Applications in Plant Biotechnology Focus on Plant

Secondary Metabolism and Plant Molecular Pharming, 1st

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iv Applications in Plant Biotechnology

biotechnological, prophylactic and therapeutic purposes. The eleventh chapter discusses

the use of plant molecular pharming to manufacture biologics against HIV. It addresses
progress in the development of plant-derived HIV vaccines, the risks and challenges
associated with vaccine production in plants, as well as the benefits of vaccine production
in plants compared to other expression systems.
In summary, this book covers two increasingly growing fields of plant biotechnology,
by drawing on some of the most prominent research groups in their respective fields as co­
authors. The research described in this book should be helpful for any who are interested
in both current and future aspects of plant biotechnology for many years to come.
 Contents

Preface iii
Acknowledgment of Reviewers vii

I-Plant Secondary Metabolism

1. Withania somnifera: A Future Pharma Factory 3
Tarun Halder, Subrata Kundu and Biswajit Ghosh
2. Biotechnological Approaches for Tropane Alkaloids Production 45
Ryad Amdoun, Boualem Harfi, Asma Moussous, Abdullah Makhzoum
and Lakhdar Khelifi
3. Anticancer Mechanisms of Plant Secondary Metabolism 61
David O Nkwe
4. In silico and Computational Analysis of Plant Secondary Metabolites 74
from African Medicinal Plants
Smith B Babiaka, Pascal Amoa Onguéné, Boris Davy Bekono
and Fidele Ntie‑Kang
5. Botany, Phytochemistry, Pharmacology, and Toxicity of the 91
Southern African Strychnos Species
Willie Marenga, Abdullah Makhzoum and Gaolathe Rantong
6. Preference of Agrobacterium rhizogenes Mediated 119
Transformation of Angiosperms
Kamogelo M Mmereke, Aaqib Javid, Sukanya Majumdar, Ipshita Ghosh,
Sonia Malik, Abdullah Makhzoum and Sumita Jha

II-Plant Molecular Pharming

7. Plant Molecular Pharming: Methods, Tools, Challenges Ahead for 165
Production of Recombinant Proteins, and Potential Solutions
Shahram Shokrian Hajibehzad, Azadeh Mohseni, Mohammad Dolati,
Saeid Malekzadeh Shafaroudi, Kathleen Hefferon and Abdullah Makhzoum
8. Plant-based Vaccines against Livestock Diseases: Way to Achieve 204
Several Sustainable Development Goals
Mohammad Tahir Waheed, Muhammad Suleman Malik, Kiran Saba,
Iqra Younus, Niaz Ahmad, Bushra Mirza and Andreas Günter Lössl
vi Applications in Plant Biotechnology

9. Chloroplast Biotechnology Tools for Industrial and 239

Clinical Applications
Kwang‑Chul Kwon
10. Plant Virus-Based Expression Vectors and their Applications 276
in Foreign Protein/Antigen Expression
Srividhya Venkataraman, Erum Shoaeb and Kathleen Hefferon
11. Plant Molecular Pharming of Biologics to Combat HIV 296
Goabaone Gaobotse, Jocelyne Trémouillaux‑Guiller, Srividhya Venkataraman,
Mohammed Kamil Sherif, Abdullah Makhzoum and Kathleen Hefferon
Index 349
About the Editors 355
 Acknowledgment of Reviewers

Reviewers
Dr. Marina Clemente
Instituto Tecnológico de Chascomús (INTECH)
CONICET-UNSAM
Intendente Marino Km 8,2; CC 164 (B7130IWA)
Chascomús; Provincia de Buenos Aires; Argentina
Tel:+54-2241-430323; FAX:+54-2241-424048
E-mail: [email protected]
https://intech.conicet.gov.ar/
www.iib.unsam.edu.ar/iib-unsam/investigacion.html
Professor Sumita Jha
Department of Botany, University of Calcutta’35 Ballygunge Circular Road, Kolkata
700019, India
Professor Sergio Rosales Mendoza
Faculty of Chemical Sciences
Center for Research in Biomedicine and Health
Office Address: Manuel Nava 6 CP. 78210 San Luis Potosi
Telephone: (+52) 4448262440
Email: [email protected]
Website: https://www.researchgate.net/profile/Sergio_Rosales-Mendoza
ORCID: 00-0003-2569-7329
Dr. Mohammad Tahir Waheed
Assistant Professor
Course Coordinator
Department of Biochemistry
Quaid-i-Azam University
45320, Islamabad, Pakistan
Member: International Society of Plant Molecular Farming (ISPMF)
Member: European Plant Science Organization (EPSO)
Member: Asian Council of Science Editors (ACSE)
Associate Editor:
1. BMC Plant Biology
2. BMC Developmental Biology
3. American Journal of Biochemistry and Biotechnology
Tel: 0092-51-90643207
viii Applications in Plant Biotechnology

Cell: 0092-333-9964114
https://scholar.google.com.pk/citations?user=5cYbyykAAAAJ&hl=en&oi=ao
https://www.researchgate.net/profile/Mohammad_Waheed2
https://bmcplantbiol.biomedcentral.com/about/editorial-board
https://bmcdevbiol.biomedcentral.com/about/editorial-board
https://thescipub.com/journals/ajbb/editors
Dr. Mohamed Elhiti
Associate Professor
Department of Botany, Faculty of Science,
Tanta University, Tanta, 31527, Egypt
Professor Ed Rybicki
Director, Biopharming Research Unit
Molecular & Cell Biology Department
University of Cape Town
Dr. Sarah Bushra Nasir
Abdus Salam School of Sciences,
Department of Life Sciences,
Nusrat Jahan College,
Chenab Nagar, Pakistan.
Abdulbaset Azizi
Department of Plant Protection
University of Kurdistan,
Sanandaj, Iran
Dr. Emmanuel Margolin
Postdoctoral Scientist
Bioharming research Unit (Department of Molecular and Cell Biology), University of
Cape Town
And Viral Vaccine Development Group and Human Papillomavirus Research Group
(Department of Pathology), Institute of Infectious Diseases and Molecular Medicine,
Faculty of Health Scienc-es, University of Cape Town
Dr. Mohammed Kamil Sherif
Central Medical Centre
Palapye, Botswana
E-mail: [email protected]
I-Plant Secondary
Metabolism
 Chapter 1

Withania somnifera:
A Future Pharma Factory
Tarun Halder, Subrata Kundu and Biswajit Ghosh*

Introduction
Humans have used plants accessible within their territory for diet and curative purposes
since their civilization began. This traditional system of medicine based on belief
and practices has been used as a therapy for most human ailments for over hundreds
of centuries (Winters 2006, Kulkarni and Dhir 2008, Mirjalili et al. 2009a, Rayees
et al. 2012, 2013). Irrespective of the progression in drug development methods, several
species of plants are unparalleled as major ingredients of current medicine. Due to our
existing industrialized lifestyle, we are continuously exposed to an over abundance of
chemicals and different environmental pollutants that ultimately intensified the incidence
of complicated neurodegenerative, cardiovascular diseases, and cancers. Perceptible
antagonistic effects of modern drugs used in the management of complicated ailments
ultimately resulted in renewed attention towards herbs and medicinal plants as an
alternative source.
Withania somnifera (L.) Dun, is one of the important medicinal plants in India
belonging to the Solanaceae family. It is commonly known as ‘Ashwagandha’, Winter
Cherry, Indian ginseng. In the monographs of the World Health Organization (WHO),
Ashwagandha has been considered as one of the important medicinal plants and also
has been included in the list of top thirty two prime concerned medicinal plants by the
National Medicinal Plant Board of India (http://www.nmpb.nic.in) owing to its huge
demand in both domestic and international markets (Mirjalili et al. 2009a, Singh et al.
2015). In Ayurveda and other traditional systems of medicine, it is one of the most
valued medicinal plants and it has been in use for more than 3000 years. It is widely
used in traditional Indian medicine systems for curing a variety of ailments. It

Plant Biotechnology Laboratory, Post Graduate Department of Botany, Ramakrishna Mission Vivekananda
Centenary College, Rahara, Kolkata-700118, India.
Emails: [email protected]; [email protected]
* Corresponding author: [email protected]
4 Applications in Plant Biotechnology

possesses adaptogenic, tonic analgesic, antipyretic, anti-inflammatory, and abortifacient

properties and is one of the most extensively used plants in various systems of medicine
(Chopra et al. 1958). There are several reports along with numerous clinical trials
that support the use of W. somnifera for hepatotoxicity (Bhattacharya et al. 2000a),
anxiety (Bhattacharya et al. 2000b), cognitive (Bhattacharya et al. 1995), neurological
disorders (Kuboyam et al. 2005, Pandey et al. 2018), inflammation (Al-Hindawi et al.
1992, Noh et al. 2016), hyperlipidemia (Visavadiya and Narasimhacharya 2007)
and Parkinson’s disease (Ahmad et al. 2005). The leaves are also known to act as an
insect repellent (Schmelze et al. 2008). The steroidal lactones known as withanolides
(a group of biologically active oxygenated ergostane type steroidal lactones) are found both
in leaves and roots parts of the plant (Kaushik et al. 2017). These compounds have been
intensely investigated because of their pronounced anti-tumor properties and novel steroidal
structure. Alkaloids constitute another major group of components that have been isolated
from W. somnifera. Some alkaloids have also been isolated from the roots of W. somnifera,
among them, withanine is the main alkaloid comprising 38% of the total alkaloid material
(Atal et al. 1975). The major withanolides withaferin A, withanolide A show antitumor
and cytotoxic activities (Mondal et al. 2010, Siddique et al. 2014, Kuboyama et al. 2014).
W. somnifera is predominantly propagated through seeds (Rao et al. 2012). Its traditional
cultivation has been limited due to a low percentage of seed viability, poor germination
and seedling survival, and low yield of withanolides from natural sources (Vakeswaran
and Krishnasamy 2003). In addition, infestation with pathogens and pests poses a serious
challenge in its commercial cultivation and improvement (Sharma et al. 2011). In recent
years, there is enormous demand for the enhanced synthesis of pharmacologically
important metabolites through biotechnological interventions (Singh et al. 2015, Ray et al.
2019). Unfortunately, the concentration of pharmacologically important withanolides in
W. somnifera is quite low, ranging from 0.001 to 0.5% of dry weight (DW) (Mirjalili
et al. 2009b). Nevertheless, the chemical synthesis of withanolides is unwieldy and
there is limited information on biosynthesis and regulation of secondary metabolites.
In this regard, cell or organ cultures are found to be of immense potential for the mass
production of secondary metabolites. Cell suspensions usually synthesize metabolites
at a faster rate owing to their uniform and active growth. Apart from these techniques,
genetic transformation (both Agrobacterium tumefaciens and A. rhizogenes mediated
transformation) has emerged as a powerful tool for engineering the plants for
overexpressing desired metabolites and to decipher molecular functions of selected
genes. The production of withanolides in the plant could be monitored through seasonal
changes or growth periods. In this chapter, we have analyzed detailed exploration on
this plant carried out by different researchers from various locations globally and their
remarkable influences on research from some of the available literature to illuminate the
pharmacological importance of W. somnifera.

Historical Background
W. somnifera has been widely used as a remedy in the Ayurvedic system of medicine
in India that can be traced back to the years’ BC (Singh et al. 2011). In Sanskrit,
ashwagandha means “horse’s smell”, probably originating from the odor of its root that
resembles the sweat of horses. The species name ‘somnifera’ means “sleep bearing” in
Latin. Traditional uses of Ashwagandha among tribal peoples in Africa include fevers
 Withania somnifera: A Future Pharma Factory 5

and inflammatory conditions. In the Indian Ayurvedic system of medicine, this plant
is described as a powerful rejuvenating herb. This plant was labeled in ayurvedic text,
i.e., the “Charaka” and the “Sushruta Samhitas”. Nevertheless, the root of Ashwagandha
has been regarded as a tonic, aphrodisiac, narcotic, diuretic, anthelmintic, astringent,
thermogenic, and stimulant. Due to its Ayurvedic history, it has been used as a
“Rasayana”, a treatment that releases emotional tension and physical discomfort, and
creates a foundation of wellness and stability in the body. Ashwagandha has been used for
thousands of years to target everything from the negative effects of stress in the human
body to strengthening the immune system throughout the seasons. In Africa, traditional
uses of Ashwagandha among tribal peoples include fevers and inflammatory diseases. In
Yemen, the dried leaves are ground into a paste that is used for treating burns and wounds,
as well as for sunscreen. For external healing, the berries and leaves have been applied to
tumors, tubercular glands, carbuncles, and ulcers. Indeed, Ashwagandha has become the
backbone of many multi ingredient Ayurvedic and other systems of medicine that have
been used for a wide assortment of ailments.

Different Plant Parts Use for Medicinal Purpose

The leaves of W. somnifera are a little bitter and are recommended to treat fever and
painful swellings. The crude preparation of the plant has also been found to be very
active against several pathogenic bacteria. Sore eyes, ulcers, and swellings can be cured
using a fomentation of the leaves (Table 1). The leaves are also used as a compelling
and an anthelmintic, can be crushed of the tissue and applied to tumors and ulcers, it is
also consumed as a vegetable and used as livestock fodder (Kirtikar and Basu 1991).
Other uses of the leaves are to heal open as well as septic and inflamed wounds and
abscesses and to treat inflammation, hemorrhoids, rheumatism, and syphilis. Major
active compounds of withanolides like withaferin A are found mostly in the leaves of
this plant, first of all, withaferin A under the withanolides group have been elucidated
from the leaves by Lavie’s group (Lavie et al. 1965). Israel chemotypes of Ashwagandha,
production of withaferin A from the intact plants are about 0.2–0.3% based on DW
of leaves (Abraham et al. 1968) compared to Indian chemotypes and were compared
with withaferin A based on DW and it is absent in roots, stems and fruits (Gupta et al.
1996). The common effects of W. somnifera root extracts have cumulative properties on
humoral immune responses. In the animal blood system, the roots are accomplished to
increase the number of white blood cells. It helps to relieve insomnia by the processing
of nervous cells and possesses a slight tranquilizing activity that promotes sound sleep.
It also helps regulate long term blood sugar levels and is also a beneficial treatment for
weight loss. The roots of W. somnifera are a natural alterative, aphrodisiac, removing
obstructions, diuretic, narcotic, sedative, and restorative. According to different levels of
experiments based on research reports, Ashwagandha roots powder can be an effective
herbal supplement from the traditional as well as a modern system of medicine for the
treatment of various types of diseases. Ashwagandha root is a popular male sex tonic in
India. It helps to cure erectile dysfunction and improve male sperm count. It also helps to
reduce bad cholesterol levels which are responsible for hypertension and cardiovascular
problems. Recent studies revealed that the ashwagandha root contains steroidal properties
which can be effective in treating inflammation. It is also used to treat low back pain and
sciatica. It is generally safe to use, as it is purely natural and free from side effects. The
6 Applications in Plant Biotechnology

Table 1: Extracts of different plant organs of W. somnifera extracted by various solvents and their
different pharmacological activities.

Plant Extract types Action on target cells References

parts
Root Chloroform Anti-cancer (Liver, breast, colon), Siddique et al. 2014
Prostate cancer
Chloroform Against chemically induced Biswal et al. 2013
Fatigue
Alcoholic Anti-tumor Khazal et al. 2013
Alcoholic Antimicrobial Khanchandani et al. 2019
Aqueous Antimicrobial Kumari et al. 2020
Ethanolic Anti-cancer (Breast cancer) Maliyakkal et al. 2013
Ethanolic Anti-cancer (Prostate cancer) Kim et al. 2020
Ethanolic of N-118 Neuroprotectant (Against Ahmad et al. 2015
cerebral stroke)
Ethanolic of 101R, Immunomodulatory Kushwaha et al. 2012a,
118R and 128R Kushwaha
et al. 2012b
Ethanolic Anti-cancer (Cervical) Jha et al. 2014
Aqueous Anti-cancer (Cervical) Nile et al. 2019
Aqueous Anti-stress (reduced in T-cell Khan et al. 2006
population and up-regulated Th1
Cytokines in chronically stressed mouse)
Aqueous Anti-stress Jain and Saxena
2009
Aqueous Alzheimer’s disease Sehgal et al. 2012
Aqueous Neuroprotective (Enhanced memory Baitharu et al.
and attenuated hippocampal 2013, 2014
neurodegeneration through inducing
glutathione biosynthesis)
Methanolic Hepatoprotective role in acetaminophen- Devkar et al. 2016
intoxicated rats
Methanolic Parkinson’s disease De Rose et al. 2016
Methanolic Neuroprotective effects Bhattarai and Han
2014
Leaf Methanolic Anticancer (TIG1, U2OS, and HT1080) Widodo et al. 2008
by activating p53, apoptosis pathway &
cell cycle arrest)
Methanolic Hypoglycemic (Increased uptake of Gorelick et al.
glucose in myotubes and adipocytes) 2015
Methanolic Neuroprotection through activation of Konar et al. 2011
neuronal proteins, oxidative stress and
DNA damage

Table 1 contd. ...

 Withania somnifera: A Future Pharma Factory 7

...Table 1 contd.

Plant Extract types Action on target cells References

parts
Methanolic Anticancer (Neuroblastoma) Kuboyama et al.
2014
Methanolic Antimicrobial (against methicillin Bisht and Rawat
resistant Stephylococcus aureus and 2014
Enterococcus sps.)
Methanolic Antiproliferative (against MCF-7, Alfaifi et al. 2016
HCT116 and HepH2 cell lines)
Methanolic Antimicrobial activity Dhiman et al. 2016
Methanolic Antibacterial (triagainst Salmonella typhi) Alam et al. 2012
and Antioxidant
Ethanolic Antimicrobial activity Dhiman et al. 2016
Hydroalcoholic Anti-cancer (Breast Cancer) Nema et al. 2013
Aqueous Anti-cancer Wadhwa et al. 2013
Stem Methanolic Cytotoxic activity against cancer Srivastava et al.
cell lines MDA-MB-231 (Human breast 2015
cancer cell lines)
Ethanolic Cytotoxic activity against Cancer cell Srivastava et al.
lines MDA-MB-231 (Human breast 2015
cancer cell lines)
Fruit/ Ethanolic Alzheimer’s disease Jayaprakasam et al.
berry 2010
Methanolic Antioxidant and antibacterial activities Alam et al. 2012

root contains an alkaloid somniferine. Roots are also used to prepare the herbal remedy of
ashwagandha, which has been traditionally used to treat various symptoms and disorders.
The roots of the plant are categorized as Ramayana’s, which are reputed to promote
health and longevity by augmenting defense against disease, arresting the aging process,
revitalizing the body in debilitated conditions, increasing the capability of the individual
to resist adverse environmental factors and creating a sense of mental wellbeing. The
medicinal importance of bioactive constituents from different parts of W. somnifera are
represented in Table 1.

Pharmacological Importance
Pharmacology is the science of drugs, including the study of those substances that interact
with living systems through chemical processes, by binding to regulatory molecules
and activating or inhibiting normal body processes. Although all the development in
synthetic chemistry and drug development progresses, plants are still a significant source
of modern medicinal preparations. Many species of plants are known for their medicinal
values and are used to treat several human ailments and diseases. Medicinal properties of
these plants are linked with their roots, seeds, flowers, leaves, fruits, or the whole plant
itself. It has been mentioned that the medicines from plants are better in bioactivity and
8 Applications in Plant Biotechnology

are found to possess curative properties against various infectious and non-infectious
diseases when compared to chemical and synthetic medicines (Itokawa et al. 2008,
Ezzat et al. 2019). Therefore the bioactive compound is the fundamental molecule that
acts like a drug. These bioactive compounds are mostly derived from plant secondary
metabolites, and many naturally occurring pure compounds have become medicines.
Active lead compounds can also be further modified to enhance the biological profiles
and developed as clinical trial candidates (Itokawa et al. 2008). It is fact that natural plant
product research and development (R&D) potentially plays a vital innovative role in drug
discovery.
The plentiful therapeutic applications of W. somnifera are linked to the occurrence
of natural phytochemicals primarily withanolides and also some other secondary
metabolites, which can be found at different levels in various plant parts like roots, stems,
and leaves and are together responsible for the different pharmacological activity. Several
withanolides have been obtained and characterized over the past five decades. This type
of steroid has attracted significant attention from numerous researchers, not only because
of its complex structural features but because of its multiple bioactivities and potential
in drug research and development (Ichikawa et al. 2006, Misra et al. 2008, Chen et al.
2011, Dar et al. 2015, Ahmad and Dar 2017). More than 12 alkaloids, 40 withanolides,
and quite a few sitoindosides have been reported from this plant species (Mishra et al.
2000, Mirjalili et al. 2009a). Withanolides are a group of steroidal lactones with atoms
C-22 and C-26 bridged by D-lactone functionality and are considered potential active
pharmacological compounds specific to this species (Abou‐Douh 2012). The chemical
diversity among different withanolides is created due to glycosylation, hydroxylation
or the creation of additional rings along with the addition of side chains in the steroid
backbone (Fig. 1). Ichikawa et al. (2006) and Misra et al. (2008) reported that the
therapeutic potential of the species W. somnifera is mainly ascribed towards the presence
of several withanolides, especially the noted compound withaferin A and withanolide
A. These withanolides have demonstrated significant pharmacological activities
(Fig. 2). Withania somnifera contains a spectrum of varied bioactive secondary metabolites
which allows it to have a wide range of biological importance. In preclinical studies, it
has revealed antimicrobial, anti-inflammatory, anti-tumor, anti-stress, neuroprotective,
cardio-protective, and anti-diabetic properties. Moreover, it has confirmed the ability to
reduce reactive oxygen species, modulate mitochondrial function, regulate apoptosis,
reduce inflammation and improve endothelial function (Dar et al. 2015).
W. somnifera contains a spectrum of diverse phytochemicals enabling it to have a
broad range of biological implications. In pre-clinical studies, it has shown antimicrobial,
anti-inflammatory, antitumor, anti-stress, neuroprotective, cardio-protective, and anti-
diabetic properties. Additionally, it has demonstrated the ability to reduce reactive oxygen
species, modulate mitochondrial function, regulate apoptosis, and reduce inflammation
and enhance endothelial function. In view of these pharmacologic properties, W. somnifera
is a potential drug candidate to treat various clinical conditions, particularly related to the
nervous system. In the field of plant science, W. somnifera has always been the center
of attraction within the research community for various pharmacological activities such
as anti-diabetic, cardio-protective, antimicrobial, anti-inflammatory, neuroprotective, etc.
(Tripathi and Verma 2014, Dhar et al. 2015). Furthermore, it has the potentiality to cure a
wide range of cancer cells such as breast, colon, cervical, lung, prostrate, etc. (Fig. 3). The
utilization of active compounds on cancer cells acts as multiple modes of action on target
cells in different ways such as induced cell cycle arrest, apoptosis, cytotoxicity, inhibition of
 Withania somnifera: A Future Pharma Factory 9

Figure 1: Chemical structure of various withanolides from W. somnifera.

Figure 2: Different pharmacological activities of W. somnifera.

10 Applications in Plant Biotechnology

Figure 3: Mechanisms of action of withanolides on cancer cells.

angiogenesis and also overwhelming various oncogenic pathways (Palliyaguru et al. 2016).
Adaptogenic or the stress response regulating effects of W. somnifera has been reported
as a pharmacological description in the 1980s (Kumar et al. 2017). Since then, several
observations of this plant have revealed ginseng like effects on various animal models,
now it is often referred to as Indian ginseng (Kulkarni and Dhir 2008).
Apart from the medicinal properties and various preclinical studies performed on W.
somnifera or its ingredients, for different pharmacological actions, several clinical studies
have also been done by various authors (Table 2). W. somnifera was found to recover
the seminal plasma levels of antioxidant enzymes, vitamins A, C, and E and corrected
fructose in infertile men. Moreover, the treatment also significantly increased serum T
and LH and reduced the treatment of male sexual dysfunction and infertility levels of
FSH and PRL, which is useful to evaluate the spermatogenic activity indication of healthy
semen oligospermic patients (Ahmad et al. 2010, Ambiye et al. 2013). It was studied by
different researchers on infertile men where the rate of sperm apoptosis and cytoplasmic
ROS levels were significantly higher on the different age groups in comparison with
control subjects, likewise, the seminal plasma levels which contain the essential metal
ions like Zn2+, Fe2+, Cu2+ and Au2+ were low. In another case, a double blind, placebo
controlled study performed on 50 patients with ICD-10 anxiety disorders, it was observed
that by the end of the first month of treatment with an ethanol extract of Withania 250
mg twice a day, 72% of patients showed moderate to excellent improvement and in
about half of these cases, benefits were observed within the first fortnight (Andrade et al.
2000). The group of patients who received naturopathic therapy involving deep breathing
relaxation technique, a standard multi vitamin and W. somnifera (300 mg b.i.d.), were
found to recover effectively (Cooley et al. 2009). A study conducted on breast cancer
patients undergoing combination chemotherapy with oral W. somnifera (2 g every 8 h)