1

PHYTOCHEMICAL STUDIES ON THE CONSTITUENTS OF OLIVE

PLANT (Olea europaea) AND DEVELOPMENT OF THEIR HERBAL
DOSAGE FORM

AMMARA RAFIQ

MSF1600038

SESSION 2016-2018

Division of Science and Technology,

University of Education,Township Campus,

Lahore

2018
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Phytochemical Studies On The Constituents Of Olive Plant (Olea europaea)

And Development Of Their Herbal Dosage Form

AMMARA RAFIQ

MSF1600038

SESSION 2016-2018

This thesis submitted in partial fulfillment of the requirements for the award
of the degree of Master of Philosophy (Chemistry)

Division of Science and Technology,

University of Education, Township Campus,

Lahore

2018
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AMMARA RAFIQ MS 2018

I hereby declare that I have read this thesis and in my opinion this thesis is sufficient in terms of
scope and quality for the award of the degree of MS (Chemistry).

Signature:

Name of supervisor: Dr. Sajid Mahmood

Date:
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Declaration

I declare that this thesis entitled “Phytochemical Studies On The Constituents Of Olive Plant
(Olea europaea) And Development Of Their Herbal Dosage Form” is the result of my own
research except as cited in the references. This thesis has not been accepted for any degree and
is not concurrently submitted in candidature for any other degree. At any time, if my statement is
found to be incorrect ever after award of MS CHEMISTRY, the university have right to
withdraw my MS CHEMISTRY Degree.

Signature : ________________________________

Name : Ammara Rafiq

Date : ____________________________________
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PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis entitled “Phytochemical Studies On
The Constituents Of Olive Plant (Olea europaea) And Development Of Their Herbal Dosage
Form” is solely my research work with no significant contribution from any other person. Small
contribution/help wherever taken has been duly acknowledged and that complete has been
written by me.
I understand the zero-tolerance policy of the HEC and University of Education, Lahore towards
plagiarism. Therefore, I as an author of the above titled thesis declare that no portion of my
thesis has been plagiarized and any material used as reference is properly referred /cited.
I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after
award of MS degree, the university reserves the rights to withdraw/revoke my MS degree and
that HEC and the university has right to publish my name on the HEC/University websites on
which names of students are placed who submitted plagiarized thesis.

Signature:

Name: AMMARA RAFIQ

Date:
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CERTIFICATE OF APPROVAL
This is to certify that the research work presented in this entitled “Phytochemical Studies On The
Constituents Of Olive Plant (Olea europaea) And Development Of Their Herbal Dosage Form”
was conducted by AMMARA RAFIQ under the supervision of ASSISTANT PROF. DR. SAJID
MAHMOOD.

No part of this thesis has been submitted anywhere else for any other degree. This thesis is
submitted to the Division of Science and Technology, Univeristy of Education,Lahore in partial
fulfillment of the requirement for the degree of Master of Philosophy in the field of Chemistry.

Student Name : AMMARA RAFIQ Signature : ……………………

Examination Committee :

1. External Examiner 1

Name: ________________ Signature: ________________

(Designation & Office Address)

__________________________

__________________________

2. External Examiner 2

Name: ________________ Signature: ________________

(Designation & Office Address)

__________________________

___________________________

Supervisor Name : Dr. Sajid Mahmood Signature:____________________

Dean/HOD Name : Dr. Asad Gulzar Signature:____________________

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OFFICE OF THE CONTROLLER OF EXAMINATIONS

NOTIFICATION
No.--------------------- Date. ---------------------

It is notified for the information of all concerned that Mr./Miss. AMMARA RAFIQ MS
student of Department of Chemistry, Division of Science and Technology, of University
of Education has completed all the requirements for the award of MS degree in the
discipline of (Chemistry)/ MS as per detail given hereunder:

MS in (Chemistry) Cumulative Result

Credit Hours
Registration Scholars Fathers Course Research Total Cumulative
No. Name Name Work Work Grade Point
Average
CGPA

Research Topic “Phytochemical Studies On The Constituents Of Olive Plant (Olea

europaea) And Development Of Their Herbal Dosage Form”

Supervisor Name: Dr. Sajid Mahmood

Foreign/External Examiner:
a. Name:
University: --------------------------------------------------------
Address: ------------------------------------------------------------
b. Name: -----------------------------------------
University: --------------------------------------
Address: ----------------------------- Signed by
Controller of Examination
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DEDICATION
TO
MY BELOVED FATHER
MY LOVING AND CARING MOTHER
AND
MY UNCLE
GHULAM MUSTAFA SANDHU

ACKNOWLEDGEMENT
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First and Foremost, I pay my gratitude to ALMIGHTY ALLAH who is most

Gracious, most Merciful and Beneficent. He is the entire source of knowledge and
wisdom endowed to mankind. Then, I pay my all respect and compassionate
endowments for the greatest man of the world, the HOLY PROPHET
MOHAMMAD (Peace Be Upon Him),a lofty personality, source of guidance,
knowledge and blessings for the humanity as a whole.

I feel it my profound pleasure to pay my gratitude and heartiest thanks to my

inspiration, worthy teacher and supervisor DR. SAJID MAHMOOD, Assistant
Professor, Department of Chemistry, University of Education, Lahore, for his kind
love, positive thoughtful, keen personal interest, guidance, cooperation, sincere
advice, vital instructions and close supervisor throughout the period of my studies
and research.

I express my last but not the least, sincerely and honestly gratitude to my dearest
father MUHAMMAD RAFIQ and my mother for their constant prayers
assurance, tolerance, love, without which my existence would have no bearings.

AMMARA RAFIQ

ABSTRACT
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Present dissertation focused on the Phytochemical Studies On The Constituents Of Olive Plant
(Olea europaea) And Development Of Their Herbal Dosage Form. There were majorly three
phases. In first phase there was performed phytochemical screening and in-vivo, in-vitro study of
Olea europaea. Which confirmed the presence of lead compounds in this plant. In second phase,
isolation and purification of known compounds was done successfully by column
chromatigraphy and thin layer chromatography. The third phase was fruitfully completed by
formulation of its dosage form against diabetes. It provided an effective anti diabetic syrup. The
whole research work was performed following the standard methods. This study would be
helpful for future studies as it provides a complete pathway of medicine formulation, from the
plant to the dosage form.

TABLE OF CONTENTS
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Sr No. Content Page No.

1 Declaration iv

2 Plagiarism Undertaking v

3 Certificate of Approval vi

4 Office of the controller notification vii

5 Dedication viii

6 Acknowledgement ix

7 Abstract x

8 Table of contents xi

9 List of tables xii

10 List of figures xiii

11 Introduction 1

12 Literature Review 15

13 Research Methodology 21

14 Result and Discussion 40

15 Conclusion 54

16 References 55

LIST OF TABLES
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Table No. Title Pg No

1.1 Some important phytochemicals 4

3.1 Plant and excipient profile 36

3.2 Formulation ingredients 37

4.1 Phytochemical screening of Olive plant 43

4.2 In-vitro Glucose Bound test values 44

4.3 In-vitro Glucose Diffusion values 44

4.4 Blood Glucose Measurement for EtOAc extract 45

4.5 Blood Glucose Measurement for n-hexane extract 46

4.6 Blood Glucose Measurement for Aqueous extract 47

4.7 Blood Glucose Measurement for Diabetic group 48

4.8 Blood Glucose Measurement for Glimepiride group 49

4.9 Evaluation parameters 50

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LIST OF FIGURES

Figure No. Title Pg No

1.1 Olive plant 6

1.2 Extraction and purification scheme of plants 10

1.3 Syrup manufacturing process 12

1.4 Tablet manufacturing process 12

3.1 Fractionation 24

3.2 Male albino rats and rotary 31

3.3 Dose, Handling, Blood sugar measurement 32

3.4 Packing, Elution of column and Purification 34

3.5 Compound I 35

3.6 Compound II 36

4.1 Blood Glucose Measurement for EtOAc extract 45

4.2 Blood Glucose Measurement for n-hexane extract 46

4.3 Blood Glucose Measurement for Aqueous extract 47

4.4 Blood Glucose Measurement for Diabetic group 48

4.5 Blood Glucose Measurement for Glimepiride group 49

4.6 Compound I 51

4.7 Compound II 53
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in-vivo and in-vitro

study for anti-diabetic
activity
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PHASE-11
Fractionated extract

Column chromatography
(Classical method)

Separation of compound

Purification by TLC

MS
Spectral solvation
Spectoral techniques 1 Known compounds
H-NMR

13
C-NMR
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PHASE-III
Crude extract

Nutraceutical herbal syrup

Dispensing

Transfer of active
drug agent

Formulation of flavor
soln.

Composition of vehicle Formulation of syrup Stability test

soln.
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CHAPTER-I
INTRODUCTION

1.1 NATURAL PRODUCTS

Natural products are the substances obtained from plants or animals. These can either be
produced from a natural source or obtained through multiple synthetic schemes. These
substances are essential for our medicine industry, which is always striving for improving drugs
and their delivery systems. Natural products have found their way in the field of cosmetics, food
and dietary supplements. These substances show interesting characteristics like biological
activity, complex structure and absorption properties either in pure or in a mixture form
(Somuelson et al, 1999).

Different synthetic methods were developed in late 1800’s, to synthesize natural compounds. For
this purpose natural extracts were first separated into their component compounds then
purification and analysis of every compound was carried out (Cutler et al, 2000). Some famous
examples of natural products includes morphin, nicotine, cocaine, camphor etc.

1.2 NATURAL PRODUCTS CHEMISTRY

This branch deals with the biologically active substances, found in living entities and their
formulation in a drug form. From the ancient times, natural products are used to cure diseases
and improving health care. Written evidences had been found in Chinese, Indian and North
African civilizations to prove the use of natural compounds to cure and prevent ailment For
example

Sumerian clay tablet is documented four thousand years ago to cure various ailments.

 Turmeric was used for blood clotting.

 Raw garlic used for circulatory disorders.
 Mandrake relieved pain.
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1.3 PHYTOCHEMISTRY

Phytochemistry deals with the study of chemicals found in plants. Specially the phyto-drugs or
medicinal plants are studied to identify active compounds. It is widely used in Chinese
pharmaceutical industry to formulate herbal medicine. It ensures the identification of bioactive
components present in plants such as alkaloids, phenols, tannins, saponins, flavonoids etc. these
compounds are identified in a plant by using a number of modern analytical techniques, such as
combination of HPLC and various detectors is used to get good results. These detectors may be
refractive index detector (RID) or mass spectrometric detector (MSD), (Dwivedi et al, 2007).

Phyochemistry helps to find secondary metabolites in plants which imparts protective

characteristics in plants against insects attacks as well as in humans (Arnason et al, 2013).

1.4 PHYTOCHEMICALS
Word “Phyto” is derived from Greek word which means plants so we can say phytochemicals
means plant-chemicals. By carefully studying the distribution of phytochemicals among plants, it
can be seen that about twenty five thousand phytochemicals are found in plants. Which includes
about 10,000 alkaloids and 4000 flavonoids exists in kingdom Planteae (Soraya, 2010).

Phytochemicals play an important function in plants such as protection against insects, diseases,
harmful solar radiations etc. Plants are also a major part of human diet such as food, nutritional
supplements, and drugs, so the phytochemicals thus consumed by humans, improves the function
of CNS. These psycho active components are actually secondary metabolites, which are not
primarily needed by plants (Kennedy et al, 2011).

1.5 PHYTOCHEMICALS OF OLIVE (Olea Europaea)

As all the plants are full of Phytochemicals, Olea europaea is also having a bulk of flavonoids,
steroids, secoiridoid, benzoic acid, sugars etc (Hashmi et al, 2015).
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1.6 SOURCES
Every herb, seed, fruit, vegetable like broccoli, oranges, flax seeds, grapes, carrots provide us
with phytochemicals which are needed to improve our health. As Digitalis is good for heart and
Quinine is used to treat malaria. Some phytochemicals regulate the hormones and some are
essential for the release of neurotransmitters required for emotional and mental stability. Some
are considered anti-cancer, anti-mutagenic, anti-oxidative, and anti-inflammatory. Historically
willow tree was used for the treatment of fever. Salicin was used as pain reliever and was
extracted from the bark of the white willow tree (Sneader, 2000; Landau, 2010).

1.7 METABOLITES

The intermediate products of metabolic reactions are called metabolites, which are catalyzed by
different enzymes present in the cell. Metabolites are mainly of two types i.e. primary and
secondary.

1.7.1 Primary Metabolites

The primary metabolites are actually very important components to maintain normal
physiological processes. These metabolites basically affect the growth of a person. These are
produced during the growth phase due to energy metabolism, so these are also called as central
metabolites. Carbohydrates, nucleic acids, proteins and lipids are some common examples.

1.7.2 Secondary Metabolites

Secondary metabolites are produced by modification of primary metabolites. These are organic
compounds and do not play any vital role in growth, development and reproduction as that of
primary metabolites. These chemicals perform their ecological functions in defense mechanism,
as antibiotic and also produce pigments. Alkaloids, resins, tannins, steroids, lignins phenolics are
included in secondary metabolites (Hanson, 2003).

1.8 CLASSIFICATION OF NATURAL PRODUCTS

Thousands of phytochemicals are isolated from plants. These are classified in different groups on
the basis of their constitutional atoms. Some are given as
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Table 1.1 some important phytochemicals

# Phytochemica Sources Solubility Activity Application Structure

ls
1 Terpenes Citrus fruits, ▪Soluble in ▪Anti- ▪Treatment
water due to inflammatory of ovarian
cherries,
their sugar ▪Anti- and breast
roots, group secretory cancer.
▪Soluble in ▪Spasmolytic ▪Perfumery,
Green
oil and high activity food
foods, percentage of insecticidal flavouring
EtOH and
grains,
medicine.
beans, ▪Increase the
production
Green tea,
of anti
Stem bark carcinogenic
enzymes.
2 Flavonoids Grapes, red ▪soluble in ▪Antioxidants, ▪Provide
water ▪Anti- pain relief
wine,
inflammatory, from joint
berries, ▪Anticancer, and muscle
Strengthen pain.
walnuts,
capillaries ▪Heart OH

green seeds, (Rao et al, diseases, HO O

2008) Cancer and
cucumbers,
stroke.
OH
oranges, ▪shielding
the eyes OH O
limes,
from Kaempferol

parsley cataracts.
(Galeotti et
lemons, tea,
al, 2008)
peaches,
apples,
3 Alkaloids Potatoes, ▪Solubility ▪Anti Responsible
depends on bacterial for
tomatoes,
pH of ▪Antifungal physiologica
egg plants solution ▪Anticancer l effect on
▪Antimalarial man or
▪Antihyperten animals.
-sive
▪Anti tussive
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Morphine
5 Saponins ▪Soya beans ▪Soluble in ▪Interferes ▪Saponins
water and with DNA have a
and soy HO

insoluble in replication, characteristi O

products ether. preventing c feature of HO O OH

▪like cancer cells frothing, so

▪Dried
glycosides, from used as
HO OH
salicin

beans and On multiplying. soap.

hydrolysis it ▪Hypolipidem .
peas
gives -ic. ▪Presence of
aglycones. ▪Anticancer saponins is
▪Phenoside, necessary
insulin for activity
releasing of cardiac
substance. glycosides.
▪Haemolytic
▪Anti
inflammatory

6 Lignan Sesame oil ▪Generally ▪Anthelmintic

soluble in ▪Anti
acetone/wate inflammatory
r. ▪Anti-tumor
H 2CO
▪Flaxseeds ▪Anti-HIV OH
OH
are not ▪Antifungal HO

soluble in oil. (Meskin et al,

2002) OCH2

OH
Secoisolariciresinol

7 Glycosides Generally ▪Anti ▪Digoxin is

water and inflammatory used for
ethanol ▪Antioxidant heart
soluble ▪Antimicrobi- treatment.
al
▪Anticarcino-
genic

1.9 FAMILY OLEACEAE

This family contains 600 or more species and its genus is olea. Its texa are about 40 which
contain shrubs.its subgenera is olea, tetrapilus and paniculateae. The subgenera olea contains the
subspecies olea europaea.(Ray et al, 2015).
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1.9.1 Texonomic Scheme Of Olea europaea

Kingdom: Plantae

Division : Magnoliophyta

Class: Rosopsida

Order: Lamiales

Family: Oleaceae

Genus: Olea

Species: europaea (Chiappetta et al,)

1.10 DESCRIPTION OF OLIVE PLANT

Its genus is Olea and species is O.europaea. there are about thirty species of genus Olea.it is also
been served as a food (Hashmi et al, 2015).it is one of the older plants. It has two major types i.e.
wild and cultivated. Its oil shows medicinal and culinary usefulness. Olea europaea is a
monoecious plant (Bracci et al, 2011)

Figure 1.1: Olive Plant

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1.10.1 Description of Olive tree

The olive tree is much branched, evergreen. The leaves are shortly stalked, narrow, oblong,
thick, leathery, pale green and oppositely arranged. Stomata are present only abassial surfaces of
leaves, which greatly prevent the plant from drought (Chiappetta et al,).

The flowers are in bunches and white in colour. It normally produce 20-25 flowers, according to
region. Its fruit shape is oval and ripe fruit is grey in colour. The fruit is small in wild plants than
in orchard cultivates. It belongs to family Oleaceae (Chiappetta et al,)

1.10.5 Occurance

Olea europea is majorly distributed from South Africa, through Africa, the Middle East,
Pakistan, India to China.Its flowering season is August to September (Hashmi et al, 2015).

1.10.6 Uses
Olea europea extract is used to cure migraines, insomnia, diarrhea, dysentery, fever, piles,
fistula. Historically olive leaves had been used to treat fever and malaria. In Tunisian folk
medicines, olive leaves have been used to cure inflammatory disorders, bacterial infections,
hypertension, and diabetes. Olive leaves in hot olive oil and salt is used for the treatment of
earache. If olive leaves are chewed, it cures the tooth pain and lip irritation. Decoction of olive
leaves functions as liquid mouth wash and also cures aphthous, gingivitis, and glossitis. Its
leaves juice is effective for treating trachoma (Nafiu et al, 2013).

1.11 ECONOMICAL IMPORTANCE

Olive (Olea europea) has been cultivated in the Mediterranean for more than 5000 years. It has
greatly affected the culture and outlook of the region. This region is producing about 97% of
world olive oil supply. It has become the most valuable crop of the region because its fruits, oil,
leaves have been used for fuel, medicine, embalming and edibles. (Ponti et al, 2014)
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1.12 ANTIDIABETIC ACTIVITY

Continous hyperglycemic condition or uncontrolled diabetes can show renal, heart and vision
failure. It also cause severe pain in arms and legs of the patient. There are many herbal and
allopathic medicines to cure it, but exercise and morning walk are the best remedy (Choudhury et
al 2017]. The antidiabetic effect of the alcoholic extract of olea europea has proven to be more
effective than glibenclamide, a traditional medicine (Eidi et al, 2009).

1.13 PLANT EXTRACTION AND FRACTIONATION

Extraction is a process in which effective and non effective constituents of plant or animal body
are separated. Suitable solvents are used for this purpose. The products achieved after extraction
can be in any form i.e. liquid, solid or in the form of paste. These extraction products can be
administered in raw form to a patient or a medicine can be formed.as plants are full of active
materials but sometimes a chemist need a plant constituent in pure for. For this purpose a special
treatment is needed for plant, it is called fractionation. By this fractionation, different medicinal
products are made either in raw form or in the form of dose. This all depend on the method
adopted for fractionation. The extraction is completed in following steps.

1. Plant is dried, chopped, and dipped in a solvent

2. Solvent is used which may be
a. very Polar
b. Medium polar
c. Nonpolar
3. Suitable method is adopted
Fractionation is a separation technique in which a mixture is further devided into smaller parts on
the basis of its physical properties. After extraction the crude extract obtained is actually a
combination of different compounds. So a single isolation technique cannot be applied to obtain
individual specie i.e. Column chromatography (CC) can be used for this purpose.
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1.14 BIOLOGICAL ACTIVITY

Traditionally, olive tree leaves are used for the treatment of temperature. Olive leaf also shows
anti-depression affectivity. It lowers body glucose. It also cures the urinary tract problems. It alo
shows some of biological properties such as it lowers the cholesterol, kills miro-organism and
acts as antioxidant (Silva et al, 2006).

1.15 ISOLATION, PURIFICATION AND STRUCTURAL ELUCIDATION

OF NATURAL PRODUCTS

Isolation is termed as removal of coextractives from bioactive compound by using different

techniques. As we know many common techniques but there are many sophisticated and modern
methods such as HSCCC(high-speed counter current chromatography) can be used to get pure
compounds. Purification is needed to obtain a compound separated from its relative compounds.
Recrystallization, sublimation or distillation may be used to purify the compound. HPLC
symmetrical peaks show a compound with purity but this technique is not suitable for
compounds with similar polarities or with enantiomers. Moreover, recrystallization at specific
melting point of eluted material give a sample mean of purity.

The pure compounds so obtained are then used for the structure elucidation and biological
activity determination. Molecular structure elucidation and chemical structure confirmation
determine the atom connectivity and composition of active compounds. Elucidation of molecular
structure is necessary to confirm the structural identity of a chemical compound during chemical
research or product development. (Sasidharan et al, 2010). In the process of structure elucidation
data are collected from different sources and assemble into a chemical structure which show all
information about structure. UV/visible, IR, NMR, MS and HPLC-MS, HPLC-NMR, HPLC-
DAD-MS-NMR etc are basic and developed techniques used for rapid structure analysis. In the
discovery of components of known or unknown structure, it is useful to compare with standard
or confirmed by literature. To avoid any ambiguity, unknown compound is modified with
specific functional group or disintegrate the structure into known one. X-rays diffraction study
involves or utilizes the compound in crystalline form, which is irradiated by X-rays. It will give
3-D structure along with its stereochemistry for optically active molecule and also provide
information about shape, bond length etc.
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The use of NMR and MS for structure elucidation is more fascinating. Other techniques such as
IR spectroscopy is excellent for determine specific functionalities and UV spectroscopy explains
the extent of conjugation in molecule. These methods are applied to confirm the structure
derived by such technique.

Figure 1.2: Extraction and purification scheme of plants (Sasidharan et al, 2010).
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1.16 TYPES OF MEDICINES

Medicines are majorly of three kinds i.e. herbal, pharmaceutical a nutraceutical. Herbal
medicines are prepared from plant extracts or its grindings. These medicines show minimum side
effects as compared to pharmaceutical medicines (Choudhury et al, 2017). Pharmaceutical or
allopathic drugs are manufactured either only of bioactive chemicals or their combination with
herbs. Nutraceutical medicines are used as food supplement in case of mineral or vitamin
deficiency, which cannot be otherwise fulfilled by daily food. These are also called medical
foods.

1.17 DOSE

A fixed quantity or volume of medicinal drug used to combat a physical ailment following a time
interval is called dose. In order to cure a disease by any type of medication, there is always
required a specific amount of drug intake after a specific time duration (Wood, 1986).

1.18 DOSAGE FORM

It is actually a suitable presentation of medicine so it can be administered easily and securely. It

is made off two material i.e. active part and inactive part. These are used according to the
requirement of dosage form i.e syrup is in liquid form and tablet is in the form of solid mass. It
also depend upon the treatment or diagnosis of a disease. For its long lasting effect a dosage form
is needed to be protected from environmental gases and moisture, with its foul odour and bitter
taste masked. A herbal, pharmaceutical or nutraceutical dosage form may be in many forms like
tablets, capsules, ointments etc (Wood, 1986).

1.18.1 Syrup

Syrup is a solution of medicinal substance, sugar, and water which is conditionally combined
with flavoring agents. In addition to purified water and active drug, syrup also composed of
sweetening agent, colorants, antimicrobial preservatives, flavorants, viscosity modifiers etc.
Manufacturing process of syrup is given in steps as follows;
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Figure 1.3: Syrup Manufacturing Process (Niazi)

1.18.2 Tablet

Tablet is a solid dosage form of a compressed drug with or without excipients added. In addition
to active salt, a tablet is also composed of inert components such as diluents, binders,
disintegrents, lubricants, colouring agents, flavoring agents and sweetening agents etc. Tablets
offer the greatest dose precision and least content variability. There are three general methods for
tablet formation i.e. Direct Compression, Dry Granulation, and Wet Granulation. General
Manufacturing process of tablet is given in steps as follows;

Figure 1.4: Tablet Manufacturing Process (Niazi)

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1.19 STATEMENT OF THE PROBLEM

It is to carried out the In-vitro and In-vivo study of plant extract of Olea europaea regarding its
antidiabetic activity. The methanolic extract will be further partitioned into different fractions.
These fractions will be subjected to chromatographic techniques for the isolation and
purification. Purification of the active compound will be carried out by thin layer
chromatography. For the sack of structure elucidation different techniques such as MS, UV, IR,
H1-NMR, C13-NMR and other advance chromatographic techniques will be used. The biological
active crude extract will be formulated and evaluated to pharmaceutical herbal dosage form. The
finished product will be analyzed under GLP parameters (physical parameters, chemical
parameters, and stability studies).

1.20 OBJECTIVES OF THE STUDY

The objective of the current research work is the isolation, purification, structure elucidation and
to analyze the In-vivo and In-vitro study of Olea europaea by development of its herbal dosage
form.

1.21 SCOPE OF THE STUDY

Our current work will be supportive in the field of pharmacology, pharmacognosy,

pharmaceutics. In future we have planned the following.

1. Pharmatochemical screening of medicinal plants from flora of Pakistan can be done.

2. In-vivo/In-vitro studies on the said biologically active plants and herbs can be
performed.
3. Design, development, and evaluation of tablet dosage form, syrup dosage form,
ointment and dried powder (sachet) dosage form in oral drug delivery system and in
parenteral routes can be modified.
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1.21 SIGNIFICANCE OF THE STUDY

1. This research work would be supportive in pharmaceutical sector.

2. Anti-diabetic study of plant extract would be fruitful to the affected people of
Pakistan.
3. Phytochemical investigation on the constituent of this plant would provide the
discovery of new compounds with pharmacological activity.
4. Formulation and evaluation of herbal dosage from crude as well as fractionated
extract of Olea europaea will be carried out. This will be marketed at economical
source in our society.
5. Training of an MS scholar in the field of natural product chemistry and its
applications.
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CHAPTER-II

LITERATURE REVIEW

Literature survey shows that about 35-40% authors had worked out on Olea europaea to
investigate its activity against microbes, hepatic disorder and diabetes etc using different
techniques and validation methods. And about 20% has investigated its phytochemicals by
extraction. A wide range of literature is available regarding the comparative study of Olea
europaea activity. But following kind of research pathway has not been adopted by anyone so
far.

Sedef( )stated that in European and Mediterranean countries the olive leaves are used in
traditional medicine. These leaves are used in form of powder, herbal teas, and in human diet in
form of extract. The olive leave extract contain many active compounds having antidiabetic,
antioxidant, anti-inflammatory, hypocholesterolemic properties. Oleuropein is a secoiridoid
compound found in olive leaves. This is about 6-9% of olive leaves. On the other hand there is
also present flavonoids, triterpenes, and other secoiridoids. Other clinical benefits of olive leaves
extract are also discussed in this study.

Claudio(2018) stated that Olive oil industry is a huge industry in the world, which produces
750,000-500,000 tons of olive plant leaves to burn for power generation. These olive leaves are
full of oleanolic acid which is a triterpenic acid and is very important in pharmaceutical and
nutraceutical fields. This research work is basically about getting oleanolic acid from olive plant
by surface-active ionic solvent. On the other hand imidazolium based ionic solvent with different
functionalities are also used for oleanolic acid extraction. Thus this method provide a good
extraction technique for triterpenic acid from natural material with a good yield.
 33

Putnik(2017) used the Pressurized liquid extraction to extract the phytochemicals from food by-
products. The basic aim behind was to getting knowledge about the effectiveness of pressurized
liquid extraction to extract the phytochemicals from food by-products. For this purpose there
were used Croatian olive leaves (olea europaea, cv. Oblica) with its application on industrial
level i.e. pharmaceutics. Pressurized liquid extraction was carried out in different cycles, static
time and temperatures. characterization of resulting extracts was done in various natural products
i.e. total flavonoids, total polyphenols etc. All the results were within limits, showing the
significant effectiveness of PLE in green technology to extract polyphenols from olive plant
leaves (pp.19-28)

Attar(2017) investigated that Antidiabetic activity of olive leaves extract using streptozotocin
induced diabetic rats. For this purpose animal models were divided into six groups i.e. control
normal, control diabetic, diabetic, and non-diabetic groups. Diabetic rats were given the same
low to high doses. All the parameters i.e. blood sugar, insulin and protein level, albumin, low and
very low density lipoprotein cholesterol etc were raised but the high density lipoprotein
cholesterol , SOD, GSH, and CAT level were lowered significantly in control diabetic group.
The second group of diabetic control rats showed low functioning of liver receptors, and many
pathogenic problems in liver. Both of these problems were cured by olive leaves extract. So the
results showed that olive leaves extract plays a protective role for liver and acts as antioxidant.

Navarro(2017) stated that Advanced Glycation End products (AGEs) cause age affected
disorders like cardiovascular problems, neurodegenerative disorders and diabetes mellitus. By
lowering the content of these products the chronic diseases can be prevented. Characterization of
acidic extract of olive leaves and two of its fractions was done by LC-MS/MS. Antidiabetic
activity of olive leaves extract was studied in vitro and it efficiently prevented the AGEs product
formation. Each fraction of olive leave extract showed different level of activity against
hyperglycemic conditions. Results showed that olive leaves extract has antidiabetic effect
(pp.56-63).
 34

Attar(2017) stated that plants had been used to cure diseases for a long time. Many plants are
proven to have compounds, which are effective against diabetes. Recent researches showed that
many plants have active compounds effective against diabetes mellitus. following study showed
the hepatic and renal protection properties of olive leaves extract, which was performed on
hyperglycemic animals. In this study it was resulted that untreated rats showed elevated level of
blood sugar, alanine, bilirubin, creatinine, uric acid, blood urea etc. But the blood superoxide
dismutase, and also catalase level was reduced. Other side effects were also shown in those rats
like change in hepatic and renal structures. When treated with low and high doses of olive leave
extract, the rats with diabetes results in hepatorenal recovery and bodily protection. Good clinical
results against diabetes in diabetic rats were shown by treating with high to low dose of olive
leaves extract. This study also showed that the olive leaves extract also mitigate the hepatic and
renal disorders caused by diabetes. Results shows that olive leaves extract not only treat diabetes
but also cure and prevent its side effects.

Meelaj(2017) investigated that Activity of oleuropein, phenolic compound and ethanolic extract
of olive fruit for its hepatoprotective and renal protection function. Hepatic and renal toxicity
was produced in Wistar rats by deltamethrin, which is a synthetic pyrethroid. After giving 30
days treatment, the renal and hepatic tissue specimens were taken to check the results. After
giving deltamethrin the rats showed an increase in blood biomarks and hepato-renal lipid
peroxidation. Meanwhile a decrease occur in antioxidant, catalase and superoxide dismutase
properties. The renal and hepatic toxicity was proved by cox-2, bcl-2, p53 and histological
investigation. Results showed that olive fruit ethanolic extract and oleuropein treatment on
DEM-effected rats proved the phenolic content of olive fruit extract to be an effective
hepatorenalprotective agent (pp.455-465).

Hashmi(2015) stated that He compiled all the information about olive plant from botanical point
of view, its phytochemicals, and pharmacological uses to find out its clinical importance and
application in the field of research. All of this information was collected through electronic
media and libraries. Olive plant is used worldwide for the treatment of many diseases. Its
 35

phytochemical studies shows that many secondary metabolites are present in olive leaves like
flavonoids, triterpenes, flavanones, biphenols etc. The olive plant extract and it isolated products
contain various clinical benefits such as hypoglycemic, improving inflammation and immunity,
relieves pain and depression, kills bacteria, and hepatorenal protective effects. So this
information can help modern researchers in the field of pharmacology and phytochemistry
(pp.29).

Xynos(2014) investigated that Extraction product, antioxidant activity, and content of oleuropin,
by Pressurized liquid extraction using environmental friendly solvents i.e. water and ethanol.
Different parameters were also studied for evaluation. This study was based upon two steps, (i)
screening and (ii) optimization. Extraction products were compared to get the optimum
extraction. HPLC was used to find out the quantity of oleuropin. Parameters adopted significant
antioxidant activity. Results showed the RSM to be a good technique for extraction of olive
leaves (pp.323-330).

Cecchi(2013) stated that Its pure oil is famous for its uses from a very old time. This study may
be useful for determining the industrial, and analytical properties of extra virgin olive oil. From
Italy, eleven monocultivar extra virgin olive oil samples were subjected to Headspace solid phase
microextraction (HS-SPME) to get volatile compounds. Characterization of forty eight
compounds was done through GC-MS. Some compounds were common in EVOOs but some
were new, so not have found in any EVOO before. Basic volatile compound, which was found,
was C6 compound from polyunsaturated fatty acid. Results of this investigation showed that
volatile compounds formation strictly depends upon genetic makeup. The geographic abundance
and genotype of the EVOO depends upon terpene hydrocarbons. In this study some newly found
volatile compounds were also presented (pp.2025-2035)

Alok(2013) reported that Now a day’s kidney stones are becoming a common problem. It may
happen due to our modern lifestyles, poor diet and industrialization. These stones may present in
 36

kidneys, ureters, urinary bladder. So the size and location of stones may vary. With the passage
of time many techniques have been developed to remove these kidney stones . As most of the
people worldwide like to adopt herbal treatment, because it is safe, have less side effects and
traditional uses. So herbal medicines gives us a good option to replace the synthetic mode of
medication with herbal medicine. In the following investigation it is suggested that herbal
medicines may be used to remove urinary stones (pp. 496-504)

Wainstein(2012) reported that Olive tree (olea europaea L.) leaves have many bioactive
compounds, which are effective against hyperglycemia. To examine its hypoglycemic activity, a
tablet containing 500mg olive leaves extract was given to adults having type 2 diabetes (T2DM).
For the 14 weeks a random clinical trial was made on 79 adults by treating with a single tablet
daily. Measurement of plasma insulin and Hba1c was done to analyse the glucose homeostasis
and was compared with treatment assignment. To examine the starch digestion and its absorption
in its presence, animal trials were made on sand rats, untreated rats and glycemic rats. The
treated group showed good results of olive leaves extract in lowering Hba1c and level of fasting
plasma insulin. But postprandial plasma insulin level was not lowered. On the other hand sand
rats, untreated rats and streptozotocin (STZ) glycemic rats on treatment with olive leaves extract,
showed lowered starch digestion and its absorption when compared with the trial group without
olive leaves treatment. Although olive leaves extract was effective but not up to the targeted
results in case of sand rats. So this study showed that olive leaves extract has been proven to be
effective in controlling and lowering glucose level in humans and also in animal models
suffering from diabetes (pp.1-6).

Youssef(2011) investigated that Headspace solid phase microextraction (HS-SPME) to be a good

technique to analyse the volatile compounds of virgin olive oils. It was applied to Oueslati type
related to different geographical origins. To investigate the formation of volatile compounds in
the olive fruits, and the factors affecting them, there was taken olive fruits at the same maturation
stage from the seven areas of country. Characterization of 27 compounds was carried out through
GC-MS and GC-FID. So the characterization was done on the basis of hydrocarbon classes of
 37

compounds, such as alcohols, ketones, esters, aldehydes etc. The results showed that a
remarkable difference was present in the level of volatile compounds, present in oils taken from
different areas. So it was concluded that along with the genetic makeup, the environmental
factors are also involved in formation of volatile compounds (pp.1770-1776).

Eidi(2009) compared that Antidiabetic effect of olive leaves alcoholic extract with a famous drug
glibenclamide. An oral dose of alcoholic extract of olive leave extract was given to untreated and
diabetes rats. It was done in two weeks. and alcoholic extract of olive leaves lowered the blood
glucose, uric acid, AST (aspirate amino transferase), ALT (alanine amino tranferase),and
Cholestrol level. It also enhance the blood insulin level in streptozotocin indused rats but not
effective in untreated animals. By this study the alcoholic portion was shown good against
diabetes as compared with glibenclamide (pp.347-350).

Sato(2007) studied that Antihypertensive and hypoglycemic properties of olive plant. He stated
that only oleuropein in olive leaves was thought to be effective compound against the above
discussed ailments but now another triterpene i.e. oleanolic acid was studied for its effectiveness
as antihypertensive and hypoglycemic agent. it was isolated and identified and proven to be
effective for causing reduction in diabetes, and enhancing the strength. Results confirmed that
oleuropein and oleanolic acid, both are effective for diabetes and treating metabolic disorders
(pp.793-798).
 38

CHAPTER-III

EXPERIMENTAL WORK

God has gifted Pakistan with enormous variety of medicinal plants. Out of these only 20% have
been evaluated so far, for their therapeutic potentials. Therefore there is a need of coordinated
and well-organized efforts in the area of indigenous medicinal plants. The present research work
deals with thee pharmaceutical studies on an indigenous medicinal plant of Pakistan namely
Olea europaea. The work is therefore, presented in the following three parts:

Phase I- Phytochemical screening and biological activity

Phase II- isolation, purification and structure elucidation

Phase III- formulation of neutraecutical dosage form

The research work was carried out on plant Olea europaea in Pakistan Council Of scientific and
Industrial Research (PCSIR), Alpha Neutraceutical Research Laboratories Kot Lakhpat Lahore
and chemistry laboratory of department of Science and Technology in University of Education
Township Campus Lahore.

3.1 CHEMICALS AND SOLVENTS USED

●Distilled water ● Methanol

● Ethanol ● Chloroform
● n-hexane ● Ethyl acetate
● Acetone ● Conc.H2SO4
● Dilute HCl ● Dilute Ammonia
● CuSO4 ● Fehling’s solution
● NaOH ● Dil. Acetic acid
● KOH ● KI
● Wagner’s solution ● Lead acetate
● Hager’s solution ● Draggondorf’s solution
 39

● FeCl3 ● Molish’s solution

● HgCl2 ● Mayer’s solution
● Ninhydrin reagent ● Benedict’s solution
● Millon’s reagent ● Starch solution

3.2 APPARATUS AND INSTRUMENTS USED

● Beakers ● Stirrer
● Conical flasks ● Measuring cylinder
● Whatmann’s Filter paper No.1 ● Funnel
● Tripod stand ● Dialysis tubes
● holder ● stand
● Scale ● Lead pencil
● Spray gun ● Ice bath
● Burner ● Wire gauze
● Gloves ● Droppers
● Forceps ● Ring clamp and stand
● Pipettes ● Glass column
● glass viols ● Electronic weighing balance
● Pestle mortar ● Cutter
● Scissors ● Spatula
● Separating funnel ● Aluminum foil

3.3 PHASE I-

PHYTOCHEMICAL SCREENING AND BIOLOGICAL ACTIVITY

The following study mainly comprises of three major steps which are explained briefly here.

3.3.1 Collection Of Samples

 40

The whole plant was collected from Alpha Neutraceutical Research Laboratories Kot Lakhpat
Lahore and identified as Olea europaea by Mr. Tanveer Ahmad at Alpha Neutraceutical
Research Laboratories Lahore. A proof plantlet #302 is deposited in the botany department of
University of Education Township Campus Lahore. The olive plant was shade dried for 20 days
at ambient temperature of 250C. Dried plant was chopped and finally ground on pestle mortar. To
get the sample into powder form it was repeatedly grinded in pestle mortar.

3.3.2 Preparation of Methanolic Extract

The 80 g olive plant powder was dipped in 200 ml methanol, stirred and kept for 3 days. After 3
days the methanol was drained out and again there was added 100 ml fresh methanol. After 1 day
the second solvent was again drained out and both solvents were then left for drying. When that
methanol got dried the left over residue was called as crude extract.(hedya jemai 2009)

3.3.3 Fractionation

There was added 20ml distilled water in crude extract and was shifted in separating funnel. To
get its n-hexane fraction there was added 30 ml n-hexane in it. After thorough agitation of
separating funnel again and again, two layers were formed i.e. aqueous layer and non-polar layer.
n-hexane fraction were separated. The aqueous layer was again taken in separating funnel to get
chloroform fraction and ethyl acetate fraction respectively. Finally we got four fractions i.e.
aqueous, n-hexane, chloroform and ethyl acetate.
 41

Figure 3.1 n-hexane part, chloroform part, ethyl acetate part and aqueous part respectively

3.3.4 Preparation of Reagents for Phytochemical Screening

1. Mayer’s Reagent

To prepare Mayer’s reagent 60 ml distilled water was taken and poured in it 1.358 g of HgCl 2
then 5 g of KI was dissolved in water, taken 10 ml in a beaker. Both of these were combined and
distilled water was filled to 100 ml

2. Wagner’s Reagent

5 g water was taken in a flask and added in it 1.27 g iodine. It was mixed well and added 2g of
KI then water was added to make the volume 100ml.

3. Molish’s Reagent

2.0 ml of filtrate was taken and three drops of alcoholic α-naphthol were mixed. Then drop by
drop 1 ml H2SO4 was added. This mixture was shaken and there was slowly added 1 ml of
sulphuric acid with the sides of test tube.

4. Fehling Solution (A)

 42

500 ml flask was taken and half filled with water. Then 34.66 g CuSO 4 was added and fill the
flask to 500ml.

Fehling solution (B)

20 ml water was taken and added 173 g KNa C4 H4 O6.H2 O. After thorough mixing 50 g NaOH
was also added and again mixed well. Then filled the volume to 500ml

5. Benedict Reagent

In a clean 1000 ml beaker, there was taken 500 ml clean water and added in it the 173 grams Na 3
C6 H5 O7. When it got mixed, there was poured 100 grams Na 2 CO3. When it got fully mixed, it
was warmed thoroughly until it got clear. Then we mix in it a solution of 17.3 grams CuSO 4 in
water. Finally the total volume was filled to 1000 ml.

6. Millon Reagent

Fuming HNO3 was taken 9 ml. there was added 1ml Hg in it. With thorough agitation 10 ml
clean water was mixed in this.

7. Ninhydrin solution

There was carried 200ml (CH3)2 CO in a 250ml flask. Then 10mg ninhydrin was mixed in this.

8. Ferric chloride

There was taken 95ml clean water in a flask and then we poured 5 grams FeCl 3 in this. These
were agitated.

.9. Lead acetate

10 grams Pb(C2 H3 O2)2 was taken in a clean flask and then we poured 100ml clean water in this
flask and mixed.

10. cerric sulphate

2 g of cerric sulphate was dissolved in 15 % aqueous sulfuric acid to form a saturated solution.
 43

for the phytochemical screening of all the four fractions, different tests were performed that are
given below.

3.3.5 Detection Test for Phytochemicals

1. Test for Alkaloids

Dry crude extract was taken 50mg in 5ml H 2SO4 and after filtring it the left over was used for
following tests.

(i) Dragendorff test

Filtrate was taken 2ml and dragendorff solution was poured in this.

(ii) Wagner test

Filtrate was taken 2ml and Wagner solution was poured in this.

2. Test for carbohydrates

Clean water was taken 5ml and in this there we mixed 50mg plant extract. Then we filtered it
and used it for next step.

(i) Benedict’s test

Filtrate was taken 1 ml and mixed Benedict reagent in it. Then warm but not on direct flame.

(ii) Molish test

Residue after filtration was taken 1ml and we poured 1ml molish reagent in this. On the other
hand 2ml H2SO4 was taken and the last formed was added in this drop by drop.

3. Detection of sterols/ steroids

(i) Salkowaski test

CHCl3 was taken 2ml and then we add extract in this weighted 10mg. from the wall 2ml H 2SO4
was also poured.
 44

(ii) Liebermann-Burchard test

CHCl3 was taken 1ml and then we add extract in this weighted 10mg. from the wall 2ml H 2SO4
was also poured.

4. Test for Tanins

(i) With Ferric chloride

Water was taken 10ml and there was poured 50mg extract in this. Then it was warmed and its
filtration was done. In filtrate there was added FeCl3.

5. Detection of Phenols

(i) Ferric chloride Test

Water was taken 05ml and there was poured 50mg extract in this. In there was added 5%
neutralized FeCl3..

6. Test for Terpenoids

(i) Chloroform Test

CHCl3 was taken 2ml and in this we poured extract weighed 2ml. then warmed until it got dried
and poured conc. H2SO4 and again heat for 2m.

7. Detection of Flavonoids

(i) Lead acetate Test

Extract solution in distilled water was taken and 1ml Pb(C2 H3 O2)2 was mixed in this.

8. Detection of Saponins

(i) Lead acetate Test

Extract solution in distilled water was taken and 1ml Pb(C2 H3 O2)2 was mixed in this.
 45

(ii) Foam formtion

Extract was taken 50mg and then added 20ml clean water in it. Then agitate it for 10minutes.

9. Detection of Reducing sugar

(i) Fehling test

Filtrate was taken 1ml and 1ml fehling solution was poured in this. Then we warm it but not on
flame.

10. Detection of Oils and Fats

(i) Spot Test

A drop of extract was dropped on filter paper. After sometime spot was observed.

(GA ayoola 2008)

3.3.6 Biological Activity

3.3.6.1 In-vitro Study

Material Used

 Glucose
 Distilled water

Apparatus Used

 Beakers
 Dialysis tube
 Dialyzer
 Electric stirrer
 Hot plate
 46

i Glucose Bound Test

Glucose solutions having different concentrations was made i.e. 5, 10, 20, 50, 100 m mol/L. Out
of this solutions 25 ml of each was taken in a round bottom flask and plant extract was added in
it. This mixture was then thoroughly stirred using an electric stirrer on a hot plate, at 37 0C for 8
hours. It was then centrifuged at 4800 rpm for 15 minutes. To determine its glucose content, its
UV examination was done.(international journal of pharmacy and 2008)

ii Glucose Diffusion

For the glucose diffusion test, dialysis was carried out. For the dialysis, 30 ml of 20 m mol/L
glucose solution was taken in a dialysis tube and there was added 1 ml of plant extract in it.
Dialysis tube was then sealed and placed in to a glass beaker containing 50 ml distilled water. It
was then kept on an orbital shaker with 100 rpm at room temperature. The glucose concentration
was measured after every 20 minute for two hours by doing its UV examination. This process
was done three times.

3.3.6.2 In-vivo Study

Material Used

 Male albino rats

 Streptozotocin
 Tween
 Distilled water
 Glimepiride

Apparatus Used

 Feeding syringes
 Feeding tubes
 Restrainer
 Gloves
 47

 Glucometer
 viols
 Prickers

Experimental work

The in vivo study of Olea europaea extract was performed at ACRC Department of PCSIR
Lahore. Three herbal extracts out of four were selected for biological activity study i.e. aqueous
extract, n-hexane extract and ethyl acetate extract. Albino rats were chosen as animal model
regarding the quantity of herbal extracts. 30 adult, male albino rats were purchased from
University of Veterinary and Animal Sciences Lahore. On the day 1 all the rats were weighed,
showing an average weight of 170 g. Five experimental groups were made i.e. ethyl acetate
extract animals, n-hexane extract animals, aqueous extract animals, diabetic group and
glimepiride group. Group four and five i.e. glimepiride and diabetic group served as positive
control and negative control respectively. Each group received 6 Albino rats. Blood glucose of
all the 5 groups was measured using On Call EZ II glucometer which came out to be 77±6 mg/dl.
Animals were kept in animal house of PCSIR, under normal room temperature and untreated for
the next 2 days. After 2 days their blood glucose (BG) was again measured and found to be
normal i.e. 70-80 mg/dl.

1. Dose preparation

For the formation of dose, 4% tween was used. As ethyl acetate and n-hexane fraction cannot be
given to animals directly so, these should be dissolved in such a medium which get them
dissolved and do not harm the rats if swallowed. So the tween solution was made in distilled
water. With respect to 1ml dose per day per rat, there was needed 126ml dose volume. So 5.04
ml of tween was dissolved in 120.6ml distilled water. Calculated and recommended dose was
dissolved in tween solution as given below for each fraction.
 48

Figure 3.2: Male albino rats and rotary

i Ethyl acetate dose

For the preparation of 21 days dose of Ethyl acetate, 6300 mg 0f ethyl acetate fraction was
dissolved in 126ml of tween solution. So that 1ml dose given daily, would contain 50mg. 126 ml
of this prepared dose was stored in a cool place.

ii n-hexane dose

For the preparation of 21 days dose of n-hexane, 6300 mg 0f n-hexane fraction was dissolved in
126ml of tween solution. So that 1ml dose given daily, would contain 50mg. 126 ml of this
prepared dose was stored in a dry and cool place.

iii Aqueous dose

For the preparation of 21 days dose of crude extract, 25,200 mg 0f crude extract fraction was
dissolved in 126ml of tween solution. So that 1ml dose given daily, would contain 200mg. 126
ml of this prepared dose was stored in a dry and cool place.

2. Diabetes Induction

Diabetes mellitus was produced in adult albino rats by intra-peritonealy injecting Streptozotocin
(STZ). For this purpose STZ was dissolved in 10mM sodium citrate buffer with PH 4.5. rats
 49

were kept fasting overnight and then 50mg/Kg body weight STZ was given through single intra-
peritoneal injection. STZ-induced rats with Random Blood Sugar (RBS) (˃250 mg/dl) were
chosen for study. Following study selected lower dose of STZ because literature survey showed
lower STZ tolerance in albino rats. Experimental diabetes was induced within 48 hours which
was confirmed by measuring blood glucose (BG) by glucometer. 10% of STZ injected rats
showed mortality during the experimental study.

3. Blood glucose measurement

Glucose was measured after 1 hour of given dose. For the measurement of glucose each rat was
kept in a restrainer with its tail out. The rat’s tail was washed with lukewarm water and was
shaved to get a clear view of blood vein. With the help of a pricker, a blood drop was taken on a
glucometer strip and glucose was measured and noted.

Figure 3.3: dose giving, handling, blood glucose measurement

3.4 PHASE-II

ISOLATION AND PURIFICATION

 50

3.4.1 Column Chromatography

For the sake of isolation there was used wet method column chromatography. A glass column
was securely clamped in the stand. A dry funnel was taken on the upper edge of the glass column
to safely pour the solvent containing silica gel. A dry flask was placed under the nozzle of glass
column to receive the extra solvent during the packing. Silica gel dipped in the n-hexane was
then poured in to the glass column, while the solvent was running through the nozzle. When
column was filled with silica gel about half of its length, the solvent was passed again and again
to get the stationary phase settled evenly in the column. Solvent was filled about 5 cm above the
level of stationary phase i.e. column silica. Then tapping of packed column was done to remove
any bubble, if trapped in the stationary phase. To load a column, slurry of ethyl acetate extract
was made in minimum amount of column silica, which was then dripped on the surface of
stationary phase. A small amount of column silica was again poured in the column so that it
cover the slurry and not let it disturbed whenever the solvent is added. Then different polarity
systems were ran through the column to get the compounds isolated.

3.4.2 Thin layer chromatography

TLC was used to get the compound in pure form. Pre-coated silica gel on aluminium plates were
used. A solvent system was set up containing different ratios of n-hexane, ethyl acetate and
methanol. First combination of solvents used was 1:2:8 of methanol, ethyl acetate and n-hexane
respectively. After filling 55 viols, the ratio was changed to 2:3:5 and again filling 35 viols. This
change of ratio was kept on until it got to ratio 9:1 of ethyl acetate and methanol. Emerging of
compounds from the column was checked by taking samples on TLC cards repeatedly and
checking it under the UV lamp. To check the presence of isolated compound the viols were first
dried and then washed with a minimum drops of respective solvents to get a concentrated
material in the viol. Then 1-2 drops were injected on a TLC card by using a capillary jet and after
drying, it was checked under UV lamp. Rf values of spots on each TLC card were noted. Those
viols were compiled whose TLC cards showed the same Rf values. After compilation of about
155 viols we got 14 viols. All those 14 viols were checked again by TLC to confirm the presence
of isolated compound. Viol# 1 of those 14 viols showed an oily layer. When this oily material
 51

was checked by TLC under UV lamp, it showed an isolated compound. Viol was sent for its UV,
IR, MS and other examination.

Figure 3.4: Packing of column Elution of column Purification

3.4.3 Structural Elucidation of Known compounds of Olea europaea

3.4.3.1 Compound-I

β-amyrin (I) formed shiny needles from EtOH melted at 197-198 0C. it shows the presence of
triterpenes and showed molecular ion peak with m/z 426.3825 in its HRMS spectrum
corresponding to C30 H50 O (calcd for C30 H50 O, 426-3861).

The IR result for hydroxyl group were (3430 cm-1),for trisubstituted double bond were (3045,
1600, 815 cm-1). In the EIMS result of (I) showing peak with m/z 257, 218, 207, 203, and 189
were confirming amyrin structure having ∆12 unsaturation [213, 214].

The EIMS spectrum of (I) exhibited eight tertiary methyls being centered at δ 1.08, 1.02, 1.01,
0.99, 0.95, 0.88, 0.85, 0.80, (all signals). The carbinylic proton resonated at δ 3.21 (1H dd, Jax,eq

= 10.0 Hz , Jax,eq = 4.5 Hz) confirming its α and axial orientation and a multiplet at δ 5.11 was
showing the olefinic proton.
 52

The 13C-NMR spectrum (BB and DEPT) revealed the presence of thirty carbon atoms including
eight methyl, ten methylene, five methane and seven quaternary carbons. Comparing this
information with that showed in documents [213, 214 ]confirmed compound (I) as β -amyrin

Figure 3.5: Compound I

3.4.3.2 Compound-II

The compound (II) gave indication of triterpenes. The compound (I)showed absorption bands
for hydroxyl group (3400 cm-1), carboxyl group (1700) and trisubstituted double bond (1660,
820 cm-1). Molecular ion peak established in HREIMS at m/z 456.3610, corresponding to
molecular formula C30 H48 O3 (calcd for C30 H43 O3, 456-3603). Besides the molecular ion peak
the EIMS showed other prominent ions at m/z 248, 203, 133, which confirmed amyrin skeleton
with ∆12 unsaturation [213, 214].

The 1H-NMR spectrum of (II) exhibited seven methyls being centered at δ 0.89, 0.90, 0.91, 0.97,
0.98, 1.03 and 1.12. The signal at δ 5.24 (1H dd, J = 3.4 Hz) was indicative of the olefinic

proton. While the proton geminal to the hydroxyl group was shown at δ 3.60 ( dd, J = 4.1, 9.9
Hz)

The 13C-NMR spectrum (BB and DEPT) revealed the presence of thirty carbon atoms including
seven methyl, ten methylene, five methane and eight quaternary carbons. Comparison of data
was confirming the reported in literature [213, 214, 221, 222].
 53

Figure 3.6: Compound ii

3.5 PHASE-III

PREPARATION OF PHARMACEUTICAL DOSAGE FORM

3.5.1 Plant and Excipients Profile

Table 3.1: plant and excipients profile

Sr. Plant and composition Uses Chemical structure

no.
1 Olive plant (Olea Anti-microbial, anti-
europaea) active drug bacterial, anti-cancer --
and anti-diabetic
2 Glycerol (exp.) Humectant
Solvent
Lubricant
laxative
3 Hydroxyl ethyl cellulose Gelling agent
(exp.) Viscosifying agent
Dissoluting agent
n
 54

4 Peppermint oil Topical analgesic

(exp.) Flavouring agent
Anti-pruritic
Nasal decongestant

5 Sodium benzoate Preservative

(exp.) Hyperammonemia
treatment

3.5.2 Preparation of Herbal Syrup

Table 3.2: Formulation Ingredients

Sr # Ingredients Typical quantities

1 Plant extract 100 ml
2 Hydroxyl Ethyl Cellulose 2g
3 Glycerol 150ml
4 Propylene Glycerol 30ml
5 Peppermint Oil 0.2ml
6 Sodium benzoate 1g
7 Purified water 1000 ml

3.5.3 Manufacturing Process

3.5.3.1 Active Solution

Dispersed 2g Hydroxyethyl cellulose was added in 300 ml purified water and was kept on room
temperature. After 30 minutes 1.0g Sodium benzoate was added and kept stirring on a hot plate
at 80 0C for two hours. after cooling this solution, 100 ml of plant extract was added and again
start stirring for one hour at room temperature.
 55

3.5.3.2 Flavour Solution

Propylene glycol was weighted 5 g and then 0.2 ml peppermint oil was added in it with thorough
mixing.

3.5.3.3 Vehicle Solution

In a 1000ml mixing beaker, there was added 150 g of glycerol. Flask containing glycerol was
rinsed with 30-40 ml of purified water into the beaker and was thoroughly mixed.

3.5.3.4 Syrup Formation

The active solution of herbal extract was then added into the mixing beaker. Container was
thoroughly rinsed with 20 ml purified water into the mixing beaker. This mixture was stirred for
20 minutes and then it was cooled down at room temperature. Vehicle solution was added at the
end and again container was rinsed with 20 ml purified water into the mixing beaker. The
mixture was filled to 1000ml with purified water.

3.5.4 Evaluation Parameters

3.5.4.1 Colour Test

Five ml of manufactured syrup was taken in a watch glass and placed on a white paper under
white tube light. The colour was observed carefully.

3.5.4.2 Odour Test

Three ml of manufactured syrup was poured in a clean watch glass then smelled by 8-10 people
repeatedly.

3.5.4.3 Taste Test

Half ml of manufactured syrup was tasted by 8-10 people repeatedly

 56

3.5.4.4 pH Test

Manufactured syrup was poured in a 100 ml flask then its volume was made up to 100 ml with
water. Sonication of mixture was done for 15 minutes. After sonication its pH was tested with
digital pH meter.

3.5.4.5 viscosity Test

Viscosity of prepared syrup was checked by Oswald Viscometer.

 57

CHAPTER IV
RESULTS AND DISCUSSION
Phytochemical screening as carried out on all the four fractions of Olea europaea i.e. ethyl
acetate fraction, n-hexane fraction, aqueous fraction and chloroform fraction respectively.
Freshly prepared reagents were used to identify the phytochemicals in each fraction. The results
are shown in the table 4.1, which reveals the phytochemicals in each fraction. According to
results n-hexane fraction confirmed the presence of sterols, terpenoids and lipids. Similarly ethyl
acetate fraction showed the presence of alkaloids, carbohydrates, phenols, saponins and lipids.
The aqueous fraction showed the alkaloids, carbohydrates, flavonoids, and reducing sugar.
Chloroform fraction showed sterols, flavonoids, and saponins.

In view of the above discussion, it is concluded that aqueous and ethyl acetate fractions are rich
with phytochemicals.

This research work revealed that Olea europaea is rich with lead compounds, which has been
proved by literature. However an attempt has been made to check its biological activity and the
active ingredient present in it.

4.1 PHYTOCHEMICAL SCREENING

4.1.1 Detection Test results for Phytochemicals

1. Detection of Alkaloids

(i) Dragendorff’s test

Orange-brown precipitate indicated the alkaloids.

(ii) Wagner test

There was formed brown precipitate indicating the alkaloids.

 58

2. Detection of carbohydrates

(i) Benedict’s test

Red precipitate showed the carbohydrates.

(ii) Molish test

Formation of a purple layer confirmed carbohydrates.

3. Detection of sterols/ steroids

(i) Salkowaski test

On shaking red colour was observed, which show steroids..

(ii) Liebermann-Burchard test

Development of dark reddish colour in chloroform layer and green fluorescence by acid layer
showed steroids/sterols.

4. Test for Tanins

(i) Ferric chloride Test

Intense green or deep blue colour showed tannins.

5. Detection of Phenols

(i) Ferric chloride Test

Intense green colour showed the phenolic compounds.

6. Detection of Terpenoids

(i) Chloroform Test

Light black colour confirmed terpenoids.

 59

7. Detection of Flavonoids

(i) Lead acetate Test

Flocculent creamy precipitate showed flavonoids.

8. Detection of Saponins

(i) Lead acetate Test

Creamy precipitate showed saponins.

(ii) Foam Test

Formation of layer of foam showed saponins.

9. Detection of Reducing sugar

(i) Fehling’s test

Red precipitate showed carbohydrates.

10. Detection of Oils and Fats

(i) Spot Test

An oily mark confirmed the lipids.(GA ayoola 2008)

 60

Table 4.1: Phytochemical Screening results Of Olive Plant (Olea Europaea)

Sr# Content Test n-hexane Chloroform Ethyl Aqueous

fraction fraction acetate fraction
fraction
1 Alkaloids Dragendorff’s - - + +
Wagner’s - - - +
2 carbohydrates Benedict’s test - - + +
Molish - - + +
3 Sterol/steroids Salkowaski test + + - -
4 Tanins 5% FeCl3 test - - - +
5 Phenols 5% FeCl3 test - - + +
6 Terpenoids Chloroform test + - + -
7 Flavonoids Lead acetate - + + +
8 Saponins 1% Lead acetate test - - + +
Foam formation - - - -
9 Reducing Fehling test
suger - - + +
10 Lipids Spot test + - + +
 61

4.2 GLUCOSE BOUND TEST

Five glucose solutions, with different concentration were made i.e. 5, 10, 20, 50, 100 μg/mL. 25
ml of this solution was mixed with plant extract and well mixed by using electric stirrer and hot
plate. Results are given below.

Table 4.2: in-vitro Glucose Bound Test Values

Sr # Concentration of Glucose Absorbance of

μg/mL UV
Standard Test
1 5 0.171 0.165
2 10 0.165 0.151
3 20 0.142 0.138
4 50 0.127 0.131
5 100 0.157 0.125

4.3 GLUCOSE DIFFUSION TEST

Glucose solution was taken in a dialysis tube i.e. 30 ml and one ml plant was mixed well in it. It
was then placed in a dialyzer, having 50 ml distilled water. It was set on 100 rpm. The diffusion
of glucose was measured after a fixed time interval by UV test.

Table 4.3: in-vitro Glucose Diffusion Values

Sr # Concentration of Glucose Absorbance of

μg/ml UV
Standard Test
1 5 0.165 0.151
2 10 0.142 0.140
3 20 0.127 0.120
4 50 0.112 0.105
5 100 0.98 0.88
 62

4.4 ETHYL ACETATE DOSE

Ethyl acetate dose was made for 21 days i.e. 126 ml solution prepared. It was stored in a cool and
dry place.

Table 4.4: Blood Sugar Measurement for Ethyl acetate Extract

Fractio No of Random Blood Sugar Values (RBS)

n Rats mg/dl
Day 0 Day 7 Day 14 Day 21
1 308 248 191 106
2 314 252 187 94
Ethyl 3 302 245 186 100
Acetate 4 304 250 193 98
5 309 249 183 104
6 311 244 188 98
Means 308 248 188 100
S.D 4.427189 3.03315 3.577709 4.38178
RBS

350

300

250

200 Day 0
Day 7
150 Day 14
Day 21
100

50

0
1 2 3 4 5 6
Rats

Animal models

Figure 4.1: Blood Sugar Measurement for Ethyl acetate Extract

4.5 N-HEXANE DOSE

 63

For the preparation of 21 days dose of n-hexane, 6300 mg 0f n-hexane fraction was dissolved in
126ml of tween solution. So that 1ml dose given daily, would contain 50mg. 126 ml of this
prepared dose was stored in a dry and cool place.

Table 4.5: Blood Sugar Measurement For n-hexane Extract

Fraction No of Rats Random Blood Sugar Values (RBS)

mg/dl
Day 0 Day 7 Day 14 Day 21
1 311 266 232 197
2 305 262 237 193
n-hexane 3 303 264 227 190
4 299 260 236 199
5 310 268 230 192
6 302 265 230 187
Means 305 264 232 193
S.D 4.690416 2.857738 3.847077 4.427189

RBS

350

300

250

200 Day 0
Day 7
150 Day 14
Day 21
100

50

0
1 2 3 4 5 6
Rats

Figure 4.2: Blood Sugar Measurement for n-hexane Extract

4.6 AQUEOUS DOSE

 64

For the preparation of 21 days dose of crude extract, 25,200 mg 0f crude extract fraction was
dissolved in 126ml of tween solution. So that 1ml dose given daily, would contain 200mg. 126
ml of this prepared dose was stored in a dry and cool place.

Table 4.6: Blood Sugar Measurement For Aqueous Extract

Fraction No of Rats Random Blood Sugar Values (RBS)

mg/dl
Day 0 Day 7 Day 14 Day 21
1 297 258 208 151
2 300 260 213 157
Aqueous 3 304 254 204 147
4 306 261 210 145
5 299 259 203 150
6 294 256 210 156
Means 300 258 208 151
S.D 4.427189 2.607681 3.84708 4.774935
RBS

350

300

250

200 Day 0
Day 7
150 Day 14
Day 21
100

50

0
1 2 3 4 5 6
Rats

Figure 4.3: Blood Sugar Measurement for Aqueous Extract

4.7 DIABETIC GROUP

 65

Diabetes mellitus was induced in adult albino rats by intraperitonealy injecting Streptozotocin
(STZ). For this purpose STZ was dissolved in 10mM sodium citrate buffer with PH 4.5. rats
were kept fasting overnight and then 50mg/Kg body weight STZ was given through single
intraperitoneal injection. STZ-induced rats with Random Blood Sugar (RBS) (˃250 mg/dl) were
chosen for study.

Table 4.7: Blood Sugar Measurement For Diabetic Group

Fraction No of Rats Random Blood Sugar Values (RBS)

Day 0 Day 7 Day 14 Day 21
1 296 306 312 321
2 302 310 320 309
Diabetic 3 290 314 315 312
Group 4 300 309 325 315
5 299 311 323 320
6 289 310 325 313
Means 296 310 320 315
S.D 5.4037 2.60768 5.44059 4.69042 RBS

330

320

310
Day 0
300 Day 7
Day 14
Day 21
290

280

270
1 2 3 4 5 6
Rats

Figure 4.4: Blood Sugar Measurement for diabetic group Extract

4.8 GLIMEPIRIDE GROUP

 66

This is a positive control group. As this group is also an induced diabetic group, it was also
treated with streptozotocin. But after induction of diabetes, this group was not treated with any
fraction. it was given a standard anti diabetic namely glimepiride for the sake of comparison.

Table 4.8: Blood Sugar Measurement for Glimepiride Group

Fraction No of Rats Random Blood Sugar Values (RBS)

Day 0 Day 7 Day 14 Day 21
1 301 221 157 98
2 295 229 152 86
Glimepiride 3 289 225 147 92
Group 4 296 228 150 88
5 299 225 156 91
6 290 222 150 97
Means 295 225 152 92
S.D 4.77493 3.16228 3.84708 4.77493

RBS

350

300

250

200 Day 0
Day 7
150 Day 14
Day 21
100

50

0
1 2 3 4 5 6
Rats

Figure 4.5: Blood Sugar Measurement for glimepiride group

 67

4.9: EVALUATION PARAMETERS

To check the quality of syrup formed, there are some parameters which should be followed. It
includes the colour, taste, viscosity, smell and pH. All these parameters were check and found to
be within standard limits. The table regarding this information is given below.

Table 4.9: Evaluation Parameters

Sr # Evaluation Parameters Standard Syrup Test Syrup

1 Colour Dark Yellow Light Yellow
2 Odour Slightly Bitter Aromatic
3 Taste Intensely Bitter Slightly Bitter
4 pH 6.52 5.5
5 Viscosity 0.98 2.5

4.10 STRUCTURAL ELUCIDATION OF KNOWN COMPOUNDS OF

OLEA EUROPAEA

3.4.3.1 Compound-I

Physical data:

Colourless needles (36mg)

MP: 197-198 0C

[α]25D: +100o (c=0.21, CHCl3)

IR (CHCl3) νmax cm-1: 3430, 3045, 1600, 815.

1
H NMR (CDCl3, 300 MHz):

δ 5.11 (m, H-12), 3.21(dd, J=10.0, 4.5 Hz, H-3), 1.08, 1.02, 1.01, 0.99, 0.95, 0,88, 0.85 and 0.80
(3H, each s, Me)
 68

13
C NMR (CDCl3, 75 MHz):

δ 139.41 (C-13), 124.12 (C-12), 78.82 (C-3), 59.02 (C-18) 55.43 (C-5), 47.72 (C-9), 42.32 (C-
14), 40.91 (C-8), 41.61 (C-22), 39.91 (C-19), 39.74 (C-20), 39.04 (C-4), 39.03 (C-1), 37.01 (C-
10), 34.06 (C-17), 33.22 (C-7), 31.42 (C-21), 28.92 (C-15), 28.07 (C-28), 28.05 (C-27), 27.41
(C-2), 26.55 (C-16), 23.31(C-29), 21.32 (C-30), 18.51 (C6), 17.42 (C-11)

HR-EIMS m/z:

426.3825 (calcd. For C30 H50 O, 426.3861).

EIMS m/z (rel. int.):

426 [M]+ (15), 411 (18), 408 (16), 393 (32), 257 (20), 218 (100), 207 (10), 203 (40) and 189 (55).

Figure 4.6: Compound i

3.4.3.2 Compound-II

Physical data:

Colourless needles (36mg)

 69

MP: 305-306 0C

[α]25D: +78.9o (c=0.07, CHCl3)

IR (KBr) νmax cm-1: 3400, 2640, 1700, 1660 and 820.

1
H NMR (CDCl3, 300 MHz):

δ 5.24 (1H, t, J=3.45 Hz, H-12), 3.60 (1H, dd, J=4.1 9.9 Hz, H-3), 1.12, 1.03, 0.98, 0,97, 0.90
and 0.89 (3H, each s, Me)

13
C NMR (CDCl3, 75 MHz):

δ 183.1 (s, C-28), 143.6 (s, C-13), 122.7 (d, C-12), 79.0 (d, C-3) 55.2 (d, C-5), 47.6 (d, C-9), 46.5
(s, C-17), 45.9 (t, C-19), 41.6 (s, C-14), 41.0 (d, C-18), 39.1 (s, C-8), 38.7 (s, C-4), 38.04 (t, C-
19), 41.6 (s, C-14), 41.0 (d, C-18), 39.1 (s, C-8), 38.7 (s, C-4), 38.4 (t, C-1), 37.1 (s, C-10), 33.8
(t, C-21), 33.0 (q, C-29), 32.6 (t, C-7), 32.4 (t, C-22), 30.6 (s, C-20), 28.1 (q, C-23), 27.7 (t, C-
15), 27.2 (t, C-2), 25.9, (q, C-27), 23.5 (q, C-30), 23.4 (t, C-11), 23.4 (t, C-16), 18.3 (t, C-6), 17.1
(q, C-26), 15.6 (q, C-24), and 15.3 (q, C-25).

HR-EIMS m/z:

456.3610 (calcd. For C30 H48 O3, 456.3603).

EIMS m/z (rel. int.):

456 [M]+ (4), 248 (98), 208 (12), 203 (60), and 133 (53).
 70

Figure 4.7: Compound ii

 71

CONCLUSION

Present study comprised of phytochemical screening, in-vivo / in-vitro study, isolation of natural
product and preparation and evaluation of neutraceutical syrup formation. Therefore the present
work consists of three phases. First phytochemical screening confirmed the presence of lead
compounds present in Olea europaea. It was also confirmed by in-vivo, in-vitro studies . second
two known compounds namely α-amyrin and β-amyrin. These compounds were isolated for the
first time and were confirmed by their characterization. In the third phase on the basis of
phytochemical screening and biological activity, the neutraceutical dosage form can successfully
be applied in the manufacturing process of pharmaceutical sector.
 72

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