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 (Gurib-

Fakim, 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).

2. AIM AND OBJECTIVES Page 13 of 113

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.

3. REVIEW OF LITERATURE

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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

micro-organisms have played and continue to play, a dominant role in the discovery of

Page 15 of 113

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.

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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

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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 S-

transferase enzyme, all of which are important in the detoxification of carcinogens as

well as mutagens. Moreover, it also significantly elevated extra-hepatic glutathione S-

transferase 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).

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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, 12-

dimethylbenz[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 Whole plant Ju et al., 2004

Boscia senegalensis Capparidaceae Leaves Ali et al.,2002

Catalpa bignonioides Bignoniaceae Seeds Muñoz et al., 2003

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Celastrus orbiculatus Celestraceae Root Jin et al.,, 2002

Clerodendrum

myricoidesVerbenaceae Root bark

Kamuhabwa et al .,

2000

Clematis chinensis Ranunculaceae Whole plant Qiu et al ., 1999

Crocus sativus Iridaceae Stigma Nair et al ., 1995

Croton palanostigma Euphorbiaceae Sap Sandoval et al ., 2002

Cunuria spruceane EuphorbiaceaeRoot, Root

bark

Gunasekera et

al ., 1979

Cupressus lusitanica Cupressaceae Leaves Lopéz et al ., 2002

Dendrostellera lessertii Thymelaeaceae Leaves Sadeghi et al ., 2003

Dioscorea birmanica Dioscoriaceae Whole plantWoerdnbeg et al.,

1986

Emblica officinalis Euphorbiaceae Fruits Jose et al., 2001

Emilia sonchifolia Compositae Whole plant Shylesh, et al., 2000

Eurycoma longifolia Simaroubaceae Leaves Jiwajinda et al ., 2002

Garcinia atroviridis Guttiferae Stem bark Mackeen et al., 2000

Helixanthera parasitica Loranthaceae Whole plantLirdprapamongkol

K, et al., 2003

Hippophae salicifolia Elaeagnaceae Fruit Uniyal et al., 1990

Humulus lupulus Cannabaceae Whole plant Goun et al., 2002

Iris germanica Iridaceae Bulbs Bonfils et al., 2001

Jatropha elliptica Euphorbiaceae Tuber Calixto et al., 1987.

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Kigelia Africana Bignoniaceae Rootbark Msonthi et al., 1983

Leptadenia hastate Asclepiadaceae Bark Aquino et al., 1995

Leptospermum

scopariumMyrtaceae Aerial parts Mayer et al., 1993

Lithraea molleoides Anacardiaceae Whole plant Ruffa et al., 2002

Melastoma

malabathricumMelastomataceae Flowers

Mohandoss et al.,

1993

Moringa oleifera Lam Moringaceae Whole plant Dhawan et al., 1980

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 Gálvez et al., 2003

Psittacanthus

calyculatusLoranthaceae Leaves

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 Lee et al., 1997

Terminalia chebula Combretaceae Fruits Saleem et al., 2002

Verbascum Thapsus ScrophulariaceaeLeaves,caps

ulesTurker et al., 2002

Virola bicuhyba Myristicaceae Seed Plotkin et al., 1990

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Xeromphis obovata Rubiaceae Rootbark Sibanda et al., 1989

Zanthoxylum oxyphyllum Rutaceae Fruit Suwal, 1970.

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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, plant-

derived 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 anti-

breast cancer potential (Amin et al., 2005). In that study, we have shown that Fenugreek

can significantly protect rats against drug-induced breast cancer.

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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).

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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. Water-

soluble 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-L-

arabinofuranosyl)-(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.,

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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 HT-

29 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).

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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 PS-

AI & PS-A II, isolated from flowers, constitute galactose, glucose, arabinose and

glucoronic acid (Prajapati et al., 2003; Chandra et al.,2001). it also contains oleanane-

type 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)

Page 30 of 113

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 570-

895 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, Anti-

rheumatoid, 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), reversed-

phase 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 low-

melting 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 1,1-

diphenyl-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/2009/14-

16).

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 Page 41 of 113

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, TIT-

Pharmacy, 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.

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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.

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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

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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 5-

fluorouracil (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 15 th 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

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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

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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).

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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.

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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

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

Page 58 of 113

Plant Name Types of AshPercentage of

Ash(w/w)

Madhuca longifolia

Total ash 10.5

Acid insoluble 3.7

Water soluble 1.7

Adina cordifolia

Total ash 8.1

Acid Insoluble 3.5

Water soluble 1.3

Sida veronicaefolia

Total ash 9.1

Acid Insoluble 3.3

Water soluble 1.5

Solvent

used

% Yeild

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

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.

Constituents Tests Pet.ether extract

CHCl3

extractAcetoneextract

Ethanolic extract

Aqueous extract

CarbohydrateMolisch’s test - - - + +Fehling’s test - - - + +

Glycosides

Legal’s test - - - - -Borntrager’s

test - - - - -

Baljet test - - - - -

Fixed oil and Fats

Spot test + + - - -Saponification

test + + - - -

Proteins and Amino Acids

Millon’s test - + - + +

Ninhydrin test - + - + +Biuret test - + - + +

Saponins Foam test - - + + +

Phenolic Comp. and

Tannins

FeCl3 test - - - - -

Lead acetate test - - - - -

PhytosterolsSalkowski test + - + + +Libermann-

Bucchard test + - + + +

Page 60 of 113

Alkaloids

Dragendorff’s test - - + + +

Mayer’s test - - + + +Wagner’s test - - + + +Hager’s test - - + + +

Gums and Mucilage

Froth test + - - - +

Alcoholic test + - - - +

Flavonoids

Lead acetate test - - + + +

Con. H2SO4

test - - + + +

FeCl3 test - - + + +Table 6.4: Preliminary phytochemical evaluation of leaves of A. cordifolia

Constituents Tests Pet.ether extract

CHCl3

ExtractAcetoneextract

Ethanolic extract

Aqueous extract

CarbohydrateMolisch’s test - - - + +

Fehling’s test - - - + +

Glycosides

Legal’s test - - - - -Borntrager’s

test - - - - -

Baljet test - - - - -

Fixed oil and Fats

Spot test + + + + -Saponification

test + + + + -

Proteins and Amino Acids

Millon’s test - + - + +

Ninhydrin test - + - + +Biuret test - + - + +

Saponins Foam test - - - + +

Phenolic Comp. and

Tannins

FeCl3 test - - + + +

Lead acetate test - - + + +

PhytosterolsSalkowski test + - + + +Libermann-

Bucchard test + - + + +

Page 61 of 113

Alkaloids

Dragendorff’s test - - - - -

Mayer’s test - - - - -Wagner’s test - - - - -Hager’s test - - - - -

Gums and Mucilage

Froth test + - - - +

Alcoholic test + - - - +

Flavonoids Lead acetate test - - + + +

Con. H2SO4

test - - + + +

FeCl3 test - - + + +Table 6.5: Preliminary phytochemical evaluation of leaves of S. veronicaefolia

Constituents Tests Pet.ether extract

CHCl3

ExtractAcetoneextract

Ethanolic extract

Aqueous extract

CarbohydrateMolisch’s test - - - + +Fehling’s test - - - + +

Glycosides

Legal’s test - - - - -Borntrager’s

test - - - - -

Baljet test - - - - -

Fixed oil and Fats

Spot test + + - + -Saponification

test + + - + -

Proteins and Amino Acids

Millon’s test - + - + +

Ninhydrin test - + - + +Biuret test - + - + +

Saponins Foam test - - - - -

Phenolic Comp. and

Tannins

FeCl3 test - - + + +

Lead acetate test - - + + +

PhytosterolsSalkowski test + - + + +Libermann-

Bucchard test + - + + +

Page 62 of 113

Alkaloids

Dragendorff’s test - - + + +

Mayer’s test - - + + -Wagner’s test - - + + -Hager’s test - - + + -

Gums and Mucilage

Froth test + - - - +

Alcoholic test + - - - +

Flavonoids

Lead acetate test - - + + +

Con. H2SO4

test - - + + +

FeCl3 test - - + + +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

Page 63 of 113

Fig 6.1: In-vitro cytotoxic activity of extract of M. longifolia by MTT assay

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

Page 64 of 113

Fig 6.2: In-vitro cytotoxic activity of extracts of A. cordifolia by MTT assay

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

Page 65 of 113

Fig 6.3: In-vitro cytotoxic activity of extracts of S. veronicaefolia by MTT

assay

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

Page 66 of 113

Fig 6.4: In-vitro cytotoxic activity of extracts of M. longifolia by NR cytotoxic

assay

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)

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CEML treated (200 µg/ml) AEML treated (200 µg/ml)

EEML treated (200 µg/ml) AQEML treated (200 µg/ml)

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

Page 68 of 113

Fig 6.6: In-vitro cytotoxic activity of extracts of A. cordifolia by NR cytotoxic

assay

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) PEAC treated (200µg/ml)

CEAC treated (200µg/ml) AEAC treated (200µg/ml)

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EEAC treated (200µg/ml) AQEAC treated (200µg/ml)

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

Page 70 of 113

Fig 6.8: In-vitro cytotoxic activity of extracts of S. veronicaefolia by NR

cytotoxic assay

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)

Page 71 of 113

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.

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

B) Acute toxicity studies of A. cordifolia

Page 72 of 113

S No. No. of Animals Extract Dose mg/kg Results

1 3

AEML

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

EEML

5 No death

7 3 50 No death

8 3 300 No death

9 3 2000 No death

10 3 5000 No death

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

Page 73 of 113

S No. No. of Animals Extract Dose mg/kg Results

1 3

AEAC

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

EEAC

5 No death

7 3 50 No death

8 3 300 No death

9 3 2000 No death

10 3 5000 No death

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

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.

Page 74 of 113

S No. No. of Animals Extract Dose mg/kg Results

1 3

AESV

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

EESV

5 No death

7 3 50 No death

8 3 300 No death

9 3 2000 No death

10 3 5000 No death

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 TreatmentTumor

Volume (ml)

Tumor

weight (gm)

Tumor cell count

Viable cells X

107/ml

Nonviable

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

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.

Page 77 of 113

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.

D) Effect of selected plant extracts on hematological parameters of tumor

bearing mice

Page 80 of 113

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

Parameter NormalTumor

control

5 FU (20

mg/kg)AEML EEML AEAC EEAC AESV EESV

Hb(g/dl) 14.3±0.10 8.35±0.09* 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*,$

RBC (million/mm3) 4.68±0.06 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*,$

WBC(million/mm3) 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*,$

Neutrophils % 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*,$ 38±1.78*,$

Lymphocytes % 68.5±0.42 30±0.57* 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*,$

Monocytes % 1.16±0.16 2.16±0.16# 1.33±0.21 ns 1.8±0.3ns 1.7±0.33ns 1.86±0.33ns 1.56±0.28ns 1.5±0.22 ns 1.83±0.30 ns

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. CONCLUSIONIt 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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