Download The flowers, seeds and seed oil of madhuka have

1. INTRODUCTION
1.1 Herbs for health
Using herbs and plants for medicinal purposes has a long tradition. In India and
China, these traditions date back thousands of years. Once thought of as "traditional
medicine" used by native or ancient cultures, herbal medicine has emerged as a popular
alternative or supplement to modern medicine. According to the World Health
Organization, 4 billion people, almost 70 % of the world population, use herbal medicine
for some aspect of primary health care (Abramov, 1996). It is estimated that in the United
States alone, botanical dietary supplements exceed $3 billion per year (The U.S. Food
and Drug Administration, 1999).
Forty percent of Americans take dietary supplements. About half of these people
take vitamin and mineral supplements, a third take some type of herbal product, and the
rest take other ergogenic aids, such as amino acids or protein powders (Industry
Overview, 1999). The herbal market is growing steadily at about 20 % in every year (The
U.S. Food and Drug Administration, 1999). People take herbs for many reasons and
many conditions. One of the biggest reasons is that herbs are considered natural and
therefore healthier and gentler than conventional drugs (Ironically, many prescription
drugs are of herbal origin). Some people take them for overall health and well-being, not
for any specific condition. For others, herbal use is grounded in traditions passed down
from generation to generation or recommended by folk healers.
Medicinal herbs are significant source of synthetic and herbal drugs. In the
commercial market, medicinal herbs are used as raw drug, extract or tincture. Isolated
active constituents are used for applied research. For the last few decades,
phytochemistry has been making rapid progress and herbal products are becoming
popular. Ayurveda, the ancient healing system of India, flourished in the Vedic Era in
India. According to historical facts, the classical texts of Ayurveda, Charaka Samhita and
Sushruta Samhita were written around 1000 B.C. The Ayurvedic Materia Medica
includes 600 medicinal plants along with therapeutics. Herbs like turmeric, fenugreek,
ginger, garlic and holy basil are integral part of Ayurvedic formulations. The
formulations incorporate single herb or more than two herbs (poly-herbal formulations).
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Medicinal herb is considered to be a chemical factory as it contains multitude of
chemical
compounds
like
alkaloids,
glycosides,
saponins,
resins,
oleoresins,
sesquiterpene lactones and oils (essential and fixed). Today there is growing interest in
chemical composition of plant based medicines. Several bioactive constituents have been
isolated and studied for pharmacological activities.
1.2 Herbs as a Traditional medicine
The World Health Organization (WHO) defines traditional medicine (TM) as "the
total combination of knowledge and practices, where explicable or not, used in
diagnosing, preventing or eliminating physical, mental or social diseases which may rely
exclusively on past experience and observation handed down from generation to
generation, verbally or in writing" (WHO Africa, 2000). WHO also specifies traditional
African medicine as "the sum total of practices, measures, ingredients and procedures of
all kinds whether material or not which from time immemorial had enabled African to
guard against diseases, to alleviate his suffering and to cure himself" (WHO Geneva,
1978). TM has been utilized by the majority of the world population for thousands of
years. Until the beginning of the 19th century, all medicines were traditional. Yet, in
many developing countries, it is true that for the majority of rural population, TM is the
only primary or any other kind of health care available (Koita, 1990). For more than 80%
of the population in Africa, they are using traditional medicine. In recognition of this fact,
WHO underlined the potential role that TM may play in reinforcing the health care
through the primary health care approach in developing countries (WHO Geneva, 1978).
1.3 History of traditional medicine
Guided by taste and experience, early societies developed a means of healing by
using plants, animal products and minerals that were not mostly among their usual diet.
The physical evidence of herbal remedies goes back some 60,000 years to a burial site of
a Neanderthal man uncovered in 1960 in a cave in Northern Iraq. In this cave, scientists
found what appears to be the remains of an ordinary human bones, and analysis of the
soil around these revealed extraordinary quantities of pollen that could not have been
introduced accidentally at the burial site. Rather, it is assumed that someone from the
cave community had consciously made eight species of plants to surround the dead body,
seven of which are medicinal plants still used throughout the herbal world (Jin-Ming et
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al., 2003). One of the earliest records of the use of herbal medicine is that of
Chaulmoogra oil from species of Hydnocarpus gaertn, which was known to be effective
in the treatment of leprosy. Such use was recorded in pharmacopoeia of the Emperor
Shen Nung of China between 2730 and 3000 B.C. Similarly, seeds of opium poppy
(Papaver somniferum L.) and castor oil seeds (Ricinus communis L.) were excavated
from some ancient Egyptian tombs, which indicated their use in that part of Africa as far
back as 1500 B.C. Suffice it to say that some 5000 years back, man was well aware of
medicinal properties of some plants growing around him (Sofowora, 1982).
The Arab medicine known as Unani system of medicine had its origin in the fifth
and fourth centuries B.C under the patronage of Hippocrates in Greece and later
expanded by the great teachers such as Aristotle, Theophrastus, Dioscorides, and Galen,
etc. Then, this body of knowledge moved to Rome, Alexandria and to the Arab countries
and got the name "The Arab (Unani) or Greco-Arab system of medicine". In the
Ayruvedic medical system that is believed to have been in practice for 2000 years mainly
in India, 582 herbs and 600 remedies were described in the early book on internal
medicine and in the book of surgery, respectively.
According to medical history, Hippocrates born in 460 B.C. was the first Greek to
regard medicine as a science and he is now referred to as the father of medicine. His
material medica consisted essentially of herbal recipes, some 400 simple remedies having
been combined and 4 described by him. Theophrastus of Athens was another famous
Greek, who was born in 370 B.C. produced a number of manuscripts including the
famous Historic plantarium. Both these early doctors administered various vegetable
drugs including myrrh and frankincense. At that time preparation of aromatic roots and
flowers were also used for treating many ailments (Jin-Ming et al., 2003; Sofowora,
1982). In the middle ages, the writings of Galen (Born in 131 A.D.) became popular. He
is considered today to be the most distinguished physician of antiquity after Hippocrates.
He treated diseases essentially by the use of herbs, and those who followed his methods
eventually developed the sect known as "Eclectics" who employed herbal as well as
mineral substances in treating the sick. Allopathic as well as homeopathic systems of
medicine today are based on doctrines expatiated by Galen (Sofowora, 1982). The use of
many medicinal plants in Europe in the 14th century was based on the doctrine of
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signature or similars developed by Paracelsus (1490-1541), a Swiss alchemist and
physician. According to this doctrine, healing herbs have features made by God
identifying the plant with specific disease or part of the body. For example, plants with
heart shaped leaves were good for treating heart disease (Sofowora, 1982).
1.4 Global perspectives of traditional medicine
Trends in the use of traditional and complementary medicine are on the increase
in many developed and developing countries. In the USA, it was estimated that 42.5
million visits were made to herbalists in 1990, contrasting with the 388 million actual
visits to primary health care physicians. In 1992, 20 million patients in Germany used
homeopathy (Jin-Ming et al., 2003), acupuncture as well as chiropractic and herbal
medicine as the most popular forms of complementary medicine. In Australia in 1998,
about 60% of the population used complementary medicine, 17,000 herbal products had
already been registered and a total of US $650 million was spent on complementary
medicine (WHO Africa, 2000).
The herbal medicine market has expanded tremendously in the last 15 years and
the total annual sale of herbal medicines is still growing over the counter sales of herbal
medicines in the USA and Canada during which it showed growth rate of 15%. In
Europe, the sales of herbal products have been referred as "Europe's growth market"
which amounted to USD 1.4 billion in 1992. In Malaysia, it is estimated that about US
$500 million is spent every year on traditional medicine, compared to only about US
$300 million on modern medicine. In 1996 the total annual sale of herbal medicines
reached US $14 billion worldwide (WHO Africa, 2000).
In China traditional medicines account for 30 – 50% of total medicinal
consumption and the total sales of their herbal medicines amounted to USD 2.5 billion in
1993. In addition, China exported medicinal herbs in 1993 with an estimated value of
USD 40 million. Within China the traditional systems of health care are incorporated into
the formal component of national health care. In 1991, there were 530,000 medical and
technical personnel in traditional Chinese medicinal field. There were more than 2,000
hospitals of traditional Chinese medicine, and 170,000 beds within the hospitals. Also,
there were more than 160 scientific research institutions of traditional Chinese Materia
Medica, forming a scientific research system. There were more than 2,000 factories of
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manufacturing medicinal herbs, producing more than 4,000 kinds of ready-made Chinese
herbal medicine every year (Xiang, 1990).
In India, where 75% of the populations depend on herbal preparations in 1991,
540 plant species were reported to be used in different formulations (Bhat, 1990). In
1995, there were 250,000 registered traditional medicine practitioners, the majority
having received training in degree graduating college.
1.5 Ethnopharmacology in drug discovery
Ethnopharmacology as a specifically designated field of research has had a
relatively short history. The term was used in 1967 as a title of a book on hallucinogens
“Ethnopharmacologic search for psychoactive drugs” and is nowadays much more
broadly defined: “The observation, identification, description, and experimental
investigation of the ingredients and the effects of the ingredients and the effects of such
indigenous drugs is a truly interdisciplinary field of research which is very important in
the study of traditional medicine. Ethnopharmacology is thus defined as the
interdisciplinary scientific exploration of biologically active agents traditionally
employed or observed by man”. This definition draws attention to the evaluation of
indigenous uses and does not explicitly address the issue of searching for new bioactive
drugs (drug discovery). Here we look at different processes involved in drug discovery.
The discovery process is composed of several stages. The first stage must be the reported
use of a naturally occurring material for some purpose, which can be related to a
medicinal use. Consideration of the cultural practice associated with it is important in
deciding possible bases of the reputed activity. If there is an indication of genuine effect,
then the material needs to be identified and characterized according to scientific
nomenclature. It can then be collected for experimental studies, usually comprising some
tests for relevant biological activity linked with isolation and structure determination of
any chemicals present, which might be responsible for the observed activity.
A) Information sources
The most reliable type of information arises from in-depth studies carried out by
field workers living in that particular community of a particular ethnic group on the use
of local plants and other materials. This usually comprises frequent communication with
the local population, preferably in their own language. It should be noted, however, that
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an extensive knowledge of TM may reside with only a few people and a focus on this
group would yield greater results. Before such knowledge can be investigated
scientifically, the information provided will often need clarification and translation into
scientific terms of particular importance. The correct identification of the species used
can be very difficult due to a lack of or poor quality sample specimens. Illustrations as
well as language difficulties can also be additional barriers. However, data on the part
used, time of collection, method of preparation of formulation and methods of application
are also necessary since they all affect the nature and amount of any biologically active
compounds. Any restriction on use due to time of year may be important since they may
indicate low levels or high levels of active compounds. Similarly, any type of individuals
excluded from being treated may indicate groups at risk due to age, gender or occupation
(Cox et al., 1994; Heinrich et al., 2001).
B) Extraction
The extract used for testing should approximate as closely as possible to that
obtained from the traditional process. In many cases, this will be simple extraction with
hot water. But a variety of other solvents as well as various additives may be used in the
treatment of materials before use. In most instances however, it is likely that fairly polar
compounds will be extracted, although the solubility of less polar substances may be
increased considerably due to solubilizing compounds (Cox et al., 1994; Heinrich et al.,
2001).
In most instances of modern drug discovery carried out by industrial and
academic research groups, a particular assay, or series of in vitro bioassays, designed on
the basis of the biochemistry or molecular biology of the disease, is used to test the
extract. In these situations, the ethnopharmacology has little relevance to the tests used
except that it provides a number of screening samples selected on the basis of their
traditional use for the disease in question (Cox et al., 1994; Heinrich et al., 2001).
C) Chemical examination
Chemical examination should be linked with tests for biological activity and it is
probably only a happy accident of history that the many alkaloidal drugs were developed
from traditional medicines, without the need for bioassay guided fractionation because
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the alkaloids were present in fairly high amounts and they were relatively easy to obtain
in a purified state.
For many other traditional medicines, where activity is not due to alkaloids, it has
been much more difficult to separate the activities from all the other compounds
(Heinrich et al., 2001). Chemotaxonomic approach increases the proportion of plants that
screen positively, thus saving research time and money. Specific secondary metabolites,
such as flavonoids, are often restricted in distribution, being found only in groups of
related plants. For example, isoflavnoids are common in species of the Fabaceae, but are
found in few other plant families. Of the over 5500 types of alkaloids known, many are
confined to a single genus or subfamily. Only a single alkaloid has been found in the
many species of Bombacaceae tested so far, but the Solanaceae, Rubiacea and
Ranunculaceae are the source of hundreds of distnict forms (Martin, 1995).
The presence of different secondary metabolites in a plant can be screened by the
use of appropriate chromogenic reagents after separation (Heinrich et al., 2001). A
typical example of success reported in drug discovery based on ethnopharmacological
approach is the discovery of artemisinin. Artemisia annua is a plant which was recorded
during 281-340 AD for treating malaria (Samuelsson, 1987). In 1976, artemisinin
compounds were identified and their mechanism of action elucidated. Artemisnin acts
against malarial parasite in a very different way from quinine and most of the synthetic
quinoline antimalarials. Several large trial studies have shown the efficacy of artemisinin
but the more soluble analogue artemether and artesunates are now widely used and are
recommended by WHO as antimalarias in chloroquine resistant areas (WHO China,
2001; WHO Geneva, 2001). The principles underlying herbal medicines are relatively
simple, although they are quite distinct from conventional medicine and herbal medicine.
Often overlooked distinction exists between herbal medicine (the practice) and the plant
based remedies used in the practice of herbal medicine. India is a rich source of medicinal
plants and a number of plant extracts are used against diseases in various systems of
medicine such as ayurveda, unani and siddha. Only a few of them have been scientifically
explored. Plant derived natural products such as flavanoids, terpenes, and alkaloids and
soon has received considerable attention in recent years, due to their diverse
pharmacological properties including cytotoxic and cancer chemo preventive effects.
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Plants have a long history of use in the treatment of cancer (Ramakrishna et al.,
1984). Extensive research at Sandoz laboratories in Switzerland in the 1960s and 1970s
led to the development of etoposide and teniposide as clinically effective agents which
are used in the treatment of lymphomas, bronchial and testicular cancer (Bholin et al.,
1999). Of 2069 anticancer trials recorded by the NCI as being in progress as of July 2004,
over 150 are drug combinations including etoposide against a range of cancers (National
Cancer Institute, 2009).
1.6 General overview of cancer and its treatment
The human adult is comprised of about 1015 cells, many of which are required to
divide and differentiate in order to repopulate organs and tissues which require cell
turnover (Bertram, 2001). The ability of the body to control cell multiplicity is achieved
by a network of overlapping molecular mechanisms which direct cell proliferation and
death. Any alteration in this balance (birth and death of cells), has a potential, if
uncorrected, to alter the number of cells in an organ or tissue. Such changes may result in
cancer, a disease that is manifested in many forms depending primarily on the organ from
which it evolves. Characteristically, cancer is defined as the uncontrolled proliferation of
cells which become structurally abnormal and possess the ability to detach themselves
from a tumor and establish a new tumor at a remote site within the host (National Cancer
Institute, 2009). Globally, cancer is one of the leading causes of death. According to the
American Cancer Society (ACS), an estimation of about 1,500,000 new cases and over
500,000 deaths are expected to be recorded in the US in 2009. South Africa experiences
one of the highest incidence rates of cancer in Africa (Mqoqi et al., 2004). Every one in
four males and six females have the potential of developing cancer. The current statistics
by the National Cancer Registry of South Africa indicate that cancers of the bladder,
colon, breast, cervix, lungs and melanoma are among the most common (Mqoqi et al.,
2004).
The existing strategy of eradicating cancer after detection has resulted in mortality
that may have been preventable if caution was taken against the causative agents (Doll et
al., 1981). Although, the etiology of cancer remains unknown to an extent, epidemiology
has suggested the hypotheses that multiple causative factors may be operating. These
factors (exogenous and endogenous) exert their specific effects at different times in the
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life of the patient. The impact of such effects might be cumulative or synergistic. The
main predictors of the incidence of cancer fall largely into two broad categories:
environmental and positive family history (Parkin et al., 2005). A number of other risk
factors exist from a wide range of studies in various populations and geographic
locations.
The progress in research on the etiology of cancer has revealed the evidence that
dietary patterns, nutrients and food constituents are closely associated with the risk of
several types of cancer (Doll et al., 1981). Fats have been the focus of nutritional studies
on cancers of the prostate, breast and colon more than any other dietary component
(National Research Council, 1989). Several studies in countries consuming high fat diets
have consistently shown higher incidence and mortality rates for breast, colon and
prostate cancers (National Research Council, 1989; Hursting et al., 1990). Studies of
specific environmental influences have suggested an increased risk of developing various
forms of cancer with exposure to particulate air pollutants and fertilizers. Substances such
as asbestos, aniline dye, uranium and nickel have been implicated as environmental
carcinogens (Monson et al., 1997).
A) The role of inflammation in the initiation of cancer
The association between inflammation and tumor has long been known (Balkwill
et al., 2001). Since then, inflammation is increasingly recognized as an important
component of several cancers, although the mechanisms involved are not fully
understood (Ben, 2006). A vast body of evidence has indicated that inflammatory
leucocytes contribute to cancer development either directly by the release of vesicle
stored growth and survival factors and diverse proteolytic enzymes, or indirectly via the
activation of cell signaling cascades as a result of altered pericellular matrix remodelling
activity (Van Kempen et al., 2006). Products of inflammation such as growth factors,
cytokines and transcription factors, like nuclear factorkappa B (NF-κB), control the
expression of cancer genes and key inflammatory enzymes such as inducible nitric oxide
(iNOS) and cyclooxygenase-2 (COX-2) (Hofseth et al., 2006).
Bacterial, viral and parasitic infections, chemical irritants and non-digestible
particles are some of the causes of chronic inflammation. The longer this inflammation
persists, the higher the risk of associated carcinogenesis (Shacter et al., 2002). Chronic
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inflammation occurs due to environmental stress around the tumor, thus generating a
shield protecting the tumor from the immune system (Assenat et al., 2006). Recent
demonstrations have shown that microenvironment of tumors highly resemble an
inflammation site, with a significant tendency for tumor progression (Assenat et al.,
2006). In addition, this micro-environment apart from its significant role in cancer
progression and protection has a considerable adverse effect on the success of the various
current cancer treatments (Assenat et al., 2006). The pro-cancerous outcome of chronic
inflammation are increased DNA damage, increased DNA synthesis, cellular
proliferation, the disruption of DNA repair pathways and cellular milieu, the inhibition of
apoptosis, the promotion of angiogenesis and invasion (Hofseth et al., 2007). Therefore,
inflammation plays a major role in the initiation and progression of cancers.
Inflammatory-related ailments are treated mainly with non-steroidal anti-inflammatory
drugs (NSAIDs). These drugs are used to reduce the consequences of inflammation
(Vane et al., 1996). Indomethacin, an NSAID, for example has been found to block
carcinogenesis in animals by reducing the production of inflammatory cytokines
(Federico et al., 2007). A lower risk of cancer incidence has also been found in people
regularly taking NSAIDs (Fosslien, 2000).
B) Treatment options for cancer
The treatment option for cancer is influenced by several factors, such as the
specific nature of the cancer; the status of the patient (age and health); and whether the
goal of treatment is eradication of the tumor, control of the local tumor growth,
prolongation of survival or palliation of cancer symptoms (National Cancer Institute,
2009). Depending on these factors, treatment options such as surgery, chemotherapy,
radiation and hormonal therapy could be used. More than half of all people diagnosed
with cancer are treated with chemotherapy because it is considered a systemic treatment.
The cancer-fighting drugs circulate in the blood to parts of the body where the cancer
may have spread and can kill or eliminate cancers cells at sites of great distances from the
original cancer. The side effects observed with these treatments may be severe, thus
reducing the quality of life, compromising treatment and sometimes limiting the chance
for an optimal outcome from treatment. Common side effects includes anaemia,
depression, fatigue, hair loss, infections, low blood counts, nausea and vomiting and long
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term effects such as cardiac toxicity, growth problems and sterility (National Cancer
Institute, 2009).
C) Phytotherapy for cancer treatment
Despite the major scientific and technological progress in the treatment and
management of cancer, no reliable and definitive cure has been found (Richardson et al.,
1999). This has led to an increase in the dependence of patients on unconventional
medical therapies (Alschuler et al., 1997). All over the world, the traditional use of plants
in the treatment of ailments has been on the increase especially in developing countries
where there is invariably poor availability of primary health care (Alschuler et al., 1997).
Plants are a viable source of biologically active natural products which have served as
commercial drugs or as lead structures for the development of modified derivatives
possessing enhanced activity (Cordell et al., 1991). Extracts of plants have a long history
of use in the treatment of cancer (Hartwell, 1969). Over 60% of the currently used
anticancer agents are derived in one way or the other from natural sources including
plants and marine organisms (Cragg et al., 2005a). For example, the breakthrough for
cancer treatment was achieved by the discovery and development of the vinca alkaloids,
vincristine and vinblastine isolated from Catharanthus roseus in the early 1950’s
(Chadwick et al., 1994). The discovery of these chemicals led to other research where
compounds such as podophyllotoxin derivatives, etoposide and teniposide from the root
of various Podophyllum species (Gurib-Fakim, 2006) and paclitaxel from the bark of
Taxus brevifolia (Cragg et al., 2005b) were isolated. Other examples include the
camptothecin derivatives (topotecan, irinotecan and 9- aminocamptothecin) isolated from
Camptotheca acuminata, homoharringtonine from Cephalotaxus harringtonia var
drupaceae and elliptinium from several genera in the Apocyanaceae family (Wall, 1998;
Tingali, 2001). Since then, various studies have been undertaken to discover more natural
sources of drugs for the treatment of cancer.
D) Efficacy and safety of medicinal plants in cancer treatment
The traditional use of plants in the treatment of ailments has been on the increase
both in developing countries, where there is poor availability of primary health care, and
also in the developed world. Herbal medicines are in great demand in the developing
world for primary health care not only because they are inexpensive but also for better
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cultural acceptability, better compatibility with the human body and minimal side effects.
This is primarily due to the general belief that herbal medicines are relatively safe
because they are natural (Gesler, 1992). The knowledge of the healing virtues of
medicinal plants has been passed on from ancient times. History tells of medicinal plants
such as Catharanthus roseus G. Don, Digitalis purpurea Linn, Rauwolfia serpentina
Plum ex Linn, Willow (Salix species), Physostigma venenosum Balf. and a host of other
plants which have been used for centuries for the treatment of diseases such as cancer,
cardiovascular diseases, hypertension, depression, pain, glaucoma and an array of other
diseases that have plagued the world. Since then, plants have served as viable sources of
biologically active natural products which are either used as commercial drugs or as lead
structures for the development of modified derivatives possessing enhanced activity
(Alschuler et al., 1997). Modern medicine as a result of civilization led to the reduced
importance of medicinal plants to human survival. This was not because these plants
were ineffective but because they were not economically profitable as the newer synthetic
drugs (Tyler, 1999). However, in recent times, the concerns over the serious adverse
effect of conventional drugs and the movement towards a more natural living has brought
about a resurgence in the use of herbal products (Pal et al., 2003).
The number of patients seeking herbal approaches for therapy has grown
exponentially (Cordell et al., 1991). In France and Germany, the medical doctors
regularly prescribe herbal medicine to 70% of their patients. Available records have
illustrated the growth of the herbal medicine market in the European Union countries. In
1991, sales were about $ US 6 billion, with Germany accounting for $ US3 billion,
France $ US 1.6 billion and Italy $ US 0.6 billion while in the US, herbal medicine
market was about $ 4 billion in 1996 (Pal et al., 2003). India boasts about $ US 80
million for the exportation of herbal crude extracts (Kamboj, 2000). The resurgence and
popularity of herbal medicines have led to an increase in the number of medicinal plant
products in the market (Gupta et al., 1998). Unfortunately, the increased dependence on
phytotherapy, without concern for efficacy and safety has resulted in preventable serious
adverse effects (Gurib-Fakim, 2006).
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a) Efficacy of medicinal plants
With the slight increase in the randomized controlled trials to evaluate the
efficacy of herbal medicines, an estimate of about 39% of all 520 new approved drugs
were natural products or derived from natural products in 1983-1994 (Cragg et al., 1997).
The study of Harvey (1999), reported that 60-80% of antibacterial and anticancer drugs
were derived from natural products (Harvey, 1999). The antimalaria quinine from
Cinchona officinalis, analgesics codeine and morphine from Papaver somnifera,
antihypertensive reserpine from Rauwolfia serpentina and cardiac glycoside digoxin from
Digitalis pupurea are some of the many drugs derived from medicinal plants that have
been in use. The fact remains that plant substances constitute the basis for a very large
proportion of medications used today for the treatment of diseases of the liver and heart,
cancer, hypertension, depression and other ailments. This is the result of an increase in
the scientific studies carried out to validate the traditional claims of these plants (GuribFakim, 2006).
b) Safety of medicinal plants
Recent findings indicate that herbal medicines may not be safe and severe
consequences have arisen from the use of certain products (Gurib-Fakim, 2006; Bush et
al., 2007). Information obtained from health centres and hospital emergency rooms have
shown that 5 % of patients receiving complementary therapies report side effects
(Molassiotis et al., 2005). The true frequency of the incidence of side effects from herbal
remedies may be several folds higher than this, (Ernst, 2004) because the lack of
surveillance systems which are less extensive than for conventional drugs have limited
these reports (Bent et al., 2004). For example, acute poisoning as a result of herbal
medicines is estimated to cause anywhere from 8,000 to 20,000 deaths annually in South
Africa (Thomson et al., 2000). These side effects may occur through several different
mechanisms, including direct toxic effects of the herbs, effects of contaminants, and
interactions with drugs or other herbs (Ernst, 2004; Bent et al., 2004; Niggemann et al.,
2003). The risk of herbal remedies producing side effects depends not only on the herb
and the dose consumed, but also on the health status and age of the patient and the
concurrent use of other drugs (De Smet et al., 1995).
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2. AIM AND OBJECTIVES
Researchers are constantly making efforts to discover new drugs and design better
protocols for cancer. Synthetic anticancer drugs kill the cancer cells but they are also
harmful to the normal cells. Since, increase in the use of these drugs in cancer therapy
leads to many side effects and undesirable hazards, there is a worldwide trend to go back
to natural resources, i.e., traditional plant preparations which are not only therapeutically
effective but are actually acceptable and economically within the reach of even the
neediest people. An alternative solution of this problem is the use of medicinal plant
preparation to arrest the insidious character of the disease. Therefore it is imperative that
more attention is focused to control the carcinogenesis. It may be easier to control the
spread of cancer, if appropriate steps are taken before the initiation of the disease. The
most important imaginative approach to reduce the cancer cases worldwide could be the
inhibition of induction of carcinogenesis or cancer by the use of herbal technology. Many
naturally occurring substances have been tested for anticancer activity on experimental
animals resulting in the presence availability of some 30 effective anticancer drugs
(Ramakrishna et al., 1984).
Cytotoxicity screening models provide important
preliminary data to help select plant extracts with potential antineoplastic properties for
future work (Bohlin et al., 1999).
Both ancient experience from traditional Chinese herbal medicine and modern
studies have demonstrated that herbal medicine could be effective remedy for cancer
treatment and to improve outcome of chemotherapy. Hence, the present study is
undertaken to evaluate the anticancer activity of Madhuca longifolia, Adina cordifolia,
Sida veronicaefolia in mice.
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3. REVIEW OF LITERATURE
3.1 Anti-Cancer plants – A Review
Natural Products, especially plants, have been used for the treatment of various
diseases for thousands of years. Terrestrial plants have been used as medicines in Egypt,
China, India and Greece from ancient time and an impressive number of modern drugs
have been developed from them. The first written records on the medicinal uses of plants
appeared in about 2600 BC from the Sumerians and Akkaidians. The “Ebers Papyrus”,
the best known Egyptian pharmaceutical record, which documented over 700 drugs,
represents the history of Egyptian medicine dated from 1500 BC. The Chinese Materia
Medica, which describes more than 600 medicinal plants, has been well documented with
the first record dating from about 1100 BC (Cragg et al., 1997). Documentation of the
Ayurvedic system recorded in Susruta and Charaka dates from about 1000 BC (Kappor,
1990). The Greeks also contributed substantially to the rational development of the herbal
drugs. Dioscorides, the Greek physician (100 A.D.), described in his work “De Materia
Medica” more than 600 medicinal plants. Phytochemicals have been proposed to offer
protection against a variety of chronic ailments including cardiovascular diseases,
obesity, diabetes, and cancer. As for cancer protection, it has been estimated that diets
rich in phytochemicals can reduce cancer risk by 20%.The compounds that are
responsible for medicinal property of the drug are usually secondary metabolites. Plant
natural product chemistry has played an active role in generating a significant number of
drug candidate compounds in a drug discovery program. Recently, it has been reported in
the literature that approximately 49 % of 877 small molecules that were introduced as
new pharmaceuticals between 1981 and 2002 by New Chemicals Entities were either
natural products or semi-synthetic analogs or synthetic products based on natural product
models.
Plants have a long history of use in the treatment of cancer. Hartwell, in his
review of plants used against cancer, lists more than 3000 plant species that have
reportedly been used in the treatment of cancer. It is significant that over 60% of
currently used anticancer agents are derived in one way or another from natural sources,
including plants, marine organisms and micro-organisms. Indeed, molecules derived from
natural sources (so called natural products), including plants, marine organisms and
Page 15 of 113
micro-organisms have played and continue to play, a dominant role in the discovery of
leads for the development of conventional drugs for the treatment of most human
diseases. The search for anti-cancer agents from plant sources started in earnest in the
1950s with the discovery and development of the vinca alkaloids, vinblastine and
vincristine, and the isolation of the cytotoxic podophyllotoxins. These discoveries
prompted the United States National Cancer Institute (NCI) to initiate an extensive plant
collection program in 1960. This led to the discovery of many novel chemotypes showing
a range of cytotoxic activities, including the taxanes and camptothecins (Cragg et al.,
2005a).
More than 50% of all modern drugs in clinical use are natural products, many of
which have the ability to control cancer cells. A recent survey shows that more than 60%
of cancer patients use vitamins or herbs as therapy (Sivalokanathan et al., 2005; Creemer
et al., 1996).
Many of these medicinal plants have been found effective in experimental and
clinical cases of cancers. Attempts are being made to isolate active constituents from
natural sources that could be used to treat this very serious illness. The first agents to
advance into clinical use were the isolation of the vinca alkaloids, vinblastine and
vincristine from the Madagascar periwinkle, Catharanthus roseus (Apo-cynaceae)
introduced a new era of the use of plant material as anticancer agents (Cassady et al.,
1981). They were the first agents to advance into clinical use for the treatment of cancer.
Vinblastine and vincristine are primarily used in combination with other cancer
chemotherapeutic drugs for the treatment of a variety of cancers, including leukemias,
lymphomas, advanced testicular cancer, breast and lung cancers, and Kaposi’s sarcoma
(Cassady et al., 1981).
The discovery of paclitaxel from the bark of the Pacific Yew, Taxus brevifolia
Nutt. (Taxaceae), is another evidence of the success in natural product drug discovery.
Various parts of Taxus brevifolia and other Taxus species (e.g., Taxus Canadensis, Taxus
baccata ) have been used by several Native American Tribes for the treatment of some
noncancerous cases (Cragg et al., 2005a). Taxus baccata was reported to use in the Indian
Ayurvedic medicine for the treatment of cancer. Paclitaxel is significantly active against
ovarian cancer, advanced breast cancer, small and non-small cell lung cancer.
Page 16 of 113
Camptothecin, isolated from the Chinese ornamental tree Camptotheca acuminate
(Nyssaceae), was advanced to clinical trials by NCI in the 1970s but was dropped
because of severe bladder toxicity. Topotecan and irinotecan are semi-synthetic
derivatives of camptothecin and are used for the treatment of ovarian and small cell lung
cancers, and colorectal cancers, respectively (Harvey, 1999; Bertino, 1997).
Epipodophyllotoxin is an isomer of podophyllotoxin which was isolated as the
active antitumor agent from the roots of Podophyllum species, Podophyllum peltatum and
Podophyllum emodi (Berberidaceae).Etoposide and teniposide are two semi-synthetic
derivatives of epipodophyllotoxin and are used in the treatment of lymphomas and
bronchial and testicular cancers (Cragg et al., 2005b).
Combretastatins were isolated from the bark of the South African tree Combretum
caffrum (Combretaceae). Combretastatin is active against colon, lung and leukemia
cancers and it is expected that this molecule is the most cytotoxic phytomolecule isolated
so far (Ohsumi et al., 1998; Petit et al., 1987).
Betulinic acid, a pentacyclic triterpene, is a common secondary metabolite of
plants, primarily from Betula species (Betulaceae) (Cichewitz et al., 2004). Betulinic acid
was isolated from Zizyphus species, e.g. Zizyphus mauritiana, Zizyphus rugosa and
Zizyphus oenoplia and displayed selective cytotoxicity against human melanoma cell
lines (Pisha E et al., 1995).
The Podophyllum species (Podophyllaceae), Podophyllum peltatum (commonly
known as the American mandrake or Mayapple), and Podophyllum emodii from the
Indian subcontinent, have a long history of medicinal use, including the treatment of skin
cancers and warts. Podophyllum peltatum was used by the Penobscot Native Americans
of Maine for the treatment of cancer (Cragg et al., 2002).
Camptothecin isolated from Camptotheca acuminata (Nyssaceae), also known as
tree of joy in China is a possible source of steroidal precursors for the production of
cortisone. The extract of Camptotheca acuminata was the only one of 1000 of the plant
extracts tested for anti-tumor activity which showed efficacy and camptothecin was
isolated as an active constituent (Cragg et al., 2002).
Other plant derived agents in clinical use are homoharringtonine isolated from the
Chinese tree, Cephalotaxus harringtonia (Cephalotaxaceae), and elliptinium, a derivative
Page 17 of 113
of ellipticine isolated from species of several genera of the Apocynaceae family including
Bleekeria vitensis, a Fijian medicinal plant with reputed anti-cancer properties. Several
Terminalia species have reportedly been used in the treatment of cancer. The
combretastatins are a family of stilbenes which act as anti-angiogenic agents causing
vascular shutdown in tumors and resulting in tumor necrosis (Cragg et al., 2002).
Dragon's blood is the popular name for a dark red viscous sap produced by
Croton lechleri. This herb is used in folk medicine as an anti-inflammatory, antimicrobial
and anticancer (Pieters et al., 1993; Hartwell, 1969; Lopes, 2004). Crude extracts from
plants like Colubrina macrocarpa, Hemiangium excelsum and Acacia pennatula have
been shown to possess a selective cytotoxic activity against human tumor cells (Popoca et
al., 1998).
In Saudi Arabia, aerial parts of Commiphora opobalsamum are commonly used to
treat various diseases. However, its potential use in stomach problems and cancer has
been reported only recently (Howiriny et al., 2005).
Some Astragalus species are used to treat leukemia and promote wound healing
(Calis et al., 1997). Salvia officinalis is the most popular herbal remedy in the Middle
East to treat common health complications. Salvia species (Labiatae) are known for their
antitumor effects (Liu et al., 2000).
Phytochemically, the whole plant contains several antioxidants that protect
against cellular peroxidative damage. Lantana camara possesses several medicinal
properties and is commonly used in folk medicine for its antipyretic, antimicrobial and
antimutagenic properties (Fernanda et al., 2005).
Solanum nigrum is a common herb that grows wildly and abundantly in open
fields. It has been used in traditional folk medicine because of its diuretic and antipyretic
effects. More specifically, it has been used for a long time in oriental medicine to cure
inflammation, edema, mastitis and hepatic cancer (Lee et al., 2003).
Evaluation of the in-vitro anticancer effects of bioflavonoids, viz. quercelon,
catechin, luteolin and rutin against human carcinoma of larynx (Hep-2) and sacroma 180
(S-180) cell lines showed that only luteolin and quercelon inhibited the proliferation of
the cells. Luteolin caused depletion of glutathione in the cells and a decline in DNA
Page 18 of 113
synthesis, as seen by 3H thymidine uptake studies, thus demonstrating its anticancer
potential (Elangovan et al., 1994).
The anti-tumor effect of the crude extract of Centella asiatica as well as its
partially purified fraction was studied in both, In-vitro short and long term
chemosensitivity test systems and in vivo tumor models. The purified fraction inhibited
the proliferation of transformed cell lines of Ehrlich ascites tumor cells and Dalton’s
lymphoma ascites tumor cells more significantly than the crude extract. It also
significantly suppressed the multiplication of mouse lung fibroblast cells in long term
culture. In-vivo administration of both extracts retarded the development of solid and
ascites tumors and increased the lifespan of the tumor bearing mice. Triturated thymidine,
uridine and leucine incorporation assays suggest that the purified fraction acts directly on
DNA synthesis (Babu et al., 1995).
Fresh root suspension of Janakia arayalpathra exhibited strong anti-tumor effects
in mice challenged with Ehrlich ascites carcinoma (EAC) cells. It prolonged the survival
of all mice and protected a number of mice from tumor growth, probably by enhancing
the activity of the immune system (Subramanian et al., 1996).
Withaferin A, a steroidal lactone isolated from the roots of Withania somnifera,
reduced survival of V79 cells in a dose-dependent manner. The applicability of this drug
as a radiosensitizer in cancer therapy needs to be explored (Devi et al., 1996).
Banerjee et al., 1996, have studied the modulatory influence of the alcoholic
extract of leaves of Ocimum sanctum on various enzyme levels in the liver, lung and
stomach of mouse. Oral treatment with the extract significantly elevated the activities of
cytochrome P450, cytochrome b5, arylhydrocarbon hydroxylase and glutathione Stransferase enzyme, all of which are important in the detoxification of carcinogens as
well as mutagens. Moreover, it also significantly elevated extra-hepatic glutathione Stransferase and reduced glutathione levels in the lever, lung, and stomach. These
observations suggest that the leaf extract or its active principles may have a potential role
in the chemoprevention of chemical carcinogenesis (Banerjee et al., 1996).
Petroleum ether extract of Hygrophilic spinosa exhibited anti-tumor activity in
Ehlrich ascites carcinoma and sacroma 180 bearing mice (Mazumdar et al., 1997).
Page 19 of 113
Aqueous extract of Podophyllum hexandrum, a herb from the Himalayas,
demonstrated significant antitumor effects when drug was tested in strain ‘A’ mice
carrying solid tumors developed by transplanting Ehlrich ascites tumor cells.
Radioprotective effects were also seen when the drug was administrated to mice before
whole body lethal irradiation of 10 Gy (Goel et al., 1998).
The chemopreventive efficacy of Trianthema portulacastrum L. Aizoaceae was
tested in male Sprague-Dawley rats. Hepatocarcinogenesis was induced by the potent
carcinogen diethylnitrosoamine (DENA). Treatment of the rats with aqueous, ethanolic
and chloroform fractions of the plant extract at a dose of 100 mg/kg once daily reduced
the incidence, numerical preponderance, multiplicity and size distribution of visible
neoplastic nodules. Morphometric evaluation of focal lesions showed a reduction in
number of altered liver cell foci per square centimeter as well as of average area of
individual lesion. A decrease in the percentage of liver parenchyma occupied by foci
seems to suggest the anticarcinogenic potential of the plant extract in DENA-induced
hepatocarcinogenesis (Bhattacharya et al., 1998).
Pretreatment with Ocimum sanctum leaf extract followed by the addition of 7, 12dimethylbenz[a]anthracene (DMBA) significantly blocked the formation of DMBA-DNA
adducts in primary cultures of rat hepatocytes invitro. The viability of the cells was not
adversely affected by the extract (Prashar et al., 1998).
Table 3.1: List of plants reported for their anticancer activity
Species
Family
Part used
Reference
Allium sativum
Liliaceae
Bulbs
Hirsh et al., 2000
Aristolochia triangularis
Aristolochiaceae
Bark
Mongelli et al., 2000
Barringtonia racemosa
Lecythidaceae
Bark
MacKeen et al., 1997
Betula platyphylla
Cupuliferae
Boscia senegalensis
Capparidaceae
Leaves
Ali et al.,2002
Catalpa bignonioides
Bignoniaceae
Seeds
Muñoz et al., 2003
Whole plant Ju et al., 2004
Page 20 of 113
Celastrus orbiculatus
Clerodendrum
myricoides
Celestraceae
Root
Verbenaceae
Root bark
Kamuhabwa et al.,
2000
Clematis chinensis
Ranunculaceae
Crocus sativus
Iridaceae
Stigma
Croton palanostigma
Euphorbiaceae
Sap
Cunuria spruceane
Jin et al.,, 2002
Euphorbiaceae
Whole plant Qiu et al., 1999
Root, Root
bark
Nair et al., 1995
Sandoval et al., 2002
Gunasekera et
al.,1979
Cupressus lusitanica
Cupressaceae
Leaves
Lopéz et al., 2002
Dendrostellera lessertii
Thymelaeaceae
Leaves
Sadeghi et al., 2003
Dioscorea birmanica
Dioscoriaceae
Whole plant
Woerdnbeg et al.,
1986
Emblica officinalis
Euphorbiaceae
Fruits
Emilia sonchifolia
Compositae
Eurycoma longifolia
Simaroubaceae
Leaves
Garcinia atroviridis
Guttiferae
Stem bark
Jose et al., 2001
Whole plant Shylesh, et al., 2000
Jiwajinda et al., 2002
Mackeen et al.,
2000
Lirdprapamongkol
Helixanthera parasitica
Loranthaceae
Whole plant
K, et al., 2003
Hippophae salicifolia
Elaeagnaceae
Humulus lupulus
Cannabaceae
Fruit
Uniyal et al., 1990
Whole plant Goun et al., 2002
Page 21 of 113
Iris germanica
Iridaceae
Bulbs
Bonfils et al., 2001
Jatropha elliptica
Euphorbiaceae
Tuber
Calixto et al., 1987.
Kigelia Africana
Bignoniaceae
Rootbark
Msonthi et al., 1983
Leptadenia hastate
Asclepiadaceae
Bark
Aquino et al., 1995
Myrtaceae
Aerial parts
Mayer et al., 1993
Leptospermum
scoparium
Lithraea molleoides
Melastoma
malabathricum
Anacardiaceae
Whole plant Ruffa et al., 2002
Mohandoss et al.,
Melastomataceae
Flowers
1993
Moringa oleifera Lam
Moringaceae
Nyssa sinensis Oliv.
Nyssaceae
Rootbark
Luo et al., 1991
Oenanthe javanica
Umbelliferae
Entire plant
Duke et al., 1985
Oldenlandia diffusa
Rubiaceae
Entire plant
Wong et al., 1993
Phyllanthus amarus
Euphorbiaceae
Aerial parts
Joy et al., 1998
Plantago afra
Plantaginaceae
Leaves
Loranthaceae
Leaves
Psittacanthus
calyculatus
Whole plant Dhawan et al., 1980
Gálvez et al., 2003
Zee Cheng et al.,
1997
Psoralea corylifolia
Fabaceae;
Seeds
Yang et al., 1996.
Rhus longipes
Anacardiaceae
Root
Chhabra et al., 1991
Solanum lyratum
Solanaceae
Aerial parts
Terminalia chebula
Combretaceae
Fruits
Lee et al., 1997
Saleem et al., 2002
Page 22 of 113
Leaves,caps
Verbascum Thapsus
Scrophulariaceae
Virola bicuhyba
Myristicaceae
Seed
Plotkin et al., 1990
Xeromphis obovata
Rubiaceae
Rootbark
Sibanda et al., 1989
Zanthoxylum oxyphyllum
Rutaceae
Fruit
ules
Turker et al., 2002
Suwal, 1970.
Page 23 of 113
3.2 Anti-Cancer Phytochemicals – A Review
A) Flavonoids
Despite the tremendous advancements in the understanding and treatment of
cancer, there is no sure-fire cure for a variety of cancers to date. Therefore, natural
protection against cancer has recently been receiving a great deal of attention not only
from cancer patients but, surprisingly, from physicians as well. Phytoestrogens, plantderived secondary metabolites, are normally divided into three main classes: flavonoids,
coumestans and lignans. Flavonoids are found in almost all plant families. Flavonoids are
present in different plant parts including the leaves, stems, roots, flowers and seeds and
are among the most popular anti-cancer candidates worldwide. Flavonoidic derivatives
have a wide range of biological actions such as antibacterial, antiviral, anti-inflammatory,
anticancer, and anti-allergic activities. Some of these benefits are attributed to the potent
antioxidant effects of flavonoids, which include metal chelation and free-radical
scavenging activities (Amin et al., 2007).
Flavonoids are the most abundant active ingredients in plants species. Wide
varieties of plant derived flavonoids are naturally present in the human diet or are
normally consumed for medicinal reasons. Flavonoids are reported to inhibit specific
enzymes, which include hydrolases, oxidoreductase, DNA synthases, RNA polymerases,
lipoxygenase and gluthation S-transferase. They also block several digestive enzymes,
including α-amylase, trypsin and lipase (Koshihara et al., 1984; Griffiths, 1986 Reddy et
al., 1994; Sadik et al., 2003). As a result, a rising number of authorized physicians are
prescribing pure flavonoids to treat many important common diseases.
Dragon's blood is the popular name for a dark-red viscous sap produced by
Croton lechleri. This herb is used in folk medicine as an anti-inflammatory (Pieters et al.,
1993), anti-microbial (Ubillas, 1994) and anticancer (Hartwell, 1969; Lopes, 2004).
Similarly, crude extracts from plants like Colubrina macrocarpa, Hemiangium excelsum
and Acacia pennatula have been shown to possess a selective cytotoxic activity against
human tumor cells KB, HCT-15 COLADCAR and UISO-SQC-1 (Popoca et al., 1998).
Another member of the family Leguminosae has been shown to have significant antibreast cancer potential (Amin et al., 2005). In that study, we have shown that Fenugreek
can significantly protect rats against drug-induced breast cancer.
Page 24 of 113
Both the whole plant and the roots of Plumbago zeylanica L. are commonly used
in the treatment of rheumatic pain, dysmenorrhea, carbuncles, contusion of the
extremities, ulcers and elimination of intestinal parasites (Chopra et al., 2006).
Pharmacological studies carried out by several workers have also indicated that P.
zeylanica L. extract possesses antiplasmodial (Simonsen et al., 2001), antimicrobial
(Durga et al., 1990), antihyperglycemic (Olagunju et al., 1999), insecticidal (Kubo et al.,
1983) and antiallergic (Dai et al., 2004) properties. P. zeylanica L. extract also stimulates
the central nervous system (Bopaiah et al., 2001) and is cytotoxic to tumor cells (Lin et
al., 2003).
In the Palestinian and Israeli territories, extracts of Teucrium polium and Pistacia
lentiscus, among others, are known to treat liver disease, jaundice, diabetes, fertility
problems and cancer. Most recently, extracts of these two plants have been shown not to
be toxic in addition to effectively suppress Fe2+-induced lipid peroxidation. As the aerial
parts of Teucrium polium and Pistacia lentiscus are rich in flavonoids, it was concluded
that the ability of these plants to suppress Fe2+-induced lipid peroxidation was mediated
by flavonoids (Ljubuncic et al., 2005). In Saudi Arabia, aerial parts of Commiphora
opobalsamum (L.) (Balessan) are commonly used to treat various diseases. However, its
potential use in stomach problems and cancer has been reported only recently.
Flavonoids, saponins, volatile oil, sterol and triterpenes have all been revealed in
Balessan and thus might contribute to its anticancer activity (Howiriny et al., 2005).
Among many other effects, Apium graveolens L. [celery, family: Umbelliferae], is
particularly known for its anti-cancer (Sultana et al., 2005) and antioxidant effects
(Momin et al., 2002). Phytochemical investigations of celery seeds revealed the presence
of terpenes like limonene, flavonoids like apigenin and phthalide glycosides. Apigenin is
an antioxidant that was documented as one of the major celery's active principals in
Apium graveolens (Miean et al., 2001). The efficacy of celery as an anti-cancer remedy
may then be attributed to the presence of flavonoids, particularily apigenin in its extract
(Hamza et al., 2007).
Apigenin is a widely distributed plant flavonoid that was recently reported as an
antitumor agent. Apigenin inhibits the growth of human cervical carcinoma cells by
activating apoptosis, Confirmation of induction of apigenin-induced apotosis in HeLa
Page 25 of 113
cells was confirmed by DNA fragmentation assays and induction of sub-G1 phase by
flow cytometry. Recent findings suggest that apigenin is a strong candidate for
development as an anti-cervical cancer agent. Apigenin’s preventive effect is shown to
be mediated through induction of p53 expression, which causes cell cycle arrest and
apoptosis (Duthie et al., 2000; Pei-Wen et al., 2005).
Butein is another polyphenolic compound, which can be extracted from Rhus
verniciflua or the heartwood of Dalbergia odorifera. It induces apoptosis in HL-60 cells
(Kim et al., 2001) and B16 melanoma cells (Iwashita et al., 2000) by regulating BCL-2
family proteins. Other properties, such as anti-inflammatory activities (Chan et al.,1998),
antinephritic effects (Hayashi et al., 1996), antioxidant properties (Lee et al., 2002), have
been documented as well. Only recently have butein's potential as a pharmacological
agent been extended to the modulation of estrogen metabolism. This effect could be
essential in the prevention and treatment of estrogen-responsive breast cancers (Wang et
al., 2005).
B) Tannins
Tannins, phenolic phytochemicals, which are natural constituents of green tea, are
considered to have cancer-preventive properties (Lambert et al., 2003; Keil et al., 2004).
Condensed tannins, isolated from black beans, did not affect the growth of normal cells,
but induced cell death in cancer cells in a dose-dependent manner. This cell death was
associated with a concentration-dependent decrease of ATP and a deterioration of cellular
gross morphology (Swami et al., 2003; Bawadi et al., 2005). Sorghum is a rich source of
various phytochemicals including tannins. Relative to other cereals or fruits, sorghum
fractions possess high antioxidant activity. These fractions may offer similar health
benefits commonly associated with fruits. Sorghum was adapted to grow in the U.A.E.
environment, and was found to contain reasonable levels of dietary fiber, minerals and
antioxidants to replace part of wheat flour in wheat-based food products (Ragaee et al.,
2005). Available epidemiological evidence suggests that Sorghum consumption reduces
the risk of certain types of cancer in humans compared to other cereals. The high
concentration of phytochemicals in sorghum may be responsible for its protective effects
(Rooney et al., 1983; Awika et al., 2004).
Page 26 of 113
C) Alkaloids
Historic medicinal practice used Cat's Claw, also known Uncaria tomentosa, as
an effective treatment for several health disorders, which include chronic inflammation,
gastrointestinal dysfunction such as ulcers, tumors and infections. The efficacy of Cat's
Claw was originally believed to be due to the presence of oxindole alkaloids. Watersoluble Cat's Claw extracts were shown not to contain significant amounts of alkaloids
(<0.05%), and yet still were shown to be very effective. Most recently, the active
ingredients of a water-soluble Cat's Claw extract were shown to inhibit cell growth
without cell death, thus providing enhanced opportunities for DNA repair, immune
stimulation, anti-inflammation and cancer prevention (Blumenthal et al., 2003; Sheng et
al., 2005). These active ingredients were chemically defined as quinic acid esters and
were also bioactive in vivo as quinic acid.
D) Saponins
An Iranian experimental study with mice indicated that saffron (Crocus sativus
L.) stigma and petal extracts exhibited antinociceptive effects in chemical pain tests and
acute and/or chronic anti-inflammatory activity. It was suggested that these effects of
saffron extracts might be due to their content of flavonoids, tannins, anthocyanins,
alkaloids, and saponins. Studies in animal models and with cultured human malignant
cell lines have demonstrated both the antitumor and cancer preventive activities of
saffron and its main ingredients. Many possible mechanisms for these activities have
been proposed. On-going clinical trials that use actual reduction of cancer incidence as
the primary endpoint may soon provide a direct evidence of the anticancer effectiveness
of saffron (Abdullaev et al., 2004).
The aqueous root extracts of some Astragalus species are used to treat leukemia
and promote wound healing (Bedir et al., 2000). The roots of Astragalus species
(Fabaceae) are known to be rich in polysaccharides and saponins (Yesilada et al., 2005).
Astragalus L., the largest genus in the family Leguminosae is represented by thirty-two
species in Egypt. Some species of this genus have been reported as having
immunostimulant, cardiovascular and antiviral activities (Rios et al., 1997). Extracts of
Astragalus kahiricus have been shown to have a reproducible cytotoxicity against the A
2780 ovarian cancer cell line, with an IC50 value of 25 μg/mL (Radwan et al., 2004).
Page 27 of 113
Tribulus terrestris, a member of the family Zygophyllaceae, is widely distributed in the
entire Gulf region. Saponin from Tribulus is also known for its hypoglycemic effect. In
our recent study, ethanolic extract of Tribulus has shown a significant antioxidant activity
against STZ-induced diabetes (Amin et al., 2006). In a search for new anticancer agents,
a novel compound polyphyllin D (PD) (diosgenylα-L-rhamnopyranosyl-(1→2)-(α-Larabinofuranosyl)-(1→4)]-[β D glucopyranoside) has been identified was shown to
induce DNA fragmentation in a hepatocellular carcinoma cell line (HepG2) derivative
with drug resistance (R-HepG2). PD is a saponin originally found in a tradition Chinese
medicinal herb Paris polyphylla. It has been used to treat liver cancer in China for many
years. PD has been reported as a potent anticancer agent that can overcome drug
resistance in R-HepG2 cells and elicit programmed cell death via mitochondrial
dysfunction (Henry et al., 2002; Cheung et al., 2005). Five saponins (diosgenin,
hecogenin, tigogenin, sarsasapogenin, smilagenin) have been tested for their biological
activities on human 1547 osteosarcoma cells. All examined saponins have shown a
significant role on tested cell line in term of proliferation rate, cell cycle distribution and
apoptosis induction (Cheung et al., 2005).
A bacterial metabolite of ginseng saponin (20-O-(--Glucopyranosyl)-20(S)protopanaxadiol; IH901) is suggested to be a potential chemopreventive agent. IH901
induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early
activation of both procaspase-3 and caspase-8. Available data suggest that the activation
of caspase-8 after early caspase-3 activation might act as an amplification loop necessary
for successful apoptosis. Primary hepatocytes isolated from normal Sprague–Dawley rats
were not affected by IH901 (0–60 M). The very low toxicity in normal hepatocytes and
high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising
experimental cancer chemopreventive agent (Bosch et al., 1999; Oh et al., 2004).
E) Sterols/Triterpines
Phytosterols, especially -sitosterol, are plant sterols that have been shown to exert
protective effects against cardiovascular diseases and many types of cancer
(Moghadasian, 2000; Awad et al., 2004). They have been reported to protect against
cancer development, however, the mechanism of this protection remains unknown even
though several different mechanisms have been proposed (Raicht et al., 1980; Rao et al.,
Page 28 of 113
1992; Awad et al., 2000a; Awad et al., 2000b). Prostatic 5-reductase and prostatic
aromatase activities were decreased in rats supplemented with phytosterols (Mettlin,
1999; Awad et al., 1998) indicating that they may suppress prostate metabolism and
growth. In independent studies, sitosterol has been shown to alter tumor growth (Hannun
et al., 1994; Awad et al., 1996). The incorporation of sitosterol in the membranes of HT29 cells resulted in a significant decrease in sphingomyelin and an increase in
phosphatidylcholine (Hannun et al., 1994). Thus, the inhibition of tumor growth could be
explained by the effect of phytosterols on the sphingomyelin cycle and increased
production of ceramide, which suggest an alteration of signal transduction pathways
(Leikin et al., 1989; Tapiero et al., 2003).
Previous studies on the cancer chemopreventive effects of natural sources
(Nakamura et al., 2002a) have shown gallic acid and methyl gallate, which were isolated
from Juca fruits of Caesalpinia ferrea (Leguminosae), as the active constituents. These
studies were conducted using the Epstein–Barr virus early antigen activation assay
(Ito et al., 1981; Nakamura et al., 2002b).
Page 29 of 113
3.3 Selected plants – A Review
A) Madhuca longifolia L
Plant Name: Madhuca longifolia
Family: Sapotaceae
Common Name: English: Indian Butter Tree
Hindi : Mahua
Bengali: Maul
Marathi: Kat-illipi
Malayalam: Illupa
Fig 3.1: Leaves of Madhuca longifolia
Telugu: Ippa
Synonyms: Bassia latifolia, Illipe latifolia, Madhuca indica, Madhuca latifolia
Parts used: Leaf, root, seed and bark.
Habitat: The plant grows in all the plains and lower hills of India up to 1200 meters, and
is at certain places, a chief constituent of the forest vegetation. It is a large deciduous tree
with rather shorter bole, but larger crown. It grows 13-16 meters in height, and bark
grayish black, scaly. The leaves, 10-20 cm long, thick leathery, pointed at tip, with 10-12
prominent veins. The flowers strongly musk-scented, falling at dawn, fleshy, pale or dull
white, in clusters near the ends of branches. The fruits, 2.5-5 cm long, ovoid berries,
yellow when ripe. The tree blooms in the summer and bears fruits in rainy season.
Chemical Constituents: The seeds contain 55% stable oil. From the flowers, liquor is
obtained by distillation. Since centuries, the flowers are used in Ayurvedic Pharmacy in
manufacturing various asavas and aristas (herbs, eigher in their fresh juice – arista, or
their decoction – asava. From fruits, sucrose, sitosterol, a sterol glucoside from nuts, and
amyrin acetate, capryloxyerythridiol and capryloxyoleanolic acid isolated. From the bark
lupeol acetate, amyrin acetate, spinasterol, erythrodiol monocaprylate, betulinic acid and
oleanolic acids caprylates, rhamnose, glucose and galactose isolated. Polysaccharides PSAI & PS-A II, isolated from flowers, constitute galactose, glucose, arabinose and
glucoronic acid (Prajapati et al., 2003; Chandra et al.,2001). it also contains oleananetype triterpene glycosides (Kazuko et al., 2000).
Properties: Madhuka is sweet in taste, sweet in the post digestive effect and has cold
potency. It alleviates vata and pitta doshas. It possesses heavy and oily (snigdha)
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attributes. The dried flowers have hot potency. The fruits alleviate kapha and vata doshas.
It has anabolic and rejuvenative properties and is used in diseases like tuberculosis, blood
diseases, asthma, burning sensation and thirst.
Uses: The flowers, seeds and seed oil of madhuka have great medicinal value. Externally,
the seed oil massage is very effective to alleviate pain (Chandra et al.,2001). In skin
diseases, the juice of flowers is rubbed for oleation. It is also beneficial as a nasya (nasal
drops) in diseases of the head due to pitta, like sinusitis. The seed oil is used in
manufacturing of soaps and is used as an edible also.
Internally, madhuka is used in vast range of diseases (Dahake et al., 2010a). The
decoction of the flowers is a valuable remedy for pitta diseases. As a general tonic, the
powder of flowers works well with ghee and honey. The decoction of flowers quenches
the thirst effectively. Because of its astringent property, madhu karista is salutary in
diarrhea and colitis. In raktapitta, the fresh juice of flowers is used with great benefit to
arrest the bleeding. The flowers play an important role in augmention the breast milk in
lactating mothers and in boosting the quantity of seminal fluids also. Madhuka is
benedicial in urinary ailments like burning micturition and dehydration, fever,
tuberculosis etc. The combination of the powders of the bark skin of madhuka, pippali
and marica fruits, rhizomes of vaca and salt in equal parts is used in the form of nasal
drops, in the treatment of epilepsy, with excellent benefit. Madhuka is the best nervine
and salutary in the diseases due to vata. The nasya-nasal therapy is useful in hysteria,
cough and sinusitis. The bark skin powder is given along with ghee and honey to improve
the vitality and sexual vigor. The plant is also used in the hyperglycemic condition
(Dahake et al., 2010a; Ghosh, 2009 ).
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B) Adina cordifolia L
Plant name: Adina cordifolia
Family: Rubiaceae
Common name:
Bengali : Keli-kadam
Hindi
: Haldu
Sanskrit : Dharakadanba
Fig 3.2: Leaves of Adina cordifolia
Synonyms: Haldina cordifolia, Adina ledermanii (hallealedermannii), Adina pilulifera
(Cephalanthus), Adina rubella, Nauclea cordifolia.
Parts used: Leaf, root, seed and bark.
Habitat: A moderate sized deciduous tree grows up to 35 m in height. Leaves large,
cordate, abruptly acuminate. Flowers yellow in globose pedunculate heads; fruits
capsules, splitting into two dehiscent cocci, seeds many, narrow, small, and tailed. It
occurs frequently but scattered in deciduous forest in the lowland and lower hills. In
Burma (Myanmar) and Thailand it is often associated with teak (Tectonagrandis L.f.)
India, Sri Lanka, Burma (Myanmar), Indo-China Southern China, Thailand and
Peninsular Malaysia (very rare) (Asolkar et al., 1992).
Chemical Constituents:-
It contains 10-deoxyadifoline, 10-deoxycordifoline indole
alkaloid, cordifoline, adifoline. Di-OH-tetra-OMe flavone has been isolated from defatted
heartwood. Oleoresin obtained from incision of trunk yields essential oil (5.2- 6.8%).
Stem contains yellow coloring matter, napthaquinone and adinin (Asolkar et al., 1992).
The leaves contain ursolic acid and quercetin. It also contains 7-hydroxycoumarin
(umbelliferone), D-glucosylcoumarin (skimmin).
Properties: A. cordifolia is a medium-weight to heavy hardwood with a density of 570895 kg/m cunic at 15% moisture content. Yellow when fresh, turning pale yellow or
reddish-brown on exposure.
Uses:- It has been used in oriental medicine since ancient times as an essential component
of various antiseptic and febrifuge prescriptions (Chopra et al., 2006b). The bark is acrid
and bitter and is used in biliousness. The roots are used as an astringent in dysentery
(Chadha et al., 1985). The A. cordifolia stem has been evaluated for its antiulcer
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potential. It is also used as Febrifuge, Antiseptic, Anti-fertility, Anti-inflammatory, Antirheumatoid, Bitter tonic, Anti-cancer, Anti-microbial.
C) Sida veronicaefolia L
Plant name
:
Sida Veronicaefolia
Family
:
Malvaceae
Common name :
Bengali
: Junka
Hindi
: Bhiunli
Tamil
: Palampasi
Synonyms: Rajbala, Bhumibala, Farid buti,
Shaktibala etc.
Fig 3.3: Leaves of Sida veronicaefolia
Habitat: It is a straggling way side herb found very often growing in shady places. It
grows mainly in clearing in the forest and as weeds in the over grown grass of public
parks and gardens (Lutterodt, 1988b; Warrier et al., 1996).
Chemical constituents: Phenenthylamines, quinazoline, gossypol, sterculic acid, linoleic
acid etc. It has muscarine like active principle (Lutterodt, 1988a; Warrier et al., 1996).
Uses: It has haemostatic, analgesic and wound healing properties. Paste of either root or
leaves is used in bleeding disorders and wounds. Being a nervine and brain tonic, it is
useful loss of memory and vata disorders.Unctuous, laxative. Useful in acid-peptic
disorder and constipation (Warrier et al., 1996). It is effective in cough. dyspnoea,
bronchitis, tuberculosis and hoarseness of voice. Aphrodisiac and useful in semen
debility. Being diuretic, it is used in retention of urine, dysuria and gonorrhea. Useful in
fevers. Being a tonic it is useful in general debility and muscle wasting. Soup of this plant
is taken in the last days of pregnancy. It has a capability to remove the three doshas from
the body, and to provide strength and glow to the body (Lutterodt, 1988a).
D) Literature Review of the Selected Plant
Kazuko Yoshikawa et al (2000) isolated four new oleanane-type triterpene
glycosides, madlongisides A−D, from the seeds of Madhuca longifolia, and their
structures were elucidated on the basis of extensive NMR experiments and chemical
methods.
They also obtained in this investigation, were the known compounds
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mimusopside A, Mi-saponins A, B, and C, and 3-O-β-D-glucopyranosyl protobassic acid
(Kazuko et al., 2000).
R. Ghosh (2009) studied the anti-hyperglycemic activity of madhuca longifolia in
alloxan -induced diabetic rats. The hydroethanolic extract of the leaves of madhuca
longifolia was administered orally to alloxan–induced diabetic rats and investigated for
its antidiabetic properties (Ghosh, 2009). Administration of 150 mg/kg and 300 mg/kg
extract (once a day, for thirty consecutive days) significantly lowered blood glucose
levels. Furthermore, the activity of glucose-6-phosphate dehydrogenase, serum
triglycerides, HDL and total cholesterol levels showed marked improvement which
indicates that the hydroethanolic extract possesses antihyperglycemic activity.
Gaikwad et al (2009) studied the Anti-inflammatory activity of ethanol extract
and saponin mixture of madhuca longifolia using acute (carrageenan-induced
inflammation), sub-acute (formaldehyde-induced inflammation), and chronic (cotton
pellet granuloma) models of inflammation in rats. Saponins alone seem to be responsible
for the anti-inflammatory activity in the studied models. MLEE (Madhuca longifolia
ethanol extract) at a dose level of 10 and 15 mg/kg and Madhuca longifolia saponin
mixture (MLSM) at a dose level of 1.5 and 3 mg/kg significantly reduced the edema
induced by carrageenan in acute model of inflammation, inhibiting both phases of
inflammation. Both the extracts had a more effective response than the reference drug
diclofenac sodium in the sub-acute inflammation model. Results indicated a significant
anti-inflammatory activity by Madhuca longifolia saponins in cotton pellet granuloma
(Gaikwad et al., 2009).
Mangesh Khond et al., (2009) were evaluated Antimicrobial activities of 55
plant extracts against twelve microbial strains using macrobroth dilution assay. Twenty
one extracts exhibited antimicrobial activity against the tested microorganisms in range
of 0.20 to 6.25 mg/ml. Extracts from Madhuca longifolia, Parkia biglandulosa,
Pterospermum acerifolium showed highest antimicrobial potential among the tested
plants (MIC 0.20-12.5 mg/ml). Bio-assays showed presence of multiple specifically
active compounds at different R values in various plant extracts. Acetone and ethanol
extract of M. longifolia, P. biglandulosa; P. acerifolium shows greater antibacterial
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activity as compared to their water extracts and could be the potential source to develop
new antimicrobial agents (Khond et al., 2009).
Akash P. Dahake et al (2010) studied the anti-hyperglycemic effects of
methanolic extract of Madhuca longifolia bark in normal, glucose loaded and
streptozotocin induced diabetic rats. All three animal groups were administered the
methanolic extract of Madhuca longifolia at a dose of 100 and 200 mg/kg body weight
(p.o.) and the standard drug glibenclamide at a dose of 500 μg/kg. Serum glucose level
was determined on days 0, 7, 14 and 21 of treatment. The extract exhibited a dose
dependent hypoglycemic activity in all three animal models as compared with the
standard antidiabetic agent glibenclamide. The hypoglycemia produced by the extract
may be due to the increased glucose uptake at the tissue level and/or an increase in
pancreatic β-cell function, or due to inhibition of intestinal glucose absorption. The study
indicated the methanolic extract of Madhuca longifolia to be a potential antidiabetic
agent, lending scientific support for its use in folk medicine (Dahake et al., 2010a).
Marikkar et al., (2010) characterized the seed fat from Madhuca longifolia
known as Mee fat and its solid and liquid fractions with the objective of distinguishing
them. A sample of Mee fat was partitioned into solid and liquid fractions using acetone as
the solvent medium. The isolated fractions were compared to the native Mee fat sample
with respect to various physico-chemical parameters using standard chemical methods as
well as instrumental techniques such as, gas liquid chromatography (GLC), reversedphase high performance liquid chromatography (RP-HPLC), and differential scanning
calorimetry (DSC). Basic analyses indicated that there were wide variations between the
native sample and its fractions with respect to iodine value (IV), and slip melting point
(SMP). The cloud point (CP) of the liquid fraction was found to be 10.5 degrees C. Fatty
acid compositional analyses showed that the proportion of saturated fatty acids (SFA)
such as palmitic and stearic went up in the high-melting fraction (HMF) while in lowmelting fraction (LMF) the proportion of unsaturated fatty acid (USFA) such as oleic and
lenoleic increased. According to the HPLC analyses, Mee fat had a tiacyl glycerol (TAG)
sequence similar to that of palm oil. After fractionation, the solid and liquid fractions
obtained were found to have TAG profiles very much different from the native sample.
Thermal analyses by DSC showed that Mee fat had two-widely separated high and low
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melting thermal transitions, a feature which was beneficial for the effective separation of
solid and liquid fractions. The thermal profiles displayed by the fractions were clearly
distinguishable from that of the native sample (Marikkar et al., 2010).
Akash P. Dahake et al., (2010) studied the antioxidant activity of the methanolic
extract of the bark of Madhuca longifolia by free radical scavenging activity using 1,1diphenyl-2-picryl-hydrazil (DPPH), reducing power assay and superoxide scavenging
activity. The results of the assay were then compared with a natural antioxidant ascorbic
acid (vitamin C) and gallic acid. The ethanolic extract of the bark of Madhuca longifolia
is a good source of compounds with antioxidant properties while the extract also
exhibited significant free radical scavenging activity, reducing power activity and
superoxide scavenging activity (Dahake et al., 2010b).
Srirangam Prashanth et al., (2010) was explore the antihyperglycemic and
antioxidant potential of ethanolic bark extract of Madhuca longifolia (ML) in healthy,
glucose loaded and streptozotocin induced diabetic rats. All three animal groups were
administered with the ethanolic extract of Madhuca longifolia at a dose of 100 and 200
mg/kg body weight (p.o.) and the standard drug glibenclamide at a dose of 500 μg/kg.
Serum glucose level was determined on days 0, 7, 14 and 21 of treatment. The extract
exhibited a dose dependent hypoglycemic activity in all three animal models as compared
with the standard antidiabetic agent glibenclamide. The antioxidant activity of the bark
was evaluated by free radical scavenging activity using 1, 1-diphenyl-2-picrylhydrazil
(DPPH), reducing power assay and superoxide scavenging activity. The results of the
assay were then compared with a natural antioxidant ascorbic acid (vitamin C). The
hypoglycemia produced by the extract may be due to the increased glucose uptake at the
tissue level and/or an increase in pancreatic β-cell function, or due to inhibition of
intestinal glucose absorption and a good source of compounds with antioxidant
properties. Finally the study indicated the ethanolic extract of Madhuca longifolia to be a
potential antidiabetic and antioxidant properties and the extract also exhibited significant
free radical scavenging activity and superoxide scavenging activity (Srirangam et al.,
2010).
Smita Sharma et al., (2010) studied the wound healing activity of ethanolic
extracts of leaves and bark of Madhuca longifolia .Ethanolic extract of leaves and bark of
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Madhuca longifolia was examined for wound healing potential in the form of 5%w/w
ointment in the excision wound created on the dorsal side of experimental animals, the
5% w/w extract should considerable difference in wound models and the result were
compatable to that of the standard drug Betadine (5% w/w) in terms of wound contracting
ability and wound closure time. Antibacterial activity of ethanolic extract of the plant was
also carried out as a supporting evidence for its wound healing potential. The mean
percentage wound closure was calculated on the 8th, 11th, 13th, 15th and 19th wounding
days. The extract treated animals showed tastes epithelisation of wound (17.86 ± 0.19 and
14.81±0.67) bark and leaves respectively then the control. The period of epithelisation
11.8±037 in case of standard drug 5% betadine ointment (Sharma et al., 2010).
Goutam Kumar Jana et al., (2011) evaluated the Pharmacological Potentials of
methanolic leaf Extract of Madhuca longifolia (Sopteacae) against Pyrexia. They
investigate the antipyretic potential of methanolic extract of Madhuca longifolia leaf in
normal, yeast induced rats. All three groups of animals (n=6) were fasted over night.
Group-I received standard drug, Group-II received vehicle only while Group-III was
administered the methanolic extract of Madhuca longifolia at a dose of 250 mg/kg body
weight (oral) and the standard drug paracetamol at a dose of 30 mg/kg in 0.5% w/v of
SLS. The study indicated the methanolic extract of Madhuca longifolia to be a potential
antipyretic agent, proving its scientific bases for its use in folk medicine (Jana et al.,
2011).
Sabir and Razdan (1970), studied the anti-fertility activity of leaf extract of
Adina cordifolia (Sabir et al., 1970).
Srivatsava and Gupta (1983), isolated a new flavanone from Adina cordifolia
(Srivatsava et al., 1983).
Rao et al., (2002), studied about the isolation and structural elucidation
of 3,4’,5,7 - tetra acetyl quercetin from the heart wood of Adina cordifolia (Rao et al.,
2002).
GD Lutterodt (1988) founds abortifacient properties of an extract from Sida
Veronicaefolia. A fraction from an alcoholic extract of Sida veronicaefolia, previously
reported to be a potent oxytocic, was studied for its abortifacient effects in pregnant rats.
Oral doses producing the abortifacient effects were greater than or equal to 32 ml/kg
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when administered from the 15th-17th day of pregnancy. Similar effects were produced by
intravenous doses of greater than or equal to 3 ml/kg. At the minimum effective oral dose
of 32 ml/kg, those animals that carried the conceptuses to term (40%) had litters with
reduced average number/litter and weight. At twice this dose, only 10% delivered and the
litters were sickly. The effects of intravenous administration of the extract were similar
but more pronounced and included also some unique acute effects (Lutterodt, 1988b).
Manisha Pandey et al., (2009) founds the Sida Veronicaefolia as a Source of
Natural Antioxidant The antioxidant activity of hexane, chloroform, hydro-alcoholic and
aqueous extract of whole plant of Sida veronicaefolia (family Malvaceae) was evaluated
using in-vitro models, DPPH free radical scavenging,scavenging of hydrogen peroxide
and reducing power method (Pandey, 2009).
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4. PLAN OF WORK
 Selection of the plants.
 Procurement and authentication of the plants.
 Extraction of plant material with different solvents in their increasing order of
polarity.
 Preliminary phytochemical studies of the plant extracts.
 Carrying out the In-vitro cytotoxicity studies of the extracts of selected plants.
 Carrying out the acute toxicological studies of the plants taken under consideration.
 Carrying out the pharmacological studies on the selected extracts of medicinal plants
for its anticancer activity.
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5. MATERIALS AND METHODS
5.1 Selection of the plant
The present study is to evaluate the anticancer activity of M. longifolia, A.
cordifolia and S. veronicaefolia based on the literature review and discussion with the
traditional medical practitioners of the Ujjain, Bhanpura and Bhopal (M.P.), India.
5.2 Collection and authentification of the plant
The Leaves of M. longifolia, A. cordifolia and S. veronicaefolia were collected
from National Botanical Research Institute, Lucknow and Sanjivini Botanical Garden,
Bhopal, India in month of June-July 2009. The plant materials were authenticated by Dr.
Sayeeda Khatoon, chemotaxonomist and the voucher specimens were deposited in the
departmental herbarium of TIT - Pharmacy for future reference (TIT-PY/HREB/2009/1416).
5.3 Preparation of crude drug for extraction
The selected plant leaves were used for the preparation of the extract. The plants
leaves were collected and dried under shade and then coarsely powdered with the help of
mechanical grinder. The powder was passed through sieve No. 16 and stored in an
airtight container for the extraction (Farnsworth et al., 1966).
5.4 Physico-chemical evaluation
The dried and stored powder of plant leaves were subjected to standard procedure
for the determination of various physicochemical parameters
A) Determination of ash values
The determination of ash values is meant for detecting low-grade products,
exhausted drugs and sandy or earthy matter. It can also be utilized as a mean of detecting
the chemical constituents by making use of water-soluble ash and acid insoluble ash
(Khandelwal, 2004).
1) Total ash value
Accurately about 3 gm of air dried powder of plants leaves were weighed in a
tared silica crucible and incinerated at a temperature not exceeding 4500C until free from
carbon, cooled and weighed and then the percentage of total ash with reference to the air
dried powdered drug was calculated (Khandelwal, 2004).
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2) Acid insoluble ash
The ashes obtained in the above method were boiled for 5 minutes with 25ml of
dilute HCl. The residue was collected on ash less filter paper and washed with hot water,
ignited and weighed. The percentage of acid insoluble ash was calculated with reference
to the air dried drug (Khandelwal, 2004).
3) Water soluble ash
The ash obtained in total ash was boiled for 5 minutes with 25 ml of water. The
insoluble matter was collected on an ash less filter paper, washed with hot water and
ignited to constant weight at a low temperature. The weight of insoluble matter was
subtracted from the weight of the ash. The difference in weights represents the water
soluble ash. The percentage of water soluble ash with reference to the air dried drug was
calculated (Khandelwal, 2004).
B) Determination of extractive values
1) Procedure
5 gm of coarsely powdered air dried drug was macerated with 100 ml of solvent
(petroleum ether, chloroform, acetone, ethanol and water) in a closed flask for 24 hour,
shaking frequently for six hours and allowed to stand for eighteen hours.
It was then filtered rapidly taking precaution against loss of alcohol. 25 ml of the
filtrate was evaporated to dryness in tared flat bottomed shallow dish, dried at 1050c and
weighed.
The percentage of alcohol soluble extractive was calculated with reference to the
air dried drug (Khandelwal, 2004).
5.6 Extraction of dried leaves by using various solvents of increasing polarity
The collected, cleaned and powdered leaves of Madhuca longifolia, Adina
cordifolia and Sida veronicaefolia were used for the extraction purpose. 500 gm of
powdered material was evenly packed in the soxhlet apparatus. It was then extracted with
various solvents from non-polar to polar such as petroleum ether, chloroform, acetone
and ethanol. The solvents used were purified before use. The extraction method used was
continuous hot percolation and carried out with various solvents, for 72 hrs. The aqueous
extraction was carried out by cold-maceration process. The extracts were concentrated by
vacuum distillation to reduce the volume to 1/10; the concentrated extracts were
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transferred to 100ml beaker and the remaining solvent was evaporated on a water bath.
Then they were cooled and placed in a dessicator to remove the excessive moisture. The
dried extracts were packed in airtight containers and used for further studies (Kokate et
al., 2008).
5.8 Preliminary phytochemical studies (Gothoskar et al., 1971; Kokate et al., 2008;
Khandelwal, 2004)
A) Tests for Carbohydrates and Glycosides
A small quantity of the extracts was dissolved separately in 4 ml of distilled water
and filtered.
The filtrate was subjected to various tests to detect the presence of
Carbohydrates.
1) Molisch’s test
Filtrate was treated with 2-3 drops of 1% alcoholic  - napthol solution and 2 ml
of Con sulpuric acid was added along the sides of the test tube. Appearance of brown
ring at the junction of two liquids shows the presence of carbohydrates.
Another portion of the extract was hydrolysed with hydrochloric acid for few
hours on a water bath and the hydrolysate was subjected to Legal’s and Borntrager’s test
to detect the presence of different glycosides.
2) Legal’s test
To the hydrolysate 1 ml of pyridine and few drops of sodium nitropruside
solutions were added and then it was made alkaline with sodiumhydroxide solution.
Appearance of pink to red colour shows the presence of glycosides.
3) Borntrager’s test
Hydrolysate was treated with chloroform and then the chloroform layer was
separated. To this equal quantity of dilute ammonia solution was added. Ammonia layer
acquires pink color, showing the presence of glycosides.
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B) Test for Alkaloids
A small potion of the solvent free alcoholic and aqueous extracts were stirred
separately with few drops of dilute hydrochloric acid and filtered. The filtrate was tested
with various reagents for the presence of alkaloids.
1) Dragondorff’s test
To a small amount of the filtrate, add 1ml of Dragendorff’s reagent. Appearance
of reddish brown precipitate indicates the presence of alkaloids.
2) Wagner’s test
To a small amount of filtrate, add 1ml of Wagner’s reagent. Appearance of
reddish brown precipitate indicates the presence of alkaloids
3) Mayer’s reagent
To a small amount of filtrate, add 1ml of Mayer’s reagent. Appearance of cream
coloured precipitate indicates the presence of alkaloids
C) Test for Proteins and Free Amino Acids
1) Million’s test
Small quantities of the extracts were dissolved in few ml of water and treated with
Millon’s reagent. Appearance of red color shows the presence of proteins and free amino
acids.
2) Ninhydrin test
Small quantities of the extracts were dissolved in few ml of water and treated with
Ninhydrin reagent. Appearance of violet color shows the presence of proteins and free
amino acid.
3) Biuret’s test
The extracts were dissolved in a few ml of water and equal volumes of 5%
sodium hydroxide solution & 1% copper sulphate solution were added. Appearance of
pink or purple color shows the presence of proteins and amino acids.
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D) Test for Phenolic Compounds and Tannins
1) Ferric chloride test
Small quantities of the extracts were dissolved in water and dilute Ferric chloride
solution (5%) was added. Appearance of violet or blue color indicates presence of
phenolic compounds and tannins.
2) Gelatin test
Small quantities of the extracts were dissolved in water and 1% solution of gelatin
containing 10% sodium chloride was added. Formation of white precipitate indicates
presence of phenolic compounds and tannins.
3) Lead acetate test
Small quantities of the extracts were dissolved in water and 10% lead acetate
solution was added. Formation of white precipitate indicates presence of phenolic
compounds and tannins.
E) Test for Flavonoids
1) Sodium hydroxide test
Small quantities of each extracts were dissolved separately in aqueous sodium
hydroxide solution. Appearance of yellow to orange indicates presence of flavonoids.
2) Sulphuric acid test
To a portion of the extract, add Conc. sulphuric acid. Appearance of yellow
orange colour shows the presence of flavonoids.
3) Shinoda’s test
Small quantities of the extract were dissolved in alcohol, to them piece of
magnesium followed by Conc. hydrochloric acid dropwise added and heated.
Appearance of magenta color shows the presence of flavonoids
F) Test for Saponins
1) Foam Test
Place 2ml of the solution of the extract in water in a test tube and shake well.
Formation of stable foam (froth) indicates the presence of Saponins.
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G) Test for Fixed oils and Fats
1) Spot test
Small quantities of various extracts were separately pressed between two filter
papers. Appearance of oil stain on the paper indicates the presence of fixed oils and fats.
2) Saponification test
Add few drops of 0.5N alcoholic potassium hydroxide to a small quantity of the
extract and heat on a water bath for 1-2 hrs. Formation of soap or partial neutralization of
tha alkali shows the presence of fixed oils and fats.
H) Test for Phytosterols
Small quantities of various extracts were dissolved separately in 5ml of water.
Then this solution was subjected to the following tests.
1) Salkowski test
The solution was treated with few drops of conc.sulphuric acid. Formation of red
colour indicates presence of phytosterols.
2) Libermann- Bucchard’s test
The solution was treated with few drops of acetic anhydride, boil and cool. Then
add conc. Sulphuric acid through the sides of the test tube. Formation of brown ring at the
junction of two layers indicates the presence of phytosterols.
I) Test for Gums and Mucilage
1) Alcohol test
A little of the extract is treated with alcohol. If it is not soluble in alcohol it shows
presence of gums and mucilage.
2) Precipitation test
The extract solution was added to picric acid solution. Formation of yellow
precipitate shows the presence of gums and mucilage.
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5.9 Pharmacological Evaluation
A) In-vitro cytotoxicity study
1) Cell Line
Elrish Acectic Carcinoma cell line was obtained through the courtesy of Amala
Cancer Research Center, Thrissur and maintained at Pharmacology Department, TITPharmacy, Bhopal in Dulbecco’s Modified egale medium (DMEM) at 37◦C and 5% CO2
using standard cell culture methods.
2) Maintenance of cell line
The maintenance of cell line was involved in the following stages (Borenfreund et
al., 1984):
a) Preparation of cell medium
i) Ingredients
DMEM
10gm
Sodium bi carbonate
2.2gm
HEPES
10ml
Antibiotics
10ml
FBS
100ml
Autoclaved water to make the volume up to 1 lit.
ii) Method of Preparation
10ml of HEPES was added to 850 ml of autoclaved water and mixed well.Then
DMEM and Sodium bi carbonate was dissolved. Finally 10 ml antibiotics and 100 ml FBS
was added in the mediumand volume was made up to 1 litre with autoclaved distilled water.
The medium was filter twice and stored at 40 C
b) Passaging of cell line
Cell passaging or splitting was a technique that enables an individual to keep cells
alive and growing under cultured conditions for extended periods of time. Cells should be
passaged when they were 90%-100% confluent (Borenfreund et al., 1984).
Hands were washed with ethanol and hood was clean with ethanol. T flasks were
removed from the incubator and were seeing under microscope to confirm that the cells were
90%-100% confluent. Media and Trypsin were warmed in 37C water bath. Then culture
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media was removed from T flasks and T- flasks were washed with PBS twice to remove the
dead cells. Then 4 ml trypsin was added to T-flask. Then cells were checked under
microscope to confirm that cells were detached from the surface. Finally culture media was
added to trypsinised cell suspension and divided into 2 or more flasks depend on the number
of cells. Medium was changed after every 24 hrs, until the T flask become confluent.
3) Seeding of cells
Hands were washed with ethanol and hood was clean with ethanol. T flasks were
removed from the incubator and were seeing under microscope to confirm that the cells were
90%-100% confluent. Media and Trypsin were warmed in 37C water bath. Then culture
media was removed from T flasks and T- flasks were washed with PBS twice to remove the
dead cells. Then 4 ml trypsin was added to T-flask for detachedment. Cells were counted by
using hemocytometer. Then cell suspention was dilute with culture medium so that each
100µl of diluted cell suspention contained 2500 to 5000 cells. Then cells were seeded in 96
well plate, each well was contained 100µl of cell suspention. These plates were incubated for
24, 48 & 72 hrs respectively in CO
2
incubator. Confluent seeded plates were used for
screening of drug (Borenfreund et al., 1984).
4) Preparation of extract solution
2g powdered extracts were dissolved in 100 ml DMSO, to got the stock solution with
concentration 20mg/ml. Then 10 ml of extract solution was taken and dissolved in 90 ml
culture media, the final concentration is 2mg /ml (Borenfreund et al., 1984).
5) Treatment with extract solution
Seeded cell plates were taken out from the incubator, and culture media was
discarded from the plates. Then culture media was replaced by the 100µl of extract
containing culture media. Then the plates were incubated in CO2 incubator for 24 hrs for the
drug action. After 24 hrs the plates were taken out from the incubator and activity of drug
was evaluated by different cytotoxic assay (Borenfreund et al., 1984).
6) In-vitro Cytotoxic Assays
These were the following cytotoxic assays which were used to evaluate the
cytotoxicity of extracts to the cancer cells.
Page 47 of 113
a) MTT Assay
MTT assay was standard colorimetric assay, which measures changes in color, for
measuring the activity of enzymes that reduce MTT to formazan, giving a purple color
(Mosmann, 1983). This mostly happens in mitochondria of living cells, and as such it is in
large a measure of mitochondrial activity. It can also be used to determine cytotoxicity of
potential medicinal agents and other toxic materials.
A solubilization solution usually either dimethyl sulfoxide, an acidified ethanol
solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid is
added to dissolve the insoluble purple formazan product into a colored solution. The
absorbance of this colored solution can be quantified by measuring at a certain wavelength
usually between 500 and 600 nm by a spectrophotometer. The absorption maximum is
dependent on the solvent employed.
This reduction takes place only when mitochondrial reductase enzymes were active,
and therefore conversion is often used as a measure of viable (living) cells. When the amount
of purple formazan produced by cells treated with an agent is compared with the amount of
formazan produced by untreated control cells, the effectiveness of the agent in causing death,
or changing metabolism of cells, can be deduced through the production of a dose-response
curve (Mosmann, 1983).
i) Procedure
25mg of MTT powder was dissolved in 5ml PBS then filtered it with the help of
10ml syringe and syringe filter. Incubated cell plates were taken out from the incubator, and
discard the culture media from the plates. Culture media was replaced by the extract
containing culture media. Then the plates were incubated in CO2 incubator for 24 hrs for the
action of extracts. 5 hours before the end of the incubation, add 20µl of MTT solution to each
well containing cells. Incubate the plate at 37ºC for 5 hours. Remove media and add 200µl of
DMSO to each well and pipette up and down to dissolve crystals. Transfer to plate ELISA
reader and measure absorbance at 550nm to get optical density. Then calculate the %
inhibition using the formula
% inhibition = [(OD of untreated)-(OD of drug Treated) /(OD of untreated)] 100
OD:- Optical Density.
Page 48 of 113
b) Neutral red uptake cytotoxicity assay
The neutral red (NR) cytotoxicity assay procedure is a cell survival/viability
chemosensitivity assay, based on the ability of viable cells to incorporate and bind neutral
red, a supravital dye. NR is a weak cationic dye that readily penetrates cell membranes by
non-ionic diffusion, accumulating intracellularly in lysosomes, where it binds with anionic
sites in the lysosomal matrix. Alterations of the cell surface or the sensitive lysosomal
membrane lead to lysosomal fragility and other changes that gradually become irreversible.
Such changes brought about by the action of xenobiotics result in a decreased uptake and
binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells,
which are the basis of this assay (Triglia, 1991; Morgan, 1991; Fautz, 1991).
The neutral red uptake assay provides a quantitative estimation of the number of
viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical
and environmental applications. Most primary cells and cell lines from diverse origin may be
successfully used.
i) Preparation of NR stock solution
NR dye (3.3gm) was dissolved in 100 ml of double distilled water and then this stock
solution was filtered by using syringe filter. It was stored at room temperature and used
within 6 months.
ii) Preparation of working solution
1 ml of NR stock solution was dissolved in the 99 ml of culture media to got the final
concentration 0.33%.
iii) Procedure
Incubated cell plates were taken out from the incubator, and discard the culture media
from the plates. Culture media was replaced by the extract containing culture media. Then
the plates were incubated in CO2 incubator for 24 hrs for the action of extracts. The extract
containing culture media was then replaced with NR-containing medium. Plates were again
placed to incubator for 4-8 hours depending on cell type and maximum cell density. At the
end of the incubation period, the medium was carefully removed and the cells were quickly
washed with PBS. The washed solution was removed and the incorporated dye was then
solubilized in a volume of Neutral Red Assay Solubilization Solution (ethanolic acetic acid)
Page 49 of 113
equal to the original volume of culture medium. The plates were allowed to stand for 10
minutes at room temperature. Gentle stirring in a gyratory shaker or pipetting up and down
(trituration) enhanced mixing of the solubilized dye. The background absorbance was
measured at 540 nm using ELISA reader to get optical density and pictures were captured
using microscope. Then calculate the % inhibition using the formula
% inhibition = [(OD of untreated)-(OD of drug Treated) /(OD of untreated)] 100
OD:- Optical Density.
7) Statistical Analysis
The results of the study were expressed as mean ± SEM. ANOVA (Gennaro et
al., 1995) was used to analyze and compare the data, followed by Dunnet’s (Dunnet et
al., 1964) test for multiple comparisons.The value of probability less than 5% (P < 0.05)
was considered statistically significant (Amin et al., 2006).
B) Acute toxicity studies
Organization for Economic co-operation and Development (OECD) regulates
guideline for oral acute toxicity study. It is an international organization which works
with the aim of reducing both the number of animals and the level of pain associated with
acute toxicity testing (OECD, 1996)
Following are the main type of guideline followed by OECD

Guideline 420, fixed dose procedure. ( 5 animals used )

Guideline 423, acute toxic class. ( 3 animals used )

Guideline 425, up and. down method. (1 animal used)
1) Guideline 423
a) Principle
Acute toxic category method is a method for assessing acute oral toxicity that
involves the identification of a dose level that causes mortality.
This test involves the administration of a simple bolus dose of test substances to
fasten healthy young adult rodents by oral gavage, observation for upto 15days after
dosing and recording of body weight and the necropsy of all the animals. In this method
pre-specified fixed doses of the test substances were used ie., 5mg/kg, 50mg/kg,
300mg/kg, 2000mg/kg and the mortality due to these doses were observed. Generally
Page 50 of 113
female animals were used for this study and each dose group should consist of 3 animals.
b) Animals
Female Wistar albino rats (150-200 g) of approximately the same age, was
procured from Central Drug Research Institute, Lucknow, and were used for acute
toxicity studies. They were housed in polypropylene cages and fed with standard rodent
pellet diet (Hindustan Lever Limited, Bangalore) and water ad libitum. The rats were
exposed to alternate cycle of 12hrs of darkness and light each. Before the test, the rats
were fasted for at least 12 hrs; the experimental protocols were subjected to the
scrutinization of the Institutional Animals Ethical Committee and were cleared by the
same. All experiments were performed during morning according to CPCSEA guidelines
(CPCSEA, 2003) for care of laboratory animals and the ethical guideline for
investigations of experimental pain in conscious animals. The standard orogastric cannula
was used for oral drug administration in rats.
c) Procedure
Fig. 5.1 Protocol for determining acute toxicity in rats
 The overnight fasted female rats were weighed and selected.
 Acetone and Ethanol extracts were dosed in a stepwise procedure, with the initial
dose being selected as the dose expected to produce some signs of toxicity and
Page 51 of 113
were observed for a period of two weeks.
 The toxic doses were selected based on the above chat.
C) In-vivo Anti-cancer Activity
1) Animals
Swiss Albino mice (20-25gm) of either sex and of approximately the same age,
procured from Institute of Animal Health and Vetarnary Biological, Mhow, Indore,
Madhya Pradesh, and were used for In-vivo anticancer study. They were housed in
polypropylene cages and fed with standard rodent pellet diet (Hindustan Lever Limited,
Bangalore) and water ad libitum. The animals were exposed to alternate cycle of 12hrs of
darkness and light each. Before each test, the animals were fasted for at least 12 hrs; the
experimental protocols were subjected to the scrutinization of the Institutional Animals
Ethical Committee and were cleared by the same. All experiments were performed during
morning according to CPCSEA guidelines (CPCSEA, 2003) for care of laboratory
animals and the ethical guideline for investigations of experimental pain in conscious
animals. The standard orogastric cannula was used for oral drug administration in
experimental animals.
2) Sources of cell line
EAC cell line was obtained from Amala Cancer and Research Institute, Thrissur,
Kerala and were maintaind by weekly intraperitoneal inoculation of 1×106 cells/mouse.
3) Experimental Design
The animals (Swiss albino mice weighing 20-25 g) were divided into 9 groups
consisting of 12 animals in each. Animals were fed with basal diet and water throughout
the experimental period. All the groups were injected with EAC cells except control
group. From day 1st, normal saline (5 ml/kg) was given in group I and group II, which
was serve as a normal control and tumor control group respectively, whereas 5fluorouracil (20mg/kg) was given to group III. All other groups (Group IV to IX) were
treated with selected plant extract as given below for 14 days. On 15th day six mice from
each group were sacrificed for the determination of tumor volume, tumor weight,
hematological parameters, etc, and rest were kept with food and water ad libitum to check
Page 52 of 113
the increase in the life span of the tumor hosts and body weight (Kuttan et al., 1990;
Mazumder et al., 1997).
Group I
:
Control animals were received normal saline.
Group II
:
Mice were inoculated with 1×106 cells per mouse intraperitoneally.
Group III
:
Mice were injected 5-fluorouracil (20mg/kg) intraperitoneally
along with EAC cells (1×106cells/mouse) treatment.
Group IV
:
Mice were treated with AEML (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
Group V
:
Mice were treated with EEML (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
Group VI
:
Mice were treated with AEAC (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
Group VII
:
Mice were treated with EEAC (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
Group VIII
:
Mice were treated with AESV (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
Group IX
:
Mice were treated with EESV (500mg/kg) orally along with EAC
cells (1×106cells/mouse) treatment.
a) Effect of selected plant extracts on tumor volume and tumor weight of
tumor bearing mice
On 15th day, after 24h of dose, 6 mice from each group were dissected and the
ascetic fluid was collected from peritoneal cavity. The volume was measured by taking it
in a graduated centrifuge tube. The tumor weight was measured by taking the weight of
mice before and after collection of ascetic fluidfrom peritoneal cavity (Kuttan et al.,
1990; Mazumder et al., 1997).
b) Effect of selected plant extracts on tumor cell count of tumor bearing mice
The ascetic fluid was withdrawn from the peritoneal cavity of the mice and
diluted 100 times with normal saline. A drop of a diluted cell suspension was placed on
the neubauers chamber and the number of cells in the 64 square was counted. The
viability and non viability of cells was checked by tryphan blue method. On staining
Page 53 of 113
viable cells did not take the dye whereas the non viable cells were stained blue (Kuttan et
al., 1990; Mazumder et al., 1997).
c) Effect of selected plant extracts on Mean survival time of tumor bearing
mice
Animals were inoculated with 1 X 106 cells/mouse on day ‘0’ and treatment with
all extracts started 24 h after inoculation, at a dose of 500 mg/kg/day p.o. The control
group was treated with the same volume of 0.9% sodium chloride solution. All the
treatments were given for 14 days. The mean survival time (MST) of each group,
consisting of 6 mice was noted. The antitumor efficacy of acetone and ethanol extract
was compared with that of 5- fluorouracil (Dabur Pharmaceutical Ltd, India). The MST
of the treated groups was compared with that of the control group using the following
calculation ((Kuttan et al., 1990; Mazumder et al., 1997)):
ILS (%) = [(MSTof treated group/ MST of control group)-1] x 100
Mean survival time = [1st Death + Last Death] / 2
d) Effect of selected extracts on body weight of tumor bearing mice
All groups (except Group I and III), consisting of six mice each were transplanted
intraperitoneally with 1×106 EAC cells. After 24 h, the groups were orally treated
Extracts. The group II, serving as the control, received normal saline (0.9%w/v).
Treatments were continued for 14 days. Body weights were recorded every 5th day till 40
days of treatment or till the death of the animal (Kuttan et al., 1990; Mazumder et al.,
1997).
e) Effect of selected plant extracts heamatological parameters of tumor
bearing mice
All the treatments were given for 14 days to each group (except group III), on the
15th day, blood was drawn by retro orbital plexus method. WBC count, RBC count,
heamoglobin, protein and packed cell volume were determined (D’Amour et al., 1965;
Lowry et al., 1951). Cells smear was prepared in slide and stained with Lieshman stain
solution (Docie et al., 1958).
Red blood cells (RBC), White blood cells (WBC) and Heamoglobin (Hb) were
estimated with the help of MS-09 heamatology analyzer (France).
Page 54 of 113
f) Effect of selected extracts on peritoneal cells in normal mice
Thirteen groups of normal mice (n= 6) were used for the study. First six groups
were treated with 500 mg/kg, p.o. of acetone and ethanol extracts only once for a single
day and other six groups received the same treatment for two consecutive days. The
untreated group was used as control. Peritoneal exudates of ethanolic and Acetone extract
groups were collected after 24hr and 48hr of treatment by repeated intraperitoneal wash
with normal saline (0.9% w/v) and the cells were counted in each of the treated groups
under WBC newbauer’s chamber and compared with those of normal control (Sur et al.,
1994).
i) Experimental deign
Group I
:
Animals were treated with normal saline.
Group II
:
Animals were treated with AEML (500mg/kg) for single
day.
Group III
:
Animals were treated with AEML (500mg/kg) for two
consecutive days.
Group IV
:
Animals were treated with EEML (500mg/kg) for single
day.
Group V
:
Animals were treated with EEML (500mg/kg) for two
consecutive days.
Group VI
:
Animals were treated with AEAC (500mg/kg) for single
days.
Group VII
:
Animals were treated with AEAC (500mg/kg) for two
consecutive days.
Group VIII
:
Animals were treated with EEAC (500mg/kg) for single
days.
Group IX
:
Animals were treated with EEAC (500mg/kg) for two
consecutive days.
Group X
:
Animals were treated with AESV (500mg/kg) for single
days.
Page 55 of 113
Group XI
:
Animals were treated with AESV (500mg/kg) for two
consecutive days.
Group XII
:
Animals were treated with EESV (500mg/kg) for single
days.
Group XIII
:
Animals were treated with EESV (500mg/kg) for two
consecutive days.
g) Statistical analysis
The results of the study were expressed as mean ± SEM. ANOVA (Gennaro et
al., 1995) was used to analyze and compare the data, followed by Dunnet’s (Dunnet et
al., 1964) test for multiple comparisons.The value of probability less than 5% (P < 0.05)
was considered statistically significant (Amin et al., 2006).
Page 56 of 113
6. RESULTS AND DISCUSSION
6.1 Selection of the plant
On the basis of literature review and discussion with the traditional medical
practitioners of the Ujjain, Bhanpura and Bhopal (MP), India, M. longifolia, A. cordifolia
and S. veronicaefolia were selected for evaluation of the anticancer activity.
6.2 Physicochemical analysis of crude drug
A) Determination of Ash Values of selected plants
The physicochemical analysis of powdered leaves of selected plants were carried
out i.e, total ash, acid insoluble ash and water soluble ash, were determined.
The total ash value were found to be 10.5%, 8.1% and 9.1% w/w for leaves of M.
longifolia, A. cordifolia and S. veronicaefolia respectively, was indicating that
considerable amount of inorganic matter were present.
The acid insoluble ash values were found to be 3.7 %, 3.5% and 3.3% w/w for
leaves of M. longifolia, A. cordifolia and S. veronicaefolia respectively.
The water soluble ash values were found out to be 1.7 %, 1.3% and 1.5% w/w for
leaves of M. longifolia, A. cordifolia and S. veronicaefolia respectively. The results were
represented in Table 6.1
Table 6.1: Determination of ash values of selected medicinal plants
Plant Name
Madhuca longifolia
Adina cordifolia
Sida veronicaefolia
Types of Ash
Percentage of
Ash(w/w)
Total ash
10.5
Acid insoluble
3.7
Water soluble
1.7
Total ash
8.1
Acid Insoluble
3.5
Water soluble
1.3
Total ash
9.1
Acid Insoluble
3.3
Water soluble
1.5
Page 57 of 113
B) Determination of extractive value of selected medicinal plants
Extractive values were determined and reported in Table 6.2
Table 6.2: Determination of extractive value of selected plants
% Yeild
Solvent
used
M. longifolia
A. cordifolia
S. veronicaefolia
Pet. Ether
1.67
2.13
1.89
Chloroform
2.23
1.54
3.86
Acetone
17.1
15.5
14.2
Ethanol
9.21
14.8
9.6
Water
10.2
19.2
13.7
Page 58 of 113
6.3 Preliminary phytochemical evaluation of selected plants
The phytoconstituents were determined by chemical tests, which showes the
presence of various constituents in different extracts. The results showed that extracts of
leaves of M. longifolia, A. cordifolia and S. veronicaefolia contains flavonoids, alkaloids,
phytosterols and phenolic compounds and were reported in Table 6.3, 6.4 and 6.5.
Page 59 of 113
Table 6.3: Preliminary phytochemical evaluation of leaves of M. longifolia.
Tests
Pet.ether
extract
CHCl3
extract
Molisch’s test
-
-
-
+
+
Fehling’s test
-
-
-
+
+
Legal’s test
Borntrager’s
test
-
-
-
-
-
-
-
-
-
-
Baljet test
-
-
-
-
-
Spot test
Saponification
test
+
+
-
-
-
+
+
-
-
-
Millon’s test
-
+
-
+
+
Proteins and
Amino Acids
Ninhydrin test
-
+
-
+
+
Biuret test
-
+
-
+
+
Saponins
Foam test
FeCl3 test
-
-
+
-
+
-
+
-
Lead acetate
test
-
-
-
-
-
Salkowski test
+
-
+
+
+
+
-
+
+
+
-
-
+
+
+
-
-
+
+
+
Wagner’s test
-
-
+
+
+
Hager’s test
Froth test
+
-
+
-
+
-
+
+
Alcoholic test
+
-
-
-
+
-
-
+
+
+
-
-
+
+
+
-
-
+
+
+
Constituents
Carbohydrate
Glycosides
Fixed oil and
Fats
Phenolic
Comp. and
Tannins
Phytosterols
Alkaloids
Gums and
Mucilage
Flavonoids
LibermannBucchard test
Dragendorff’s
test
Mayer’s test
Lead acetate
test
Con. H2SO4
test
FeCl3 test
Acetone Ethanolic Aqueous
extract
extract
extract
Page 60 of 113
Table 6.4: Preliminary phytochemical evaluation of leaves of A. cordifolia
Constituents
Tests
Pet.ether CHCl3 Acetone Ethanolic Aqueous
extract Extract extract
extract
extract
Molisch’s test
-
-
-
+
+
Fehling’s test
-
-
-
+
+
Legal’s test
Borntrager’s
test
-
-
-
-
-
-
-
-
-
-
Baljet test
-
-
-
-
-
+
+
+
+
-
+
+
+
+
-
Millon’s test
-
+
-
+
+
Proteins and
Amino Acids
Ninhydrin test
-
+
-
+
+
Biuret test
-
+
-
+
+
Saponins
Foam test
FeCl3 test
-
-
+
+
+
+
+
Lead acetate
test
-
-
+
+
+
Salkowski test
+
-
+
+
+
+
-
+
+
+
-
-
-
-
-
-
-
-
-
-
Wagner’s test
-
-
-
-
-
Hager’s test
Froth test
+
-
-
-
+
Alcoholic test
+
-
-
-
+
-
-
+
+
+
-
-
+
+
+
-
-
+
+
+
Carbohydrate
Glycosides
Spot test
Fixed oil and
Saponification
Fats
test
Phenolic
Comp. and
Tannins
Phytosterols
Alkaloids
Gums and
Mucilage
Flavonoids
LibermannBucchard test
Dragendorff’s
test
Mayer’s test
Lead acetate
test
Con. H2SO4
test
FeCl3 test
Page 61 of 113
Table 6.5: Preliminary phytochemical evaluation of leaves of S. veronicaefolia
Constituents
Tests
Pet.ether CHCl3
extract Extract
Acetone Ethanolic Aqueous
extract
extract
extract
Molisch’s test
-
-
-
+
+
Fehling’s test
-
-
-
+
+
Legal’s test
Borntrager’s
test
-
-
-
-
-
-
-
-
-
-
Baljet test
-
-
-
-
-
+
+
-
+
-
+
+
-
+
-
Millon’s test
-
+
-
+
+
Proteins and
Amino Acids
Ninhydrin test
-
+
-
+
+
Biuret test
-
+
-
+
+
Saponins
Foam test
FeCl3 test
-
-
+
+
+
Lead acetate
test
-
-
+
+
+
Salkowski test
+
-
+
+
+
+
-
+
+
+
-
-
+
+
+
-
-
+
+
-
Wagner’s test
-
-
+
+
-
Hager’s test
Froth test
+
-
+
-
+
-
+
Alcoholic test
+
-
-
-
+
-
-
+
+
+
-
-
+
+
+
-
-
+
+
+
Carbohydrate
Glycosides
Spot test
Fixed oil and
Saponification
Fats
test
Phenolic
Comp. and
Tannins
Phytosterols
Alkaloids
Gums and
Mucilage
Flavonoids
LibermannBucchard test
Dragendorff’s
test
Mayer’s test
Lead acetate
test
Con. H2SO4
test
FeCl3 test
Page 62 of 113
6.4 In-vitro cytotoxic studies
A) In-vitro cytotoxic activity of extracts of M. longifolia by MTT assay
The result showed that % inhibition of PEML, CEML, AEML, EEML and
AQEML were 7.06 ± 0.81, 13.72 ± 3.16, 80.0 ± 2.28, 82.0 ± 3.12 and 20.86 ± 3.59
respectively and were reported in Table 6.6 and Fig 6.1.
Table 6.6: In-vitro cytotoxic activity of extracts of M. longifolia by MTT assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3660
0.00 ± 1.31
2
PEML
200 µg/ml
0.3401
7.06 ± 0.81ns
3
CEML
200 µg/ml
0.3157
13.72 ± 3.16*
4
ACML
200 µg/ml
0.0732
80.0 ± 2.28**
5
EEML
200 µg/ml
0.0658
82.0 ± 3.12**
6
AQEML
200 µg/ml
0.2896
20.86 ± 3.59**
8 wells /group OD at 550 nm,
*P<0. 01 Vs control,
**P<0.001 Vs control.
Values are expressed as mean ± SEM
Fig 6.1: In-vitro cytotoxic activity of extract of M. longifolia by MTT assay
Page 63 of 113
B) In-vitro cytotoxic activity of extracts of A. cordifolia by MTT assay
The result showed that % inhibition of PEAC, CEAC, AEAC, EEAC and
AQEAC were 19.56 ± 3.16, 19.94 ±1.31, 91.34 ± 4.56, 89.11 ± 2.97 and 14.03.± 2.74
respectively and were reported in Table 6.7 and Fig 6.2
Table 6.7: In-vitro cytotoxic activity of extracts of A.cordifolia by MTT assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3660
0.00 ± 1.31
2
PEAC
200 µg/ml
0..2943
19.56 ± 3.16**
3
CEAC
200 µg/ml
0..2930
19.94 ±1.31**
4
AEAC
200 µg/ml
0.0317
91.34 ± 4.56**
5
EEAC
200 µg/ml
0.0399
89.11 ± 2.97**
6
AQEAC
200 µg/ml
0.3146
14.03.± 2.74*
8 wells /group OD at 550 nm,
*P<0. 01 Vs control,
**P<0.001 Vs control.
Values are expressed as mean ± SEM
Fig 6.2: In-vitro cytotoxic activity of extracts of A. cordifolia by MTT assay
Page 64 of 113
C) In-vitro cytotoxic activity of extracts of S. veronicaefolia by MTT assay
The result showed that % inhibition of PESV, CESV, AESV, EESV and AQESV
were 14.48 ± 5.74, 13.04 ± 3.42, 93.83 ± 3.48, 95.71 ± 3.45 and 19.94 ± 1.74 respectively
and were reported in Table 6.8 and Fig 6.3
Table 6.8: In-vitro cytotoxic activity of extracts of S. veronicaefolia by MTT
assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3660
0.00 ± 1.31
2
PESV
200 µg/ml
0..3130
14.48 ± 5.74*
3
CESV
200 µg/ml
0,3182
13.04 ± 3.42*
4
AESV
200 µg/ml
0.0226
93.83 ± 3.48***
5
EESV
200 µg/ml
0.0157
95.71 ± 3.45***
6
AQESV
200 µg/ml
0..2930
19.94 ± 1.74**
8 wells /group OD at 550 nm,
*P<0. 05 Vs control,
**P<0.01 Vs control.
***P<0.001 Vs control,
Values are expressed as mean ± SEM
Fig 6.3: In-vitro cytotoxic activity of extracts of S. veronicaefolia by MTT
assay
Page 65 of 113
D) In-vitro cytotoxic activity of extracts of M. longifolia by NR cytotoxic assay
The results showed that AEML and EEML were remarkable cytotoxic against
EAC with % inhibition of 78.2 ± 2.29 % and 81.7 ± 1.53 % receptively. The results were
reported in Table 6.9, Fig 6.4 and 6.5.
Table 6.9:
In-vitro cytotoxic activity of extracts of M. longifolia by NR
cytotoxic assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3800
0.00 ± 1.88
2
PEML
200 µg/ml
0.346
8.94 ± 0.78*
3
CEML
200 µg/ml
0.3361
11.57 ± 2.09**
4
ACML
200 µg/ml
0.1060
78.2 ± 2.29**
5
EEML
200 µg/ml
0.0940
81.7 ± 1.53**
6
AQEML
200 µg/ml
0.2852
25.12 ± 3.21**
8 wells /group OD at 550 nm, *P<0.01 Vs control. **P<0.001 Vs control.
Values are expressed as mean ± SEM
Fig 6.4: In-vitro cytotoxic activity of extracts of M. longifolia by NR cytotoxic
assay
Page 66 of 113
Fig 6.5: In-vitro cytotoxic activity of extracts of M. longifolia by NR cytotoxic
assay
The effect of M. longifolia extracts (200 µg/ml) on EAC cells was reported in Fig
6.5. In the normal control there was no vacant space and no cell death whereas extract
and standard drug treated group were shown, that indicates the cells were dead.
Normal control (No treatment)
PEML treated (200 µg/ml)
CEML treated (200 µg/ml)
AEML treated (200 µg/ml)
EEML treated (200 µg/ml)
AQEML treated (200 µg/ml)
Page 67 of 113
E) In-vitro cytotoxic activity of extracts of A. cordifolia by NR cytotoxic assay
The results showed that AEAC and EEAC were remarkable cytotoxic against
EAC with % inhibition of 93.36 ± 4.56% and 90.00 ± 3.64% receptively. The results were
reported in Table 6.10, Fig 6.6 and 6.7.
Table 6.10:
In-vitro cytotoxic activity of extracts of A. cordifolia by NR
cytotoxic assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3800
0.00 ± 1.88
2
PEAC
200 µg/ml
0.3091
18.68 ± 4.13*
3
CEAC
200 µg/ml
0.3482
08.42 ± 3.79ns
4
AEAC
200 µg/ml
0.0252
93.36 ± 4.56**
5
EEAC
200 µg/ml
0.0380
90.00 ± 3.64**
6
AQEAC
200 µg/ml
0.2523
33.68 ± 1.96**
8 wells /group OD at 550 nm, *P<0.01 Vs control, **P<0.001 Vs control.
ns
Not significant, Values are expressed as mean ± SEM
Fig 6.6: In-vitro cytotoxic activity of extracts of A. cordifolia by NR cytotoxic
assay
Page 68 of 113
Fig 6.7: In-vitro cytotoxic activity of extract of A. cordifolia by NR cytotoxic
assay
The effect of A. cordifolia extracts (200 µg/ml) on EAC cells was reported in Fig
6.7. In the normal control there was no vacant space and no cell death whereas extract
and standard drug treated group were shown, that indicates the cells were dead.
Normal control (No treatment)
CEAC treated (200µg/ml)
EEAC treated (200µg/ml)
PEAC treated (200µg/ml)
AEAC treated (200µg/ml)
AQEAC treated (200µg/ml)
Page 69 of 113
F) In-vitro cytotoxic activity of extracts of S. veronicaefolia by NR cytotoxic
assay
The results shows that AESV and EESV were remarkable cytotoxic against EAC
with % inhibition of 94.19 ± 3.48 % and 93 .72± 3.45 % receptively. The results were
reported in Table 6.11, Fig 6.8 and 6.9.
Table 6.11: In-vitro cytotoxic activity of extracts of S. veronicaefolia by NR
cytotoxic assay
S No
Extract
Concentration
Optical density
% inhibition
1
-
No treatment
0.3800
0.00 ± 1.88
2
PESV
200 µg/ml
0.285
25.00 ± 3.09**
3
CESV
200 µg/ml
0.3140
23.42 ± 1.23**
4
AESV
200 µg/ml
0.0221
94.19 ± 3.48**
5
EESV
200 µg/ml
0.0239
93 .72± 3.45**
6
AQESV
200 µg/ml
0.3310
13.15 ± 4.89*
8 wells /group OD at 550 nm, *P<0.01 Vs control. **P<0.001 Vs control.
Values are expressed as mean ± SEM
Fig 6.8: In-vitro cytotoxic activity of extracts of S. veronicaefolia by NR
cytotoxic assay
Page 70 of 113
Fig 6.9: In-vitro cytotoxic activity of extracts of S. veronicaefolia by NR
cytotoxic assay
The effect of S. veronicaefolia extracts (200 µg/ml) on EAC cells was reported in
Fig 6.9. In the normal control there was no vacant space and no cell death whereas
extract and standard drug treated group were shown, that indicates the cells were dead.
Normal Control (No Treatment)
PESV treated (200µg/ml)
CESV treated (200µg/ml)
AESV treated (200µg/ml)
EESV treated (200µg/ml)
AQESV treated (200µg/ml)
The results of the acetone and ethanol extracts of M. longifolia, A. cordifolia and
S. veronicaefolia shows the remarkable cytotoxic activity and these extracts were selected
for acute toxicity studies (Dose determination) and In-vivo anti-cancer activity.
Page 71 of 113
6.5 Acute toxicity studies
A) Acute toxicity studies of M. longifolia
The acetone and ethanol extracts of leaves of M. longifolia were screened for
acute toxicity study by OECD guideline no. 423. The results showed that both extracts
belonging to category-5(>5000).
The result was reported in Table 6.12.
Table 6.12: Acute toxicity studies of extracts of M. longifolia
S No.
No. of Animals
Extract
Dose mg/kg
Results
1
3
5
No death
2
3
50
No death
3
3
300
No death
4
3
2000
No death
5
3
5000
No death
6
3
5
No death
7
3
50
No death
8
3
300
No death
9
3
2000
No death
10
3
5000
No death
AEML
EEML
Page 72 of 113
B) Acute toxicity studies of A. cordifolia
The acetone and ethanol extracts of leaves of A. cordifolia were screened for
acute toxicity study by OECD guideline no. 423. The results showed that both extracts
belonging to category-5(>5000).
The result was reported in Table 6.13.
Table 6.13: Acute toxicity studies of extracts of A. cordifolia
S No.
No. of Animals
Extract
Dose mg/kg
Results
1
3
5
No death
2
3
50
No death
3
3
300
No death
4
3
2000
No death
5
3
5000
No death
6
3
5
No death
7
3
50
No death
8
3
300
No death
9
3
2000
No death
10
3
5000
No death
AEAC
EEAC
Page 73 of 113
C) Acute toxicity studies of S. veronicaefolia
The acetone and ethanol extracts of leaves of S. veronicaefolia were screened for
acute toxicity study by OECD guideline no. 423. The results showed that both extracts
belonging to category-5(>5000).
The result was reported in Table 6.14.
Table 6.14: Acute toxicity studies of extracts of S. veronicaefolia
S No.
No. of Animals
Extract
Dose mg/kg
Results
1
3
5
No death
2
3
50
No death
3
3
300
No death
4
3
2000
No death
5
3
5000
No death
6
3
5
No death
7
3
50
No death
8
3
300
No death
9
3
2000
No death
10
3
5000
No death
AESV
EESV
Page 74 of 113
6.6 Anticancer activity of extracts of M. longifolia, A. cordifolia and S.
veronicaefolia
A) Effect of selected plant extract on tumor volume, tumor weight and tumor
cell count of tumor bearing mice
There was reduction in the tumor volume, tumor weight and tumor cell count of
mice treated with AEML, EEML, AEAC, EEAC, AESV, EESV(500 mg/kg/day, p.o.)
and 5-FU (20 mg/kg) (P<0.001). Tumor volume of control animals were 6.70 ± 0.16 ml,
whereas the extract-treated group was 3.46 ± 0.07, 3.12 ± 0.08, 2.55 ± 0.11, 2.25 ±0.09,
2.15 ±0.09, 2.56 ±0.10 and 1.01 ± 0.10 ml of AEML, EEML, AEAC, EEAC, AESV,
EESV and 5-FU, respectively.
Tumor weight of control animals were 6.87 ±0.21 g and the extract-treated group
were 3.54 ±0.31, 3.23 ±0.10, 2.68 ±0.30, 2.34 ±0.37, 2.25 ±0.25, 2.71 ±0.31 and 1.1
±0.06 g of AEML, EEML, AEAC, EEAC, AESV, EESV and 5-FU respectively.
The viable tumor cell count was decreases and increases in non viable tumor cell
were found. These results were reported in Table 6.15 and Fig 6.10, 6.11 and 6.12.
Table 6.15: Effect of selected plant extract on tumor volume, tumor weight
and tumor cell count of tumor bearing mice
S No
Treatment
Tumor
Tumor
Volume (ml)
weight (gm)
Tumor cell count
Viable cells X
Nonviable
107/ml
cells X 107/ml
1
Tumor Control
6.70 ± 0.16
6.87 ±0.21
9.83 ±0.3
0.33 ±0.21
2
5- FU (20mg/kg, i.p)
1.01 ± 0.10*
1.1 ±0.06*
0.83 ±0.3*
1.67 ±0.33**
3
AEML (500 mg/kg, p.o)
3.46 ± 0.07*
3.54 ±0.31*
3.66 ±0.21*
2.5 ±0.22*
4
EEML (500 mg/kg, p.o)
3.12 ± 0.08*
3.23 ±0.10*
3.16 ±0.3*
2.3 ±0.21*
5
AEAC (500 mg/kg, p.o)
2.55 ± 0.11*
2.68 ±0.30*
2.83 ±0.3*
2.6 ±0.42*
6
EEAC (500 mg/kg, p.o)
2.25 ±0.09*
2.34 ±0.37*
2.66 ±0.21*
2.5 ±0.5*
7
AESV (500 mg/kg, p.o)
2.15 ±0.09*
2.25 ±0.25*
2.33 ±0.21*
1.8 ±0.3**
8
EESV (500 mg/kg, p.o)
2,56 ±0.10*
2.71 ±0.31*
2.83 ±0.16*
2.1 ±0.3**
*P<0.001 Vs tumor control, **P<0.01 Vs tumor control
n=6 animals in each group, No of days = 14, Values are expressed as mean ± SEM.
Page 75 of 113
Fig 6.10: Effect of selected plant extracts on tumor volume of tumor bearing
mice
Fig 6.11: Effect of selected plant extracts on tumor weight of tumor bearing
mice
Page 76 of 113
Fig 6.12: Effect of selected plant extracts on tumor cell count of tumor
bearing mice
Page 77 of 113
B) Effect of selected plant extracts on the mean survival time (MST) of tumor
bearing mice
The MST for the control group was 21.50 ± 2.73 days and 30.33 ± 4.7, 32.83 ±
3.25, 34.33 ± 3.2, 34.83 ± 3.9, 35.16 ±2.8, 34.16±4.5 and 40.16 ± 2.13 days for the
groups treated with AEML, EEML, AEAC, EEAC, AESV, EESV (500 mg/kg/day, p.o.)
and 5-FU (20 mg/kg/day, i.p.) respectively.
The % increase in the lifespan of tumor-bearing mice treated with AEML, EEML,
AEAC, EEAC, AESV, EESV and 5-FU was found to be 41.06, 52.69, 59.67,62.00,
63.50, 58.88 and 86.79% respectively (P< 0.01) as compared to the control group.
The results were shown in Table 6.16 and Fig 6.13.
Table 6.16: Effect of selected plant extracts on Mean Survival Time (MST) of
tumor bearing mice
S No
Treatment
Mean Survival Time (Days)
Increase in life span (%)
1
Tumor Control
21.50 ± 2.73
-
2
5- FU (20mg/kg, i.p)
40.16 ± 2.13*
86.79 %
3
AEML (500 mg/kg, p.o)
30.33± 4.7*
41.06 %
4
EEML (500 mg/kg, p.o)
32.83 ± 3.25*
52.69 %
5
AEAC (500 mg/kg, p.o)
34.33 ± 3.2*
59.67 %
6
EEAC (500 mg/kg, p.o)
34.83 ± 3.9*
62.00 %
7
AESV (500 mg/kg, p.o)
35.16 ±2.8*
63.50 %
8
EESV (500 mg/kg, p.o)
34.16±4.5*
58.88 %
n=6 animals in each group,
*P<0.01 Vs control,
Days of treatment = 14,
Values are expressed as mean ± SEM
Page 78 of 113
Fig 6.13: Effect of selected plant extracts on Mean survival time of tumor
bearing mice.
Page 79 of 113
C) Effect of selected extracts on body weight of tumor bearing mice
There was a significant decrease in weight gain of extract and standard drug
treated group when compared to weight gain of tumor control group. The result were
reported in Table 6.17.
Table 6.17: Effect of selected plant extracts on body weight of tumor bearing
mice
Treatment/dose
7th Day
14th Day
21st Day
28th Day
35th Day
Normal
22.00±0.77
23.00±0.51
24.16±0.54
27.66±0.66
31.83±0.83
Tumor Control
27.83±0.79*
40.33±0.76*
50.16±0.65*
-
-
5-FU (20mg/kg, i.p)
23.33±0.61
24.50±0.34$
26.83±0.6 $
29.33±0.66
32.83±0.47
AEML(500 mg/kg, p.o)
24.33±0.33**
29.50±0.76#
30.5±0.88$,#
31.00±0.25# 35.83±0.30#
EEML(500 mg/kg, p.o)
25.0±0.25**
30.66±0.33$,#
32.3±0.66 $,#
34.33±0.33# 37.33±0.33#
AEAC(500 mg/kg, p.o)
25.06±0.18** 29.68±0.37$,# 32.7±0.53 $,#
34.27±0.66# 37.58±0.39#
EEAC(500 mg/kg, p.o)
25.33±0.56$
28.5±0.21#
30.43±0.33$,# 31.6±0.38#
35.4±0.28#
AESV(500 mg/kg, p.o)
24.33±0.33$
29.5±0.56$,#
30.5±0.88$,#
31.5±0.35#
34.83±0.30#
EESV(500 mg/kg, p.o)
25.43±0.33$
28.5±0.76#
30.5±0.88$,#
31±0.25#
35.83±0.30#
n= 6 in each group.
* P< 0.001 Vs Normal control,
$ P< 0.001 Vs Tumor control,
# P<0.001 Vs Standard,
** P<0.01Vs Tumor control
Values were expressed as mean± SEM.
Page 80 of 113
D) Effect of selected plant extracts on hematological parameters of tumor
bearing mice
The hematological parameters of tumor-bearing mice showed significant changes
on 14th day, when compared with the normal mice. In tumor control, the total WBC
count, proteins and PCV were found to increase with a reduction in the hemoglobin
content of RBC. The differential count of WBC showed that the percentage of
neutrophils increased (P<0.001) while that of lymphocytes decreased (P<0.001), whereas
AEML, EEML, AEAC, EEAC, AESV, EESV (500 mg/kg/day, p.o.) and 5-FU (20mg/kg,
i.p) treatment significantly altered all the parameters, near to normal and were able to
reverse the changes in the haematological parameters consequent to tumor inoculation.
The results were reported in Table 6.18.
Page 81 of 113
Table 6.18: Effect of selected plant extracts on hematological parameters of tumor bearing mice
Tumor
5 FU (20
control
mg/kg)
14.3±0.10
8.35±0.09*
RBC (million/mm3)
4.68±0.06
WBC(million/mm3)
Parameter
Normal
AEML
EEML
AEAC
EEAC
AESV
EESV
Hb(g/dl)
14.0±0.05*,$
12.4±0.4*,$
12.1±0.21*,$
12.94±0.12*,$
13.1±0.15*,$
13.2±0.10*,$
12.91±0.10*,$
2.6±0.07*
4.11±0.04*,$
3.18±0.3*,$
3.06±0.32*,$
3.78±0.06*,$
3.14±0.05*,$
3.13±0.06*,$
3.88±0.04*,$
7.48±0.03
27.19±0.07*
8.23±0.02*,$
9.6±0.7*,$
9.22±0.10*,$
9.05±0.05*,$
9.54±0.09*,$
9.58±0.02*,$
9.09±0.03*,$
Proteing %
8.21±0.06
13.95±0.2*
8.65±0.04*,$
9.2±0.1*,$
9.1±0.13*,$
9.13±0.03*,$
9.64±0.09*,$
9.6±0.05*,$
9.1±0.03*,$
PCV (mm)
16.5±0.42
31.5±0.42*
19.5±0.42*,$
26.2±0.1*,$
25.2±0.33*,$
21.7±0.33*,$
24.4±0.43*,$
24.5±0.42*,$
21.3±0.33*,$
30.83±0.60 68.83±0.60* 31.83±0.47*,$ 38.1±2.2*,$ 44.32±0.78*,$
38.3±1.75*,$
42.11±0.68*,$ 42.16±0.60*,$
64.66±0.42*,$ 50.3±2.1*,$ 51.10±0.52*,$
59.2±0.45*,$
54.10±0.67*,$
54.0±0.68*,$
59.5±0.42*,$
1.86±0.33ns
1.56±0.28ns
1.5±0.22 ns
1.83±0.30 ns
Neutrophils %
Lymphocytes %
68.5±0.42
30±0.57*
Monocytes %
1.16±0.16
2.16±0.16#
1.33±0.21 ns
1.8±0.3ns
1.7±0.33ns
38±1.78*,$
n= 6 in each group,
* P< 0.001 Vs Normal control,
P< 0.001 Vs Tumor control,
# P<0.05Vs Normal Control,
ns – not significant,
Values are expressed as Mean  SEM.
Page 82 of 113
E) Effect of selected plant extracts on peritoneal cells in normal mice
The average number of peritoneal exudates cells in normal mice was found to be
5.8±0.1×106. Single day treatment with AEML, EEML, AEAC, EEAC, AESV, EESV
(500 mg/kg/day, p.o.) enhanced peritoneal cells to 7.21 ± 0.7 ×106 and 7.38 ± 0.12 ×106,
while two consecutive day treatments enhanced the number to 10.21±0.06 ×106 and
10.31±0.19 ×106, respectively, (P< 0.001) as reperted in Table 6.19.
Table 6.19: Effect of selected extracts on peritoneal cells in normal mice
Group
Treatment
Peritoneal cell count(×106)
1
Normal control
5.8 ± 0.01
2
AEML (500 mg/kg, p.o) treated once
7.38±0.12*
3
AEML (500 mg/kg, p.o) treated twice
10.31±0.19*
4
EEML (500 mg/kg, p.o) treated once
7.21 ± 0.07*
5
EEML (500 mg/kg, p.o) treated twice
10.21±0.06*
6
AEAC (500 mg/kg, p.o) treated once
9.23±0.23*
7
AEAC (500 mg/kg, p.o) treated twice
12.89±0.56*
8
EEAC (500 mg/kg, p.o) treated once
9.42±.023*
9
EEAC (500 mg/kg, p.o) treated twice
13.26± 0.34*
19
AESV (500 mg/kg, p.o) treated once
9.7±0.37*
11
AESV (500 mg/kg, p.o) treated twice
14.3±0.27*
12
EESV (500 mg/kg, p.o) treated once
9.1±0.1*
13
EESV (500 mg/kg, p.o) treated twice
13.2±0.2*
n= 6 in each group,
* P< 0.001 Vs Normal control, Values are expressed as Mean  SEM.
Page 83 of 113
7. CONCLUSION
It is well established that plants have been a useful for the treatment of tumor
(Kamuhabwa et al., 2000). There are different approaches for the selection of plants that
may contain new biologically active compounds (Cordell et al.,1991). One of the
approaches used is ethnomedical data approach, in which selection of a plant is based on
the prior information on the folk medicinal use of the plant. It is generally known that
ethnomedical data provides substantially increased chance of finding active plants
relative to random approach (Chapuis et al., 1988). However, as for cancer, the disease is
complicated and heterogeneous, which makes it difficult to be well diagnosed, especially
by traditional healers. Traditional Indian and Chinese medicinal herbs have been used in
the treatment of different diseases in the country for centuries. There have been claims
that some traditional healers can successfully treat cancer using herbal drugs. Indeed,
some traditional healers who were interviewed recently in the country stressed that they
have successfully treated patients presented with cancer or cancer related diseases.
Cancer chemoprevention has been defined as a process facilitated by blocking
induction of neoplastic process or preventing transformed cells from progression to
malignant phenotypes by administration of one or more chemical entities, either as
synthetic drugs or naturally occurring phytoconstituents. Chemotherapy is the standard
and accepted treatment tool for cancer, alone or in conjunction with elective surgery and
radiation, as the case may be. Chemotherapeutic drugs are cytotoxic by design, and thus
most of the formulation drugs that are available can effectively kill the cancerous cell,
they also damage the DNA of normal cell and are able to cause serious dose limiting
adverse effects at therapeutics doses. Taking into account the side effect of synthetic
drug, natural plants drug interplay as chemotherapy for various type of diseases and in
particular against carcinogenesis.
Recent studies on tumor inhibitory compounds of plant origin have yielded an
impressive array of research on medicinal plant. The efficacy of M. longifolia, A.
cordifolia and S. veronicaefolia against Ehrlich ascetic carcinoma provide the beneficial
rational about efficacy as anticancer activity.
In anticancer activity of the selected plant extracts, it was observed that group of
animals treated with test drugs (500 mg/ kg) showed significant decrease in the tumor
Page 84 of 113
volume, tumor weight, tumor cell count, body weight, and brought back the
hematological parameters to more or less normal levels. In EAC-bearing mice, a regular
rapid increase in ascites tumor volume was noted. Ascites fluid is the direct nutritional
source for tumor cells and a rapid increase in ascites fluid with tumor growth would be a
means to meet the nutritional requirement of tumor cells (Prasad et al., 1994 ). It is being
clear that the reliable criterion for evaluating the value of any anticancer drug is the
prolongation of the life span of animals (Clarkson et al., 1965; Oberling et al., 1954). As
these extracts decreased the ascites fluid volume, viable cell count, and increased the
percentage of life span of the animals therefore they can be considered as important
source for the treatment of such deadly diseases.
Usually, in cancer chemotherapy the major problems that are being encountered
are of myelosuppression and anemia (Price et al., 1958; Hogland et al., 1982). The
anemia encountered in tumor bearing mice is mainly due to reduction in RBC or
hemoglobin percentage, and this may occur either due to iron deficiency or due to
hemolytic or myelopathic conditions (Fenninger et al., 1954). In EAC control group, a
differential count the presence of neutrophils increased, while the lymphocyte count
decreased,
the
observed
leucocytopenia
indicates
a
common
symptom
of
immunosuppression in many types of cancers (Rashid et al., 2010; Ropponen et al.,
1997) and one of the causes of neutrophilia is myeloid growth factors which are produced
in malignant process as part of a paraneoplastic syndrome. In addition to this another
factor granulocyte colony stimulating factor produced by the malignant cells has also
been attributed to be the cause of neutrophilia because of its action on bone marrow
granulocytic cells in cancer. After the repeated treatment, all of these extracts were able
to reverse the changes in altered neutrophils and lymphocytes count. Treatment with
these extracts brought back the hemoglobin content, RBC, and WBC count more or less
to normal levels and this indicates that these extracts posses protective action on the
hematopoietic system (Ulich et al., 1990; Uchida et al., 1992).
A significant enhancement of peritoneal cell count was observed. The effect of
extracts treatment on the peritoneal exudate cells of normal mice is an indirect method of
evaluating its inhibitory effect on tumor cell growth (Rajkapoor, 2003). Normally, a
mouse contains about 5 x 106 peritoneal cells, 50% of which are macrophages. Extracts
Page 85 of 113
treatment were found to enhance peritoneal cells count. These results demonstrate the
indirect inhibitory effect of these extracts on EAC cells, which is probably mediated by
the enhancement and activation of either macrophage or cytokine production (Rajkapoor,
2003).
In-vitro cytotoxicity is the method by which the cells are directly treated with the
extract and their % inhibition is evaluated. Selected extracts showed significant %
inhibition activity against the cell lines. This result was showed the anticancer activity of
selected extract.
Preliminary phytochemical screening of the selected plants ascertains the
presence of flavonoids, phenolic compound, tannins, alkaloids and phytosterols,
trierpenoids etc. It is being already reported that compounds such as flavonoids, tannins
etc, possess significant antimutagenic (Brown JP, 1980) and antimalignant activity
(Hirano T et al., 1989). They play an important role as a chemopreventive agent in cancer
because of their effects on signal transduction in cell proliferation (Weber et al., 1996)
and angiogenesis (Fotsis et al., 1997). Such pharmacologically active phytoconstituents
induces cell death of cancer cells by concentration-dependent decrease of ATP and a
deterioration of cellular gross morphology (Swami et al., 2003; Bawadi et al., 2005 ).
They also inhibit growth of cancer affected cell (Lambert et al., 2003; Keil et al., 2004)
and avoids death of normal cell therefore, they may considered as a efficient candidate to
enhanced opportunities for DNA repair, immune stimulation, anti-inflammation and
cancer prevention (Blumenthal et al., 2003; Sheng et al., 2005). Similarly they can inhibit
the tumor growth by an alteration in signal transduction pathways(Leikin et al., 1989;
Tapiero et al., 2003). Depending on such a important pharmacological activity posses by
these phytoconstituents, it might be clear that they are responsible for providing a
significant anticancer activity. As M. longifolia, A. cordifolia and S. veronicaefolia
contains these phytoconstituents, therefore we can conclude that anticancer activity
shown by these plant may be because of these pharmacologically active
phytoconstituents.
From above studies it was concluded that M. longifolia, A. cordifolia and S.
veronicaefolia are very much effective in preventing EAC in mice and possess significant
anticancer activity against EAC.
Page 86 of 113
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LIST OF PUBLICATION
 In-vitro Cytotoxic activity of leaves of Madhuca longifolia against Ehrlich
Ascites Carcinoma (EAC) Cell line. International Journal of Drug Discovery
and Herbal Research, 2011, 1(2): 55-57.
 Antitumor activity of Sida Veronicaefolia against Ehrlich Ascites Carcinoma in
mice. Journal of Pharmacy Research, 2012, 5(1): 315-319.
 Anti-Tumor Effect of acetone extract of Madhuca longifolia against Ehrlich
Ascites Carcinoma (EAC) in mice. Phytopharmacology 2012, 3(1): 130-136.
 Anticancer effect of ethanol extract of Madhuca longifolia against Ehrlich Ascites
Carcinoma. Molecular and Clinical Pharmacology.2012 2(1): 12-19.
 Anticancer activity of Adina Cordifolia against Ehrlich Ascites Carcinoma in
mice. Continental Journal of Pharmacology and Toxicology Research, 2012,
5(1): 7-16.
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