other
unrelated families, but are not characteristic of them, as they are present
sporadically.
Cyanogenetic Compounds
These are distributed in about 80 families of angiosperms, a few gymnosperms
and several fungi. They hold some systemic significance in chemotaxonomy
because of their limited distribution and also ease of their detection. In fact, they
are extensively studied for this purpose.
The presence of the same biogenetic type of cyanogenetic glycosides in closely
related plant species establishes the familial relationship between them. For
example, only phenyl alanine-derived glycosides, such as prunasin and vicianin,
are present in fern, while only the tyrosine-derived taxiphyllin is found in
gymnosperms.
The original order, Perietales, was taxonomically divided into two orders –
Guttiferales and Violates. This division appears to be in accordance with
chemotaxonomic principles A number of families belonging to the sub-order
Flacoutinae of Violates are rich in cyanogenetic glycosides; but no genera of the
family Violaceae, whose inclusion in this order is doubtful, has them. Sixteen
families of Guttiferales also do not contain them.
The family Rosaceae is known for the presence of cyanogenetic glycosides.
Earlier, it was believed that the presence of phenyl alanine-derived glycosides,
such as prunasin and amygdalin, is characteristic of this family. Later, it was
proved true only in the case of two subfamilies, as the other subfamily was
reported to contain heterodendrin, and cardeospermin–p-hydroxybenzoate,
cardeospermin–p-hydroxycinnamate or dhurrin. Therefore, the division of this
family into subfamilies is in accordance with the concept of chemotaxonomy
using cyanogenetic glycosides as the taxonomical marker.
Terpenoids
These are a varied group of plant constituents formed by the condensation of
active isoprene units. Irrespective of their complex structures, some of them like
phytol, carotene and sterols are found in all phyla of the plant kingdom as they
are physiologically vital. They therefore lack systemic significance in
chemotaxonomy.
 Carotenoids are associated with all photosynthetic tissues, and therefore, occur
with chlorophyll in higher plants. The correlation between the nature of the
carotenoid content in various algae of the proposed evolutionary development,
which is based on conventional assessment is presented in Table 14.1.
Table 14.1 Relationship between the nature of carotenoid content and different
classes of algae

In higher plants, the conversion of chloroplasts into chromoplasts occurs during

ripening with the formation of products like β-carotene oxidated products. These
are devoid of any significance, except a in a few, like capsanthin (capsicum).
The extensive work that was carried out worldwide revealed the link between
the formation of triterpenes and phylogeny. The details are given in Table 14.2.
Table 14.2 Relationship between the nature of terpenes and status of plants in
plant evolution

Many important sesqui-terpene lactones are reported in the family Compositae,

and their nature and distribution have been studied in detail. These studies
revealed that the subdivisions of this family are characterized by the distinct
types of the sesqui-terpene lactones they biosynthesize.
Irridoids
These are another important group of terpenes, mostly monoterpene lactones.
Their presence in 50 families is correlated with certain of their morphological
features. Therefore, all these can be brought into the same group
chemotaxonomically. However, certain unrelated families also contain them,
which indicates that their formation in them is probably independent of the
evolutionary process of angiosperms.

14.3.5 Semantides and Macromolecules

Semantides are the genetic information-carrying molecules. They are classified
as

Primary semantide: DNA

Secondary semantide: RNA
Tertiary semantide: Proteins

Owing to their large molecular size as well as complex structure, the isolation
and comparative study of semantides is very difficult. In view of this, special
techniques like DNA–DNA hybridization, serology, electrophoresis, amino acid
sequencing etc are required for gaining relevant evidence in the case of
semantides.
The sequence of nucleosides, in case of nucleic acids, provides the necessary
information for chemotaxonomy. These techniques are discussed here.
DNA–DNA Hybridization
Charles Sibley and John Ahlquist were the pioneers of this technique, which is
still widely used in microbiology to identify bacteria. This molecular biology
technique is a fundamental technique available in comparative phytochemistry;
it involves the comparison of DNA molecules from different species.
In this technique, the degree of genetic similarity is determined to find out the
genetic distance between two species. DNA sequencing and computational
comparisons of sequences is now the generally adopted method for determining
genetic distance.
 DNA exists as a double helix and its two complementary strands (helices) can
be separated (dissociated) from each other by heating to 100° C and cooling
rapidly. Under suitable conditions, they come together again and combine to
form a complete molecule (re​associated DNA). Re-association depends on the
collision rate i.e. concentration.
In the procedure, DNA from the two species to be compared is extracted,
purified and cut into short pieces of 600–800 base pairs. The DNA of one
species is labelled, and then mixed with the unlabelled DNA of the second one.
The mixture is incubated to allow the DNA strands to dissociate and re-anneal,
forming hybrid double-stranded DNA.
The technique of ‘DNA melting ’ is utilized for comparing one species with
another. ‘DNA Melting ’ is a measure of the energy or temperature required for
the separation of the strands of DNA. It represents the thermal stability of re-
associated DNA molecule. The degree of base pair matching along the double
strands of the re-associated DNA molecule determines the thermal stability.
Hybridized sequences with a high degree of similarity will bind more firmly, and
therefore, require more energy to separate them. Therefore, native DNA will
have higher stability because of perfect matching than the hybridized DNAs
which have some degree of base pair mismatching.
In this way, species, varieties etc of plants can be compared on the basis of the
extent of their DNA Hybridization.
Proteins
These are probably only next to phenolic compounds in providing the data
required for chemotaxonomy. Proteins, though widely occurring in plants and
animals, still possess some chemotaxonomical importance, as they differ from
species to species. The comparison of a protein mixture provides the basis for
the possible chemotaxonomic grouping of such species.
The methods employed for obtaining the chemotaxonomic data are

Serology
Electrophoresis
Amino acid sequence

14.4 PLANT SEROLOGY AND SEROTAXONOMY

The data required for comparing the proteins of different plants can be procured
from serological experiments. The study of the origin and properties of
antiserum is known as ‘serology’. The technique of serology relies on the
immunological reactions shown by mammals when they are invaded by foreign
proteins (antigens).
Proteins are the most widely used antigens in serotaxonomy because they are

1. ubiquitous
2. possess high antigenesity and are
3. preservable

Serological procedures involve production of antiserum, followed by the

initiation of an immunological (antigen–antibody) reaction.

14.4.1 Production of Antiserum

When a plant extract containing proteins or purified proteins or mixed proteins is
injected into a mammal (usually a rabbit), it initiates the formation of antibodies.
These are proteinous and specific to that antigen, and are thus capable of
rendering it non-functional. The antibodies formed are extracted (antiserum) and
standardized by testing for sensitivity against a serial dilution of antigen. This
‘standard’ antiserum is used in sero-taxonomical experiments. Water-soluble
seed proteins are extensively used for this purpose.

14.4.2 Initiation of an Immunological Reaction

Antiserum gives a precipitation reaction with the plant extract (antigen–antibody
reaction). Similarity of other species to the first one can be assessed by
measuring the amount of coagulation it causes. For example, the extracts of
related plants will react similarly, although probably to a lesser extent. But, the
extracts from unrelated plants will fail to give a reaction, as they do not contain
the same or similar protein.
The quantitative precipitation in solution is frequently replaced by the more
convenient ‘Gel diffusion method’.

14.4.3 Gel Diffusion Method

In this procedure, antigen and antibodies may be allowed to react in a gel. The
gel diffusion method has several advantages which include:

physical separation of antigen from antibodies

testing of several extracts of different species, side by side, in the same gel
testing of various ratios of antigen and antibodies

Different methods (single or double diffusion) may be followed. In the single

diffusion method, the antiserum is incorporated into the gel, whereas in the
double diffusion method, both reactants are allowed to diffuse. In this procedure,
discs are cut from a layer of agar in plates and antiserum is placed in one cavity
and antigenic extract (plant) in another. The liquids diffuse towards one another
and react to form a precipitation line or series of lines depending on whether it is
of the same, similar or different antigenic nature. These are indicated by the
degree of physical fusion and the number of precipitin lines. These are illustrated
in Figures 14.2a, b, c.
Other methods are:
1. Absorption
2. Immunoelectrophoresis
3. Radio- immunoassay
4. Enzyme Linked Immunosorbent Assay (ELISA)

14.4.4 Absorption
Protein mixtures of different plants often contain some common proteins. In this
method, the removal of the antibodies for such common proteins from the
antiserum is carried out. As the antiserum now contains only those antibodies
which can react with specific proteins, logical comparisons can be drawn from
precipitation reactions.
In this procedure, antiserum is saturated with the antigenic extract of the
second plant and allowed to diffuse towards the original antigen. The formation
of precipitation lines indicates the presence of the specific toxins in the original
plant, but not in the second. A spur on the precipitation line between antigenic
extract and its antiserum is seen if it is suitably placed in relation to the antigenic
extract of the second plant.
Fig. 14.2a Identical antigens: Complete fusion of precipitin lines (As =
Antiserum; 1–6 Plant extracts)

Fig. 14.2b Similar antigens: Partial fusion of precipitin lines (As=Antiserum; A

and B=Plant extracts)

Fig. 14.2c Different antigens: Complete crossing of precipitin lines

(As=Antiserum; A and C=Plant extracts)

14.4.5 Immunoelectrophoresis
This is a combination of serological and electrophoresis procedures. Initially, the
antigens are separated by electrophoresis on a gel and then allowed to diffuse
towards the antiserum. This method is much more specific, as better antigenic
separations occur.

14.4.6 Radio-immuno Assay

In this procedure, the antigens or antibodies are labelled with radioactivity,
which facilitates their identification, even though they are in small
concentrations.

14.4.7 Enzyme Linked Immunosorbent Assay (ELISA)

Enzymes couple their catalytic activity with a specific immunoglobulin, which
forms the basis for the estimation. This is used for example in tests like ELISA
(Enzyme Linked Immunosorbent Assay), which involve the labelling of either
the antigens or antibodies and linking this with enzymes for detecting them even
in minute quantities.

14.4.8 Significance and Limitation of Serotaxonomy

Serological studies have provided a correlation between serotaxonomy and the
classical chemotaxonomic scheme in a number of instances. These studies
mainly denote the relationship between different species and genera. However,
they fail to specify the evolutionary relationships, because which antigen is the
most primitive is unknown.
Electrophoresis
The elecrophoretic separation of proteins relies on their amphoteric properties,
where they are charged positively and negatively to various extents according to
the pH of the medium. These charged ions move across a voltage gradient
through a gel at various speeds. After a suitable separation time, the current is
switched off and the proteins are located by dyes.
Amino Acid Sequence
Amino acids from polypeptide chains are cleaved one by one and identified by
the chromatographic method. In this way, the amino acid sequence of a protein is
determined and compared to the proteins of other plant species.
14.5 CHEMOTAXONOMY IN LOWER PLANTS
With reference to secondary metabolite formation, the four organisms – plants,
fungi, lichen fungi, and actinomycetes – are considered good producers, while
yeasts, protozoa, and animals are regarded as being less efficient. Therefore,
secondary metabolites tend to be important chemotaxonomically, mainly in plant
and fungal taxonomy.
Though bacteria, fungi and algae, are capable of biosynthesizing compounds
via acetate and shikimic acid pathways, apparently, they seldom combine them.
Therefore, flavonoids have never been reported in any of these primitive
organisms.

14.5.1 BACTERIA
Bacteria differ from all other prokaryota in some aspects, like the range of
chemical characters, RNA base sequences, and intermediary metabolism and
also in cell wall composition. Chemotaxonomy, so far has been neglected in
bacteriology.

14.5.2 FUNGI
Fungi, while growing on a given substratum, produce different types of
compounds including toxins, antibiotics and extracellular compounds. Many
filamentous fungi have been screened for a wide range of constituents such as
terpenes, polypeptides, non-ribosomal peptides, and combinations of all these.
The findings reveal that the distribution of secondary metabolites throughout the
fungal kingdom is inconsistent. Thus, they are not apt for interpreting the
phylogeny, but effective for the identification and classification of fungi.
Successful chemotaxonomical interpretations could be made so far in large
Ascomycete genera such as Penicillium, Aspergillus, Fusarium, and in a few of
basidiomycete genera, but not in Zygomycota and Chytridiomycota.

14.5.3 ALGAE
Most algae are still aquatic in their habitat, and hence, differ considerably from
those plants which have adapted to a terrestrial life. Early workers classified
algae into the green, brown and red forms. According to classical taxonomy,
algae have been classified into eleven divisions. These divisions differ
characteristically, in terms of chlorophylls and other pigment groups. Among
these groups, Chlorophyta and Euglenophyta share a similar set of pigments,
which are also found in all higher plants. This provides conclusive evidence for
the common origin of higher plants and green algae.
Bilieproteins in Blue Green Algae
Phycobiliproteins are water-soluble fluorescent proteins, formed of a complex
between proteins and covalently-bound phycobilins. The phycobiliproteins are
the most important constituents of the phycobilisomes, which are subcellular
structures in many algae. They have the ability of capturing light and then
passing it on to chlorophylls during photosynthesis. They act as chromophores
(the light-capturing part), thus, they serve as accessory or antenna pigments for
photosynthetic light collection.
Chemotaxonomic Significance of Phycobiliproteins
The distribution of bilieproteins is primarily restricted to blue green algae. They
are also present in cyanobacteria and certain eukaryotic algae like Rhodophytes,
Cryptomonads and Glaucocystophytes.
Phycobiliproteins are classified on the basis of their colour into two large
groups, namely, the phycoerythrins (red) and the phycocyanins (blue). All
phycobiliproteins contain either phycocyanobilin or phycoerythrobilin
chromophores. These large groups have been subdivided on the basis of λmax
variations sand the specific shape of the absorbance spectrum among the
proteins. Initially, these subdivisions were identified by prefixes to the
phycobiliprotein name. For example, the name, C-phycocyanin, abbreviated C-
PC, indicates the taxa of the organisms from which the pigments were isolated.
Similarly, R-PE stands for R-phycoerythrin, which was first isolated from
Rhodophyta.
Subsequent investigations showed that they can be produced by the organism

at different stages of its life cycle

under different growth conditions
 on certain occasions

Also, different organisms produce them. Therefore, specific phycobiliprotein

types are not always restricted to specific taxa. But the different species or
strains of blue green algae can be distinguished by the differences in
phycobiliproteins formation when grown in monochromatic light. The influence
of quality of light (wave length) on the formation of phycobiliproteins in blue
green algae is referred to as ‘chromatic adaptations’. Such phenomenon is
reported widely in the literature. For example, the blue green algae, Nostac sp.,
showed a manifold increase in its phycoerythrin content when grown in
florescent light. But the same organism could produce higher phycocyanin
content than phycoerythrin when grown in red light. Another group of algae,
Mastigocladus, had shown only a phycocyanin peak under fluorescent light.
However, it produced a higher proportion of phycocyanin under red light, but no
phycoerythrin in green light. Chromatic adaptations, therefore, present vital
clues for the differentiation of blue green algae.

14.5.4 Bryophytes
Lignins and flavonoids occur commonly in almost all plant groups higher than
bryophytes in the plant kingdom Lignins are of taxonomic interest, as they
exhibit characteristic chemical differences depending upon the source i.e.
gymnosperms, mono- and di-cotyledons.
Flavonoids first appear in Bryophyta The earliest report of the existence of
flavonoids in bryophytes appears to be that of Paul (1908), who noticed
anthocyanin-like pigment in a number of Sphagnum species. Flavonoids are by
far the most investigated secondary metabolite of them all. Their utility as
chemical markers is well recognized.
Taxonomically, bryophytes are divided into three classes, namely Hepaticae
(liverworts), Anthocerotae (homed liverworts) and Bryopsida (mosses).
Homworts were previously considered a class within the division of bryophytes,
but, now they are favoured to have their own class called Anthocerotophyta, as
this group appears paraphyletic.
Class: Broyopsida (Mosses)
The class Bryopsida constitutes the largest class of mosses, containing 95% of
all moss species. Nearly half of the screened 200 species of the more than 75
genera of mosses is reported to contain various amounts of flavonoids.
 The class Broyopsida is distributed into three subclasses, namely Sphagnidae,
Andreaeidae and Bryidae. No flavonoid ,except Sphagnombins, was reported
from species of Sphagnidae and Andreaeidae. Among Bryophytes, the subclass
Bryidae appears to containe most flavonoids, but the data could not be correlated
between flavonoid formation and taxonomic data.
Class: Hepaticae (Liverworts)
This division of Bryophytes is commonly referred to as hepatics or liverworts;
it consists of 6000 to 8000 species of liverworts. Markham et al (1965) presented
proof of the presence of flavonoids in liverworts and isolated two apigenin di-C-
glycosides from Hymenophyton flebellatum. They seem to be more frequent in
hepaticae than in mosses. Nearly all investigated species of Merchantiales
possess flavonoids, whereas in Jungermanniales, these compounds are rare.
They are also not distributed equally in each group.
Study of flavonoids chemistry in liverworts is extensive and systematic.
According to Markham and Porter (1978), the composition of flavonoids is so
specific that it is a promising chemical tool for this group of plants.
Distribution of Flavonoids
At the level of families: The only family of the order Blasiales is Blasiacae. The
two genera of this family, Blasia and Cavicularia were screened for flavonoids.
A total of 13 flavonoids were isolated, out of which 10 were totally and three
were partially identified. Though all of them were flavones, only 2 were
glycosides and the remaining were aglycones. Cavicularia was reported for 5
flavonoids, which were flavones.
At the level of genera: The distribution of flavonoids in the 4 species of genus
Mylia is interesting. All these species had shown the presence of flavone-di-C–
glycoside, but no O- glycoside flavone. The flavone-di-C–glycosides of Mylia
are commonly observed in other species of Bryophytes. The presence of 3 to 4
flavonoid glycosides is chemotaxonomically significant. Vicenin-II and
leucenin-II are present not only in many species of liverworts but also in a few
mosses. The distribution of stellavin-II is restricted to only a few; in fact, they
have been detected so far in two bryophytes. The fourth, flavone di-C- glycoside
tricetin 6,8, di-C-glycoside was reported in trace amounts in only one species of
Mylia i.e., M. verrucosa.
 At the level of species: Flavonoids patterns are species specific and are not
influenced by external conditions such as temperature, light, humidity, seasons
etc. They are therefore precious markers for the discrimination of the Bryophyte
species. Some liverworts are reported for highly \ariable flavaonoid content and
pattern. For example, Metzyeria lureata and another of the same variety vulvula
show highly variable flavonoids pattern. Therefore, a cautious approach, like the
comparison of several samples of a species, is required prior to drawing a
conclusion.

14.5.5 Gymnosperms
Cycades are as ancient as dinosaurs. Cycasin and related cyanogenetic
glycosisdes are reported only in the order Cycadales. This makes them valuable
taxonomic markers in the chemotaxonomy of gymnosperms.
The genera ‘Pinus' known for resinous extracts like gum turpentine, comes
under another important order, Coniferales. In most of the classifications, the
genus Pinus is divided into two major subgenus. They are Strobus (haploxylon
or soft pines, with one fibrovascular bundle in the needle) and Pinus (diploxylon
or hard pines, with two fibrovascular bundles in the needles). This division is
consistent with data from wood anatomy and secondary chemistry i.e. by the
presence or absence of gum turpentine. This division is also well supported by
recent molecular phylogenetic studies.

14.6 PROSPECTS AND PROBLEMS OF CHEMOTAXONOMY

Chemotaxonomy, in spite of having a few shortcomings, offers many
advantages. These are:

14.6.1 Advantages
Chemical constituents are less easily influenced by modifying factors, as
they appear to be closer to genes. Thus, they act as invaluable markers for
classification purposes.
It is entirely independent of the classical biological methods, which is
perhaps its greatest virtue.
It provides a meeting ground for taxonomists and chemists.
 Phytochemists can assist botanists to point out as well as verify the
problems in taxonomy and also to draw conclusions logically.
It facilitates a more precise understanding of biologic evolution and natural
relationships.
The principles of chemotaxonomy can be applied to determine the potential
sources of known drugs and also to find out the areas of the plant kingdom
in which new drugs are most likely to be discovered. These are the most
vital points from the perspective of a pharmacognosist.

14.6.2 Limitations
Like all other plant classifications, it is also not free of a few limitations. These
are as discussed below.

Chemotaxonomy is very useful at the genera and lower levels, but is of very
limited value at the higher taxonomic levels.
At present, structures of only a few thousand natural products have been
established. This indicates that a vast number is yet to be investigated.
Apart from this, their distribution in the plant kingdom is very little
understood. Thus, utilizing chemical facts for classification has its own
inherent constrictions.
Another retarding factor is the difficult as well as tedious procedures
involved in the isolation and structural elucidation of phytoconstituents.
Relying totally on the evidence obtained from a single source often leads to
wrong conclusions.

14.6.3 Problems
A few problems pertaining to chemotaxonomical studies are mentioned below.
1. Many common chemical constituents in plants are parts of different
metabolic pathways and their presence in two taxa might be due to the
presence of quite different sets of enzymes. Therefore, the best way is to
study chemical pathways rather than chemical substances as the pathways
indicating a series of enzymes involved rather than a single compound.
2. If the molecule is more complex and is further away from a common
metabolic pathway, then it is less likely to be polyphyletic.
 3. Difficulty in the detection of metabolic products, as they are often present
in low quantities.
4. Non-availability of complete chemical information due to incomplete
investigation.
5. Significant variations in the chemical composition of the plants due to the
influence of factors such as parts of the plant, growing conditions, the stage
of the life cycle or season, the time and method of collection, storage
conditions, method of extraction of its chemical constituents etc.

14.6.4 Reasons for Development

In spite of the shortcomings and problems associated with it, chemotaxonomy is
rapidly expanding due to the following reasons.

The activity and therapeutic use of drugs are based on chemical

constituents, so chemical classification is preferred.
Gathering evidence by new techniques (especially chromatography and
electrophoresis) is now much faster and simpler and also requires less plant
samples.
The vital metabolic pathways are similar, but there are a lot of variations in
the less vital pathways between plants.
 15

The Role of Literature Search in

Medicinal Plant Research

With major advances in biotechnological and phytochemical techniques,

academic and industrial interest in discovering and developing bioactive, natural
products has been increasing. Medicinal plants are treated as a subject of serious
study and intense research all over the world. Accurate research is the
cornerstone of decision making for the development of herbal drugs, and it is
increasingly important as the number of herbal-based treatments for specific
conditions increase. Herbal drug research is at the forefront in the present
therapeutic scenario. Researchers have to search the literature for medicinal plant
information and often, they find it a highly confusing prospect. This chapter
focuses on the basic plan to be adopted before starting a literature search on the
worldwide web; it also outlines the specific methods to be used to obtain correct
information [Mueen Ahmed. K K et al 2006].

15.1 DEFINITION
Literature search involves the systemic search of various formal and informal
publications used to disseminate academic research, in order to find items
relevant to a particular area of interest. The primary focus should be on formal
material, which includes books from recognized academic publishers, articles
published in referred academic journals, conference papers and theses, all of
which have had some form of peer or editorial review. An effective literature
search will establish that the piece of research that the researcher intends to
undertake has not been undertaken by someone else already; it will also help
locate existing research that has already been done in related areas.
15.2 Purpose of Literature Search
Any investigation will involve reading and reviewing what other people have
written about the researcher’s area of interest. This is done for a number of
reasons, primarily to give support and offer justification for his/her work, and to
ensure that what others have done is not being repeated. It also helps the
researcher to look around the problem for helpful ideas, and compare his work to
prior research.
As a part of the written report, the literature review will ground the reader in
the area and explain why the research that is being undertaken, fills a gap. It
usually forms at least one chapter of the report. In order to review the literature,
you need to find out what has been published in your field, and this is where the
literature search comes in.
A literature search has to be systematic and focused – you are not looking to
read everything in a broad area, only those articles of information that are
relevant to your work. A literature search must also be evaluative – each
reference that is found will have to be critically assessed to determine if it is
worth pursuing. Searching for and reviewing literature is a skill that can be
learnt, but it takes some practice. Conducting a literature search involves the
following steps:
1. Planning a search strategy
2. Searching bibliographic indexing databases that are relevant.
3. Refining search strategy until it produces relevant results.
4. Evaluating the results produced from research.
5. Saving useful results
6. Recording the results
7. Repeating the process using different databases.
8. Evaluating all saved literature references.
9. Finding the literature that is required.
10. Reading it thoroughly.
15.3 THE INTERNET AS AN INFORMATION SEARCH TOOL IN
PHYTOPHARMACOLOGICAL RESEARCH
The Internet is a global computer network, allowing communication with
millions of computer users; it provides access to resources from around the
world. No matter what type of computer is used for connecting to the Internet, a
virtually limitless wealth of resources is available for everyday use. The Internet
is also a world wide computer network which allows communication with
millions of other internet users and enables the use of web resources throughout
the world.
Natural products have been a major source of drugs for centuries. With major
advances in biotechnological and phytochemical techniques, academic and
industrial interest in discovering and developing bioactive natural products has
been growing. Medicinal plants are treated as a subject of serious study and
intense research all over the world. The immense surge of scientific interest in
natural products as a potential source of drugs has contributed to the
development of phytopharmacology research. However, scientific information is
found in intimidating quantities in various types of databases, virtual libraries,
journal sites, all of which often tend to confuse the prospective researcher.
Sadly, many of the articles published in India are but minor variations of work
already done elsewhere and the reason for this unfortunate situation is that a
thorough search of the literature may not have been done before planning the
experiments. Ironically, the presence of so much information may itself make
scientific research elusive. Most academicians and researchers agree that a
rational drug discovery can be initiated, if a review of existing information can
be conducted in a systematic and objective manner. Today, the internet has
presented the researcher with more effective tools for carrying out a systematic
literature search.
Simple as it may seem, acquiring information on the World Wide Web is not
easy. Beginners in internet search often restrict their search only to search
engines like Google, Yahoo, Infoseek and MSN search, which results in a narrow
outcome of the required information. A compilation of available sources of
information has been done with a view to address these difficulties of beginners
(Fig. 15.1 and Table 15.1).
Fig. 15.1 The Internet and Access to Information.
Table 15.1 Classification of Sources for Literature Search

15.4 PLANNING A SEARCH

Before beginning a search, define what exactly is being looked for.

15.4.1 Step 1
Write out your search as a statement. For example, the topic could be: ‘The role
of Azadirachta indica in the treatment of diabetes mellitus’

15.4.2 Step 2
Identify the important words/concepts. Sentences should be translated into
keywords, by thinking of all possible synonyms (words that mean the same) and
variant spellings, so that relevant articles show up in the search. For example, in
the case above, the keywords would be: Azadirachta indica, Diabetes mellitus,
Apocynaceae, Hypoglycemic
Redundant words: Some words, in sentences, will be redundant because they
are another way of expressing the same concept, or they can be determined from
reading the actual material. For example, instead of searching for the term,
‘novel methods’. search for all types of methods, then decide which are novel,
by reading the articles.
Spelling: It is important to spell the search terms correctly. For example,
search for Azadirachta (correct spelling), not Azadrachta (which is incorrect).
Phrases: If there is a particular group of words that are always used to describe
a particular concept, treat them as a phrase – this means results are possible,
where the words are used together in the given order. For example, “sugar
reducing”.
Truncation: Use truncation for a single term for related words that express the
same concept. The truncation symbol is usually * or $ – the ‘ help’ for each
database will provide this symbol. For example “optim*” will find optimize;
optimized; optimizing; optimal; optimum. It will also find all the
American/British spelling of these words for more related words and synonyms.
The online websites of dictionaries and thesauruses is worth using (www.m-
w.com). The following example shows how a sentence can be broken down into
keywords and grouped by concept. It also shows how they relate to one another.
Terms searched for: “Antihyperglycemic activity of Neem”
Key words

Azadirachta indica
Neem
Diabetes mellitus
Medicinal plant
Blood glucose lowering activity
Hypoglycemic activity

15.4.3 Step 3: Think about ways in which one could limit the search
There will be more published information than you will be able to handle with
regard to some subjects or terms. To deal with this, one can limit the search in
other ways.

Date – restrict search only to items published in the last few years.
Language – restrict the search only to references in English.
Geography – restrict search only to a specific place or research published in
a particular country.
Medicinal plants – restrict search only to specific genus or species of a
particular plant.
Type of publication – restrict search only to references in journal articles,
books or theses.

15.5 ELECTRONIC DATABASES

The ones used for literature searches are bibliographic indexing databases, which
hold records containing details of publications, including books, journal articles,
conference papers and proceedings, theses etc; You can use these databases to
find useful references on specific subjects. The other type of electronic database
is a full-text collection. This means than they hold the full-text of all the articles
from specific journals. Databases are accessible in a number of ways.
Via Electronic Resources (www)
Databases are collections of citations published in indexed journal articles, using
storage devices such as online servers, hard disks and compact discs. Online
databases enable a precise query and help retrieve a particular set of items by
matching the words searched for to words from the field of citations.
Some libraries subscribe to a range of bibliographic databases: some cover all
subject areas (for example, Web of knowledge), some cover particular types of
publication (for example, index to theses), some cover specific subject areas (for
example, Embase, PsycINFO).

15.6 SOME PLANT-RELATED DATABASES

15.6.1 List of Databases: Medical Plants and Alternative Medicine
Resources
BIOSIS provides vital sources of information for life scientists.
http://www.bioisis.org/
CISOM Database- http://www.rccm.org.uk/cisc.htm
CRIPS (Computer Retrieval of Information on Scientific Projects) -
Database of federally funded biomedical research projects conducted at
universities, hospitals and other research institutions.
http://crips.cit.nih.gov/
Chinese Medicine: Traditional Chinese Medicine Database system.
http://www.cintcm.com/index.htm
Dr. Duke’s Phytochemical and Ethno botanical Databases: An
Agricultural Research Service from the US Department of Agriculture.
http://www.ars – grin.gov/duke/index.html.
EMBASE: An international database to citations covering biomedical
pharmacological and drug literature. http://www.embase.com/
HerbMed: A herbal database http://www.herbmed.org/
HPPD (Herbal: Past and Present Database)
http://www.extra.hu/hbock/dbase/index.html
Indian System of Medicine: A gateway for information on the Indian
Systems of Medicine and Homoeopathy maintained by the Department of
ISMH, Ministry of Health and Family Welfare, Government of India; it also
links up with the Indian Ayurvedic Pharmacopoeia http://
Indianmedicine.nic.in/
The Medicinal and Aromatic Plants Abstracts (MAPA): A bimonthly
abstracting journal by the National Institute of Science Communication and
Information Resources (NISCAIR), provides coverage of the global
literature on all aspects of medicinal and aromatic plants. Till date, 81,000
abstracts have been published in 24 volumes, covering the period from
1979 to 2002. About 54,000 abstracts (from 1988 onwards) are available in
database form.
Contact address: The Director, National Institute of Science
Communication and Information Resources, Dr. K.S. Krishna Road, New
Delhi -110012, India.
Email: [email protected] Reprints of full text articles can be obtained
from the Indian Medlars Centre - http.//www.uncat.nic.in/
Manual, Alternative and Natural Therapy (MANTIS) Database
(formerly CHIROLARS): A coverage for healthcare disciplines, not
significantly represented in the major biomedical databases, references
from more than 1,000 journals, with preference given to peer-reviewed
journals. http://www.healthindex.com/ MANTISAbout.htm/
Dialog Corporation http://library ;
dialog.com/bluesheets/html/b10091.html
Health Index http://www.healthindex.com/
OVID Databases http://www.ovid.com/
MEDLINE Database: is the best interface known as PubMed from the
National Library of Medicine, Bethesda, USA
http://www.ncbi.nlm.nih.goventrez/
Medicinal and Poisonous Plant Databases http://www.biologie.uni-
hamburg.de/b-online/ibc99/poison/
MICROMEDEX Complementary and Alternative Medicine (CAM)
Series:- A series of databases covering four areas – herbal medicine and
dietary supplements, clinical protocols, patient education, and herbal and
dietary supplement toxicology http://www.micromedex.com/products /
healthcare/cam/
NAPRALERT (Natural Products ALERT from STN International):
Contains bibliographic and factual data on natural products, including
information on the pharmacology, biological activity, taxonomic
distribution, ethno-medicine and the chemistry of plant, microbial, and
animal (including marine) extracts. In addition, the file contains data on the
chemistry and pharmacology of secondary metabolites that are derived
from natural sources and that have a known structure. The NAPRALERT
file contains more than 100,000 records from 1950 to the present.
Approximately 50% of the file is from a systematic survey of the literature
from 1975 to the present. The remaining records were obtained by selective
 retrospective indexing, dating back to 1950.
http://Infor.cas.org/ONLINE/DBSS/napralertss.html,
http:/stneasy.cas.org/html/English/login1.html
Patent database – United States Patent and Trademark Office: This is a
tool to locate registered patents in complementary and alternative medicine,
http://www.uspto.gov/patft/index.html

15.7 SEARCHING TECHNIQUES

There are a number of important methods that can be used to make sure you
search efficiently and effectively; to ensure that only relevant references are
found; and that you find all of them.

1. Truncation/wild card symbols

2. Search operators
3. Phrase searching
4. Searching using indexes

15.7.1 Truncation/Wild card Symbols

Most databases will only retrieve records that are an exact match for what is
typed in. Truncation and wild card symbols enable the researcher to overcome
this limitation. These symbols can be substituted for letters to retrieve variant
spellings and word endings.
A wild card symbol replaces a single letter: This is useful to retrieve
alternative spellings and simple plurals. For example, wom?n will find woman
or women.
A truncation symbol retrieves any number of letters: This is useful to find
different word-endings based on the root of a word. For example,. Africa* will
find Africa, African, Africans, Africaans, Agricultur* will find agriculture,
agricultural, agriculturalist.
Check the online help screens for details of the symbols recognized by the
database you are searching – not all databases use the ? and * symbols.

15.7.2 Search Operators

Use search operators (also known as Boolean operators – AND, OR, NOT) to
combine your search words. The records retrieved are indicated by the shaded
areas on the diagrams.
“AND” retrieves records containing both words. It narrows your search.
Some databases automatically connect words typed with AND or OR and
retrieve records containing either word. It broadens your search. You can use it
to include synonyms in your search.
“NOT” retrieves your first word, but excludes the second. Beware! By using
this operator you might exclude relevant records, because you will lose those
records that include both words.

15.7.3 Phrase Searching

Phrase searching is a useful technique that can increase the relevance of search
results. Sometimes the search may be made up of common words which, when
combined in an ‘AND’ search, retrieve too many irrelevant details and records.
Databases use different techniques to specify phrase searching – check the online
help.
Examples of phrase searching.
Web search engines – many allow you to specify a phrase using inverted
commas, for example, ‘agricultural development’, ‘foot and mouth’
Some databases allow you to specify a phrase by putting ‘adj’ between your
search words. For example, agricultural adj development.
Some databases automatically perform a phrase search if you do not use any
search operators.

15.7.4 Searching using Indexes

It is possible to search some databases using indexes. These are usually
alphabetical lists of authors or subjects. Indexes are particularly useful for
searching for authors: there may be inconsistencies in the way an author has
been cited. By browsing the index, you will be able to select all possible variant
forms of the name to ensure that relevant references are not missed.

15.7.5 Saving Search Results

Databases usually provide several ways of saving search results.

Emailing records to oneself is the most common option.

 Save on to flash drive or another directory on the PC or network directory.
Print copy
Export into bibliographic management software, for example, Refworks,
Endnote, Papyrus.

In recent years, the Internet has revolutionized the way in which many
researchers get information, making available, a vast array of resources. The
explosive growth in biomedical literature has made it difficult for researchers to
keep up with advancements, even in their own narrow specializations. In turn,
the Internet is an important tool for increasing cutting-edge access in medicinal
plant research.
 16

Patenting of
Herbal Drugs

A patent is a set of exclusive rights granted by a state to an inventor or his

assignee for a limited period of time in exchange for the disclosure of an
invention.
The procedure for granting patents, the requirements placed on the patentee
and the extent of the exclusive rights vary widely between countries, according
to national laws and international agreements. Typically, however, a patent
application must include one or more claims defining the invention which must
be new, inventive and useful or industrially applicable. In many countries,
certain subject areas are excluded from patents, such as business methods and
mental acts. The exclusive right granted to a patentee in most countries is the
right to prevent others from making, using, selling, or distributing the patented
invention without permission.
Under the World Trade Organization’s (WTO) Agreement on Trade-
Related Aspects of Intellectual Property Rights (TRIPS), patents should be
available in WTO member states for any invention, in all fields of technology,
and the term of protection available should be a minimum of twenty years.
Different types of patents may have varying patent terms (i.e., durations).
The word patent originates from the Latin patere, which means “to lay
openâ€​ (i.e., to make available for public inspection), and more directly, as a
shortened version of the term letters patent, which originally denoted ‘an open
for public reading’ royal decree, granting exclusive rights to a person. In
short, we can say that it is the grant of sole rights to make or sell.

16.1 DEFINITION
Patents are applied to the inventions of new producers and processed in scientific
fields such as pharmacology, biotechnology, electronics, machinery and
climates.
The term patent usually refers to the right granted to anyone who invents or
discovers any new and useful process, machine, article of manufacture, or
composition of matter, or any new and useful improvement thereof. The
additional qualification utility patents are used in the United States to distinguish
them from other types of patents, but this should not be confused with utility
models granted by other countries. Examples of particular species of patents for
inventions include biological patents, business method patents, chemical patents
and software patents.
Some other types of intellectual property rights are referred to as patents in
some jurisdictions: industrial design rights are called design patents in some
jurisdictions (they protect the visual design of objects that are not purely
utilitarian), plant breeder’s rights are sometimes called plant patents, and
utility models or Gebrauchsmuster are sometimes called petty patents or
innovation patents. The word patent is related primarily to the patent for an
invention, although so-called petty patents and utility model may also be granted
for inventions.

16.2 BENEFITS OF PATENT PROTECTION

The research-based pharmaceutical industry relies heavily on patent protection
to sustain its development of new medicines. The benefits of patent protection
for pharmaceuticals, including vaccines, have been well documented.

1. Over 90% of prescription medicines and vaccines in the market today are
the result of industry research, which would not have been made without
the incentive of market exclusivity offered by patent protection.
2. Patent protection encourages researchers to take risks to develop new
producers and new therapies.
3. Patent protection strikes the right balance between an inventor’s
legitimate concerns about compensation, and society’s need for more
scientific developments.
4. With high levels of patent protection, it is more difficult for manufacturers
 to produce substandard pharmaceuticals, which can result in serious adverse
health effects in many countries.
5. Effective patent protection provides producers the time to educate medical
professionals about the proper use of new medical therapy.
6. Patent protection is an indispensable element of economic development and
growth.
7. Patent protection encourages local inventors to disclose and commercialize
their inventions.
8. Patent protection improves a country and its credibility and may lead to its
requisition as a dependable supplier.
9. Patent protection for pharmaceutical products is an essential element in the
provision of more cost-effective healthcare.
10. It will result in flexibility for the Government to ensure the availability of
drugs for national emergencies and disasters.

16.3 PATENT APPLICATION

A patent application is a request pending at a patent office for the grant of a
patent for the invention described and claimed by that application. An
application consists of a description of the invention (the patent specification),
together with official forms and correspondence relating to the application. The
term patent application is also used to refer to the process of applying for a
patent, or to the patent specification itself (i.e. the content of the documents filed
with a view to initiating the process of applying for a patent).
In order to obtain the grant of a patent, a person, either legal or natural, must
file an application at a patent office with jurisdiction to grant a patent in the
geographic area over which coverage is required. This will often be a national
patent office, but could be a regional body, such as the European Patent Office.
Once the patent specification complies with the laws of the office concerned, a
patent may be granted for the invention described and claimed by the
specification.
The process of “negotiatingâ€​ or “arguingâ€​ with a patent office for the
grant of a patent, and interaction with a patent office with regard to a patent after
it has been granted is known as patent prosecution. Patent prosecution is distinct
from patent litigation, which relates to legal proceedings for the infringement of
a patent after it is granted.

16.4 PLANT BREEDERS€™ RIGHTS

Plant breeders’ rights (PBR) are the rights granted by Government to a plant
breeder or originator or owner of a new variety, to exclude others from
producing for commercializing the propagating material for a minimum period
of 15–20 years. A protected variety must fulfill some requirements which are

1. It should be new
2. It should be distinct
3. It should be uniform
4. It should be stable
5. There should be no recourse to short cuts

The PBR is extended to all the plants as it helps in extending the breeding
work to more crops, which in turn will contribute to the development of greater
crop diversity.
A wide range of agricultural and horticultural methods and products can be
patented, provided these are inventions and represent novelties. For example:

1. Bio-pesticides and bio-insectides

2. Novel techniques of micro propagation
3. Plant tissue culture methods to prepare useful secondary metabolites.
4. Hybrid patent for molecular genetic research.
5. Development of herbicide-resistant cotton, insect-resistant tobacco and
virus-resistant potato through genetic engineering.
6. Production of antibodies through hybridoma technology.

16.4.1 Benefits of PBR

1. It encourages private companies to invest in plant breeding activities.
 2. It makes it convenient to develop varieties which have been developed in
foreign countries, which are protected by IPR.
3. The imposition (the action of unfair) of PBR in India will lead to the
following problems:
The cost of seeds will increase.
There will be a delay in the spread of new varieties to the farmers.
The benefit of new varieties will be restricted to a small segment of
farmers.

16.4.2 Disadvantages of PBR

1. PBR will encourage monopoly in genetic material for specific trade.
2. It suppresses the free exchange of genetic material and may encourage
unhealthy practices.
3. The farmer’s privilege to reserve the seed may became diluted or may
be eliminated gradually.

1 6 .5 IMPORTANT POINTS TO BE KEPT IN MIND WHILE

DRAFTING AND FILING A PATENT
A patent is not a right to practice or use the invention. Rather, a patent provides
the right to exclude others from making, using, selling, offering for sale, or
importing the patented invention for the term of the patent, which is usually 20
years from the filing date, subject to the payment of maintenance fees. A patent
is, in effect, a limited property right that the government offers to inventors, in
exchange for their agreement to share the details of their inventions with the
public. Like any other property right, it may be sold, licensed, mortgaged,
assigned or transferred, given away, or simply abandoned.
The rights conveyed by a patent vary from country to country. For example, in
the United States, a patent covers research, except “purely philosophicalâ€​
inquiry. A US patent is infringed by any “makingâ€​ of the invention, even a
making that goes toward the development of a new invention – which may itself
become the subject of a patent.
A patent being an exclusionary right does not, however, necessarily give the
owner of the patent the right to exploit the patent. For example, many inventions
are an improvement of prior inventions which may still be covered by someone
else’s patent. If an inventor takes an existing, patented mouse-trap design,
adds a new feature to make an improved mouseÂ​trap, and obtains a patent on
the improvements, he or she can only legally build his or her improved mouse-
trap with permission from the patent holder of the original mouse-trap, assuming
the original patent is still in force. On the other hand, the owner of the improved
mouse-trap can exclude the original patent owner from using the improvement.
Some countries have “working provisionsâ€​ which require that the
invention be exploited in the jurisdiction it covers. The consequences of not
working an invention vary from one country to another, ranging from revocation
of the patent rights to the awarding of a compulsory license awarded by the
courts to a party wishing to exploit a patented invention. The patentee has the
opportunity to challenge the revocation of license, but is usually required to
provide evidence that the reasonable requirements of the public have been met
by the working of the invention.

16.5.1 Enforcement
Patents can generally only be enforced through civil lawsuits (for example, for a
US patent, by an action for patent infringement in a United States federal court),
although some countries (such as France and Austria) have criminal penalties for
wanton infringement. Typically, the patent owner will seek monetary
compensation for past infringement, and will seek an injunction prohibiting the
defendant from engaging in future acts of infringement. In order to prove
infringement, the patent owner must establish that the accused infringer practices
all of the requirements of at least one of the claims of the patent (noting that in
many jurisdictions the scope of the patent may not be limited to what is literally
stated in the claims, for example due to the “doctrine of equivalentsâ€​).
An important limitation on the ability of a patent owner to successfully assert
the patent in civil litigation is the accused infringer’s right to challenge the
validity of that patent. Civil courts hearing patent cases can, and often do,
declare patents invalid. The grounds on which a patent can be found invalid are
set out in the relevant patent legislation and vary between countries. Often, the
grounds are a sub-set of the requirements for patentability in the relevant
country. While an infringer is generally free to rely on any available ground of
invalidity (such as a prior publication, for example), some countries have
sanctions to prevent the same validity questions being re-litigated. An example is
the UK Certificate of contested validity.
However, the vast majority of patent rights, are not determined through
litigation, but are resolved privately through patent licensing. Patent licensing
agreements are effectively contracts, in which the patent owner (the licensor)
agrees to forgo his right to sue the licensee for infringement of the licensor’s
patent rights, usually in return for a royalty or other compensation. It is common
for companies engaged in complex technical fields to enter into dozens of
license agreements associated with the production of a single product. Moreover,
it is equally common for competitors in such fields to license patents to each
other under cross-licensing agreements in order to share the benefits of using
each other’s patented inventions.
The United Nations Statistics Division reports that the United States was the
top market for patents in force in 2000, closely followed by the EU and Japan.

16.5.2 Ownership
In most countries, both natural persons and corporate entities may apply for a
patent. In the United States, however, only the inventor(s) may apply for a
patent, although it may be assigned to a corporate entity subsequently and
inventors may be required to assign inventions to their employees under their
contract of employment. In most European countries, ownership of an invention
may pass from the inventor to their employer by rule of law, if the invention was
made in the course of the inventor’s normal employment duties.
The inventors, their successors or their assignees, become the proprietors of
the patent when and if it is granted. If a patent is granted to more than one
proprietor, the laws of the country in question and any agreement between the
proprietors may affect the extent to which each proprietor can exploit the patent.
For example, in some countries, each proprietor may freely license or assign
their rights in the patent to another person, while the law in other countries
prohibits such actions without the permission of the other proprietor(s).
The ability to assign ownership rights increases the liquidity of a patent as
property. Inventors can obtain patents and then sell them to third parties. The
third parties then own the patents and have the same rights to prevent others
from exploiting the claimed inventions, as if they had originally made the
inventions themselves.
1 6 .6 INDIAN AND INTERNATIONAL PATENT LAWS AND
PROPOSED AMENDMENTS
The grant and enforcement of patents are governed by national laws, and also by
international treaties, where those treaties have been given effect to in national
laws. Patents are, therefore, territorial in nature.
Commonly, a nation forms a patent office with responsibility for operating
that nation’s patent system, within the relevant patent laws. The patent office
generally has responsibility for the grant of patents, with infringement being the
purview of the national courts.
There is a trend towards global harmonization of patent laws, with the World
Trade Organization (WTO) being particularly active in this area. The TRIPs
Agreement has been largely successful in providing a forum for nations to agree
on an aligned set of patent laws. Conformity with the TRIPs agreement is a
requirement of admission to the WTO, and so, compliance is seen by many
nations as important. This has also led to many developing nations, which may
historically have developed different laws to aid their development, enforcing
patents laws in line with global practice.
A key international convention relating to patents is the Paris Convention for
the Protection of Industrial Property, initially signed in 1883. The Paris
Convention sets out a range of basic rules relating to patents, and although the
convention does not have direct legal effect in all national jurisdictions, the
principles of the convention are incorporated into all notable current patent
systems. The most significant aspect of the convention is the provision of the
right to claim priority: filling an application in any one member state of the Paris
Convention preserves the right for one year to file in any other member state,
and receive the benefit of the original filing date. Because the right to a patent is
intensely date- driven, this right is fundamental to modem patent usage.
The authority for patent statutes in different countries varies. In the United
States, the Constitution empowers Congress to make laws to “promote the
Progress of Science and useful Arts..... â€​ The laws Congress passed are codified
in Title 35 of the United States Code and created the United States Patent and
Trademark Office. In the UK, substantive patent law is contained in the Patents
Act 1977 (as last amended by the Copyright, Designs and Patents Act 1988).
In addition, there are international treaty procedures, such as the procedures
under the European Patent Convention (EPC) [administered by the European
Patent Organization (EPOrg)], and the Patent Cooperation Treaty (PCT)
(administered by WIPO and covering 137 countries), that centralize some
portion of the filing and examination procedure. Similar arrangements exist
among the member states of ARIPO and OAPI, the analogous treaties among
African countries, and the nine CIS member states that have formed the
European Patent Organization.

1 6 .7 THE TRADE-RELATED ASPECTS OF INTELLECTUAL

PROPERTY RIGHTS (TRIPS)
TRIPs provide minimum standards for intellectual property rights, including
patents, which each member of the World Trade Organization (WTO) must
incorporate into its national patent statute. Therefore, all WTO members will
provide at least these minimum standard intellectual property rights, with good
enforcement provisions, including a ban on the importation of infringing goods.
The TRIPs Agreement is supplemental to the contribution of WIPO in the
global intellectual property scene. The WTO, including TRIPs, coexists with
WIPO, with TRIPs setting provisions, which shall be found in the patent statutes
of all WTO members.
The more important provisions set by TRIPs include

Patents shall be available and patent rights enjoyable without discrimination

as to the place of invention, the field of technology and whether products
are imported or locally produced.
A patent for a product shall prevent an unauthorized third party from
making, using, offering for sale, selling or importing the product for such
purposes.
The patent term shall be a least 20 years from the date of filing the patent
application.
Compulsory licenses will only be available if certain very limited
provisions are fulfilled – after prior negotiations of reasonable terms, for
predominant supply to the local market, on payment of adequate
remuneration taking into account the economic value of the license, and
subject to appeal.
Procedure for infringement actions and remedies available against the
proven infringement and to prevent infringement, including interlocutory
injunctions.
Tansitional arrangements
 Glossary

Acetous: Producing or resembling vinegar

Alicyclic: An organic compound that is both aliphatic and cyclic
Amphoteric: Able to react to both acid and base; having both acidic & basic
properties
AUC: Area under the plasma concentration–time curve
Binomial classification: The usage of two names—the generic and the specific
—in classifying living things.
BP: British pharmacopoeia
BPC: British Pharmaceutical Codex
Castorium: Oil extracted from beaver secretion
Chemotaxonomy: Classification of organisms based on differences at the
biochemical level, espeÂ​cially in the amino acid sequences of common proteins.
Conchoidal: Of, relating to, or being a surface characterised by smooth, shell-
like convexities and concavities
Cure (food): To treat food (such as meat, cheese or fish) by one of several
methods in order to preÂ​serve it
Cure (polymer) : A process of hardening a polymer material
Cytogenetics: The branch of biology that deals with heredity and cellular
components
Disc pulveriser: A disc type grinder, designed to grind virtually any material to
produce a fine mesh sample in one operation.
DMEM: Dulbecco’s modified Eagle’s medium
DMSO: Dimethyl sulphoxide
DPA: Diphenyl amine
Ebers payprus: The text of the document on Egyptian medicine, containing
more than 800 formulae and 700 different drugs obtained from plant, animal and
mineral origin.
Ebullition : The action of boiling
EDTA: Ethylenediamine tetraacetic acid
Eicosanoids: A class of oxygenated hydrophobic molecules.
Embryology : The branch of biology that deals with the formation, early growth,
and development of living organisms
Enforcement: Compelling influence
FBS: Foetal bovine serum
Flocculent: A substance that causes a dispersed colloidal system (such as clay)
to coagulate and form flocs (coagulated masses of particles in a liquid).
GMP: Good manufacturing practises
Gooch crucible: A crucible with a bottom perforated with small holes, designed
specifically for use in filtration, especially for gravimetric analysis.
Hammermill: A machine used to shred material into fine particles
HE: A substance consisting of haemotoxylin and eosin used to stain tissues for
histopathological studies.
High proof spirits: Alcoholic proof is a measure of how much ethanol is in an
alcoholic beverage, and is approximately twice the percentage of alcohol by
volume. Wines top out at about 28 to 32 proof while other spirits are much
higher, meaning they have a higher alcoholic content.
Hypertonicity: Having the higher osmotic pressure of two solutions.
i.d.: Inner diameter
IP: Indian pharmacopoeia
ISH & M: Indian Systems of Medicine and Homoeopathy
Kieselgur: Diatomaceous earth, a powder made of the desiccated shells of
diatoms, used as a filtering agent, adsorbent, and abrasive in many chemical
operations.
Lineal concept: The theory that a plant has a particular line of descent.
Materia medica: The aspect of medical science which deals with origin and
preparation of drugs, their doses and their mode of administration.
Menstruum: Solvent
Nestorian Church : Christian community of Iraq, Iran, and Malabar, India.
Neutral red: Otherwise known as toluylene red is a dye used for staining in
histology.
OD540: Optical density at 540 nm
Panchakarma: The Ayurvedic therapeutic system used for cleansing of the body
and mind of accuÂ​mulated waste. It includes Vaman, Virechan, Basti,
Anuvasana and Nasya.
PBS: Phosphate buffered saline
Pharmacokinetics: The study of the bodily absorption, distribution, metabolism,
and excretion of drugs
Phylogeny: The evolutionary development and history of a species or higher
taxonomic grouping of organisms
Polynology: The discipline of science that focuses on the study of pollen grains
and spores from both living and extinct plants.
Polyol: An alcohol having many hydroxyl radicals including polyethers, glycols,
polyesters and castor oil.
Ppt.: Precipitate
Probit: Inverse cumulative distribution function.
Proteolysis: The direct degradation of proteins by cellular enzymes.
q.s.: Sufficient quantity
RMPI-1640: Media developed at the Roswell Park Memorial Institute utilising a
bicarbonate bufferÂ​ing system and alterations in the amounts of amino acids and
vitamins.
Tanenia: A tapeworm of the genus taenia
TCA: Trichloroacetic acid
The Reformation: The Protestant Reformation was a movement which emerged
in the 16th century as a series of attempts to reform the Roman Catholic Church
in Western Europe.
The Valley of the Queens: The Valley of the Queens is located on the West Bank
at Luxor (ancient Thebes). There are between 75 and 80 tombs in the Valley of
the Queens, or Biban al-Harim belongÂ​ing to the queens of the 18th, 19th and
20th Egyptian Dynasties.
Triturate: Grind to a fine powder
TTE: Tris-HCl (pH: 8.0), 0.5% Triton X-100, and 0.1 mM EDTA in double-
distilled water.
Veda: Science of knowledge
Xenograft: A graft of tissue from one species to another.
 Glossary of Botanical Terms

Abaxial:

i. Applied to an embryo which is out of the axis of the seed as a result of the
one-sided thickness of the albumen.
ii. The side of a lateral organ away from the axis.

Acetals: Compounds with one hydrogen atom and two alkoxy groups attached to
the same carbon atom.
Achenes: Dry fruits that contain one seed that is attached to the wall, or the
pericarp.
Acicular: Long, narrow and cylindrical leaf i.e. needle shaped.
Actinomorphic: Flowers that are radially symmetrical
Acuminate: Tapering gradually to a point
Acute: Distinctly and sharply pointed.
Adaxial: The side or face next to the axis; ventral.
Adelphous: Said of androecium with the filaments of stamen united Adnate:
Joined together
Amphitropous: (of a plant ovule) Partly inverted; turned back 90 degrees on its
stalk Anatropous: Where the ovule is reversed with the micropyle close to the
side of the hilum and the chalaza at the opposite end.
Androecium: Collectively, all of the stamens in the flower of a seed plant
Angiosperms: Plants having their seeds enclosed in an ovary Apetalous: Without
petals
Apocarpous: Consisting of carpels that are free from one another Appressed:
Closely flattened
Arcuate: Bent, curved or arched like a bow.
Aril: A fleshy coloured covering on the seed.
Asvattha: Pipal tree
Axile placentation: Said of an ovary having the ovules attached to the tissues of
the central axis Basal: Starting from the base of the plant
Basifixed: Attached by its base (as certain anthers to their filaments or stalks)
Bilabiate: Two-lipped
Bract: A reduced leaf, particularly one subtending a flower Caducous:
(botanical) Opposite of persistence, falling readily Caducous: Dropping off
early.
Calyx: The usually green, outer whorl or series of whorls surrounding flower
petals Campylotropous: (of a plant ovule) Curved with the micropyle near the
base almost touching its stalk Capitulum: A racemose inflorescence with the
axis flattened to form a disc.
Caryopsis: A dry, indehiscent, one-seeded fruit in which the seed coat is fused to
the pericarp Caudex: A trunk or stock
Cauline: Starting from the stem of the plant
Chalaza: The part of the ovule or seed where the nucellus joins the integuments.
Channelled: Hollowed like a gutter
Cocci: Mericarps; one-seeded portions of a fruit which splits up at maturity
Comose: Having tufted hairs
Confluent: Blending, running into one another
Connate: Closely joined or united
Cordate: Heart shaped
Corolla: The inner, usually coloured or otherwise differentiated, whorl or whorls
of the flower Corymb: A raceme with the lower flower-stalks longer than those
above, so that all the flowers are at the same level.
Corymbose: Arranged in corymbs, a flat-topped or merely convex and open
flower cluster of the indeÂ​terminate or centripetal order.
Cremocarp: A dry, indehiscent, bi-chambered fruit, composed of two one-
seeded carpels invested by an epegynous calyx, separating when ripe into
mericarps. Cremocarp is the characteristic fruit of umbelliferae, e.g. coriander,
fennel.
Culms: Stem of a grass
Cuneate: Wedge-shaped
Cupule: A cup surrounding the fruit
Cyme: An inflorescence in which the terminal bud is a flower bud Cypsela: A
one-seeded fruit formed from a syncarpous inferior ovary Cystolith: Mineral
concretions, usually of calcium carbonate on a cellulose stalk, occurring chiefly
in special cells of the urticaceae.
Darbha: Cogon grass
Decidous: Falling in season, as petals fall after flowering or leaves in autumn.
Decumbent. Lying flat, but having the tip growing upwards Decurrent.
Descriptive of leaves whose edges run down onto the stem as two wings
Decussate: Said of leaf arrangement, leaves are opposite, but each pair is at right
angles to the one above and below it Dichasial cyme: A cyme with each branch
giving rise to two other branches Didynamous: Of an androecium, consisting of
four stamens, two being distinctly Dioecious: Having the male and female
reproductive organs borne on separate individuals of the same species.
Distichous: Arranged in two vertical rows
DNA: Deoxy ribonucleic acid
Dorsiftxed: Said of anther when filament is attached firmly to one point of the
dorsal surface Drupe: Where the pericarp is fleshy or leathery, containing a stone
with a kernel.
Emarginate: Having the margin notched
Emersed: Amphibious
Epigynous: Flower with receptacle fused to the pistil and the stamens, petals and
sepals attached to the receptacle above the pistil Epipetalous: Borne on the
petals or corolla.
Epiphyllous: Growing upon, or inserted into, the leaf.
Epiphytic: An epiphytic plant is one which naturally grows upon another plant
but does not derive any nourishment from it.
Etaerio: An aggregate or cluster fruit
Exfoliation: The falling away in flakes, layers or scales Exstipulate: Lacking
stipules
Fascicle: A tuft of leaves crowded on a short stem
Fistular: Hollow
Follicle: A many-seeded dry fruit, derived from a single carpel and splitting
longitudinally down one side at dehiscence Fusiform: Elongated and tapering
towards each end
Gamopetalous: Having the petals of the corolla more or less united
Gamosepalous: Having the sepals in a flower fused
Glabrous: Hairless
Glume: One of a pair of dry bracts, at the base of and enclosing the spike-let of
grasses Grana: Stacked membranous structures within a chloroplast that contain
the chlorophyll and are the sites of the light reactions of photosynthesis
Granular. (Botanical) Composed of grains or divided into little knots or
tubercles.
Gynandrous: Having the stamens and pistil united to form a column Gynobasic:
Said of style which arise near the base of the carpels Hasted: Spear shaped.
Helicoid monochasial. A monochasial cymose inflorescence, repeatedly
branching on the same side of the pedicel (plants probably with paired lateral
bracteoles), so forming a spiral when viewed from above.
Hesperidium: A partitioned berry with a leathery, removable rind Hilum:
(i) The scar left on a seed where it was formerly attached to the funicle or
placenta.
(ii) The central point of a starch granule, which the ring-like markings seems to
surround.
Hispid: With rough bristly hair
Hyaline: Thin and translucent
Hybridoma: Culture of cells produced by the technique hybridization.
Hypanthium : An enlargement or development of the torus under calyx.
Hypogynous: A flower with the stamens, petals and sepals attached to the
receptacle below the pistil Idioblast: A special cell in a tissue which markedly
differs Lorn the rest in form, size or contents Imbricate: Overlapping
Immersed: (Botanical) Embedded in the tissues of the plants Indehiscent: Fruits
which do not open spontaneously upon maturity and drying Integuments: Cell
layers enveloping the nucellus of an ovule, and which will become the seed coat.
Involucre: A set of bracts surrounding a flower cluster Latex: An unorganised
milky substance formed by the secretory tissues of certain plants which oozes
out from the cut surface and immediately coagulates upon exposure to air.
Constituents like sugars, proteins, alkaloidal salts, resins, gums, starch and
minerals are present in the latex eg opium.
Lenticular: Having the shape of a double-convex lens
Leucoplast: Specialised colourless protoplasmic granule Lignin: An incrusting
or impregnating substance on the cell wall, producing woody tissue; it is inÂ​-
soluble in water or ether, soluble in alcohol and alkalis, and is the residue after
the cellulose has been removed by chemical means.
Loculicidal: Said of a fruit which splits open along the mid-ribs of the carpels
Lomentum: A fruit, usually elongated, which develops constrictions as it
matures, finally breaking across these into one-seeded portions.
Lorate: Shaped like a strape
Lyrate: The shape of the leaf is that of a lyre i.e. lobe and some smaller lateral
lobes, as in radish.
Lysigenous: An intercellular space derived by the dissolution of cells.
Male fern: Aspidium; filix-mass
Medullary rays: The plates of parenchyma or cellular tissue radiating from the
pith to the cortex.
Megasporangia: Sporangium (structure containing asexual spores) in which
macrospores (female spores) are produced Meristem:
(i) Nascent tissue, capable of being transformed into special forms, as cambium,
etc.
(ii) Actively dividing cell tissue.
Merous: Greek suffix, referring to the parts in each circle of flower organs,
generally with a numerical prefix, such as 2-merous Micropyle: The aperture in
the skin of the seed, formerly the formation of the ovule; it marks the poÂ​sition
of the seed.
Monochasial cyme: A cyme in which each flowering branch bears one other
flowering bud in its turn Monodilphous: Related to or being stamens with all the
filaments united into a single tube-like group.
Monoecious: Having unisexual reproductive organs or flowers, with the organs
or flowers of both sexes borne on a single plant, as in com and pines.
Mucilage: A thick gummy substance, often produced by certain plants
Mucronate: With a short, sharp point at the end.
Muricate: Having the surface roughened by short, sharp points Napiform:
Turnip shaped or rooted.
Netted-veined: Reticulated, net-veined with any system of irregularly
anastomosing (connecting by cross-veins and forming a network) veins.
Nutlets: Small, dry, one chambered and one seeded fruit developing from a
superior, bi-or polycarpellary ovary, with the pericarp hard and woody.
Obcordate: Heart-shaped, with attachment at or near the narrow end (inverted
heart shape) Obovate: A roughly elliptical shape with the terminal half broader
than the basal.
Obtuse: Blunt or rounded at the end.
Orthotropous: Said of an ovule which is straight
Palmate: Of a leaf shape, divided into three or more lobes, leaflets, or veins of a
leaf that diverge from a common point Panicle: A branched inflorescence with
each branch bearing a raceme of flowers Pappus: A ring of fine hair developed
from the calyx and covering the fruit Parietal. Joined to the wall
Pellucid: (Botanical) Wholly or partially transparent.
Peltate: Of a leaf shape; round, with the stem attached near the centre of the
lower surface rather than the margin Pepo: A fleshy fruit with a firm rind
Perfoliate: Said of a leaf base which surrounds a stem completely so that the
latter appears to pass through it.
Perianth: The floral envelope, consisting of the calyx and corolla, literally
meaning “around the antherâ€​
Periclinal: A plane of division or cell wall establishment which is parallel with
the surface of the organ Perigynous: A flower in which the receptacle forms a
cup surrounding, but not fused to the pistil and the stamens; petals and sepals are
attached on the edge of the cup Persistent: (Botanical) Retained, not shed
Phanerogam: A seed-bearing plant
Phytogeny : The evolutionary development of a species through a succession of
forms Polyphyletic: Descent from more than one line
Piliferous:
(i) Bearing hairs or tipped with them.
(ii) The young superficial tissue of roots, producing root hairs, when present.
Pinnate: Of a leaf shape, divided up into many small leaflets, arranged in rows
along either side of a mid-rib Placentation : Arrangement of placenta in a
syncarpous ovary Plano-convex: Plane on one side and convex other.
Plasmodesmata: Connecting threads of protoplasma passing through pores in
the cell walls.
Polyandrous: (Botanical) Having a large number of stamens Polypetalous:
Having a corolla composed of many separated or distinct petals Polysepalous:
Having the sepals separate from each other Pome: A fruit from a compound
inferior ovary enclosed in thick flesh Pubescent: Covered with fine hair
Raceme: An inflorescence with the main axis bearing stalked flowers Raphe:
(i) In a more or less anatropous ovule, the card or ridge of fibrovascular tissue
connecting the base of the nucellus with the placenta and the adherent funicle; it
may occur on the side of the ovule turned to the axis (ventral), or on the external
face of the ovule (dorsal).
(ii) In diatoms, the median line or rib of a valve; it may be heteropolar or
isopolar.
(iii) The stucture between the carpels in umbelliferae.
Regma: A kind of dry fruit, consisting of three or more cells, each of which at
length breaks open at the inner angle Reniform: Kidney-shaped.
Repent: Prostrate and rooting
Retuse: A shallow notch at the rounded apex
Rhytidoma: The formation of plates of cellular tissue.
RNA: Ribonucleic acid
Rosettes: A cluster of leaves or other organs in a circular form.
Sagittate: In the shape of an arrow-head
Samara: Single-seeded dry indehiscent fruit, having wing-like extension of
pericarp Satavari: Wild asparagus (Asparagus racemosus ) Scabrous: Having or
covered with scales or small projections and rough to the touch.
Scandent: Climbing
Scapose: A leafless floral axis or peduncle arising from the ground.
Scarious: Dry, thin, membranous, non-green, more or less translucent.
Schizocarp: Seeds which split on maturity into its constituent carpels
Schizogenous: Intercellular spaces formed by the separation of cell walls of
adjacent cells along their middle lamellae Sclerotium: The hard, dark body of a
fungi consisting of a mass of hypal threads Scorpioid monochasial: A
monochasial cyme in which the lateral branches develop on only one side, each
successive segment branching on the side Scorpoid cyme: A cyme in which the
branches develop alternately to the left and right Sepaloid: Resembling or
characteristic of a sepal
Sessile: Lacking a stalk
Siliqua: A fruit that is long and narrow, more than three times as long as broad
Spathulate: Paddle or spoon shaped
Staminode: An abortive stamen
Stele: An axial cylinder of tissue passing from the plerome (core of the
meristem) into the older tissues, in which the vascular tissue is developed.
Stipule: A small outgrowth at the base of the leaf stalk Stolon: A sucker, runner
or any basal branch which is disposed to root or a slender creeping stem with
minute leaves Syncarpous: Composed of united carpels. A syncarp is an
aggregate fruit Syngenesious: Said of stamen united by their anthers to form a
tube around the style Tabulate: Horizontally flattened
Taxon: A taxonomic category or group, such as a phylum, order, family, genus,
or species.
TCA cycle: Tricarboxylic acid cycle
Terete: Cylindrical
Testa: The outer coat of the seed, usually hard and brittle.
Torus: Receptacle of the flower
Triquetrous: Having 3 angles and 3 concave surfaces
Umbel: An inflorescence with pedicels or flower stalks or both, nearly equal in
length and arising from a common point; umbrella-shaped.
Unilocular. Having one cavity
Urticle: A small fruit with the pericarp free from the seed Utricle: A little sac or
vesicle
Valvate: Having non-overlapping margins
Varuna: Three leaved caper
Versatile: Said of anther which is attached at tip of filament by a small area on
the dorsal side, so it turns freely in the wind.
Zygomorphic: Divisible in half by only one longitudinal plane
 Glossary of Medical Terms

Abhyanga: Oil massage therapy

Abortifacient: A substance that causes pregnancy to end prematurely and causes
an abortion
Actinomycosis: A chronic infection that produces abscesses and open draining
sinuses
Adenoma: A benign tumour that develops from epithelial tissue.
Adrenergic drugs: Drugs activated by or capable of releasing epinephrine or an
epinephrine-like subÂ​stance, especially in the sympathetic nervous system
Amygdala: Almond-shaped groups of neurons located deep within the medial
temporal lobes of the brain in complex vertebrates, including humans.
Angani svedana: Induction of perspiration without the use of Agni (fire)
principle. The patients are advised to perform physical exercise.
Anoxia: Lack of oxygen to the brain
Anthelmintic: Substance used to destroy parasitic worms
Antibody: A protein produced in the body which reacts with a given antigen.
Anti-epinephrine: A drug which antagonises the receptor action of admaline and
related drugs
Anti-fibrillatory: An agent which acts against fibrillation, which is the rapid
contraction of muscles especially abnormal action of the heart muscle.
Antigen: A foreign substance which, when introduced into the body, stimulates
the production of antibodies.
Antileprotic: A substance used against leprosy
Anti-muscarinic: Substance that inhibits or prevents the action of muscarine or
muscarine-like agents or drugs which brings parasympathetic stimulation at the
neuroeffector junction.
Antineoplastic: Drug acting to prevent, inhibit or halt the development of a
tumour.
Anti-protozoal: A drug which destroys protozoa or inhibits their growth and
ability to reproduce.
Anti-pruritic: A drug which counter-acts itching
Anti-pyretics: Drugs that reduce elevated body temperature
Anti-sialagOgue: An agent that diminishes or arrests the flow of saliva
Antitussive: Any substance administered to suppress a cough.
Aphrodisiac: A drug or other agent that stimulates sexual desire
Apoptosis: A type of cell death in which the cell uses specialised cellular
machinery to kill itself.
Ascites: An accumulation of serous fluid in the peritoneal cavity;
hydroperitoneum
Asphyctic seizures: A seizure which stops breathing activity
Astringent: A drug which causes precipitation of surface proteins, arrests
secretion and bleeding.
Ataxia: Unsteady and clumsy motion of the limbs or trunk due to a failure of the
gross coordination of muscle movements.
Atherosclerosis: Process of fatty substances, cholesterol, cellular waste products,
calcium and fibrin (a clotting material in the blood) building up in the inner
lining of an artery.
Bahya: External application of oils and medicated cream
Bioavailability enhancer. A substance which acts synergistically to increase the
rate at which cells, including microorganisms, absorb phytotoxins (poisonous
substances derived from plants)
Bronchitis'. Inflammation, acute or chronic, of the bronchial tubes or any part of
them.
Cannula: A flexible tube which when inserted into the body is used either to
withdraw fluid or insert medication.
Carminative: A drug or agent that induces the expulsion of gas from the
stomach or intestines.
Catarrhal ailments: Minor illness due to inflammation of the mucous membrane
of the nose, throat etc
Cathartic: An agent for purging the bowels, especially a laxative.
Cauda: Of, at, or near the tail or hind parts
CD97: Convulsive dose in 97% animals
Cholagogue: Substance which promotes the discharge of bile from the system
Cholinergic drugs: Drugs activated by or capable of liberating acetylcholine,
especially in the paraÂ​sympathetic nervous system.
Choorna: The fine sieved powder of a well-dried drug
Clofibrate: A biochemical anti-cholesterolemic in nature having the molecular
formula C12H15ClO3.
Clonus: Repetitive, rhythmic contractions of a muscle when attempting to hold
it in a stretched state.
CNS: Central nervous system
Condylomata acuminatium: Formation of small, pointed papilloma of viral
origin, usually occuring on the mucous membrane or skin of the external genitals
or in the perianal region.
Corpora lutea: Yellow, progesterone-secreting masses of cells that forms from
an ovarian follicle after the release of a mature egg.
Crop milk: A secretion from the lining of the crop of pigeons and doves with
which the parents feed their young by regurgitation.
Deepana: Substance which increases the appetite
Dementia: Mental and emotional deterioration caused by organic brain disease,
characterized by madness.
Demulcent: A soothing, usually mucilaginous or oily substance, used especially
to relieve pain in inÂ​flamed or irritated mucous membranes.
Diaphoretic: Drug causing/increasing perspiration.
Distal. Sites located away from the centre or mid-line of the body
Dysmenorrhoea: Pains in the lower abdomen, stemming from uterine cramps,
during a menstrual period.
Dyspepsia: Gastric indigestion
Dyspnoea : Shortness of breath
Dysuria: Pain or discomfort when urinating
Ecbolic: A drug which helps child birth or causes abortion
Eczema: An inflammatory disease of the skin, characterised by the presence of
redness, itching, minute papules and vesicles, weeping, oozing and crusting and
later by scaling, lichenification and often pigmentation
ED50: Effective dose for 50% of population
Emmenagogue: Substance which has the ability to promote menstruation
Emollients: Substances which soften and soothe the skin.
Endotracheal intubation: A procedure by which a tube is inserted through the
mouth down into the trachea (the large airway from the mouth to the lungs).
Enteric coating: It is a barrier applied to oral medication that controls the
location in the digestive system where it is absorbed.
Eosinophilia: Condition in which abnormally high amounts of eosinophils are
found in either the blood or in body tissues.
Epididymis: Coiled, ductular structures located along border of testicles;
location of sperm storage and maturation.
Epilepsy: A disorder of CNS, manifesting as brief episodes of loss or disturbance
of consciousness with or without characteristic body movements, sensory or
psychiatric phenomena.
Epileptogenesis: The process that lead to the development of epilepsy.
Erythema: Redness of the skin caused by increased blood flow to the capillaries
Euglycemia/ normoglycemia: normal level of glucose in the blood
Excipient: An inactive substance that is used as a vehicle or a medium for a
drug.
Exsanguinate: To deprive or drain of blood.
Exudate: A liquid discharged through incision or pores.
FSH: follicle stimulating hormone
Fundus: Base of an organ.
Galactagogue: Agent that induces milk secretion
Gastritis: Inflammation of the mucous membrane and glands of the stomach.
Gavage: force-feeding
Genes: Units of heredity, representing the characteristics in the offspring G7T:
Gastrointestinal tract
Glaucoma: The group of eye diseases characterised by an increase in intra-
ocular pressure, causing pathological changes in the optic disc and typical visual
field defects.
Glycosuria: excess sugar in the urine
Granuloma: Localised nodular inflammation found in tissues.
Haematuria: Appearance of blood in the urine
Haemophilia: A medical condition in which the ability of the blood to clot is
severely reduced, causing severe bleeding from even a slight injury. A
haemophiliac is a person with haemophilia.
Haemorrhoids', (often known as Piles) enlarged and engorged blood vessels in
or around the back passage (anus).
HDL: High density lipoprotein
Hepatic ‘“firstpass" effect: A phenomena observed in drugs taken orally
whereby they pass through the digestive system, the liver and only then to the
systemic circulation.
Hilum: A depression or fissure where vessels or nerves or ducts enter a bodily
organ (in the book it is the kidney)
Hodgkin’s disease: A cancer that starts in lymphatic tissue
Hydrogogue: Substance which promotes watery evacuation of bowels.
Hyperalgesia: Extreme sensitivity to pain
Hypercalciuria: Excessive urinary calcium excretion
Hyperglasia: An increase in the number of the cells of an organ or tissue causing
it to increase in size.
Hyperglycemia: Increase in blood glucose
Hyperoxaluria: Excretion of excessive amounts of oxalate in the urine.
Hyperparathyroidism: Overactivity of the parathyroid gland.
Hyperplasia: An increase in the number of the cells of an organ or tissue causing
it to increase in size.
Hypoglycaemia: Decrease in blood glucose
I.d. : Intraduodenal (adminstration of drugs)
i.m.: Intramuscular (adminstration of drugs through)
i.p.: Intraperitoneal (administration of drugs through)
Iatrogenic disease: Disease which occur due to the therapeutic effort itself
Idiopathic. Arising spontaneously or from an obscure or unknown cause
Intercostal space: The space between two adjacent ribs
Intra hippocampal injection: An injection through the lateral ventricle of the
brain
Ischaemic heart disease: Disease caused due to insufficient supply of blood to
the heart
Ischemia: A condition in which blood flow (and thus oxygen^ is restricted to a
part of the body.
Kaolin: A pure white clay, known also as china-clay, since it is an essential
ingredient in the manufacÂ​ture of china, or porcelain
Ketoacidosis: A life-threatening condition in which ketones, which result from
the breakdown of fat for energy, accumulate in the blood stream and the pH of
the blood decreases
Ketonemia : An abnormal increase of ketone bodies in the blood
Lacrimation: Secretion of excessive tears
Lanolin: (also called wool wax, wool fat, or wool grease) A greasy yellow
substance from wool bearing animals
Laparotomise: Surgical incision of the abdominal wall
LDL: Low density lipoprotein
Leucorrhoea: A condition in which there is a whitish discharge from the vagina.
LH: Luteinising hormone
Limbus: A distinctive border or edge, such at the junction between the cornea
and the sclera of the eyeball.
Linea alba: A fibrous structure that runs down the mid-line of the abdomen.
Microtubules: Structural components within cells which are involved in many
cellular processes inÂ​cluding mitosis, cytokinesis, and vesicular transport.
Mydriatic: A substance causing dilatation of pupil of eye.
Nauseant: A substance or an agent that is used to generate the tendency to
vomit.
Necropsy: Autopsy
Necrosis : Death of tissue in the body caused by not enough blood supplied to
the tissue, whether from injury, radiation, or chemicals.
Nephrectomy : Surgical removal of a kidney
Nephrolithiasis: Formation of kidney stones
Neuralgia: Sharp, severe paroxysmal pain extending along a nerve or group of
nerves
Nuclear cataract: A cataract in which the centre of the lens hardens and
becomes opaque.
Occlude: To obstruct
Organoleptic: Relating to perception by a sensory organ
Ovarectomise: Removal of ovaries
Ovulation point: The point on the surface of the ovary where ovulation occurs
Oxytocic: A drug used for hastening or facilitating childbirth, especially by
stimulating contractions of the uterus.
Pachana: Substance which helps in digestion
Parasympathomimetic: A substance which produces actions similar to that of
acetylcholine, either by directly interacting with cholinergic receptors or by
increasing availability of actylcholine at these sites.
Parturition: Childbirth
Pedicle: Stalk
Perfusion: The injection of fluid into a blood vessel in order to reach an organ or
tissues, usually to supply nutrients and oxygen.
Peya: Medicated liquid formulation
p.o.: Per oral
Polyploidy: Possession of more than two sets of homologous chromosomes.
Poultice: A drug which exerts an emollient, relaxing or stimulant counter-irritant
effect upon the skin and underlying tissues.
Preneoplastic lesions: Lesions appearing before the formation of a tumour.
Priming: The act of making an organism ready (here) for reproduction
Proteinuria: A kidney disorder resulting in an abnormally high amount of
protein in the urine.
Pylorus. The region of the stomach which connects to the duodenum.
Quatha: Decoction
Rasayana drugs: Drugs which rejuvinate the body
Refrigerant: Substance used for cooling
Renal tubular acidosis: Defective hydrogen-ion excretion in the renal tubules,
resulting in inadequate acidification of the urine.
Rheumatism: Any painful disorder of the joints or muscles or connective tissues
marked by inflamÂ​mation, degenration or metabolic derangement
Rubefaciant: An external application which produces redness of the skin by
hyperemia.
s.c: Subcutaneously (adminstration of drug)
Sagni svedana: Induction of perspiration with the use of Agni (fire) principle
like fomentation, steam etc.
Saluretic: A drug that promotes excretion of salt in the urine.
Saphenous veins: The principal veins running superficially (near the surface) up
the leg.
Sarcoidosis: Inflammation that produces tiny lumps of cells in various organs of
the body.
Sarcolemma: Cell membrane of a muscle fibre
Sedative: An agent which subdues excitement and calms the subject without
inducing sleep, though drowsiness may be produced.
Septa: PI of septum; Thin partition or membrane dividing two cavities or soft
masses of tissue in an organism.
SGPT: Serum glutamic pyruvic transaminase
Sib-mating: Mating involving two individuals of the same parentage
Spasmolytic: A drug used to relieve or prevent spasms
Spermatorrhea: Involuntary discharge of semen without orgasm
Squamous: Scaly
Stereotypy: A behavioural condition characterised by a lack of variation in
patterns of thought, motion and speech; by repetition of said patterns; or both.
Sternotomy : Incision of the entire sternum
Stomachic: Substance used for promoting appetite and assisting digestion
Stratum corneum: The outer-most layer of the epidermis.
Stratum germinativum: The layer of skin that lies at the base of the epidermis
immediately above the dermis
Sugar cataract: Cataract caused by diabetes
Sympatholytic: A substance opposing the physiological effects caused by
stimulation of the sympaÂ​thetic nervous system
Sympathomimetics drugs: A class of drugs whose effects mimic those of a
stimulated sympathetic nervous system
Tachycardia: An abnormally rapid beating of the heart.
Teratogenicity : Causing malformations of an embryo or foetus
Thorcotomy : Opening of the chest
Trabecular meshwork: A group of tiny canals through which most of the fluid in
the eye drains
Tranquilisers: An agent which reduces mental tension and produces calmness
without inducing sleep or depressing mental faculties.
Ulcus cruris: Ulceration of the leg from knee to foot, due to chronic venous
deficiency
Uremia: The presence of excessive amounts of urea in the blood, which may be
a sign of kidney disease or failure.
Urolithiasis: The process of forming stones in the kidney, bladder, and/or urethra
(urinary tract).
Vagotomy: The surgical cutting of the vagus nerve (the simulation of this nerve
causes the stomach to produce acid)
Vajeekarana drugs: Drugs which have aphrodisiac properties
Vati: Drugs prepared in the form of tablets
Vesicant: A substance that causes tissue blistering
Vibrissae: Whiskers
Vis Medicatrix Naturae: The tendency of nature to heal itself
Warts: The epidermal lesion with a horny surface caused by a human
papillmavirus or epidermal proliferations of non-viral origin.
Xiphoid process: A small cartilaginous extension to the lower part of the
sternum.
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