Potent Anticancer Medicinal Plants

POTENT ANTICANCER
MEDICINAL PLANTS
Secondary Metabolite Profiling,
Active Ingredients, and Pharmacological Outcomes
POTENT ANTICANCER
MEDICINAL PLANTS
Secondary Metabolite Profiling,
Active Ingredients, and Pharmacological Outcomes
Edited by
Deepu Pandita
Anu Pandita
First edition published 2024
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Library and Archives Canada Cataloguing in Publication

Title: Potent anticancer medicinal plants : secondary metabolite profiling, active ingredients, and pharmacological outcomes/
edited by Deepu Pandita, Anu Pandita.
Names: Pandita, Deepu, editor. | Pandita, Anu, editor.
Description: First edition. | Includes bibliographical references and index.
Identifiers: Canadiana (print) 20230445446 | Canadiana (ebook) 20230445497 | ISBN 9781774913116 (hardcover) |
ISBN 9781774913123 (softcover) | ISBN 9781003431190 (ebook)
Subjects: LCSH: Medicinal plants. | LCSH: Antineoplastic agents.
Classification: LCC QK99.A1 P68 2024 | DDC 581.6/34—dc23
Library of Congress Cataloging-in-Publication Data
CIP data on file with US Library of Congress
ISBN: 978-1-77491-311-6 (hbk)

ISBN: 978-1-77491-312-3 (pbk)
ISBN: 978-1-00343-119-0 (ebk)
About the Editors
Deepu Pandita
Senior Lecturer, Government Department of
School Education, Jammu, Union Territory of
Jammu and Kashmir, India
Deepu Pandita is working as a Senior Lecturer
in the Government Department of School
Education, Jammu, Union Territory of Jammu
and Kashmir, India. Deepu Pandita has more
than 20 years of teaching experience and has
done her Masters in Botany (MSc) from the
University of Kashmir, Jammu and Kashmir, India and Master of Philosophy
(MPhil) in Biotechnology from the University of Jammu, Jammu and
Kashmir, India. Deepu Pandita has a number of international and national
courses to her credit. She has qualified for fellowships, such as JRF-NET
and SRF from the Council of Scientific and Industrial Research (CSIR),
New Delhi India; Biotechnology Fellowship, Government Department of
Science and Technology, Jammu & Kashmir, India; and IAS-INSA-NASI
Summer Research Teacher Fellowship, India. Deepu Pandita has presented
her research work at both national and international conferences and was
awarded Best Oral Presentation Award at an International Conference on
Biotechnology for Better Tomorrow, Avtar Krishan Award of Cytometry
in 12th Indo-US Cytometry Workshop on Clinical Research; a Women
Researcher Award; and a Research Excellence Award from two professional
associations in India. She is a life member of various scientific societies.
Deepu Pandita is also a reviewer, associate editor, and editor of a number of
journals of international repute. She has published a number of editorials,
book chapters (Springer, CRC, and Elsevier), reviews, and research articles
in various journals of national and international repute, including Cells,
Frontiers in Plant Sciences, Journal of Fungi, Frontiers in Sustainable Food
Systems, and Frontiers in Physiology. Deepu Pandita has several books
currently under production.
vi About the Editors
Anu Pandita
Senior Dietician, Vatsalya Clinic,
New Delhi, India
Anu Pandita is working as a Dietician at
Vatsalya Clinic, E-5/11, Krishna Nagar, New
Delhi, India. Previously, she worked as a
Lecturer at Bee Enn College of Nursing, Talab
Tillo, Jammu, India, and Dietician at Ahinsa
Dham Bhagwan Mahavir Charitable Health
Centre, New Delhi, India. Anu Pandita has
done her MSc internship and a course in the Dietetics Department of Post
Graduate Institute of Medical Education & Research (PGI), Chandigarh,
India. She conducted a case study at the Pediatric Gastroenterology Ward
at Nehru Hospital, PGI, Chandigarh, India, on a patient suffering from
chronic liver disease. She has done a Certificate in Food & Nutrition as
well. Anu Pandita has presented her research work at both national and
international conferences. She has a number of trainings, refresher courses,
and workshops to her credit. She is a life-time member of the Indian Dietetic
Association and Indian Science Congress Association, Kolkata, India. She
has published several book chapters in Springer and CRC journals and a
number of reviews and research articles in various journals of national
and international repute, including Cells, Journal of Fungi, Frontiers in
Sustainable Food Systems, and Frontiers in Physiology. She has several
books under production currently.
Contents
Contributors.............................................................................................................ix
Abbreviations ...........................................................................................................xi
Preface .................................................................................................................... xv
1. Garcinia indica ................................................................................................1

Tewin Tencomnao
2. Centella asiatica Linn....................................................................................19

Sagar Barge, Dhananjay Jade, and Narayan Chandra Talukdar
3. The Anticancer Activity of Nigella sativa on Hepatocellular

Carcinoma......................................................................................................39
Iffat Zareen Ahmad and Heena Tabassum
4. Boswellia serrata Roxb..................................................................................61

Manohar M. V., Anu Pandita, Amogha G. Paladhi, Bhoomika Inamdar,
Sugumari Vallinayagam, Deepu Pandita, and K. M. Srinivasa Murthy
5. Catharanthus roseus.....................................................................................89
Ramachandra Reddy Pamuru, Rajagopal Reddy S., Ambedkar, Chandrasekhar T.,
Madhusudhana Reddy A., and Chandramathi Shankar P.
6. Withania somnifera (L.) Dunal................................................................... 111

Pulala Raghuveer Yadav, Lepakshi M. d. Bhakshu, K. Venkata Ratnam,
Anu Pandita, Deepu Pandita, and K. M. Srinivasa Murthy
7. Camptotheca acuminata Decne...................................................................131

Amogha G. Paladhi, Anu Pandita, Manohar M. V., Bhoomika Inamdar,
Sugumari Vallinayagam, Deepu Pandita, and K. M. Srinivasa Murthy
8. Taxus Baccata..............................................................................................151
Rohit Sam Ajee and Shuchi Kaushik
9. Panax Ginseng.............................................................................................181
Shalini Gurumayum, Sagar Barge, and Jagat C. Borah
viii Contents
10. Tinospora cordifolia (Thunb.) Miers..........................................................203

Lepakshi M. d. Bhakshu, Pulala Raghuveer Yadav, K. Venkata Ratnam,
11. Taxus brevifolia (Nutt.) Pilger.....................................................................223

K. Venkata Ratnam, Lepakshi Md. Bhakshu, Pulala Raghuveer Yadav,
12. Glycyrrhiza glabra......................................................................................247

Vijay Kumar Veena, B. Uma Reddy, Adhikesavan Harikrishnan, and
Ramasamy Shanmugavalli
13. Ocimum sanctum ........................................................................................267

Sharmistha Banerjee, Rajesh Singh Tomar, and Shuchi Kaushik
Index .....................................................................................................................283
Contributors
Madhusudhana Reddy A.
Department of Botany, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Iffat Zareen Ahmad

Department of Bioengineering, Integral University, Dasauli, Lucknow, Uttar Pradesh, India
Rohit Sam Ajee

Independent Researcher and Alumni Amity Institute of Biotechnology, Amity University,
Madhya Pradesh, India
Ambedkar
Department of Biochemistry, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Department of Biotechnology, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Sharmistha Banerjee
Biomedical Engineering and Bioinformatics, University Teaching Department, Chhattisgarh
Swami Vivekanand Technical University, Newai, Bhilai, India
Sagar Barge
Chemical Biology Lab I, Institute of Advanced Study in Science and Technology, Paschim Boragaon,
Guwahati, Assam, India
Lepakshi M. D. Bhakshu
Department of Botany, PVKN Government College (Autonomous), Chittoor, India
Jagat C. Borah
Shalini Gurumayum
Adhikesavan Harikrishnan
Department of Chemistry, School of Arts and Science, Vinayaka Mission Research Foundation-AV
Campus, Chennai, India
Bhoomika Inamdar
JSS Medical College (Deemed to be University), Mysuru, Karnataka, India
Dhananjay Jade
Biomedical sciences, University of Leeds, Leeds LS2 9JT, UK
Shuchi Kaushik
State Forensic Science Laboratory, Madhya Pradesh, India
K. M. Srinivasa Murthy
Department of Microbiology and Biotechnology, Jnanabharathi Campus Bangalore University,
Bengaluru, India
x Contributors
Manohar M. V.
Chandramathi Shankar P.
Department of Biotechnology, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Amogha G. Paladhi
Christ (Deemed to be University), Bengaluru, Karnataka, India
Ramachandra Reddy Pamuru

Department of Biochemistry, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Anu Pandita
Vatsalya Clinic, Krishna Nagar, New Delhi, India
Deepu Pandita
Government Department of School Education, Jammu, Jammu and Kashmir, India
K. Venkata Ratnam
Department of Botany, Rayalaseema University, Kurnool, India
B. Uma Reddy
Department of Studies in Botany, Vijayanagara Sri Krishnadevaraya University, Ballari, Karnataka, India
Rajagopal Reddy S.
Department of Botany, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Ramasamy Shanmugavalli
Department of Chemistry, School of Arts and Science, Vinayaka Mission Research Foundation-AV
Campus, Chennai, India
Chandrasekhar T.
Department of Environmental Sciences, Yogi Vemana University, Kadapa, Andhra Pradesh, India
Narayan Chandra Talukdar

Assam Down Town University, Panikhaiti, Guwahati, Assam, India
Heena Tabassum
Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Pune, Maharashtra, India
Department of Bioengineering, Integral University, Lucknow, Uttar Pradesh, India
Tewin Tencomnao
Natural Products for Neuroprotection and Anti-ageing Research Unit, Chulalongkorn University,
Bangkok, Thailand
Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University,
Bangkok, Thailand
Rajesh Singh Tomar

Amity Institute of Biotechnology, Amity University Madhya Pradesh, Maharajpura Dang, India
Sugumari Vallinayagam
Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India
Vijay Kumar Veena

Department of Biotechnology, School of Applied Sciences, REVA University, Bangalore, India
Pulala Raghuveer Yadav

Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Telangana State, India
Abbreviations
AA asiatic acid
ACh acetylcholin
ADA D-Adenosine deaminase
AM acrylamide
ALP alkaline phosphatase
ALT alanine aminotransferase
AST aspartate aminotransferase
ATM ataxia telangiectasia mutated
BA boswellic acid
BAP Benzylaminopurine
BSA bovine serum albumin
CA Centella asiatica
CCA Cholangiocarcinoma
CAT catalase
CPT camptothecin
DR death receptors
DTQ dihydrothymoquinone
EAC Ehrlich ascites carcinoma
EGFR epidermal growth factor receptor
ELSD evaporative light scattering detection
EMT epithelial to mesenchymal transition
ER endoplasmic reticulum
ERK extracellular signal-regulated kinase
FAK focal adhesion kinase
FasL factor associated-suicide ligand
FCM flow cytometry
FGF fibroblast growth factor
FPS farnesyl pyrophosphate synthase
FT-IR Fourier transforms infrared spectroscopy
GBD glabridin
GC gas chromatography
GGPP geranylgeranyl diphosphate
GI Garcinia indica
GPS geranyl diphosphate synthase
xii Abbreviations
GPS ginseng polysaccharide

GST glutathione-S-transferase
HCC hepatocellular carcinoma
HGF hepatocyte growth factor
HPLC high-performance liquid chromatography
HPTLC high-performance thin layer chromatography
HRMS high-resolution mass spectrometry
HSCCC high-speed counter-current chromatographic
HSP heat shock proteins
IAA iso-angustone A
IAP inhibitor of apoptosis protein
IFP interstitial fluid pressure
IL interleukin
LC liquid chromatography
LCM licocoumarone
LG Liquiritigenin
MAA methacrylic acid
MAP microtubule associated protein
MAPKs mitogen-activated protein kinases
MDR multidrug resistance
MeJA methyl jasmonate
MIP molecular imprinting
MMPs matrix metalloproteinases
MS mass spectrometry
MS multiple sclerosis
MVA mevalonate
NAA naphthalene acetic acid
NAC N-acetylcysteine
NCAM neural cell adhesion molecule
NMR nuclear magnetic resonance
NMR nuclear resonance spectroscopy
NO nitric oxide
NOS nitric oxide synthases
NPC nasopharyngeal carcinoma
OSCs oxidosqualene cyclases
PARP poly ADP ribosyl polymerase
PBD polo-box domain
PBMC peripheral blood mononuclear cells
PCNA proliferative cell nuclear antigen
Abbreviations xiii
PDA pancreatic ductal adenocarcinoma

PDGF platelet derived growth factor
PMA phorbol myristate acetate
PMK phosphomevalonate kinase
PSA prostate-specific antigen
RNAi RNA interference
ROS reactive oxygen species
RSM response surface methodology
SAR structure-activity relationships
SLE systemic lupus erythematosus
SOD superoxide dismutase
SRE serum response factor
SS squalene synthase
THQ thymohydroquinone
THY thymol
TME tumor microenvironment
TNBC triple negative breast cancer
TLC thin-layer chromatography
TQ thymoquinone
UV ultraviolet
VEGF vascular endothelial growth factor
WGOS water-soluble ginseng oligosaccharides
WHO World Health Organization
Preface
Cancer is the leading cause of death worldwide, and the quest for effective
treatments has been ongoing for decades. While conventional treatments like
chemotherapy, radiation therapy, and surgery have improved survival rates,
these often come with severe side effects that can reduce the quality of life
for cancer patients.
In recent years, there is an increasing interest in using medicinal plants
as a complementary or alternative treatment for cancer. Many studies have
shown that certain plant compounds can inhibit the growth and spread of
cancer cells, induce apoptosis (cell death) in cancer cells, and even enhance
the effectiveness of chemotherapy and radiation therapy. Herbal medicines
play a critical role in the prevention as well as the treatment of various forms
of cancer which is the foremost cause of death worldwide. Synthetic drugs
have prolonged toxic side effects, and substitute panacea is the economical
and natural medicinal plants.
This book aims to provide an in-depth compilation of most vital plant
genera and species, namely Garcinia indica, Centella asiatica Linn, Nigella
sativa, Ocimum sanctum, Boswellia serrata Roxb, Catharanthus roseus,
Withania somnifera (Linn.) Dunal, Camptotheca acuminata Decne, Taxus
baccata L., Panax ginseng, Tinospora cordifolia (Wild) Miers, Taxus
brevifolia, and Glycyrrhiza glabra, which have potent anticancer activity.
These anticancer medicinal plants are bestowed with novel and essential
cradle of chemotherapeutic complexes, biologically active molecules, and
secondary metabolites like taxol, vinblastine, vincristine, camptothecin,
topotecan, etc. with promising properties to cure cancer and have the potential
for new drug discovery in the era of modern medicine to fight against cancer.
This book also covers the evidence for their effectiveness in preclinical and
clinical studies, as well as any potential side effects or interactions with other
medications.
We hope that the information in this book will prove to be a treasured
resource for researchers, healthcare professionals and residents in
pharmacy and medicine, oncologists, biotechnologists, medicinal chemists,
pharmacists, pharmacologists, phyto-chemists and members of biomedical
and pharmaceutical sciences working in the areas of cancer treatment for the
welfare of human society and anyone interested in using natural remedies
xvi Preface
for cancer prevention and treatment and those seeking a comprehensive

understanding of the potential of medicinal plants in the fight against cancer.
We hope that this book will contribute to the growing body of knowledge on
medicinal plants and inspire further research and development in this field.
It is important to note that while medicinal plants may hold promise as
a complementary or alternative cancer treatment, they should never be used
as a substitute for conventional medical treatment. Cancer patients should
always consult their healthcare providers before using any herbal remedies
or supplements.
Few Lines on Anticancer Medicinal Plants

In gardens, fields and forests, they quietly thrive and grow,
Nature’s wonders, to help fight the foe, with a healing flow.
Potent anticancer plants with healing power and strength so rare,
To ward off the cancer, hour by hour and power to spare.
With healing powers to mend and renew,
Anticancer properties to see us through.
With ancient knowledge and modern research,
Their benefits for health, we can now perch.
In ancient wisdom, they were known,
To aid in healing, to strengthen and hone.
And now science has shown their worth,
As potent allies in the fight on earth.
From Boswellia’s boswellic acid to Catharanthus’s vincristine and
vinblastine a potent blend,
They hold the key to health, longevity and cancer prevention,
on their help we depend.
These plants of life with rainbow of color, we must treasure,
A natural cancer-fighting crew, we cannot measure.
The rose periwinkle, a wondrous flower, with its vibrant hue,
Holds a secret medicinal power to cure cancer, that could save me and you.
The Withania somnifera, a beacon of light,
In the darkness of cancer’s plight.
Panax Ginseng, a medicinal wonder, its roots hold the secret key,
Its benefits we cannot ponder, to fight cancer, a remedy free.
Tinospora cordifolia, a herb so pure,
Its anticancer compounds we can’t ignore.
Taxus, a plant with healing power,
Its anticancer compounds we can’t devour.
Preface xvii
Glycyrrhiza glabra, a herb so pure,

Its anticancer compounds we cannot ignore.
Ocimum sanctum, a herb so divine,
Its anticancer compounds we must incline.
Garcinia indica, a plant so rare,
Its anticancer compounds we must share.
Centella asiatica, a herb so bright,
Its anticancer compounds shine in the light.
Nigella sativa, a herb so divine,
Its anticancer compounds we must incline.
Camptotheca acuminata, its name,
Holds the key to a cancer-fighting game.
Their bark, leaves and roots, a source of great hope,
Contain alkaloids, flavonoids and novel compounds that help us cope.
With leukemia and lymphoma, they can fight,
And slow the spread of cancer’s might.
Against cancer cells, they can prevail,
And slow their growth without fail.
And the list goes on and on,
A multitude of plants to call upon.
May we continue to learn and explore,
And find new ways to fight cancer’s core.
Let us honor these plants with a heart of gold, for all what they provide,
Their healing properties are a source of hope for those who have cried.
With compounds that can heal and cure,
And offer hope in times unsure.
In the face of cancer’s deadly grip,
These plant offers a chance to slip.
May these greens continue to heal and inspire,
Until cancer is conquered, we never tire, and all can aspire.
CHAPTER 1
Garcinia indica
TEWIN TENCOMNAO1,2
1
Natural Products for Neuroprotection and Anti-Ageing Research Unit,
Chulalongkorn University, Bangkok, Thailand
2
Department of Clinical Chemistry, Faculty of Allied Health Sciences,
Chulalongkorn University, Bangkok, Thailand
ABSTRACT
Garcinia indica (GI), a plant native to India commonly known as kokum,

belongs to the Clusiaceae family. GI has been famous for a long time due
to its various benefits such as culinary, pharmaceutical, and industrial
applications. GI is recognized as a natural antioxidant, thus making the
GI extract so valuable for both research and application. Interestingly, GI
was found to possess numerous activities such as anti-inflammatory, anti-
infectious, antineurodegenerative, and anticancer effects. Emerging roles
of garcinol, the main bioactive compound of GI, have been grown since it
exhibits various pharmacological effects, particularly anticancer. Anticancer
mechanisms of garcinol have been documented vastly depending on the
cancer cell types. These mechanistic targets of garcinol include cell cycle,
proliferation, migration, invasion, metastasis, apoptosis, angiogenesis, stem
cell-like phenotype, epigenetics, miRNAs, and so forth. Therefore, garcinol
may represent an attractive lead compound for further anticancer drug
development.
Potent Anticancer Medicinal Plants: Secondary Metabolite Profiling, Active Ingredients, and
Pharmacological Outcomes. Deepu Pandita and Anu Pandita (Eds.)
© 2024 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
2 Potent Anticancer Medicinal Plants
1.1 BOTANICAL DESCRIPTION AND TRADITIONAL USES OF

GARCINIA INDICA
Commonly recognized as kokum, Garcinia indica (GI), a plant in the

mangosteen family (Clusiaceae), is a slender, evergreen, and fruit-bearing
tree with drooping branches, which can grow approximately 15 m tall with a
dense canopy of green leaves and red-tinged tender emerging leaves (Singh,
1993). The fruits of GI are spherical with diameters of 2.5–5.0 cm (Yella
Reddy and Prabhakar, 1994). This plant is found widely in tropical regions,
especially in Asia and Africa. GI grows widely on the western coast of India
(Padhye et al., 2009).
To date, GI has been known for numerous benefits to humans including
culinary, pharmaceutical, and industrial purposes. As revealed in many
reports, this particular plant species has long been known for culinary uses
(Chate et al., 2019) and industrial uses (Maheshwari and Reddy, 2005). In
addition, the health benefits of GI include medicinal and cosmetics properties
(Baliga et al., 2011).
1.2 MEDICINAL PROPERTIES OF GARCINIA INDICA
Furthermore, certain studies on the functional properties of GI demonstrated

its antioxidant effects (Mishra et al., 2006; Panda et al., 2012; Jayakar et al.,
2021), anti-inflammatory effects (Khatib et al., 2010; Panda et al., 2013) and
anti-infectious effects (Lakshmi et al., 2011; Varalakshmi et al., 2011; Sutar
et al., 2012; Tharachand et al., 2015).
GI extract is well-classified as a natural antioxidant. Since oxidative
stress is contributed to the development of a vast array of diseases such
as age-related, inflammatory, neurodegenerative, and noncommunicable
diseases, antioxidants play a critical role in alleviating these diseases. For
example, numerous investigations have revealed that antioxidants play
a crucial role in neurodegenerative diseases such as Alzheimer’s disease,
and Parkinson’s disease. As depicted in Figure 1.1, the neuroprotective
effect against 6-hydroxydopamine (6-OHDA) neurotoxicity for striatal
dopaminergic neurons in the rat model was demonstrated using GI extract, thus
implicating its potential benefit for Parkinson’s disease (Antala et al., 2012).
Furthermore, GI was found tofight against both brain ischemic reperfusion
in the rat model (Ahmed, 2020) and depression via monoaminergic pathway
(Dhamija et al., 2017).
Garcinia indica 3
FIGURE 1.1 The protective effects of GI extract on Parkinson’s disease, Alzheimer’s disease,
and depression.
1.3 CHEMICAL COMPOSITION OF GARCINIA INDICA
Various medicinal and cosmetics propertiesof GI fruitrinds and leaves were

uncovered to link with the chemical constituents of GI (Baliga et al., 2011).
GI was found to possess protein, tannin, pectin, sugars, fat, organic acids
including (−)-hydroxycitric acid, hydroxycitric acid lactone, and citric acid;
the anthocyanins, cyanidin-3-glucoside, and cyanidin-3-sambubioside; and
the polyisoprenylated phenolics garcinol and isogarcinol (Krishnamurthy et
al., 1981; Baliga et al., 2011; Kaur et al., 2012; Biersack, 2016; Jayakar et al.,
2020). Secondary metabolites, garcinol and isogarcinol, are found in both rinds
and leaves of GI. The chemical structures of both natural polyisoprenylated
benzophenone derivatives garcinol and isogarcinol are shown in Figure 1.2.
In line with the biological activity spectra of GI extract, the activity spectra of
garcinol and isogarcinol include anticancer, antibiotic, antioxidant, and anti-
inflammatory effects (Hemshekhar et al., 2011; Schobert and Biersack, 2019).
1.4 ANTI-CANCER ACTIVITIES OF GI OR ITS CHEMICAL

COMPOUNDS
Herein, anti-cancer effects exhibited by GI and its chemical constituents are

presented and discussed according to numerous previous reports. GI leaf
extract was demonstrated to exert a cytotoxic effect to A498 human renal

carcinoma cells, but the finding was based on the MTT assay only (Jayakar
et al., 2021). As far as the molecular mechanisms of cancer are concerned,
the association between endoplasmic reticulum stress signaling and cancer
has been firmly established (Oakes, 2020). Interestingly, there was a study
demonstrating thatGI extract standardized for 20% garcinol could reduce
endoplasmic reticulum stress in both cultured cells and adipose tissue, thus
suggesting the medical effects of GI for not only diabetes and obesity, but
cancer as well (Majeed et al., 2020). Furthermore, GI extract was shown to
possess an inhibitory effect on cultured 3T3 mouse fibroblasts (Varalakshmi
et al., 2011).
FIGURE 1.2 Structures of the secondary plant metabolites garcinol and isogarcinol.
In fact, the activities of cancer suppression were mainly found due to

the main active substance, garcinol, but not due to the GI extract. Aggarwal
et al. (2020) discussed a variety of anticancer mechanisms of garcinol, but
a few interesting examples are addressed in this book chapter. Table 1.1
summarizes the anticancer mechanisms of garcinol as reported in numerous
publications.
Antiproliferative and anti-invasive effects of garcinol on gallbladder
carcinoma cells and the inhibitory mechanism of garcinol were associated
with the suppression of Stat3 and Akt signaling pathways, which might
TABLE 1.1 Anti-Cancer Mechanisms of Garcinol in Various Cancer Types.
Garcinia indica
Types of cancer cells Proposed signaling pathway involved (mechanisms of action) References
ISH and HEC-1B cells Garcinol exhibited cell cycle arrest and activated JNK/c-JUN signaling Zhang et al. (2021)
(endometrial cancer cells) pathway to cause apoptosis
U-87 MG and GBM8401 cells Garcinol showed antiproliferative effect by inhibiting the activation of STAT-3, Liu et al. (2019)
(glioblastoma cells) 5 by inducing the expression of Hsa-miR181d
HGC-27 cells (gastric cancer cells) Garcinol inhibited the invasion of cancer cells through the downregulation of Zheng et al. (2020)
PI3K/AKT signaling pathway
OVCAR-3 cells (ovarian cancer cells) Garcinol alone or in combination with cisplatin showed apoptotic effect by Zhang et al. (2020)
inhibiting the activation of PI3K/AKT, NF-κB pathway
KYSE150 and KYSE450 Garcinol inhibited metastasis by downregulating p300 and p-Smad2/3 Wang et al. (2020)
(Esophageal cancer cells)
CAL-27, SCC-15 cells Garcinol inhibits ATP production, mitochondrial respiration, and basal Zhang et al. (2019)
(oral squamous cell carcinoma cells) respiration and subsequently affected the energy-producing pathway in cancer
cells. It also reflexively boosted glycolysis apart from the upregulation of
glucose transporter 1 and 4, and HIF-1α, AKT, and PTEN
A549 and H1299 cells Garcinol affects epithelial to mesenchymal transition by modulating miR-200b Farhan et al.
(nonsmall cell lung carcinoma cells) to sensitize the carcinoma cells to chemotherapy (2019)
Hela and SiHa cells Garcinol showed antiproliferative activity by attenuating PI#/AKT pathway Zhao et al. (2018)
(human cervical cancer cells) and inhibited cell migration and invasion by suppressing MMP-2,9 and
inducing T-cadherin
Caki cells (renal carcinoma cells) Garcinol upregulated the expression of DR5 and downregulated the expression Kim et al. (2018)
of c-FLIP, thereby inducing apoptosis of the TRAIL-negative cells
A549 and H441 cells (nonsmall cell lung Garcinol diminished the ability of the NSCLC cells to form spheres and form Huang et al.
carcinoma cells) colonies, by impairing phosphorylation of LRP6, dysregulating the Wnt/β- (2018)
catenin pathway, and reducing the Dvl2, Axin2, and cyclin D1 expressions
5
TABLE 1.1 (Continued)
6
A549 cells Garcinol enriched DNA damage-inducible transcript 3 (DDIT3), altered Wang et al. (2017)
(nonsmall cell lung carcinoma cells) DDIT3-CCAAT-enhancer-binding proteins β (C/EBPβ) interaction resulting in
the attenuation of the prognostic cancer cell marker aldehyde dehydrogenase 1
family member A1 (ALDH1A1) expression
4T1 cells (breast cancer cells) and female Garcinol sensitized the cells toward Taxol treatment and improved the efficacy Tu et al. (2017)
Balb/c mice of the drug in mice through the suppression of caspase-3/iPLA2 and NF-κB/
Twist1 signaling pathways.
HT-29 cells (colorectal cancer cells) Garcinol exhibited an antiproliferative effect and antiangiogenic effect by Ranjbarnejad et al.
suppressing of HIF-1α and VEGF expression (2017)
SCC-4 cells Garcinol exerted antiproliferative, pro-apoptotic, cell-cycle regulatory, and Aggarwal and Das
(oral squamous cell carcinoma cells) anti-angiogenic effects by inhibiting the expression of STAT-3, c-Src, JAK1, (2016)
JAK2, NF-κB, COX-2 and VEGF
PC-3 cells (prostate cancer cells) Garcinol inhibited autophagy and exhibited apoptosis through the activation of Wang et al. (2015)
p-mTOR and p-PI3K/AKT
Potent Anticancer Medicinal Plants

PANC-1 SP cells (Pancreatic cancer cells) Garcinol suppressed the stem-like properties and metastasis by attenuating the Huang et al.
Notch1 signaling pathway through the upregulation of miR-200c (2017)
UMSCC1 cells (head and neck squamous Garcinol enhanced the apoptotic effect of cisplatin by attenuating surviving, Li et al. (2015)
cell carcinoma cells) and HNSCC Bcl-2 and suppressed tumor growth through the downregulation of Ki-67 and
Xenograft mouse model CD-31
MCF-7 cells (breast cancer cells) Garcinol exhibited anti-proliferative activity against 17β-estradiol and Ye et al. (2014)
promoted cell cycle arrest and apoptosis by suppressing ac-p65/NFκB pathway
HepG2, HUH-7 cells (hepatocarcinoma Garcinol exhibited apoptotic and anti-tumor growth effects by inhibiting Sethi et al. (2014)
cells) and athymic nu/nu female mice dimerization, phosphorylation, and acetylation of STAT3
p53 wild type H460 and p53-null H1299 Garcinol induced cell cycle arrest by inducing CDK inhibitors p21Waf1/Cip1 and Yu et al. (2014)
cells (nonsmall cell lung carcinoma cells) exhibited apoptosis through p38-MAPK pathway
Garcinia indica
Panc-1 (Pancreatic cancer cells) Garcinol sensitized the cells toward gemcitabine treatment and induced apoptosis Parasramka et al.
by altering the miRNA profile involved in the oncogenic signaling pathway (2013)
MDA-MB-231, BT-549 cells (Breast Garcinol inhibited epithelial to mesenchymal transition through the upregulation Ahmad et al.
cancer cells), and xenograft mice model of miR200b, miR-200c and regulating NFκB, Wnt signaling pathways (2012)
Hep3B cells (hepatocellular carcinoma Garcinol elevated ROS levels and caused caspase-3-mediated apoptosis Cheng et al. (2010)
cells)
HT-29 cells (colorectal carcinoma cells) Garcinol inhibited cell invasion, decreased tyrosine phosphorylation, and Liao et al. (2005)
prevented activation of the Src, MAPK/ERK, and PI3K/Akt signaling
pathways with an increase in caspase-3 activity to promote apoptosis
HeLa cells (cervical cancer cells) Garcinol inhibited HAT activity, thereby repressing chromatin transcription Balasubramanyam
and inducing apoptosis by altering global gene expression involved in et al. (2004)
oncogenic signaling
7
contribute to the downregulation of the molecular targets such as matrix

metalloproteinase 2 (MMP2) and MMP9 (Duan et al., 2018).
Garcinol was found to suppress cancer stem cell-like phenotype via inhibi
tion of the Wnt/β-catenin/STAT3 axis signaling pathway in human nonsmall
cell lung carcinomas, thus suggesting the roles of garcinol in blocking cancer
recurrence and metastasis (Huang et al., 2018).
In addition to the modulation of an increased hsa-miR-181d/STAT3
and hsa-miR-181d/5A ratio by garcinol, Liu et al. (2019) reported that
garcinol treatment significantly suppressed the proliferative, invasive, and
migratory potential of U87MG or GBM8401 glioblastoma cell lines in a
dose-dependent fashion and the anticancer effect by garcinol significantly
attenuated the glioblastoma stem cell-like phenotypes, as reflected by the
reduced capability of both cell lines to form colonies and tumorspheres, and
suppressed expression of OCT4 and SOX2.
Using the human colorectal cancer cell line, HT-29, as a cell model,
garcinol was revealed to modulate tyrosine phosphorylation of focal adhesion
kinase (FAK) and subsequently induce apoptosis through downregulation
of Src, ERK, and Akt survival signaling (Liao et al., 2005). Also, they
demonstrated that garcinol significantly inhibited the expression of MMP-7
in interleukin 1β (IL-1β)-induced HT-29 cells, thus suggesting that garcinol
reduced cell invasion and survival through the inhibition of FAK’s downstream
signaling. Garcinol was also reported to exhibit anti-proliferative activities by
targeting microsomal prostaglandin E synthase-1 in human colon cancer cells
(Ranjbarnejad et al., 2017).
Regarding the effects of garcinol on breast cancer cells, antiproliferative
and apoptosis-inducing actions were reported and the apoptosis-inducing
effect of garcinol was mediated by the NF-κB signaling pathway (Ahmad et al.,
2010). Consistently, the same might be true for the antiapoptotic mechanism
of garcinol in other cancer types. In particular, garcinol was proven to inhibit
tumor cell proliferation, angiogenesis, and cell cycle progression and induce
apoptosis via NF-κB inhibition in oral cancer (Aggarwal and Das, 2016).
As an acetyltransferase inhibitor, garcinol was demonstrated to suppress the
proliferation of breast cancer cell line MCF-7 stimulated by 17β-estradiol,
thus indicating the epigenetics mechanism of the action exerted by garcinol
(Ye et al., 2014). Although the data pertaining to precise toxicity, dosages,
routes of administration, and safety for clinical applications are still limited,
garcinol seems to serve as a promising anti-cancer epigenetic drug (Kopytko
et al., 2021).
Garcinol was shown to dose-dependently suppress cell viability, colony
formation, invasion, migration, and cell cycle progression, and promoted cell
Garcinia indica 9
apoptosis in cervical cancer cell lines, as well as inhibited tumor growth in

a xenograft model, and its mechanism of action was found to be associated
with activating T-cadherin/P13 K/AKT signaling pathway (Zhao et al., 2018).
Garcinol was shown to inhibit the proliferation of endometrial cancer cells by
inducing cell cycle arrest, and following the garcinol treatment, the expression
levels of p53 and p21 were increased, while the expression levels of CDK2,
CDK4, cyclin D1, and cyclin B1 were gradually decreased in a dose-dependent
manner in endometrial cancer cell lines (Zhang et al., 2021).
In each cancer cell type, garcinol may participate to fight against cancer
in more than one signaling pathway. For example, a single strategy for
successful cancer treatment in certain cancer types may be very rare. Integra
tive therapeutic approaches may shed light on the success of cancer manage
ment. For example, pancreatic cancer represents one of the most aggressive
types of malignancy due to its high resistance toward most clinically avail
able treatments. Therefore, research in this field has been continued and
made natural product research significant and intriguing. It has been reported
that garcinol modulates specific MicroRNA (miRNA) biomarkers associated
with the sensitization of pancreatic cancer cells to gemcitabine treatment,
thus attenuating the drug-resistance phenotype (Parasramka et al., 2013).
More recently, garcinol was found to suppress the stem-like properties of
human pancreatic cancer cell line and inhibit the metastatic potential by
downregulating the expression of Mcl-1, EZH2, ABCG2, Gli-1, and Notch1
(Huang et al., 2017). Remarkably, garcinol treatment led to the upregulation
of several tumor suppressor microRNAs, and downregulated notch1 via
modulating mir-200c by garcinol was observed.
1.5 ISOGARCINOL AS AN IMMUNOSUPPRESSANT
Unlike garcinol, isogarcinol extracted from the Garcinia species has been
shown to exert many attractive activities, predominantly as an immunosup
pressive agent. Searching for immunomodulatory drugs from natural sources
is very critical in the pharmaceutical industry because immunosuppressants are
used for post-transplant organ rejection. It has been reported that isogarcinol
could inhibit calcineurin, a ubiquitous protein phosphatase that is involved in
several physiological roles such as apoptosis, T-cell activation, and cell cycle
control as well as in other pathological conditions including neurodegenerative
disease, thus serving as an immunosuppressant (Cen et al., 2013). Enzyme
kinetic analysis demonstrated that inhibition of calcineurin by isogarcinol was
competitive, and it bound directly to calcineurin in vitro (Cen et al., 2015).
Psoriasis is an incurable, long-term (chronic) inflammatory skin condition

featured by keratinocyte hyperproliferation of epidermis. HaCaT keratinocyte
cell line has been a proper cell model for antipsoriatic drug research. Certain
Thai medicinal extracts were shown to possess an antipsoriatic effect via
the modulation of NF-B signaling markers in HaCaT cells (Saelee et al.,
2011). Isogarcinol was reported to improve imiquimod-stimulated psoriasis-
like skin lesions in mice and suppress inflammatory factor expression in
lipopolysaccharide-induced HaCaT cells, thus considered an antipsoriatic
agent (Chen et al., 2017).
Using collagen-induced arthritis mice as a model, the oral administra
tion of isogarcinol was revealed to reduce not only serum inflammatory
cytokines, but the erosion of cartilage and bone as well, thus making it an
antiarthritic agent (Fu et al., 2014). Furthermore, the in vitro study of Fu et
al. demonstrated that isogarcinol downregulated iNOS and COX-2 mRNA
expression and NO content by suppressing the NF-B signaling pathway.
A previous study has shown isogarcinol as a promising therapeutic
agent due to its ability to reduce proteinuria, corrected the abnormal serum
biochemical parameters, lowered the levels of serum antibodies, alleviated
the abnormal activation of CD4 T cells, and decreased the inflammation in
a murine model of systemic lupus erythematosus (SLE)-like disease (Li et
al., 2015).
Using a murine model of multiple sclerosis (MS), isogarcinol improved
clinical scores, reduced inflammation, lowered demyelination of the spinal
cord, and decreased intracranial lesions, and isogarcinol appeared to inhibit
T helper cell differentiation (Wang et al., 2016). Therefore, isogarcinol was
established as a promising candidate for treating MS.
1.6 ANTI-CANCER ACTIVITIES OF GARCINIA INDICA AND GARCINOL
Herein, anti-cancer effects exhibited by GI and its chemical constituents are

presented and discussed according to numerous previous reports. GI leaf
extract was demonstrated to exert a cytotoxic effect on human renal A498
carcinoma cells, but the finding was based on the MTT assay only (Jayakar
et al., 2021). As far as the molecular mechanisms of cancer are concerned,
the association between endoplasmic reticulum stress signaling and cancer
has been firmly established (Oakes, 2020). Interestingly, there was a study
demonstrating that GI extract standardized for 20% garcinol could reduce
endoplasmic reticulum stress in both cultured cells and adipose tissue, thus
suggesting the medical effects of GI for not only diabetes and obesity, but
Garcinia indica 11
cancer as well (Majeed et al., 2020). Furthermore, GI extract was shown to

possess an inhibitory effect on cultured 3T3 mouse fibroblasts (Varalakshmi
et al., 2011).
In fact, the activities of cancer suppression were mainly found due to
the main active substance, garcinol, but not the GI extract. Aggarwal et al.
(2020) discussed a variety of anti-cancer mechanisms of garcinol, but a few
interesting examples are addressed in this book chapter. Table 1.1 summarizes
the anti-cancer mechanisms of garcinol as reported in numerous publications.
Garcinol was shown to suppress the Stat3 and Akt signaling pathways
as well as downregulated the expression of matrix metalloproteinases 2
and 9, thereby exerting antiproliferative and anti-invasive activities against
gallbladder carcinoma cells (Duan et al., 2018).
Garcinol was found to suppress cancer stem cell-like phenotype via inhi
bition of the Wnt/-catenin/STAT3 axis signaling pathway in human nonsmall
cell lung carcinomas, thus suggesting the roles of garcinol in blocking cancer
recurrence and metastasis (Huang et al., 2018).
In addition to the modulation of an increased hsa-miR-181d/STAT3
and hsa-miR-181d/5A ratio by garcinol, Liu et al. (2019) demonstrated the
anti-proliferative, anti-invasive, and anticancer effects of garcinol against
glioblastoma cell lines (U87MG or GBM8401) in a dose-dependent fashion
garcinol was revealed to modulate tyrosine phosphorylation of FAK and
subsequently induced apoptosis of human colorectal HT-29 cancer cell line
through the downregulation of Src, ERK, and Akt signaling (Liao et al.,
2005). Also, they demonstrated that garcinol markedly mitigated MMP-7
expression in HT-29 cells, suggesting that garcinol reduced cell invasion and
survival through the prevention of FAK’s downstream signaling. Garcinol
was also reported to exhibit anti-proliferative activities via the modulation
of microsomal prostaglandin E synthase-1 in human colon cancer cells
(Ranjbarnejad et al., 2017).
Regarding the effects of garcinol on breast cancer cells, antiproliferative
and apoptosis-inducing action were reported and the apoptosis-inducing
effect of garcinol was mediated by NF-B signaling pathway (Ahmad et al.,
2010). Consistently, the same might be true for the anti-apoptotic mecha
nism of garcinol in other cancer types. In particular, garcinol was proven
to inhibit tumor cell proliferation, angiogenesis, and cell cycle progression,
thereby inducing apoptosis via NF-B inhibition in oral cancer (Aggarwal
and Das, 2016). As an acetyltransferase inhibitor, garcinol was demonstrated
to suppress the proliferation of breast cancer MCF-7 cell line stimulated by
17-estradiol, thus indicating the epigenetics mechanism of the action exerted
by garcinol (Ye et al., 2014). Although the data pertaining to precise toxicity,
dosages, routes of administration, and safety for clinical applications are still
limited, garcinol seems to serve as a promising anti-cancer epigenetic drug
(Kopytko et al., 2021).
Garcinol was shown to promote cell apoptosis in cervical cancer cell lines,
as well as prevented tumor growth in a xenograft model, and its mechanism
of action was found to be associated with activating T-cadherin/P13K/AKT
signaling pathway (Zhao et al., 2018). Garcinol treatment was shown to
increase the expression levels of p53 and p21, while decreased the expression
levels of CDK2, CDK4, cyclin D1 and cyclin B1, resulting in the inhibition
of endometrial cancer cells in a dose-dependent manner (Zhang et al., 2021).
In each cancer cell type, garcinol may participate to fight against
cancer in more than one signaling pathway. For example, a single strategy
for successful cancer treatment in certain cancer types may be very rare.
Integrative therapeutic approaches may shed light on the success of cancer
management. For example, pancreatic cancer is one of the most aggressive
types of malignancy because it shows increased resistance to most of the
cancer treatments available today. Therefore, research in this field has been
continued and made natural product research significant and intriguing. It
has been demonstrated that garcinol modulates specific miRNA biomarkers
associated with the sensitization of pancreatic cancer cells to gemcitabine
treatment, thus attenuating the drug-resistance phenotype (Parasramka et al.,
2013). More recently, garcinol was found to suppress the stem-like properties
of human pancreatic cancer cell line and inhibit the metastatic potential by
the downregulation of Mcl-1, EZH2, ABCG2, Gli-1, and Notch1 (Huang et
al., 2017). Remarkably, garcinol treatment led to the upregulation of several
tumor suppressor microRNAs, and downregulated notch1 via modulating
mir-200c by garcinol was observed.
To search for the mechanisms of natural products for anticancer
chemotherapy, scientists have attempted to utilize many different strategies
which are known to participate in any cellular process such as autophagy.
Autophagy is a degradative process during stressful conditions intracellu
larly. Interestingly, autophagy has been linked to both tumor suppression
and promotion (Yun and Lee, 2018; Chavez-Dominguez et al., 2020).
Garcinol was demonstrated to inhibit autophagy in human prostate cancer
cells through the activation of p-mTOR and p-PI3 Kinase/AKT (Wang et
al., 2015). However, it was proven to induce autophagy in osteosarcoma
cells by inhibiting acetyltransferase and increasing the expression of LC3-II
(Pietrocola et al., 2015). Hence, future investigations should be focused to
unravel the modulatory roles of garcinol in autophagy and cancer for anti
cancer drug discovery.
Garcinia indica 13
1.7 CONCLUSION AND FUTURE PERSPECTIVE
Although it grows extensively in India, GI has been recognized worldwide

for its various benefits to humans such as culinary, medicinal, and indus
trial applications. GI has been shown to possess many pharmacological
effects including antioxidant, anti-inflammatory, anti-neurodegenerative,
and antitumor effects. Nevertheless, its secondary metabolite garcinol has
been mostly researched so far in terms of anti-cancer activities. As shown
in Figure 1.3, the anticancer mechanisms of garcinol are presented as
promising anti-cancer drug candidates. Interestingly, the emerging roles of
garcinol are extensively documented. However, elucidation of anti-cancer
mechanisms exerted by garcinol is still needed for each type of cancer using
different study designs and experimental models as well as various cutting-
edge technologies. Recognized as a multifactorial disease, the mechanisms
of cancer are not straightforward since the contribution of genetic factors
and environmental factors toward cancer risk is distinct for each cancer
type. More studies on garcinol can be achieved to fill gaps of knowledge,
thus hoping to reach the phase of the clinical trials soon. It should be noted
that researchers find difficulty in gaining a large quantity of garcinol from
nature. So, improved extraction and purification are the key success factors
in garcinol research since the insufficient quantity of garcinol hinders our
progress nowadays.
FIGURE 1.3 Anticancer effects of garcinol.

ACKNOWLEDGMENTS
The author wishes to thank the Rachadapisek Sompote Fund, Chulalongkorn

University for supporting the work of Natural Products for Neuroprotection
and Anti-Ageing Research Unit. Also, this chapter would have not be feasible
without the images created by Dr. James M. Brimson and Ms. Chamaiphorn
Wongwan. Finally, the author would like to thank Dr. Phaniendra Alugoju
for his proofreading and English editing.
KEYWORDS
• Garcinia indica
• plant extract
• garcinol
• anti-cancer
• molecular mechanisms
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CHAPTER 2
Centella asiatica Linn

SAGAR BARGE1, DHANANJAY JADE2, and
NARAYAN CHANDRA TALUKDAR1,3
1
Chemical Biology Lab I, Institute of Advanced Study in Science and
Technology, Paschim Boragaon, Guwahati, Assam, India
2
Biomedical Sciences, University of Leeds, Leeds, UK
3
Assam Down Town University, Panikhaiti, Guwahati, Assam, India
ABSTRACT
Natural products are gaining a lot of attention in medicine due to their

medicinal value. Natural products are mainly secondary metabolites and used
for drug discovery for many years. Centella asiatica (CA) is a folk medicinal
plant used for different phytotherapeutic treatments across southeast Asia.
Centella asiatica L. is a herb-like plant traditionally assigned to the Apiaceae
family. In India, species are locally known for their different names, such as
Brahmi, mandukaparni, Indian pennywort or Spadeleaf, and Gotu kola in
Southeast Asian countries, including China. Many tribal groups have been
using traditional leaves, stem, and flower parts of the CA plant for hundreds
of years. The records of the use of CA plants in both Indian Ayurveda
and Chinese traditional medicine are available in the treatment of various
medical emergencies. CA was further subjected to mechanistic and clinical
investigation in modern medical research based on traditional knowledge.
Several compounds have been isolated from CA, including polyphenolic,
triterpenoids, saponins, and essential oils. Pharmacological reports reveal the
efficacy of the CA plant against the antimicrobial, antiviral, antidepressive
antidiabetic, wound-healing, memory-enhancing, and antioxidant activities.
Compound, namely, asiatic acid (AA), asiaticoside has anticancer potential

against Breast, lung, and ovarian cancer. These anticancer agents mainly
affect the receptors, enzymes, transcription factors, cell signaling cascade,
and apoptotic proteins. In the biosynthesis of compounds, plant hormones
play an essential role in plant metabolism via the upregulation of genes
related to secondary metabolites, and scientific efforts for manipulating these
metabolic processes in order to increase the biosynthesis of these anticancer
agents, have received much attention.
2.1 INTRODUCTION
The genus Centella consists of 53 species of flowering plants, including CA

one of the popular medicinal herbs. The genus Centella initially belongs
to the family of Araliaceae and Mackinlayoideae as a subfamily but the
molecular studies assigned the genus to the Apiaceae family. CA is a green
leafy vegetable medicinal plant known as Indian pennywort, with many
nutritional values. Due to its high medicinal value, it is cultivated in many
countries (Matsuda et al., 2001) for hundreds of years, including India,
China, Sri Lanka, and Malaysia, for traditional purposes (Brinkhaus et al.,
2000b). CA is reported as “Sushruta Samhita” in ayurvedic medicine, as
ancient Indian text commonly called mandukparni (Diwan, 1991), which is
commonly used for various diseases such as mental disorders, skin problems,
liver ailments, epilepsy, asthma, hair loss, and tetanus (Meulenbeld and
Wujastyk, 2001). In China and Indonesia, CA, also known as Gotu kola, and
used as “miracle elixirs of life” (Diwan, 1991). The plant species’ leaves are
edible, grow in orbicular or oblong–elliptic shapes, and are yellowish–green
(Chopra et al., 1956). The plant species are consumed as leaf juice, drink,
and other food products. and it is most famous for its use as a “brain tonic.”
Gotu kola is also beneficial for the growth of connective tissues and wound
healing (Somboonwong et al., 2012).
CA mainly consists of many pentacyclic triterpenoids saponins, also called
centelloids. These include madecassic acid, asiatic acid (AA), brahmoside,
brahminoside, madecassoside, sceffoleoside, and asiaticoside (Singh and
Rastogi, 1968; Sun et al., 2020; Yu et al., 2006). Pharmacological evidence
suggests that these compounds are used against many diseases (Sun et al.,
2020), such as antidiabetic (Legiawati et al., 2020; Oyenihi et al., 2019),
anticancer (Tariq et al., 2015), anti-inflammatory and antioxidant (Ariffin et
al., 2011; Hafiz et al., 2020; Kumari et al., 2016; Ratz-Łyko et al., 2016),
Centella asiatica Linn 21
neuroprotective (Gray et al., 2018; Lokanathan et al., 2016; Yadav et al.,

2019), etc. Cancer has caused many deaths globally, and it is estimated that
by the end of the year 2025, there will be approximately 20 million new
cancer cases globally (Sevgi and Nazım, 2019). However, natural product has
played a vital role in cancer treatment and provided many natural drugs (Seca
and Pinto, 2018). Therefore, many researchers mainly studied the anticancer
effect of CA and its bioactive compound.
In many countries, including India, China, and South Africa, traditional
healers’ consultation and use of medicinal plants in disease is common health
care practice. There are 80% of the population uses medicinal plant which
is homegrown in their diet. Moreover, the treatment of various diseases with
medicinal plants increases its market demand which caused harvesting and
overexploitation of wild medicinal plants (Barbhuiya et al., 2009; Vasisht et
al., 2016). To reduce the overexploitation of the CA plant, biotechnological
manipulation of such plants using plant cell culture and organ culture is the
best alternative source of natural products (Tan et al., 2010).
The manipulation of these metabolic pathways can be studied using plant
metabolomics analytical techniques. These analytical techniques allow us
to study and identify the changes in metabolic pathways upon treatment.
This chapter has discussed the biosynthesis of secondary metabolites and
metabolic profiling of the compounds present in the CA. We also highlight
the omics approach, namely, metabolomics, by which almost the entire range
of metabolites can be analyzed for bioactivity in an organism at a specific
time and under particular conditions. Also, we will discuss the mechanism of
action of anticancer agents present in the CA.
2.2 METABOLITE PROFILING OF CENTELLA ASIATICA

PHYTOCONSTITUENTS
Metabolic profiling involves detecting the role of metabolites in a biochemical

pathway, and metabolomics is the qualitative and quantitative analysis of
metabolites in a plant crude extract (Bhalla et al., 2005). Metabolite profiling
or metabolomics was used in various fields with various technical methods
to identify and characterize secondary metabolites (Cerny et al., 2020;
Sumner et al., 2007). The plant extracts consist of many complex secondary
metabolites with different polarities in various concentrations; therefore,
various techniques are employed to characterize and detect these compounds.
This technique includes liquid chromatography–mass spectrometry (LC/
MS), ultrahigh-performance liquid chromatography (UHPLC), thin-layer

chromatography (TLC), and nuclear magnetic resonance (NMR).
2.2.1 TLC AND HPLC-BASED METABOLITE PROFILING
The secondary metabolites from the CA plant have been identified and
quantified by simple analytical techniques such as TLC and high-performance
liquid chromatography (HPLC). In early 1991, high-speed counter-current
chromatographic (HSCCC) techniques were introduced to separate
madecassoside and asiaticoside from the CA plant (Diallo et al., 1991). Further,
in 2004, the same technique separates pentacyclic triterpene aglycones and
glycosides (Du et al., 2004). Gunther and Wagner, in 1996, quantified and
characterized the AA, asiaticoside, madecassoside, and madecassic acid, the
triterpene from CA plant using an acetonitrile/water as a mobile phase on
RP18 column in a reverse phase separation system (Günther and Wagner,
1996). Later on, the separation and identification of triterpenes achieved by
TLC were also reported (Brinkhaus et al., 2000b). Further, Bonfill et al. in
2006 identified four triterpenes using TLC and mass spectrometry matrix-
assisted laser desorption/ionization-time of flight (MALDI-TOF) (Bonfill et
al., 2006). Although during early studies of metabolite profiling, the HPLC
was equipped with ultraviolet (UV) detection and used to characterize and
quantify compounds but failed to detect some compounds due to a lack
of solid UV-absorbing triterpenes. Therefore, some UHPLC systems are
coupled with evaporative light scattering detection (ELSD) to better detect
centellosides. Recently, metabolic profiling of ethanolic and aqueous extract
of CA by HPLC analysis revealed that the presence of a flavonoid group of
compounds such as rutin, gallic acid, quercetin, kaempferol, catechin, and
luteolin (Mohammad Azmin and Mat Nor, 2020).
2.2.2 LC/MS-BASED METABOLITE PROFILING AND METABOLOMICS

STUDY
The metabolomics approach allows for an analysis of the different and

multiple metabolites present in the biological system. The LC-MS is the
technique that tends to identify and analyze the multiple metabolites with
different polarities. Metabolic profiling through LC-MS and gas chromatog
raphy–mass spectrometry (GC-MS) also addresses large group metabolites
about the targeted metabolic pathway and its related compound (Eugster et
al., 2011). High-resolution mass spectrometry (HRMS) is often used coupled

with the LC to separate the compounds and the compounds and perform
the analysis of compounds present in the crude extract. Metabolic profiling
of CA plant extract has been studied extensively recently; screening of CA
plant extract and fractions reveals the 22–33 flavonoid group of compounds
through LS-MS and GCMS analysis (Ondeko et al., 2020). Similarly, 117
compounds are identified, and 24 are quantified through phytochemical
analysis using a quadrupole time of flight analyzer coupled with an opti
mized HPLC separation (Alcazar Magana et al., 2020).
Furthermore, to analyze the changes during the biosynthesis of compounds
by manipulating the pathways, the LCMS and GCMS techniques were
employed. For example, changes in the metabolite profile were analyzed upon
methyl jasmonate treatment to produce triterpenoids in the CA plant (James
et al., 2013; Tugizimana et al., 2015). The CA plants’ undifferentiated and
differentiated leaf cells were also analyzed for their presence of 18 metabolites
in methanolic extract using ultrahigh-performance liquid chromatography–
quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) (Ncube et
al., 2017).
2.2.3 NMR-BASED METABOLITE PROFILING AND METABOLOMICS

STUDY
Secondary metabolites with structures are elucidated by NMR. NMR analysis

for metabolite profiling is a simple and reproducible technique that provides
direct detailed structural information, but this technique has the limitation that
it has relatively low sensitivity, meaning that it generally enables profiling
only of major constituents.
Different types of NMR are specifically used to identify the 2 dimensional
or 1-dimensional structure of the compounds. For example, the two flavonoid
molecules were isolated from the whole plant of CA and were identified as
castilliferol 1 and castillicetin 2 using spectral analysis by 1D and 2D NMR
(Subban et al., 2008). Similarly, metabolites from three different species
of Centella, including CA, were analyzed using a 1H NMR spectroscopy
in various lighting conditions, which reveals the accumulation of certain
secondary metabolites in some species making them more medicinal than
other species (H et al., 2012). Further, in 2011, the researcher first time isolated
the Arabic acid from the cell-cultured CA plant by MS/NMR spectroscopic
analysis to confirm its structure (Antognoni et al., 2011).
2.3 SECONDARY METABOLITES OF CENTELLA ASIATICA AND ITS

BIOSYNTHESIS
Centella asiatica consists of many significant phytoconstituents mainly found

in the aerial and root parts of the plants (Brinkhaus et al., 2000a; James and
Dubery, 2009). These secondary metabolites are involved in the defensive
interaction between plants and the environment, including substances like
attractants phytoalexins, antifeedants, and pheromones (“The biosynthesis
of secondary metabolites,” 2003; Yang et al., 2018). Therefore, studies on
biosynthesis and the biological activity of these compounds are increased
due to their importance in medicine and industry (Schäfer and Wink, 2009).
Many medicinal plants consist of similar compounds, but their biochemical
studies reveal different biosynthesis pathways based on their environmental
conditions (Li et al., 2020).
2.3.1 BIOSYNTHESIS OF CENTELLA ASIATICA TRITERPENES AND

TERPENOIDS
CA contains a high amount of triterpenoids; these are pentacyclic in

nature and have also been called centelloids. The biosynthesis and level of
saponins triterpenes change with the geographical origin and environmental
conditions (“The biosynthesis of secondary metabolites,” 2003). For the
synthesis of saponins and terpins, they follow the isoprenoid pathway. This
pathway mainly consists of three precursors: isoprenoid, isomeric 5-carbon
precursors, isopentenyl diphosphate, and dimethylallyl diphosphate. In the
first step, the prenyltransferase synthase enzyme converts these three C5
precursors into farnesyl diphosphate (C15) to create an assembly. In the next
step, squalene synthase catalyzed the two molecules of farnesyl diphosphate
to give squalene (C30).
At last, presence of O2 and nicotinamide adenine dinucleotide phosphate
(NADPH), squalene (C30) gets converted into the squalene 2,3-epoxide
(2,3-oxidosqualene), which is the precursor for the synthesis of both sterol
and triterpenoids. 2,3-oxidosqualene further cyclizes through cationic
intermediates and converts into triterpenoids of CA via oxidosequalene
cyclases enzyme, including α and β amyrin synthase. α-amyrin synthase
is mainly involved in the synthesis of AA and the action of cycloartenol
synthase engaged in the synthesis of sterol via protosteryl cation (Azerad,
2016; Gallego et al., 2014; Kim et al., 2005). Sterol and terpene biosynthesis
general pathways are discussed by many researchers elsewhere (Collin,

2001; Gershenzon and Kreis, 1999; Kalinowska et al., 2005).
FIGURE 2.1 Pathways involved in biosynthesis of CA centelloids.
2.3.2 BIOSYNTHESIS OF CENTELLA ASIATICA FLAVONOIDS AND

PHENOLS
CA contains many flavonoids, including catechin, epicatechin, kaempferol,

quercetin (Idris and Mohd Nadzir, 2021). The biosynthesis of flavonoids
occurs in CA plants via the shikimate pathway and acetate pathway: Both
pathways use p-cinnamoyl-CoA and malonyl-CoA units as initial precursor
molecules for biosynthesis. Further Claisen-type reaction forms a naringenin
chalcone with the help of chalcone synthase, resulting in the formation of A
ring of the naringenin and flavanone. Naringenin, via enzymatic reactions,
gives flavons-like compounds called apigenin and flavonols (Dewick, 2002;
Winkel-Shirley, 2001). From methanolic extract of the CA plant, Subban et
al. have isolated a castilliferol, also known as kaempferol-3-p-coumarate,
and castillicetin, also called quercetin-3-caffeate, esterified flavonoid and
derivative of hydroxycinnamic (Subban et al., 2008).
Chlorogenic acids are another group of polyphenol-rich in CA plant

formed by cinnamic acid derivative via esterification of quinic acid (Ncube
et al., 2016). During the biosynthesis of chlorogenic acids, catalysis of
L-phenylalanine occurs to eliminate the E2 ammonia by the enzyme called
phenylalanine ammonia-lyase to produce trans-cinnamic acid further
produces p-coumaric acid with the help of 4-cinnamic acid hydroxylase
enzyme. Further hydroxycinnamic acid derivatives caffeic acid are
formed via hydroxylation and methylation reactions. Additional hydroxyl
groups on L-quinic acid get esterified, and caffeic acid carboxyl function
yields caffeoylquinic acids. Finally, the ester of caffeoyl acid-containing
3-hydroxyl position of L-quinic acid produces chlorogenic acid (Xie et al.,
2011).
2.4 MANIPULATION OF METABOLIC PATHWAYS FOR THE

PRODUCTION OF PENTACYCLIC TRITERPENOIDS
Pentacyclic triterpenoids are pharmacologically active compounds with

a complex structure, making the chemical synthesis of such compounds
expensive and time-consuming. Therefore, increasing the production of
such compounds in the plant via the manipulation of biosynthetic path
ways is essential (Baltz, 2016; Birchfield and McIntosh, 2020). Secondary
metabolite synthesis occurs during the specific time of plant develop
ment, and it is tissue-specific and also regulated by plant growth (Aziz
et al., 2007; Khare et al., 2020). The metabolic interaction is essential
during the Secondary metabolite production between the root and other
plant parts (Pott et al., 2019). Plant cell culture is the approach used to
manipulate biosynthetic pathways in CA by adding precursors in the cell
culture medium (Bouhouche et al., 1998), but this approach has some
limitations after prolonged culture, the cell’s ability to produce compounds
gets declined. Another approach is to increase the enzyme levels by using
plant-specific molecules such as methyl jasmonate. A similar approach was
applied to increase the asiaticoside levels in CA where the plant is treated
with methyl jasmonate, and the sterols synthesizing enzymes are inhibited
from increasing the synthesis of asiaticoside (Mangas et al., 2006). Another
study reported that following treatment of methyl jasmonate in roots of CA
inhibited the expression of cyclases responsible for synthesizing sterols
and activated the expression of β-amyrin synthase responsible for AA
production (Kim et al., 2005).
2.4.1 MANIPULATION OF BIOSYNTHESIS PATHWAY USING

ELICITORS IN VITRO PLANT CULTURE
Elicitors are the external factors that stimulate physiological reactions and
induce the stress response to protect the plants against disease, resulting in
more production of secondary biological metabolites (Baenas et al., 2014).
The plant tissue culture techniques use different types of elicitors such as
CuCl2, MeJA, yeast extract, and fungal to increase the production of asiati
coside in CA.
The elicitors such as MeJA and yeast extract showed positive effects (Kim
et al., 2004). In order to increase the yield of asiaticoside and madecassoside
in CA, Kim et al. have performed various studies using cell culture
techniques and treatment with methyl jasmonate. His studies demonstrated
that the transformation of hairy root culture and methyl jasmonate treatment
for 12h to 14 days increased gene expression of β-amyrin synthase, which
significantly enhanced the production of asiaticoside. Further, in his other
study, he raised the production of asiaticoside by using elicitors and yeast
extract. (Kim et al., 2007, 2004). Methyl jasmonate (MeJA) about 100 µm
along with cytokinin TDZ (0.025 mg/L) supplementation in plant cell culture
increased 82% of the production of asiaticoside in leaves (Yoo et al., 2011).
The fungal elicitors have also been shown to positively affect biomass
production and asiaticoside in CA plant shoot culture. The results revealed
the use of fungal elicitors such as mycelial extract of Colletotrichum linde
muthianum, Fusarium oxysporum, and Trichoderma harzianum culture
filtrate 3% v/v enhanced both asiaticose and biomass production (Prasad et
al., 2013). Similarly, the fungus Piriformospora indica cocultivation with
roots of CA plant shows an increase in the production of asiaticoside (2-fold)
and biomass (Satheesan et al., 2012).
2.4.2 IN VITRO SECONDARY METABOLITE PRODUCTION IN CA
During CA tissue culture studies, manipulation of growth hormones such

as auxin and cytokinin can increase centellosides production in CA via the
influence of callus formation, somatic embryogenesis, and whole plantlet
regeneration (Nirmal et al., 2007; Prasad et al., 2012). For example, increase
in the concentration of 6-benzylaminopurine (BAP) and 1-naphthalene acetic
acid (NAA) results in an enhanced axillary bud formation from nodal explant,
which helped in the production of asiaticoside (Mukundan et al., 2004; Nath
Tiwari et al., 2000). Similarly, the callus and the cell suspension systems have
increased the production of asiaticoside in vitro culture systems (Nath and
Buragohain, 2004). Further different culture conditions are also necessary for
the secondary metabolite production manipulation of these conditions, such
as Ph and carbon source, resulting in metabolite production and increased
expression of genes responsible for centelloside pathways. Leaves of the CA
are an excellent source of asiaticoside; around 82.6% of the asiaticoside is
produced from leaves compared to the whole plant culture (Kim et al., 2004).
Similarly, the triterpenoids (asiaticoside and madecassoside) localization
was tissue-specific, and the highest amount of triterpenoids are present in
leaves as compared to the transformed roots and undifferentiated callus;
these observations are made comparing two Malaysian phenotypes of CA
for root transformation and callus culture (Aziz et al., 2007). The study by
James et al. in 2008 also shows that the triterpenoid content is higher in
the leaves of callus, and cell suspensions in South African CA plants than
the undifferentiated callus and cell suspensions (James et al., 2008). These
reports suggest that the manipulation of growth hormones can increase the
production of secondary metabolites.
2.5 ANTICANCER EFFECT OF CA INGREDIENTS
Cancer is commonly defined as the uncontrolled growth of abnormal and

genetically unstable cells in the body and infecting nearby cells. CA poses
many biological activities, including anticancer activity (Sun et al., 2020).
The report suggests that triterpenoids in the ethanolic extract of the CA plant
have an inhibitory action on the A549 cancer cells upon ionizing radiation IR
induction in the lungs (Han et al., 2020). AA is a major compound found in
the CA plant and is effective against various cancer. There are many reports
on AA that were published showing its effect against lung cancer. AA is also
effective against Nasopharyngeal carcinoma (NPC) by inducing apoptosis in
the NCP cells via p38 phosphorylation (Liu et al., 2020). Similarly, AA has
antiproliferative and apoptotic activity in KKU-156 and KKU-213 human
cancer cells (Sakonsinsiri et al., 2018).
Furthermore, the AA treatment inhibits the growth of lung cancer tumor
cells via mitochondrial-associated damage, which results in the formation
of reactive oxygen species (ROS) in lung cancer cell lines such as A549 and
H1299 cells also in C57BL/6J mice (Wu et al., 2017). AA also tends to in vitro
downregulate the p-glycoprotein by reducing the expression of the MDR1
gene in human lung adenocarcinoma A546/DDP cells; the p-glycoprotein is
responsible for multidrug resistance (MDR) during chemotherapy (Cheng et

al., 2018). Kim et al., 2014, reports that the AA increases the expression of
MicroRNA-1290 in A549 lung carcinoma cells resulting in apoptosis of cells
(Kim et al., 2014).
FIGURE 2.2 Asiaticoside (C48H78O19) CAS RN 16830-15-2.

Source: https://scifinder-n.cas.org/
FIGURE 2.3 Asiatic acid (C30H48O5) CAS RN 464-92-6.

Source: Ref. https://scifinder-n.cas.org/
FIGURE 2.4 Madecassoside (C48H78O20) CAS RN 34540-22-2.

Source: Ref. https://scifinder-n.cas.org/
FIGURE 2.5 Madecassic acid (C30H48O6) CAS RN 18449-41-7.

Source: Ref. https://scifinder-n.cas.org/.
AA was also effective against colon cancer cells such as SW480 and
HCT116 via the PI3K/Akt/mTOR/p70S6K signaling pathway by regulating
Pdcd4 in cells. These pathways regulate cellular migration, proliferation, and
apoptosis in cells; these cellular processes are found to be inhibited upon
AA treatment in colon cancer cells (Hao et al., 2018). Similarly, AA induces
apoptosis in the SW480 human colon cancer cells (Tang et al., 2009). AA
was also influential on SK-MEL-2 skin cancer cells by inducing apoptosis
and increasing ROS in cells (Park et al., 2005). AA exerts its effect on human
ovarian cancer by (PItf3K)/Akt/mTOR cascade in vitro in SKOV3 and
OVCAR-3 cells it causes 50% toxicity in the cells (Ren et al., 2016).
Another ingredient from the CA plant called asiaticoside has an anti
cancer effect on hepatocellular carcinoma (HCC); results have shown that
the asiaticoside treatment has an antiproliferative effect on HCC cell lines
and also induces apoptosis via G1 cell cycle growth arrest in the cells (Ma
et al., 2020). Madecassoside major triterpene glycosides from CA plants
have shown their in vitro bioactivity against HCC in HepG2 and SMMC-77
cancer cells. The evidence suggests that it works via the upregulation of the
cMET-PKC-ERK1/2-COX-2-PGE2 pathway and inhibits the cells’ hepato
cyte growth factor (HGF) induced proliferation. These reports suggest that
the major ingredients present in CA plants, such as centellocides are mainly
involved in anticancer activity.
2.6 CONCLUSION
CA has a wide range of medicinal values in the market; due to that, the
demand for the cultivation of CA plants increased in many countries. The
excessive harvesting of CA plants from the wild makes them endangered
species, and the conservation of such species is essential. Therefore, an
alternative for producing secondary metabolites such as centellocides was
achieved by genetic manipulation of metabolite pathways using in vitro plant
tissue culture. The biosynthesis of metabolites includes using elicitation
agents such as CuCl2, MeJA, yeast extract, and fungal elicitors to induce
the production of secondary metabolites. Hairy root cultures are also effec
tive for the biosynthesis of triterpenoids. The plant tissue culture techniques
further manipulate the growth hormones such as auxin and cytokinin in
different cultures like callus culture and cell suspension cultures to produce
metabolites. These manipulations in metabolic pathways are mainly tracked
using metabolomics studies that identify the changes in the manipulated
pathways upon elicitation and also helped in metabolite profiling. The
produced secondary metabolites were then characterized using different

analytical techniques such as TLC, HPLC, LCMS, and NMR analysis. These
techniques also help in the qualitative and quantitative identification of new
compounds from the plant.
Centellosides are the enriched compounds in the CA plant that shows
many biological activities, including anticancer activity. AA was majorly
involved in vitro and in vivo regulation of pathways associated with anti
cancer activity.
KEYWORDS
• Centella asiatica L.
• anticancer
• omics
• asiatic acid
• metabolomics
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CHAPTER 3
The Anticancer Activity of Nigella sativa

on Hepatocellular Carcinoma
IFFAT ZAREEN AHMAD1 and HEENA TABASSUM1,2
Department of Bioengineering, Integral University, Dasauli, Lucknow,
1
Uttar Pradesh, India

Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Pune,
2
Maharashtra, India
ABSTRACT
Nigella sativa L. is a medicinal seed, commonly used as spice in many

Indian cuisines including pickles, sauces, pulao, lentils, and vegetables. Its
seed is a rich storehouse of several medicinal bioactive compounds, of which
thymoquinone and thymol are the dominant ones. It has been shown to be
highly effective in almost all the ailments and disorders. Since it is used in food,
it is an important nutraceutical product and a source of dietary antioxidants.
Hepatocellular carcinoma is among the leading cancers all around the globe
and its mortality rate is very high. Besides, there is no treatment in advanced
stages and whatever treatments are available, they show severe side effects.
Several natural products have shown effects on hepatocellular carcinoma as
both preventive and curative drugs. N. sativa has been explored extensively
as an antihepatocellular cancer agent and it has a high potential to inhibit the
growth. The studies have shown that N. sativa has a prospective effect against
liver reperfusion and also on cholestatic liver damage; therefore, it could
be a powerful cancer preventive agent. The researchers have given several
mechanisms of its action on liver cancer cells. The receptors present on the
liver cancer cells have been targeted to develop drugs. There is a huge scope
for the thymoquinone in drug designing and development so that it can be

established into a commercialized drug that can specifically target the liver
cancer cells.
3.1 INTRODUCTION
Nigella sativa is a herbal plant that produces flowers and grows up to 20–30
cm annually. It has finely divided green leaves with delicate pale blue and
white blossoms. It is commonly grown in Middle Asia, Middle East, and
North Africa for its aesthetic and ornamental value. The scientific term for
the seeds is derived from Latin “Niger” meaning “black” (Aggarwal et al.,
2009). Both the seed and its oil are exploited for the cure of various ailments
including that of diuretic, lactagogue, vermifuge, and carminative.
3.2 NIGELLA SATIVA AND ITS BIOACTIVE MOLECULES
According to several studies, the seeds and oil obtained from N. sativa
contain over 100 different bioactive compounds with healing potential. Out
of these 100 compounds only 69 are well identified and characterized. It has
been reported that the combined effect of these compounds is responsible
for the promotion of good immune response. It is a noteworthy resource of
carbohydrates, proteins, fatty acids, minerals, and vitamins. The N. sativa
seeds comprise large quantities of sterols; particularly beta-sitosterol that is
well recognized anticancer compound (Khan, 1999) as it is known for the
presence of unsaturated esters of fatty acids along with alcohol of terpene.
Moreover, alkaloids are reported which belong to two categories of alkaloids,
that is, isochinoline alkaloids (nigellimin-N-oxide and nigellimin) and
pyrazol alkaloids (nigellicin and nigellidin) (Yessuf, 2015). The compound,
TQ, was recognized as the pivot component reported to be the constituent
of the essential oil (about 0.5% of average with maximum 1.5%) besides
pinene, p-cymene, diTQ, and thymohydroquinone. Similarly, some of the
terpene derivatives such as limonene, carvacrol, citronellol, carvone, and
4-terpineol were found only in trace amounts. The seeds of N. sativa are the
essential wellspring of unsaturated fatty acids chain in highest concentration,
commonly including linoleic acid (50–60%), followed by about 20% of oleic
acid, dihomolinoleic acid (10%), and eicodadienoic acid (3%). However, the
amount of saturated fatty acid is to be present around 30%, with TQ as an
The Anticancer Activity of Nigella sativa 41
ingredient of the essential oil which is responsible for a peculiar aroma and
flavor. The seeds yield yellowish-brown volatile oil steam distillation with a
characteristic smell and are reported to have the constituents of d-limonene,
nigellone, and carvone (Yessuf, 2015).
Thymoquinone (TQ), thymohydroquinone (THQ), and thymol (THY)
are three natural phytochemical compounds having tremendous medicin6+al
significance as reported in previous research (Hussain and Hussain, 2016).
Saponin, alkaloids, protein, and both essential and fixed oils are found in
the blackseed (Ali and Blunden, 2003). Ghosheh et al. (1999) described the
determination of certain medicinally significant constituents in blackseed oil,
containing THY, diTQ, TQ, dihydrothymoquinone (DTQ), and THQ, using
HPLC (High Performance Liquid Chromatography). Alkaloids, antioxi
dants, flavonoids, α-hederin, and fatty acids are among the other therapeutic
compounds present in these seeds.
3.3 PHARMACOLOGICAL PROPERTIES OF THYMOQUINONE AND

THYMOL FOR LIVER DISEASES
TQ is known to possess medicinal and toxic effects (Ali and Blunden, 2003).
It shows exceptional anti-inflammatory and antioxidant properties. It also
exhibits incredible anticancer potential. It has the capability to act together
with a range of proteins and it can also inhibit protein–protein interaction
(Reindl et al., 2008). It has the capacity to bind with the phosphoserine/
phosphothreonine, α1-acid glycoprotein (orosomucoid), human bovine
serum albumin (BSA), and recognition site of polo-box domain (PBD) (Yin
et al., 2012). It inhibits the activity of polo-like kinase 1 (Plk1) and therefore
prevents its localization by stopping the PDB-dependent association (Reindl
et al., 2008). Some significant bioactivities of TQ and THY are highlighted
in this chapter. Their mechanisms of action on hepatocellular carcinoma
have also been discussed.
3.4 HEPATOCELLULAR CARCINOMA (HCC)
Hepatocellular carcinoma (HCC) is the most prevalent type primary cancer

around the globe; however, high incidence of alcoholism, hepatitis infection,
aflatoxin, and its side effects are the common reasons of liver cancer-related
death. More than 100 out of 100,000 people have been affected by HCC
in various regions of Asia and Africa. Secondary liver cancers are more
common in Europe and the United States, whereas in Asia and Africa,
primary liver cancer is more frequent. HCC is at number two and is most
commonly associated with cancer-related death around the globe, with
746,000 deaths. It occurs more frequently in male rather than in females
(with the ratio of about 2.4:1) (Torre et al., 2015). The diagnosis for liver
malignancy is extremely poor with a total frequency of mortality of about
0.95, and in this way, the geological examples on the basis of occurrence
and death are similar. Therefore, more effective approaches for the treat
ment of cancer are urgently required. The most promising approach is the
application of the well-researched medicinally active compounds from plant
sources (Cragg and Pezzuto 2016). Traditionally, medicinal plants have been
the valuable source for many presently available cancer therapeutic agents
such as vincristine, paclitaxel (Taxol®, Bristol-Myers Squibb Company, NY,
USA), docetaxel, and topotecan (Newman and Cragg 2007). Of late, another
natural compound that has been extensively studied and has been of interest
to researchers is thymoquinone because of its broad-spectrum biological
activities (Lutterodt et al., 2010), hepatoprotective, anticancer, antitumor,
antimutagenic, and antibacterial (Woo et al., 2013).
Plant-derived compounds have potential anticancer activity, and can
be developed into commercial drug (Banerjee et al., 2010). N. sativa is a
flowering annual plant indigenous to the Mediterranean, India, and Pakistan
(Poonia et al., 2016). In the Arabian region, its seed oil has been used conven
tionally as a natural medicine for the cure of lung-related illness, arthritis,
and hypercholesterolemia (Pathak and Raghuvanshi, 2015). N. sativa has
been reported to have antihypertensive, analgesic, diuretic, antibacterial, and
liver-protective activities (Ahmad et al., 2003). However, the detailed scien
tific investigations are deficient. Previous research reported the inhibitory
capacity of the extract of the N. sativa to inhibit the progression and spread
of liver cancer cell (HepG2). It is also demonstrated that N. sativa employs its
anticancer properties through various mechanisms of action involving cell
growth inhibition, apoptosis induction, arrest of cell cycle at different stages,
ROS, as well as the fundamental molecular mechanism causing death of
cells (Banerjee et al., 2010). Thymoquinone, the most active component of
N. sativa, is consumed generously as a flavoring agent in many cuisines. In
case of human breast cancer cell line, TQ has shown to suppress the growth
and progression by inducing p38 and ROS signaling (Woo et al., 2013).
Liver carcinoma is one of the most familiar types of disease around the
globe, and HCC comprises the bulk of them (70%– 90%) of them (Venook
et al., 2010). HCC is a disease that is a challenging health problem due

to the gradual rising rate of occurrence universally (Venook et al., 2010).
The investigations by scientists have shown a significant reperfusion effect
against hepatic cells, thereby making it notably a potential agent toward the
cancer prevention (Yildiz et al., 2008). The researchers discovered in animal
models that N. sativa had a possible effect of cholestatic liver damage (Coban
et al., 2010). In CCl4-administered rats, N. sativa and Urtica dioica L show
suppressed level of lipid peroxidase and liver enzymes and were effective
cancer preventative agents (Kanter et al., 2005). A study has also reported
the protective ability of Thymoquinone (TQ) on I/R in liver and decreased
SSAT and CYP3A1 gene expression damage through a cancer preventive
system (Abdel Wahab, 2013). It has been reported N. sativa is involved in
stimulating the apoptosis cell signaling pathway by inducing the target p38
(Iyoda et al., 2003).
3.5 EFFECT OF BIOACTIVE MOLECULES ON HEPATOCELLULAR

CARCINOMA
3.5.1 THYMOQUINONE
TQ contributes actively to the regulation of biochemical reactions and

physiological processes that result in the ROS generation (Yu and Kim,
2015). It is an efficient scavenger of free radicals (Mansour et al., 2004).
After interacting with amino acids and forming semiquinone, it undergoes
numerous redox reactions that involve 1e-reduction and the other 2e-reduction
with thymohydroquinone.
It undergoes several redox reactions after reacting with amino acids and
with the synthesis of semiquinone: 1e- reduction and thymo hydroquinone
and 2e− reduction (Cremer et al., 1987). It also behaves as a potent anti
oxidant and effectively inhibits lipid peroxidation, producing radicals of
superoxide (Woo et al., 2012; Banerjee et al., 2010). It effectively increases
the activity of many antioxidant enzymes including superoxide dismutase
(SOD), catalase (CAT), glutathione transferase, and glutathione (GSH). TQ
produces two powerful antioxidants: glutathionyl-dihydro thymoquinone
(DHTQ-GS) and DHTQ, upon reaction with antioxidant enzymes (GSH,
NADH and NADPH). Taking into consideration the cellular microenviron
ment, TQ plays an essential function as both a pro-oxidant and an anti
oxidant. According to a study conducted by Badary et al. (2003), TQ was
shown as a potent superoxide anion scavenger. TQ is capable of reversing

the expression of mRNA and diethylnitrosamine-associated reduction in
various antioxidant enzyme levels (Sayed-Ahmed et al., 2010). Extensive
investigations have been aimed at determining the effect of TQ on tripeptide
glutathione, which is widely used in the prevention of the damage caused
by reactive oxygen species (ROS) and in the detoxification of medications
(Pompella et al., 2003). TQ has also been shown to be helpful in the recovery
of female Lewis rats after they had suffered from allergic encephalomyelitis
(Mohamed et al., 2003).
TQ extracted from N. sativa incited caspase-3 via stimulation of
cytochrome C release from mitochondria in squamous cell cancer (Das et
al., 2012). Similar outcomes were observed in HL60 cells during apoptosis
elicited by TQ (El-Mahdy et al., 2005). According to a study, TQ showed the
decrement in the gene expression of SSAT and CYP3A1 while demonstrating
a defensive activity against renal reperfusion-related injury or ischemia by
an antioxidant defense system (Yildiz et al., 2008). The protective effect
by a cancer preventive agent on renal ischemia/reperfusion-associated
injury was shown (Yildiz et al., 2008). TQ also showed protection against
tertbutyl hydroperoxide toxicity as reported in rodent hepatic cells. As a
result of these observations, it was discovered that pretreatment of hepatic
cells with TQ decreased the transit of cytoplasmic proteins, ALT, and
AST (Daba and Abdel-Rahman, 1998). Owing to its antioxidant property,
the in vivo administration of a single TQ dose to male albino rat initiated
a protective effect toward CCl4-induced hepatotoxicity (Sayed-Ahmed
et al., 2010). In another study, the defensive role of TQ was evaluated on
aflatoxin B1-associated hepatic toxicity in mice. This study showed that TQ
totally diminished the level of ALP, ALT, MDA, and AST. This protective
impact might be facilitated through a decrease in lipid peroxidation and also
an increased tolerance to oxidative stress (Nili-Ahmadabadi et al., 2011).
TQ demonstrated defensive activity against lipopolysaccharide-induced
endotoxemia because of its anti-inflammatory, antioxidant, and anti-
apoptotic activities (Helal, 2010). The protective activity of TQ on sodium
fluoride-associated hepatic toxicity and oxidative stress in animals improved
the antioxidant state and decreased the changes in biochemical factors. Oral
administrations of TQ ameliorated the sodium fluoride-induced toxicity and
stress in rats’ liver probably by reducing the level of peroxidation and/or
improving the activities of enzymatic and nonenzymatic antioxidants of the
liver (Abdel-Wahab, 2013). Studies also showed that TQ markedly reduced
the tamoxifen-induced depletion of hepatic glutathione and regulated SOD
activity (Suddek, 2014). TQ worked against the hepatotoxicity induced by

the acetaminophen and the studies reported reduced toxicity toward the liver
by further decreasing the ALT activity in serum which was dependent on
dose. Enhancement in tolerance to oxidative and nitrotative stress, as well
as improved energy generation in mitochondria, was shown to assist the
protective impact of TQ on hepatic cell viability (Nagi et al., 1999). The
activity of ethanolic extracts of Nigella sativa, Zingiber officinale, and their
combination in individuals infected with the hepatitis C virus were shown
in clinical trials (HCV). TQ showed potential activity against I/R injury
and neuronal damage. In addition to this, TQ also inhibited histological
impairment, inflammation, and oxidative stress. Interestingly, it decreased
the ER stress parameters expression that included caspase-12, CHOP, and
GRP78. Along with this, it also increased mitochondrial functions and
restricted the expression of apoptotic parameters. Additionally, TQ enhanced
phosphorylation of ERK and P38 (Bouhlel et al., 2017). TQ reduced the
combination of TNF-alpha, MCP-1, Cox-2, and interleukin (IL)-1b in
pancreatic ductal adenocarcinoma (PDA) cells substantially, which is both
dose and time dependent. TQ fully suppressed the production of the cytokines
listed above, as well as the intrinsic activity of the MCP-1 promoter. It also
inhibited NF-kB activation and decreased NF-kB transport to nucleus from
the PDA cells (Chehl et al., 2009). TQ-induced apoptosis, as determined
by flow cytometry and colorimetric estimate of Caspases 3 and 9, showed
that an early G1/S arrest was typical for apoptosis (Hassan et al., 2010).
The hepatoprotective effect of TQ on damaged liver through AMP-activated
protein kinase (AMPK) signaling in HSCs was demonstrated. The data
of in vitro study showed the time-dependent attenuation of Liver kinase
B-1 (LKB1) by TGF-β and AMPK phosphorylation, which slowed down
by pretreatment with an AMPK activator including 5-Aminoimidazole
4-carboxamide ribonucleotide (AICAR) and TQ. It can also considerably
enhance the expression of MMP-13 while suppressing α-SMA, collagen-Ι,
and tissue inhibitor metalloproteinase-1 (TIMP-1), to prevent the initiation
of human HSCs induced via TGF-β. Furthermore, TQ increased the
expression of the peroxisome proliferator activated receptor-γ (PPAR- γ),
which was inhibited by a genetic deletion of the AMPK gene. In in vivo
research on mice fed with ethanol, TQ significantly reduced elevated serum
hepatic triglyceride and amino transferase levels, while also significantly
increasing AMPK phosphorylation and LKB1 levels and further inducing
an enhanced expression of the sirtuin 1 (SIRT1) gene (Yang et al., 2016).
Different mechanisms have been shown in Figure 3.1.
FIGURE 3.1 The molecular targets of thymoquinone on hepatocellular carcinoma cells.
3.5.2 THYMOL
Thymohydroquinone and TQ exhibited restricted inhibition of specifically

COX-2 which pointed to the role of the N. sativa constituents (diTQ, THQ,
THY, and TQas anti-inflammatory agents) (Marsik et al., 2005). With an
IC50 value of 0.2 μM, THY shows the best inhibitory activity than TQ and
thymohydroquinone against COX-1 with 0.1 and 0.3 μM of IC50, respectively.
THQ showed cytotoxicity against human hepatic cells, that is, HepG2 cells
(Stammati et al, 1999), P815 mastocytoma cells (Jaafari et al., 2007), HepG2,
Caco-2, and V79 cells (Slamenova et al., 2007). THY showed antioxidative
and cytotoxic potential against P388 cell line (Gedara, 2008; Hirobe et al.,
1998). Jayakumar and collaborators (Jayakumar et al., 2012; Ozkan and
Erdogan, 2011) demonstrated THY to be toxic to HepG2, K562 cells, and
colonic Caco-2 cells, believed to be associated with antioxidant action rather
than the effect of DNA damage. HepG2 cells were treated with both carvacrol
and thymol, while the refence drug was taken at various doses (N-acetylcystene
(NAC)). The data revealed that acetaminophen (APAP) effected the growth
of HepG2 cells by inflammation and oxidative stress. Carvacrol and THY
treatment of HepG2 cells by exposing to acetaminophen for 24 hours reduced
oxidative stress and inflammation. In addition, antioxidant enzymes such as
ALT and LDH were studied in HepG2 cells and reported to provide protection
toward the toxicity induced through APAP by improving antioxidant activity
and decreasing pro-inflammatory cytokines, such as IL 1β, and TNF-α. In
APAP toxicity, high-dosage THY and carvacrol were nearly identical to NAC
therapy and shown increased effectiveness when compared to carvacrol
(Palabiyik et al., 2016). A schematic representation of the effects of THY in
different experimental models of cancer is shown in Figure 3.2.
3.6 MECHANISMS OF HEPATOPROTECTIVE ACTIVITY OF

N. SATIVA AND ITS IMPORTANT BIOACTIVE COMPOUNDS
3.6.1 THE SIGNALING MECHANISMS UNDERLIE THE ANTICANCER

PROPERTIES OF N. SATIVA AND ITS PRINCIPAL COMPONENT
THYMOQUINONE
The disease of the liver encompassed several other cancerous cell signaling
pathways including Notch (Strazzabosco and Fabris, 2012; Villanueva et
al., 2012; Viatour, 2011), insulin-like growth factor 1/ IGF1 Receptor (IGF/
IGFR) (Enguita-Germán and Fortes, 2014; Sprinzl et al., 2015), c-Met/
HGF (hepatocyte growth factor) (You et al., 2011; Goyal et al., 2013), and
epidermal growth factor receptor (EGFR) signaling pathway (Berasain and
Avila, 2014). The biochemical mechanisms behind TQ’s defence against
HCC were unknown until it was discovered that the Notch signaling pathway
is significantly suppressed by TQ (Ke et al., 2015). Overexpression of Notch
1 (NICD), the notch receptors resulted in the enhanced TQ’s inhibitory effect
on proliferation of the cells. Such evaluations were corroborated both in vivo
FIGURE 3.2 Schematic representations of the effects of thymol on different experimental

models of hepatocellular carcinoma.
and in vitro studies in HCC patients where the notch pathway was targeted
(Ke et al., 2015). Furthermore, TQ’s efficacy was further investigated in
murine leukemia, which further revealed the dose- and time-dependent
manner reduction of the viability of WEHI-3 cells and also reported to induce
apoptosis at an early stage accompanying arrest at the phase of G1/S of cell
cycle, Bcl-2 downregulation, and Bax upregulation resulting in an enhanced
ratio of the Bax/Bcl-2 (Salim et al., 2014). When administered orally to
BALB/c mice, TQ substantially decreased neoplastic cells proliferation and
elevated apoptosis in the liver and spleen, according to in vivo research. The
activation of caspase 3 and the downregulation of XIAP showed that TQ had
a strong pro-apoptotic activity. It also resulted in higher expression of p53
and p21, which caused arrest in G2/M and sub-G1 cell cycle phases. N. sativa
oil along with TQ was also reported to significantly decrease 5-LO activity,
which results in the powerful activity against cancer (El-Dakhakhny et al.,
2002; Houghton et al., 1995; Mansour and Tornhamre, 2004; El-Mezayen
et al., 2006). TQ has also shown to be an effective HDAC inhibitor, as

evidenced by the activation of target genes of HDAC such as Maspin, p21,
and Bax, and further reported to downregulate Bcl2 expression, as these are
involved in inducing programmed cell death and arresting the cell cycle.
The experiments were carried out, both in vivo and in vitro to evaluate the
prospective of TQ along with Tamoxifen, to modulate the signaling of the
control AKT in the cancer cells of breast (Rajput et al., 2013).
TQ-treated PEL cells were reported to associate with structural variation
in Bax and further lead to MMP loss and excretion of Cyt C from the
mitochondria to cytosol which results in elevated activity of Caspase-3/9,
which further leads to the PARP cleavage, while the presence zVAD-FMK
or NAC repels its effect (Hussain et al., 2011). A dose-dependent expression
of prostaglandin E2 (PGE2) and COX-2 expression was reported to be
induced in the MDA-MBA-231 cell line (Yu and Kim, 2013). SB203580
and LY294002 replicated the TQ’s effect, shows a significant inhibition of
p38 and P13K, respectively, which is related to the positive regulation of
both PGE2 and COX-2 (Yu and Kim, 2013).
In another study a combination of paclitaxel and TQ demonstrated a
significant triple activity in the cell lines of breast both in vitro in in vivo
models (Sakalar et al., 2016). Another work suggested upregulation of genes
involved in tumor suppressor (like BRCA1, hic 1 and p21) and increases
caspases and PARP levels by TQ. TQ-induced autophagic cell death was
halted by p38 and JNK inhibitors, indicating that TQ activated p38 and JNK
signaling to cause the death of LoVo cells via autophagy (Chen et al., 2015).
TQ-mediated autophagy was proven to be the result of caspase-independent
activity, since co-treatment of the inhibitor of caspase-3 (z-DEVD-FMK) had
no effect on cell death via autophagy induced by TQ (Chen et al., 2015).
These results showed caspase-dependent apoptosis as the main type of cell
death at low TQ treatment and it switches to caspase-independent autophagy
at high concentrations of TQ treatment. Although TQ was shown to be the
main anticancer compound it was established that some compounds other
than TQ also significantly demonstrate the anticancer effects of N. sativa
against HepG2 cells (Samarakoon et al., 2010). Also, saponins, α-hederin, and
a pentacyclic triterpene are all isolated from N. sativa seeds and reported to
exhibit strong anticancer activities both in vitro and in vivo (Swamy and Tan,
2000, Cheng et al., 2014). Other bioactive compounds present in N. sativa
which have demonstrated good cytotoxic and anticancer potential include
thymol (THY), Thymohydroquinone (THQ), dithymoquinone (DTQ),
carvacrol, nigellidine, nigellicine, and nigellimine-N-oxide (Randhawa and
Alghamdi, 2011; Horvathova et al., 2014).
3.6.2 p53 SIGNALING PATHWAY
The anticancer and pro-apoptotic activities of TQ have been provided

which involves its capability to alter the expression of many target proteins
of cell cycle. The molecular mechanisms and signaling pathways involved
in the TQ-associated anticancer activity have been revealed. It was shown
that noncytotoxic concentrations of TQ markedly reduced the growth of
papilloma and spindle carcinoma cells derived from mouse keratinocytes
(Gali-Muhtasib et al., 2004). Treatment with TQ treatment was related to
suppression of Bcl-2 expression and upregulation of p53 and its target p21,
resulting in less CDK2 activity. Pifithrin-alpha (PFT-alpha) showed the role
of p53 in TQ-induced cell cycle arrest and programmed cell death and the
action of TQ on the expression level of Bcl2, p21, and p53 was studied
on HCT-116 cells (Gali-Muhtasib et al., 2004). It was also shown that TQ
was active on human breast cancer MCF-7/DOX cells by forcing G2/M
phase cell cycle arrest resulting in damage of the DNA and thus triggering
apoptosis (Arafa et al., 2011). The pro-proliferative and anti-apoptotic
activity associated with TQ causes the enhanced p21 and p53 expression
along with elevated PARP cleavage and caspase activation (Arafa et al.,
2011). Furthermore, the activity also involves the downregulation of p53,
BRCA1, and BRCA2 (Linjawi et al., 2015) along with the upregulated
expression of Bax and a downregulated of Bcl-2, resulting in increase of
Bax/Bcl-2 ratio (Arafa et al., 2011).
3.6.3 MAPK SIGNALING PATHWAY
TQ-induced apoptosis in five distinct human colon cancer cell lines

was investigated for the direct connection between oxidants and MAPK
signaling pathways (LoVo, HCT-116, Caco-2, HT-29, and DLD-1). TQ,
interestingly, reduced the development of all cell lines of cancer examined
with no impact on normal intestinal cell lines of human (El-Najjar et al.,
2010). TQ-induced apoptosis was further shown by the substantial produc
tion of ROS, as treatment with N-acetyl cysteine (NAC), a powerful ROS
scavenger, nearly entirely abolished TQ-induced apoptosis (El-Najjar et al.,
2010). TQ increased activation of JNK and ERK but not p38 MAPK, and
treatment with specific JNK and ERK inhibitors (SP600125 and PD98059)
inhibited TQ-induced apoptosis (El-Najjar et al., 2010). Similarly, TQ treat
ment of HepG2 cells resulted in a substantial rise in ROS levels as well as
the expression of NQO1 and HO-1 (oxidative stress-related genes), leading

to apoptosis (Ashour et al., 2014). As a consequence of TQ use in HTLV-1
negative cells of Jurkat leukemia causes depletion of glutathione, cytochrome
c release, loss of MMP, increase in ROS levels, activation of caspase-3/9,
and PARP cleavage (Dergarabetian et al., 2013). TQ was found to potently
promote apoptosis and ROS production in a time- and dose-dependent
manner, with NAC co-treatment completely removing these effects (Yu and
Kim, 2013). Furthermore, TQ administration increased the activity of JNK,
p38, PI3K, and ERK1/2 (Yu and Kim, 2013). The findings indicated that
TQ primarily targets MAPK and PI3K signaling pathways to mediate its
pro-apoptotic actions.
3.6.4 HEPATOPROTECTIVE DISEASES AND REACTIVE OXYGEN

SPECIES (ROS)
The liver is the body’s central metabolic organ and is accountable for a
variety of vital activities. The danger to the body’s metabolic system may
be life-threatening if the liver is damaged or injured. Antioxidants have been
shown to delay hepatotoxicity by inhibiting lipid peroxidation, producing
ROS, and lowering the activity of alkaline phosphatase (ALP), alanine
aminotransferase (ALT), and aspartate aminotransferase (AST) (Yen et al.,
2007; Park et al., 2012; El-Shafey et al., 2015). Antioxidant activities have
been found in the bioactive components of N. sativa seed. The majority of
liver damage is caused by the production of ROS, which depletes antioxidant
enzyme sources and reduces the capacity of cells to resist harm (Muriel,
2009). The three enzymes that generate ROS in the body are myeloperoxi
dases, nitric oxide synthases (NOS), and NADPH oxidases. Different kinds
of ROS generated as by-products of cellular metabolism include hydroxyl
radical (OH-), superoxide anion radical (O2-), hypochlorous acid (HOCl-),
peroxynitrite (ONOO-), and hydrogen peroxide (H2O2). Oxidative stress is
a condition caused by an imbalance in the quantity of ROS and antioxidants
in the body. Free radicals may interfere with cellular activities by disrupting
the structural properties of polyunsaturated lipids and sulfhydryl groups
(Muriel, 2009). Because the liver’s metabolism is so fast, it is very vulnerable
to oxidative stress and free radical damage. The negative consequences of
ROS may be avoided by reducing the formation of free radicals, scavenging
free radicals, or improving the antioxidant defense system (Tsukamoto and
Lu 2001).
3.7 FUTURE PROSPECTS OF NIGELLA SATIVA SEEDS IN DRUG

DISCOVERY
N. sativa seed is extensively studied for a broad range of medicinal

properties (Darakhshan et al., 2015). It has shown high medicinal potential
for the prevention and cure of many ailments (Ahmad et al., 2013). It has
favorable characteristics of a potent drug candidate which is in the process
of establishment as a new well-established drug (Dajani et al., 2016). Its
bioactive compound, thymoquinone, is a widely studied compound and has
crossed many critical steps involved in drug development like preclinical
and clinical research and it has been reported by many scientists working on
this compound (Goyal et al., 2017; Almajali et al., 2021).
Many analogs of thymoquinone have been developed in different studies
that have shown incredible efficiency toward various classes of cancers.
Recently, several cytotoxic terpenes, monoterpenes, and sesquiterpenes
were combined with novel TQ analogues to see whether they might improve
cancer-fighting efficacy (Banerjee et al., 2010).
Pharmacokinetics studies of thymoquinone have been done comprehen
sively on different animal models. Different doses of thymoquinone were
administered to animals, by various routes including intraperitoneal, intrave
nous, and intragastric routes to study its efficacy in disease models (Alkharfy
et al., 2015). The different factors of pharmacokinetics study in the blood
samples were evaluated using high-performance chromatography extracted
from rabbits (Alkharfy et al., 2015). Thymoquinone bound to proteins at a
rate of 99%, with a sophisticated quick eradication and reduced absorption
after oral administration. By considering the overall analysis, thymoquinone
has demonstrated a substantial prospective for its clinical usage as an impor
tant drug for pharmaceutical development and commercialization. Moreover,
thymoquinone has shown good solubility and bioavailability also which a
common limitation in drug discovery and development is. So, it could be an
effective drug if targeted drug delivery is done by adopting newer technolo
gies, mainly nanotechnology (Elmowafy et al., 2016).
Concisely, it can be said that the wide range of medicinal properties,
good pharmacokinetics, security and low toxic effects, the lipophilic nature,
high therapeutic index, efficiency, and bioavailability make thymoquinone
a favorable compound for the drug development. Apart from these, the
exceptional probable benefit of this compound is its natural origin and its
clinical trials will lead to the transformation of the experimental data into
actuality in human being.
3.8 CONCLUSIONS
Autophagy is a significant mechanism of cell death that interacts closely

with other prevalent programs of cell death that include apoptosis, to
produce a cascade of events. It is a promising, but challenging, medication
target for cancer, because autophagy plays a critical role in cell death. As a
result, researchers tried to figure out whether autophagy and apoptosis were
involved in the cancer cell inhibitory process. HepG2 cells were treated
with a 5d sprout extract of N. sativa-loaded NLC, which caused cell cycle
arrest, autophagy, mitochondrial membrane depolarization, and apoptosis.
The data suggested that understanding of conventional medicinal systems
can be used to investigate, recognize, develop, and take new medicinal
compounds to commercial level that can be used safely and efficiently.
Research into the potential benefits of nano-formulation in target-based
curative uses such as anticancer drugs may further strengthen its use in
these applications. As the mechanism of action, apoptosis or autophagy
appears to be necessary machinery for cell death, and it is currently an
objective in cancer treatment.
There has been considerable interest in the use of natural products as
potential therapeutic agents for the treatment of human malignancies, owing
to their low toxicity toward normal cells and the fact that they are nontoxic
to cancer cells. In this respect, herbal extracts from various sections of plants
have been thoroughly investigated for their possible anticancer properties
(Zhou et al., 2009). TQ is the most important biological component extracted
from Nigella sativa. The presence of active phytoconstituents such as TQ
and THY in the methanolic extracts may explain the activity shown by the
extracts. TQ and THY are found in the methanol soluble part of N. sativa
oil, which is derived from the plant (Abou Basha et al., 1995). When TQ
was shown to be the primary active component of black seed essential oil,
researchers were encouraged since it has been shown to exhibit potential
antineoplastic growth inhibition against a variety of tumor cell lines in vitro
and in vivo, respectively (Salomi et al., 1992; Worthen et al., 1998; Shoieb
et al., 2003; Gali-Muhtasib et al., 2004; Edris, 2004; El-Mahdy et al., 2005;
Gali-Muhtasib et al., 2008). This action may be related to the drug’s capacity
to induce apoptosis as well as its inhibitory effects on the development of
malignant cells (Gali-Muhtasib et al., 2004).
KEYWORDS
• Nigella sativa
• thymoquinone
• thymol
• hepatocellular carcinoma
• drug discovery
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CHAPTER 4
Boswellia serrata Roxb

MANOHAR M. V.1, ANU PANDITA2, AMOGHA G. PALADHI3,
BHOOMIKA INAMDAR1, SUGUMARI VALLINAYAGAM4, DEEPU PANDITA5,
and K. M. SRINIVASA MURTHY6
1
2
3
4
Department of Biotechnology, Mepco Schlenk Engineering College,
Sivakasi, Tamil Nadu, India
5
Government Department of School Education, Jammu,
6
Department of Microbiology and Biotechnology, Jnanabharathi Campus
Bangalore University, Bengaluru, Karnataka, India
ABSTRACT
Boswellia serrata Roxb belongs to the family Burseraceae. There are mentions
of this plant in various ancient texts and its Sanskrit name “Gajabhakshya” and
Salai guggal. In this chapter, anticancerous property of Boswellia serrata Roxb
is discussed. The alkaloids Boswellia serrata Roxb has various components
like oils, triterpenoids, and resins among which 25–35% is β-boswellic acid
and also acetates of AKBA and ABA. Diene derivatives of boswellic acids
like 11-dehydroxy β-boswellic acid, α-amyrin, 11-diene-24-oicacid are
found. There are various tricyclic, tetracyclic components found in the extract
of oleo gum resin that also shows a little anticancer activity. Understanding
of chemistry of boswellic acid is the core knowledge to know the molecular
activity of boswellic acid and its molecular interactions in various inhibition

pathways. Boswellia serrata is known for multiple pharmacological effects.
Experiments have shown that it can be helpful in treating various cancers
like brain, CNS, breast, gastro-intestinal cancers like hepatic, colo-rectal
cancer, prostate cancer, leukemia, and various other cancers. Molecular
targets of boswellic acids are known to be topoisomerases-1 and -2, different
apoptosis inducing cascades. Various clinical and preclinical analyses are
also being mentioned, which show the rate of success of boswellic acids and
its derivatives as treating agents of cancers and multiple myelomas. These
experiments have reported inhibitions of certain pathways and also execution
of apoptotic pathway that helps in curing of cancer and also in reducing
the side effects of the tumor. Boswellia serrata extracts are known to show
zero side effects in its natural form, thus making it a potential compound for
therapeutic use of cancer treatment.
4.1 INTRODUCTION
Boswellia serrata Roxb is a tree that is usually found in the tropical parts of
Asia and Africa. In India, it is found in dry hill forests like Madhya Pradesh,
Rajasthan, Gujarat, Assam, Bihar, Orissa, and other regions. This tree secrets
gum when incisions are made on tree trunks and the obtained gum are used
in various fields like cosmetics, pharmaceuticals, etc. The obtained gum
tastes bitter and is pleasant in flavor. The Frankinscence is used by different
ancient civilizations of Greeks, Romans, and Egyptians as incense, various
aromatic purposes, and fumigants. Currently, it is used in the production
of incense sticks and incense powders. The preparations of oleo gum resin
of Boswellia serrata have found a significant place in Unani and Ayurveda
for its properties to treat various health conditions like asthma, cough,
bronchitis, issues related to throat and digestive system. It is also used in
treatment of cephalytes and pulmonary diseases. It acts as a stimulant of
various processes to keep the system healthy. The gum is prescribed I cases
of dysentery, dispenscia, diarrhea, jaundice, and hemorrhoids and can also be
administered in weak and unhealthy subjects.
As cancer is an issue worldwide causing majority of morbidity and
mortality, the interests in therapeutic using natural products contain bioactive
constituents with anticancer and anti-inflammatory activity. Oleo gum resin
of Boswellia serrata has found its importance. Common names of Boswellia
serrata are Olebanum, Salai guggal, Kundur, Loban, and Gajabhakshya,
oleo gum resin from Boswellia serrata consists of essential oil, resins, and
Boswellia serrata Roxb 63
gums that can cure various health conditions that have adversely affected the
individual. The essential oil of Salai guggal has a mixture of mono, di, and
triperpenes containing 33 essential components. The gum contains arabi
nose, galactose, and xylose containing some digestive enzymes. Resins are
the most important essential compounds of Boswellia serrata Roxb extracts
composed of pentacyclic triterpenic acids. The derivatives of this are found
to be β-boswellic acids like 3-o-acetyl-β-boswellic acid, 3-o-acetyl-11-
keto-β-boswellic acid, and 11-keto-β-boswellic acids. The residues of oleo
resin present in Salai guggal possess anti-diarrohoeal, anti-hyperlipidemic,
anti-asthematic, immunomodulatory, hepatoprotective, anti-inflammatory,
antimicrobial, hypoglycemic, and anticancerous in nature. Various
conducted experiments have shown that there are no side-effects found due
to the administration of Boswellia serrata extracts and are also found to be
administered in daily basis. Various groups of scientists have reported the
use of AKBA isomer gives the more potent and efficient results (Nerkar,
2020). The boswellic acids are found to be actively involved in the inhibition
of cell-proliferation by inducing various apoptotic pathways like caspase-3
activity, Bcl2, BAX. Despite cancer therapeutics, Boswellia serrata has also
found its use in avoiding adverse effects of tumors like edema, necrosis,
angiogenesis, etc. There are different nanoparticle-based studies that have
evaluated boswellic acid for treatment of various purposes like cytotoxicity,
anti-invasive activities, and cell-specific drug-resistant activities. Because
boswellic acids are sourced from Boswellia serrata of various cultivators, the
bioavailability of the extracts must be known to formulate and standardize to
use them in clinical protocols.
Taxonomical hierarchy (Serrata et al., 2020)

Kingdom—Plantae
Subkingdom—Tracheobionta
Division—Magnoliophyta
Class—Magnoliopsida
Order—Sapindales
Family—Burseraceae
Genus—Boswellia
Species—serrata
Subspecies—Roxb
4.2 CHEMISTRY OF BOSWELLIC ACID
The alkaloids of Boswellia serrata Roxb mostly consist of essential oils,

resins, and triterpenoids. The prime constituent of monoterpenoids is α-
pinene (73.3%) and others include p-cymene (1.0%), limonene (1.42%),
myrcene (1.71%), trans-pinocarveol (1.80%), β-pinene (2.05%), cis-verbenol
(1.97%), verbenone (1.71%), borneol (1.78%), thuja-2,4 (10)-diene (1.18%),
whereas α-copaene is the only sesquiterpene that has been recognized in the
oil (Grant, 2009). Terpenoids are the major constituents of oleo gum resin and
the fraction is found to be 25–35% including mainly β-boswellic acid [BA,
1] that also consists of 11-keto-b-boswellic acid along with its corresponding
acetates AKBA and ABA. Tschirh et al. in 1898 first extracted terpenoids
from boswellic acid (BA) (Sudharsan et al., 2005). After this first isolation of
terpenoids from BA, many works have been done for structural elucidation
of boswellic acid. Crystallographic X-ray studies were made to elucidate the
structure of boswellic acid constituents (Syrovets et al., 2005a, 2005b). One
of the boswellic acids, β-boswellic acid, belonging to the group of ursanes
among triterpenic acids is lipophilic in nature that encompasses only one
α- hydroxyl as the functional group. To vary the solubility and the acidic
property of terpenes, it was subjected to modify the structural orientation
by replacing a carboxyl functional group with an amino functional group,
to make it a week basic compound (Garg and Deep, 2015). Boswellic acids
are generally seen along with diene derivatives like with 3-O-acetyl-9 by the
dehydration of 3-O-11-hydroxy-b-boswellic acid; a derivative is obtained
namely 11-dehydro-beta-boswellic acid. NMR spectroscopy is known to
elucidate the structures of all penta-cyclic triterpenes including boswellic
acid and diene derivatives. X-ray crystallographic studies have also shown
the structures of ABA and methyl esters of boswellic acid. 11-dien-24-oic
acid, 3-hydroxy-urs-9, and α-amyrin were elucidated from the gum resins
of Boswellia serrata Roxb. Various other tetracyclic terpenoids found in
Boswellia serrata Roxb are found to be 3-keto-tirucall-8, 24-dien- 21-oic acid;
3a-acetoxy-tirucall-8, 24-dien-21-oic acid; 3a-hydroxy-tirucall-8, 24-dien
21-oic acid and 3b-hydroxytirucall-8, 24-dien-21-oic. Various other triterpe
noids like urs-12-ene-3a, 24-diol, and 2, 3-dihydroxy-urs-12-ene-24-oic acid
were found in Boswellia serrata Roxb that were structurally elucidated from
the acidic and neutral fractions of the gum. urs-12-ene-3b,24-diol is present
as an isomeric diol (Aktionen, 1967). Boswellia serrata extractions consist
of gum resins containing around 50–60% of α- and β-boswellic acids. 1–3%
of the total extracts are mostly bioactive AKBA fractions (Sterk et al., 2004).
The anticancerous activity of Boswellia serrata extractions is known

to cure various cancers like prostrate, leukocytes, colon, liver, colo-rectal,
brain, and CNS (central nervous system). The inhibition of tumor formation
by AKBA acts on nuclear factors like NFκB kappa B, signal transducers
and also induces activator of transcription-3 (STAT-3)-related pathways, and
thus helps in apoptosis to avoid cell proliferation and inhibits angiogenesis
to prevent neoplastic cell proliferation (Liu et al., 2006; Borrelli et al., 2006;
Lu et al., 2008; Pang et al., 2009).
FIGURE 4.1 3-o-acetyl-11-keto-β-boswellic acid (AKBA).
FIGURE 4.2 3-o-acetyl-β-boswellic acid (ABA).

4.3 APPLICATIONS
Indian frankincense or Salai guggal (Boswellia serrata) is known to be

used in various medicinal practices like Ayurveda and Unani as a traditional
medicine to treat inflammatory diseases since ages. References of use of
Boswellia serrata can be seen in various ancient texts related to Ayurveda.
“Gajabhakshya” is the Sanskrit name of the plant. The name is thus given
because it is mostly consumed by elephants. In the original texts of Susruta
Samhita, Astanga Hridaya, Astanga Sangraha, and Charaka Samhita the use
of this plant is mentioned signifying its medicinal values. In Susruta Samhita
and Charaka Samhita the reference of this plant is in the treatment of arthritis
and other inflammatory diseases. In systems like Ayurveda and Unani, the
internal and external administration of this plant is seen in the treatment of
various inflammatory conditions (Moussaieff and Mechoulam, 2009). Later,
based on these mentions, the resins and gums of these plants were evaluated
for its medicinal value for conditions like arthritis, asthma, colitis, and other
inflammatory issues (Moussaieff and Mechoulam, 2009). The gum resins
usually contain triterpenoids, essential oils, and carbohydrates out of which
fundamental constituent of oleo gum resin are boswellic acids like acetyl
11-keto-β-boswellic acid, 11-keto-β- boswellic acid, and β-boswellic acid
(Kirste et al., 2011).
4.3.1 LEUKOTRIENE INHIBITION
Studies have shown that gum resins extracted from Boswellia serrata Roxb
inhibit leukotriene B4 formation of peritoneal neutrophils. Leukotrienes
are forms when phagocytosis is stimulated particularly in neutrophils.
Neutrophils are responsible for inflammation in body. Thus, BAs can also
be used as nonsteroidal anti-inflammatory drug that is free from side effects
(Upaganlawar and Ghule, 2009).
4.3.2 ANALGESIC AND PSYCHO-PHARMACOLOGICAL EFFECTS
Menon et al. have experimented the use of gum resin on animals. They found
the extracts of Boswellia serrata had a sedative effect and analgesic activity
on animals. The sedative effects of gum resins are due to its property of
reducing spontaneous motor activity (Upaganlawar and Ghule, 2009).
4.3.3 ANTI-INFLAMMATORY AND ANTI-ARTHRITIC EFFECT
Extracts of Boswellia serrata are known to cure edema in mouse and also
help in curing mycobacterium adjuvant-induced poly-arthritis in mice, thus
decreasing paw swelling (Singh and Atal, 1986).
4.3.4 IMMUNO-MODULATORY ACTIVITY
Experiments have shown that gradual degranulation of mast cells when it is

administered in different doses, thus resulting in stabilizing the activity of
mast cells (Pungle et al., 2003).
4.3.5 HYPO-LIPIDEMIC AND HEPATO-PROTECTIVE ACTIVITY
The extracts of Boswellia serrata when used to study activities on lipidemia,

the experiments resulted in decreased total cholesterol about 38–40% and
also increased HDL, thus helping the movement of fats from the place of
deposition to peripheral tissues. This causes decrease in liver damage and
is known by reduced SGPT, SGOT anti-transferases and serum enzyme
activities (Upaganlawar and Ghule, 2009).
4.3.6 HYPOGLYCEMIC ACTIVITY
Oleo gum resins extracted from Boswellia serrata are reported to show anti-
diabetic activity in conditions like non-insulin-dependent diabetes mellitus. In
rat models with induced diabetes, significant reduction of blood-glucose level
is seen after the administration of this extract (Upaganlawar and Ghule, 2009).
4.3.7 ANTI-DIARRHOEAL ACTIVITY
Acetylcholin (ACh) and Barium chloride (BaCl2) are toxic in nature due to
which the peristalsis of the intestine increased causing inflammatory bowel
syndrome which causes diarrhoea. Boswellia serrata extractions are found
to be the inhibitors of ACh that inhibits rapid gastro intestinal movements.
The same is also effective to inhibit castor oil-induced diarrhoea (Borrelli et
al., 2006).
4.3.8 ANTIMICROBIAL ACTIVITY
Proteus mirabilis UCH 28, E. coli LASUTH 54, and S. aureus OGSUTH 108
were significantly inhibited by essential oils of Boswellia serrata and are also
known to inhibit gram-positive and gram-negative bacteria (Upaganlawar
and Ghule, 2009).
4.3.9 ANTI-ASTHMATIC ACTIVITY
A study was conducted by Gupta et al. in 1998 and the results were reported
showing the patients with prolonged history of asthma were cured. Symptoms
like dyspnea were inhibited due to increase in number of healthy bronchi
and increased stimulation of MAPK (Mitogen Activated Protein Kinase)
by the mobilization of intracellular calcium Ca2+ thus decreasing number of
asthmatic attacks (Upaganlawar and Ghule, 2009).
4.4 ANTICANCER ACTIVITY
The extracts of Boswellia serrata induce anticarcinogenicity on various

tumors. The mode of action by which it inhibits cell proliferation and cell
growth is by inhibiting the biosynthesis of activities of RNA, DNA, and
formed proteins (Tsukada et al., 1986). Boswellic acid when administered in
particular doses is known to inhibit number of cancers by inhibiting tumor
formation (Upaganlawar and Ghule, 2009). Boswellic acids are known to
induce apoptosis by inhibiting protein synthesis and thus help to inhibit cell
proliferation in patients of different cancers. The inhibitory on glioblastoma
patients is primarily studied (Upaganlawar and Ghule, 2009).
4.4.1 ANTICANCER ACTIVITY IN VIVO AND IN VITRO
Various extracts and phytochemicals from Boswellia are being examined for
its anticancerous and anti-inflammatory activities in comparison with other
Frankinscence. Multiple experimental investigations were used in deter
mining the above properties. It is found that standardization of Frankinscence
extracts has a very significant role in phytotherapy to cure cancer. Thus, it can
serve as standardized drug. Synthetic drugs with synthetic compounds can
also be used once these phytochemicals are completely analyzed. Preclinical
experimental analysis of Boswellia extracts and isolated phytochemicals are

found to actively inhibit various types of cancer malignancies like fibrosar
coma, cancers of liver, lung, bladder, breast, pancreas, prostate, glioblastoma,
leukemia, multiple myeloma, and other meningioma. These experiments
confirm that phytochemicals of Boswellia serrata possess the efficiency to
cure cancer. The experimental analysis of boswellic acids actions clearly
shows that the molecular inhibitions made by these phytochemicals have
reduced oxidative stress and increase stress on proteins from endoplasmic
reticulum (Hussain et al., 2009). Boswellia phytochemicals are known to
cause de-methylation that leads to epigenetic changes by decreasing the
action of DNMT1 (DNA methyl transferase-1) and increases histone protein
activity (Thorsteinsdottir et al., 2011). Various signal transductor proteins
and transcription factors were affected by inhibitory actions of Boswellia
serrata extracts. This inhibition includes STAT-3, AMPK (AMP-activated
protein kinase inhibition), protein kinase b (AKT) expression and phos
phorylation, wingless int (WNT)/β-catenin, SRC (sarcoma tyrosine kinase),
dephosphorylation of ERK1/2, ribosomal protein S6 kinase (RPS6), FAK
(focal adhesion kinase) (Kunnumakkara et al., 2009) (B. Park et al., 2011).
It also induces unfolding of proteins in endoplasmic reticulum by activating
the signal transduction in unfolded protein response pathway (Saleh and
Trinchieri, 2010). Boswellic acid causes modulations in NFκB and inhibitory
factor κB-α (IκB-α) activity (Kunnumakkara et al., 2009; Zhao et al., 2003).
It also inhibits the activity of transcription factors like mTOR, specificity
protein 1 transcription factor (SP1) cellular myelocytomatosis protooncogene
(c-MYC), HIF-1 (hypoxia-inducible factor 1) (Agrawal et al., 2011; Morad et
al., 2013; Syrovets et al., 2000). Growth inhibition takes place due to induced
apoptosis by p53 signaling pathway and DNA damage induced by proteins
and p21 gene expression and also arrests the cell cycle in different phases
thus inhibiting cell proliferation (Kunnumakkara et al., 2009; Syrovets et al.,
2000). Cell proliferation is also inhibited by inhibition of few factors like
proliferation cell nuclear antigen (PCNA) expression and mitosis, inhibition
of Kiel proliferation antigen 67 (Ki-67), decreased fork-head box M1 protein
(FOXM1) expression, inhibition of cyclin B/D, and cyclin-dependent kinases
2, 4, and 6 (CDK2/4/6), decreased DNA topoisomerases 1 and 2 (TOPO1/2A)
expression, inhibition of Aurora A/B and Polo-like kinase 1, dephosphory
lation of CDK1, decreased dual specificity phosphatase 25A (CDC25A)
expression (Frank et al., 2009; Kunnumakkara et al., 2009; Park et al., 2011).
Derivatives of Boswellia serrata inhibit inflation and also inflammatory
markers like 5-LOX, TNF-α, leukotriene synthesis, COX-1/-2 IL6 signaling,
PGE2, and monocytic differentiation and increase the activity of IL12 expres
sion (Conti et al., 2018; Weber et al., 2006). This elevates the expression of
SMAD14, SRC homology region 2 domain-containing phosphatase 1 (SHP1),
sphingomyelin phosphodiesterase 3 (SMPD3), glutathione-depleting ChaC
glutathione-specific γ-glutamyl cyclotransferase 1 (CHAC1), homocysteine-
inducible endoplasmic reticulum stress-inducible ubiquitin-like domain
member 1 (HERPUD1), sestrin 2 (SESN2), cystathionine γ-lyase (CTH),
tribbles homologues 3 (TRIB3) and decreases the activity of serum response
factor (SRE), Praja ring finger ubiquitin ligase 2 (pJaC2), transglutaminase
2, endothelin 1 (EDN1), cell migration-inducing protein hyaluron-binding
protein (CEMIP), inhibitor of DNA binding 1 (ID1), SRY box 9 protein
(SOX9) (Lakka et al., 2011; Saleh and Trinchieri, 2010; Thorsteinsdottir et
al., 2011).
Bosweelia serrata extracts and isolated phytochemicals induce apoptosis,
metastasis, cell-proliferation, invasion, angiogenesis by increasing p53
upregulated modulator of apoptosis (PUMA) signaling and dynamin-related
protein 1 (DRP1) translocation to mitochondria) and decreasing mitochondrial
membrane potential, BCL-2 (B-cell Lymphoma protein), BH3 interacting
domain death agonist (BID), BCL extra-large protein (Bcl-xL), inhibitor of
apoptosis protein (IAP), survivin expression, activation of caspases 3, 8, and
9, increased BCL-2-associated X protein (BAX) expression, activation of
caspases-3/-8/-9, cleavage of poly(ADP-ribose) polymerase (PARP), DNA
fragmentation, increased expression of death receptor 4 and 5 (DR4/5),
cytosolic release of pro-apoptotic factors (i.e., cytochrome c, Diablo
IAP-binding mitochondrial protein (SMAC/DIABLO)), C/EBP homologous
protein (CHOP), TNF receptor 1 (TNF-R1), inhibitor of caspase-activated
DNAse (ICAD) expression (Kunnumakkara et al., 2009; Lakka et al., 2011;
Xia et al., 2005).
4.4.2 CLINICAL INVESTIGATIONS
Preclinical analysis of Boswellia serrata has shown amusing results against

tumor and cancer acting via inhibition of cell proliferation and induction of
apoptosis. Thus, the compound was clinically investigated on human subjects
for cancer therapy considering control and test. The way of approach in
clinical investigations is different and varies when compared with laboratory
analysis where the administration of compounds is either oral or intravenous.
Thus, there is a bit of difficulty for the body to absorb these compounds in
few patients. Boswellic acids when treated in various inflammatory condi

tions have reportedly cured the patients with various cancers or also found
to inhibit rapid proliferation by increasing longevity of life. There is a long
history of traditional use of Boswellia serrata for the conditions like cancers
and tumors in Ayurveda and Unani as this also is considered as clinical
trials when administered in particular dosages for certain time. Recent
clinical investigations with various modes of administration have resulted in
decreased response when compared to native medicines. Recently, analogues
of boswellic acids are used in cancer therapy to cure the adverse effects
of radiotherapy, chemotherapy, etc. where edematous and other inflamma
tory conditions are very well cured (Volm and Efferth, 2015). The effect
of edema curing and tumor and antitumor response by Boswellia serrata
extracts is confirmed when investigated on 29 glioma patients when admin
istered with increasing doses consequently for a week (Kirste et al., 2011).
There is another study of clinical treatment where 19 children were treated
with 126mg/kg of boswellic acid for their condition of brain tumor showed
very positive results and also reduced neurological symptoms with healthy
weight gain, reduction in edema, improved muscle strength, and also general
health improvements (Lalithakumari et al., 2006). In one of the studies,
the patient suffered due to breast cancer with brain metastasis resistant to
standard cancer treatment. Thus, boswellic acid administration was made
which suppressed and controlled the re-occurrence of brain metastasis, for
almost four years due to the activity of inhibition of lipoxygenase pathway
(Singh et al., 2012). Thus, proper approach in the clinical administration
in the treatment of cancer can remarkably cure the cancers and its adverse
inflammatory effects and also symptoms. Boswellia serrata extracts thus
possess properties of tumor regression and control the proliferation of cancer
of almost any type of cancer (Efferth and Oesch, 2020).
4.4.3 BRAIN TUMOR
LN-18 and LN-229 were considered and treated with the ethanolic extract of
Boswellia serrata gum resin. This experiment resulted in proving cytostatic
and cytotoxic effect due to the induced apoptotic activity by the use of
boswellic acid (Nand et al., 2016).
In brain tumor, a severe effect of edema is seen. This edematous condi
tion when gets severe will be morbid to the life of patients. Various effects
of Boswellia serrata include anti-inflammatory and antitumor activities and
this principle can be used in clinical trials to treat edematous conditions in

brain tumors. Studies have been conducted, where the patients were treated
with Boswellia serrata extracts showing significantly low edematous condi
tion than others with various comparative drugs (Kirste et al., 2011).
4.4.4 GLIOMA
Various boswellic acids like ABA (acetyl-β-boswellic acid), AKBA, and

β-boswellic acid show anticancerous activity due to its property of cytotoxicity
on malignant glioma cells. Even the low-concentration administrations of
boswellic acids show anticancerous activity basically by induction of apoptosis
and also apoptosis induced by BA is independent of formation of free radicals.
Boswellic acids are known to induce the expressions of various tumor depressant
genes like p21 that does not depend on p53 to cause apoptosis. BA-induced
p21expression does not alter the levels of BCL-2 and BAX proteins (Glaser et
al., 1999). Further investigations were made on BCA; an in vivo study reported
the efficiency of BA to reduce perifocal edema. The results of treatment were
remarkable in varied concentrations of boswellic acid dosages (Winking et al.,
2000). Another experiment was conducted to check the growth-inhibiting activity
of extracts of Boswellia serrata was made in which immunocompromised
mice contain C6 glioma tumor xenograft as targets. A derivative of boswellic
acid containing a modified A ring (2 cyano, 3 enone), CEMB (cyanoenone of
methyl boswellates) were administered that showed high-growth inhibition,
pro-differentiative, anti-inflammatory, and antitumorous activity that cured the
glioma of the subject (Ravanan et al., 2011).
4.4.5 BREAST
Suhail et al. (2011) showed that the essential oil extracts of Boswellia
serrata reduce cell viability and increase cell death in tumor tissues, thus
showing anticancerous activity in all human breast cancer cell-lines (Khan
et al., 2016). Studies on the effect of AKBA to inhibit breast cancer were
made and it is found that AKBA inhibits cell proliferation and invasion of
tumors and confirmed by reduced CXCR4 protein levels (Park et al., 2011).
A cream made of boswellic acid was used to study the prevention of breast
cancer caused by radiations and it was found that it effectively reduced skin
superficial symptoms and erythema (Khan et al., 2016).
4.4.6 GASTRO INTESTINAL CANCERS
Gastrointestinal system constitutes various glands and muscle systems which

can be infected by cancer due to various reasons that are termed gastroin
testinal cancers. The prevention and cure of such cancers using Boswellia
serrata extracts are studied, such as hepatic, gastric, pancreatic, and colo
rectal cancers (Garcea et al., 2003).
4.4.7 HEPATIC CANCER
Hep G2 cells were subjected to administration of keto-β-boswellic acid,

β-boswellic acid, and AKBA to study the pathways of inhibition of cell
proliferation and apoptotic efficiency of Boswellia serrata extractions of
these cell-lines. Studies showed that β-boswellic acids inhibit DNA histone
complex formations at high concentration and keto-β-boswellic acid and
AKBA increases the activity of caspase cascade (caspase-3, caspase-8, and
caspase-9) which are associated with PARP cleavage (Khan et al., 2016).
4.4.8 COLO-RECTAL CANCER
Various in vitro and in vivo studies are conducted to prove the anticancerous
properties of Boswellia serrata are also effective against colon cancer.
HT-29 colo-rectal cancer cell-lines were subjected to the administration of
the acetyl-keto-β-boswellic acid (AKBA), keto-β-boswellic acid (KBA), and
β-boswellic acid (β-BA), to study apoptotic and antiproliferative efficiency
where BA induces the activation of caspase-8-dependent apoptotic pathway
and also Fas/Fas ligand-independent action in these cell-lines (Liu et al.,
2002).
AKBA is investigated for anticancerous study using the xenograft model,
where it was able to arrest cell cycles in certain stages thus preventing colo
rectal cancer (Toden et al., 2015).
4.4.9 PROSTATE
The chemiresistant androgen independent PC-3 cell-lines of prostate cancer

were administered with 3-α-acetyl-11-keto-α-boswellic acid (AKBA) extracted
from Boswellia serrata in vitro and in vivo, to study cytotoxic and anticancerous
activity respectively. AKBA is found to cause toxicity in mitochondrial activity
by releasing cytochrome c, causing DNA fragmentation, and also inhibiting
NF-κB signaling (Syrovets et al., 2005b). The same is investigated using a
chick chorioallantoic membrane with transplanted xenograft of prostate cancer
cells (Büchele et al., 2006; Khan et al., 2016).
4.4.10 LEUKEMIA
Leukemia is one of the commonly seen cancers which is a global concern.

Leukemia is known to be more incident in males than in females and also 10x
higher in adults than in children. Various leukemia cell-lines of humans like
(THP-1HL-60, K-562, MOLT-4, U-937) were administered using gum resin
extract of Boswellia serrata to study its cytostatic, cytotoxic, pro-apoptotic,
and anticancerous activity using flow cytometry (Khan et al., 2016).
The administration of AKBA is known to activate caspase-8 either by
inducing activity of caspase-3 or Bid, thus causing the decrease in mitochon
drial membrane potentials (Xia et al., 2005).
The triterpenediol induces nitric oxide formation that cleaves Bcl-2
and causes translocation of Bax in mitochondrial membrane leading to the
loss of membrane potential thus releases cytochrome c. this action induces
expression of death receptors like TNF-R1 and DR4 that activates caspase-8.
Thus, the above study shows that triterpenediol induces apoptosis and also
increases oxidative stress in both intrinsic and extrinsic apoptotic pathways
(Bhushan et al., 2007).
4.5 MECHANISM OF ACTION AND MOLECULAR TARGETS OF

BOSWELLIC ACID
Boswellic acids being anti-inflammatory and anticancerous have many molecular

targets like enzymes, receptors, transcription factors, growth factors, and other
biomolecules that are involved in cell growth and proliferation. Experiments
have shown that boswellic acids and its analogues regulate apoptosis through
inhibitory and supportive actions on the mentioned molecular targets, thus
inhibiting tumor formation. Experiments show AKBA (acetyl-11-keto-β—
boswellic acid) when administered to meningioma cells to study cytotoxic
activity; it is found that the phosphorylation of extracellular signal induced
kinase-1 and -2 prohibition and also it impairs the motility of meningioma
cells that are induced by platelet-derived growth factors (Park et al., 2002).
When HCT-116 was administered with boswellic acid as a treatment of colon
cancer, it leads to the reduction in cyclin E, cyclin D, and Cyclin-dependent
kinases like CDK2 and CDK4 and also reduces (pRb) phosphorylated Rb) (Liu
et al., 2006). Syrovets and coworkers studied the effect of boswellic acid when
administered to NF-κB which is a transcription factor and when inhibited it
causes downregulation of activated human monocytes (Syrovets et al., 2005b).
Boswellic acid also targets the activation of cell transducers, transcription
factors STAT-3 (Signal Transducers and Activators of Transcription-3) and
also regulates cell proliferation, survival, chemo resistance, and angiogenesis
in tumor cells and MM (Multiple Myeloma). AKBA affects the genes, thus
inhibiting cell proliferation and angiogenesis by targeting various factors
(Kunnumakkara et al., 2009).
FIGURE 4.3 Types of cancers that can be cured by BA.

Molecular targets of AKBA in prostate cancer include DR5 (Death

Receptor 5) androgen receptor, VEGFR2 (Vascular Endothelium Growth
Factor Receptor 2) leading to the inhibition of cell proliferation by causing
induced apoptosis and decreased angiogenesis (Lu et al., 2008; Yuan et al.,
2008; Pang et al., 2009).
FIGURE 4.4 Molecular targets of Boswellic acid.
4.5.1 INHIBITOR OF HUMAN TOPOISOMERASES I AND II
Human topoisomerases are the enzymes that control the molecular structures
and arrangements of DNA (Syrovets et al., 2000). Topoisomerases are
the enzymes that act as ligases and lyases on DNA double strands during
DNA recombination, replication fork formation, transcription, etc. DNA
topoisomerases-1 causes DNA strands by opening the twists of DNA by
changing the torsion. They also create torsions to pair up and wind the DNA.
Rapidly dividing cells should generally have high DNA-replicating activities,
where DNA topoisomerases are much needed. This condition is usually seen
in tumor formation and cancerous condition. Boswellic acids are known to
inhibit DNA topoisomerase activity. Thus, they can be pharmacologically
used to inhibit DNA replication that helps in inhibiting self-proliferation
(Nerkar, 2020).
4.5.2 TOPOISOMERASE II DNA CLEAVAGE COMPLEX
Topoisomerase II is the enzyme responsible for DNA supercoiling and

entangling. Supercoiling and entangling inhibition is known to be target site
to inhibit cancer cell-proliferation. These enzymes are ATP dependent. They
pass through DNA acting on the strands using a set of internal interfaces or
gates that are dissociable in nature. Topoisomerases have various analogues
that can cleave single stranded DNA by unifying two essential enzyme
families involved in cleavage mechanisms. This chromosomal detangling
machine regulates breakage of DNA to avoid mutations and helps in
cytotoxicity. These structures are analyzed by clearing the water molecules
and co-catalyzed by a haem-ligand (Nerkar, 2020).
4.5.3 PRECLINICAL ACTIVITY
Hoernlein et al. (1999) discovered and reported their works on inhibition of

topoisomerase enzyme by AKBA which is an extract of Boswellia serrata
Roxb. AKBA in various laboratory experiments has reportedly exhibited the
activity of constitutive signal transducer and transcription 3 activator inhibi
tion (STAT-3) in human multiple myeloma cells. Thus it induces separa
tion of STAT-3 by interleukins. AKBA inhibits phosphorylation of STAT-3
pathway. This leads to the separation of cyclin D1, Bcl-xL, VEGF separation
(Kunnumakkara et al., 2009). Choi et al. have reported the inhibitory activity
of extracts of Boswellia serrata that were examined to inhibit induced migra
tion of PDGF (Platelet-Derived Growth Factor) and cell proliferation (Choi
et al., 2009).
4.6 SAFETY AND BIOAVAILABILITY ENHANCEMENT OF

BOSWELLIA SERRATA
Based on various clinical and preclinical activities of Boswellia serrata,

activity on various animals and cell-lines of humans there are absolutely
no mortality, side-effects, or adverse effects of this plant extract that
was noticed (Singh et al., 2012). Boswellia is traditionally consumed as
decoction in various doses, in later days; it can be considered taking in the
form of tablets and capsules orally. The balance should be maintained in
the doses for the medicine to be effective; the quantity and concentrations
must be considered as suggested. Because Boswellia serrata is sourced from
various places the quality may vary, considering this factor the effectiveness
of the extracts from Boswellia serrata of different suppliers differs. Thus,
the standardization becomes more complex. Clinical results cannot be
compared when the extracts from different suppliers are used (Giles et al.,
2005). Number of methods and way of approaches were used to discover
the potential of pharmacological use of various analogues of boswellic acids
to maximize their bioavailability (Du et al., 2015). Studies have also shown
that the use of Boswellia serrata as a constituent of regular meal also helps in
the effective mode of supplementation. The activity of anticancerous activity
will be elevated when administered with anionic drugs (Hüsch et al., 2013;
Skarke et al., 2012). There are various recent studies that are conducted
which use nanoparticles, lipid carriers, micelles, poly (lactic-co-glycolic
acid) nanoparticles, liposomes, and emulsions as vectors to help the effective
utilization of the administered compound (Aqil et al., 2013).
4.7 STUDIES ON SEMISYNTHETIC BOSWELLIC ACID DERIVATIVES
Various studies have been conducted to assess semi-synthetic assay and alkyl
derivatives of boswellic acid, BA145 (3-O-α-butyryl-11-keto- β-boswellic
acid) as an efficient anticancerous agent that is toxic due to the inhibition of
STAT proteins and NF-κB (Kumar et al., 2012).
Further, more studies conducted by Pathania et al. in 2013 showed that
boswellic acids are highly potent in certain concentration ranges causing
induced apoptosis via downregulation of PI3K/Akt and Erk. The experiment
also proved that BA145 analogue induced autophagy which is a signifi
cant mechanism that can regulate angiogenesis and causes cell protection
(Pathania et al., 2013).
Ravanan et al. (2011) used boswellic acid with a modified ring structure
to develop cyanoenone modified methyl boswellates as an analogue to
inhibit nitric oxide production caused by interferons in mice macrophage. It
is found as a potent agent to induce apoptosis and to inhibit DNA synthesis
when administered in doses to treat C6 rat glioma cells in mouse xenograft
model (Ravanan et al., 2011).
Another experiment was conducted on a synthesized derivative of BA,
butyl-2-cyano-3, 11-dioxours-1, 12-dien-24-oate and is witnessed to cause
anti-proliferatory effect by upregulating apoptotic pathway by the activation
of p53-p21-PUMA in HeLa cells. This is also known to inhibit cell signaling
cascades like p-AKT and NF-κB (Khan et al., 2011). Khan et al., in 2012,
also found that the cleaving activity of butyl-2-cyano-3,11-dioxours-1,12
dien-24-oate in PARP1 also induces caspase-3 cascade activity (Khan et
al., 2012). The study of another derivative propionyloxy, that is, 11-keto-β-
boswellic acid showed the inhibition of growth and proliferation of HL-60
pro-myelocytic leukemia cell-lines. It is known that the derivative inhibits
the activity of topoisomerase-1 and -2 and also it is also found to induce
apoptosis and is confirmed by the molecules that showed apoptotic activity
when morphologically analyzed. The induced apoptosis is caused by the
activation of caspase cascade and PARP cleavage by the derivative (Chashoo
et al., 2011).
Boswellic acid semi-synthetic derivatives like 3-α-acet-oxy-4-β-amino-
11-oxo-24-norurs-12-ene and 3-α-acetoxy-4-β-amino-24-norurs-12-ene are
found to show efficient inhibition of histone deacetylases, thus arresting cell
cycles at a certain concentration G1 phase and also causing loss of mitochon
drial membrane potentials (Raina et al., 2014).
The semi-synthetic analogues of boswellic acids showed induced apop
tosis and cytotoxicity in various cancer cell-lines. It is found that triterpenoid
ring actively induces apoptosis by targeting mitochondria-dependent path
ways in HL-60 cells and also induces DNA fragmentation, cell shrinkage,
condensation of chromatin, fragmentation of nucleus, and membrane bleb
bing (Qurishi et al., 2010).
The experiments conducted showed that β-boswellic acids containing
acyl substituents result in elevated property of chemotherapeutic actions.
Another semi-synthetic triterpenoid derivative was synthesized, that is,
3-cinnamoyl-11- keto-β-boswellic acid which has its effect on rapamycin as
a target and also induces apoptosis in both prostate and breast cancer. This
compound induces antiproliferation and pro-apoptotic activities through
inhibition of TOR signaling in a certain range of concentration (Morad et al.,
2013). 11-keto-β- boswellic acid induces the formation of endoperoxide that

can be evaluated as an anticancer activity biomarker in many human cancer
cell-lines by using sulforhodamine B assay (Csuk et al., 2010).
4.8 DISCUSSION
Boswellia serrata has found a significant importance in the field of medicine

in various medicinal fields like Ayurveda, Unani, and other ethnopharmaceau
ticals. Various experiments provide evidence to the properties like antitumor,
anticancer, anti-inflammatory, etc. Boswellia serrata is one of the indigenous
species and also recently listed out as endangered species. Thus, the use of
Boswellia serrata for the extractions of its components can be reduced by
the use of artificial derivation and analogues of its components like oleo gum
resin, boswellic acids. There are analogues of boswellic acid available in
the market that can be manipulated accordingly for pharmacological uses.
Various plant sources also contain boswellic acid and can be obtained from
them. Increasing the number of dedicated plantation for extraction purposes
can be done as an agricultural practice also increases socioeconomical per
capita development of farmers as individuals. The current source of informa
tion on Boswellia serrata is obtained from a limited number of experiments
conducted. Thus, scholars should continue more number of experiments
regarding the inhibitory pathways of boswellic acids by which it inhibits
cancers and other inflammations. Investigations on particular cancer during
are more suggested by clinicians. Preclinical analysis has shown excellent
results of anticancerous activity by the extracts of Boswellia serrata Roxb,
but the clinically available data is very less known in current-day medicine
practice. More investigations on cellular pathways in which boswellic acid
inhibits cancer must be known. Molecular targets in induced apoptosis are
clearer in the use of Boswellia serrata anticancer agents. Standardized extrac
tion procedures should be improvised as various standards give different
percentage of efficiency in the action of boswellic acids. Ethno medicines
should be considered source where the information of properties of boswellic
acids is already being mentioned and practiced since ancient times. Boswellic
acids are found to have zero-toxic and no severe side effects except few mild
symptoms like diarrhea. These can be used in the field of chemotherapeutic
administration. Boswellia serrata is thus known as a potent, natural, and safe
component to be administered.
4.9 CONCLUSIONS
Boswellia serrata Roxb is well known for gum resin secretions belonging
to a certain class of plants known as Frankinscence. It is elucidated that
pentacyclic triterpenes like boswellic acids and diene derivatives are respon
sible for its anticancerous activity. Other than triterpenoids there are also
acidic and neutral fractions of the gum that assured 1–3% of boswellic acid
constituents, out of which AKBA fractions are bioactive. They are known
to cure cancers like Liver, Brain, Prostrate, Colon, and other Gliomas by
inhibiting the formation of tumor by reducing the activities of DNA by
inhibiting topoisomerases. They also act in apoptotic pathways to prevent
neoplastic cell proliferation and also inhibit angiogenesis in which capillaries
and blood vessel formation lead to the nourishment of these tumors that are
inhibited. The mentions of this activity of Salai Guggal as Gajabhakshya
in Sanskrit are found in the original texts of Susruta Samhita, and Charaka
Samhita as references in systems like Ayurveda and Unani. Various effects
of these gum extracts are known and can be listed as immunomodulatory
anti-inflammatory and leukotriene inhibitors. Boswellic acid has also been
reported as hypolepidemic and hepatoprotective in nature that helps in the
reduced activity of tumor forming due to fat accumulation in hepatocellular
cancers. These components also reduce Cyclin E, D, and cyclin-dependent
kinases that help in apoptosis, and thus reduce the target tumors. Molecular
targets of AKBA in apoptosis are death receptor-induced androgen receptors
like VEGFR2; this also inhibits angiogenesis. The preclinical studies are
conducted in various xenograft models in mice and rabbits and also in human
cell-line studies, where the mode of action varies in every single experiment.
The results show that almost every human-related cancer can be treated by
using boswellic acids and its analogues. In in vitro conditions, boswellic
acid analogues show its maximum potency in avoiding inflammation and
cell-proliferation as per the known knowledge by ancient texts but when
administered in clinical purposes, the activity of these acids is very low in
nature. Thus, the way of approach in clinical treatments must be modulated
so that the efficiency of the drug increases and also the utilization of the
administered boswellic acid can be increased. The extractors should consider
the bioavailability of the compound and the plant source and also should
maintain balance. Various approaches of administration are seen in different
systems of medication, considering the effectiveness the standardization
should be varied accordingly. Due to its various properties, this is also

suggested to be included in everyday meal or various lipid emulsions, lipid
nanoparticles can also be used as carriers to reach the compounds to target
organs. The use of Boswellia serrata extracts is also seen in various cosmetic
products like perfumes, creams, lotions, soaps, and detergents (Serrata et
al., 2020). In the above-mentioned studies, information about phytochemical
analysis of gum portions of Boswellia serrata has reported very insuf
ficient data. In later years, various pentacyclic triterpene compounds that
are analogues of Boswellia serrata extracts are found, which needs more
analysis in both structure and property-based data. Thus, special attention
should be given on the clinical trials to know its therapeutic value that has
tremendous potential to cure cancer.
KEYWORDS
• Boswellia serrate
• boswellic acid
• pharmacological effects
• cancer
• apoptotic pathway
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S0361-090X(02)00170-8
CHAPTER 5
Catharanthus roseus
RAMACHANDRA REDDY PAMURU1, RAJAGOPAL REDDY S.2,
AMBEDKAR1,3, CHANDRASEKHAR T.4, MADHUSUDHANA REDDY A.2,
and CHANDRAMATHI SHANKAR P.3
1
Department of Biochemistry, Yogi Vemana University, Kadapa,
Andhra Pradesh, India
2
Department of Botany, Yogi Vemana University, Kadapa,
3
Department of Biotechnology, Yogi Vemana University, Kadapa,
4
Department of Environmental Sciences, Yogi Vemana University,
Kadapa, Andhra Pradesh, India
ABSTRACT
Cancer, an alarming disease, occurs in humans without clear explanations

causing deaths worldwide. Bioactive compounds are promising drugs to
treat the cancer with less side effects compared with chemotherapy and
radiotherapy in cancer patients. Several bioactive compounds are extracted
from various plant sources with anticancerous activity. One of such plant
holding a plethora of anticancer compounds is Catharanthus roseus. Over
past decades the anticancer properties of this plant have been used for cancer
treatment as traditional medicine and were later identified as the bioactive
compounds. Moreover, producing bioactive anticancer pharmaceutical drugs
is cost effective with no toxicity to the environment and less side effects
in the patients treated with these drugs. An attempt is made to review the
Catharanthus, a magic plant for its biology, distribution, phytochemicals,

and anticancer properties along with the production of these compounds in
submerged cultures in the present chapter.
5.1 INTRODUCTION
Cancer is the second highest mortality chronic disease worldwide. Uncon

trolled growth takes up in cancerous tissue and later it spreads to other
parts of the body. According to World Health Organization (WHO) about
10 million cancer deaths were recorded during the year 2020. Reasons
for cancer are not clear and prevalence is high at Western countries. The
available treatment methods of cancer are surgery, chemotherapy, and
radiotherapy that cost more and cause severe side effects in patients. None
of the scientific group invented/identified a correct drug for complete treat
ment of cancer without side effects. Still research groups are in search
of a perfect drug to kill the tumors. However, out of all therapies natural
plant-based medicine could be an alternative method of treating cancer.
Several plant-based drugs have been extracted from various plant species
and many were tested for their anticancer activity. Natural drugs that are
used first time for cancer therapy are vincristine and vinblastine (Costa
et al., 2008). Still the identified plant drugs are not so effective and were
found with side effects.
Catharanthus roseus (C. roseus), a well-known plant available all over
the world, belongs to family Apocynaceae. Purple flower is the meaning in
Greek for the name of this plant Catharanthus. This plant is also commonly
named as periwinkle and originated in Madagascar. This plant is a common
medicinal and ornamental plant in many places and used in traditional
medicine in India, China, and Africa. Common diseases like diabetes,
Hodgkin’s, and malaria are treated with extracts of Catharanthus. Several
alkaloids extracted from this plant are in use as clinical medicine for its
antispasmodic and antihypertensive properties (Nejat et al., 2015; Sain and
Sharma, 2013).
The most important and potential anticancer plant identified is C. roseus.
The most predominant alkaloid compounds that show antitumor function
from this plant are vincristine and vinblastine (Das and Sharangi, 2017).
Moreover, above 150 phytochemicals are identified and extracted from
Catharanthus (Kabesh et al., 2015). There are studies finding the antitumor
function of other compounds extracted from this plant. There are five drugs
Catharanthus roseus 91
from this plant which are commercially available in the market for the treat
ment of cancer and other diseases. Combination of nanotechnology with
bioactive compounds is another potential area to improve the efficacy of
drugs against diseases. The importance Catharanthus bioactive compounds
and their anticancer properties are presented in the present chapter.
5.2 CATHARANTHUS ROSEUS
Catharanthus roseus (C. roseus) common plant grows in Asia, Africa,

Europe, and United States continents and is famous as Vinca rosea, Ammo
callis rosea, Lochnera rosea, and Pervinca rosea (Plaizier, 1981). But it is
endemic to Western Indian Ocean, that is, Madagascar. This plant has many
common names and varies from one region/language to other. Some of the
common names for this plant are cape periwinkle, Madagascar periwinkle,
bright eyes, graveyard plant, pink periwinkle, old maid, rose periwinkle,
everyday Jasmin, etc. (USDA-NGPS, 2021). In India this plant has many local
names like sadabahar, sadaapushpa, nityapushpa, shavamnaari, sadaphuli,
nayantara, kumtluang, nayantora, billaganneru, baahrama, asephool, etc.
Catharanthusa flowering ornamental plant belongs to dicotyledon and
grows elsewhere.
5.2.1 BOTANY
The evergreen plant Catharanthus is a small herb or shrub grows up to 60

cm. The leaves of this plant are long oval to oblong hairless shiny green with
short petiole and pale midrib. Leaves are oppositely arranged and approxi
mately 2.5–9.0 cm long and 1.0–3.5 cm broad. After germination Cathar
anthus grows very fast and reaches adult flowering stage in 15–25 days
in normal conditions. Flowers hold basal tube (2.5–3.0 cm long) attached
with five lobes like petalsin two colors (white or rosy-purple to dark pink)
with centered dark red. Self- or insect-mediated (butterflies) pollination is
common fertilization methods in this plant. Paired pod fruits of length of
about 2.0–4.5 cm and width of 3.0 mm are seen in Catharanthus. The whole
Catharanthus plant along with flowers and fruit is presented in Figure 5.1.
Taxonomical classification of Catharanthus is as follows; Kingdom-Plantae,
Division-Magnoliophyta, Class-Magnoliopsida, Order-Gentianales, Family-
Apocynaceae, Genus-Catharanthus, and Species-roseus.
FIGURE 5.1 Catharanthus roseus complete plant, two types of flowers and fruit.
5.2.2 GEOGRAPHIC DISTRIBUTION OF PLANT
Catharanthus is a worldwide well-distributed ornamental tropical or

subtropical plant and is a native of Madagascar, a large island located in
Western Indian Ocean which is located near Africa. It mostly grows during
rainy seasons and propagates widely through seeds at a soil pH 5.5–6.0.
Leaves of this plant have thick vax coat which facilitates this plant to
tolerate high abiotic stress (dry, frost-free, humid, and saline) and has no
boundaries to grow even at different altitudes of land (0–900 m heights),
during summers under sunlight, under shades and well-drained soils. It is
a common plant to find everywhere in the Asian and African continents.
Because of its easy growth and distribution in different habitats such as
grass lands, dry waste lands, sandy soils, inland riverbanks, shrub lands,
dunes in savannas, houses, beaches, roadsides, limestone rocks, deserts, and
different other places, it was introduced into many countries and traveled
across continents. Moreover, after the discovery of Catharanthus medicinal
properties are cultivated widely as a commercial crop in countries like India,
Africa, United States, Spain, Australia, Southern Europe (PROTA, 2011;

Łata, 2007; Joy et al., 2008). It survives in warm climates throughout the
year, but it may not survive in cool springs, wet soils, and watery soils due
to fungal and bacterial diseases (Thomas and Latimer, 1996; Whiting et al.,
2011). Moreover, in many countries like India, South Africa, China, Mexico,
and Malaysia Catharanthus is used as a medicinal and/or ornamental plant
(Patel et al., 2012; Ong et al., 2011).
5.3 PHOTOCHEMISTRY AND MEDICINAL PROPERTIES
Phytochemicals are the secondary metabolites of plants. C. roseus is a trea

sure for phytochemicals. Since it is a common plant all over the world it is
used as medicine for treating various ailments. All the parts of Catharanthus
such as leaves, roots, stem, flowers, and fruits are used as medicine. Appli
cations of modern biological methods are in use for developing traditional
medicine industrially.
5.3.1 PHYTOCHEMICALS
Chemicals that are isolated from various parts of plants using modern
biophysical techniques are collectively called phytochemicals. These
chemicals are having high potentiality in treating various chronic diseases
including cancer. Phytochemicals of Catharanthus are known and pharma
ceutically important. The two major cytotoxic dimeric alkaloids isolated
from this plant are vincristine and vinblastine. The amount of these two
chemicals varies from different parts of the plant. Very low amounts were
identified in the leaves of Catharanthus (Sevestre-Rigouzzo et al., 1992).
There are different types of compounds such as steroids, polypehnols,
flavonoids, anthocyanins, iridoid glucosides, and glycosides identified in
different parts of C. roseus (Mustafa and Verpoorte, 2007). Natural indicator
for acid–base testing has been isolated from the flowers of Catharanthus
(Candido and Martinez, 2009). However, the compounds present in stems
and leaves are similar, but they differ with petals and seeds (Ferreres et al.,
2008). The two major compounds isolated from Catharanthus are alkaloids
and phenolics. Figure 5.2 shows the structure of various phytochemicals
isolated from C. roseus.
Vincristine Vinblastine 3,4–Anhydro vinblastine
Serpentine Vinopocetine Ajmalicine
Reserpine Yohimbine Ajamaline
Serpentine Catharanthine Vinflunine

FIGURE 5.2 (Continued)
Vindoline Vindolicine Vindolinine
Catharoseumine Tabersonine Tryptamine
Catharanthamine 17-Deacetoxycyclovinblastine Vinorelbine
Cycloleurosine 17-Deaceto xyvinamidine Vinposidin

FIGURE 5.2 (Continued)
Catharine Leurosidine Leurosine
Caffeoylquinic acid Isorhamnetin Kaempferol
Quercetin Perivine Tabersonine

FIGURE 5.2 Identified Catharanthus roseus phytochemicals and their structure.
5.3.1.1 ALKALOIDS
An amount of 130 indole terpenoid alkaloids has been extracted from various
parts of Catharanthus (Daniel, 2006; Hisiger and Jolicoeur, 2007; Renault
et al., 1999; Wang et al., 2012). Majority of these alkaloids appear in high
content during flowering stage which are holding distinct medicinal proper
ties (Schmelzer and Gurib-Fakim, 2008). Many alkaloids that are extracted/
isolated from this plant are presented in Table 5.1. Hisiger and Jolicoeur (2007)
reported that out of all only five Catharanthus alkaloids isolated are commer
cialized such as vincristine, vinblastine, serpentine, 3,4–anhydro vinblastine,
and ajmalicine. The two velbabanamine group alkaloids (vinorelbine and
vinflunine) derived from precursor molecules vindoline and catharanthine
are holding highest pharmacological role, compared to vinblastine that shows
structurally with these molecules (Nirmala et al., 2011).
5.3.1.2 PHENOLICS
Another set of major phytochemicals along with alkaloids identified in

Catharanthus are phenolic compounds. Phenolics are major secondary
metabolites holding one to many phenolic groups and are found in all
plant species. Flavonoids, cinnamic acid derivatives, and anthocyanins
belong to phenyl-propanoids and 2,3-dihydroxy-benzoic acid is holding
radical scavenging ability found in Catharanthus (Mustafa and Verpoorte,
2007). Table 5.1 shows various phenolic compounds isolated/extracted
from C. roseus.
5.3.2 CATHARANTHUS IN TRADITIONAL MEDICINE
In the regions of India and Africa Catharanthus has been used since ancient
days as medicinal plant for treating various ailments. This plant is well
recognized in indigenous Indian Ayurveda for the treatment of cancer and
diabetes due to its antioxidant, antimicrobial, antidiabetic, antitumor, and
antimutagenic principles in leaves, roots, stem, and flowers (Chopra et
al., 1956; Grover et al., 2002). Since the 1700s this plant is grown as an
ornamental plant in Europe and later it became popular folk medicine with
wide applications. Europeans also believe that it is a magic plant to ward off
evil spirits. Violet of the sorcerers is referred by French people for this plant.
A wide variety of diseases/disorders are treated using Catharanthus plant
as traditional medicine and some are ocular inflammation, insect stings,
tranquilizer, homeostasis (fever), diabetes, cancer, lowers hypertension, etc.;
they are also used as disinfectant, to stop bleeding, comfort lung congestion,
sore throats, eye infections and irritations, etc. The boiled leaves of this plant
TABLE 5.1 Alkaloids and Phenolic Compounds Isolated/Extracted from Catharanthus roseus.
98
S. Part of the Plant Method of extraction/ Phytochemicals References
no. Identification
1 Protoplast-derived Yeast-elucidated extraction Vinblastine and vincristine Maqsood and Abdul (2017)
tissue
2 Flower petals UPLC-Q-TOF Vinblastine and vincristine Schweizer et al. (2018)
3 Dried whole plant Diode array detector HPLC Vinblastine, vincristine, vindoline, yohimbine and Liu et al. (2016)
catharanthine
4 Roots, stem, and HPLC-Ultraquadrupole Vindoline, vincristine, ajmalicine, serpentine, Jeong and Heung (2018)
leaves vinblastine, catharanthine
5 Cambium culture CMCs-ultraviolet extraction Vincristine, vindoline, catharanthine, and vinblastine Moon et al. (2018)
6 Leaves, roots and Ultra HPLC-Tandem MS Vincristine, vindoline, reserpine, vindesine, ajamaline, Kumar et al. (2018)
stem ajmalicine, and vinblastine
7 Leaves IR, UV, NMR and MS Vindolicine, perivine, vindolidine, vindoline, serpentine, Tiong et al. (2015)
and vindolinine

8 Dried whole plant qRT-PCR, HPLCand UV-C Vinblastine, vindoline, vincristine, Moon et al. (2017)
catharanthineandvindogentianine
9 Hairy root cultures Reverse HPLC with UV Vinblastine, vincristine, and catharanthine Hanafy et al. (2016)
detector
10 Crude extracts of Chromatographical Vindoline and catharanthine Kotland et al. (2016)
aerial parts centrifuge
11 Flowers, roots, DAD-HPLC and analysis Vindolinine, ajmalicine, vincristine, anhydrovinblastine, Pan et al. (2016)
stem and leaves serpentine, catharanthine, and vinblastin
12 Dried whole plant HPLC with NF-κβ and JNK Dimeric indole alkaloids and cathacunine Wang et al. (2016)
Catharanthus roseus
S. Part of the Plant Method of extraction/ Phytochemicals References
no. Identification
13 Stem Imaging MS and single-cell Terpenoid Indole Alkaloids Yamamoto et al. (2016)
MS
14 Flowers, roots, Reviewed on methods of Terpenoid Indole Alkaloids Almagro et al. (2015);
stem, and leaves extraction Pham et al. (2020)
HPLC, High-Performance Liquid Chromatography; MS, Mass Spectrometry; NF-κβ, Nuclear Factor Kappa light chain enhancer of activated B
lymphocytes; JNK, c-Jun N-terminal Kinase; DAD, Diode Array Detector; UV, Ultra-Violet; IR, Infra-Red; NMR, Nuclear Magnetic Resonance;
qRT-PCR, quantitative Reverse Transcriptase-Polymerase Chain Reaction; UPLC-Q-TOF, Ultra HPLC-quadruple time of MS.
99
are believed to control diabetes effectively, which turns its role in modern
medicine in the 1950s.
Besides its use in traditional medicine by various societies Catharanthus
has been identified with a wide number of applications in pharmaceuticals. It
is found with more than 120 medicinal/pharmaceutically important alkaloid
compounds holding terpenoid and indole structures (Van der Heijden et al.,
2004). Many phytochemicals of Catharanthus are isolated and characterized.
Some are anticancer vincristine, vinblastine, antihypertensive ajmalicine,
sedative serpentine, etc. (Sottomayor and Ros Barcelo, 2005). Though the
vincristine and vinblastine are low in concentration in Catharanthus, they
are widely used as drugs in combination with other chemicals used for the
treatment of leukemia and lymphomas (Gidding et al., 1999).
5.4 ANTICANCEROUS ACTIVITY OF CATHARANTHUS ROSEUS
A significant improvement in medical oncology is the use of cytotoxic

medications for cancer chemotherapy. Although these drugs are used to
target tumor cells, the majority of them can also induce genotoxic, carci
nogenic, and teratogenic outcomes in nontumor cells (Chung et al., 1998;
Philip, 2005). These harmful consequences limit the application of chemo
therapeutic agents despite their high effectiveness in the killing of target
cancerous cells. Therefore, the search for alternative or corresponding drugs
that are successful on cancer cells while showing insignificant toxicity to
normal cells is an operational area of research (Tang et al., 2003). Many
of these investigations are plant-based, folkloric medicine from various
civilizations around the world. Moreover, a report from WHO (World Health
Organisation, 1996) stated that about 80% of the world population is wholly
or moderately dependent on plant-based drugs.
5.4.1 RESEARCH ON CATHARANTHUS
Anticancerous activity of biological compounds, especially alkaloid, mainly

depends on the presence of antineoplatic elements like nitrogenous atoms.
Catharanthus, a proven anticancerous plant, is rich with compounds holding
antineoplastic atoms. Many studies have been focused on determining
the anticancerous activity of natural C. roseus all over the world. Several
indole alkaloids commonly named vinka alkaloids are widely used to stop
the cell mitotic division which ultimately helps in controlling the growth of
cancer cells (Almagro et al., 2015). Sottomayor et al. (2006) identified the
two anticancer compounds vinblastine and vincristine from Catharanthus.
But, Moudi et al. (2013) identified vindesine, vinflunine, and vionorelbine,
the derivatives of the above two compounds. About six compounds isolated
from Catharanthus showed potential anticancer activity and are released
commercially for treating different cancer types (Table 5.2). The commercial
names are oncovin (vincristine), velban (vinblastine), Navelbine (vinorelbine),
vinflunine, cathachunine, and catharanthine. The first patented Catharanthus
compound is vinposidin/leurosidin, an antimitotic molecule by Eli Lilly
Company in 1974 (Keglevich et al., 2012).
5.4.2 DOSAGE AND ANTICANCER ACTIVITY
Studies confirmed that the anticancerous activity of Catharanthus alkaloids

also emphasized on the dose and time of exposure-related function of these
compounds. In general, an optimum dose of compounds can block the
microtubule function effectively and causes apoptosis within a short span
of time, whereas at a low concentration apoptosis may be induced after a
long period of exposure (Attard et al., 2006). Almagro et al. (2015) and Van
der Heijden et al. (2004) tested the anticancerous efficacy of vinblastine,
vincristine, and vinflunine against human cancer xenografts and murine
cancer tissue and found that vinflunine has great binding affinity toward
calmodulin than vincristine and vinblastine which shows highest binding
with tubulins. In vitro cytotoxic effects of catharoseumine holding unique
peroxide bridge have been reported against human cancer cell lines (HL-60)
(Wang et al., 2012).
5.4.3 MECHANISM OF ANTICANCER ACTIVITY
Microtubules of the cellular cytoskeletal components are responsible for

mitotic/meiotic spindle formation during cell division. Spindle fibrils separate
the chromosomes during anaphase. Besides this, microtubules are also held
responsible for transportation, cell structure maintenance, and different other
processes in the cell. Alkaloids of Catharanthus aggravate programmed cell
death and lower the cell division through altering the microtubular dynamics
(Wang et al., 2012). Microtubule formation and dissociation depends on
polymerization and depolymerization of its basic heterodimeric units α- and
β-tubulin. Potential binding of tubulins for the formation of microtubules is
102
TABLE 5.2 Anticancer Compounds of Catharanthus roseus and Their Testing.
S. no. Compounds of Catharanthus Method of testing Type of cancer References
1 Vinblastine In vivo Chronic Lymphocytic Leukemia Leukemia Bates et al. (2013)
2 Vincristine In vitro B cell lymphoma Lymphomas Qiu et al. (2018)
In vitro EA hy926 h-umbilical vein cells; K562 Leukemia Wang et al. (2016)
h-chronic myelogenous leukemia cells; HL60
h-acute promyelocytic leukemia cells.
3 Catharanthine In vitro HCT116 h-colorectal cancer cells Colorectal cancer Siddiqui et al. (2010)
4 Vinflunine In vivo human patients Urethra cancer Schinzari et al. (2018)
C Lung carcinoma Krzakowski et al. (2010.)

5 Vinorelbine In vivo clinical testing Lung carcinoma Nazir et al. (2016)
In vivo clinical testing Breast carcinoma
In vivo clinical testing Solid carcinoma Bahleda et al. (2018)
6 Cathachunine NF-κβ and JNK pathways Leukemia Wang et al. (2016)
NF-κβ, Nuclear Factor Kappa light chain enhancer of activated B lymphocytes; JNK, c-Jun N-terminal Kinase.
maintained by guanosine triphosphate. Moreover, the stability of tubulins in

the cellular system is maintained by electrostatic and van der Wall bonding
and these can be easily destabilized by external molecules when they bind to
it (Coderch et al., 2012). Jordan (2002) explained the destabilization of micro
tubule, which leads to programmed cell death in cancerous tissue. Moreover,
the presence of depolymerizing agents causes no assembly of tubulins, thereby
restricting microtubule formation (Perez, 2009). Cell apoptosis is induced in
the cancerous tissue by Catharanthus alkaloids and their derivatives through
depolymerization of microtubules when these bind with the surface of microtu
bular heterodimers near the GTP-binding site (Bolanos-Garcia, 2009). Gigant
et al. (2005) reported the potential anticancerous functions of Catharanthus
alkaloids through depolymerization of microtubules. Among tested alkaloids
they ranked them as vincristine > vinblastine > vinorelbine > vinflunine for
their anticancer activity. Schutz et al. (2011) and Barbier et al. (2014) reported
a new mechanism for the action of alkaloids against cancer growth. They
identified that calmodulin after binding with microtubule-associated protein
can inhibit the protein synthesis through blocking internal amino acid biosyn
thetic pathways. However, the interactions of Catharanthus alkaloids with
calmodulin are also reported. The mechanism of action of other Catharanthus
alkaloids that are holding anticancerous activity is unknown.
5.4.4 IN VITRO CULTURES AND ANTICANCEROUS COMPOUNDS
Bioavailability of potent anticancerous compounds of Catharanthus is

limited and immense interest has been created in producing these compounds
using different biotechnological methods (Arora et al., 2010). Moreover,
isolation and purification of these compounds from the plant is not cost
effective. The effective method of producing high amount of quality product
is found as plant cell cultivation in vitro. Due to its advantages in producing
a large quantity of secondary metabolites into the culture medium, simple
extraction process and well-controlled sterile systems plant tissue culture
became the best for producing plant-based drugs within a short span of time.
Initially, standard tissue culture methods were followed to produce secondary
metabolites from the source materials. Later it is commercialized to produce
large quantities of these products by cultivating plant cells in high-volume
bioreactors. Submerged culture systems are favorable in producing high-
value medicinal compounds from Catharanthus (Mujib et al., 2014). Verma
et al. (2012) produced catharanthine, ajmalicine, and serpentine in a large
scale using Catharanthus hairy root cultures at bioreactors. But for large
production of vinblastine and vincristine stirred tank-type bioreactors were

found suitable under controlled conditions. The only limitation in these
processes is high amounts of excretory compounds may arrest the growth
of cultured cells. This can be achieved by maintaining controlled conditions
like aeration, pH, temperature, flow of nutrients in the case of continuous
cultures, addition of antifoaming agents etc. during the fermentation process.
Taha et al. (2014) observed the production of alkaloids vincristine (13.47
folds) and vinblastine (7.94 folds) is higher than the normal production
of these alkaloids from intact plant. Chemical agents that are added to
bioreactor can help to reduce the toxicity of secondary metabolites on the
growth of culture cells and production of desired compounds may also
increase. Sometimes, the added chemical may cause toxicity to the cultured
cells if it is not appropriate for cultures. One of such chemical chromium
(10–100 µg) was reported by Rai et al. (2014) for its cellular toxicity, while
producing vincristine and vinblastine production. Furthermore, Fatima et al.
(2015) noticed elevated enzyme activities related to stress with the addition
of sodium chloride in the bioreactor cultivation of Catharanthus cells for its
alkaloids. In contrast to chemicals, Maqsood and Abdul (2017) performed
suspension cultures of Catharanthus with the addition of year extract (1.5
g/L) and found improved production of vincristine and vinblastine. Further,
coculturing of Aspergillus flavus with Catharanthus cells elevated its growth
and alkaloid production (Tonk et al., 2016).
5.5 CONCLUSIONS
Cancer treatments such as chemo and radiotherapies are costly and showing
many side effects on patient’s health. Alternative found best is bioactive
compounds of holding antitumor functions. Catharanthus, a wonderful plant,
is having more than 150 bioactive compounds and holding anticancerous
activity. Major anticancer compounds found in this plant are vincristine and
vinblastine which are available in low content in the plant. Simple extraction
and enhanced quantity need to be improved to meet the demands of bioac
tive anticancerous drugs with an affordable price to the ever-growing human
cancer patients worldwide. In vitro culture methods were found promising
to produce high amounts of Catharanthus alkaloids and were upgraded
to commercial bioreactor level. By-product inhibition of cell growth is
one of the greatest obstacles to producing high amounts of Catharanthus
alkaloids in large-scale fermenters. Addition of chemicals may improve
this but can also create cellular toxicity. One of the promising methods to
improve Catharanthus anticancerous alkaloids production is coculturing of

other suitable organisms in the culture media or addition of biological-based
compounds/extracts. Further, studies in this direction are essential to find
promising organisms and biological extracts/compounds.
ACKNOWLEDGMENTS
The authors are highly thankful to the academic and administrative support
provided by Yogi Vemana University, Kadapa, Andhra Pradesh, India, in
completing this book chapter. The authors are also grateful to the departmental
facility and support of staff for successful completion of this project. The
authors are grateful to PubChem for the structure of Catharanthus alkaloids
and other compounds.
CONFLICT OF INTEREST
The authors declared to have no conflict of interest and have contributed

their part in this book chapter.
KEYWORDS
• cancer
• Catharanthus
• biology
• phytochemicals
• anticancer compounds
• tissue culture
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CHAPTER 6
Withania somnifera (L.) Dunal

PULALA RAGHUVEER YADAV1, LEPAKSHI M. D. BHAKSHU2,
K. VENKATA RATNAM3, ANU PANDITA4, DEEPU PANDITA5, and
K. M. SRINIVASA MURTHY6
1
Department of Biotechnology, Indian Institute of Technology Hyderabad,
Kandi, Telangana State, India
2
Department of Botany, PVKN. Government College (Autonomous),
Chittoor, Andhra Pradesh, India
3
Department of Botany, Rayalaseema University, Kurnool, Andhra Pradesh,
India
4
5
6
Department of Microbiology and Biotechnology,
Jnanabharathi Campus Bangalore University, Bengaluru, India
ABSTRACT
Among noncommunicable diseases in humans, the second most cause of

death is cancer. Vital organs are affected by cancer and various cancers are
increasing worldwide, accompanied by the death rate. So there is a need for
effective drugs for cancer treatment. Chemoprotective drugs are routinely
used but have side effects, whereas plant-based medications have minimal
side effects. In Ayurveda, plants with medicinal properties have been utilized
to cure ailments for centuries. Therefore, plant-based products may reduce
adverse side effects in cancer treatment. Withania somnifera is a shrub, erect
with lengthy tuberous roots, from the Solanaceae family commonly called
Indian winter cherry/Indian ginseng/Ashwagandha, a familiar Ayurvedic
medicinal plant. In Ayurveda, extracts of root/leaf from Withania somnifera
are included in formulations in India. Withania somnifera is used as antioxi
dant, anti-inflammatory, antistress, antiepileptic, antiarthritic, antidepressant,
anticoagulant, antidiabetic, antipyretic, rejuvenative, immunomodulatory,
and antiangiogenesis. The existing research shows that the plant extracts and
their active components have demonstrated anticarcinogenic activity. The
anticarcinogenic activities of Withania somnifera inhibit cancer cell survival,
proliferation, motility, angiogenesis, metastasis, cell cycle arrest, apoptosis
and autophagy induction, etc., on cancers in the last 20 years. Withaferin A
is the most studied drug for anticancer effects in various cancers due to its
therapeutic effect. Herein, we review the anticancer properties of Withania
somnifera extracts and their active components, which have shown inhibi
tory properties against several cancers and their mechanism of action in vitro
and in vivo.
6.1 INTRODUCTION
Cancer is a non-communicable disease in humans and is the second

reason of death. Cancer affects various human organs in the body. There
is a development in the field of drugs toward cancer treatment, but various
cancers are increasing worldwide along with the death rate. Chemoprotective
drugs have side effects, whereas drugs from plants show fewer side effects.
In Ayurveda the Withania somnifera extracts of root/leaf are included in
formulations in India. Withania somnifera (L.) Dunal is a shrub, which is
erect with lengthy tuberous roots and comes from the Solanaceae family
commonly called Indian winter cherry/Ashwagandha/Indian ginseng
known for its several beneficial health activities in India from olden days
which is shown in Figure 6.1. Withania somnifera is used as antioxidant,
anti-inflammatory, antistress, antiepileptic, antiarthritic, antidepressant,
anticoagulant, antidiabetic, antipyretic, rejuvenative, immunomodulatory,
and antiangiogenesis (Subbaraju et al., 2006). Withania somnifera and its
components have an anticancer effect.
Withania somnifera is still under investigation for its anticancer effects
inhibiting cell survival, proliferation, motility, angiogenesis, metastasis, cell
cycle arrest, apoptosis, autophagy induction, etc. Withania somnifera bioac
tive components are shown in Figure 6.2.
Withania somnifera (L.) Dunal 113
FIGURE 6.1 Withania somnifera (L.) Dunal.
FIGURE 6.2 Withania somnifera bioactive compounds.
The withanolides are the bioactive compounds, C28-steroidal lactones

and Withaferin A is the most studied drug for the anticancer effects in various
cancers due to its therapeutic effect. The active component structures (Tao et
al., 2015; Motiwala et al., 2013) involved in the anticarcinogenic effect are
shown in Figure 6.3.
FIGURE 6.3 Structures of active constituents of Withania somnifera against cancer.
The chemistry of the therapeutic effect of Withaferin A is yet to be

understood. Withaferin A chemical structural analysis suggests alkylation
reactions at nucleophilic sites, probably at three positions. Structure–activity
relationship experiments propose Ring A α, β-unsaturated ketone moiety,
OH group at C27 and C5(6) epoxide involvement in the biological activity
of Withaferin A (Berghe et al., 2012). This chapter reviews the Withania
somnifera extracts and their components anticancer effects on various
cancers and their mechanisms of actions.
6.2 BREAST CANCER
Breast cancers were diagnosed in 2.3 million women in 2020 and there were
685,000 deaths worldwide. In the last 5 years 7.8 million were diagnosed
with breast cancer. These data reveal breast cancer as the most prevalent
cancer in the world. Worldwide breast cancer is a severe ailment in women
affecting lakhs of women. Though there are medications, there is still a need
to explore new therapeutic and preventive ways required to reduce the deaths
and suffering from this disease. The medicinal plants and their constituents
are researched for therapy and/or chemoprevention of breast cancer. Here,
we discuss the Withania somnifera and its component’s anticancer effects on
cellular processes and molecular targets.
Kuttan (1996) reported Withania somnifera methanol root extract (Witha
nolide sulfoxide) treatment on breast cancer cell lines suppressed NF-κB
activation. Dar et al. (2019) investigated Withania somnifera aqueous root
extract administration which led to induction of apoptosis in MDA-MB-231
breast cancer cells. Alfaifi et al. (2016) explored Withania somnifera meth
anol leaf extract administration which led to cell cycle arrest and apoptosis in
breast cancer cell lines. Srivastava et al. (2016) investigated the cytotoxicity
of breast cancer cell lines on administering Withania somnifera methanol
and ethanol extracts. Wadhwa et al. (2013) studied Withania somnifera water
extract treatment on MCF-7 breast cell lines, which resulted in the inactiva
tion of p53 and pRB, cyclin B1 reduction and cyclin D1 rise, and downregu
lation of MMP-3 and 9. Stan et al. (2008a) reported the MDA-MB-231 cells
implanted orthotopically in mice treated with Withaferin A for 2.5 weeks.
The in vivo efficacy of Withaferin A demonstrated a significant reduction
in cell proliferation and apoptosis rise in the treated mice. The Withaferin
A-treated mice (P<0.05) showed average tumor volume ~1.8 fold lesser than
control mice. Nagalingam et al. (2014) reported the Withaferin A antitumor
effect in MDA-MB-231 cells. Kim et al. (2016) demonstrated Withaferin A
inhibitory effect in vivo in genetically modified MDA-MB-231 cells with
Notch2 knockdown. Thaiparambil et al. (2011) proved the antitumor activity
of Withaferin A in the orthotopic mouse. Here, Withaferin A at two doses 2
and 4 mg/kg were given intraperitoneally on alternate days for a month.
Liu et al. (2019) reported Withaferin A efficacy in the MDA-MB-231
xenograft model. Samanta et al. (2017) reported MNU-induced Luminal
type breast cancer in the rat model and Withaferin A administration was done
intraperitoneally at two doses 4 and 8 mg/kg from the second week after
N-Methyl-N-nitrosourea induction for five times in a week till the 10th week.
Tumor incidence, multiplicity, and weight showed a decrease. Withaferin
A treatment in MCF-7 and MDA-MB-231 cells for 24 hours inhibited cell
viability with an IC50 between 1.5 and 2.0 µmol/L being reported by Stan
et al. (2008a). Thaiparambil et al. (2011) investigated vimnetin cytoskeleton
inhibition by Withaferin A. Vimnetin overexpression has a relationship with
the induction of EMT. Imaging studies explained the perinuclear vimnetin
accumulation preceded by quick vimnetin depolymerization by Withaferin A.
Withaferin A at less than/equal to 500 nM retained an effective anti-invasive
effect. Structure–activity relationships explained that the expected vimnetin
binding region of Withaferin A is essential to induce ser56 phosphorylation
in vimnetin. Withaferin A showed an antimetastatic activity in breast cancer
via its effects on vimnetin and vimnetin ser 56 phosphorylation. Hahm et al.
(2011a) investigated the MCF-7 cells proliferation by Estrogen stimulation,
inhibited by Withaferin A. Stan et al. (2008b) reported the increase in the
G2-M fraction in a dose- and time-dependent manner on the treatment of
Withaferin A in breast cancer cell lines; concomitantly, there was a decrease
in CDC 25B and/or CDC 25 C, cyclin-dependent kinase 1 causing a buildup
of Tyrosine15 phosphorylated (inactive) Cdk1. Withaferin A-mediated arrest
at the G2-M phase in MDA-MB-231 cells was protected partially by the
overexpression of cell division cycle 25 C. Zhang et al. (2011) reported the
G2/M arrest in breast cancer cells from the Withaferin A treatment. Antony et
al. (2014) showed Withaferin A arrest at the G2 and mitotic phase in MCF-7,
SUM 159, and SK-BR-3 cell lines and linked with reduction of β-tubulin
levels. Withanone and Withanolide A is C6, C7-epoxy analogs of Withaferin
A could not cause mitotic arrest in these cells. Withaferin A dislocated the
spindle morphology. In MCF-7 cells, the Withaferin A bounded covalently
to Cys303 of β-tubulin. The molecular docking studies explained that the
β-tubulin has the binding pocket (Hydrophobic floor & wall with hydro
philic entrance) on its surface for the Withaferin A. Samanta et al. (2018)
investigated the cell cycle arrest on the administration of Withaferin A in
the MCF-7 cell line. The role of peptidyl-prolyl cis/trans isomerase 1 is cell
cycle arrest. Lee et al. (2012) showed a Withaferin A inhibitory effect on cell
migration in breast cancer. Withaferin A treatment in MDA-MB-231 cells
inhibited invasion ability and there was a decrease in the gene expression
profiles of ADAM8, PLAT, uPA (Extracellular matrix-degrading proteases),
pro-inflammatory mediators (ANGPTL2, TNFSF12, CSF1R, IL6) and cell
adhesion molecules (Integrins, Laminins) (Szarc et al., 2014).
Withaferin could inhibit experimental EMT and cell migration. Treatment
of TNF-α and TGF-β could partially reverse the process (Lee et al., 2015).
Withaferin A induces death in breast cancer cell lines by ROS-mediated parap

tosis (Ghosh et al., 2016). MNU induced Luminal-type breast cancer in the rat
model. Withaferin A administration was done intraperitoneally at two doses, 4
and 8 mg/kg, from the second week after N-Methyl-N-nitrosourea induction,
five times until the 10th week. There was an increase in TUNEL-positive
apoptotic cells (Samanta et al., 2017). There was no change in the complex
III assembly in MDA-MB-231, but complex III decreased in SUM159 and
MCF-7 cells as analyzed by gel electrophoresis. The fusion process involved
proteins, their expression was decreased, with the other proteins OPA1,
mitofusion 1, mitofusion 2 also after Withaferin A treatment. The protein
DRP1 was also decreased after the Withaferin A treatment (Sehrawat et al.,
2019). Withaferin A treatment against MDA-MB-231 xenografts and breast
cancer cell lines in vivo induce apoptosis. ROS production led to suppres
sion of oxidative phosphorylation and complex III activity after Withaferin
A treatment. Breast cancer cell lines Rho-0 variants (Mitochondrial DNA-
deficient) were resistant to ROS induced by Withaferin A. A new insight, that
is, ROS production and activation of Bax/Bak is seen into the mechanism of
Withaferin A-induced apoptosis (Hahm et al., 2011b). One of the transcription
factors of the forkhead box, FOXO3a, is identified in breast cancer cells as a
mechanistic target and its knockdown in MCF-7 cells resulted in protection
against Withaferin A-mediated apoptosis involving target Bim (Stan et al.,
2008a). The progression of proliferation, metastasis, and chemoresistance in
breast cancer has been implicated by overexpression and constitutive activa
tion of STAT3 (Ma et al., 2020).
Withaferin A inhibits the STAT3 transcription factor in breast cancer cells.
It is observed that Withaferin A suppresses constitutive and/or inducible
activation of STAT3 along with phosphorylation of Janus activated kinase 2
(Upstream regulator) in breast cancer cell lines. Withaferin A administration
in breast cancer cells furthermore affected inhibition of a. STAT3 transcrip
tional activity with or without stimulation of IL-6; b. dimerization of STAT3
in any case in MDA-MB-231 cells; and c. nuclear translocation of STAT3
(phosphorylated) in both cells. In MDA-MB-231 cells, facilitated activa
tion of STAT3 by IL-6 conferred limited defense counter to cell invasion
inhibition by Withaferin A (Lee et al., 2010). 17 β -estradiol (E2) attenuated
the Withaferin A treatment in MCF-7 cells. In the MDA-MB-231 cell line,
overexpression of ER-α conferred limited but statistically significant defense
against Withaferin A-mediated apoptosis, but not the arrest of G2/M phase
(Hahm et al., 2011a). Zhang et al. (2011) investigated downregulation of
ER α protein expression in MCF-7 cells after Withaferin A treatment. Hahm
et al. (2011b) reported Withaferin A treatment in MCF-7 cells induced p53

activation and Ser 15 phosphorylation. Withaferin A induced apoptosis in
MCF cells and protection was due to RNA interference of p53. Withaferin
A induces apoptosis and various molecular changes in human breast cancer.
Withaferin A facilitated extracellular signal-regulated kinase (ERK) hyper
phosphorylation but not p38 MAPK/c-jun NH-terminal Kinase, and this
change was partially protected by manganese superoxide dismutase overex
pression. Withaferin A-induced apoptosis in MCF-7 cells was prominently
increased by the suppression of p38 MAPK and ERK. On the other hand in
MCF-7 cells were partly attenuated by JNK inhibition (Hahm et al., 2014).
6.3 CERVICAL CANCER
Cervical cancer is seen in women, and the Human papillomavirus expressing

onco proteins E6 and E7 is the cause. 311,000 approximately died from the
disease in 2018 and in women cervical cancer is the fourth common cancer.
Withaferin A inhibits the CaSki cervical cancer cells proliferation. The
Withaferin A mechanism involved the decrease in the E6 and E7 oncoproteins
expression. There were changes like induction of p53 accumulation and change
in the p53 levels-mediated apoptotic markers-cleaved PARP, caspase-3, Bax,
Bcl2, rise in p21cip1/waf1. Withaferin A interaction with PCNA causes G2/M
phase arrest and the variation of cdc2, cyclin B1, p34 and PCNA levels and
STAT3 and its phosphorylation at Tyr705 and Ser727 decreases. The in vivo
studies in athymic nude mice results showed a similar trend to that of in vitro
in manipulating molecular markers (Munagala et al., 2011).
6.4 OVARIAN CANCER
Ovarian cancer is seen in women and the seventh most common cancer.
Ovarian cancer has the highest death rate among gynecologic cancers. In
2018, ovarian cancer deaths percentage constituted 4.4% of total mortality
from cancers in women. The efficacy of Withaferin A in the treatment of
ovarian cancer is proved. The combination of Withaferin A and Doxorubicin
against A2780/CP70, A2780, and CaOV3 epithelial cancer cells expresses a
dose- and time-dependent synergistic effect on inhibition and induction of
cell death, thus reducing ill effects and dosage requirement of Doxorubicin.
ROS generation showed a noticeable improvement, leading to induction
of autophagy, massive DNA damage as observed under TEM. Analysis of

cleaved caspase 3 explained the increase in LC3B (autophagy marker)
expression and led to cell death. The combination therapy was validated
in 3 dimension (3D) tumor model and xenograft model of ovarian cancer;
the results showed a reduction of 70–80 % in tumor growth compared with
control or animals treated with Doxorubicin or Withaferin A alone (Fong et
al., 2012). Withaferin A and Cisplatin showed efficacy in the treatment of
refractory ovarian cancer. Withaferin A at 1.5 µM concentration prominently
reduced the spheroid formation in the A2780 ovarian cancer cell line and not
by Cisplatin. Combination of Withaferin A and Cisplatin prominently reduced
spheroid formation comparatively to control, Withaferin A only, and Cisplatin
alone treatment. In orthotopic ovarian tumors bearing nude mice, Cisplatin
treatment increased cancer stem cells. In contrast, Withaferin A administra
tion prominently decreased Aldehyde Dehydrogenase cancer stem cells
and the combination resulted in the reduction of Aldehyde Dehydrogenase
Cancer stem cells. Withaferin A and Cisplatin alone/in combination treatment
in orthotopic ovarian tumors bearing nude mice caused complete metastasis
inhibition and 70–80% decline in tumor growth (Kakar et al., 2014).
6.5 PROSTATE CANCER
Second common cancer in men is prostate cancer and worldwide it is the fifth
cause of mortality. There were about 358,989 deaths from prostate cancer
in 2018. Withania somnifera extracts and Withaferin A are used in prostate
cancer for the treatment. Moselhy et al. (2017) reported that Withaferin A
showed a chemopreventive effect in Pten conditional knockout mice with
constitutively activated AKT signaling. Das et al. (2016) investigated
Withaferin A delaying tumor progression in prostate cancer by activating
prostate apoptosis-4. Rah et al. (2015) reported in prostate cancer cells
about the switching from autophagy to apoptosis by pro-apoptotic protein
PAWR-mediated suppression of BCL-2 after the administration of 3-azido
derivative of Withaferin A. Withaferin A administration resulted in cell
death in PC-3 and DU-145 androgen-independent cancer cells (Nishikawa
et al., 2015). Balakrishnan et al. (2017) said Withania somnifera extract in
androgen-independent prostate cancer cell lines (PC3) for the inhibitory
effects on metastasis. The expression of COX-2 and IL-8 in PC3 cells in 24
hours is prominently suppressed by Withania somnifera extract, controlling
disease advancement from androgen-dependent to androgen-independent.
6.6 PANCREATIC CANCER
Pancreatic cancer is more common in men than in women and is the seventh
leading death-causing cancer worldwide. Yu et al. (2010) reported Witha
ferin A inhibition efficacy and the mechanism of Hsp90 in pancreatic cancer.
It has shown the antiproliferative effect in BxPc3, MiaPaCa2, and Panc-1
pancreatic cancer cell lines with ICs of 2.78, 2.93, and 1.24 µM. Withaferin A
induced apoptosis in Panc-1 cell line in a dose-dependent way and is verified
by Annexin V staining. The Hsp90 chaperone effect to promote the break
down of Hsp90 proteins was suppressed by Withaferin A and evidenced by
Western blot. Pancreatic Panc-1 xenografts tumor growth was inhibited by
administering Withaferin A at 3, 6 mg/kg by 30 and 58%, respectively. Thus,
Withaferin A binds to Hsp90, suppresses its chaperone activity, and shows
anticancer activity against pancreatic cancer. Oxaliplatin is limited in use
for the treatment of pancreatic cancer. Li et al. (2015) reported Oxaliplatin
induced growth inhibition, apoptosis, and its enhancement of this activity
by Withaferin A in pancreatic cells. This growth inhibition and apoptosis
involve PI3K/AKT pathway inactivation and mitochondrial dysfunction.
Combination therapy leads to an antitumor effect better than the single drug
in pancreatic cancer treatment.
6.7 LUNG CANCER
Lung cancer is the leading cause of cancer death worldwide. In 2020, the
deaths were 1.80 million and were the highest among all cancers. Choudhary
et al. (2010) reported the compounds Withaferin A, chlorinated steroidal
lactone and diepoxy withanolide inhibited the growth and had cytotoxic
activity in NCI-H460 lung cancer cell line. Withaferin A has been the most
significant (GI = 0.18 µg/mL and LC = 0.45 µg/mL). Cai et al. (2014) studied
the activity of Withaferin A in non-small cell lung cancer A549 and observed
the inhibition of cellular growth and apoptosis by PI3K/AKT pathways
inactivation. Withaferin A is screened insilico as a significant antilung cancer
and antilung cancer stem like cell agent and showed cytotoxicity toward
various lung cancer cells, along with induction of apoptosis and autophagy.
ROS activation plays an upstream role in mediating Withaferin A-elicited
effects. Withaferin A suppressed the growth of lung cancer stem like cell and
spheroid forming capacity, reducing side population. Withaferin combined
with Cisplatin and Pemetrexed showed synergistic effects on suppressing
EGFR wild-type lung cancer (Hsu et al., 2019). Withaferin A treatment

in non-small cell lung cancer (NSCLC) A549 and H1299 NSCLC cells
demonstrated time- and dose-dependent cytotoxicity. After this, the cells
were treated with ≤ 0.5 µM Withaferin A for ≤ 4 h to decrease cytotoxicity
and examine its effects on EMT, cell adhesion, motility, migration, and
invasion. The pretreatment of cells with Withaferin A showed inhibition to
cell adhesion, migration, and invasion of A549 and H1299 cells. Withaferin
A inhibited Epithelial to mesenchymal transition (EMT) in A549 and H1299
NSCLC cells, induced by TGFβ1 and TNFα. The phosphorylation and
nuclear translocation of Smad2/3 and NF-κB in A549 and H1299 cells are
inhibited by Withaferin A. Hence, Withaferin A is a potential therapeutic
agent against metastasis in NSCLC (Kyakulaga et al., 2018).
6.8 COLORECTAL CANCER
Colorectal cancer is second common cancer seen in women, and third in men.
There were 935,000 deaths in 2020 from colorectal cancer. Kuttan (1996)
reported Withania somnifera methanol root extract (withanolide sulfoxide)
treatment on cell lines suppressed TNF-induced NF-κB activation. Alfaifi et
al. (2016) showed cell cycle arrest and apoptosis in breast cancer cell lines by
the administration of Withania somnifera methanol leaf extract. Withaferin
A induced apoptosis in colorectal cancer cells as verified by Flow cytometry
and western blot results. Withaferin A stimulates production of ROS and its
accumulation, which causes the mitochondrial membrane potential loss and
mitochondrial dysfunction (Xia et al., 2018). Koduru et al. (2010) reported
in the colon cancer cells that the Withaferin A suppressed Notch-1 signaling
and downregulated prosurvival pathways. There was also downregulation
of the expression with p4E-BP1and pS6K, the rapamycin signaling compo
nents of mammals. Withaferin A activated apoptosis via c-Jun-NH-kinase in
colon cancer cells.
6.9 OTHER CANCERS
6.9.1 LEUKEMIA
Mondal et al. (2010) reported in leukemia patients, derived lymphoid and

myeloid cells accompanied by primary cells, administered with WithaD,
improved the accumulation of ceramide, and induced apoptosis. Senthil et al.

(2007) reported in promyelocytic leukemia HL-60 cell lines about the induc
tion of apoptosis by the treatment of Withania somnifera methanolic extract
of leaf and withanolide. There was caspase 9,8,3 activation and release of
cytochrome c.
6.9.2 MYELOID LEUKEMIA
The mechanism of action by Withaferin A causing cytotoxicity in cancer

cell lines is not apparent. It induced reactive oxygen species production
and mitochondrial membrane potential loss, subsequently the release of
cytochrome c, Bax mitochondrial translocation, and factor inducing apoptosis
to cell nuclei, concomitantly, caspases 9, 3 activation and cleavage of PARP.
The extrinsic pathway activated by Withaferin A is evident from the hike in the
levels of caspase 8 activity in a time-dependent way next to overexpression
of TNFR-1. Thus in human leukemia cell lines HL-60, Withaferin A induced
ROS and mitochondrial dysfunction elicit actions responsible for apoptosis
in mitochondrial-dependent and independent pathways (Malik et al., 2007).
6.9.3 LYMPHOBLASTIC AND MYELOID LEUKEMIA
Withaferin A treated lymphoblastic and myeloid leukemia patients’

primary cells and leukemic cell lines from humans showed a potent growth
inhibitory effect. Withaferin A induced programmed cell death (apoptosis)
as evidenced by the activation of p38 MAPK signaling cascade, that is, the
rise in phosphorylated p38MAPK. The phosphatidylserine externalization
demonstrates apoptosis, mitochondrial transmembrane potential loss, and
the release of cytochrome c. Along with a surge in Bax/Bcl-2 ratio on time,
caspases 3, 9 activations, DNA fragmentation showed the buildup of cells in
sub-G0 (Mandal et al., 2008).
6.9.4 SKIN CANCER
Mayola et al. (2011) reported in human melanoma cells that Withaferin A

induced apoptotic cell death. The mechanism involved the Bcl-2 down-
regulation, translocation of Bax to mitochondria, the release of cytochrome c
transmembrane dissipation, caspase 9,3 activation, and DNA fragmentation.

Swiss albino mice induced by 7, 12-dimethylbenz(a)anthracene resulted
in skin cancer. The mice have been treated with the Withania somnifera
hydroalcoholic root extract at 400 mg/kg po (high dose). The extract had
a chemopreventive effect against skin cancer (Prakash et al., 2002). Halder
et al. (2015) reported Withania somnifera root extract was tested in A375
cells and has a potent cytotoxic effect, DNA fragmentation, nuclear blebbing
and cell morphology supported by MTT assay, Agarose gel electrophoresis,
DAPI staining, and fluorescence microscopy.
6.9.5 GLIOBLASTOMA
Shah et al. (2009) reported the Withania somnifera alcoholic extract of leaf,
its constituents and their blends to show the ability to induce growth arrest
and differentiation in YKG1 and C6 glioma cell lines. The analysis at the
molecular level resulted in cell migration delay, glial fibrillary acidic protein
and neuronal cell adhesion molecules enhanced expression. Kataria et al.
(2011) investigated the Ashwagandha leaf extract treatment in the glioma
cells showing antiproliferative and differentiation activity. The presence of
these activities in the Ashwagandha leaf extract is supported by the Neural
cell adhesion molecule, Mortalin (Hsp 70) and Glial fibrillary acidic protein
expression levels observed changes, the MTT assay and wound scratch
assay. The Ashwagandha water extract treatment in the orthotopic glioma
allograft in the rat model reduced intracranial tumor volumes and inhibited
the cyclin D1, Hsp70, pAKT, NF-κB, VEGF, PSA-NCAM. The upregulation
of mortalin and NCAM and decrease in GFAP indicate the Ashwagandha
water extract antiglioma efficiency in vivo (Kataria et al., 2016).
6.9.6 NEUROBLASTOMA
The study revealed Ashwagandha water extract to decrease cell prolifera

tion and induce differentiation in IMR-32 neuroblastoma cells of humans.
Induced expression of NCAM and polysialylation decrease explained the
Ashwagandha water extract antimigratory activity. Along with MMP 2 and
9 activity downregulation (Kataria et al., 2013), Caputi et al. (2018) reported
methanol root extract of Withania somnifera treatment on Neuroblastoma
cell lines alters gene expression of opioid receptors.
6.9.7 DALTON’S ASCITIC LYMPHOMA
Withania somnifera has shown an anticarcinogenic effect in the Swiss albino

mice with Dalton’s Ascitic Lymphoma. Withania somnifera ethanolic root
extract showed a surge in lifespan, a fall in tumor weight and cancer cell
number. There was a hematological improvement (Christina et al., 2004).
6.9.8 MOUSE EHRLICH ASCITES
Devi et al. (1995) investigated Withania somnifera aqueous root extract treat
ment on Mouse Ehrlich ascites carcinoma showing a radio sensitizing effect.
6.9.9 RENAL CARCINOMA
Um et al. (2012) investigated the Caki human cancer cells that were adminis
tered with Withaferin A. Withaferin A induced apoptosis mediated by the STAT3
signaling pathway (Bcl-xL, Bcl-2, Survivin, and cyclin D1) downregulation.
6.9.10 LIVER CANCER
Alfaifi et al. (2016) reported cell cycle arrest and apoptosis after the administration
of Withania somnifera methanol leaf extract on breast cancer cell lines. Ashwa
gandha water extract showed antioxidant and anticancer activity in the HepG2
hepatocellular carcinoma cell line. The levels prominently were increased in
Glutathione S-transferase, glutathione reductase and total antioxidant, caspase
3,8 and 9 activities, Fas-ligand and the level of TNF-α decreased significantly.
The HepG2 cells treated were arrested at the G/G and G/M phases (Ahmed et
al., 2018). Zhou et al. (2016) reported Withaferin A inhibition in Hepatocellular
carcinoma cells proliferation. Along with these changes: (1) there was a rise
in apoptosis rate and arrest at G1 phase, (2) rise in p53, bax expression, and
reduction in bcl-2 expression, and (3) upregulation of p21 and downregulation
of CDK2 and cyclin D1.
6.10 CONCLUSIONS
Traditional medicines can be the best alternative medicines as they are reason
ably priced and effective. For these herbal medicines to be an alternative,
proper studies have to be conducted to look into safety, effectiveness, and

superiority. The research studies of Withania somnifera and its components
in human cancer cells and rodent models have indicated the anticancer
activity. Hence, Withania somnifera can be used as an additional therapy to
decrease the side effects of chemo and radiotherapy and as a combination
therapy with conventional treatments to improvise the effects of chemo and
radiotherapy. Finally, clinical studies are to be done for the validation before
translation into clinical application.
KEYWORDS
• Withania somnifera
• withaferin A
• anticancer effects
• mechanism of anticancer activity
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CHAPTER 7
Camptotheca acuminata Decne

AMOGHA G. PALADHI1, ANU PANDITA2, MANOHAR M. V.3,
BHOOMIKA INAMDAR3, SUGUMARI VALLINAYAGAM4,
DEEPU PANDITA5, and K. M. SRINIVASA MURTHY6
1
2
3
4
Department of Biotechnology, Mepco Schlenk Engineering College,
Sivakasi, Tamil Nadu, India
5
6
Bangalore University, Bengaluru, India
ABSTRACT
Camptotheca acuminata Decne belonging to the family Nyssaceae is a

native flora of Asian countries like South China, India, Pakistan, etc. In the
early 1960s, the fruits of Camptotheca acuminata Decne were used in folk
medicine against cancer. In the 1970s, curiosity led to an accidental discovery
of a group of alkaloids named Camptothecin from the plant Camptotheca
acuminata Decne. Extractions were made to analyze the therapeutic
properties of this alkaloid against tumors. The cause of previously known
anticancerous property of C. acuminata is the alkaloid named Camptothecin.
In the late 1980s, SAR studies were made to learn more about Camptotheca
which showed a primitive anticancer property in its native form of planar
pentacyclic ring structure. Analogues of this component were prepared in

various experiments to improve the property of Camptothecin like solubility
and antitumor properties. Efforts were made to extract Camptothecin from
various parts of the plant leading to the comparative studies of concentration
of Camptothecin in various stages of its life. Camptothecin enters the stages of
cell cycle wherein the enzyme DNA TOP1 acts for replication and winding of
DNA. Camptothecin inhibits DNA replication by binding itself to DNATOP1
by forming covalent complexes. The Camptothecin derivatives are created
by various groups of scientists to treat a very wide range of malignancies like
Adeno-carcinoma of Thyroid, Hepatocellular, Colo-rectal, Ovarian, Lung,
Leukemia, and many more. Topotecan, Irinotecan, and Exatecan are widely
used analogues in preclinical and clinical trials. To improve the efficiency
of Camptothecin, approaches were made with the use of nanostructures like
Poly (methacylic acid-co-methyl methacrylate) nano-formulations. Thus,
alkaloids like Camptothecin and its analogues from Camptotheca acuminata
Decne can be used in anticancerous activity in most human malignancies.
7.1 INTRODUCTION
Camptotheca acuminata Decne is one of the native floras of South China,
Punjab, Pakistan, and other South Asian countries. Camptotheca acuminata
Decne is commonly called Chinese Happy Tree (Martino et al., 2017). It is
one of the plants used in native medicine therapies as the alkaloids in the
plant are of high medicinal value and an interesting genus of ethnobotany.
In the late 1980s, one of its alkaloids belongs to a group of drugs known
as camptothecins. Camptothecin (CPT) is known to be a chemotherapeutic
drug. The natural alkaloid obtained from various parts of Camptotheca
acuminata Decne is 20-(S)-camptothecin (CPT).
Camptothecin is known to be an anticancer drug for its property to
break the strands of DNA in mammalian cells by efficiently inhibiting DNA
topoisomerase 1 (Gong et al., 2018). Due to this property, with the goal of
increasing its efficiency to treat cancer, various derivatives or analogues
have been developed till date (Guo and Yuan, 2016). In the 1960s, revolution
in screening of various plants was made in search of steroid compounds.
The screenings for steroids were made to test its properties like antitumor,
antibacterial, and antiviral activities. One of those plants was Camptotheca
acuminata of South China, and the alkaloid from this tree was first extracted
from its bark. It was found that the alkaloid belonged to the group of
Camptothecin which is an anticancer drug with its property of specifically
inhibiting the activity of DNA TOP1 enzyme, which was capable of DNA
Camptotheca acuminata Decne 133
replication. This led to a milestone in the discovery of anticancer drugs as

the root cause of cancer that can be treated by camptothecins. The extract
of Camptotheca acuminata Decne was first tested in vivo to treat mouse
leukemia and its first discovery of naturally obtained alkaloid Camptothecin
being an antitumor agent (Wani et al., 1987). In the 1970s, it was found
that camptothecins are able to inhibit DNA and RNA replication in various
organisms (Chabner, 1992). In cancerous tumor, the root cause of cell
differentiation and proliferation is replication of its genetic material due to
the increased activity of DNA TOP1. As this process increases the number
of copies of DNA leading to karyokinesis and cytokinesis, it causes cell
division. The increased action of the enzyme TOP 1 is due to the formation
of covalent complex between substrates (DNA) and enzymes. The action
of TOP1 is found to be winding an unzipping of replication forks in DNA,
where these sites of interests are the target for analogues of Camptothecin to
act on. Camptothecin action leads to the formation of noncovalent complex
that drastically decreases and inhibits DNA replication, and also prevents
recombination of double-stranded helix. DNA is found to be stable and its
double-helix model, due to the enzyme inhibition action of CPT; the DNA,
once divided into single strands, is not capable of winding torsionally by
itself leading to the death of cell. Various experiments have shown that
the activity of CPT is directly proportional to the concentration of DNA
TOP1 (Hsiang et al., 1985; Redinbo et al., 1998). DNA TOP1 is found to be
hypersensitive to its CPT-induced cytotoxicity, meaning that the higher the
concentration of enzyme, the easier for the CPT to inhibit its action causing
cell death (Dancey and Eisenhauer, 1996). In various studies on cancer,
where it is found that due to the high number of dividing cells in tumor, the
concentration of the enzyme DNA TOP1 is very high, because it is known
that cancer cells are highly dividing regardless of requirement (Martino et
al., 2017). It is easier for CPT and its analogues to efficiently act on the
enzyme due to its availability as a substrate in cancer tissues. Thus, CPT is
known to be a leading anticancer drug in recent decades (Champoux, 1978).
In later discoveries, CPT is found to have very less solubility; this is a
limitation of CPT to be used as a naturally available form. Thus, discoveries
regarding improvisation of solubility of CPT were made leading to the
development of its analogues. The analogues are found to be more water
soluble which can be clinically used.
SAR studies suggested that the anticancer activity of camptothecin
depends on the structural orientation and presence in a number of rings,
meaning that the least number of rings that must be present for CPT to
act against cancer is 5 (planar pentacyclic ring structure). Further studies
showed that lesser number of rings in CPT makes the molecule very unstable
and useless. Addition of benzene ring to its structure makes it hexacyclic
in nature showing an elevated anticancer activity. As there were developed
SAR studies, it is found that the synthesis of CPT analogues with the addi
tion of benzene ring is more efficient; lactone is one of the core molecules
from which various derivatives were obtained, thus adding a sixth ring to the
planar structure making it hexacyclic (Chabner, 1992). These SAR studies
led to the discovery of analogues like topotecan, irinotecan, and exatecan.
Topotecan and Irinotecan are approved for clinical use by FDA. In later
studies, Exatecans showed very promising and efficient anticancer activities
in preclinical studies (Dancey and Eisenhauer, 1996).
7.2 PHYLOGENY
The genus Camptotheca is well known as a source for camptothecin (CPT),
an anticancer drug. The genus was first termed in 1983 and was placed
under the family Cornaceae along with other genus like Davidia, Nyssa,
and Cornus, based on its morphology, anatomy, palynology, embryology,
cytology, pollination, phytochemistry, phylogenetic position, and paleobi
ology. However, some taxonomists placed Camptotheca under the family
Nyssaceae. The most recent APG (angiosperm phylogeny Group) system of
classification that includes molecular phylogenetic analysis confirmed that
Camptotheca can be placed under the family Nyssaceae along with other
genus Davidia, Nyssa, Cornus, Diplopanax, and Mastixia. Nyssaceae is
now placed as a sister clade comprising loasaceae, hydrostachyaceae, and
hydrangeaceae (Gong et al., 2018).
7.3 CAMPTOTHECIN (CPT)—AN ANTICANCER DRUG

Camptothecin (CPT) is an alkaloid that is produced by a native tree of South
China called Camptotheca acuminata Decne. It is a quinoline alkaloid having
a pentacyclic ring system consisting of a quinolinic portion, pyrrolidine,
a lactam, a stereo center configured on α-hydroxy lactam system with
delocalized aromatic moiety (Wall et al., 1966). In the late 1980s, it was found
that this group of camptothecin drug can be used to treat various cancerous
conditions. Initially, it was used in various clinical trials against colon
carcinoma, thus evaluating CPT as an anticancer drug with the approval of
FDA (Food and Drug Administration) (Martino et al., 2017). SAR (Structural
Activity Relationship Studies) has led to the discovery of various derivatives
of CPT that are more eligible and efficient to treat cancer. Analogues of
CPT with a planar pentacyclic system are the only component that helps in
anticancer treatment, while other cyclic systems like bi, tri, and tetracyclic are
not found to have any biological activity (Wall et al., 1966; Wani et al., 1980).
There are structures of CPT that contains more than five ring planar
structures that also show anticancerous activity in a conserved manner.
Hexacyclic and heptacyclic show more activities than pentacyclic, thus
showing a planar structure of CPT with five rings and more have elevated
biological activity while the lesser ones are inert (Wani et al., 1980). The
replacement of lactam-containing benzene ring might lead to the complete
loss of anticancer activity in CPT (Nicholas et al., 1990) and also the opening
of the ring structures of α-hydroxy lactone by hydrolysis shows very low
antitumor activity results in the reduction in the activity of CPT molecule,
if the spatial arrangement of structures forming the complex is disoriented
(Jaxel et al., 1989; Rowinsky et al., 1992; Wani et al., 1987).
7.3.1 CAMPTOTHECIN ANALOGUES

Camptothecin was later modified to increase its therapeutic value due to
its low solubility. Efforts were made to develop the derivatives with more
stability and solubility than that of the naturally obtained alkaloid. Various
synthetic and semisynthetic derivatives associated with anticancerous
property were brought into action. Studies have reported that the in vivo and
in vitro evaluation resulted in increased activity of camptothecin analogues
that were derived. Various solubilizing groups were added to the planar
ring structure of CPT to increase its solubility leading to the discovery of
highly efficient therapeutic derivatives called Topotecan (Date et al., 2002),
Irinotecan (Fukuda et al., 1996), and Exatecan (Pizzolato and Saltz, 2003).
7.3.1.1 TOPOTECAN
Topotecan is one of the first found derivatives of CPT. Topotecan is a
water-soluble analogue approved by FDA (Food and Drug Administration)
for clinical use. The structure of topotecan is 9-[(dimethylamino)methyl]-
10-hydroxycamptothecin. Various in vitro experiments and preclinical trials
were performed that showed an excellent anticancer property. Later, Topote
cans were put to use in treating adeno-carcinomas of colon, ovary, and breast
cancer. The tumors of CNS (central nervous system) and sarcomas were also
treated well and cured by the administration of the same (Pizzolato and Saltz,
2003).
FIGURE 7.1 Structure of camptothecin.
FIGURE 7.2 Structure of topotecan.
FIGURE 7.3 Structure of irinotecan.
7.3.1.2 IRINOTECAN
Kawoto et al. in 1991 discovered the activity of Irinotecan which is a water-
soluble analogue of CPT. It is found that irinotecan is 1000 times more
efficient than the native camptothecin. Irinotecan is an active compound
that shows anticancerous property in various types of carcinoma cell lines,
both in in vitro and in vivo. Irinotecan is otherwise scientifically named as
7-ethyl-10-(4-[1-piperidino]-1-piperidino) methyl-10-hydroxycamptothecin
or just CPT-11. SN-38 is the irinotecan analogue of CPT which was used by
Kawoto et al. that resulted in the inhibition of topoisomerase- I activity, also
showing anticancerous activity in HT-29 human colon carcinoma cells in in
vitro. Irinotecan is known to be the most biologically active anticancerous
drug found in its array of analogues (Dancey and Eisenhauer, 1996).
7.3.1.3 EXATICAN
Exatican mesylate (C25H26FN3O7S) is one of the recently known DNA TOP1
inhibitors. Exatican is a derivative analogue of CPT which is water soluble
in nature. Clinical trials were known to be experimented on Colo-rectal

cancer in earlier days. The studies have shown that Exaticans resulted in the
maximum activity on biliary (bile duct and pancreatic) cancer (Dancey and
Eisenhauer, 1996).
7.4 EXTRACTION OF CAMPTOTHECIN FROM VARIOUS PARTS OF

CAMPTOTHECA ACUMINATA DECNE
7.4.1 BARK
As alkaloid Camptothecin is known to exhibit efficient antileukemic activity,
Wall et al. considered the extraction of the alkaloid from the stem wood
of Camptotheca acuminata Decne as they quoted stem wood was extracted
by a standard method. They tried to crystallize camptothecin by methane-
insoluble material from chloroform to crystallize the alkaloid resulting in the
formation of pale yellow needles of camptothecin (Wall et al., 1966).
7.4.2 FRUIT
The alkaloids form Camptotheca acuminata was mostly extracted from the
fruits of the plant. It was found that folk medicine also used fruits of this
plant as therapy against various malignancies. Liu et al. in 2013 extracted
camptothecin from the fruit and used it in clinical trials (Y. Liu et al., 2013).
An aqueous extract from the fruits of Camptotheca acuminata can inhibit
the growth of cancer cells and this was experimented by Lin et al. in 2014
(Lin et al., 2014). Guo et al. considered using dried fruits of Camptotheca
acuminata, 50% ethanol at 80 ̊C was used to extract camptothecin from the
coarse powder (He et al., 2019).
7.4.3 LEAF
Wang et al. separated and extracted the alkaloids that were major secondary
metabolites in the leaves of Camptotheca acuminata (Wang et al., 2015).
Experiments have shown that younger leaves contain more camptothecin
when compared with older leaves. It was found that the concentration of
camptothecin was decreased by 11% as it gets older by every month. The
concentration of camptothecin in extraction depends on the way of approach
it is extracted. Oven-dried leaves showed 27% less CPT concentration than
freeze-dried leaves (Z. Liu et al., 1998).
7.5 STRUCTURE–ACTIVITY RELATIONSHIPS (SAR) OF

CAMPTOTHECINS
Various studies have been made to examine the structure of quinoline alkaloid
revealing Camptothecin, a pentacyclic planar ring structure that consists of
pyrrolo (3,4-β) quinoline moiety and also consists of an α-hydroxy lactone
ring within which an asymmetric center is present (Wall et al., 1966). It is
suggested that the planar pentacyclic ring is the most capable structure of
camptothecin which has the capability to inhibit the activity of DNA TOP1.
If this ring is removed or disoriented, the structure of CPT gets distorted and
loses the property of its activity. Thus, it is concluded that CPT has a deprived
activity in bi, tri, and tetracyclic compounds (Cesare et al., 2000). SAR also
showed that the activity of CPT is increased in its structure containing more
than five ring compounds suggesting that a pentacyclic ring system is required
for anticancerous property of CPT.
Earlier SAR studies have also concluded that an oriented structure of CPT
with intact α-hydroxy lactone group is one of the most essential structures
(Venditto and Simanek, 2010) that have been identified to denature DNA
TOP1 as a target for its antitumor activity (Hsiang et al., 1985; Jaxel et al.,
1991, 1989; Kjeldsen et al., 1988; Thomsen et al., 1987).
The interest of cure in cancer has been prioritized in poisoning TOP1
as it is the most efficient enzyme that helps in DNA duplication and other
biological activities of DNA. Thus, showing derivatives of camptothecin
obtained from Camptotheca acuminata Decne with the combination of
various other compounds showed an excellent anticancerous property
(Hsiang and Liu, 1988; Hertzberg et al., 1989).
7.6 CPT SYNTHESIS, SEMISYNTHETIC DERIVATIVES

CPT was natively extracted from C. acuminata which is native species of
South China. As this species is found to be endangered and belongs to red
line species, if the extraction increases leading to the exploitation then it
would result in the extinction of the species. Efforts were made to chemi
cally synthesize this alkaloid synthetically and semisynthetically (Martino
et al., 2017). Stork et al. in 1971 were the first to synthesize the synthetic
form of CPT (Martino et al., 2017). This procedure consisted of 15 steps
in which prior nine were molecules chemically synthesized by Danishefsky
in 1993 (Shen et al., 1993). Curran et al. in 1994–1996 developed radicalic
pathway (Martino et al., 2017). Comins in 2001 first advanced an optimized
procedure consisting of six steps of total synthesis (Comins et al., 2001).
7.7 MODE OF ACTION

Camptothecin (CPT) obtained from Camptotheca acuminata Decne is found
to be a blocker or inhibitor of an enzyme known as DNA Topoisomerase-I in
mammalian cells (Martino et al., 2017). It is a profound anticancer activity
where CPT poisons DNA Topoisomerase-I in tumor cells. It has various
anticancerous activities, but these are limited due to its poor solubility and
high degradation rate (Gigliotti et al., 2017). Researches are conducted to
assist the use of camptothecin by manipulating it using the encapsulation of
b-cyclodextrin-nanosponges (CN-CPT) that helps in increasing the solubility
of CPT. It increases the rate of uptake in tumor or cancer cells and also to
reduce the toxicity of the tissue by degrading and inhibiting the growth factors
of tumors in both in vitro and in vivo conditions (Gigliotti et al., 2017). Due
to its low solubility, experiments were carried out to set up the discovery in
the use of camptothecin (CPT) by increasing its property of effectiveness
and water solubility by deriving it synthetically and semisynthetically.
Structure–activity relationship studies (SAR) were clinically conducted with
various numbers of small and large derivatives of CPT. Mainly, topotecan
(7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) and
irinotecan (9-[(dimethylamino)methyl]-10-hydroxy-camptothecin) are the
two SAR derivatives of CPT that are in current clinical use (Venditto and
Simanek, 2010).
As CPT is well known for its DNA Top1 inhibition activity, the enzyme
is mostly involved in various significant biological activities of DNA like
repair, recombination, replication, and transcription which are the core stages
for cell replication and proliferation that happens in normal cell (Hertzberg
et al., 1989; Hsiang et al., 1985). This principle of DNA TOP1 inhibition
like CPT is used to damage the enzyme which in turn inhibits the above
stages of DNA. This process, when experimented on tumor cells, inhibits
cell division and proliferation thus inhibiting the tumor and also helps in
curing the existing cancerous conditions. DNA is a stable nucleic acid and
also genetic material of all mammals that exist in stable double helix, double
strand. When the helix is untangled and the DNA is spilt into single strand,
DNA TOP1 helps to maintain and restore the integrity of DNA double
helix by rotating each antiparallel single strands together (Chabner, 1992;
Pommier, 2006; Stewart et al., 1998).
Core of DNA TOP1 enzyme directly acts on DNA while the other subdo
mains of enzymes like I and III help in the stabilization of DNA and enzyme
complex by the formation of salt bridges. The spatial arrangement of the
DNA/enzyme complex allows Tyr123 to form covalent phosphodiester bonds
in 3'-end of DNA at active sites of enzymes, thus activating enzyme catalysis

(Stewart et al., 1998). The action of camptothecin was discovered in the
late 1980s which breaks the interaction of enzyme catalysis. The core target
of camptothecin and its derivatives are DNA TOP1 where they act to form
a noncovalent bond complex between DNA and Topoisomerase-I enzyme
(Hsiang et al., 1985; Redinbo et al., 1998). This results in the breaking of
DNA double-stranded helix and leading to the irreversible process causing
cell death. Results of various experimental approaches have shown that the
toxicity of CPT is directly proportional to the concentration of enzyme in the
tumor. This means that higher levels of enzymes present in cancerous cells
show hypersensitivity to the cytotoxic activity of CPT. The above explana
tion also demonstrates the abnormally high availability of enzymes results in
the formation of cancer cells, also helping them to replicate and proliferate
(Dancey and Eisenhauer, 1996). The equilibrium has to be maintained in
between the presence of free TOP and DNA TOP1 complex in the cell. If
the equilibrium is disrupted by shifting either of the entities from normal to
high, it results in cancer formation. To inhibit this action, a tertiary complex
is formed that inverts the free CPT into bound form by the formation of a
TOP1-DNA complex (Pizzolato and Saltz, 2003).
The formation of this tertiary complex leads to the shifting of free TOP
that results in greater DNA replication leading to the formation of new cells.
When such conditions are treated with CPT, it breaks the complex resulting
in inhibiting tumor formation.
In contrast, the 3-hydroxy group-induced Camptothecin is used reversibly
to interact with an enzyme that unbounds DNA where the cell is damaged
due to the inability of DNA to form double helix by the inhibited activity
of TOP1 by CPT. 20-hydroxy group is required for DNA TOP1 complex
stabilization and also shows anticancerous activity (Hertzberg et al., 1989).
7.7.1 EFFECT OF CAMPTOTHECIN IN VARIOUS INCIDENCES
7.7.1.1 IN VITRO ANTICANCER ACTIVITY
Studies that are made on the treatment of human colon cancer cell-line,
Caco2 by using CN-CPT (cyclodextrine nano-sponges) or camptothecin
encapsulated with poly-nano-formulations in in vitro to analyze the antican
cerous activity. When greater quantities like >1000 μg/ml of camptothecin
were encapsulated using poly (methacylic acid-co-methyl methacrylate)
FIGURE 7.4 Mode of action.

nano-formulations resulted in enhanced anticancerous activity of camptoth

ecin on Caco2 cell-line (Manikandan and Kannan, 2017).
FIGURE 7.5 Inhibition of DNA-TOPI activity.

7.7.1.2 IN VIVO ANTICANCER ACTIVITY
Studies were made on rats considering body weight as a criterion to detect

the capability of camptothecin encapsulated with poly (methacylic acid
co-methyl methacrylate) nano-formulations as an anticancerous drug. The
above is compared with other batches of rats that were administered with
DMH (1,2 dimethylhydrazine). The rats that were treated with camptothecin
poly-nano-formulations showed excellent results of increased weight gain
denoting increased health of rats. This showed colon carcinoma in rats was
cured better by CN-CPT when compared with DMH (Manikandan and
Kannan, 2017).
7.7.1.2.1 Effect of Camptothecin on ACF Incidence
Agner et al. conducted an experiment on groups of rats to demonstrate the

treatment of CPT to cure colo-rectal carcinoma by considering colonic ACF
(aberrant cryptic foci) as a biomarker that shows the intensity of occurring
tumor. This experiment demonstrated the use of camptothecin encapsulated
poly (methacylic acid-co-methyl methacrylate) nano-formulations signifi
cantly decreased the levels of colonic ACF, resulting in 100% cure in rats
suffering from colo-rectal carcinoma (Agner et al., 2005).
7.7.1.2.2 Effect of Camptothecin on Tumor Incidence
Hsiang et al. demonstrated an experiment using Burkit’s lymphoma cell-

line (Kjeldsen et al., 1988), where they reported increase in the concentra
tion of camptothecin by 50 µM in every trial to demonstrate the action of
camptothecin that is known to bring the equilibrium of TOP1 and DNA
by torsionally relaxing the coiled DNA (Thomsen et al., 1987). Various
other experiments also showed the cleavage reaction in the DNA–TOP1
complex (Thomsen et al., 1987). Around 70% of DNA molecules were
cleaved at the site of recognition sequences. This shows specificity in
the sequence of enzymes in varied ratios of enzyme and substrate. The
equilibrium dissociation remains a constant, portraying the action of
camptothecin helps in the cure of tumors and also the influence of camp
tothecin on the cleavage properties of human topoisomerase-I (Thomsen
et al., 1987).
7.7.1.3 CN-CPT INHIBITS CELL PROLIFERATION IN VITRO
Experiments were conducted to record the anticancerous activity of CN-CPT

when compared with naturally occurring form of camptothecin in cell-line
studies. In in vitro conditions, animal cancer cell models were cultured in
various titrated amounts to obtain a viable number of cells and assessed
using MTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-
carboxanilide) assay. The results of the above studies confirm that CN-CPT
has a very high efficiency in inhibiting the growth of cultured cell-lines when
compared with free CPT. Very little differences were seen when tested for
different cell-lines due to the effect of time and concentration. Variable effects
of inhibition by effective doses were also observed. To confirm the results,
the cells were cultured for more number of days to increase the number of
cells. CN-CPT and CPT were again administered where CN-CPT showed
the inhibition of multiple colonies being a better derivative of Camptothecin
than the free form (Gigliotti et al., 2017).
7.8 TARGET ORGANS
7.8.1 THYROID CANCER CELLS
Anaplastic carcinoma of thyroid is a very lethal cancer that is malignant in

nature and must be effectively cured by stopping the proliferation (Gigliotti
et al., 2017). CN-CPT is used in various in vitro, in vivo experiments and
clinical trials. This resulted in inhibiting the proliferation of endothelial
cells adhesive to anaplastic carcinoma of Thyroid and also it inhibits the
migration of these cells. The CN-CPT activity is studied where it regulates
the formation of adhesive cells leading to lamellipodia and increased cell
motility (Kim et al., 2013; Occhipinti et al., 2013).
7.8.2 HEPATOCELLULAR CARCINOMA
Stotz et al. discovered that hepatocellular carcinoma (HCC) being a malignant

tumor condition with high mortality rate can be treated using CPT (Stotz et
al., 2015). In the later stages of human hepatocellular carcinoma, the chances
of surgical removal of tumor are very less. Simultaneously, an indeed option
was found to be chemotherapy (Hu et al., 2013). By this, surgeries can be
avoided due to the therapeutic use of CPT in treating human HpG2 and
Hep3B cell-lines which showed more positive results compared with other
modes of treatment (He et al., 2019).
7.8.3 COLO-RECTAL CANCER
Tubular adenomas are cancers that are macroscopically sessile that have been
induced in a batch of cell-lines; later it was compared with the treatment of
DMH and CPT. As CPT does not show much expected results, CPT was
encapsulated using poly (methacylic acid-co-methyl methacrylate) nano
formulations and the cell-lines were treated, thus depicting the anticancerous
activity of CPT (Manikandan and Kannan, 2017; Pizzolato and Saltz, 2003).
7.8.4 OVARIAN CANCER
Ovarian cancer being a very lethal condition for woman must be treated.
The use of CPT derivatives like Topotecans helps in avoiding DNA replica
tion and cell proliferation in Ovary that helps in the maintenance of a static
number of cells and also helps in curing the tumor (Pommier, 2006).
7.8.5 LUNG CANCER
Derivatives or analogues of CPT are used in the treatment of lung cancer.

Exatecan is intravenously administered in human subjects suffering from
lung cancer. Topotecan is also one of the CPT derivatives used in its treat
ment. The mode of action remains the same in almost every cancer treat
ment. The motto is to inhibit the binding of DNA TOP 1 enzyme that helps
in winding and unwinding of DNA that has torsional rotation, thus helping in
chemotherapeutical treatment of lung cancer (Pommier, 2006).
7.8.6 LEUKEMIA
Wall et al., in one of their works, have used leukemia as a subject to be

treated using camptothecin. As camptothecin is found to be insoluble, it was
standardized using various derivatives and ring structures. The analogues
of camptothecin exhibited antileukemic property that cured leukemia in
samples. They used X-ray diffraction studies to determine the molecular

activity of camptothecin derivatives (Wall et al., 1966).
7.9 LIMITATIONS
Camptothecins and its derivatives despite being beneficial in treatments

of cancers also have some limitations that are inevitable. Maintenance of
cleavage complexes of DNA and TOP1 is one of the due concerns of CPT
administration. The TOP1ccs (cleavage complexes) should be maintained in
tumor cells until the camptothecin converts the complex into damaged DNA
by its action of inhibition. This leads to a prolonged infusion to maintain the
cleavage complexes persistently until DNA is damaged. Another unexpected
limitation of camptothecin administration procedure by infusion is that it
might cause leucopenia, but this limitation can be resolved by limited
administration of camptothecin. Irinotecans when administered have been
studied for off-target effects due to the induced diarrhea. The derivatives
of camptothecin obtained from lactone like α-hydroxylactone E-ring have
a very short shelf-life and been converted into carboxylate that is inactive
against DNA TOP1 which binds to serum albumin (Pommier, 2006).
7.10 CONCLUSION
To conclude with, various studies and experiments that are conducted in

vitro and in vivo on various animal and human carcinoma modules and
cell-lines have shown that camptothecin and its derivatives can efficiently
be used as therapeutic drug in cancer. X-ray crystallographic studies have
shown that TOP1 is an enzyme that cleaves the DNA and also helps in the
replication. In experiments, CPT has shown anti-topoisomerase activity by
the action of inhibition. Studies have shown that the camptothecin is more
efficient in the presence of Cu (II) and long wavelength UV radiations.
Various camptothecin analogues were used in the anticancer treatment and
experiments that also showed the similar but elevated activity in forming a
noncovalent complex of DNA-TOP1 or inhibiting the enzyme from binding
DNA. Free camptothecin and 10-hydroxycamptothecin together are known
for its anticancer activity.
Camptothecin has unique properties that act only against the enzyme
TOP1 in any given cells with DNA. Studies have confirmed the above, by
experiments in which the TOP1 gene is removed from the cells. It is seen
that if the cell-lines show single-point mutation in TOP1 gene, such cells
show resistance to camptothecin. Thus, SAR studies led to the discovery
of analogues of CPT that showed efficient antitumor activities against
tumor cells. Tumor cells being highly replicable have increased activity of
TOP1 due to which DNA duplication takes place. CPT has a main role in
inhibiting this enzyme thus stopping tumor cell proliferation. Studies have
shown that Camptothecin and its derivatives have the capability of curing
almost any cancer by its inhibition activity that poisons TOP1. Cancers like
adenocarcinoma of thyroid, hepatocellular carcinoma, colo-rectal, ovarian,
lung, leukemia, etc. increase the activity of camptothecin; the molecules
were encapsulated using poly (methacylic acid-co-methyl methacrylate)
nano-formulations.
KEYWORDS
• Camptotheca acuminata Decne

• Camptothecin
• anticancer drug
• antitumor activity
• Topoteca
• Irinotecan
• Exatica
• Chinese Happy Tree
• DNA TOP1
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https://doi.org/10.1021/jm00179a016
CHAPTER 8
Taxus Baccata
ROHIT SAM AJEE1 and SHUCHI KAUSHIK2
1
Independent Researcher and Alumni Amity Institute of Biotechnology,
Amity University Madhya Pradesh, India
2
State Forensic Science Laboratory, Madhya Pradesh, India
ABSTRACT
Cancer is the perfect example of an abnormality developed in the cells

leading to disastrous effects on overall wellbeing of the organism. It is the
result of certain factors which are not always controllable, resulting in a
disorder which can afflict almost any part of the human body and also has the
ability to spread. Modern science has been looking for a cure to this ailment
for several decades with some progress. Current cancer treatment options
include the use of toxic compounds or invasive techniques to immobilize and
terminate cancerous cells. However, these treatments are also responsible for
severe side effects, some of which are almost as deadly as the original disease
as well. The desire to treat this debilitating condition with minimal side
effects has driven science towards nature to look for an answer. There have
been several plants and other biological systems, which have been studied
exclusively for their ability to treat cancers. Taxus baccata is one such plant.
The European Yew, as the plant is commonly known, has been shown to
contain some phenolic metabolites which express significant antioxidant as
well cytotoxic properties which could aid in tumor treatment. Several studies
have also been carried out which used the plant extract as encapsulating
material for anticancer nanoparticles as well as the plant for green synthesis
of the said nanoparticles. However, the ace in the hole for Taxus baccata is
its ability to produce the anticancer agent known as Paclitaxel. Although
conventionally, Taxus brevifolia is used for the production and harvesting

of taxol, recent studies have focused on alternate plants for the same. This
study focuses on the characteristic medicinal properties of Taxus baccata
with special reference to anticancer efficacy.
8.1 BASIC INTRODUCTION TO CANCER, ITS TYPES, AND

PLANT-BASED TREATMENT OPTIONS
A rapid, unregulated cellular division that starves the surrounding tissues

to death while also occasionally having the ability to infiltrate and traverse
to different parts of the body is one way to briefly sum up the biological
disorder known as cancer. A lot of research and experiments are done related
to this topic for a diverse number of reasons such as to identify the under
lying cause of cancer, to discover potential cures, to identify the key features
of cancerous cells and tumors, to fully understand the disruptive effects it has
on the body, etc. While we have made progress, it is by no means an issue
we have completely understood yet. In 2011, Hanahan and Weinberg talked
about the Six Hallmarks of Cancer in their report. The hallmarks according
to them are (Hanahan and Weinberg, 2011):
• Continuous Proliferative Signaling
• Escaping from the growth Suppressors
• Opposition to Cell Death
• Facilitating Replicative Immortality
• Prompting Angiogenesis
• Stimulating Invasion and Metastasis.
The pair elaborately describes a hypothetical cell which undergoes
neoplastic transformations and gradually accrues each of the hallmarks
mentioned above that eventually results in malignant tumor mass. In
addition to attaining the final neoplastic state, the report also mentions that
the tumors seem to become more than just a bunch of rapidly proliferating
cells. They express a complex arrangement with the inclusion of multiple,
distinct cell types that interact with each other and surrounding tissues to
further propagate itself. This revelation was further built upon to create
the concept of the Tumor Microenvironment (TME). Earlier studies
identified genetic (and eventually epigenetic) changes as the sole factor
responsible for cancer development and proliferation. However, research
on the matter has revealed that in addition to Genetic and Epigenetics,
Taxus Baccata 153
Tumor Microenvironment (TME) is just as substantial an element in tumor

progression (Baghban et al., 2020). The neoplastic cells within the tumor
communicate with surrounding nonmalignant cells and extracellular matrix
components like the collagen, laminin, fibronectin, and immune cells like
macrophages and lymphocytes via complex signaling pathways. Once
control has been obtained, the tumor cells force the surrounding cells to
further stimulate tumor growth. The nature of interaction between a tumor
and the TME establishes the bond as a critical means of tumor progression.
Consequently, the TME can be assumed as an ideal target for disrupting and
potentially even terminating tumor development.
To this end, Nia et al. have recently published a study in which the four
physical traits associated with most tumors and their TME were discussed
upon. As the tumor develops and starts interacting with surrounding
noncancerous cells, they induce physical alterations to the microenviron
ment. These alterations may then affect the biology of the cancer and their
response to cures (Nia et al., 2020). One may view the traits mentioned
in this review as the physical counterpart (addition even) to the biological
hallmarks that were established in Hannagan and Weinberg’s review. These
physical abnormalities along with the biological hallmarks of tumors
provide a more complete understanding of the cellular as well as physical
nature of cancer.
• Elevated Solid Stress
• Elevated Interstitial Fluid Pressure (IFP)
• Increased Stiffness and Altered Material Properties, and
• Altered Microarchitecture
These are the four physical hallmarks of cancer.
Now that we have basic understanding as to the nature of cancer, let us
explore some treatment options for it. Classical cancer treatment options
generally involve invasive and at times potentially harmful options like
chemo and radio therapy and surgery. With time however, science and
research has started focusing on more holistic options. Plant-based cancer
treatment is one such field that has massive potential. Several plants exist
in nature that contains active compounds which can aid curing or treating
cancer in one form or another. It is up to the scientific community to identify,
extract, and enhance these compounds and put them to use. As indicated
by extensive research work, there are numerous anticancer drugs clinically
recognized and are suggested for the malignancy therapy (Stark, 2006;
Berman et al., 2000).
8.1.1 CURRENT CANCER THERAPY VIA PHYTOCHEMICALS: A NOVEL

APPROACH
Since primordial age, use of phytochemicals to treat and cure a number

of medical conditions had been a regular practice. Traditional Indian and
Chinese medicine systems relied almost extensively on plants and plant-
based phytochemical extracts to alleviate sickness. However, due to the lack
of written records and general mistrust towards ancient medicine systems,
science generally disregarded these cures. All that changed however with the
discovery and isolation of morphine from Papaver somniferum. This single
finding was more than enough to generate curiosity regarding the presence
of medicinal compounds in plants, which fleshed out over the years. With
time, a wide range of medicinally useful compounds and drugs that were
extracted from plants were approved for use (Newmann and Cragg, 2016).
Some of these compounds have been found to possess significant
antitumor properties as well, making them suitable for cancer treatment.
Based on the property of the compound and the origin of extraction, these
complexes show a variety of curative properties and target specific path
ways or products involved in the carcinogenic process. While some help in
preventing the proliferation of cancerous cells (Yan et al., 2018), others delay
the progress of the tumors by neutralizing free radicals (Lee et al., 2013) or
interact with tumor activator/suppressor proteins to help in regulating the
process (Adams et al., 2010). The following section will briefly talk about a
few phytochemical agents with potential anticancerous properties.
A study regarding the efficiency and ability of Allicin, a major biological
compound present in garlic, to suppress the proliferation of Cholangiocarci
noma (CCA) indicated that the compound was able to significantly suppress
proliferation (Chen et al., 2018). The compound was able to do so by
influencing the STAT3 signaling pathway. The organic sulfur compound was
found to be capable of inhibiting the STAT3 signaling pathway which then
prevents tumor cells from migrating or invading other tissues. The study was
also able to identify that allicin induces apoptosis of the tumor cells. These
results were obtained from both in vitro as well as in vivo studies. What
makes allicin even more attractive is the fact that CCA is largely resistant
to conventional cancer therapy treatments like chemotherapy, which neces
sitates the formulation of effective alternate treatment options.
Apigenin, a phytochemical belonging to the flavonoid class, has been
recently targeted for its anticancerous properties. It has potential use in
chemotherapy as a substitute to conventionally used agents due to its low
Taxus Baccata 155
toxicity and high antitumor property (Madunic et al., 2018). For cancers
that affect the head and neck regions of the body, apigenin was found to
promote apoptosis by upregulating the expression of Tumor Necrosis Factor
Receptor pathway. It has shown similar activity in the inhibition of breast,
prostate, pancreatic, skin, and colorectal cancers. Another factor that works
in favor of apigenin is that it is able to positively interact with other drugs
and compounds used in conventional chemotherapy. The flavonoid is able
to augment the beneficial properties of the drugs while decreasing their
harmful effects. For example, apigenin and paclitaxel have a synergistic
interaction wherein apigenin promoted the absorption and half-life of the
other compound. Apigenin loaded nanoparticles were used to study the
effects it had on hepatocellular carcinoma in rats (Bhattacharya et al., 2018).
The study was able to conclude that apigenin loaded nanoparticles were able
to slow down the progress of hepatocellular carcinoma in both in vivo and in
vitro studies. The presence of the carrier increased the accuracy of transmis
sion as well biodistribution of the compound in the tumor-specific site. The
controlled release of the drug ensures optimal delivery resulting in higher
cytotoxicity and improved cancer suppression as compared to free apigenin.
Resveratrol is yet another phytochemical derived from Polygonum
cuspidatum which was found to express considerable antitumor properties.
When the polyphenolic compound was administered to female Sprague–
Dawley rats to test its effects in controlling mammary carcinogenesis, it
was observed that the phytochemical was significantly suppressing tumor
expression (Banarjee et al., 2002). The compound was able to do so by
downregulating the expression of the transcription factor, NF-κB. Over
the recent years, the role of NF-κB as a tumor-promoting element has been
cemented via multiple studies (de Martin et al., 1999). Resveratrol’s ability
in inhibiting the expression of this key component in tumorigenesis greatly
inhibits overall tumor spread and progression. Furthermore, in vitro studies
revealed that resveratrol was able to inhibit the spread of the breast cancer in
a concentration-dependent manner.
Gingerol is a type of flavonoid with antioxidant potential and is commonly
found in fresh ginger. The compound is shown to exhibit significant anti-
inflammatory and anticancerous properties (de Lima et al., 2018). In vitro
studies conducted on the action of gingerol on triple negative breast cancer
(TNBC) showed that the phytochemical was able to induce concentration-
dependent cell death in both human and mouse cell lines (Martin et al.,
2017). The study was also able to identify that gingerol initiated cellular
apoptosis by influencing the activation of caspase-3 and related pathways.
Quite surprisingly, the study also concludes that the phytochemical is capable
of preventing or at the very least, inhibiting the spontaneous metastasis in
subjects where the primary tumor was removed.
Taxus baccata has, for quite some time, been under interest because of
its alkaloid parts. Despite the fact that its alkaloids share normal primary
similitudes with some others, they have a one of a kind taxane ring. These
compounds function by advancing and balancing out microtubule arrange
ment and thus inhibiting mitosis. Microtubules are keys for cell division, and
tubulins polymerize to frame microtubules within the sight of microtubule-
associated protein (MAP) and GTP. It appears to be conceivable that taxol
targets microtubules’ dimeric proteins. Subsequently, microtubules become
nonfunctional, which meddles cell separating and obstructs cell cycle.
Because of strange groups shaped along these lines, nonfunctional microtu
bules get distributed (Manish et al., 2005). Albeit some atomic instruments
are assumed for the activity of taxus species in the disease interaction, it
appears to be very conceivable that there may be some different components
obscure yet. In this way, further examinations are required.
8.2 BRIEF REVIEW ON TAXUS BACCATA: THE WONDERFUL PLANT

WITH MULTIFARIOUS ACTIVITIES
The European yew or Taxus baccata is found across various parts of the
northern side of the equator, mostly in temperate regions. Aside from the
organic product, it is a small to medium-sized evergreen tree that generally
has been utilized for weapon production and making medicines (Abella,
1996). The Genus Taxus has the following classification system:
Class: Pinopsida
Order: Taxales
Family: Taxaceae
Species: T. baccata (European or English yew), T. brevifolia (Pacific yew
or Western yew), T. canadensis (Canadian yew), T. chinensis (Chinese yew),
T. cuspidata (Japanese yew), T. floridana (Florida yew), T. globosa (Mexican
yew) and T. wallichiana (Himalayan yew)
Cross breeds: Taxus × media = T. baccata × T. cuspidata and Taxus ×
hunnewelliana = T. cuspidate × T. canadensis.
Because the species are so similar, they are regularly simpler to isolate
geologically than morphologically (Cope, 1998).
Taxus Baccata 157
A group of researchers assessed the potential impacts of Taxus baccata

extract on D-Adenosine deaminase (ADA) action in harmful and noncan
cerous human gastric and colon tissues to explain its anticancer potential.
D-Adenosine deaminase (ADA) is a chemical (EC 3.5.4.4) associated with
purine digestion. It is required for the breakdown of adenosine and for the
turnover of nucleic acids in tissues. It is available practically in every single
mammalian cell and its essential capability in organism is the development
and support of the immune system (Kingston, 2005). However, the full
physiological function of ADA isn’t yet totally understood (Wilson et al.,
1991). ADA affiliation has additionally been seen with epithelial cell separa
tion, neurotransmission, and incubation maintenance (Cristalli et al., 2001).
It has likewise been recommended that ADA, notwithstanding adenosine
breakdown, animates arrival of excitatory amino acids and is vital for the
coupling of A1 adenosine receptors and heterotrimeric G proteins (Wilson
et al., 1991).
According to a scientific viewpoint, utilization of ADA inhibitors has
helped much in understanding the component activity of adenosine metabolites
and analogs. ADA inhibitors have additionally prompted the comprehension
of the administrative cycles related to immunodeficiency portrayed by an
absence of ADA, and development of the invulnerable response (Glazer,
1980). One of them, pentostatin (Nipent) is a nucleoside simply having
the capacity to restrain ADA chemical. Hindrance of ADA impedes the
deamination responses in the purine rescue pathway, consequence of which
is the restraint of ribonucleotide reductase. Subsequently, this cycle exhausts
the nucleotide pool and limits DNA synthesis (Brown et al., 2008).
Furthermore, taxol forestalls spread of metastatic disease cells by
repressing cell relocation. Paclitaxel essentially acts at the G-2/M-stage
intersection. Nonetheless, docetaxel acts primarily in S-period of mitosis.
These compounds are not harmful to non-dividing cells since they don’t
need the mitotic spindle. Hence, they act just on multiplying cells (Manish
et al., 2005). Additionally, as the microtubule framework is fundamental for
the arrival of different cytokines, balance of cytokine discharge by this drug
might assume a significant part in its anticancer activity (Smith et al., 1995).
8.3 TAXANE AND OTHER ACTIVE COMPOUNDS PRESENT IN

TAXUS BACCATA
Taxus baccata, or the European Yew which is its common name, has been a
subject of interest to the scientific community for quite a while now. These
TABLE 8.1 Pharmacological Activity of Taxanes Against Different Types of Cancer.
158
Taxane type Study system Important outcomes of study
Paclitaxel laden nanostructured In vitro: HepG2 liver In vitro: Enhancement of ROS generation in addition to dose-dependent cell
lipidic carriers carcinoma cells viability decrease. Chromatin condensation, condensed as well as fragmented
In vivo: Wistar rats nuclei were observed all of which points to apoptosis.
In vivo: Enhanced absorption of paclitaxel (Harshita et al., 2019)
Docetaxel encapsulated in gold In vitro: HepG2 liver In vitro: The docetaxel nanoparticles lowered cell viability in a
nanoparticles carcinoma cells concentration-dependent manner. Evidence indicating apoptosis like cell
conglomeration and bleb and bulge development were observed.
Apatite carrier In vivo: Mice In vivo: When treated with docetaxel nanoparticles, mice were observed to
revert to standard live architecture (Wan et al., 2018).
Docetaxel In vitro: Hepatic stellate cells, In vitro: Dose-dependent decrease in the percentage of cell viability of
carboxymethylcellulose Hep3B, and HLF Human hepatic stellate cells, HCA-1, Hep3B, and HLF cells.
nanoparticles carcinoma cells Molecular level: Dose-dependent downregulation of α-smooth muscle actin
In vivo: Male C3H/ (α-SMA). Additionally, collagen I at protein and mRNA levels was also
HeNCrNarl mice observed to be downregulated.

In vivo: Inhibition of tumor growth (Chang et al., 2018)
Cabazitaxel In vitro: SK-hep-1, SM- In vitro: A time and dose-dependent inhibition of Sk-hep-1, Huh-7, SMMc
MC7721, Huh-7, HCC-LM3, 7721, and HCC-Lm3 cell lines.
Huh-TS-48, and SK- sora-5 Cell cycle arrest: SK-hep-1 and Huh-7 cell lines treated with cabazitaxel
showed an increase in the number of cells in the G2-M stage.
Molecular level: Decrease in the expression of Cdc2, pCdc2, Cdc25c, and
cyclin B1 protein levels.
Induction of apoptosis: Increased ratio of apoptotic cells in SK-hep-1 and
Huh- 7 cell lines along with a decline in antiapoptotic protein Bcl-2 and a
stronger cleavage of poly ADP-ribose polymerase (PARP) (Chen et al., 2018)
Taxus Baccata
Paclitaxel and survinin In vitro: A549, A549 lung In vitro: A549 cells showed increased levels of nuclear fragmentation,
siRNA encapsulated into cancer cells. cytotoxicity, chromosomal abnormalities, and G2/M cell cycle arrest.
polyethyleneimine-block Animal model: BALB/c nude In vivo: Effective tumor inhibition (Jin et al., 2018)
polylactic acid mice
Caffeic acid with paclitaxel In vitro: Non-small cell lung Antiproliferative action: NSCLC H1299 showed decreased proliferation.
cancer (NSCLC) H1299 cells H1299 cells were arrested in the sub G1 phase of the cell cycle and
and normal Bease-2b cells approached apoptosis.
Molecular level: Bax and Bid were activated, and PARP was cleaved
downstream. Phosphorylation of extracellular signal-regulated c-Jun
NH2-terminal protein kinase1/2 and kinase1/2 was also observed. MAPK
pathway involved in apoptosis was activated (Min et al., 2018).
Micelle delivery system (TP-M) In vitro: Caco-2, A549, and In vitro: Due to improved uptake of PTX-TP-M compared to regular PTX,
loaded with paclitaxel (PTX) Lewis lung cancer cells higher toxicity was observed against A549 and Lewis cells.
with Plasdone S-630 Copovidone In vivo: Male Sprague– In vivo: Treatment with PTX-TP-M yielded higher tumor inhibition. Tumor
(PVPS630) and vitamin E-TPGS Dawley rats and male cell volume and mitotic cells were reduced (Hou et al., 2017).
(TPGS) as carriers C57BL/6 mice
PEGylated liposomes containing In vitro: A549 lung cancer In vivo: Cotreatment with docetaxel and telmisartan showed significant
docetaxel with telmisartan cell tumor inhibition and mostly intact lung integrity in mice.
In vivo: Sprague–Dawley Molecular level: Antiapoptotic marker expression was decreased (survinin) and
rats, athymic Nu/nu mice metastasis markers MMP9 and MMP2 were downregulated (Patel et al., 2016).
Noscapine and paclitaxel In vitro: LNCaP and PC-3 In vitro: Increased apoptosis in cancerous cell lines coupled with decreased
prostate cancer cells cell viability
Molecular level: mRNA expression of Bcl-2 was significantly decreased
and mRNA expression of Bcl-2-associated X protein Bax, and Bax/Bcl-2
159
ratios in LNCaP and PC-3 cells were increased. Prostate-specific antigen and
androgen receptor expression was also decreased (Rabzia et al., 2017).
160
Docetaxel with desmopress In vitro: PC3 prostate cancer In vivo: Significant mice tumor volume reduction (Bass et al., 2019)
cell
In vivo: Mice
Combination of Impressic acid In vitro: VCaP prostate cancer In vitro: Strong antiproliferative activity was expressed in the form of
and Acankoreanogenin with cell apoptosis in VCaP cells.
docetaxel Molecular level: Reduced nuclear factor-κB (NF-κB) activity, decreased
expression of Bcl-2, NF-κB, p-Akt, and phosphorylated signal transducer
and activator of transcription 3 (p-Stat 3) and increased expression of
phosphorylated c-Jun N-terminal kinase (p-JNK) (Jiang et al., 2018)
Docetaxel-loaded nanoparticles In vitro: LnCaP and PC3 In vitro: Increase in lactate dehydrogenase release in the culture medium of
prostate cancer cells prostate cancer cells indicating concentration-dependent cell death (Gallego-
Yerga et al., 2017)
Cabazitaxel In vitro: Human prostate In vitro: Reduced proliferation in both the cancer cell lines
cancer LNCaP and PC-3 cells Molecular level: Androgen receptor and androgen receptor-associated factors

HSP90α, HSP40, and HSP70/HSP90 organizing protein levels were lowered.
Activity of antiapoptotic factor HSP60 was also suppressed (Rottach et al.,
2019).
Cabazitaxel and silibinin In vitro: PC-3 and DU-145 In vitro: Dose-dependent inhibition of cell migration, cellular growth, and
coencapsulated cationic prostate cancer cells induction of apoptosis
liposomes Cell cycle arrest: Upon treatment G2/M cell cycle arrest and prevention of
colony formation was observed.
Nucleus morphology: Evidence indicating apoptosis like cell blebbing,
membrane shrinkage, and nuclear granulation were observed
(Mahira et al., 2019).
Taxus Baccata
Bone targeted cabazitaxel In vitro: PC-3 and C4-2B In vitro: Along with high affinity towards bones, the survival percentage
nanoparticles luciferase prostate cancer of PC-3 and C4-2B prostate cancer cells was determined by the dose and
cells concentration.
In vivo: Tumor weight was significantly reduced in addition to reduction in
the appearance of bone lesions (Gdowski et al., 2017).
Paclitaxel nanoparticles (nab- Human pancreatic cancer cell Antitumor activity in SUIT2 cancer model: Ascites and distant metastasis
PTX) bound to albumin and (SUIT2-GLuc) was efficiently suppressed. Also showed best survival rates for the
S-nitrosated human serum combination therapy
albumin dimer (SNO-HSA
Dimer)
Docetaxel nanoparticles in In vitro: AsPC-1 and BxPC-3 In vitro: Treatment with docetaxel combined with radiotherapy resulted in
combination with radiotherapy pancreatic cancer cells decreased cell colonies, increased tubulin polymerization as well as apoptosis.
In vivo: Mice Molecular level: Caspase 3 expression levels were boosted
In vivo: AsPC-1 and BxPC-3 derived tumors were suppressed from growing
(Rivera-Franco and Leon-Rodriguez, 2018).
Combination of sorafenib with In vitro: MDA-MB-231 In vitro: Breast cancer cells proliferation was inhibited with respect to the
paclitaxel and radiation therapy breast cancer cells dose coupled with increased sub-G0-G1 phase.
In vivo: BALB/c nude mice Molecular level: p21, CHOP, BAX, Apaf-1, and cleaved caspase-3 levels
were heightened and (B-cell lymphoma) Bcl-2 levels were lowered which
points to caspase cleavage and inhibition of the Bcl-2 pathway ultimately
resulting in cell cycle arrest.
Cellular level: Cytochrome C levels were heightened in the cytosol which
points to a cytochrome c-dependent apoptotic cycle.
In vivo: Caki1 and MB-231 cell xenograft tumors were suppressed (Foglietta
161
et al., 2018).
162
Paclitaxel containing In vitro: Human breast cancer In vitro cytotoxicity: Concentration-dependent inhibition of cell proliferation
cell-penetrating peptide cell MCF7. In vivo antitumor efficiency: Reduction in tumor weight indicates that there
producing nanoliposomes In vivo: BALB/c nude mice is significant inhibition of tumor in the MCF7 tumor-bearing mouse model
(Choi et al., 2019).
Anti-EGFR anchored immune In vitro: MDA-MB-468 In vitro cytotoxicity: The viability of cancer cells was lowered.
nanoparticle bearing paclitaxel breast cancer cell In vivo antitumor efficiency: Reduction in tumor and greater accumulation of
In vivo: Athymic mice paclitaxel in the tumor plasma (Zhang et al., 2018)
Nab-Paclitaxel with Phase III trial: Human Nab-Paclitaxel with atezolizumab prolonged the progression-free survival in
atezolizumab females patients suffering from triple-negative breast cancer (Venugopal et al., 2018).
Paclitaxel and gebcitabine In vitro: 4T1, MCF-7, and In vitro: Inhibition of proliferation of 4T1, MCF-7, and MDA-MB-231 in
through methoxy-poly MDA-MB-231 breast cancer a dose-dependent manner by paclitaxel was observed. A combination of
(ethyleneglycol)-poly cells paclitaxel with gebcitabine resulted in a significant reduction in viability
(lactide-coglycolide) of 4T1, MCF-7, and MDA-MB-231 below 50%. Human breast cancer cell
polypeptide nanoparticles resistance to drugs was reversed (Schmid et al., 2018).

Paclitaxel loaded with keratin In vitro: MCF7 and Cell death: In MCF-7, the percentage of late apoptotic cells was increased
nanoparticles MDA-MB-231 breast cancer after 48 h of treatment and in MDA-MB-231, the percentage of early
cells apoptotic cells was increased after 24 h of treatment.
Molecular level: Cleaved caspase-3 (CC3) protein and proapoptotic BAX
gene expression was heightened (Dong et al., 2018).
Docetaxel loaded in folic acid In vitro: MDA-MB-231 In vitro: Nanoparticles loaded with docetaxel showed better cytotoxicity
and thiol-decorated chitosan breast cancer cell when compared to docetaxel.
nanoparticles Ex vivo: Successful transportation and improved oral bioavailability (Sajjad
et al., 2019)
Taxus Baccata
pH-sensitive nanoparticles In vitro: 4T1 mammary In vitro: Reduction in cell viability along with G2/M cell cycle arrest
containing Dihydroartemisinin carcinoma cell Molecular level: Expression of E-cadherin was increased and expression
and docetaxel of NF-κB, p-AKT, and p65 was decreased. Additionally, Matrix
metalloproteinase-2 (MMP-2) was arrested (Tao et al., 2018).
Docetaxel with ionizing In vitro: MCF-7 breast cancer In vitro: A concentration and time-dependent decrease in viability of cancer
radiation cell cells upon cotreatment of docetaxel and ionizing radiation suggesting a
synergistic effect (Doddapaneni et al., 2016)
Docetaxel with SC-43 In vitro: MDA-MB-231, In vitro: Dose-dependent increase in antiproliferative activity and treatment
MDA-MB-468, and with docetaxel and SC-43 induced apoptotic activity.
HCC-1937 breast cancer cells Molecular level: STAT3 downstream effector cyclin D1 and p-stat 3
In vivo: NCr athymic nude expressions were lowered. Increased SHP1 expression
mice In vivo: Suppression of tumor growth and tumor weight compared to control
(Hendrikx et al., 2016)
Noscapine and docetaxel In vitro: MDA-MBA-231 and In vitro: Increased cytotoxic effect and decreased cell viability upon
MDA-MB-468 breast cancer treatment with a combination of noscapine and docetaxel
cells Molecular level: Downregulation in the expression of Bcl-2, survivin,
In vivo: Mice α-tubulin, and pAKT
In vivo: Reduction in tumor collage levels and higher intratumoral uptake of
drug-loaded liposomes (Liu et al., 2017)
Ritonavir and docetaxel In vivo: Cyp3a knockout In vivo: Docetaxel and ritonavir cotreatment resulted in reduction to ⅓ of
mice (Cyp3a) original volume.
Tumor model: K14cre; Histological observation: Mammary tumor cells expressed significant
Brca1F; p53F mouse model pleomorphism along with expanded stroma, fibrotic changes, and abundance
of apoptotic cells in addition to the absence of necrosis (Fard et al., 2017).
163
164
Cabazitaxel-loaded poly(2 In vitro: MDA-MB-231, In vitro: Toxicity of cabazitaxel to MDA-MB-231, MDA-MB-468, and
ethylbutylcyanoacrylate) MDA-MB-468, and MCF-7 MCF-7 cell lines
nanoparticles breast cancer cells Tumor growth inhibition: Tumor growth was inhibited and was completely
In vivo: Mice remissioned in mice upon treatment. CD206, a marker of M2 macrophages
(protumorigenic and anti-inflammatory activity), expression was inhibited
(Fusser et al., 2019).
Cabazitaxel and thymoquinone In vitro: MCF-7 and MDA- In vitro: MCF-7 and MDA-MB-231 breast cancer cell lines upon treatment
coloaded lipospheres MB-231 breast cancer cells with cabazitaxel-thymoquinone-loaded nanoparticles expressed a dose-
dependent decrease in viability.
Cell cycle analysis: Concentration-dependent percent increase in sub G1
population upon treatment with lipospheres loaded with combination drugs
Apoptosis: Early apoptotic cells were increased in percentage upon treatment
with combination drugs (Kommineni et al., 2018).
Hyaluronic acid-coated In vitro: MCF-7 breast cancer In vitro: Concentration-dependent decrease in cell viability upon treatment as

cabazitaxel-loaded solid lipid cells compared to controls (Zhu et al., 2017)
nanoparticles
Cabazitaxel-loaded polymeric In vitro: 4T1 metastatic breast In vitro: 4T1 cell migration was inhibited upon treatment with cabazitaxel
micelles cancer cells loaded polymeric micelles.
In vivo: Tumor growth was inhibited upon treatment with cabazitaxel-loaded
polymeric micelles (Zhong et al., 2017).
Source: Reprinted from Sinha (2020). https://creativecommons.org/licenses/by-nc-sa/3.0/#. Open access https://creativecommons.org/licenses/
by/4.0/.
Taxus Baccata 165
conifers belong to the Taxaceae family and are found in parts of Europe,
Africa, and Southwest Asia. The reason for the interest lies in the presence
of the active compound taxane in all parts of the plant (except for the arils),
which undergoes internal enzymatic synthesis to form taxol (Malik et al.,
2011). Although the anticancer nature of the compound was discovered
initially in Taxus brevifolia extracts in 1971, it was eventually figured out
that it was better from an economic and efficiency view point to extract
the compound from the leaves of T. baccata and other Taxus species. Addi
tionally, several studies have been conducted which have identified a large
amount of taxanes and their subsequent derivatives which were extracted
from T. baccata (Parmar et al., 1999, Vaishampayan et al., 1999).
As opposed to the medicinal nature of the taxane that ultimately results
in the formation of taxol, the seeds and foliage of the yew tree are found to
be quite toxic in nature. This is attributed to the presence of the taxine. This
complex alkaloid compound exhibits powerful cardiotoxic properties and
is readily absorbed in the digestive tract. Taxus baccata is also home to a
few antibacterial, antifungal, and antioxidant compounds (Erdemoglu and
Sener, 2001; Milutinović et al., 2015). A study conducted in 2014 was able
to isolate three lignan derivatives from the plant. Of the three only two were
shown to have moderate antifungal properties, while the last derivative had
antibacterial properties. None of the three lignan derivatives showed any
cytotoxic activity (Erdemoglu et al., 2004).
But the focus of this section is going to be on the compound taxane and
its antitumor derivative. Taxol, also known as paclitaxel, acts as a mitotic
inhibitor and potent antineoplastic agent (Needleman et al., 2005). Basically,
taxol interacts with the tubulin dimers of the microtubules and stabilizes them
to prevent depolymerization. It does so by binding to the β subunit of tubulin
resulting in a hyperstabilized state which prevents the cell from undergoing
standard mitotic division. Additionally, studies have also discovered that
taxol is also responsible for inducing apoptosis in neoplastic cells by binding
to the Bcl-2 protein (Haldar et al., 1996). The Bcl-2 is an apoptosis stopping
protein and when taxol binds to it, it becomes nonfunctional resulting in
cellular destruction.
8.3.1 PRODUCTION OF TAXOL
Taxol is a secondary metabolite belonging to the diterpenoid variety and as

such is derived solely from geranylgeranyl diphosphate (GGPP). The actual
process of biosynthesis of taxol involves the actions of various enzymes,
cyclization, oxidation, acylation, and hydroxylation. Due to the successful

nature of the drug, taxol is constantly in high demand. At present, the main
source of paclitaxel is still dependent on the yew tree. If one were to simply
depend on naturally extracted taxol for manufacturing the require drugs
for global consumption, then we would run out of the viable sources in a
couple of years (Ismaiel et al., 2017). This is primarily due to the very low
concentrations of taxol in every plant source. Since natural sources can
only be used in moderation, lest we eradicate an entire species of plant,
the search for alternate modes of taxol production has taken priority. As a
result, researchers are attempting to produce taxol using a variety of current
approaches, including chemical synthesis, semisynthesis, and plant tissue
culture methods. Nonetheless, these procedures offer both benefits and
drawbacks (Ismaiel et al., 2017; Shankar Naik, 2019). Although chemical
synthesis is an option, the low product yield and highly complex nature
of the reaction deters large scale production possibilities (Nicolau et al.,
1994). Another approach is the semisynthetic pathway, which focuses on
synthesizing taxol from intermediates such as 10-deacetylbaccatin III.
Kusari et al. (2012) in their study revealed that several attempts have been
carried out across the world to extract paclitaxel from sources other than the
plants, and in this fungal endophytes have been proved to be a good alternate
source (Kusari et al., 2012). By and large, isolation and identification of taxol-
producing microorganisms is a decent methodology in the creation of taxol
(Ismaiel et al., 2017). Along these lines, fostering a less expensive paclitaxel
fermentation measure by microorganisms has turned into a maintainable
arrangement (Shankar Naik, 2019; Somjaipeng et al., 2015).
Molecular imprinting (MIP) technology is a method of equipping a poly
meric material with the ability to detect a specific target molecule. With the
progression of science and innovation, molecular imprinting is presently an
advanced innovation, and various scientists convey this technology to isolate
and get the target product (Li et al., 2017).
Molecular imprinting is generally used for the detachment and advance
ment of dynamic elements of natural products (Ishkuh et al., 2014). The
application of MIP technology to the separation of dynamic elements of
natural medicinal resources has attracted an increasingly more consideration
recently. For example, using Ultra-spectrophotometry, a few analysts have
widely researched the interactions among paclitaxel and some normal
functional monomers, such as methacrylic acid (MAA), acrylamide (AM),
2-vinylpyridine (2-VP), and 4-vinylpyridine, in various solvents and tracked
down that the most stable communication among paclitaxel and 2-VP in
chloroform was seen at a proportion of 1:6 (Li et al., 2013, 2015).
Taxus Baccata 167
Ishkuh et al. (2014) have used MSP using ethylene glycol dimethacrylate
for preparing MIPs for paclitaxel with a genuine degree of crosslinking
and found that the most important confining breaking point concerning
paclitaxel was 48.4%. However, the particle sizes of the MIPs were
generally around 100 nm. Hence, in any case, inspite of its incredible
engraving impact, the MIPs couldn’t be used further for separation and
assessment (Ishkuh et al., 2014).
Contrasted and regularly utilized partition techniques like fluid extraction
and column chromatography, MIP innovation partakes in the advantages of
economy, speed, and ease (Li et al., 2017). At this point, a gigantic number
of studies have set up that paclitaxel shows high anticancer activity. Its
antitumor framework incorporates limiting to tubulin, course of action of a
consistent cylinder pack, provoking the insufficiency of agreement among
dimers, and progression of microtubule gathering polymerization (Li et al.,
2017). Consequently, the infection cells are caught in the late G or M stage,
mitosis of the danger cells is quelled, augmentation of the harm cells is
hindered, and the cells bit by bit contract and in the end die. Notwithstanding,
there are relatively few reports on other normal activities of paclitaxel, for
instance, prevention of HIV-1 viral replication activity (Shankar Naik, 2019;
Wang et al., 2015). Wang et al. (2015) checked out exercises of taxol made
by endophytic parasites Noduli sporium sylviforme HDFS4-26 with that of
taxol extracted from yew bark in controlling turn of events and impelling
apoptosis of threatening development of cells (Wang et al., 2015). Cell
morphology, cell counting kit (CCK-8), staining (HO33258/PI and Giemsa),
DNA agarose gel electrophoresis, and flow cytometry (FCM) examinations
were used to choose the apoptosis status of malignant growth of cell lines,
for instance, MCF-7 cells, HeLa cells, and ovarian infection HO8910 cells.
The parasitic taxol showed cytotoxic activity against HeLa malignant growth
cell lines in vitro, and antifungal and antibacterial potential against different
pathogenic strains (Das et al., 2017).
However, the most economically viable and large-scale suited alternate
for the production of taxol is via plant cell culture. One can further improve
the yields obtained via tissue culture by
• Utilizing cell lines with high yield potential
• Selecting and utilizing suitable phytohormones (Khosroushahi et al.,
2006)
• Using optimal carbohydrate sources: It has been observed that the
presence of fructose stimulates taxol production, while glucose does
the opposite.
• Utilizing elicitors, precursors, and other additives that enhance the

production process.
• Setting up a two-phase production chamber with in situ product
removal is shown to boost yields. This helps in preventing feedback
inhibition and the degradation of the metabolite via enzymatic path
ways, which ultimately increases overall yield.
8.4 REGULATORY ASPECTS OF HERBAL ANTICANCER DRUGS
Herbal prescriptions have been used for a long time now, with the written
records to very nearly since 5000 years ago (Swerdlow, 2000). Till 1890,
about 59% of the listing accessible on the US Pharmacopeia was connected
somehow to the home grown origins, while till as of late, it has been tracked
down that one out of five people in the USA professed to utilize natural products
(Barnes et al., 2004; Revathy et al., 2012). The American Botanical Council
revealed sales of US$ 5.3 billion in 2011, that increased to US$ 6 billion
by the end of 2013 (Lindstrom et al., 2014). Other than these, an enormous
extent of the African and Asian populace relies on customary medication and
restorative spices for their essential medical care. A part of these were taken
forward by the old frameworks of restorative information like Ayurveda,
Siddha, and Chinese medication (WHO, 2008; Pan et al., 2014).
As of late, an increase in the inclination of home grown medications along
with an adaptable administrative framework and related harmfulness has had
many inquiries on the use of these medications and enhancements (Elvin-
Lewis, 2001; Cuzzolin et al., 2006; Sahoo et al., 2010). Notwithstanding
Herbal medication's usage for a long time, there are no shared view guide
lines (Thakkar et al., 2020). While FDA (Food and Drug Administration) is
the essential body directing home grown enhancements in the USA, they
permit the herbal supplements to be sold as dietary enhancements and not as
the endorsed over-the-counter medications. This gives the producer enough
freedom to sidestep the tough course of exhibiting security and adequacy
as the law characterizes it extensively, and despite the fact that they are not
permitted to make direct claims, they can in any case make primary and
useful claims on medical advantages (Bent, 2008).
The innovative work plans for new drug are all around spread out and clear,
while the equivalent isn’t valid for natural medications. As plants seldom have
one explicit dynamic pharmacological compound, it makes it hard to report
the particular capacities (Sahoo et al., 2010). The overall focus or synthetic
profiles of the various mixtures found in a plant could fluctuate a great deal
Taxus Baccata 169
and can altogether impact the outcomes. Other than the previously mentioned
heterogeneity, there are different things to be thought about, similar to defile
ment of one or the other engineered or compound that is normal with the
exchange of therapeutic plant is one (Chan, 2003; Yee et al., 2005; Lam et al.,
2008), while others are misidentification of plants dependent on morphology
(Ramawat and Goyal, 2008; Yap et al., 2008), purposeful or unintentional
expansion of harmful substantial metals (Rai et al., 2001; Dargan et al., 2008;
Mazzanti et al., 2008; Zhang et al., 2012), and pesticides (Rai et al., 2008;
Xue et al., 2018; Xue et al., 2008) or microbial disease (Bugno et al., 2006)
might actually influence the helpful qualities and should be regulated.
Phytomedicine improvement is a multistep measure that starts with the
assortment of crude material, trailed by identification, adjustment, extractions,
quality affirmation, separation of dynamic compound, purging, fractionation,
and harmfulness assessment. All through the turn of events, process
normalization is a basic advance that relies upon many variables including,
however not restricted to, development conditions, circulation, assembling,
and advertising. Further, the biochemical profile of a plant and its auxiliary
metabolites are likewise answered to be influenced by genetics, moisture,
environment, supplement, photoperiod, capacity, collection, extraction, and
bundling. To keep the norm of medication adequacy and security, synthetic
consistency ought to be routinely checked and kept in ideal reach throughout
the process (Sahoo et al., 2010).
Because of innate intricacies and potential varieties at each progression of
the interaction, a multimethod approach incorporating plant strategy, synthetic
marker, and so on should be taken together for quality control (Mukherjee,
2019; Thakkar et al., 2020). As of late, following methods and steps are
utilized for the distinguishing proof and guideline of herbal medications:
• Qualitative investigation is done through moisture content, ash value,
extractive worth, pesticides, heavy metals, essential oils, phyto
chemical constituents, and unrefined fiber (Holst, 1973; Idu et al.,
2008; Abdelhadi et al., 2015; Akram et al., 2015; Chen et al., 2015).
• Morphological identification and macroscopic assessment include
organoleptic study, micromorphology investigation of roots, bark,
stems, rhizomes, woods, leaves, seeds, natural products, and elevated
parts (Bauer, 1998; Quettier-Deleu et al., 2000; Grubeši et al., 2005;
Smillie and Khan, 2010).
• Chemical investigations, for example, thin layer chromatography
(TLC), High-performance thin layer chromatography (HPTLC), and
High-performance liquid chromatography (HPLC) are most of the
time utilized for better identification (Lazarowych and Pekos, 1998;

Xie et al., 2006; Raclariu et al., 2017).
• Metabolite investigations are typically done through LC/MS, GC-MS,
LC-NMR, protein examination, and HPLC/MS (Alali and Tawaha,
2009; Farag et al., 2012; Trivedi et al., 2017; Bendif et al., 2020).
• Bioassay and Herbal medications assessment should be carried out in
vitro/in vivo. Moreover, one can likewise consider various proteins,
receptors, and possibly can run different high-throughput evaluates
for the medication assessment and its parasiticidal, hostile to viral,
and antimicrobial impact (Chanda and Nagani, 2013; Tiong et al.,
2013; Naz et al., 2015).
• Safety study includes harmfulness study, spice drug connection, enlist
ment, restraint of CYP450, and pharmacovigilance (Nasri, 2013).
• Quality control is generally directed through the arrangement of
specialized rules distributed by WHO throughout the long term. A
couple of them are “Quality control strategies for restorative plant
material,” “Guidelines of good agricultural and collection practices
GACP” and “WHO rules for evaluating the nature of herbal drugs
concerning impurities and buildups.” Other than these, numerous
nations like Japan, China, India, and European affiliations have their
own arrangements of rules administering the assortment, fabricating,
capacity, applications, and engendering of herbal plants (WHO, 1998,
2003, 2007; Liu et al., 2018).
8.5 TRENDS IN TRADITIONAL MEDICINE SYSTEMS AND THE

FUTURE OF ANTICANCER PLANT-BASED PRODUCTS
While there is a great amount of studies being conducted on the various

avenues for the production of anticancer agents, plant-based compounds
probably hold the most promise in the long run. Anticancer agents derived
from natural sources in general have two things going for them;
1. Since they are naturally occurring substances, they can be extracted
from viable sources, purified and then put to use.
2. Naturally sourced compounds are generally shown to have fewer
toxic and adverse effects.
These factors work in tandem to make plant-based and other naturally
sourced bioactive compounds a solid contender to their synthetic counterparts.
Taxus Baccata 171
Over the years, the research and development world has come to the same
conclusion and has shifted their attention to these natural products, particu
larly plant-based anticancer agents. This interest in natural products is only
going to increase with time as newer and more advanced technologies for
the extraction, production, and purification of these compounds are created
(David et al., 2014).
This is not to say that there are no limitations related to the field of plant-
based anticancer products. One particularly rampant problem associated with
most naturally sourced compounds is the access to biological samples in a
day and age, where natural biodiversity is being destroyed at an alarming rate.
Additionally, it is quite difficult at times to gain access to specific plants for
their bioactive compounds, especially when factors like intellectual property
rights and legal supply are thrown around. The other major limitation would
be correctly identifying the useful active compounds from natural sources.
Due to the lack of communication regarding nonessential compounds
present in plant systems, it gets increasingly hard to determine and identify
compounds of value. Of course, there are various efforts like the Collective
Molecular Activities of Useful Plants, Chinese Medicine Integrated Database,
SymMap, Encyclopedia of traditional Chinese medicines, etc. which aim to
remedy this situation. By improving the communication thread regarding
active plant compounds and related information, we can aim to improve the
identification and production of various plant-based compounds, including
anticancer agents.
8.6 CONCLUSIONS
Taxus baccata (European yew) has been perhaps the most continuous source
of taxol for investigations of the biosynthetic pathway and further developed
creation of this anticancer medication. It is by all accounts very clear that
the taxanes are continually in upgradation mode both as far as mechanistic
viewpoints and clinical perspectives. The roads of up-degree and extempo
rization of taxanes are totally open for additional investigations. In addition,
mix therapies (taxanes alongside some different medications) likewise need
to be looked upon to work on the viability and anticancer action. This further
opens the way for the investigation of new medications from nature and
regular assets plants being the lone retreat for this situation.
As per a characteristic viewpoint, development of Taxus sp. for large
scale production of taxanes requires more thought and biotechnological
approaches and can be done through tissue culture procedures. Treatment

of malignant growth would be more refined if the sensibility of the prescrip
tions is contemplated by the medication business and administrative arrange
ments. Diminishing the expenses of the prescriptions will not simply make it
more accessible to people yet moreover all the while will expect an obvious
part as a popular anticancer drug among all the other large number of medi
cines available in the market. As such, cost assessment of treatment should
in like manner outline a huge limit close by drug headway for the overall
benefit of humankind, unequivocally with respect to the quantity of patients
in underdeveloped countries where the occasion of cancers is increasing to
an alarming level, essentially due to contamination and irregular lifestyle.
Hence, taxane is among such compounds that has acquired a spot in the
medication world for the treatment of a wide bunch of malignancy threat. Its
further advancement both to the extent pharmacology and cost viability stay
absolutely open for the benefit of patients and mankind. Taxol creation in T.
baccata suspension cultures has been improved by enriching and improving
culture conditions, fundamental media, plant advancement regulators, sugar
supplements, etc., and cultures have been increased to a bioreactor level
for scaling up. A sensible system might give new comprehension into how
the taxol biosynthetic pathway is controlled, with hereditary and metabolic
planning strategies, differential hereditary articulation, record factors, and
key qualities inciting higher taxol yields. One viewpoint to consider is the
taxol release from cells, which could be improved by using a two-stage
culture structure, so far not estimated in T. baccata cell suspensions. Future
perspectives could be focused on the coordinated usage of observational
and rational systems and analyzing the two-stage culture structure to make a
biotechnological structure for high taxol creation.
KEYWORDS
• antioxidant
• biocompatible
• phenolic compounds
• secondary metabolites
• tumor
Taxus Baccata 173
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CHAPTER 9
Panax Ginseng
SHALINI GURUMAYUM, SAGAR BARGE, and JAGAT C. BORAH
Chemical Biology Lab I, Institute of Advanced Study in Science and
Technology, Paschim Boragaon, Guwahati, Assam, India
ABSTRACT
Panax ginseng C.A. Meyer (deciduous herb belonging to the family Aralia
ceae), also known as “healing herb,” has long been used by the medicinal
herbal healers of Far East Asia, including China and Korea, for curing different
types of diseases as well in many other purposes to maintain a healthy and
long life, and is mostly used as a tonic or adaptogen against hemorrhage,
weak constitution, low vitality, fatigue, etc. Apart from curing common
ailments, P. ginseng was reported to show anticancer activity. Ginsenoside
(a triterpene saponin), one of the main compounds present in ginseng, has
a steroid rigid four-ringed skeleton, containing sugar residues attaching to
a –OH group of glycones having C20 side chain, and the cancer preventing
activity gradually increases with respect to the decrease in the number of
sugar units present. Metabolomic profiling of UPLC-MS has revealed that
bioactive ginsenoside level increases and accumulates in the roots of old
ginseng plants, which supports the usage of old roots in medicines. It was
also reported that metabolite synthesized from the above-ground portion of
the plant shifts underground in the later stage, arising to the elevated levels
of ginsenosides biosynthesis across the root area. The ginsenosides are the
key players of different signaling cascades. They showed potent antitumor
efficacy through cell proliferation inhibition, cell cycle arrest, and induction
of apoptosis in tumor cells. Most of the isolated ginsenosides have a unique
potency in controlling the progression of different cancer types. A thorough
metabolomic approach to study the variation of metabolites present in the

plant according to their growing season and age will be the further perspective
to contribute to a proper agronomic as well as quality and quantity specific
strategy for effective synthesis of bioactive ginsenosides in the plant.
9.1 INTRODUCTION
Nature has been the primary source of medicine since time immemorial in
curing different types of ailments. The traditional herbal healers in other
parts of the world have already discovered numerous plant species that
are prescribed either solely or in the form of a mixture of different types
of medicinal plants (Alyaa, 2019). Mainly, the herbalist uses the plants in
various forms like boiling or crushing. So, according to the traditional knowl
edge we have gained from the herbalist, many natural products have come
up in the economy as a substitute for synthetic drugs. Natural derivatives and
their structural analogs have historically made a significant contribution to
pharmacotherapy, especially for cancer and infectious diseases (Alyaa, 2019).
Panax ginseng belongs to the family of Araliaceae, also known as Asian
ginseng, the “king of herb.” This plant is mainly grown in Asia, namely
China, Japan, and Korea, for thousands of years to treat various diseases, as
a health tonic and for the longevity of life (Chang et al., 2003). The govern
ment has approved the plant of the said countries as a part of traditional
medicine. The herbal healers prescribed this plant as an immune tonic to
improve brain function and physical aid performance (Kiefer and Pantuso,
2003; Ellis and Reddy, 2002; Yun, 2001). “Panax” was originated from
the Greek word meaning “all-healing.” Carl Anton von Meyer, the Russian
botanist, first used this word (Court, 2000). Among the genus of Panax, P.
ginseng was used primarily as indigenous ginseng, a perennial herb growing
up to 60–80 cm tall. Its roots are aromatic, fleshy, and grayish-white to amber
yellow in color, grows around 5–6 cm long deep. The ginseng stem is deep
red, simple, and erect with oval, thin, and digitate leaves. Ginseng flowers
are pink in color and give small red berries (Yun, 2001; Coates et al., 2005).
Medicinal plants are primary source of lead compounds that gave rise to
different types of drugs today. These lead compounds are found distributed
in various parts of the plant. Similarly, P. ginseng leaves, flowers, roots, and
berries can be used as medicine or dietary supplement. For example, leaves
are being used in the treatment of skin diseases (Lee et al., 2015; Kim et al.,
2013). The flowers of P. ginseng are used as tea, as a dietary supplement (Hu,
1977), and steamed roots are being consumed for various pharmacological
Panax Ginseng 183
activities (Takagi et al., 1972). Due to its high medicinal value, separation
and extraction techniques permitted the isolation of active constituents such
as ginsenosides, glycopeptides, polysaccharides, and volatile oils from PG
(Choi, 2008).
However, the quality and the quantity of the metabolites in a ginseng
plant depends on age, the season of harvest, and the habitat of the plants;
as a result, ginsenosides are distributed thoroughly throughout the various
parts of the plant in different concentrations (Song et al., 2019). So, a
thorough study of the cultivation and harvesting period of the plant to get the
maximum quantity of byproducts (ginsenosides) of high quality is required,
which is augmented by using different types of metabolomic studies (Yang
et al., 2021). This chapter will focus on the characterization of metabolites
and metabolite profiling of PG using LCMS, GC–MS, NMR, and HPLC
techniques. Metabolic pathways of ginsenoside biosynthesis also depend on
the seasonal environmental condition, which in turn affect the secondary
metabolite biosynthesis. Also, we will be discussing the mechanism of action
of anticancer agents present in the PG (“Ginseng,” 2016).
9.2 CHARACTERIZATION OF SECONDARY METABOLITES

ISOLATED FROM PG OVER THE YEARS
Recent studies have unveiled that different parts of the plant, originating
from a different geographical region with a variation in cultivation status,
gives different metabolome. Studies to widen the knowledge of the variation
of metabolite concentration in roots and leaves during their growth are a must
for exploring a thorough ginseng study (Lee et al., 2019). Metabolomic study
has become an emerging trend in the world of science and technology and
has given us the insight to take forward experiments on a deeper and more
precise level (Shyur and Yang, 2008). The main technique in metabolomic
study is to target analysis and profiling of particular metabolite in a sample
in place of a wide metabolomic analysis with the help of different types of
techniques such as gas chromatography-mass spectrometry (GC–MS) and
liquid chromatography-mass spectrometry (LC-MS). Other methods include
thin-layer chromatography (TLC), Fourier transforms infrared spectroscopy
(FT-IR), Raman spectroscopy, and nuclear magnetic resonance (NMR) (Shyur
and Yang, 2008). MS coupled with high field NMR has proven to be a user-
friendly as well an efficient system for the analysis of metabolites. Different
coupled techniques with NMR, such as HPLC–SPENMR or MALDI- TOF/
TOF MS help in the characterization of the quantity of secondary metabolites
present in crude samples as a result of the raised sensitivity of the techniques

used (Shyur and Yang, 2008).
9.2.1 METABOLITE PROFILING AND CHARACTERIZATION OF

METABOLITES USING LCMS AND NMR
The use of these techniques helps in distinguishing complex chemical struc

tures isolated from the PG. The characterization of chemical constituents
present in P. ginseng has been widely studied (Chen et al., 2015). Among the
different compounds present, ginsenosides (a triterpene saponin) are major
bioactive constituents that have a vital role in the pharmaceutical world. The
structure of ginsenosides has already been elucidated by Shibata et al. in
1963 (Shibata et al., 1963; Kim, 2012). They have basic similar structural
backbone consisting of a nucleus of dammarane steroid of C17 arranged in
rings of four (Zhu et al., 2011).
From the year 2000–2019 about 50 new ginsenosides have been isolated
where most of them possessed C17 side chain variation (Piao et al., 2020; Liu
and Xiao, 1992; Li et al., 2020). With the help of 2D-NMR, 26 metabolites
including ginsenosides Rg1, Rg2s, Rg3s, Rb2, Rb1, Re, and Rf, distinguished
from among all the isolated metabolites (Chen et al., 2015).
During a study involving metabolomics profiling of P. ginseng, with the
help of UPLC-Q-TOF, it was seen that P. ginseng can also be identified as
mountain or garden cultivated according to their ages (Xie et al., 2008; Zhu
et al., 2018). It was found that P. ginseng constitutes 93 metabolites having
surplus signals from other different isotopes, in-source fragmentation as
well as HCOO– adduct (Lee et al., 2018; Wang et al., 2017). In another
case study, using GC–MS, ginsenosides were found in different concentra
tions distributed in leaves, stems, and roots in various stages of growth.
Ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1 make up about 90% of the total
ginsenoside content of P. ginseng roots which was reported by a ginseng
evaluation program. Metabolomic studies with the help of liquid chromatog
raphy (LC), gas chromatography-mass spectrometry (GC–MS), and proton
nuclear magnetic resonance (1H NMR) showed that the metabolic analysis
of ginseng berries varies with respect to the difference in the stages of fruit
ripening as well as the different metabolites found in roots according to the
cultivation age (Wei et al., 2020; Yang et al., 2012). 1H NMR was also used
in the control of quality of ginseng products as well as for the identification
of ginseng origin (Lee et al., 2011; Yuk et al., 2013). Using HR-MAS-NMR,
ginseng processed products are screened for different secondary metabolites
Panax Ginseng 185
(Yoon et al., 2020). Abundant levels of ginsenoside have been reported

during preharvest stages in ginseng berries (Lee et al., 2020). Furthermore,
35 ginsenosides were identified using HPLC-APCI/MS method; this method
allows to identify thermolabile ginsenosides via thermal degradation of ions
in both positive and negative ionization, measuring molecular masses of
sugar and aglycone structures of ginsenosides (Ma et al., 2005).
The LCMS technique was also used to analyze the quality of ginseng
grown in various regions to improve the quality of metabolites and produc
tion of ginsenosides (Wu et al., 2018). In this regard, 60 compounds from the
ginseng were evaluated for quality upon cultivation from northeast China.
GC–MS/GC–TOF–MS was also used for the detection of the longevity of
P. ginseng seeds, which in turn helps during the sowing season and also for
differentiating metabolite differences according to cultivation years and the
stage of ripening of ginseng berries (Cui et al., 2015; Kim et al., 2016; Liu
et al., 2017; Min et al., 2019; Park et al., 2019). Studies using HPLC have
also contributed to the differentiation of different Panax species (Pace et
al., 2015). During the investigation, it has been observed that environmental
conditions and cultivation technology affected the quality of ginseng roots
and rhizomes (In et al., 2017).
A thorough investigation of the many constituents in medicinal plants
can be achieved through high-throughput metabolomic studies. Metabo
lomic studies help in the elucidation of components that are essential for
the development of pharmaceutical drugs/products that are of exceptional
quality. Moreover, this study also aids in the conservation of rare medicinal
plants that have been exhausted due to excessive use by mankind in the ever-
changing environmental conditions (Shyur and Yang, 2008).
9.3 BIOSYNTHESIS OF SECONDARY METABOLITES OF PG
The biosynthesis of ginsenosides in P. ginseng has been studied widely, as

ginsenosides are found to have potent activity from the pharmacological
point of view. More importance to ginsenoside synthesis, quantity, and
quality has been given due to its high demand (Shin et al., 2015).
9.3.1 BIOSYNTHETIC PATHWAY OF GINSENOSIDES
Ginsenosides are mostly amphipathic with an –OH group closely linked to

the backbone, which helps to interact with the phospholipid’s membrane
polar head and the β-OH group in cholesterol. The polar –OH groups are the
determinant factors of the physicochemical interactions between ginsenosides
and other neighboring lipids, so the difference in the number and sites of these
hydroxyl groups gives diversity to ginsenosides. Ginsenosides are reported to
be synthesized from 2,3-oxidosqualene.With the help of enzymes cycloartenol
synthase, dammarenediol-II synthase, and β-amyrin synthase acting upon 2,3-
oxidosqualene, it forms the different types of ginsenosides (Tansakul et al.,
2006). With the help of reactions that involves geranyl diphosphate synthase
(GPS), farnesyl pyrophosphate synthase (FPS), and squalene synthase (SS),
squalene is synthesized via the mevalonate pathway (Liu et al., 2010).
Downstream reactions with squalene epoxidase yielded 2,3-oxidosqualene
(Deng et al., 2017; Lee et al., 2019). Enzymes like oxidosqualene cyclases
(OSCs), dammarenediol-II synthase (PNA), and beta-amyrin synthase (PNY)
helps in the cyclization of 2,3- oxidosqualene into dammarenediol and beta
amyrin (Yun-Soo et al., 2009).
Ginsenosides in P. ginseng were mainly synthesized through two main
biosynthetic pathways: mevalonate (MVA) pathway in the cytosol and
2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids of the
plant (Figure 9.1). In the MVA pathway, in the cytosol, acetyl CoA is converted
into HMGCoA with the help of acetyl CoA transferase, and the latter is
further reduced to mevalonic acid (MVA). MVA is then phosphorylated to
diphosphomevalonate (MVPP) with the help of phosphomevalonate kinase
(PMK), which is then decarboxylated to IPP (Isopentenyl phosphate).
Similarly, in the plastids of the cell, G3P is converted to 2-C- methyl-D
erythritol-4-phosphate pathway (MEP), which then forms IPP. IPP being the
precursor of ginsenosides, with the help of farnesyl diphosphate synthase,
forms squalene. Squalene is converted to 2-oxidosqualene and, with the help
of two different enzymes: β-amyrin synthase and DDS, helps in the synthesis
of oleanane type ginsenoside as well as PPD and PPT type which are the
backbone of different ginsenosides found in P. ginseng (Figure 9.2) (Han et
al., 2012, 2013; Lee et al., 2017).
9.3.2 BIOSYNTHETIC PATHWAY OF OLEANOLIC ACID
Oleanolic acid was known to be synthesized in P. ginseng with the

help of β-amyrin 28-oxidase encoded by the CYP716A subfamily gene
CYP716A52v2 that helps in modifying β-amyrin and converting into
oleanolic acid, whose byproduct was confirmed using GC/MS (Han et al.,
2013). It is reported that CYP716A52v2 from P. ginseng helps the synthesis
Panax Ginseng 187
of oleanolic acid. The CYP716A52v2 gene present in yeast confirmed the

potency to synthesize oleanolic acid, which was further confirmed in vitro.
Moreover, it was also found that change in Ro ginsenoside biosynthesis
was also regulated by CYP716A52v2 overexpression and RNA interference
(RNAi) as shown by studies in transgenic ginseng roots. Experimental results
showed that CYP16A52v2 helps in the catalyzation of critically important
steps during the synthesis of ginsenoside in P. ginseng (Han et al., 2013).
FIGURE 9.1 The biosynthetic pathway of ginsenosides in P. ginseng plants where IPP
(isopentenyl phosphate), the precursor of ginsenoside, was synthesized from the mevalonate
pathway (MVA) and 2-C- methyl-D-erythritol-4-phosphate pathway (MEP), which downstream
the synthesis of oleanane type ginsenoside along with protopanaxdiol (PPD) and protopanaxtriol
(PPT) type ginsenosides.
Source: Adapted from Lee et al. (2017). https://creativecommons.org/licenses/by-nc/3.0/
9.3.3 SEASONAL AND ENVIRONMENTAL EFFECTS ON THE

BIOSYNTHESIS OF SECONDARY METABOLITES IN PG
P. ginseng is widely famous for its myriad health benefits. The harvesting
season and time of ginseng has always been the key to its bioactivity since
ancient times, and mainly the roots are being used to prepare herbal concoc
tions (Liu et al., 2017). The main reasons for these particularities have been
explained through metabolomics studies of the plant grown in different
seasons at different timelines (Yang et al., 2021). The harvesting season,
along with the age of the plant, was analyzed using different techniques.
Moreover, the berries of the ginseng plant also have a certain amount of
ginsenosides that make it a potent medicine. The distribution of ginsenosides
across various parts of the plant according to their growing season and
age is one of the most interesting studies that can help benefit the growth,
production, and maintenance of the quantity and quality of the ginseng for
commercial value. The traditional use of P. ginseng has been subdivided into
hairs, lateral, and main roots. The P. ginseng plants are usually cultivated
for about 4–6 years before harvesting. The constituents and efficacy of this
medicinal plant are highly dependent on the age of cultivation and the harvest
season. So, 6-years cultivated ginseng is more expensive and efficient than
the 4-years cultivated plant (Shan et al., 2014).
FIGURE 9.2 Different types of ginsenosides (backbone) isolated from Panax ginseng.
The quality and quantity of main root ginsenosides were distinguishably

changed during the whole growing season and were found to be spiked till the
month of May. With the help of orthogonal partial least squares discriminant
Panax Ginseng 189
analysis (OPLS-DA) it was identified that the main roots of ginseng, which
were harvested in the month of April, have elevated levels of secondary
metabolites but lower levels of glucose and sucrose as compared to those
which were harvested in the month of March. Glucose and phenylalanine
levels were higher in main ginseng roots that were harvested in June and
were found to be decreased as compared to those harvested in May; sucrose
levels were significantly elevated in main ginseng roots harvested in June.
Lateral ginseng roots have higher contents of ginsenoside compounds, FAs,
sterol, and arginine as compared to the main roots, with higher sucrose level
as compared to that in lateral roots. It was also found that less exposure
time in the sun with a high level of rainfall after May also contributed to
the inhibition of ginsenoside synthesis in ginseng roots due to a decline in
photosynthesis (Lee et al., 2019).
9.4 ANTICANCER ACTIVITY OF P. GINSENG AND ITS CONSTITUENTS
Cancer can be defined as the uncontrolled growth or proliferation of cells

defying the natural agenda of contact inhibition as the normal cells usually
do. There are two types of cancer: benign, where it is localized at a particular
place, and malignant, where the cancer cells get detached and move along
the lymph nodes or blood and get attached at another site and establish new
tumors (GM, 2000). The chromosomes of cancer cells are highly unstable
and prone to mutations like deletions and duplications and have more nucleus
content as compared to normal cells (US, 2007). Cancer, as studies showed,
arises due to both environmental factors as well as of genetic origin. With
the progress in evolution, the genetic pool also acquired different types of
recessive or dominant mutations. In recent findings, plant-based anticancer
drugs have gained considerable attention worldwide. Since ancient times,
plants have always been a source of medicine for different human ailments,
and many cancer drugs commercially available today have been derived
from plant products (Greenwell and Rahman, 2015; Rahman et al., 2020).
Currently used anticancer drugs, although effective to some point, have their
own share of side effects, and most of the cancer drugs are not effective
when it comes to higher disease progression. The drugs circulate and not
only target the cancer cells but also cause effects on other parts of the body
like rapidly dividing normal cells, including hair follicles and gastrointestinal
cells (Iqbal et al., 2017). In another different case, there arise drug-resistant
cancer cells where due to a high rate of mutation, the cancer cells develop its
own immunity against a particular drug. So, the single-target drug has also
become ineffective during such conditions and requires a combination of

drugs (Iqbal et al., 2017; Rahman et al., 2020). Plant-based medicines have
been circulating on the table as they help in providing multiple-target drugs
with lesser side effects (Greenwell and Rahman, 2015; Iqbal et al., 2017;
Rahman et al., 2020).
One of the most famous anticancer plants is Panax ginseng or Asian
ginseng (Kamei et al., 2000). Since ancient times, this plant has been used
as a cure for different types of cancer, and studies revealed that ginsenosides
are the main active ingredient that possesses anticancer activity apart from
the polysaccharides, flavanols, etc. (Ahuja et al., 2018; Alyaa, 2019; Kamei
et al., 2000; Park, 2019; Shareef et al., 2016). The activity of ginsenoside
against cancer, as already stated, differs with respect to the amount of sugar
units present; so, lesser the sugar moieties higher the anticancer activity. PPD
and PPT type (no sugar moieties) shows the highest activity in different types
of cancer (Nag et al., 2012). Ginsenosides play multiple roles in inhibiting
cancer growth following different pathways. The common pathway includes
inhibition of metastasis, cell cycle arrest, inhibition of angiogenesis, and
apoptosis (Yue et al., 2007; Ahuja et al., 2018; Chen et al., 2020) (Figure 9.3).
FIGURE 9.3 Anticancer properties of Panax ginseng.

Source: Adapted from Nag et al. (2012). https://creativecommons.org/licenses/by-nc/3.0/
The different types of ginsenosides found in P. ginseng follow different

signaling pathways that help in the inhibition of cancer progression. It
Panax Ginseng 191
helps in the activation of both intrinsic (through a mitochondrial pathway

involving the formation of apoptosomes) as well as the extrinsic apop
totic pathways through caspase activation (Chen et al., 2018). They also
act as antimetastatic as well as antiangiogenic through NF-κβ inhibition,
which helps in the downregulation of vascular endothelial growth factor
(VEGF) as well as fibroblast growth factor (FGF), which helps promote
angiogenesis (Chen et al., 2020; Chen et al., 2018; Kashfi, 2009; Lee et
al., 2019) (Figure 9.5). Ginsenosides also inhibit cancer cell growth via
cell cycle arrest by activation of tumor suppressor genes like p53, p21, and
p27 (Figure 9.4) which in turn helps in the activation of cyclin proteins that
interrupts cell cycle progression and stops further growth and division of
cells (Chen et al., 2018, 2020).
FIGURE 9.4 A simple schematic diagram of ginsenoside-induced cell cycle arrest.

Source: Reprinted from Mohanan et al. (2018). https://creativecommons.org/licenses/
by-nc-nd/4.0/
FIGURE 9.5 A schematic diagram of anticancer activity signaling pathway of ginsenosides.

Source: Adapted from Chen et al. (2018); Mohanan et al. (2018). https://creativecommons.
org/licenses/by-nc-nd/4.0/
9.5 CONCLUSIONS
Several studies have been carried out using P. ginseng both in vitro as well as
in vivo. P. ginseng, regarded as the “all healing herb,” has lived up to its name
as many of the compounds from this plant have different roles in different
types of diseases (Park et al., 2018). Due to high demand in economic market,
the ginseng plant has been regarded as a rare plant, and conservation of this
plant has also been a major challenge among ginseng experts. By using high
throughput metabolomic devices and software a thorough screening of the
plant’s growing season, age, type of cultivation, habitat as well as mode of
biosynthesis of its secondary metabolites have been closely monitored to
get a higher yield of desired plant products, which are of high quality and
quantity. The origin and the quality of the plant itself were also chosen to
benefit the evergrowing agroeconomic conditions and ensure a high quality
adulteration-free product.
TABLE 9.1 The Antitumor/Anticancer Activity of P. ginseng and Its Constituents.
Panax Ginseng
Sl.no Extract/compound Antitumor activity and signaling pathway References
1 Protopanaxdiol Inhibited growth of human lung cancer cells A549 and Bi et al. (2014)
H460, downregulate levels of β-catenin, cyclin D1,
CDK4, and c-myc
2 (20R)-12b-O- (L-chloracetyl)-dammarane-3b, Decrease growth of cancer cells, induces apoptosis Xia et al. (2014)
20, 25-triol (xl), (20R)-3b-O- (L-alanyl) through caspase signaling
dammarane-12b, 20, 25-triol (1c), and (20R)
3b-O- (Boc-L arginyl)-dammarane-12b, 20,
25-triol (8b)
3 a1-a4, b1-b4 (first class, a5, a6 b5, and b6 a5, a7, b5, and b7 show anticancer activity Qu et al. (2017)
(second class), a7, a8, and b7 (third class)
4 Water-soluble ginseng oligosaccharides (WGOS) Hinder tumor formation, helps in TNF-production, and Jiao et al. (2014)
stimulates TNF-α production
5 Ginsenoside Rk1 Inhibits telomerase activities, cell growth, and proliferation, Kim et al. (2008)
and induces apoptosis through executioner caspases
6 Ginsenoside Ro (Ro) Helps in suppression of tumor growth Zheng et al. (2019)
7 Panaxadiol from roots of Panax ginseng Helps in suppression of hypoxia-inducible factor (HIF)-1α Wang et al. (2020)
and regulates the expression of phosphoinositide 3-kinase
(PI3K) and mitogen-activated protein kinase (MAPK). It
also shows antiproliferative activity.
8 Neoginseng Inhibits tumor progression due to its anti-inflammatory as Keum et al. (2000)
well as antioxidative activity
9 Ginsenoside Rg3 Suppresses cancer cell proliferation through inhibition of Yang et al. (2017)
C/EBPβ/NF-κB signaling
193
194
10 Ethanolic extract of Asian ginseng, EAG (Panax Suppresses tumor growth along with downregulation Wong et al. (2010)
ginseng) of PCNA proliferative marker, shows cancer cell
cytotoxicity, induces MAPK and p53 signaling through
suppression of cyclin B–cdc2 complex, and induces
G2–M arrest leading to apoptosis
11 Ginseng polysaccharide (GPS) Antiproliferative and induce apoptotic pathway, Xiong et al. (2017);
transcription of p38 and JNK were elevated while Zhao et al. (2019)
that of ERK was downregulated while it suppressed
phosphorylation of ERK, NF-κB, and cyclin D1
12 Ginsenoside Rg3 Antitumor activity Keum et al. (2003)
13 Ginsenoside Rg1 Inhibited JAK2/STAT5 pathway activity, helps in Li et al. (2014);
caspase-3 and C-PAPR expression, it also shows Li et al. (2019)
anticancer activity in drug-resistant nasopharyngeal
cancer cells via activation of autophagy, cell cycle arrest
at S phase, and inhibition of PI3K/AKT pathway

14 compound K (CK), Reduces the metastatic growth by reducing colony Ming et al. (2011)
formation, adhesion, and invasion, decreases nuclear
NF-κB p65 and increases cytosolic NF-κB p65
suppressing the NF-κB pathway
15 Ginsenoside Rg3 Synergistic effect is shown by the combination of cisplatin Lee et al. (2014)
and Rg3 through alteration of G2/M phase and intrinsic
apoptotic pathway.
16 Ginsenoside Rb2, 20(R) and 20(S) Shows inhibition of metastasis of lung cancer cells Mochizuki et al. (1995)
by inhibiting cell adhesion and invasion along with
antiangiogenic effect
Panax Ginseng
17 Ginsenoside Rb1 Shows antiangiogenic effect via suppression of PEDF Leung et al. (2007)
18 Compound K Induced apoptosis through caspases 8, 9, and 3, and of Bid Cho et al. (2009)
protein
19 Ginsenoside Rh2 Induces apoptosis in nephrotoxicity via caspase mediated Qi et al. (2019)
pathway
20 Ginsenoside Rd Shows antitumor activity through G0/G1 phase Tian (2020)
21 Ginseng proteins Shows antitumor activity and induces intrinsic apoptotic Wan et al. (2019)
pathway
22 Ginsenoside Rg5 Helps in the inhibition of cancer cell proliferation via Liu and Fan (2020)
intrinsic apoptotic pathway and induction of autophagy
195
KEYWORDS
• Panax ginseng
• herbal medicines
• pharmacological uses
• metabolites
• active compounds
• metabolomics
• antitumor activity
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CHAPTER 10
Tinospora cordifolia (Thunb.) Miers.

LEPAKSHI M. D. BHAKSHU1, PULALA RAGHUVEER YADAV2,
K. VENKATA RATNAM3, ANU PANDITA4, DEEPU PANDITA5, and
1
Department of Botany, PVKN Government College (Autonomous),
2
Department of Biotechnology, Indian Institute of Technology Hyderabad,
3
Department of Botany, Rayalaseema University, Kurnool,
4
5
6
Department of Microbiology and Biotechnology,
Jnanabharathi Campus Bangalore University, Bengaluru, India
ABSTRACT
Tinospora cordifolia (Thunb.) Miers, is a woody twiner, belongs to family

Menispermaceae, with characteristic lenticels on stems. It is commonly used
in Ayurvedic medicine and popularized as Gaduchi or Amrit (heavenly elixir).
The stem, stem bark, and leaves were appraised for the human ailments like
anti-diabetic, anti-oxidant, anti-stress, anti-malarial, anti-spasmodic, anti-
inflammatory, anti-arthritic, anti-leprotic, hepatoprotective, anti-allergic,
immune-modulatory, and anti-neoplastic activities. In addition, the plant is also
scientifically proved for the treatment of various types of cancers such as IMR
32 human neuroblastoma, C6 glioma cells, and IMR-32 cells. The extracts or

compounds such as berberine, were potentially inhibited the cancer cells and
have been proved the molecular mechanism including the genetic expression
of homeostasis and senescence indicating proteins-HSP70 and Mortalin. This
plant contains glycosides like cordiosides, tinocordifolioside, tinocordiside,
palmitosides, and steroids such as ecdysterone and β-sitosterol. Alkaloids
like berberine, palmatine, 11-hydroxymustakone magnoflorine, and some
diterpenoid lactones along with aliphatic compounds as pharmacologically
active constituents responsible for its medicinal properties. The present review
focused on the utilization of extracts and their principles in the cancers as well
the leading path for the apoptosis and involvement of cytokines expression
studies in relation to cancer therapies supports the extensive use with safe and
cost-effective.
10.1 INTRODUCTION
Mother nature is blessed with curative properties of many human ailments by

gifting medicinal plants with plethora of secondary metabolites which have
to be scientifically investigated and properly utilized in human welfare. The
changing human culture might be a reason for leading to dreadful illness and
Cancer is one among them. The medicaments developed for the treatment
of Cancer, have to supplement with the herbal boosters for the improvement
of overall health during the course. In this connection, the Ancient systems
of medicines were providing such medicaments and Tinospora cordifolia
(Thunb.) Miers. (Figure 10.1) belongs to Menispermaceae family, is one
among the lifesaving herbs and believed to be safe, non-toxic, and cost-
effective. It is most commonly used as a potent herbal recipe in the Ayurveda
system of medicine, known as Amrita or heavenly elixir (Giloy).
The scientific world have appreciations and reported several bioac
tive components from T. cordifolia such as alkaloids, steroids, lactones,
glycosides, aliphatic compounds sesquiterpenoid, diterpenoid and polar
compounds, polysaccharides, and phenolics with varied pharmacological
properties. Amrit has been reported as a remedy for jaundice. The plant has
been reported as hepato-protective, immunomodulatory and immunostimu
latory, potential for diabetes and related disorders in addition to adaptogenic,
cardiotonic, anti-oxidant, anti-inflammatory, and anti-psychotic activities.
Interestingly, many of the reports were demonstrated its effectiveness on
cancerous cells.
Tinospora cordifolia (Thunb.) Miers. 205
FIGURE 10.1 Tinospora cordifolia (Thunb.) Miers.
The recent studies have evidenced for the inhibition role of the extracts
of T. cordifolia on the anti-proliferative activity in various in vitro/in vivo
cancer models supporting anti-neoplastic, cytotoxic, anti-angiogenesis, and
anti-metastatic activities (Mishra et al., 2013; Sharma et al., 2019) were
being discussed. The modern tools were provided the characterization for its
anti-cancer properties with possible mechanisms even providing evidences
at a molecular level and the present study able to focus on the sequential
roles of the T. cordifolia on the effectiveness of Cancer.
10.2 ACTIVE COMPONENTS
The extracts of T. cordifolia have tremendous medicinal potential and

the pharmacological activities are ascribed to the presence of secondary
metabolites such as terpenoids, for example, furanolactone, tinosporide,
tinosporaside, and ecdysterone, etc., glucosides like poly acetate, phenylpro
pene disaccharides, tinocordioside (Figure 10.2), cordifoliosides, cordioside,
Alkaloids such as palmatine (Figure 10.3), berberine (Figure 10.4), tinospo

rine, magnoflorine, Choline, Jatrorrhizine, lignans, steroids, and others were
characterized (Sharma et al., 2019).
FIGURE 10.2 Tinocordiside.
FIGURE 10.3 Palmatine.
10.3 ANTI-CANCER ACTIVITIES
The extracts of T. cordifolia (TCEX) obtained from the methanol and water
and methylene chloride when tested on the survival of HeLa cells at the dose
of 50 and 100 μg/mL exhibited significant killing effect when compared to
non-treated cells whereas the methylene chloride extract is more effective
(up to 6.8 times) as compared to the standard (doxorubicin) in a linear

pattern and suggested as anti-neoplastic agent for further studies (Jagetia et
al., 1998).
FIGURE 10.4 Berberine.
10.3.1 EFFECT ON SKIN CANCER
Anti-cancer efficiency of Immu-21, an Ayurvedic polyherbal formulation

containing amla, holy basil, ashwagandha, and TCEX extracts on B/T cell
mitogens, phytohemagglutinin, LPS, and concanavalin-induced cell prolifera
tion in splenic leukocytes using in vitro and K 562 cells in mice. Cells treated
with Immu-21 enhanced the division capacity of splenic leukocytes exposed
with LPS and B-cell mitogens. Further, the formulation exhibited notable
cytotoxicity in K 562 cells in mice (Nemmani et al., 2002).
The effect of TCEX on 7,12-dimethylbenz(a)anthracene (DMBA) induced
skin cancer and evaluated at two-stage levels and pretreated with 100 mg/kg
bw (TCEX) and tumor formation for 16 days was found significant reduction
in incidence and weight of the tumor when compared to the untreated mice
besides declined tumor burden, papillomas, the elevation of detoxifying
enzymes, reduction on lipid peroxides in liver and skin during the studies
strongly supported its anti-tumor effect (Chaudhary et al., 2008). Further, the
studies of Ali and Dixit (2011), supported the effect of the alkaloid obtained
from T. cordifolia on the skin cancer in the DMBA-effected skin tumor
model using response surface methodology (RSM). The cytogenic effect of
the mixed plant extracts of T. cordifolia, ashwagandha, and holy basil was
assessed on the blood samples collected from healthy volunteers, revealed
that the extracts significantly declined colchicine-induced chromosomal
abnormalities (Sharma et al., 2011).
10.3.2 EFFECT ON MULTI DRUG RESISTANT SKIN CANCER CELLS
The anti-tumor potential of ethanolic extracts of TCEX and ashwagandha

on the side population of drug-resistant cancer cells showed that TCEX
exhibited a noteworthy reduction in side population and still no effect was
observed in ashwagandha extract-treated cells. Flow cytometry results
revealed that petroleum ether and dichloromethane extracts of TC strongly
suppressed the expression of ABC-G2 and ABC-B1 genes, which play an
important role to sensitize the cancer cells to the chemotherapeutic drug
(Maliyakkal et al., 2015).
10.3.3 EFFECT ON ORAL CANCER
The anti-cancer effect of an herbal regimen made from Emblica officinalis

(EO) and T. cordifolia was evaluated in oral cancer patients (114) with buccal
and tongue cancer, revealed that the formulation showed promising effect on
reducing mucosa formation on cheeks and back of the lips and tongue (Dias
et al., 2018). TCEXAQ reported the induction of apoptosis in AW13516 oral
cancer line attenuating its potential for epithelial–mesenchymal transition in
a dose-dependent pattern and exhibited potential effect at 5 µg/mL (Patil et
al., 2021).
10.3.4 EFFECT ON PROSTRATE CANCER
The anti-cancer actions of ethanolic extract of T. cordifolia against human pros

tate cancer cell lines (LNCaP cells) for a period of 48 h experimental schedule,
demonstrated that extracts showed concentration variant stimulation and
proliferation of prostate cancer cells. The anti-androgen flutamide reversed the
ethanol extract and stimulated the secretion of prostate-specific antigen (PSA)
in cancer cells, which is similar to the standard dihydrotestosterone (DHT)
and suggested TCEX may contain androgenic compounds and stimulated the
androgen receptor proving endocrine modulation (Kapur et al., 2009).
10.3.5 ANTI-TUMOR POTENTIALITY
The polysaccharide fraction from T. cordifolia was reported significant

effects on the suppression of the passage of melanoma cells (B16F-10) and
strongly suppressed the growth of tumors in the lungs of C57BL/6 mice and
the biochemical markers of neoplastic cancer like lung collagen hydroxypro
line uronic acids secretions and hexosamines, were decreased significantly
in the TCEX-treated mice compared with the control mice in addition to
affecting serum γ-glutamyl-transpeptidase (γ-GT) and sialic acid levels
(Leyon and Kuttan, 2004).
Pretreatment of alcoholic TCEX to mice with T cell lymphoma, resulted
in a significant reduction in tumor growth in thymus. Further, TCEX
enhanced the proliferation rate of thymocytes and reduced its apoptosis.
The extract also reduced Hassal’s corpuscles number. TCEX showed its
mechanism by reducing thymocyte’s apoptosis and enhancing the secretion
of growth-promoting cytokines such as IL-2 and IF-γ in thymocytes (Singh
et al., 2005a).
Cytotoxic efficiency of the water extract of T. cordifolia stem (TCEXAQ)
and bioactive polysaccharides such as arabinogalactan (AG), were assessed
on pulmonary cancer cells and its associated cytokines in mice-induced
tumorogenesis with benzo(a)pyrene (B(a)P) and dose-dependent treatments
in different groups. The B (a) P-induced serum or plasma markers like
lactate dehydrogenase, circulating tumor DNA, tumor necrosis factor, and
carcinoembryonic antigen significantly attenuated in the TCEX treatments
which depict TCEX’s protective role and even enhanced the apoptosis in
cancer-affected cells. The apoptosis is more effective in treatments with
TCEXAQ than AG in modulating lung carcinogenesis (Mohan and Koul,
2017).
The radio-sensitizing activity of T. cordifolia dichloromethane extract
was investigated in gamma rays induced radiation in mice with Ehrlich
ascites carcinoma at five different concentrations. The results demonstrated
that mice treated with extract at 30 mg/kg b.wt. expressed dose-dependent
tumor suppression and radio-sensitizing activity and enhanced the life span
of irradiated mice. The extract showed its radio-sensitizing mechanism
by reducing glutathione S-transferase and glutathione concentrations and
increased the levels of lipid peroxidation and DNA fragmentation in cancer
cells (Rao et al., 2008).
Anti-tumor efficiency of the alcoholic extract of T. cordifolia was
assessed in in vivo mice models with bone marrow cells and DL-bearing
(Daltons Lymphoma) mice, revealed that T. cordifolia extract significantly
increased the formation of colonies of granulocyte-macrophages in mice
with bone marrow cells and enhanced the bone marrow cells proliferation
in DL-bearing mice. The treated mice showed an improved response to
LPS-activated production of IL-1 and TNF indicating the T. cordifolia role

in the differentiation of precursor of myeloid bone marrow progenitor cells
and the involvement of the macrophages (evidenced by increased number
during the TCEX treatment) in response to tumor growth in situ are directly
related to anti-tumor effect, particularly on myelopoiesis (Singh et al., 2006).
10.3.6 EFFECT ON BONE CANCER CELLS
Anti-cancer and anti-mitotic activity of methanolic extract of T. cordifolia at

three different concentrations was investigated in vivo models, that is, Swiss
albino mice and C57 Bl mice, demonstrated that the extract significantly
reduced tumor size and increased life span of animals in both the tested
animal models (Verma et al., 2011).
10.3.7 CYTOTOXICITY ON FEMININE CANCER CELL
The cytotoxic effect of various organic fractions of T. cordifolia was evaluated

in vitro on three human breast (MCF-7), ovary (IGR-OV-1), prostrate
(DU-145), and the tested cancer cell lines affected by the fractions with
moderate cytotoxic potential. The chloroform, acetone, and aqueous extracts
exhibited cytotoxic activity against the breast cancer cell lines with >50%
inhibition, while other extracts showed <50% inhibition on breast cancer
cells. The extracts demonstrated <40% cytotoxic effects against the prostate
and ovarian cancer cell lines. Paclitaxel, mitomycin-C, and adriamycin were
used as standard anti-cancer drugs as reference drugs (Mishra et al., 2013).
Further, the potent anti-tumor activity of TCEX was reported in mice with
Dalton’s lymphoma ascites tumors (Adhvaryu et al. (2007). However, the
exact mechanism suggests that decline in clonogenicity, protection from
DNA damage, suppression of glutathione-S-transferase and topoisomerase-II
activities, and induction of tumor-associated profile involved in the cytotoxic
activity (Jagetia and Rao, 2013).
The methanol and aqueous extracts of WS (WSMEX and WSAQEX)
and TC (TCEXM and TCEXAQ) were tested and exhibited the cytotoxic
and apoptotic effects on MDA MB 231and MCF7 in the concentration-
dependent pattern except aqueous extracts. Further studies revealed
hallmark patho-cascade of programmed cell death like blebbing of
membrane, condensation of nuclear material, and fragmentation of DNA
with the TCEXM and enhanced the sub-G content, however. In addition
to that WS extracts seized the cell cycle progression in the G/M phase and
the extracts not affected the normal cells (HaCaT) while affected the breast
cancer cells (Maliyakkal et al., 2013).
10.3.8 EFFECT OF BERBERINE AT GENETIC LEVEL IN CANCER CELLS
A comparative anti-cancer activity of commercial and isolated berberine was

investigated on HCA-7, human colon carcinoma cell lines in vitro methods.
The results demonstrate that out of 44 oncogenes tested in HCA-7 cell
lines, 33 genes were significantly suppressed in a time and concentration-
dependent manner. The results suggest that the presence of berberine in T.
cordifolia may be attributed to its anti-tumor activity (Palmieri et al., 2019).
10.3.9 EFFECT OF NANOPARTICLES OF TCEX ON CANCER CELLS
Cytotoxic effects of silver nanoparticles prepared in the combination of T.

cordifolia leaves, defined via numerous techniques like scanning microscopy,
Fourier Transformed infrared, X-ray diffraction, transmission microscopy,
and energy dispersive X-ray analysis were assessed on A549 cell lines. The
size and morphology of nanoparticles were confirmed with transmission
microscopy and exhibited very strong anti-tumor activity and T. cordifolia
leaves are found to be toxic against human respiratory organ glandular cancer
cell line A549 (Mittal et al., 2020).
The role of nano-formulation of TCEX stem extract in glandular carci
noma cells reported, that there’s no caspase-mediated cell death induction,
however, stops proliferation in prostate cancer cells without apoptosis and can
be regarded as an anti-proliferative agent against prostate cancer (Karuppath
et al., 2016). Anti-proliferative activity was in cervical malignant neoplastic
disease in HeLa cell lines by demonstrating that the dichloromethane and
ethyl alcohol fraction of TCEX exhibited potent cytotoxic impact within the
effective concentrations (Polu et al., 2017).
10.3.10 TCEX IMPACT ON NERVE FIBER CELLS
T. cardifolia showed a broad spectrum of immune-augmentary effects and

tumor-associated macrophages which in turn upregulates the anti-tumor
property. The differentiation of macrophages to dendritic cells was enhanced
by the TCEX. The treatment of alcoholic extract (200 mg/kg bw) for two
days, showed improvement in post-tumor transplantation through tumor
cytotoxicity and NO, TNF, and IL-1 production. The survival was extended
in tumor-bearing mice after the adoptive transfer of dendritic cells obtained
from tumor-associated macrophages into mice with Dalton’s lymphoma
(Singh et al., 2003, 2004, 2005b).
The cytotoxicity of bone marrow cells obtained by dendritic cells (BMDC)
enhances maturity by arabinogalactan (G1-4A) by T. cardifolia was due to
Nitric oxide synthase and apocynin-inhibited killing. BMDC phagocytosed
killed cells and activates cytotoxic T-cells (Pandey et al., 2014).
The T cardifolia stem extracts were used to isolate bioactive constituents
with the aid of chromatography and characterized by NMR and MS. Eight
compounds were isolated tinocordiside, cordifolioside, yangambin, magno
florine, palmatine, jatrorrhizine, and 11-hydroxymustakone, these are from
different classes. The tested phytoconstituents were effective on CHOK-1,
KB, SiHa, HT-29, and murine primary cells differentially of which some
compounds possess anti-cancer and while others exhibited immunomodula
tory properties (Bala et al., 2015).
HeLa cells were exposed to 2-Gy- γ-radiation, and were treated with
T. cardifolia dichloromethane extract for 4 h before exposure to radiation.
This led to cell death, nearly approximately 50%. There is a downfall in the
viability of HeLa cells, depending on the doses of γ-radiation. The concentra
tion of T. cardifolia dichloromethane fraction was also important along with
the concentration of γ-radiation, this combination led to a further decrease
in the cell viability. A total of 1–4 Gy γ-radiation was radiated on Hela cells
and later treated with different doses of T. cardifolia dichloromethane frac
tion demonstrated a significant decrease in cell viability. A dose-dependent
decrease in the surviving fraction was observed with the increasing dose
before irradiation. Among different doses, 4 μg/mL T. cardifolia dichloro
methane extract showed the lowest survival fraction. There was an increase
in LDH and a decrease in GST activity in HeLa cells treated with T. cardifolia
dichloromethane fraction during post-irradiation times. Lipid peroxidation
multiplied in 4 h of post-irradiation and slowly declined by 12 h of post-
irradiation (Rao and Rao, 2010).
T. cordifolia and Zingiber officinale were treated in MCF-7 cells, MTT
assay resulted in IC50 509 and 1 mg/mL, respectively. Combination of T.
cordifolia and Zingiber officinale administered in MCF-7 cells showed
IC50 of 2 μg/mL from MTT assay which indicates that combination treat
ment is more effective than alone. Combination treatment of T cordifolia
and Zingiber officinale induced apoptosis and arrest at the G0/G1 phase. T.
cordifolia and Zingiber officinale anti-cancer effect are due to MMP2, 9, and
ALOX5 major gene targets. Pharmacological network and in vitro studies
prove the synergistic effect of T. cordifolia and Zingiber officinale combina
tion in MCF-7 cells (Javir and Joshi, 2019).
10.3.11 CORRELATION WITH HEAT SHOCK PROTEINS (HSP)

EXPRESSION AND CANCER
Rashmi et al. (2017) reported the inhibitory property and regulation of heat
shock proteins and angiogenesis in certain tumor models such as Ehrlich
ascites MDA-MB-231 cell lines through the inactivation of PI3K or AKT
and related cassette of gene expression by the isolated pyrrole derivative
from the leaves and this activity was demonstrated in the in vivo and ex vivo
models. Clerodane furano diterpene glycoside was a new molecule isolated
from the stems of T. cardifolia (hydro-alcoholic extract), shown anti-cancer
activity by inducing apoptosis and autophagy in HCT116 (human colorectal
cancer) cells. Besides this, N-nitroso-diethylamine-induced liver cancer
in male Wister albino rats were treated with T. cordifolia ethanolic extract
showed a prominent increase in LPO and declining of enzymic and nonen
zymic anti-oxidants (Sharma et al., 2018).
The TCEX affected the Bcl-2 or Bax protein expression which impacts
on “mitochondrial membrane depolarization,” MPTP, and peroxidation of
cardiolipin. It evoked the cytochrome unharness of the cytoplasm, activation
of protease, leading to deoxyribonucleic acid fragmentation. The initiation of
apoptosis and modifications in cell structure was evident from the “phospha
tidylserine externalization” and increases the “subG” population.
The Ehrlich neoplasm (EAT) in mouse (in vivo) demonstrated the
effectiveness of TCEX in declining the neoplasm burden and enhanced the
chance of ~2 folds in the survival of mice reporting marginal “hepato-renal
damage.” T. cordifolia extracts reported for the effect on the ROS and restore
p50 activity and mitochondrial-mediated cell death in “MDA-MB-231 cells”
besides the activation of EAT necrobiosis, which inhibited the neoplasm
proliferation (Rashmi et al., 2019).
10.3.12 EFFECT ON CELL CYCLE
The inhibition effect of TCEX of proliferation of “KB cells” has been linked
with the arrest of G0/G1-phase in cell cycle and proved to be safe on the
normal peripheral blood mononuclear cells (PBMC) indicates its specific

effect might be beneficial in the treatment of cancer (Bansal et al., 2017).
10.3.13 EFFECT ON GLIOBLASTOMA
The extracts of T. cordifolia showed anti-cancer effect on neuroblastoma

cells (IMR-32) along with its metastasis effecting on cell cycle at G0/G1
and modulated the expression of polymer clamp slippery macromolecules
(PCNA) and cyclin D1. Further, TCEX-treated cells showed differentiation
as unconcealed by their morphology and also the expression of neural cell-
specific differentiation markers NF200, MAP-2, and NeuN in metastatic
tumor cells. The differentiated composition was related to the induction
of senescence and pro-apoptosis pathways by enhancing the expression of
senescence marker mortalin and Rel-A fractional monetary unit of nuclear
factor kappa beta (NFkB) beside decreased expression of anti-apoptotic
marker, Bcl-xl. TCEX exhibited anti-metastatic activity and considerably
reduced cell migration within the scratched space beside the downregulation
of neural cell adhesion molecule (NCAM) polysialylation and secretion of
matrix metalloproteinases (MMPs). Mishra and Kaur (2013) showed the anti-
brain cancer potential, 500th ethanolic extract effects C6 brain tumor cells
considerably elicited differentiation in C6 brain tumor cells, and reduced the
cell-proliferation (Mishra and Kaur, 2015).
10.3.14 EFFECT ON NEUROBLASTOMA
The anti-cancer effect of TCEX on Neuroblastoma (IMR-32 cells) was

established with different doses and found effective at 200 μg/mL (IC50) and
reported nontoxic on normal cells. Based on the studies of Mishra and Kaur
(2015) and the following mechanism has been provided on the Glioblastoma.
1. TCEX-arrested cell cycle at G0/G1 phase.
2. It is associated with the growth retardation and expression of regula
tory proteins in cell cycle.
3. Verified the stimulatory role in the cell cycle process from the G1/S
phase.
4. TCEX treatment inhibited the expression of cyclin-D1.
5. TCEX treatment affected the PCNA protein expression of the majority
of IMR-32 cells.
6. TCEX induces differentiation of morphology in IMR-32 Cells with

multiple and elongated processes.
7. TCEX-treatment-proceeded IMR-32 cells induce differentiation
through enhanced expression of cytoskeleton proteins of neurons
(“NF-200” and “MAP-2”).
8. Elevated NeuN only at the translational and transcriptional level.
9. TCEX-affected cells showed enhanced mitochondrial activity in
IMR-32 cells and indicated that the cell survival and differentiations
were dose-dependent.
10. TCEX induces senescence in neuroblastoma cells through the produc
tion of mortalin, a chaperone of senescence marker which exerts
prominently at 0.3 mg/mL. In addition, the HSP70 also reported as
a senescence indicator in TCEX-treated cells in association with the
phosphorylated form of Akt-1 (pSER473).
11. The suppression of Bcl-xl production significantly induces the pro
apoptotic/apoptotic pathways.
12. The TCEX affected the cell survival and differentiation and induced
senescence and preceded the apoptosis besides significant anti-
migratory potential of cells from scratched areas.
Based on the above-mentioned properties, clearly emphasizing its efficacy
on the neuroblastoma and found to safe usage in a combination of rational
therapies of neuroblastomas (Mishra and Kaur, 2013). The fractions obtained
from Chloroform and hexane of TCEX reported for the significant inhibition
of proliferation and differentiation in glioblastoma and neuroblastoma.
The non-polar fractions obtained from hexane and Chloroform affected
significantly and reduced the rate of proliferation and induced cell differ
entiation in glioblastoma and neuroblastoma, respectively, which are
correlated through the stress markers such as Mortalin and HSP-70 which
were upregulated in treated cells in addition to metastasis of glioblastoma
(U87MG and IMR-32) or neuroblastoma and declined the production of
NCAM found to be an important event in the treatment of neural carcinomas
(Anuradha et al., 2019).
10.4 MECHANISM OF ACTION OF PHYTOCOMPONENTS ON

CANCER CELLS
The phytocomponents of TCEX affected the iatrogenic malignant tumor,

have demonstrated through mitochondrial-mediated caspase-mediated cell
death, cytotoxic effect, tumor size reduction, formation of ROS, attenuate the
cell cycle, and gene expression resulting in significant inhibition of cancer
cell proliferation. The mechanism of action of TCEX depends on the phyto
components in which the “clerodane furano-diterpene” organic compound
proved for inhibition of malignant tumor through the programmed cell death
by initiating reactive chemical element agents (ROS) besides autophagy.
Additionally, the phenolics of the TCEX have geno-protective against cancer
cells (Singh et al., 2006). The ethanolic extracts showed iatrogenic apoptosis
by increasing the “sub-G0 phase” without affecting the cell cycle (Maliyakkal
et al., 2013). Arabinogalactans of aqueous TCEX initiate the immunological
and cytotoxic effects. The chemoprotective role has attributed to flavonoids
in cancer besides pyrrole-phytoconstituents induce programmed cell death
and cytotoxic effects (Rashmi et al., 2019).
Palmatine showed an increased inhibition effect by enhancing the levels
of anti-oxidant enzymes and conjointly exhibited detoxifying effect in addi
tion to declining of lipid peroxidation. The berberine and hexane fractions
proved the tumor inhibition property programme cell death through “capsase
3-activated DNase,” respectively. The epoxy-clerodane diterpene of TCEX
polysaccharides resulted in anti-neoplastic effect (Leyo and Kuttan, 2004)
besides it involved in the blockage of the metabolic activation and encour
aged in the detoxification of carcinogens. Singh et al. (2006) reported that
the TCEX showed direct tumoricidal effect. Thus, TCEX proved the anti-
carcinogenic effects through the stimulation of DNA injury, clonogenicity,
inhibition of topoisomerase-II, glutathione S enzyme activity, apoptosis, and
enhancing lipid oxidase activity.
The berberine of TCEX has demonstrated the cell cycle inhibition and
differentiation, HEP2 human speech organ neoplastic cells. The clerodane
furano-diterpene-glycoside proved for vital cytotoxic effect and iatrogenic
programmed cell death in human prostate cancer (PC-3, SF-269 [CNS], skin
cancer [MDA-MB-435], pulmonary cancer [A549], carcinoma [MCF-7]
cells, and carcinoma [HCT-116]). Singh et al. (2005a, 2005b) demonstrated
with the known synthetic resin compounds from a fungus extract of endo
phytic flora Cladosporium velox TN-9S which are isolated from the stems
of TCEX, showed a marginal geno-protective effect against DNA injury on
Chinese hamster ovary cell lines when the treatment with non-ionic water
nonylphenol. It had been conjointly noted that the endophyte’s capability
to synthesize phytocomponent was just like the host plant. Anti-cancer
potency of chemical constituents such as cordifolioside A, tinocordiside,
mangoflorine, jatrorrhizine, palmatine, N-formylannonain, yangambin, and
11-hydroxymustakone isolated from T. cardifolia using chromatography

technique was evaluated against four types of cancer cell lines. It revealed
that among the tested constituents palmatine supressed oral and colon
cancer cell lines, tinocordiside active on the ovarian and oral cancer cells
and yangambin inhibited the growth of oral cancer cells. The components
11-hydroxymustakone and N-formylannonain exhibited the highest immu
nomodulatory activity in the tested cancer cell lines (Bala et al., 2015).
In addition, the TCEX and fractions were compared the malignant tumor
growth inhibition and had been proposed that the effect is magnified due to
combinations and synergistic (Bala et al., 2015).
Rashmi et al. (2017), determined apoptotic induction using different
apoptotic markers, generation of ROS, activity of proteinase, and cell cycle
analysis and found phytocomponents of TCEX proved its anti-cancer effect
and conjointly inhibited the tumor proliferation. Though thorough studies
demonstrated with TCEX, the exact mechanism has to derive. The flavonoids
such as rutin and quercetin from TCEX showed anti-proliferative activity of
human carcinoma neuroblastoma cells through ignition of programmed cell
death, intracellular ROS and altered gene expression.
Anti-tumor efficiency of aqueous extract and arabinogalactan, bioac
tive polysaccharide of T. cardifolia stem was assessed in benzo(a)pyrene
induced tumors in lungs and its associated biochemical markers in animal
model, demonstrated that the extract and arabinogalactan significantly
attenuated benzo(a)pyrene-induced tumor markers such as TNF-alpha,
carcinoembryonic antigen, lactate dehydrogenase, and ctDNA in animal
models. The results indicated that extract was more potent that polysac
charide in reducing lung tumors in animal models (Mohan and Koul,
2018).
The phenolics of TCEX such as ellagic acid and kaempferol as geno
protective property on the fish model and found the reduction in nuclear
abnormalities (Mishra et al., 2013; Bakrania et al., 2017). Palmatine rein
forces the inhibitor catalyst levels and conjointly inhibits carcinogenesis on
singe administration (Ali and Dixit, 2013) besides berberine showed growth
remission on Swiss anomaly mice. Jagetia et al. (1998; Jagetia and Rao,
2006), reported that the malignant tumor suppression was dose-dependent
and opined that the combinatory result of the alkaloids inhibits the malignant
tumor. Leyon and Kuttan (2004) studied carbohydrate fraction from TCEX
and reported the pathologic process result on C55BL/6 mice and, therefore,
the extremely vital inhibition effect on skin cancer cell line B16F-10 has
attributed to its immune modulation.
10.5 MOLECULAR MECHANISM OF ANTI-CANCER EFFICACY
The details of the mechanism of anti-tumor activity of the Amruth and its
phytoconstituents were established on certain cell lines and were still under
the progression. However, TCEX roles have clearly proved on the certain
anti-tumor and its metastasis effects of cancers. Moreover, the TCEX trig
gers of ROS (reactive oxygen species) which elevates the inhibition effect
of growth cells through DNA damage causing mortality of cancer cells
besides reduced anti-oxidant enzymes activity, and exaggerated oxidation
of lipids and DNA damage might lead to cell death. The binding of TCEX
components with DNA topoisomerases plays a prominent role in DNA
replication especially Topoisomerase I/II may induce cytotoxic effects and
cell proliferation. Berberine of TCEX, demonstrated to suppress the DNA
topoisomerase I/II activity and stabilizes the catalyst mediated-DNA ‘‘cleav
able complex,’’ which can additionally trigger DNA damage and mortality.
In addition, Berberine arrests end elongation causing toxicity in carcinoma
cells. However, suppression of COX-II, NF–κB, and Nrf2 might have addi
tionally extended the vital role in neoplastic cell death by T. cordifolia where
the berberine and the extracts involved in the declining of anti-inflammatory
markers (Kapil and Sharma, 1997; Gao and Cai, 2008; Kapur et al., 2010;
Birla et al., 2019).
Berberine, a nitrogenous compound isolated from aerial parts of T.
cordifolia has been found to seize the progression of the G2/M phase of the
cell cycle, over the expression of cytotoxic markers Bax, CDk2, and p53. In
addition to this, it activated the caspase enzyme complex and other markers
such as PARP, involved in inducing programmed cell death (Palmeri et al.,
2019).
10.6 CONCLUSIONS
The extracts/principles of TCEX have proven for its beneficial effects on the
distinguished cancer cell lines and in the animal models (Figure 10.5). More
over, the selected plant possesses numerous medicinal properties of which
anti-cancer effect is appreciated by many scientific studies and berberine,
magnoflorine, palmatine, rutin, quercetin, and arabinogalactans may be an
important principle during the cancer treatment. More insightful efforts have
been made to develop this plant for cancer treatment since it has proved as
a best anti-diabetic, anti-inflammatory, and anti-cancer besides many physi
ological or pathogen induced ailments.
FIGURE 10.5 Effect of T.cordifolia on cancer cells.
KEYWORDS
• Tinospora cordifolia
• anti-cancer properties
• mechanism of anti-cancer properties
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CHAPTER 11
Taxus brevifolia (Nutt.) Pilger.

K. VENKATA RATNAM1, LEPAKSHI MD. BHAKSHU2,
PULALA RAGHUVEER YADAV3, ANU PANDITA4, DEEPU PANDITA5, and
1
Department of Botany, Rayalaseema University, Kurnool,
2
Department of Botany, PVKN, Government College (Autonomous),
3
Department of Biotechnology, Indian Institute of Technology, Hyderabad,
4
5
6
Bangalore University, Bengaluru, India
ABSTRACT
Taxus brevifolia (Nutt.) Pilger., known as pacific yew or mountain mahogany
of the family Taxaceae, native of North America, distributed in coastal
areas of Alaska to California. Local tribes of these areas used pacific yew
to treat lung and skin diseases. Economically the plant has been used to
make weapons, canoe paddles, and drum frames, etc. The major chemical
constituents of the pacific yew are diterpene alkaloids called as taxanes. Some
of the notable active principles of taxane family are paclitaxel, docetaxel,
and 10-deacetylbaccatin III. Anticancer activity of taxol and its derivatives
have been reported by several authors in different cancer cell lines like liver,
prostate, lung, pancreatic, and breast. The components showed its mechanism
of action in two ways, that is, mitotic action and apoptotic action. In mitotic
action, the components target the formation microtubules, which play a
crucial role in chromosome separation during cell division. In apoptotic
action, the principles inactivate the proapoptotic genes such as Bcl-2 and p53
and activate the apoptosis-induced genes, that is, caspase-3 family.
11.1 INTRODUCTION
Taxus brevifolia (Nutt.) Pilger., known as pacific yew or mountain mahogany

(family Taxaceae), native of North America and distributed in the coastal
areas of Alaska to California (Little, 1979; Munz, 1973). The species
belongs to Gymnosperm plant group. It is a slow growing coniferous tree
species. Traditionally, the wood has been used to prepare weapons, poles,
wooden boxes, drum frames, bows, and tool handles by the native Americans
(Gunther, 1945; Hartzell, 1991). The chief phytochemical constituents of the
pacific yew are diterpene alkaloids called as taxanes. Some of the notable
active principles of taxane family are paclitaxel, docetaxel, and 10-deacetyl
baccatin III. These components showed their beneficial effects on cancer
by stabilizing the microtubules present in the cells and by suppressing the
mitotic cell division of cancer cells (Sinha, 2020). In the present chapter,
we discussed anticancer activity of T. brevifolia extracts, nanoparticles, and
active principles published by reviewing some of the noteworthy publication
available online.
11.2 ANTICANCER ACTIVITY OF TAXOL/TAXANES
Taxol, a terpenoid substance, isolated from Taxus species, is widely used as

a chemotherapeutic drug to treat various types of cancers. Paclitaxel generic
name of taxol was reported to have strong tumor suppressing activity in
distinct cancer cell lines such as ovarian, lung, breast, leukemia, and malig
nant melanoma cancers (Nwafor et al., 2001). A clinical experiment study
results revealed that taxol exhibited noteworthy toxicity against leukocytes
than thrombocytes and reticulocytes in a 22 days experimental schedule
(McGuire et al., 1989). A sequence clinical therapy of taxol with cisplatin
was assessed in untreated and pre-treated solid tumor patients, showed that
sequence with cisplatin before taxol showed very less cytotoxic activity
(Rowinsky et al., 1991). Anticancer and free radical production capacity of
Taxus brevifolia (Nutt.) Pilger. 225
FIGURE 11.1 Bioactive phytoconstituents of Taxus brevifolia.
taxol was assessed against macrophages, neutrophils, and murine macro-

phages using a chemiluminescence indicator. Taxol modulated the phorbol
myristate acetate-induced chemiluminescence in neutrophils and macro-
phages but not in murine macrophages (Czuba et al., 1998). While Manfredi
et al. (1982) investigated the binding capacity of taxol to microtubules in

murine macrophages. The drug showed maximal binding capacity at higher
toxic doses. Cytotoxic efficiency of taxol along with cisplatin was studied
in cancer cells, indicated that both the tested drugs significantly suppressed
the cell proliferation by arresting the cell cycles events such as G1/S and G2
phases (Donaldson et al., 1994).
Antimitotic effect of paclitaxel was assessed in a murine xenograft
human cancer model using high resolution in vivo microscopy method. The
microscopic visuals revealed that paclitaxel altered the spindle assembly,
mitotic cell division arrest, formation of multinucleate cells, and apoptosis
(Orth et al., 2011). A comparative cytotoxic activity of taxol, cisplatin, and
Taxotere was investigated against 12 human normal bone marrow cells,
25 tumor cells, and 35 primary cultures using in vitro studies. The results
showed that taxol and docetaxel both are exhibited the potent antitumor
activity than cisplatin (Braakhuis et al., 1994). Bissery et al. (1991) reported
potent antitumor activity against B16 melanoma cells. It further cured colon
adenocarcinoma and pancreatic ductal adenocarcinoma at earlier stage.
The pre-treatment of different types of cancer cells with docetaxel for 24-h
period time, showed 50% survival capacity of cervix, gastric, and pancreatic
cell lines (Balcer-Kubiczek et al., 2006). Fabbri et al. (2006) conducted a
cytometric analysis to understand the apoptotic mechanism of docetaxel
against cancerous cells. The mechanism of action of docetaxel indicated that
it induced cell shrinkage and mitochondrial membrane transition, reduced
cell granularity, and DNA fragmentation.
The efficacy and toxicity of a new nano-formulation prepared using
paclitaxel and abraxane were investigated in B16 melanoma xenografts
growing in athymic mice, indicated that paclitaxel-based nano-formulation
strongly inhibited the tumors in mice than abraxane (Feng et al., 2010). Ferlini
et al. (2009) investigated the antitumor mechanism of paclitaxel in cancer
cell lines, revealed that paclitaxel binds to the sites of tubulin and Bcl-2
sites, but functionally mimic it as Nurr77, a member pf nuclear subfamily, its
expression and localization are closely connected with cell proliferation and
programmed cell death. Derry et al. (1995) investigated the cytotoxic effects of
taxol at different concentrations (10 nM to 1 µM) on the growth and condense
dynamics of microtubules in bovine brain cells under in vitro conditions. At
lower concentrations, taxol selectively suppressed the shortening the plus
ends of microtubules and slightly increased the mass of microtubule protein.
At intermediate concentration (100 nM to 1 µM), it inhibited the growth
and condensation of microtubules at both ends and stopped the additional
increase of microtubules mass. At higher concentrations (more than 1 µM),

microtubular mass was sharply increased and microtubular dynamics were
completely suppressed. A comparative anti-angiogenic effect of docetaxel
and paclitaxel was investigated on human umbilical vein endothelial cells,
demonstrated that endothelial cells are strongly inhibited with both the tested
drugs (Grant et al., 2003).
The anticancer efficiency of paclitaxel at seven different concentrations
was assessed against FM3A cell lines. It significantly suppressed the growth
of cells and increased dead cell number. It also induced morphological
changes like bleb formation and nuclear fragmentation (Ilgar and Arican,
2009). DNA fragmentation was confirmed with agarose gel electrophoresis.
Antitumor activity of nanocrystals of paclitaxel was investigated in cancer
cell lines and xenograft animal models. It demonstrated that nanocrystals of
paclitaxel showed better therapeutic effects over taxol in both the studied
models (Liu et al., 2010). Inoue et al. (2003) studied the docetaxel-enhanced
antitumor effect of TNP-479 in 253J B-V growing human transitional cell
carcinoma cell lines (in vivo) and HUVE cells (in vitro). Docetaxel increased
anti-proliferation activity of TNP-479 in HUVEC cells, but no effect was
observed on 253J B-V. The combination of docetaxel and TNP-479 was
significantly suppressed the intra-tumor neovascularization. The cytotoxic
activity of docetaxel-loaded lipid microbubbles was investigated on liver
tumors of VX2 rabbits. It showed that higher tumor suppression and apoptotic
index were observed in treated animals. Higher levels of caspase-3 enzymes
levels indicated its antitumor activity (Kang et al., 2010).
11.2.1 LIVER CANCER
Anticancer activity and to enhance the bioavailability of paclitaxel (PTX), a

biocompatible formulation prepared using dietary lipidic-based nanocarriers
(NLCs) was investigated in the HepG2 cell line. The formulation along with
PTX, significantly inhibited cell proliferation in a concentration-dependent
manner and enhance ROS levels in HepG2 cell lines (Harshita et al., 2019).
Wan et al. (2018) studied the anticancer properties of docetaxel and gold
nanoparticles encapsulated apatite carrier against human liver cancer
(HepG2) cells. Both docetaxel and nanoparticles showed noteworthy cell
toxicity against the tested cancer cell lines. Docetaxel carboxymethyl coated
cellulose nanoparticles strongly suppressed the tumor growth in mice. At
the molecular level, it significantly altered the expression levels of collagen
I and α- smooth muscle actin in hepatic satellite cells in a dose-dependent
manner (Chang et al., 2018). Cabazitaxel is a semi-synthetic derivative of a

natural component taxoid, significantly arrested the cell cycle progression
in Huh-7 and SK-hep-1 cell lines. At the molecular level, it suppressed the
expression levels of different types of cyclins such as Cdc2, Cdc25c, and
pCdc2 at protein levels (Chen et al., 2018).
Anticancer effects of docetaxel against human hepatocellular carcinoma
cells (SMMC-7721 HCC) were investigated. Colony-forming assay results
revealed that docetaxel exhibited strong and concentration-dependent anti-
proliferative activity. Flow cytometry and fluorescence microscopy results
indicated that docetaxel arrested the cell cycle at the G2 phase of Mitotic
division. Further, it strongly induced free radical generation and inhibited
glutathione levels in cells (Geng et al., 2003). A trial of docetaxel-based
chemotherapy was conducted in patients with advanced liver cancer,
revealed that the drug was not safe for patients with advanced hepatocellular
carcinoma (Hebbar et al., 2006).
11.2.2 LUNG CANCER
Nanoparticles encapsulated with PEG, which has detachable properties to

transport siRNA along with paclitaxel against survivin gene used in lung
cancer therapy. The nanoparticles capsulated with survivin siRNA effectively
bring down the expression of mRNA of survivin and enhanced the uptake of
nanoparticles by the cell. In the cell counting assay, nanoparticles expressed
low toxicity and paclitaxel presented good antiproliferative activity in A549
cells (Jin et al., 2018). Min et al. (2018) investigated the anticancer efficiency
of paclitaxel combined with caffeic acid using in vivo and in vitro methods
against non-small-cell lung cancer h1299 cells. Paclitaxel along with caffeic
acid exhibited noteworthy cytotoxic activity in h1299 cells by inducing apop
tosis. At the molecular level, both significantly activated protein markers
of the MAPK pathway. Anticancer activity of mixed micelles loaded with
paclitaxel was assessed in A549, Caco-2 cells, and Lewis C57BL/6 mice.
The results demonstrated that mixed micelles loaded with paclitaxel exhib
ited noteworthy cytotoxic effects against the tested cell lines models. In in
vivo studies, paclitaxel-loaded mixed micelles showed better activity than
paclitaxel alone treated Lewis C57BL/6 mice (Hou et al., 2017). Patel et al.
(2016) assessed the tumor-disrupting activity of docetaxel-loaded PEGylated
liposomes. Docetaxel-loaded PEGylated liposomes expressed high levels of
physical stability and very feeble hemolysis. It further showed higher levels
of bioavailability with significantly higher intra-tumoral uptake of the drug.
Antitumor activity of exisulind in combination with docetaxel was

carried out using in vivo and in vitro experimental models. The in vitro study
results revealed that both the tested competent increased the apoptosis in
A549 human lung cancer cells. Whereas in vivo results indicated that both
components moderately increased the survival capacity of nude rats with
A549 orthotopic lung cancer, reduced tumor formation, and increased apop
tosis in comparison with control rats (Chan et al., 2002). Anticancer effect
of docetaxel was assessed in human lung cancer cell lines H1299 and A549
to understand the mechanism of action in in vitro conditions. The p53-null
H1299 cells treated with docetaxel for 18-h period enhanced beta-tubulin
gene expression levels and strongly induced the p53 RNA levels in A549
cells (Chang et al., 2006). The efficacy and toxicity of a new nano-formula
tion prepared using paclitaxel and abraxane were investigated against NCI
H460 human lung cancer, indicated that paclitaxel-based nano-formulation
showed significant anti-tumor activity than abraxane (Feng et al., 2010).
Antitumor effect of paclitaxel at two concentrations (low concentration
35 nM and higher concentration 70 µM) against human lung adenocarcinoma
cells, revealed that paclitaxel at low concentration, it exhibited the potent
cytotoxic effect by inducing DNA fragmentation, caspase-3, phosphatidyl
serine (PS) externalization, Bax translocation into mitochondria, and cell
cycle arrest at G2/M phase. At higher concentrations, it induced vacuoles
in cytoplasm and swellings in mitochondria and paraptosis like cell death
(Guo et al., 2010). Antitumor activity of docetaxel and diindolylmethane was
investigated using in vitro and in vivo models, demonstrated the increased
apoptotic cell percentage, expression of Bax, and N-cadherin and cleaved
poly (ADP-ribose) polymerase in A549 cells. In vivo studies indicated that
the combination treatment significantly reduced the lung tumors in mice
models (Ichite et al., 2009).
11.2.3 BREAST CANCER
Anticancer efficacies of nanoparticle-encapsulated versus free cabazitaxel

were investigated in the xenograft mice model. The nanoparticle-encapsu
lated drug resulted in 75% reduction in tumors growth; whereas drug alone
gave complete remission only 22% reductions in tumors growth (Fusser et
al., 2019). Cytotoxic studies of lipospheres co-loaded with thymoquinone
along with cabazitaxel were investigated against breast cancer cell lines
like MCF-7 and MDA-MB-231. It revealed that in cell cycle analysis
combination of cabazitaxel and thymoquinone co-loaded lipospheres
significantly increased Sub G1 phase arrest and cell death in MCF-7 and
MDA-MB-231 breast cancer cells (Kommineni et al., 2018). Anti-breast
cancer efficiency of solid lipid nanoparticles loaded with cabazitaxel and
hyaluronic acid was assessed in MCF-7 cells. Increased cytotoxicity was
noted in nanoparticles-treated cells than with the cells treated with drug
(Zhu and An, 2017). Anticancer effects of cabazitaxel-loaded micelles
revealed that cabazitaxel-loaded micelles did not show much effect on the
survival capacity of 4T1 cells, but remarkably reduced the cell migration
activities (Zhong et al., 2017).
Antitumor efficiency of keratin nanoparticles loaded with paclitaxel
was investigated against breast cancer cell lines such as MDA MB 231 and
MCF-7 using 2D and 3D models. The nanoparticles significantly suppressed
the proliferation of cancer cells in 2D models. The nanoparticles showed
their anticancer mechanism by increasing the proapoptotic genes levels and
proteins (Foglietta et al., 2018). Combined anticancer effect of sorafenib,
paclitaxel, and radiation therapy was assessed in renal cell carcinoma
and breast cancer xenograft models. It revealed that combination therapy
exhibited more significant anticancer effects than cotreatment with paclitaxel
or sorafenib and radiation (Choi et al., 2019). The anticancer activity of
nanoparticles encapsulated with paclitaxel and gemcitabine, revealed that
it enhanced anticancer activity than the paclitaxel and gemcitabine treated
alone (Dong et al., 2018). Antiproliferative activity of paclitaxel on two
cancer cell lines such as MCF-7 and Hela cell lines, revealed that paclitaxel
strongly inhibited the growth of MCF-7 cells, and very less cytotoxic activity
was observed against Hela cell lines (Ryang et al., 2019).
Docetaxel nanoformulation was prepared using folic acid, thiol, and
chitosan to enhance cellular internalization and to improve oral absorption
of docetaxel in in vitro and in vivo models. The formulation notably inhibited
the cancerous cell proliferation and enhanced the successful transport of
drug across the intestine in in vivo model and significantly improved the
oral bioavailability of drug (Sajjad et al., 2019). Further, Fard et al. (2017)
studied anticancer effects of combined docetaxel therapy and radiation
therapy on MCF-7 cancer cell lines. Significant synergetic dose-dependent
cytotoxicity was observed in neoadjuvant therapy when compared to mono-
therapy models. Antiproliferative efficiency of paclitaxel-loaded selenium
nanoparticles was assessed against breast, cervical, and colon cancer cell
lines. Nanoparticles showed noteworthy antiproliferative activity against the
tested cancerous cell lines. It showed their mechanism of action by seizing
the G2 phase of the M phase leading to apoptosis (Bidkar et al., 2017).
The cytotoxic effect of taxol was carried out on MCF-7 breast cancer
and p53 null cell lines using in vitro studies. Taxol exhibited its anticancer
activity mechanism by suppressing the cyclin inhibitor p21WAF1 in both
cell lines (Blagosklonny et al., 1995). Further, they elucidated the anticancer
mechanism of taxol, showed that Bel-2 phosphorylation will occur after the
activation of Raf-1 protein. The cytotoxic activity of taxol was time and
dose-dependent (Blagosklonny et al., 1996). Brown et al. (2004a) identified a
new anticancer mechanism of action against docetaxel resistant two types of
breast cancer cell lines, that is, MCF-7 and MDA-MB-231 using the cDNA
microarray method. The reduction in p27 expression levels is associated
with acquired docetaxel-resistant cancer cells. Chan et al. (2010) evaluated
the anticancer efficiency of Bevacizumab with the combination of taxanes in
metastatic breast cancers. The combined therapy exhibited more beneficial
effects in a broad range of cancer patients than taxanes alone treatment.
A clinical study with taxane-based neoadjuvant chemotherapy was
conducted in 45 breast cancer patients with stage II/IIIA. They suggested
that patients with tumor size ≤4 cm after chemotherapy with taxanes are
best candidates for breast conservation therapy (El-Sayed et al., 2010). A
combined cytotoxic effect of docetaxel with oestrogen receptor (ICI 182,780)
was assessed against two breast cancer cell lines, that is, MCF-7 ADRr and
MDA-MB 231. Both the tested drugs showed synergetic cytotoxic effects on
tested cell lines. ICI 182,780 increased the docetaxel activity. The mecha
nism involved is inhibition of P-glycoprotein activity, seizing G2 phase of M
phase and inactivation of Bcl-2 protein and induction of apoptosis (Ferlini
et al., 1998). Tumor suppression activity of taxol, vincristine, colchicine,
and 2-Methoxyestradiol (2-ME) was investigated on C57BL/6 mice model.
2-ME and taxol exhibited moderately inhibited the expression levels of
VEGF-induced neovascularization and basic fibroblast growth factor, but
vincristine and colchicine showed no cytotoxic effect (Klauber et al., 1997).
Antiangiogenic and antitumor efficiency of paclitaxel, trastuzumab with/
without combination was evaluated on mice treated with ErbB-2-overexpressing
breast carcinoma cells. In combined therapy with paclitaxel/trastuzumab,
exhibited significant reduction in tumor volume and micro-vessel density than
control group. The antitumor activity of both the components may be mediated
via the reduction of phosphorylated Akt (Klos et al., 2003). A comparative
clinical study was conducted to evaluate anticancer activity of paclitaxel and
with or without bevacizumab in metastatic breast cancer patients. Combina
tion therapy with bevacizumab/paclitaxel extended the development of free
survival rate but not overall survival rate (Miller et al., 2007).
11.2.4 PROSTATE CANCER
Anticancer effects of paclitaxel or noscapine alone and with the combination

were investigated against prostate cancer cell (PCC) lines such as PC-3 and
LNCaP. Both the cells treated with paclitaxel and noscapine significantly
decreased the mRNA expression of B-cell CLL/Lymphoma and enhanced
the expression levels of Bcl-2-associated X protein (Rabzia et al., 2017). The
mechanism and synergetic effects of docetaxel combined with acankoreano
gein or impressic acid was studied by (Jiang et al., 2018). Anticancer poten
tial of cabazitaxel was investigated in prostate cancer cell lines. It revealed
that the component significantly suppressed the functionality of androgen
receptor and its associated proteins HSP40, HSP90α, and HSP70/HSP90
(Rottach et al., 2019). Anticancer effects of hyaluronic acid encapsulated
cationic liposomes of cabazitaxel was assessed against DU-145 and PC-3
cancer cell lines (Mishra et al., 2019). It revealed that liposomes exhibited
potent cytotoxic activity with low IC50 values by inhibiting cell migration and
inducing apoptosis. Further, the liposomes showed its synergetic cytotoxic
effects on CD44+-labeled DU-145 and PC-3 cancer cell lines.
Anticancer efficiency of nano-formulation prepared of cabazitaxel was
investigated against metastatic prostate cancer. It showed that the formula
tion significantly reduced the pain of the bone tumor and improved bone
structure (Gdowski et al., 2017). The combined anticancer efficiency of
docetaxel and desmopressin was studied against the castrate-resistant
prostate cancer cell (CRPC) lines-induced tumors in orthotopic nude mice
models. The combination therapy significantly reduced the CRPC cell lines-
induced tumor volume in mice model (Bass et al., 2019). Gallego-Yerga et
al. (2017) investigated the cytotoxic effect of nanoparticles synthesized from
docetaxel against human PCC lines PC3 and LnCap and glioblastoma rat C6
and human U87 cell lines. The nanoparticles encapsulated with docetaxel
exhibited dose-dependent antitumor effects against the tested prostate and
glioblastoma cell lines.
Combined cytotoxic effects of docetaxel- and angiostatin-mediated gene
therapy were evaluated on PCC lines LNCaP, PC3, and DU145 and human
endothelial cells. It revealed that human endothelial cells are more sensitive
to docetaxel than prostate cancer cell PCC lines. Among the tested PCC
lines, PC3 cells are more sensitive to docetaxel. Combined therapy exhibited
significant antitumor effects in PC3-grafted athymic mice (Galaup et al.,
2003). Anticancer efficiency of taxanes, that is, paclitaxel and docetaxel in
combination with methylseleninic acid were evaluated on PCC lines in in
vitro methods and in vivo method using DU145 xenograft mice model. It
showed that combination therapy exerted good cytotoxic effects on tested
PC-3 and DU145 cells. In in vivo study, the combination of paclitaxel/meth
ylseleninic acid was significantly inhibited tumors in xenograft mice (Hu et
al.,2008).
The in vitro and in vivo experiments were conducted by Jiang and Huang
(2010) to demonstrate the antitumor activity of taxol on 22Rv1 PCC lines
and 22Rv1 xenografts mice models, revealed that in vitro experiment taxol
upregulated PTEN, tumor suppressor gene, and inhibited transcripts of the
androgen receptor. In in vivo study, it induced mitotic arrest and reduced the
expression levels of the androgen receptor. The antimitotic effect of docetaxel
and all-trans retinoic acid (ATRA) was assessed in drug and hormone-resis
tant prostate cancer cell lines, DU-145. The results demonstrate that both the
tested components exhibited time and dose-dependent cytotoxic activity and
strongly reduced the expression of lymphotoxin beta-receptor, surviving,
and myeloid cell leukemia-1 at transcript levels (Kucukzeybek et al., 2008).
11.2.5 PANCREATIC CANCER
Anticancer efficiency of polymeric nanoparticles prepared using docetaxel

along with IR in pancreatic cancer therapy was assessed (Park et al., 2018).
The combination of nanoparticles along with IR showed a strong cytotoxic
effect against the tested cell lines models. They concluded that this method
is a novel and promising radio-sensitizing agent.
11.2.6 OVARIAN CANCER
Combined therapy of paclitaxel with cyclophosphamide and cisplatin was

assessed in ovarian cancer patients, revealed positive clinical benefits to
human ovarian cancer patients (Reed et al., 1995). Cytotoxic efficiency of
taxol was investigated against ovarian cancer cells such as human A2780
and hamster CHO. It revealed that taxol was more toxic to mitotic phase
cells but not interphase cells. Taxol exhibited a potent and noteworthy
cytotoxic effect against human A2780 cells than CHO cells (Lopes et al.,
1993). Antitumor effects of taxol against murine mammary and ovarian
cancer cells induced tumors in mice were studied using in vivo experiment.
The kinetic study results revealed that taxol induced antitumor effect in a
concentration-dependent manner (Milas et al., 1995). Lorigan et al. (1996)
highlighted the introduction of paclitaxel as a potent chemotherapeutic drug

to treat ovarian cancer. The anticancer effect of taxol was assessed in patients
with three stages of ovarian cancer, that is, advanced, progressed, and drug
refractory. Taxol significantly reduced cancer lesions drug refractory stage
patients (McGuire et al., 1989). Nicoletti et al. (1993) reported the antitumor
potential of taxol against ovarian cancer tumors growing in the peritoneal
cavity of mice. Taxol exhibited notable enhancement in the survival time of
mice with tumors than the reference drug cisplatin. A comparative cytotoxic
study of natural and synthetic taxol was conducted against ovarian cancer
cell lines using in vitro methods. Results revealed that taxol exhibited
concentration-dependent cytotoxic activity against the tested cell lines. No
significant difference was observed between synthetic and natural taxol
(Modarresi-Darreh et al., 2018).
A combined clinical study was carried out with taxanes with or without
platinum in 222 patients with sex cord-stromal ovarian tumors. Pre-treatment
with taxanes showed significant antitumor activity in the tested patients.
Taxanes in combination with platinum reduced the disease symptoms in
patients (Brown et al., 2004b). du Bois et al. (2009) conducted a randomized
clinical experiment with two combinations, that is, cisplatin/paclitaxel and
carboplatin/paclitaxel in 798 ovarian cancer patients. Of the two combina
tions tested, carboplatin/paclitaxel combination affords better anticancer
effects associated with the quality of life with better tolerability than cisplatin/
paclitaxel combination. The efficacy and toxicity of a new nano-formulation
prepared using paclitaxel and abraxane were investigated against 2008 human
ovarian cancer, indicated that paclitaxel-based nano-formulation showed
significant anti-tumor activity than abraxane (Feng et al., 2010).
11.2.7 CERVICAL CANCER
Microtubule suppression activity of taxol isolated from T. brevifolia was

investigated against HeLa cell lines. No effect was observed at concentrations
on DNA, RNA, and protein synthesis in cancer cell lines during the treatment
period. The component significantly promoted the assembly of calf brain
microtubules but decreased the lag time for microtubule assembly and the
critical concentration of tubulin required for assembly (Schiff et al., 1979).
Cytotoxic effects of paclitaxel assessed in HeLa cell demonstrated that
the component induced breaks in DNA into nucleosome-sized fragments,
which is a characteristic feature of apoptosis (Jordan et al., 1996). Further,
taxol inhibited the proliferation of HeLa cells at 8 nM concentration. The
mechanism of action showed that taxol altered the formation of metaphase

plate and spindle fiber during metaphase cell division (Jordan et al., 1993).
11.2.8 LEUKEMIA
Anti-proliferative activity of taxol against leukemia cell lines, that is, HL-60
and KG-1 cells, demonstrate that taxol induced internucleosomal DNA
fragmentation and apoptosis. It exhibited marked inhibition against HL-60
cells. It showed its anticancer mechanism by reducing the Bcl-2 and c-myc
transcription factor levels (Bhall et al., 1993). A combined cytotoxic effect
of docetaxel with oestrogen receptor (ICI 182,780) was assessed against two
leukemia cell lines, that is, CEM VBLr and CEM. Both the tested drugs
showed synergetic cytotoxic effects against the tested cell lines. ICI 182,780
increased the docetaxel activity. The mechanism involved is the inhibition of
P-glycoprotein activity, seizing of the G2 phase of M phase, inactivation of
Bcl-2 protein, and trigger apoptosis (Ferlini et al., 1998).
11.2.9 GASTRIC CANCER
Docetaxel and radiotherapy (hypersensitivity)-based cytotoxic effects against

gastric cancer cell lines, revealed that hypersensitivity was not correlated with
ATM kinase-dependent early G2 phase checkpoint arrest (Balcer-Kubiczek
et al., 2008). Chang et al. (2005) investigated the combined anticancer effect
of carboplatin and paclitaxel in patients with advanced gastric cancer, treated
with 5-fluorouracil and platinum. The results revealed that the combination
therapy showed beneficial effects to reduce the gastric cancer in patients.
11.2.10 EPITHELIAL CANCER
Cytotoxic activity of docetaxel was assessed in human epidermoid KB, breast

HCC1937, and colon HT-29 cancer cells. The pre-treatment of cells with
docetaxel for 48 h resulted in increased levels of RAS protein, the phosphory
lated isoforms of Erk-1/2 and Raf-1. In another study, they used R115777,
a farnesyl transferase inhibitor that suppresses RAS, along with docetaxel,
resulted in strong synergetic inhibition of the growth of the tested cancer cell
lines (Caraglia et al., 2005). Epithelial cancer (A549) cells treated with pacli
taxel demonstrated that paclitaxel at low concentrations (3–6 nM) significantly
inhibited its proliferation. It upregulated both p53 and p21 genes, which leads
to cell cycles arrest at G1 and G2 phases (Giannakakou et al., 2001).
11.2.11 COLON/ COLORECTAL CANCER
The antimitotic activity of toxoids (paclitaxel and docetaxel) and nocodazole

was evaluated using RT-PCR, immunoblot, and flow cytometry analysis assays
against HT29-D4 cell line. Both the components exhibited 30–50% inhibi
tion in c-myc induction in HT29-D4 cell cultures. Flow cytometry analysis
revealed that c-myc inhibition is not linked to the mitotic arrest (el Khyari et
al., 1997). Colon cancer (HCT 116) cells treated with paclitaxel demonstrated
that paclitaxel at low concentrations (3–6 nM) exhibited cytotoxic effects
with no significant activity (Giannakakou et al., 2001). The anti-angiogenetic
activity of non-toxic concentration of docetaxel was investigated on LS174T
Cells. It significantly suppressed the expression levels of transcripts such as
basic fibroblast growth factor, matrix metalloproteinase-2, matrix metallopro
teinase-9, and VEGF at both gene and protein levels (Guo et al., 2003).
11.2.12 HEAD AND NECK SQUAMOUS CELL CARCINOMA
Cytotoxic effects of bevacizumab combined with paclitaxel against head and

neck squamous cell carcinoma cell lines and tumor xenografts mice models,
resulted in strong inhibition of tumors in xenografts mice models when
compared to individual components (Fujita et al., 2007).
11.2.13 FIBRO SARCOMA
A comparative anti-tumor/anti-angiogenic effect of docetaxel and paclitaxel

was investigated on fibro sarcoma cells, demonstrated that docetaxel exhibited
noteworthy anti-angiogenic activity in fibro sarcoma cells (Grant et al., 2003).
11.2.14 THYROID CANCER
A combined treatment taxanes with dehydroxymethylepoxyquinomicin

(DHMEQ) on anaplastic thyroid cancer cells, demonstrate that it strongly
induced programmed cell death, and expression of poly (ADP-ribose)
polymerase, caspase 3, and survival. In the xenograft animal model, the

combined therapy significantly exhibited the greater antitumor activity
(Meng et al., 2008).
11.3 ANTICANCER ACTIVITY OF BREVIFOLIOL
Brevifoliol is another diterpenoid cyclodecane (Figure 11.2), isolated from

needles of Himalayan yew tree reported to have potent anti-proliferative
activity against all cancer cell lines than paclitaxel and it exhibited note
worthy cytotoxic activity against colon cancer cell lines than taxol. It
showed its mechanism of action by inducing apoptosis through the process
of apoptosomes formation in CaCo2 cell lines (Chattopadhyay et al., 2008;
Kaur et al., 2014).
FIGURE 11.2 Therapeutic effect of T. brevifolia components against different types of cancers.
11.4 ANTICANCER ACTIVITY OF BACCATIN III
Tumor suppression activity of baccatin III, a precursor used to synthesize

taxol, in combination with the antitumor drug gemcitabine was investigated.
The combination of gemcitabine with baccatin III was resulted in a signifi
cant reduction in tumor growth. Baccatin III alone enhanced the immune
adjuvant activity (Kim et al., 2011). A comparative antitumor effect of

standard baccatin III and biochemically produced baccatin III was against
four human cancer cell lines such as cervical, lung, skin, and liver cancers.
Enzymatically synthesized baccatin III showed its potent cytotoxic effects
against cervical cancer cell lines followed by lung, skin, and liver cancer cell
lines. Further, it suppressed the free radical formation in the tested cell lines
(Sah et al., 2020).
FIGURE 11.3 Structure of brevifoliol.
11.5 ANTICANCER ACTIVITY OF TAXUS BREVIFOLIA EXTRACTS/

NANOPARTICLES
Silver nanoparticles synthesized from T. brevifolia leaf aqueous extract were

subjected to evaluate anticancer activity against MCF-7 cell lines using in
vitro methods. It revealed that the nanoparticles showed strong antimitotic
activity on MCF-7 cell lines than standard drug taxol (Sarli et al., 2020).
A comparative cytotoxic/anticancer activity of taxol derived from T. brevi
folia and Aspergillus terreus was investigated in Ehrlich ascites carcinoma
mice. In results, taxol derived from both sources exhibited noteworthy
cytotoxic activity in mice models and further it enhanced the antioxidant
potential of mice. Histopathological studies showed that toxol significantly
reduced Ehrlich ascites carcinoma-induced oxidative stress (Zein et al.,

2019). Anti-proliferative efficiency of taxol and baccatin III isolated from
Fusarium solani was investigated against different types of cancer cell lines
like Ovcar3, HeLa, Jurkat, HepG2, and T47D. Both baccatin and taxol were
derived from the fungal source and significantly suppressed the growth of
tested cancerous cell lines in vitro conditions (Chakravarthi et al., 2013).
KEYWORDS
• Taxus breviflia
• taxol
• anticancer activity
• paclitaxel
• docetaxel
• and 10-deacetylbaccatin III
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CHAPTER 12
Glycyrrhiza glabra
VIJAY KUMAR VEENA1, B. UMA REDDY2,
ADHIKESAVAN HARIKRISHNAN3, and RAMASAMY SHANMUGAVALLI3
1
Department of Biotechnology, School of Applied Sciences, REVA University,
Bangalore, Karnataka, India
Department of Studies in Botany, Vijayanagara Sri Krishnadevaraya
2
University, Ballari, Karnataka, India

3
Department of Chemistry, School of Arts and Science, Vinayaka Mission
Research Foundation-AV Campus, Chennai, Tamil Nadu, India
ABSTRACT
Glycyrrhiza glabra, a medicinal botanical belonging to the family Fabaceae

(Leguminosae) that has been traditionally used for various diseases and
disorders worldwide. The medicinal metabolites attributed to cure or treat
the diseases are mostly belonging to the class of triterpenoids, flavonoids,
and saponins. G. glabra and its bioactive molecules, including glycyr
rhizin, glycyrrhetinic acid, isoliquiritigenin, Lic-A, B, and E, liquirtigin,
isoangustone-A, licoricidin, glycerol, licocoumarone, and β-hydroxy-DHP
are reported to exhibit the anticancer properties. Mode of anticancer action of
these molecules are different depending on the type of cancer and character
istics of these molecules. The major mode of anticancer mechanisms reported
to induce cell cycle arrest and antimetastatic potential produce the reactive
oxygen species (ROS), inhibit the protein kinases and transcription factors,
and activation of programmed cell death or autophagy through downregu
lating the various oncogenes and oncoproteins. The anti-inflammatory and
antioxidative potentials of the metabolites have an indirect anticancer mecha

nism. However, their interaction with the oncoproteins and their regulation
seems to be crucial for the bioactive molecules in G. glabra to be cancer
chemopreventive and to have anticancer properties.
12.1 INTRODUCTION
Glycyrrhiza glabra is a perennial plant that grows up to a height of 1 m

with pinnate leaves of 7–15 cm in length with pale to purple flowers in
hermaphrodite arrangement type of inflorescence with oblong legume fruit
with several seeds. The Glycyrrhiza genus is reported to have more than 30
species and some important ones are G. glabra, G. uralensis, G. inflata, G.
aspera, G. korshinskyi, and so on. Among them, G. glabra is well reported
with medicinal properties and it can fix nitrogen fixation in humid soils
(Fiore et al., 2005). The root part of the G. glabra is commonly utilized for
major medicinal and food usage, whereas the leaves are used as agrochemical
waste (Hayashi et al., 2009; Siracusa et al., 2011).
G. glabra, a medicinal plant from the family Fabaceae (Leguminosae)
is traditionally used for food additive and feeding purposes. The word
Glycyrrhiza means sweet root in Greek and commonly referred as liquorice,
licorice, glycyrrhiza, sweet-wood, etc. G. glabra is native to Mediterranean
regions and is also present in India, China, and Russia. G. glabra extracts
have been well-documented for various medicinal values. Hence, the extract
of plants is being utilized in functional food preparations, food supplements
in food industries, and pharma industries (Hayashi et al., 2016; Herrera et al.,
2009; Armanini et al., 2002). In traditional medicine of different geographical
areas of the world, the G. glabra extracts have been documented to treat the
disorders, such as cough, bronchitis, arthritis, and gastrointestinal issues. Also,
it is commonly used to treat peptic ulcers, gastritis, tremors, and respiratory
disorders in folk medicines.
Tea prepared from G. glabra roots is efficient in quenching the thirst,
the dried root powder is used as a tooth cleanser and also as food additive
(Armanini et al., 2002: Mukhopadhyay and Panja, 2008; Rizzato et al.,
2017). In the cosmetics industries, the G glabra extracts are used as pigmen
tation reducing agents in tropical uses. The root fibers are used as insulation,
wallboards, and boxboard materials in fire extinguishers, and beer industries
(Asl and Hosseinzadeh, 2008; Fiore et al., 2005).
Glycyrrhiza glabra 249
12.2 PHYTOCHEMICAL COMPOSITION OF GLYCYRRHIZA GLABRA
Phytochemical analysis of G. glabra has revealed that it is rich in proteins,

amino acids, simple and complex sugars with essential minerals, and
phytochemicals, including magnesium, calcium, iron, sodium, potassium,
phosphorous, silicon, selenium, manganese, copper, zinc, pectin, resin,
phytosterols, and gum (s) (Wang et al., 2015a, b). Metabolites, such as
estrogens, phytosterols, glycosides, tannins, coumarins, and vitamins, such
as B-complex, E, and C are reported. The major classes of phytomolecules
present in G. glabra include triterpenes, saponins, and flavonoids (Rizzato
et al., 2017). Glucuronides and glycyrrhizin, which are saponins present as
major constituents in G. glabra, are reported to be responsible for the sweet
taste (Yu et al., 2015). Oral intake of glycyrrhizin has been reported to break
down it into the 18-glycyrrhetic acid 3-O-monoglucuronide, and glycyr
rhetic acid by bacteria present in the human gut microbiome (Albermann
et al., 2010). The yellowish color of G. glabra is due to the presence of
flavonoids, such as flavones, flavanones, flavanonols, isoflavans, chalcones,
isoflavenes, and isoflavanones.
The majority of flavonoids reports include glycosides-based liquiritigenin
and isoliquiritigenin that includes liquiritins, isoliquiritin, liquiritin-apioside,
and licuraside (Rizzato et al., 2017). More recently, new flavonoids, such as
glucoliquiritin apioside, shin-flavanone, shinpterocarpin, prenyl-licoflava
none A, and 1-methoxyphaseloin have been reported from the dried roots
(Rizzato et al., 2017) of G. glabra. Glabridin is reported as a major isoflava
none in the dried root (Simmler et al., 2013). Small quantities of phenolics,
such as isoprenoid-substituted flavonoids, chromene, coumarins, comestans,
dihydroxyphenylalanine, dihyrostilbenes, and benzofurans are also reported.
Some of the volatile compounds present in the root include geraniol, hexanol,
pentanol, terpinene 4-ol, and α-terpineol which are responsible for the charac
teristics and smell of the plant. Essential oils are also reported from G. glabra
that include propionic acid, furfuraldehyde, 2, 3-butanediol, benzoic acid,
furfuryl-formate, maltol, 1-methyl-2-formylpyrrole, and trimethyl-pyrazine
(Chouitah et al., 2011).
12.3 PHARMACOLOGICAL PROPERTIES OF GLYCYRRHIZA GLABRA
The use of G. glabra dates back to a long period of traditional and folk medi
cine in various parts of the globe. The first reports are linked be in ancient
Egypt, China, and India. However, Theophrastu and Pedanius Dioscorides

have documented the G. glabra as medicinal herb that describes about the
medicinal effects (Armanini et al., 2002). Hence, it is the most important and
popular component in herbal and traditional medicine practiced worldwide.
Glabridin and G. glabra extracts have been reported to have neuroprotec
tive properties in orally administrated in vivo mice models by improving
the learning and memory through interaction with the adrenergic and
dopaminergic neurons (Hasanein, 2011; Dhingra and Sharma, 2006; Parle et
al., 2004). Similar reports have shown that G glabra extract and its purified
metabolites have been reported for their sedative (Jin et al., 2013; Cho et al.,
2010), antidepressant (Dhingra and Sharma, 2006), and estrogenic activities
(Sharma et al., 2012; Su et al., 2015; Tamir et al., 2001; Tung et al., 2014).
Glycyrrhizin of G. glabra has been reported to have antiviral activities
against the viruses, such as HIV (Sasaki et al., 2002), DHV (Soufy et al.,
2012), EV71 (Kuo et al., 2009), Hepatitis-C (Yasui et al., 2011), and influenza
virus (Michaelis et al., 2011). Glycyrrhetic acid, glycyrrhizin, glabridin, and
G. glabra extracts have been reported to possess hepatoprotective properties
(Huo et al., 2011; Hajiaghamohammadi et al., 2012; Yin et al., 2017; Liu et
al., 2017) and anti-inflammatory activities (Xiao et al., 2010; Rackova et al.,
2007; Wang et al., 2017a, b) in vivo and in vitro studies.
12.4 ANTICANCER METABOLITES FROM GLYCYRRHIZA GLABRA
Several reports have shown the anticancer potential of G. glabra extracts

and purified metabolites. The 18-β-glycyrrhetinic and glycyrrhizic acid have
shown anticancer properties by inducing the mitochondrial-mediated cell
death in the tumor cells, such as cervical and uterus cancer cells (Lee et al.,
2008). Glycyrrhizin acid and glycyrrhetinic acid have been reported to have
anticancer activities against gastric cancer. However, glycyrrhizin exhibited
the suppression of thromboxane A-2 in lung cancerous cells with less toxic
effects (Deng et al., 2017). The 18-β-glycyrrhetinic acid is reported for
anticancer properties against the ovarian, gastric, liver cancer, and leukemic
cells through the induction of ROS, RNS, and collapse of the membrane
potential of mitochondria (Hasan et al., 2016).
Derivatives of glycyrrhetic acids have been shown for the inhibition
of breast cancerous cells (MCF-7, MDA-MB-231 cells) (Li et al., 2016).
Another study has shown that these molecules have actively inhibited
the leukemic cells (HL-60 cells) through the induction of apoptotic
death through intrinsic or mitochondrial-mediated and extrinsic or ligand
receptor-mediated pathway (Huang et al., 2016). Lic-E has very efficient

than Lic-A and isoliquiritigenin in the anticancer properties (Xiao et al.,
2011; Yu et al., 2017; Park et al., 2015).
12.5 ANTICANCER MECHANISM AND MOLECULAR TARGETS
Phytochemical analysis of G. glabra especially commonly used root part

consisted of alkaloids, oils, polyamines, phenolics, triterpenes, flavones,
flavans, chalcones, flavonoids, and isoflavanoids (Kusano et al., 1991; Vaya et
al., 1997; Hatano et al., 2000). Antioxidant and anti-inflammatory properties
of G. glabra extracts and its components play a crucial role in various diseases
including cancer. The anticancer molecules reported in G. glabra include
glycyrrhizin, glycyrrhetinic acid, isoliquiritigenin. Lic-A, B and E, Liquirtigin,
iso-angustone A, licoricidin, glycryol, licocoumarones, and β-hydroxy-DHP
(Table 12.1)
Glycyrrhizin and glycyrrhetinic acid of G. glabra are reported to control
the proliferation and metastasis of skin and melanoma cancer in mice models
(Agarwal et al., 1991; Aydemir et al., 2011; Kobayashi et al., 2002). Similarly,
the anticancer potential of prostate cancer (Thirugnanam et al., 2008), gastric
cancers (Lin et al., 2014), leukemia (Hibasami et al., 2006), endometrial
cancer (Niwa et al., 2007), and breast cancers (Rossi et al., 2003).
Glabridin (GBD) have been reported to interfere in the migrative, inva
sive, and epithelial mesenchymal transition (EMT) in lung (A549), breast
(MDA-MB-231), and human endothelial (HUVEC) cells by downregulation
of FAK, Src, Akt, myosin, and Rho activities (Tsai et al., 2011; Hsu et al.,
2011). Similarly, in Huh-7 and Sk Hep 1 cancer cells, GBD decreased the
MMP, phosphorylated ERKs, NF-κB, and upregulated the MMP-1 inhibitors
(Hsieh et al., 2014).
Glycyrrhetinic acid has been shown to have anticancer activities in the
lung (A549, NCI-H460) cancer cells by inhibiting thromboxane synthase
activity without any toxicity to normal lung cells such as NCH-123 cells
and 16HBE-T bronchial epithelial cells (Huang et al., 2014a, b, c). Further,
it is known to induce G1/M cell cycle arrest and programmed cell death
in the NCI-H460 HSCLC cells with poly-ADP ribosyl polymerase (PARP)
cleavage and activation of caspases-3 with downregulation of antiapoptotic
Bcl-2 proteins and cyclin-D1-Cyclin-E proteins (Song et al., 2014). Also, the
glycyrrhetinic acid has been shown to reduce the phosphorylated forms of
protein kinase C (PKC αβ), extracellular activated proteins kinases (ERKs),
and C-Jun NH-2 terminal kinases that eventually induced cell death in the
TABLE 12.1 Various Anticancer Molecules Reported in G. glabra and Their Mode of Anticancer Action.
252
Anticancer molecules Mechanistic activity Molecular targets References
from G. glabra
Mixed Extracts Cell cycle arrest, apoptosis, Downregulation of mTOR, Agarwal et al. (1991); Aydemir et al. (2011);
autophagy, anti-metastasis upregulation of p53 and Bax Kobayashi et al. (2002)
Glycyrrhizin Cell cycle arrest, apoptosis, Downregulating the NF-κB, Thirugnanam et al. (2008); Lin et al. (2014);
autophagy, anti-metastasis, Selectin, TNF-α, and HMGB1 Hibasami et al. (2006); Niwa et al. (2007);
antiangiogenetic, and sensitization Rossi et al. (2003)
Glycyrrhetinic acid Cell cycle arrests, apoptotic cell Downregulation of NF-κB, Cherng et al. (2011); Chueh et al. (2012);
death, autophagy, anti-metastasis, PKC and GSH Song et al. (2014); Huang et al. (2014a, b, c)
differentiation, and antiangiogenetic
Isoliquiritigenin Cell cycle arrest, apoptosis, Downregulation of NF-κB and Cuendet et al. (2010); Ye et al. (2009);
autophagy, anti-metastasis, JNK Vandooren et al. (2013); Ma et al. (2013);
differentiation, antiangiogenetic, Lorusso and Marech (2013); Zhao et al.
and sensitization (2014); Takahashi et al. (2004); Hsu et al.
(2009); Bode and Dong (2015)

Lic-A Cell cycle arrests, apoptotic cell Blockade of ERKs, NF- κB and Kwon et al. (2008); Jiang et al. (2014); Yuan
death, autophagy, anti- metastasis, Ki-67); Downregulating the Bcl et al. (2013); Yo et al. (2009); Park et al.
differentiation, and antiangiogenetic 2 and Akt (2008); Kim et al. (2010); Yao et al. (2014)
sensitization
Lic-B and E Cell cycle arrest, apoptotic cell Downregulation of Bcl-2, Kwon et al. (2013); Kim et al. (2012) and Lee
death, autophagy, anti-metastasis, surviving, DNA topoisomerase, et al. (2013a, b)
and antiangiogenetic Ki67 and upregulation of Bax
Liquirtigin Apoptosis, autophagy, anti- Downregulation of Akt, Liu et al. (2011); Liu et al. (2012); Wang et al.
metastasis, differentiation, and activation of p53, and (2012); Zhou et al. (2010)
antiangiogenetic production of ROS
Glycyrrhiza glabra
Anticancer molecules Mechanistic activity Molecular targets References
from G. glabra
Isoangustone-A Cell cycle arrests, apoptotic cell Downregulation of mTOR, Huang et al. (2014a, b, c); Seon et al. (2010,
death, autophagy, anti-metastasis, JNK, and activation of death 2012); Lee et al. (2013a, b); Liang et al.
and antiangiogenetic receptors (DR) (2002); Song et al. (2013)
Glabridin Anti-metastasis, antiangiogenetic Downregulation of Rho-A, Tsai et al. (2011); Hsu et al. (2011); Hsieh et
FAK, Akt, and Src al. (2014)
Licoricidin Anti-metastasis Downregulation of uPA, Kobayashi et al. (1995)
MMP-9, and ICAM
Licocoumarone Apoptosis Increase in DNA damage Shin et al. (2011); Watanabe et al. (2002)
Glycryol Cell cycle arrests, apoptosis, and Activation of caspases-8, 9 and Shin et al. (2011); Tang et al. (2015)
autophagy FAS
β-hydroxy-DHP Apoptosis Downregulation of Bcl-2 Tang et al. (2015)
protein
253
tumor cells. However, the molecular and cellular targets of G. glabra extracts
and their components are not yet identified.
G. glabra is proven to be cancer chemopreventive in the case of
UV-irradiated skin tumor mice model, where it delayed the incidence of
tumor and reduced the tumor proliferation and size. In this study, the cells
had decreased thymidine dimers in UV-exposed skin cells, downregulated the
gene expression of proliferative cell nuclear antigen (PCNA), and exhibited
few DNA tail in terminal deoxy-nucleotidyl transferase-mediated d-UTP
nick labeling (TUNEL) assay of treated cells. The treated animals exhibited
increased p53 and p21/Cip1 levels in the epidermis (Cherng et al., 2011).
Further, the inhibition of NF-κB activity and cyclooxygenase-2 (COX-2)
activities with decreased prostaglandin E2 (PGE2) and nitric oxide (NO)
levels has also been observed. This evidence suggested that glycyrrhetinic
acid of G. glabra extract exhibited anticancer effects by blocking the
translocation of NF-κB by interrupting the degrading IκB (Cherng et al.,
2011). In another study, glycyrrhetinic acid-supplemented Wistar rats have
been shown to suppress the development of 1,2-dimethylhydrazine (DMH)
induced pre-cancerous lesions. This study also suggested the reduction in
the mast cells by reducing the gene expression of Ki67, NF-κB/p65, COX
2, iNOS, and vascular endothelial growth factor (VEGF) with increased
gene expression of p53, connexin 43, caspases 9, and activated caspases 3 to
activate the apoptosis (Khan et al., 2013). In the case of WEHI-3 leukemic
cells, glycyrrhetinic acid has been shown for the induction of G0/ G1 cell
cycle arrest and apoptotic cell death through DNA damage death-receptor,
mitochondrial-mediated, and endoplasmic reticulum (ER) stress-induced
signaling pathway (Chueh et al., 2012).
Isoliquiritigenin [(E-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-
2-en-1-one] (ILQ) chalconoids have been identified from G. glabra for its
anticancer activities. Dietary supplements of ILQ have been reported to delay
the DMBA-induced breast cancer in female Sprague-Dawly rats without any
toxic effects through aromatase inhibition (Cuendet et al., 2010; Ye et al.,
2009). But ILQ is also known to inhibit the matrix metalloproteases (MMP),
COX 2, and induced apoptosis (Vandooren et al., 2013). ILQ is also known
to downregulate the metastatic gene expressions, such as hypoxia-inducible
factor (HIF)-1 (Ma et al., 2013), VEGF (Cao, 2014), and MMP-2/-9 protein
(Kessenbrock et al., 2010) levels in breast cancerous cells. The mentioned
anticancer activity could be because of the regulation of gene expression
of PI3K, NF-κB, and p38 signaling pathways (Lorusso and Marech, 2013).
ILQ is shown to inhibit the phorbol myristate acetate (PMA)-induced gene
expression of MMPs in HuVeCs through inhibition of JNKs and p38 signaling

pathway (Kang et al., 2010). Oral supplementation of ILQ has reduced the
colon cancer proliferation, incidence and size of the tumor in azoxymethane
induced carcinogenesis in colitis-associated tumor of mice model (Zhao et al.,
2014). The same study has explained the downregulation of M2 macrophage
polarization in colon carcinogenesis through the downregulation of PGE2 and
IL- 6 inflammatory signaling pathway (Zhao et al., 2014). A majority of the
reports suggested that ILQ has induced the apoptotic cell death in cancer cells
through the upregulation of p53 and p21 levels (Zhou et al., 2014; Takahashi
et al., 2004). In the case of HeLa cervical cancer cells, ILQ has been shown to
induce G2/M cell cycle arrest and induced apoptosis (Hsu et al., 2009). This
mode of anticancer activities was observed through the activation of ataxia
telangiectasia mutated (ATM), increased phosphorylated p53, and cell cycle
regulators, including cyclin B, A, cell division cycle protein (Cdc2) cdc25C,
and checkpoint kinases (Chk2) even tyrosine kinase inhibitor resistance
NSCLC cells (Hsu et al., 2009; Bode and Dong, 2015).
Isoangustone-A (IAA), a new flavonoid-based compound has been
reported from the roots of G. glabra. IAA has been reported to have
anticancer activities mainly by interfering with the cell cycle and apoptosis.
IAA is reported to induce caspase-dependent and mitochondrial-mediated
apoptotic cell death in SW480 colorectal cancerous cells (Huang et al., 2014a,
b, c). IAA can also induce similar mechanism in hormone-resistant DU145
prostate cancerous cells and xenograft cancer BALB/c mice models through
the induction of G1 cell cycle arrest via downregulation of mTOR, Cdk-2,
Cdk-4, cyclin-A, and D1 levels (Seon et al., 2010, 2012; Lee et al., 2013a, b;
Liang et al., 2002). IAA is also known to suppress the growth of SKMEL 28
cells by direct binding to PI3K, MKK 4, and MKK 7 even in xenograft cancer
(Song et al., 2013).
Licochalcone-A (Lic-A), a major phenolic compound reported in G.
glabra is shown to exhibit antiproliferative and anti-inflammatory potentials,
but it also has strong antioxidant properties (Xiao et al., 2011). Lic-A has been
reported to induce apoptosis in breast cancer (MCF-7) and human promyelo
cytic leukemic (HL-60) cells through downregulation of Bcl-2 proteins (Rafi
et al., 2000). In combination with geldanamycin, it enhances the apoptosis
in ovarian cancer cells (OVCAR-3 and SKOV-3) (Kim et al., 2013; Lee et
al., 2013a, b). Lic-A also suppresses the proliferation of colon cancer (CT26)
implants in BAL-B/c by inducing apoptosis (Kim et al., 2010). Lic-A is also
reported to induce apoptotic cell death in the prostate cancerous (DU145)
cells (Park et al., 2014) while in human prostate cancerous cells (LNCaP),
it induced autophagy (Yo et al., 2009). An interesting result, Lic-A reduced

the viable cells in oral (KB) cancer cells with less effect on primary normal
keratinocytic cells (Kim et al., 2014). Further, it induced apoptosis through
gene expression of factor-associated suicide ligand (FasL) and eventually
the activation of caspases in KB cells (Kim et al., 2014). Others reported
that Lic-A inhibited gastric cancer cells (MKN28, AGS, and MKN45) by
inducing the G2 /M cell cycle arrest and apoptosis with increasing Rb levels
and decreasing cyclin-A/B and MDM-2 levels with increased cleavage of
PARP and activation of caspases-3 (Xiao et al., 2011; Fu et al., 2004). Few
reports suggest that Lic-A exhibits the inhibition of platelet-derived growth
factor (PDGF) induction of rat vascular smooth muscle cells (r-VSMC)
proliferations to reduce cyclin-A, D1, CDK 2/4, ERKs, and phosphorylated
Rb (Park et al., 2008).
The anticancer effect of Lic-A is partially because of its antioxidant and
anti-inflammatory potential. A study of C57BL mice with DSS-induced
colon cancer showed that Lic-A has reduced tumor formation and decreased
the expression of PCNA, β-catenin, iNOS, COX-2, and proinflammatory
levels (Kim et al., 2010). Another study suggested that when Lic-A has been
combined with cisplatin the treated mice of CT-26 have shown to inhibit the
cisplatin-induced liver and kidney damage (Lee et al., 2004).
Lic-A also has anti-inflammatory potential that inhibited the production
of NO and COX 2 in LPS-inflamed macrophage (Raw-264.7) cells (Kwon
et al., 2008). Lic-A also inhibited the human bladder cancer by generating
ROS and induction of apoptosis (Jiang et al, 2014; Yuan et al., 2013). Several
reports have suggested that Lic-A has been reported to suppress the NF-κB
and also mitogen-activated protein kinases (MAPKs) signaling pathway
(Furusawa et al., 2009). Other reports have suggested that Lic-A inhibited
the migrative and invasive potentials of lung (A549 and H460) cancerous
cells with the suppression of phosphorylated Akt (Huang et al., 2014a, b,
c). Other mechanisms reported by Lic-A is JNKs that is observed in other
diseases, such as diabetes or Parkinsons’ diseases (Yao et al., 2014).
Licochalcone-E (Lic-E) from G. glabra appears to induce the cell cycle
arrest and apoptotic cell death similar to the inactivation of NF-κB and
downregulating the antiapoptotic Bcl 2 proteins in breast cancerous cells
in mice model studies (Kwon et al., 2013). These studies also suggested
that Lic-E has an antiangiogenesis activity that reduced the migrative and
invasive potential of breast cancer cells. Like Lic-A, Lic-E and Lic-B have
anti-inflammatory and antioxidant potential as similar mechanism (Kim et
al., 2012; Lee et al., 2013a, b).
Liquiritigenin (LG), a flavonoid that possess anticancer activities in liver

cancer AMMM-7721 and HeLa cell lines through upregulation of Bax and
activation of caspase through inhibiting the Bcl-2 and survinin by the induc
tion of ROS in the cancer cells. In the lung cancer A549 cells and Hela xeno
graft mice studies, the LG inhibited the migrations and attachment of cells by
inactivating the MMP 2, reducing the phosphorylated Akt and activation of
ERKs (Liu et al., 2011; Liu et al., 2012; Wang et al., 2012; Zhou et al., 2010).
There are few reports on licocoumarone (LCM) and β-hydroxy-DHP that
are known to have antiproliferative potential through induction of apoptosis
(Shin et al., 2011; Watanabe et al., 2002). Glycryol has been shown to induce
apoptosis and cell cycle arrest by activating the Fas and caspases in Jurkat
cell lines. In the case of AGS and HCT-116, glycryol has induced autophagy
through the activation of JNK (Shin et al., 2011; Tang et al., 2015).
12.6 CONCLUSIONS
G. glabra and its molecules are reported to be used in the tradition-based

medicinal system and folk medicinal systems for long years. A literature
survey has suggested that the anticancer properties of phytochemicals present
in G. glabra had been reported to possess various modes of actions due to
various interactions of oncoproteins. Major responsible bioactive anticancer
molecules reported in G. glabra include glycyrrhizin, glycyrrhetinic acid,
isoliquiritigenin, Lic-A, B and E, Liquirtigin, isoangustone-A, licoricidin,
glycryol, licocoumarone, and β-hydroxy-DHP. The major mode of anti
cancer action includes inducing the cancerous cell cycle arrest, inducting
the apoptotic cell deaths, and antioxidant activity. Major molecular targets
to exhibit the anticancer potential from G. glabra have been identified that
include the protein markers related to cell cycle arrests, apoptosis-inducing
proteins, MMPs, COXs, GSK- β, PI3Ks, NF- κB, and MAPK that have been
evidenced by in vitro, in vivo, and in silico studies. These survey results in
G. glabra, suggest that its extract and components have no obvious toxicity
but can be chemopreventive and has anticancer potential.
ACKNOWLEDGMENT
We acknowledge Dr. Honoureen Beatrice Gamble, Department of Chemistry,

School of Arts and Science, Vinayaka Mission Research Foundation- AV
campus, Chennai.
KEYWORDS
• Glycyrrhiza glabra
• glycyrrhizin
• 18β‐ glycyrrhetinic acid
• glabrin
• isoliquiritigenin
• isoangustone-A
• Lic-A
• anticancer mechanism
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CHAPTER 13
Ocimum sanctum
SHARMISTHA BANERJEE1, RAJESH SINGH TOMAR2, and
SHUCHI KAUSHIK3
Biomedical Engineering and Bioinformatics, University Teaching
1
Department, Chhattisgarh Swami Vivekanand Technical University,

Newai, Bhilai, Chhattisgarh, India
Amity Institute of Biotechnology, Amity University Madhya Pradesh,
2
Maharajpura Dang, Madhya Pradesh, India

3
State Forensic Science Laboratory, Sagar, Madhya Pradesh, India
ABSTRACT
Ocimum sanctum (O. sanctum) is a tropical annual herb belonging to the

family Lamiaceae (Labiatae) and is popularly called Tulasi or holy basil, and
is cultivated across the whole of India. It ascends to a height of 1800 m in
the Himalayas, usually grown in courtyards and gardens. It is a Perennial
herb with multiple branching and poses a unique aroma. It is considered an
excellent drug with various essential oils and secondary metabolites and can
be used for curing dysentery, diarrhea, malaria, bronchitis, bronchial asthma,
arthritis, skin diseases, eye diseases, and chronic fever. O. sanctum also poses
antifungal, anticancerous, antifertility, antimicrobial, immunomodulatory,
anti-ulcer, anti-inflammatory, hepatoprotective, antihypertensive, antispas
modic, radioprotective, antiemetic, cardio protective, analgesic, antidiabetic,
diaphoretic and adaptogenic activity. Extracts from Ocimum sp. and phyto
chemicals or bioactive components from O. sanctum, such as rosmarinic acid,
eugenol, apigenin, β–sitosterol, carnosic acid, luteolin, oleic acid, linoleic
acid, isorientin, ocimarin, orientin, aesculectin, aesculin, chlorogenic acid,
galuteolin, citronellal, gallic acid, sabinene, camphene, dimethyl benzene,

vitamin C, ethyl benzene, and calcium are known to have several pharma
cological properties. Few among them have been reported to prevent oral,
lung, and liver cancers and chemical-induced skin cancer by enhancing the
antioxidant potential, altering genetic expressions, inducing apoptosis, inhib
iting metastasis and angiogenesis by deactivating matrix metalloproteinases.
The anticancer potential of O. sanctum extract has been tested against various
cancer cells both in vitro and in vivo, for example, human fibrosarcoma cells,
Swiss albino mice having Ehrlich ascites carcinoma (EAC), hamster buccal
pouch carcinogenesis, papillomas. This article is an attempt to study the prop
erties of secondary metabolites and bioactive components present in Tulsi and
its ability to act as a potent anticancer drug along with its mechanism.
13.1 INTRODUCTION
Tulsi also popularly known as holy basil, is extensively used in the Indian
medicine system. Another name for it is Vishnupriya which symbolizes the
one who is having the power to amuse Lord Vishnu. It is easily available and
cultivated across the whole country and its scientific name is O. sanctum.
Tulsi belongs to the family Lamiaceae/Labiatae, and in Sanskrit, it is named
as surasa. It has been used for more than 1000 years in Ayurveda for its
multiple healing ability and therapeutic potential (Kavyashree et al., 2019).
Tulsi is also referred to in the Charaka Samhita, the conventional
ayurvedic text (NIIR, 2004). Due to its astringent taste and strong smell,
it is considered a life saver in Ayurveda and is also known for increasing
the life span of individuals (Puri and Rasayana, 2002). Tulsi, the queen of
herbs, mythically unbeatable is one of the sacred and most cherished herbs
in the universe because of its immense medical and healing ability. Tulsi is
mentioned in conventional Ayurveda and the ancient Unani system of herbal
medicine and holistic health because of its immense healing potential and
a remedy for several ailments (Warrier, 1995). Fresh extract or decoction
of Tulsi leaves has been used since time immemorial for treating various
disorders (Siva et al., 2016).
Tulsi is present throughout the country and ascends to a height of 1800
m (30–60 cm) in the Himalayas, usually grown in courtyards and gardens.
It is a perennial herb with multiple branching and a peculiar aroma. Stems
and branches are usually purple in color, sub-quadrangular, sometimes
woody toward the bottom, clothed with soft spreading hairs. Leaves are 2.5
Ocimum sanctum 269
by 1.6–3.2 cm in size, Elliptic –oblong, obtuse or acute, entire or serrate,

pubescent on both sides, minutely gland—dotted, with obtuse base or acute,
petioles are 1.3–2.5 cm in length, hairy and slender (Kavyashree et al., 2019).
Inflorescence: Verticillaster. Flowers are racemes, 15–20 cm long in
close whorls, bract is nearly 3 mm long, width is mostly equal to the length,
usually ovate, long slender acuminate, ciliate; pedicels are longer than the
calyx, slender, pubescent. Fruits are basically shaped like nutlets, 1.25 mm
long, majorly ellipsoidal, nearly smooth, yellow in color with black mark
ings (Sharma et al., 2005; Basu, 1987; Kavyashree et al., 2019).
13.1.1 VERNACULAR NAMES
Vernacular names are names of the drug by which they are commonly known
in several regions of the world, and therefore helps in easy identification
of the drug globally. Vernacular names of Tulsi in different languages are
presented in Table 13.1.
TABLE 13.1 Vernacular Names of Tulsi.

Languages Vernacular names
Hindi Kalatulasi, Tulasi
Kannada Vishu tulasi, Kari tulasi, Sri tulasi, Tulashi-gida
English Holy basil
Malayalam Tulasi, Trttava, karuttarttavu, Niella tirtua, Krishna tulasi, Shiva tulasi
Telugu Tulasi, Gaggera-chettu
Tamil Tulasi, Karuttulasi
Bengali Tulasi, Krishna tulasi
Gujarati Tulasi, Talasi
Punjab Bantulsi, Tulasi
Marathi Tulasa, Tulasi
Konkani Tulsi
Source: Adapted from Siva et al. (2016).
Three varieties of Ocimum sp. are as follows:

1. Light or Rama Tulsi (Ocimum sanctum)
2. Dark or Shyama Tulsi (Ocimum sanctum)
3. Vana Tulsi (Ocimum gratissimum)
Scientific classification:
• Kingdom: Plantae
• Division: Magnoliophyta
• Class: Magnoliopsida
• Order: Lamiales
• Family: Lamiaceae
• Genus: Ocimum
• Species: O.Tenuiflorum
• Binomial name: Ocimum tenuiflorum or Ocimum sanctum L. (Siva et
al., 2016)
13.2 PROPERTIES
Decoction or extracts of Tulsi are used in the treatment of headaches, common

cold, heart disease, stomach disorders, inflammation, malaria, and several
forms of poisoning. In conventional times, O. sanctum L. is used in several
ways, as a dried powder, fresh leaf, or herbal tea. Numerous researchers have
used these extracts and have shown their antioxidant, anti-inflammatory,
anti-stress, and immunomodulatory activities (Singh et al., 1996; Mauli et
al., 1997; Mediratta et al., 2002; Sen et al., 1992). In addition to this, it
has also been known to pose anticarcinogenic and radioprotective properties
(Siva et al., 2016).
Several parts of O. sanctum L., such as flowers, stem, leaves, seeds,
root are reported to exhibit medicinal properties and have been used by
several conventional medicinal practitioners as analgesic, anticancer,
expectorant, antiemetic, antiasthmatic, antidiabetic, antistress, diaphoretic,
hepatoprotective, antifertility, hypotensive, hypolipidemic drugs. It is also
known to be used in treating bronchitis, fever, convulsions, arthritis, etc.
(Siva et al., 2016).
Tulsi is also known to have anti-fatigue characteristic, adaptogenic
property, antimicrobial, anticonvulsant, antifertility, antidiabetic, radio-
protective, anti-inflammatory cardioprotective, immunomodulatory, hepa
toprotective, anticarcinogenic, mosquito repellent, and analgesic activities
(Venkatachalam and Muthusamy, 2018).
The current article has tried to include information from several works
of literature regarding Tulsi, including synonyms, classification, chemical
composition, bioactive compounds, and anticancer properties.
Ocimum sanctum 271
13.2.1 CHEMICAL COMPOSITION
The chemical composition of O. sanctum L. bears high complexity as it

contains several nutrients and bioactive compounds. The composition and
concentration of these components are dependent on varying cultivation
regions, processing, storage, and harvesting environment. The reason and
mechanism behind this are yet unexplored completely (Pattanayak et al.,
2010; Siva et al., 2016).
13.3 BIOACTIVE COMPONENTS
The pharmacological and nutritional activities of Tulsi in its natural form are
because of the synergistic action of several phytometabolites present in it.
O. sanctum contains eugenol, volatile oil, carvacrol, urosolic acid, limatrol,
linalool, sesquiterpine, methyl eugenol, estragol and caryophyllene. Sugars
present in it contain polysaccharides and xylose (Pattanayak et al., 2010;
Kelm et al., 2000). Phytochemical analysis of leaves and stems of O. sanctum
has shown compounds, such as flavonoids, saponins, tannins, and triterpe
noids (Pattanayak et al., 2010; Jaggi et al., 2003). The herb also contains
Vitamin A, C, and minerals, such as iron, zinc, calcium, chlorophyll, and
several other phytonutrients (Anbarasu and Vijayalakshmi, 2007). Different
phytochemicals and chemical compounds present in several parts of Tulsi
are depicted in Table 13.2.
TABLE 13.2 Different Phytochemicals and Bioactive Components Present in Several Parts
of Ocimum sanctum.
S. Plant Phytochemicals Bioactive component References
No. part
1. Leaves Flavonoids, saponins, Eugenal, eugenol, carvacol, Pattanayak et al.
alkaloids, tannins, limatrol, urosolic acid. (2010); Kelm et al.
anthocyanins, sterols, linalool methyl carvicol, (2000); Jaggi et al.
phenols, terpenoids caryophyllene, anthocyans (2003)
2. Stem Phenols, flavonoids, Crisimartin, apigenin, Pattanayak et al.
tannins, triterpenoids, Romarinic acid, isothymonin, (2010); Kelm et al.
saponins isothymocin (2000)
3. Seeds Sitosterol, Fatty acids Xylose and polysaccharides Pattanayak et al.
(2010); Kelm et al.
(2000)
Source: Adapted from Siva et al. (2016). https://creativecommons.org/licenses/by-nc/3.0/
13.3.1 ANTICARCINOGENIC PROPERTY
Tulsi is a rich source of phytoconstituents and possesses an outstanding role

in medicine. These phytochemicals are not essential for its survival but are of
substantial importance to the human community to carry out various protec
tive functions in the human body.
Plant contains polyphenolic compounds such as flavonoids, tannins,
curcuminoids, gallocatechin; stilbenes like resveratrol; anthocyanidin like
delphinidin; they have a broader range of pharmacological activities and
are considered to have chemopreventive and remedial properties against
malignant growth (Singh et al., 2012). Additionally, phenolic phytochemi
cals regulate oxidation stress-related sicknesses because of their immediate
association with quenching the free radicals (Boots et al., 2008). Anticancer
activity of phytopolyphenols includes preparation of endogenous copper
potentially chromatin-associated copper and ensuing pro-oxidant activity
(Hadi et al., 2007). It has additionally been proposed that the cytotoxic move
ment of plant phenolics displays pro-oxidant action (Galati and O’Brien,
2004, Summers and Felton, 1994) and cytotoxic properties (Yamanaka et al.,
1997; Sugihara et al., 1999).
The test studies completed on biological models utilizing O. sanctum
(OS) extract on fibrosarcoma cells in culture have shown that O. sanctum
displays anticancer action (Karthikeyan et al., 1999). Younger leaves of
the plant display additional resistance, and furthermore are known to have
anticancer activity in experimental organisms (Mondal et al., 2009). O.
sanctum has additionally been shown to display restoring properties against
septic and hostile hypersensitive impacts (Godhwani et al., 1988). Tulsi has
numerous gainful properties with insignificant harmfulness and is an ideal
antistress/adaptogenic specialist for the advancement of well-being and the
counteraction and treatment of illness. Methanolic concentrate of Ocimum
varieties has been displayed to have malignant growth preventive potential
through the decrease of the overabundance measure of nitric oxide (Kim et
al., 1998). Tulsi also diminishes the frequency of benzo(a) pyrine-actuated
neoplasia and 3-methyl di-methyl amino azobenzene initiated hematomas
in trial creatures (Aruna and Sivaramarishnan, 1990). Ethanolic l extract of
Tulsi leaves administered through a topical route resulted in a significant
decrease in the upsides of growth occurrence (Paplliomas) in the skin of
abino mice (Prashar et al., 1994).
Tricyclic sesquiterpenoids 2-(hydroxymethyl)- 5,5,9-trimethylcyclo
[7.2.0.03,6]undecan-2-ol extracted from the oil of OS leaves have been
Ocimum sanctum 273
found to display antiproliferative activity against MCF-7 cell line (IC50 30

± 0.5 μM) utilizing doxorubicin as standard (IC50 9.7 μg/mL) (Singh and
Chaudhuri, 2013; Singh et al., 2014). In continuation of antiproliferative
screening against MCF-7 cell line, 5β-hydroxycaryophyllene, 4,5-epoxy
caryophyllene, and sesquiterpenes β-caryophyllene displayed IC50 esteems
4.8, 7.0, and 73.0 μg/mL, individually. Mixtures of apigenin, rosmarinic acid,
orientin, luteolin, vicenin-2, oleanolic acid, and ursolic acid are reviewed
and assayed for their anticancer activity (Nagaprashantha et al., 2011).
Flavonoids and terpenoids are the significant class of mixtures answerable
for the anticarcinogenic movement of O. sanctum.
Eugenol (1-hydroxy-2-methoxy-4-allylbenzene), the dynamic compound
present in Tulsi has been observed to be a great extent answerable for the
remedial possibilities of Tulsi in the treatment of different chronic sick
nesses including cancer (Prakash and Gupta, 2005). It was tracked down
that the ethanolic concentrate of Ocimum leaves hindered the expansion
and angiogenesis-related protein through the downregulation of Bcl-2 and
vascular endothelial development factor (VEGF) articulation and overar
ticulation of capase-3 during N-methyl-N"- nitro-N-nitrosoguanidine incited
gastric malignancy bearing rates, and with the decrease in cancer cell size, an
expansion in life expectancy of mice bearing Sarcoma-180 and Lewis-lung
carcinoma in animal models was additionally noted (Prashar et al., 1998;
Manikandan et al., 2007a, b; Nakamura et al., 2004). It is also observed that
the dynamic phytoconstituents, in particular, oleanlic acid and urosolic acid
in Tulsi can display anticancer properties (Singh et al., 2010). The alcoholic
concentrate has also been known to enhance the activity of cytochrome b5,
cytochrome p450, glutathione S-transferase, and aryl hydrocarbon hydroxy
lase that plays a significant part in the detoxification of cancer-causing
agents and mutagens (Govind and Madhuri, 2006). The test study directed
on animal models has shown that O. sanctum has the ability to diminish the
occurrence of benzo(a)pyrine instigated neoplasia of forestomach of mice
and 3-methyl-4-dimethylaminoazobenzene prompted hepatomas in rodents
(Aruna and Sivaramarishnan, 1992).
The ethanolic O. sanctum leaf concentrate represses 7, 12-dimethylbenz[a]
anthracene (DMBA)- incited oxidative stress and genotoxicity by modulating
the xenobiotic metabolizing enzymes, thus decreasing the degree of protein
and lipid oxidation and up-managing cancer prevention agents (Manikandan
et al., 2007a, b). Manikandan et al. (2008) contemplated the combinatorial
chemopreventive viability of O. sanctum (OS) and Azadirachta indica (AI)
against N-methyl-N’- nitro-N-nitrosoguanidine (MNNG)-incited gastric
carcinogenesis, in light of changes in oxidant-cancer prevention agent status,

cell multiplication, angiogenesis, and apoptosis in a rodent forestomach
carcinogenesis model and found that OS and AI mix might be interceded by
their cancer prevention agent, antiproliferative, antiangiogenic, and apoptosis
activating properties.
OS extract diminished the potency of ornithine decarboxylase, a protein
participating in the regulation of cell expansion and increase of malignant
growth. There was additionally a contemporary decrease in the Phase I
catalysts and lipid peroxidation inferring that O. sanctum interferes in the
movement of cancer-causing agents and that this causes a decline in the
development of extreme cancer-causing moiety (Jemal et al., 2007). The
anticancer potential of seed oil of Tulsi was inspected against 20-methylcho
lanthrene-induced fibrosarcoma cancers in the thigh of Swiss albino mice.
An increase of 100 μL/kg body weight (most extreme endured portion) of the
oil essentially diminished 20-methaylcholathrene-initiated cancer predomi
nance and growth volume. The mice supplemented with the O. sanctum seed
oil showed an enhanced survival rate and the incidence of tumor was also
delayed in this case (Aggarwal et al. 2006).
13.3.2 ANTIRADIATION ACTIVITY
O. sanctum can shield the DNA of the body from risky radiation (Singh,
2005; Panda and Kar, 1998; Devi and Gonasoundari, 1995). It is important
to refer that the flavonoids specifically vicenin and orientin extracted
from Tulsi leaves displayed comparatively better radioprotective impact
in comparison to commercially available chemical radioprotectors. They
have shown critical protection to the human lymphocytes against the
clastogenic impact of radiation at low, non-poisonous concentrations
(Devi et al., 2000). The blend of OS leaf extract with WR-2721 (an
engineered radioprotector) enhances higher bone marrow cell protection
and show a decrease in the harmfulness of WR-2721 at higher portions,
recommended that the blend would have promising radioprotection in
humans (Gonasoundari et al., 1998).
Bhartiya et al. (2006) explored radiodefensive effect of aqueous extract of
O. sanctum L. (40 mg/kg, for 15 days) in mice exposed to high dosages (3.7
MBq) of oral 131 iodine by examining the organ loads, lipid peroxidation,
and cell reinforcement protection compound in different target organs, such
as stomach, kidney, liver, and salivary organs at 24 h after administration.
Ocimum sanctum 275
The consequences of the investigations showed that the pretreatment with

O. sanctum L. in radioiodine-exposed group displayed a critical decrease in
lipid peroxidation in both salivary glands, kidney, and in the liver; dimin
ished glutathione (GSH) levels displayed a huge decrease after radiation
exposure while pre-therapy with O. sanctum L. has shown less exhaustion in
GSH level even after exposure to 131 iodine. Nonetheless, no such changes
were seen in the stomach. The outcomes demonstrate the chance of utilizing
aqueous extract of O. sanctum L. for improving 131 iodine-activated harm
to the salivary glands.
Subramanian et al. (2005) have seen that two polysaccharides isolated
from O. sanctum L. have the capacity to forestall oxidative harm to liposomal
lipids and plasmid DNA prompted by different oxidants, such as γ-radiation,
2,2-azobis (2-amidino-propane) dihydrochloride (AAPH) and iron. Vrinda
and Uma (2001) detailed that two water-dissolvable flavonoids, Vicenin
(Vc) and Orientin (Ot), isolated from the leaves of O. sanctum L. give huge
protection against lethality, radiation, and chromosomal distortion in vivo.
The effect of aqueous concentrate of the leaves of O. sanctum L. against
lethality, radiation, and chromosomal damage was investigated by radiation-
instigated lipid peroxidation in liver and the outcomes have displayed that
aqueous concentrate itself increased the GSH and compounds fundamen
tally above the normal level, though radiation essentially decreased all the
qualities and altogether increased the lipid peroxidation rate, arriving at a
most extreme worth at 2 h after exposure (3.5 times of control) (Devi and
Gonasoundari, 1999) In another examination, the liquid concentrate of OS
has been found to decrease the lipid peroxidation and to speed up recupera
tion to normal levels in test organisms and Ocimum flavonoids delivered
promising anti-radiation effect (Devi et al., 1998) Ganasoundari et al.
(1998) examined the radioprotective effect of the leaf extract of O. sanctum
L. (OE) in combination with WR-2721 (WR) on mouse bone marrow and
noticed a huge abatement in unusual cells just as various kinds of distortions.
The counter radiation impact of Tulsi is especially pertinent to individually
presented to abundance radiation like working with radiodetermination and
treatment (e.g., atomic medication, angiography, activity under X-beam
control), getting radiography for malignomas, working in nuclear reactors
and different units with exposure to radiation, routinely presented to high
height sunlight-based radiation (e.g., aircraft staff), persistently presented to
TV and PC screens. Subsequently, Tulsi can securely be utilized in anticipa
tion of sick impacts of radiation in people presented to different radiations
(Singh et al., 2010).
13.3.3 ANTIOXIDANT ACTIVITY
The antioxidant action of Tulsi has been accounted for by numerous researchers
(Govind, 2009). The antioxidant activity of flavonoids and their connection to
membrane protection was studied (Saija et al., 1995) Antioxidant potential of
flavonoids (vicenin and orientin) in vivo was expressed in a critical decrease
in the radiation-prompted lipid peroxidation in mouse liver (Gonasoundari
et al., 1998). OS extract has the critical capacity to scavenge profoundly
responsive-free radicals (Kelm et al., 2000). The phenolic compounds viz.,
cirsimaritin, apigenin, irsilineol, rosmarinic acid, isothymusin, and calculable
amounts of eugenol (a significant part of the unstable oil) from OS concen
trate of stem and new leaves had great antioxidant activity (Nair and Gunas
egaran, 1982). The antioxidant potential of essential oils acquired by steam
hydrorefining from O. sanctum L. was assessed utilizing high performance
liquid chromatography (HPLC)-based hypoxanthine/xanthine oxidase and
DPPH (1,1-Diphenyl-2-picrylhydrazyl) assays. It was also observed that in
hypoxanthine/xanthine oxidase assay, effective cancer prevention activity
was shown by O. sanctum L. (Trevisan et al., 2006). In another examination,
the aqueous concentrate of O. sanctum L. was identified to altogether increase
the action of antioxidants (Gupta et al., 2006). Oral intake also gives critical
aortic and liver tissue security from hypercholestrolemia-prompted peroxida
tive damage (Yanpallewar et al., 2004).
Godhwani et al. (1988) examined the immunoregulatory profile of aqueous
extract and methanolic concentrate of O. sanctum L. passes on to antigenic
test of Salmonella typhosa and sheep erythrocytes by evaluating agglutinating
antibodies utilizing the Widal agglutination and sheep erythrocyte aggluti
nation tests and E-rosette development in albino rodents. The results of the
investigation demonstrate an immunostimulation of humoral immunogenic
reaction as addressed by an increase in counteracting agent titer in both the
Widal and sheep erythrocyte agglutination tests just as by cell immunologic
reaction addressed by E-rosette development and lymphocytosis.
Banerjee et al. (1996), observed the anticarcinogenic potential of O.
sanctum against many cancer-causing agents. Decoction of fresh Tulsi leaves
has anticancer potential. Alcoholic extracts of O. sanctum act on the activi
ties of cytochrome b5, aryl hydrocarbon hydroxylase, and cytochrome P-450
in the liver and glutathione-S-transferase (GST) and a reduced glutathione
level in lungs and liver have been observed. All these cofactors and enzymes
are reported to exhibit an essential role in the detoxification of mutagens
and carcinogens. Tulsi leaves when fed to experimental rats for 10 weeks
Ocimum sanctum 277
remarkedly led to the reduction of hematoma and squamous cell carcinoma

incidences (Aruna and Sivaramakrishnan, 1992; Shiva et al., 2016).
The anticancer activity of O. sanctum has been reported against human
fibrosarcoma cells culture, wherein the alcoholic extract induced cytotoxicity
@ 50 mg/mL and above, morphologically, the cells displayed condensed
nucleus and shriveled cytoplasm. DNA was also observed to be disintegrated
upon visualization in agarose gel electrophoresis (Pandey and Madhuri,
2006; Kathiresan et al., 1999).
13.4 CONCLUSIONS
O. sanctum (Os) has been examined for the bioassay-directed isolation of

compounds and also for looking for novel particles from various concentrates.
A few synthetic classes of mixtures including flavonoids, phenolics, phenyl
propanoids, terpenoids, neolignans, unsaturated fat subordinates, coumarins,
and fixed and essential oil have been accounted for from this spice. The
essential oil present in Tulsi is a decent source of normal eugenol and is well
investigated in analytical, compound, and organic aspects because of its high
business significance in drug, beauty care products, and food industries.
Fixed oil present in Tulsi seeds is rich in ω-3 unsaturated fats and is a new
research interest, because of its wide scope of pharmacological properties
particularly in cardioprotection. Flavonoids are a significant class of mixtures
extracted from Ocimum and have been reported as the fundamental dynamic
components. The water-soluble flavonoids, that is, vicenin and orientin have
been very much investigated as far as their radiation defensive impacts at both
low and high dosages. The hydrophilic nature of both the flavonoids makes
them valuable for their antioxidant impact in detoxification just as radiation
defender in malignant growth treatment. The conventional significance of
OS as immunomodulator spice was additionally upheld by the isolation of
antistress particles, ocimumoside A and ocimumoside B, which recommend
the use of Tulsi in treating neurological disorders. Further examinations are
expected to investigate the atomic and cellular mechanism of antistress action
of ocimumoside A and ocimumoside B. Until this point, the exploration work
completed on OS is predominantly centered on the natural potential of its
various concentrates and essential oil. The literature showed that ursolic acid,
eugenol, orientin, rosmarinic acid, ocimumoside A and ocimumoside B, and
vicenin are the principle dynamic phytocomponents of Tulsi and proposing a
chance of discovering novel bioactive particles.
However, there are a few points that are expected to be investigated and
examined further, such as work on the enormous plant material to extract
adequate quantity of major and minor chemical constituents to investigate
their pharmacological potential and component for restorative potential;
investigate preclinically known compounds for clinical practices, particularly
in anti-stress and radiation security; limited work has been done for the isolation
and biological examinations on the root concentrates of O. sanctum. More
than 60 compounds have been isolated, but only a few have been investigated
for their pharmacological actions and preclinical examinations. In general,
there is a demand for additional examination of the compound parts of OS
to get new constituents with enhanced pharmacological potential. The long
history of conventional uses, wide range of pharmacological properties, and
toxicological studies recommended OS as a protected significant spice for
clinical studies. The current knowledge of the active constituents alongside
their pharmacological properties will be useful in future examinations on O.
Sanctum plant as well as in looking for new leads for drug revelation.
KEYWORDS
• secondary metabolites
• apoptosis
• angiogenesis
• matrix metalloproteinases
• metastasis
• carcinoma
• papilloma
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Index
A hepatic cancer, 73
hepatocellular carcinoma (HCC), 39, 41–43
Acetylcholin (ACh), 67
anticancer properties, 47–49
Analgesic and psycho-pharmacological
bioactive molecules, 40–41
effects, 66
hepatoprotective diseases and reactive
Anti-arthritic effect, 67
oxygen species (ROS), 51
Anti-asthmatic activity, 68
MAPK signaling pathway, 50–51
Anti-cancer activities, 68
baccatin III, 237–238 mechanisms of, 47–49
brain tumor, 71–72 NA seeds in drug discovery, 52
breast, 72 p53 signaling pathway, 50
brevifoliol, 237 principal component thymoquinone,
Catharanthus roseus (C. roseus), 100 47–49
dosage and, 101 signaling mechanisms, 47–49
mechanism of, 101–103 thymol (THY), 46–47
research on, 100–101 thymoquinone (TQ), 41, 43–46
in vitro cultures and anticancerous leukemia, 74
compounds, 103–104 Nigella sativa L., 39, 41–43
clinical investigations, 70–71 anticancer properties, 47–49
colo-rectal cancer, 73 bioactive molecules, 40–41
effects, 206–207 hepatoprotective diseases, 51
anti-tumor potentiality, 208–210 MAPK signaling pathway, 50–51
berberine, genetic level in cancer cells, mechanisms of, 47–49
211 NA seeds in drug discovery, 52
bone cancer cells, 210 p53 signaling pathway, 50
cell cycle, 213–214 principal component thymoquinone,
cytotoxicity on feminine cancer cell, 47–49
210–211 and reactive oxygen species (ROS), 51
glioblastoma, 214 signaling mechanisms, 47–49
heat shock proteins (HSP) expression, thymol (THY), 46–47
213 thymoquinone (TQ), 41, 43–46
multi drug resistant skin cancer cells, prostate, 73–74
208 taxol/taxanes, 224–227
neuroblastoma, 214–215 breast cancer, 229–231
oral cancer, 208 cervical cancer, 234–235
prostrate cancer, 208 colon/ colorectal cancer, 236
skin cancer, 207 epithelial cancer, 235–236
TCEX impact on nerve fibre cells, fibro sarcoma, 236
211–213 gastric cancer, 235
TCEX on cancer cells, 211 head and neck squamous cell
gastro intestinal cancers, 73 carcinoma, 236
glioma, 72 leukemia, 235
284 Index
liver cancer, 227–228 antimicrobial activity, 68

lung cancer, 228–229 hypoglycemic activity, 67
ovarian cancer, 233–234 hypo-lipidemic and hepato-protective
pancreatic cancer, 233 activity, 67
prostate cancer, 232–233 immuno-modulatory activity, 67
thyroid cancer, 236–237 leukotriene inhibition, 66
in vivo and in vitro, 68–70 boswellic acid, 74–76
Anti-cancer efficacy chemistry of, 64–65
molecular mechanism, 218 derivatives, studies, 78–80
Anticancer plant-based products human topoisomerases I and II,
traditional medicine systems, 170–171 inhibitor, 76–77
Anticancerous activity, 100 preclinical activity, 77
dosage and, 101 topoisomerase II DNA cleavage
mechanism of, 101–103 complex, 77
research on, 100–101 mechanism of action, 74
in vitro cultures and anticancerous safety and bioavailability enhancement
compounds, 103–104 of, 78
Anti-diarrhoeal activity, 67 taxonomical hierarchy, 63
Anti-inflammatory Boswellic acid, 74–76
and anti-arthritic effect, 67
chemistry of, 64–65
effect, 67
derivatives, studies, 78–80
Antimicrobial activity, 68
human topoisomerases I and II, inhibitor,
Anti-tumor potentiality, 208–210
76–77
preclinical activity, 77
B
topoisomerase II DNA cleavage complex,
Baccatin III, 237–238 77
Barium chloride (BaCl2), 67 Brain tumor, 71–72
Berberine Breast cancer, 115–118
genetic level in cancer cells, 211 Brevifoliol, 237
Bone cancer cells, 210
Boswellia serrata Roxb, 61 C
anticancer activity, 68
brain tumor, 71–72 Camptotheca acuminata Decne
breast, 72 camptothecin (CPT), 135, 140
clinical investigations, 70–71 ACF incidence, effect, 143
colo-rectal cancer, 73 CN-CPT inhibits cell proliferation in
gastro intestinal cancers, 73 vitro, 144
glioma, 72 exatican, 136–137
hepatic cancer, 73 irinotecan, 136
leukemia, 74 synthesis, semisynthetic derivatives,
prostate, 73–74 138
in vivo and in vitro, 68–70 topotecan, 135–136
applications, 66 tumor incidence, effect, 143
analgesic and psycho-pharmacological in vitro anticancer activity, 140–142
effects, 66 in vivo anticancer activity, 143
anti-asthmatic activity, 68 extraction of, 137
anti-diarrhoeal activity, 67 bark, 137
anti-inflammatory and anti-arthritic fruit, 137
effect, 67 leaf, 137
Index 285
limitations, 146 vitro plant culture

mode of action, 139–140 biosynthesis pathway, manipulation
phylogeny, 134 of, 27
target organs, 144 secondary metabolite production in,
colo-rectal cancer, 145 27–28
hepatocellular carcinoma, 144–145 Cervical cancer, 118
leukemia, 145–146 Colorectal cancer, 121
lung cancer, 145 Colo-rectal cancer, 73, 145
ovarian cancer, 145
thyroid cancer cells, 144 D
Cancers, 121 Dalton’s ascitic lymphoma, 124
Dalton’s ascitic lymphoma, 124 Dosage, 101
glioblastoma, 123
leukemia, 121–122 G
liver cancer, 124
lymphoblastic and myeloid leukemia, 122 Garcinia indica (GI), 1
Mouse Ehrlich ascites, 124 anti-cancer activities
Catharanthus roseus (C. roseus), 89 cancer types, 5–7
anticancerous activity, 100 chemical compounds, 3–9
dosage and, 101 botanical description and traditional uses,
mechanism of, 101–103 2
research on, 100–101 chemical composition, 3
in vitro cultures and anticancerous garcinol
compounds, 103–104 anti-cancer activities, 10–12
botany, 91–92 isogarcinol, 9–10
geographic distribution of plant, 92–93 medicinal properties, 2–3
photochemistry and medicinal properties, Glutathione-S-transferase (GST), 276
93 Glycyrrhiza glabra, 247
alkaloids, 96–97 anticancer
phenolics, 97 mechanism and molecular targets,
phytochemicals, 93–96 251–257
traditional medicine, 97–100 metabolites, 250–251
Cell cycle, 213–214 pharmacological properties, 249–250
Centella asiatica Linn (CA), 19 phytochemical composition, 249
anticancer effect, 28–31
metabolic pathways, manipulation of H
pentacyclic triterpenoids, 26 Heat shock proteins (HSP)
phytoconstituents, metabolite profiling, expression, 213
21–22 Hepatocellular carcinoma (HCC), 39, 41–43,
LC/MS-based metabolite profiling, 22–23 144–145
metabolomics study, 22–23 bioactive molecules, 40–41
NMR-based metabolite profiling, 23 thymol (THY), 46–47
TLC and HPLC-based metabolite thymoquinone (TQ), 43–46
profiling, 22 hepatoprotective activity
secondary metabolites and biosynthesis, 24 mechanisms of, 47–49
flavonoids and phenols, 25–26 important bioactive compounds
triterpenes and terpenoids, anticancer properties, 47–49
biosynthesis, 24–25 hepatoprotective diseases, 51
286 Index
MAPK signaling pathway, 50–51 p53 signaling pathway, 50

p53 signaling pathway, 50 principal component thymoquinone,
principal component thymoquinone, 47–49
47–49 and reactive oxygen species (ROS), 51
reactive oxygen species (ROS), 51 signaling mechanisms, 47–49
signaling mechanisms, 47–49 NA seeds in drug discovery, 52
NA seeds in drug discovery, 52 thymoquinone (TQ)
thymoquinone (TQ) thymol for liver diseases, 41
thymol for liver diseases, 41
Herbal anticancer drugs O
regulatory aspects of, 168–170
Ocimum sanctum, 267
Hypoglycemic activity, 67
bioactive components, 271
Hypo-lipidemic and hepato-protective
anticarcinogenic property, 272–274
activity, 67
antioxidant activity, 276–277
antiradiation activity, 274–275
I
properties, 270
Immuno-modulatory activity, 67 chemical composition, 271
vernacular names, 269–270
L Oral cancer, 208
Leukemia, 145–146 Ovarian cancer, 118–119, 145
Leukotriene inhibition, 66
Lung cancer, 120–121, 145 P
Panax ginseng, 181
M anticancer activity of, 189–192
Metabolite profiling, 21–22 secondary metabolites, 185
LC/MS-based metabolite profiling, 22–23 characterization, 183–184
metabolomics study, 22–23 ginsenosides, 185–186
NMR-based metabolite profiling, 23 metabolite profiling, 184–185
TLC and HPLC-based metabolite metabolites using LCMS and NMR,
profiling, 22 184–185
Mouse Ehrlich ascites, 124 oleanolic acid, 186–187
Multi drug resistant skin cancer cells, 208 seasonal and environmental effects,
Myeloid leukemia, 122 187–189
Pancreatic cancer, 120
N Pentacyclic triterpenoids, 26
Neuroblastoma, 123 Phytoconstituents
Nigella sativa L., 39, 41–43 metabolite profiling, 21–22
bioactive molecules, 40–41 LC/MS-based metabolite profiling,
bioactive molecules, effect 22–23
thymol (THY), 46–47 metabolomics study, 22–23
thymoquinone (TQ), 43–46 NMR-based metabolite profiling, 23
hepatoprotective activity TLC and HPLC-based metabolite
mechanisms of, 47–49 profiling, 22
important bioactive compounds Plant culture
anticancer properties, 47–49 biosynthesis pathway, manipulation of, 27
hepatoprotective diseases, 51 secondary metabolite production in, 27–28
MAPK signaling pathway, 50–51 Prostate cancer, 119, 208
Index 287
R lung cancer, 228–229

ovarian cancer, 233–234
Renal carcinoma, 124
pancreatic cancer, 233
prostate cancer, 232–233
S
thyroid cancer, 236–237
Secondary metabolites Taxus baccata, 151
biosynthesis, 24, 185 anticancer plant-based products
characterization, 183–184 traditional medicine systems, 170–171
flavonoids and phenols, 25–26 cancer, 152
ginsenosides, 185–186 herbal anticancer drugs
metabolite profiling, 184–185 regulatory aspects of, 168–170
metabolites using LCMS and NMR, review, 156–157
184–185 taxane and active compounds, 157–165
oleanolic acid, 186–187 taxol, production, 165–168
seasonal and environmental effects, types of cancer, 158–164
187–189 types and plant-based treatment options
triterpenes and terpenoids, current cancer therapy via
biosynthesis, 24–25 phytochemicals, 154–156
isolated Taxus brevifolia (Nutt.) Pilger., 223
characterization, 183–184 anticancer activity
metabolite profiling, 184–185 baccatin III, 237–238
metabolites using LCMS and NMR, of brevifoliol, 237
184–185 extracts, nanoparticles, 238–239
Skin cancer, 123, 207 taxol/taxanes, anticancer activity,
224–227
T breast cancer, 229–231
Target organs, 144 cervical cancer, 234–235
colo-rectal cancer, 145 colon/ colorectal cancer, 236
hepatocellular carcinoma, 144–145 epithelial cancer, 235–236
leukemia, 145–146 fibro sarcoma, 236
lung cancer, 145 gastric cancer, 235
ovarian cancer, 145 head and neck squamous cell
thyroid cancer cells, 144 carcinoma, 236
Taxane and active compounds, 157–165 leukemia, 235
taxol, production, 165–168 liver cancer, 227–228
types of cancer, 158–164 lung cancer, 228–229
Taxol/taxanes ovarian cancer, 233–234
anticancer activity, 224–227 pancreatic cancer, 233
breast cancer, 229–231 prostate cancer, 232–233
cervical cancer, 234–235 thyroid cancer, 236–237
colon/ colorectal cancer, 236 Thymol (THY), 46–47
epithelial cancer, 235–236 Thymoquinone (TQ), 43–46
fibro sarcoma, 236 thymol for liver diseases, 41
gastric cancer, 235 Thyroid cancer cells, 144
head and neck squamous cell Tinospora cordifolia (Thunb.) Miers, 203
carcinoma, 236 active components, 205–206
leukemia, 235 anti-cancer activities, effects, 206–207
liver cancer, 227–228 anti-tumor potentiality, 208–210
288 Index
berberine, genetic level in cancer cells, W

211
Withania somnifera (L.) Dunal, 111
bone cancer cells, 210
breast cancer, 115–118
cell cycle, 213–214
cancers, 121
cytotoxicity on feminine cancer cell,
Dalton’s ascitic lymphoma, 124
210–211
glioblastoma, 123
glioblastoma, 214
leukemia, 121–122
heat shock proteins (HSP) expression,
liver cancer, 124
213
lymphoblastic and myeloid leukemia,
multi drug resistant skin cancer cells,
122
208
mouse ehrlich ascites, 124
neuroblastoma, 214–215
myeloid leukemia, 122
oral cancer, 208
neuroblastoma, 123
prostrate cancer, 208
renal carcinoma, 124
skin cancer, 207
skin cancer, 123
TCEX impact on nerve fiber cells,
cervical cancer, 118
211–213
colorectal cancer, 121
TCEX on cancer cells, 211
lung cancer, 120–121
anti-cancer efficacy
ovarian cancer, 118–119
molecular mechanism, 218
pancreatic cancer, 120
phytocomponents on cancer cells
prostate cancer, 119
mechanism of action, 215–217
Traditional medicine, 97–100
Tulsi, 268

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POTENT ANTICANCER

© 2024 by Apple Academic Press, Inc.

Library and Archives Canada Cataloguing in Publication

CIP data on file with US Library of Congress

ISBN: 978-1-77491-311-6 (hbk)

1. Garcinia indica ................................................................................................1

2. Centella asiatica Linn....................................................................................19

3. The Anticancer Activity of Nigella sativa on Hepatocellular

4. Boswellia serrata Roxb..................................................................................61

6. Withania somnifera (L.) Dunal................................................................... 111

7. Camptotheca acuminata Decne...................................................................131

10. Tinospora cordifolia (Thunb.) Miers..........................................................203

11. Taxus brevifolia (Nutt.) Pilger.....................................................................223

12. Glycyrrhiza glabra......................................................................................247

13. Ocimum sanctum ........................................................................................267

Iffat Zareen Ahmad

Rohit Sam Ajee

Ramachandra Reddy Pamuru

Narayan Chandra Talukdar

Rajesh Singh Tomar

Vijay Kumar Veena

Pulala Raghuveer Yadav

GPS ginseng polysaccharide

PDA pancreatic ductal adenocarcinoma

for cancer prevention and treatment and those seeking a comprehensive

Few Lines on Anticancer Medicinal Plants

Glycyrrhiza glabra, a herb so pure,

Garcinia indica (GI), a plant native to India commonly known as kokum,

1.1 BOTANICAL DESCRIPTION AND TRADITIONAL USES OF

Commonly recognized as kokum, Garcinia indica (GI), a plant in the

1.2 MEDICINAL PROPERTIES OF GARCINIA INDICA

Furthermore, certain studies on the functional properties of GI demonstrated

1.3 CHEMICAL COMPOSITION OF GARCINIA INDICA

Various medicinal and cosmetics propertiesof GI fruitrinds and leaves were

1.4 ANTI-CANCER ACTIVITIES OF GI OR ITS CHEMICAL

Herein, anti-cancer effects exhibited by GI and its chemical constituents are

extract was demonstrated to exert a cytotoxic effect to A498 human renal

In fact, the activities of cancer suppression were mainly found due to

Potent Anticancer Medicinal Plants

contribute to the downregulation of the molecular targets such as matrix

apoptosis in cervical cancer cell lines, as well as inhibited tumor growth in

1.5 ISOGARCINOL AS AN IMMUNOSUPPRESSANT

Psoriasis is an incurable, long-term (chronic) inflammatory skin condition

1.6 ANTI-CANCER ACTIVITIES OF GARCINIA INDICA AND GARCINOL

Herein, anti-cancer effects exhibited by GI and its chemical constituents are

cancer as well (Majeed et al., 2020). Furthermore, GI extract was shown to

1.7 CONCLUSION AND FUTURE PERSPECTIVE

Although it grows extensively in India, GI has been recognized worldwide

FIGURE 1.3 Anticancer effects of garcinol.

The author wishes to thank the Rachadapisek Sompote Fund, Chulalongkorn

Centella asiatica Linn

Natural products are gaining a lot of attention in medicine due to their

Compound, namely, asiatic acid (AA), asiaticoside has anticancer potential

The genus Centella consists of 53 species of flowering plants, including CA

neuroprotective (Gray et al., 2018; Lokanathan et al., 2016; Yadav et al.,

2.2 METABOLITE PROFILING OF CENTELLA ASIATICA

Metabolic profiling involves detecting the role of metabolites in a biochemical

MS), ultrahigh-performance liquid chromatography (UHPLC), thin-layer

2.2.1 TLC AND HPLC-BASED METABOLITE PROFILING

2.2.2 LC/MS-BASED METABOLITE PROFILING AND METABOLOMICS

The metabolomics approach allows for an analysis of the different and

al., 2011). High-resolution mass spectrometry (HRMS) is often used coupled

2.2.3 NMR-BASED METABOLITE PROFILING AND METABOLOMICS

Secondary metabolites with structures are elucidated by NMR. NMR analysis