2. review of litrature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/33761/2/... ·...

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2. REVIEW OF LITRATURE

2.1. Free radicals

A free radical is any species capable of independent existence that contains one or

more unpaired electrons (Southern et al., 1988). A related term, reactive oxygen species is

used to describe collectively not only the oxygen derived free radicals but also the non-

radical oxidants like hydrogen peroxide and hypochlorous acid which are not unpaired

electrons. The essential prerequisite for stability of any atom or molecule is that the

electrons in its outermost orbit may carry a positive or negative charge or may be

electrically neutral but it should be paired (Brown, 1982). In presence of any unpaired

electron, the atom or molecule gets unstable and shows high reactivity in order to gain an

electron from other atoms/molecules. The salient features of free radicals are it contains an

unpaired electron, unstable in nature to gain stability by snatching electrons from

neighbouring entities, hence are reactive in nature. In doing so, they initiate a chain

reaction, which fits into the definition of oxidation. The free radical oxidation moves from

molecule to molecule, cell to cell, organelle to organelle, causing immense damage to the

human body (Hooper, 1989).

Free radicals are normally produced as a by-product of cellular metabolism. Free

radicals are capable of killing bacteria, damage biomolecules, provoke immune response,

activate oncogens, cause atherogenesis and enhance ageing process. However, in healthy

conditions, nature has endowed human body with enormous antioxidant potential. Subtle

balance exists between free radical generation and antioxidant defence system by various

enzymes and vitamins to cope with oxidative stress at cellular level which prevents the

occurrence of disease. However, factors tilting the balance in favour of excess free radicals

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generation lead to widespread oxidative tissue damage and diseases. Therefore, trouble

starts when there is an excess free radicals and the defence mechanism lags behind.

Overwhelming production of free radicals in response to exposure to toxic chemicals and

ageing may necessitate judicious antioxidant supplement to help alleviate free radical

mediated damage (Halliweli, 1989).

2.1.1. Sources of free radicals

The production of free radicals is from two sources (Sinclair, 1991).

2.1.1.1. Endogenous

The free radicals are produced during cellular metabolisms like prostaglandin

synthesis, mitochondrial electron transport, endoplasmic reticulum oxidation, enzyme

activity, oxyhaemoglobin, auto oxidation and phagocytosis.

2.1.1.2 Exogenous

Exogenous free radicals are produced due to external stimuli like pesticides, air

pollutants, smoke, radiation and drugs. It has been reported that generation of free radical

is associated with side effects of many drugs. Free radical production may also be

increased during various disease states (Richards, 1992).

2.1.2. Important free radicals

Although a large number of free radicals are formed, the following are responsible

for inducing disease and hence need to be combated at all costs (Singhal, 1988).

2.1.2.1. Superoxide radicals

This radical is the most important factor in oxygen toxicity. It is mainly derived

from the electron transport chains of mitochondria and endoplasmic reticulum. It is

responsible for damages associated with cardiac and intestinal ischemia.


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2.1.2.2. Hydroxyl radical

This is derived from ionising radiations, iron and hydrogen peroxide. This is the

most reactive radical and capable of damaging every type of molecules found in living

cells viz., carbohydrate, amino acids, phospholipids and nucleic acid. It is also responsible

for the damages done to cellular DNA and to membranes.

2.1.2.3. Transition metals

These are in a position to transit between two different states on the basis of

electron transfer. These metals are iron, copper and zinc. Amongst these, iron has been

shown to be a precursor of OH radical which is formed as a result of fenton reaction

(reaction with H 2O2). This reaction is responsible for consequences of iron overload seen

in thalassemic patients given blood transfusion. Contrary to this, copper and zinc do not

lead to the formation of hydroxyl ions since they are found in bound form. These free

radicals are responsible for widespred and indiscriminate oxidation and peroxidation of

lipid, denaturation of proteins, depolymerization of polysaccharides and break to modify

DNA causing cell death or organ damage because of huge chemical reactivity,

autocatalytic potential and low chemical specificity. The outcome of these indiscriminate

and extensive oxidative damage will be cell membrane disruption, enzymatic inactivation,

altered antigenicity and carcinogenesist (Floyd, 1990).

2.1.3. Pathological consequences of free radical formation

2.1.3.1. Lipid peroxidation

This is the most common and dangerous type of free radical oxidation. As a

consequence of interaction of free radical and lipid to form peroxide (intermediate free

radicals) subsequently leads to autocatalytic radical chain reactions, resulting in membrane

damage, as an example carbon tetrachloride induced liver damage. Lipid peroxidation


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leads to majority of human diseases, such as atherosclerosis, (Stringer, 1989), ischaemic

perfusion injury (Mc Cord, 1985) and hypertension. In tissue iscaemic hypoxia, ATP is

transformed through intermediate stages by hypoxanthines. The enzyme xanthine

dehydrogenase, which is abundant in the gut and liver normally catalyses the reaction

(Parks and Granger, 1986; Chariot et al., 1987).

2.1.4. Other pathological consequences

Free radical inhibits the production of vasodilators namely endothelium derived

relaxing factors which control microcirculation. This may contribute to the genesis of

pulmonary shock syndrome and postischaemic coronary vasoconstriction

(Chand et al., 1981). Free radicals are released by activation of macrophage. This process

is triggered by immune complexes, endotoxin and activated complement causing damage

in cases of resuscitation of critically ill patient resulting in multisystem organ failure

characterized by failure of hepatic, renal and pulmonary systems.

Further it has been reported that tocopherol and β-carotene are the antioxidants

that protect LDL molecule (Keller et al., 1985). α-Tocopherol is the principal lipid soluble,

chain breaking antioxidant in tissues and plasma. β-carotene effectively scavenges

oxidising radicals, particularly singlet oxygen. Ascorbic acid is the first line defence

against oxygen radicals in the water soluble compartment. The antioxidants may have a

useful therapeutic role with cardioprotective potential to reduce endothelial damage and

atheroma formation (Kendal et al., 1994). Free radicals are important mediators of acute

and chronic inflammatory reactions. During phagocytosis they are released into the

extracellular space where they cause direct tissue injury and alter structural

macromolecules such as elastin, collagen and hyaluronic acid. In addition they may react

with a plasma component to produce a chemotactic substance that attract more neutrophils


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to the site of inflammation, such as in rheumatoid arthritis where free radical mediated

destruction of hyaluronic acid causes synovial fluid to lose its viscosity and joint cartilage

to become eroded (Ottonello et al., 1995). Other common diseases where free radicals

have been implicated are connective tissue disease, inflammatory bowel diseases, immune

deficiency and arthritis. Recently, Helicobacter pylori causing peptic ulcer has been

reported to have powerful superoxide dismutase and extracellular catalase action of

producing severe inflammation (Mohanty et al., 1992).

2.2. Antioxidant system of the body

In a healthy condition, subtle balance exists between free radical generation and

antioxidant defence system by enzymes, vitamins and minerals at cellular level which

prevent the occurrence of disease. However, factors tilting the balance in favour of free

radical generation will lead to widespread oxidative tissue damage and disease. The free

radicals are invisible silent killers at work. It has been estimated that the DNA in each cell

of human body receives about 10,000 oxidating hits (oxidative stress) per day. However,

human body makes use of certain substances that counter the process of free radical

oxidation. This system consists of substances that provide the much needed stability to free

radical by allowing the pairing of electron. This antioxidant defence can conveniently be

classified into two groups, defence by enzymes and defence by micronutrients

(Dormandy, 1989).

2.2.1. Defence by enzymes

The antioxidant enzymes are known as free radical scavengers which remove free

radicals directly irrespective of their source. The primary intracellular enzymes which

scavenge free radicals include the following:


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2.2.1.1. Catalase: This is a haem-centered enzyme responsible for decomposition of

hydrogen peroxide to water and oxygen. This enzyme is associated in its different forms

with zinc, copper and manganese, and is a potent antioxidant, especially against the

superoxide radicals and singlet oxygen. It keeps the body healthy by mopping up the

reactive oxygen radicals and, thereby, protecting the body against oxidative stress of the

free radicals. In this enzyme catalysed reaction called dismutation, the copper and

managanese ions undergo alternate oxidation-reduction reaction, whereas zinc contributes

to the stability of the enzyme.

2.2.1.2. Glutathione peroxidase: The enzyme catalyses the oxidation of reduced

glutathione (GSSH) to its oxidised form (GSSG), at the expense of hydrogen peroxide.The

active site of the enzyme contains selenium, and in fact, several symptoms of selenium

deficiency have been explained due to lack of glutathione peroxidase. The enzyme is

found at its high activity in liver and moderate activity in heart, lung and brain. It is

predominantly present in cytosal and mitochondrial matrix.

2.2.1.3. Methionine sulphoxide reducatase: The aminoacid methoionine can be oxidised

by free radicals to methionine sulphoxide. The protein within the lens of cataract patient

contains significant amount of this substance. This is removed by the enzyme methionine

sulphoxide reductase.

2.2.2. Defense by micronutrients

These include vitamins and other micronutrients (Hendler, 1995). β-carotene, the

precursor of Vitamin A and tocopherol (Vit E) are lipid soluble oxidant scavengers that

protect biomembrane. Ascorbic acid (Vit C) and GSH are important water soluble


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antioxidants. It has been reported that people who took supplements of Vit A or

β-carotene, Vit E, Vit C, copper and selenium were 37% less likely to develop cataract and

blindness.

2.2.2.1. β-carotene: There is controversy as to wheather only β-carotene is free radical

scavenger or β-carotene and Vit A both. However, the role of β-carotene in protecting cell

membrane from inside is an established fact. It forms primary ring of protection in lipid

compartment.

2.2.2.2. Vitamin E (tocopherol): Vit E is the oldest recognised biological antioxidant. It is

hydrophobic and protects cell membrane from outside. It also protects circulating

lipoproteins. The concentration of lipoproteins in adrenal glands are higher as compared to

that in the mitochondria and Vit E protects these structures against lipid peroxidation by

reacting with lipid peroxide radicals and acts as the chain terminator. Vit E also forms

primary ring of protection in the lipid compartment.

2.2.2.3. Vitamin C (ascorbic acid): This vitamin is hydrophilic and hence forms primary

rings of protection in hydrophilic compartment. Being present in neutrophil in 50 times

more concentration than in extracellular compartment, the primary role of Vit C is

maintaining the phagocytic activity at an optimum level. It is one of the important

antioxidants of cardiovascular and respiratory systems.

2.2.2.4. Minerals: Certain minerals revive the natural antioxidant ring of protection. It has

been established that minerals such as zinc, copper, manganese and selenium are essential

for efficient functioning of human antioxidant defence, especially the enzymes. Copper,

manganese and zinc are essential components and subunits of major human natural

antioxidant enzymes. Selenium activates body's first line of antioxidant defence

i.e., glutathione peroxidase (Maxwell, 1998).


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2.3. Plants as natural antioxidants

In developing countries like India where poverty and malnutrition is rampant,

knowledge of plant derived antioxidants could reduce the cost of health care. India has a

rich history of using various herbs and herbal components for treating various diseases.

Many Indian plants have been investigated for their beneficial use as antioxidants or

source of antioxidants using presently available experimental techniques.

Wong et al., (2006) have reviewed extensively about Temella fuciformis, Alphinia

oxyphylla, Rhodiola sacra, Glycyrrhiza uralensis, Astragalus membranaceus, Polygonum

multiflorum, Psoralea corylifolia, Morus alba and several others that have antioxidant

activity and used in Chinese traditional medicine. Recently, Siriwatanametanon et al.,

(2010) have reviewed Basella alba, B. rubra, Cayratia tryfolia, Gynera pseudochina,

Muehlenbeckia platyclada, Oroxylum indicum, Pouzolzia indica and Rhinacanthus nasutus

used in Thai traditional medicine and reported to show antioxidant activity

2.4. Cancer

Free radical entity is well proven cause of a vast number of pathological disorders

or diseases affecting various systems of human body. The free radicals induce chain of

reaction leading to initiation of carcinomas, due to DNA damage and mutagenesis (Ansari,

1991).

All cancers begin in cells, which are the fundamental unit of life. These cells grow

and divide in a well-controlled set manner to produce more cells as they are needed to

keep the body healthy. As cells become older or damaged, they die and are replaced with

new cells. However, due to certain reasons this normal condition is interrupted, which

leads to an abnormal behaviour by the cells, typically in one part or organ of the body,

where the cells continue to multiply and live beyond their lifespan. These are referred to as

the cancerous cells. The term cancer is used to describe a medical condition, where there is


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abnormal and uncontrolled multiplication of the body cells. These mutant cells can migrate

and invade other parts of the body through blood and lymph. Manifestation of the disease

is in the form of a tumor, a group of mutant cells that form tissue. These can affect all

living cells in the body, at all ages and in both genders. The cause is multi-factorial and the

disease process differs at different sites.

2.4.1. Classification of cancer

‘Cancer’ is not a single disease but it refers to a group of diseases which share

similar characteristics. Classification of cancer is on the basis of the tissue from where it

originates. For example, carcinoma is a malignancy that arises in the skin, the lining of

various organs, or glandular organs or tissues. Most carcinomas affect organs or glands

capable of secretion, such as the breasts, lungs, colon, prostate and bladder. Sarcoma is a

malignancy arising in bone, muscle, or connective tissue. The most common sarcoma often

develops as a painful mass on the bone. Leukemia, a neoplastic disease of the white blood

cells, arises in bone marrow. Leukemia is commonly classified into acute and chronic

forms. These are further broken down according to the type of white blood cells affected

by the disease. Lymphoma develops in the glands or nodes of the lymphatic system, a

network of vessels, nodes, and organs that purify bodily fluids and produce infection-

fighting white blood cells or lymphocytes (Thun et al., 2011).

2.4.2. Mechanism of cancer

Cancer starts when the genetic material (DNA) of a cell can become damaged or

changed, producing changes known as mutations, which affect normal cell growth and

division. It is not always clear as to what causes mutations. Some mutations are inherited,

some may be due to diet, and others might be caused by exposure to environmental factors,

which are referred to as carcinogens such as some chemicals, tobacco, etc. When this


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occurs, cells do not obey the normal life cycle, so the old and damaged cells do not die, but

new ones are still formed leading to large number of cells than required by the body. These

excess cells form a mass of tissue, which is called a tumor. However every tumor may not

be cancerous. The medical terms to differentiate between a tumor and a cancerous tumor

are benign and malignant. Tumor that is not cancerous is referred to as benign tumor and

this can often be surgically removed. In most cases, rarely there is a recurrence of the

condition after surgery and remains contained within its organ of genesis and does not

invade or spread to other organs and other parts of the body.

Malignant tumors are cancerous. Cells in these tumors can invade nearby tissues

and spread to other parts of the body. The spread of cancer from one part of the body to

another is called metastasis. The extent of growth of cells in the originating tissue, its

invasion to nearby lymph nodes and spread to distant organs determines the seriousness,

the impact and the line of treatment of the disease. This assessment is referred to as staging

of the cancer. Usually cancer of the blood and bone marrow such leukemia do not form

tumor (Scat et al., 1996).

2.5. Apoptosis

The antitumor activity of natural products has been explained, at least in part, by

their ability to trigger cell death pathways, including apoptosis in cancer cells. Apoptosis

or programmed cell death is the cell’s intrinsic death program that plays a pivotal role in

maintaining tissue homeostasis that is highly conserved among different animal species

(Evan and Vousden, 2001). Because apoptosis is involved in the regulation of many

physiological processes, defective apoptosis signalling may contribute to a variety of

different pathological conditions. Thus, increased apoptosis is involved in degenerative

processes affecting neurons, muscle or lymphoid tissues. Conversely, disabled apoptosis is

one of the hallmarks of human cancer cells (Hanahan and Weinberg, 2000). Thus, cancer


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cells have a marked tendency to disable the mitochondrial (intrinsic) pathway of apoptosis.

Besides their vital function for cellular bioenergetics, mitochondria play a key role in the

regulation of the point of no return during apoptosis.

According to the statistics, more than 75% of cancers have an environmental origin

(Namiki, 1990; Moller et al., 1996). Genetic damages, changes in DNA sequences, gene

mutations and other changes in chromosomal structure play an important role in cancer

(Mccord, 1994). Most of mutagenic and carcinogen agents display their destructive effects.

2.6. Angiogenesis

Angiogenesis, the formation of new blood vessels from preexisting endothelium, is

a fundamental step in a variety of physiological and pathological conditions including

wound healing, embryonic development, chronic inflammation, tumor progression and

metastasis (Folkman, 1971; Folkman, 1995; Folkman, 1996; Folkman and Chesney, 1997;

Hanahan et al., 2007; Karamysheva, 2008). Complex and diverse cellular actions are

implicated in angiogenesis, such as extracellular matrix degradation, proliferation and

migration of endothelial cells, and morphological differentiation of endothelial cells to

form tubes (Goh et al., 2007). The angiogenic process is tightly controlled by a wide

variety of positive or negative regulators, which are composed of growth factors,

cytokines, lipid metabolites, and cryptic fragments of haemostatic proteins, and many of

these factors are initially characterized in other biological activities (Ding et al., 2008).

Among these, vascular endothelial growth factor (VEGF), a soluble angiogenic factor

produced by many normal and tumor cells, plays a key role in regulating normal and

abnormal angiogenesis (Asahara and Inser, 1999). VEGF is found to stimulate endothelial

cells to secrete proteases and plasminogen activator, resulting in the degradation of vessel

basement membrane, which in turn allows cells to invade the surrounding matrix


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(Bao et al., 2008; Houck et al., 1991). After subsequent migration and proliferation, the

cells finally differentiate to form a new vessel. Enhanced expression of VEGF has been

observed in many human cancers including rectal, breast, non-small cell lung and ovarian

cancers (Gasparini and Harris, 1995). High levels of VEGF have been found in a variety of

effusions accompanying pathologic disorders like edema formation in the brain, human

rheumatoid synovial fluid and malignant ascites (Stoelcker, 2000).

A balance between angiogenic and anti-angiogenic factors has given rise to a

significant interest in the use of exogenous anti-angiogenic agents for the treatment of

solid tumors and it has been demonstrated that anti-angiogenic treatment retards tumor

growth (Noonan, 1997). Although several new chemotherapeutic drugs of both synthetic

and natural origin are being discovered from time to time, disease like cancer lacks

satisfactory solutions. There has been a continuous search for compounds useful in the

prevention or treatment of cancer, especially for agents with reduced toxicity. It is well

established that neoplasms cannot grow beyond a certain size without adequate blood

supply. Formation of new blood vessels from existing vasculature or ‘angiogenesis’ is

characteristic of all cancer types and serves as a route of nutrition, oxygenation and

metastasis (Folkman et al., 1989). Therefore, anti-angiogenic therapy for cancer has been

considered as an attractive proposition.

2.6.1. Plants as anti-angiogenic agents

Since angiogenesis is important in the pathogenesis of various diseases, the

inhibition of angiogenesis is one of promising approaches in their treatment. Angiogenesis

plays a prominent role in cancer cell survival, tumor growth and metastasis and its

inhibition is considered to be an important strategy for cancer therapy (Chen et al., 2005).

Disruption in the signal pathway to angiogenesis can give rise to the blockage of


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angiogenesis (Shawver et al., 1997). Since angiostatin (O’Reilly et al., 1994) and

endostatin (O’Reilly et al., 1997) were identified to inhibit angiogenesis, there have been a

variety of antiangiogenic components isolated from natural products such as

psammaplin A from a marine sponge (Shim et al., 2004), erianin from Dendrobium

chrysotoxum (Gong et al., 2004), shiraiachrome A and 11,11′-dideoxyverticillin from

Shiraia bambusicola (Tong et al., 2004; Chen et al., 2005), epigallocatechin-3-gallate

from dried tea leaves (Fassina et al., 2004), pseudolarix acid B from Pseudolarix

kaempferi (Tan et al., 2004), withaferin A from Withania somnifera (Mohan et al., 2004),

and geniposide from Gardenia (Koo et al., 2004a,b). Some of them are known to inhibit

aminopeptidase N, suppress receptor phosphorylation, antagonize VEGF-mediated anti-

apoptosis, and disrupt endothelial tube formation. The aqueous extracts of Berberis

paraspecta, Catharanthus roseus, Coptis chinensis, Taxus chinensis, Scutellaria barbata

Polygonum cuspidatum and Scrophularia ningpoensis have been reported to have strong

anti-angiogenesis activity (Brakenhielm et al., 2001; Kimura and Okuda, 2001).

2.7. Antitumor activity

Several plant products have been tested for anticancer activities and some of them

like vincristine and taxol are now available as a drug of choice (Vandana, 2005). The rich

and diverse plant resources of India are likely to provide effective anticancer agents. One

of the best approaches in the search for anticancer agents from plant resources is the

selection of plants based on ethnomedical leads and testing the selected plant’s efficacy

and safety in the light of modern science.

Natural products have served as a source of drugs for centuries and about half of

the pharmaceuticals in use are derived from natural products (Clark, 1996). Dependence

on plants as the source of medicine is prevalent in developing countries where traditional


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medicines play a major role in health care (Austin, 1991). Several plant extracts and plant

products have been shown to possess significant anti-tumour and anti-inflammatory

activities (Bhattacharya et al., 1997). A dehydrochalcone, isolated from Pityrogramma

calomelanos was found to be cytotoxic and tumour reducing (Sukumaran and Kuttan

1991). Administration of P. amarus extract has been shown to inhibit the liver tumour

development induced by N-nitrosodiethylamine in rats and increased the life span of

hepatocellular carcinoma harboring animals (Joy and Kuttan, 1998; Rajeshkumar and

Kuttan, 2000). The partially purified component of Solanum trilobatum, known as

sobatum, was obtained from the petroleum ether:ethyl acetate (75:25) extractable portion.

It was found to be cytotoxic to Dalton’s lymphoma ascites (DLA) and Ehrlich ascites (EA)

cells. Sobatum significantly inhibited peritoneal tumour induction and was found to reduce

solid tumour growth in mice injected with DLA and EA tumour cells (Mohanan and

Devi, 1986).

2.7.1. Ehrlich ascites carcinoma

Experimental tumors have great importance for the purpose of modeling, and

Ehrlich ascites carcinoma (EAC) is one of the commonest. Originally it appeared firstly as

a spontaneous breast cancer in a female mouse (Takin, 2002; Ozaslan et al., 2001), and

then Ehrlich and Apolant in 1905 used it as an experimental tumor by transplanting tumor

tissues subcutaneously from mouse to mouse. In 1932, Loewenthal and Jahn obtained the

liquid form in the peritoneum of the mouse and named it as “Ehrlich ascites carcinoma”

due to the ascites liquid together with the carcinoma cells. EAC is referred to as an

undifferentiated carcinoma, and is originally hyperdiploid, has high transplantable

capability, non-regression, rapid proliferation, shorter life span, 100% malignancy and also

does not have tumor specific transplantation antigen (TSTA) (Kaleo lu and Li, 1977).


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The effusion, which contained neoplastic cells that are proliferated after injection

of tumor cells into the peritoneal cavity, is referred to as the “ascites”. Frequently, tumor

virulence increases via repetitious passages, while the proliferating rate of such tumors

increases gradually. However, differentiation gradually disappears, while the cells get free

growth control mechanisms, gain hetero transplantability and in the end, are converted to

the ascites form. Ascites liquid is grey-white, or sometimes has a light bloody viscose

liquid and contains 10 million neoplastic cells in 0.1cc (Aktas, 1996). The reason for its

wide usage is that the suspension contained homogeneous free tumor cells of the Ehrlich

ascites tumor, and in this way, it has a transplantable capacity for certain quantitative

tumor cells to another mouse (Klein, 1951). Therefore, it is not only the tumor cell count

that is transplanted, but also, the growing tumor size can be determined by common basic

counter systems (Ekinci, 2000). If ascites fluid injected into peritoneal cavity contains the

tumor cells, the ascites form is obtained, but a solid form is obtained when the tumor cells

are injected into muscle tissue (Zeybek, 1996; Okay, 1998). EAC cells grow in suspension

in the peritoneal cavity of mice and they do not adhere to the synthetic surface in vitro

(Lazebnik et al., 1991; Vinuela et al., 1991; Song et al., 1993; Akta, 1996). In 4 to 6 days

after passage, the ascites fluid is formed and a total of 5 to 12cc ascites fluid is

accumulated (Gümühan, 2002).

2.8. Mutagenicity

Mutations are the cause of inborn errors of metabolism leading to morbidity and

mortality in living organisms. Besides the inherited metabolic disorders, a spectrum of age

related human diseases, including cancer, are caused by mutations. Mutagenic agents may

be synthetic or natural toxic substances. Since cancer has become the number one cause of

death, much attention has been focused on the chemoprevention of cancer, with little


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success. However, less attention has been given to the substances in medicinal plants and

herbal medicines that may serve to protect against chemical mutagens or carcinogens

acting as initiators in the carcinogenic process (Shona et al., 2004).

The rich diversity of Indian medicinal plants has not yet been systematically

screened for antimutagenic activity. Many plant species are known to elicit

antimutagenesis and thus have a full range of prospective applications in human

healthcare. Even for populations which use herbs traditionally, encouraging the use of

species with chemopreventive actions could be helpful as part of the life expectancy

improvement strategies where the costs are significantly low, herbs have usually little or

no toxicity during long-term oral administration and are relatively available at large scale.

It has been suggested that regular consumption of anticarcinogens and antimutagens in the

diet may be the most effective way of preventing human cancer and search for novel

antimutagens acting in chemoprevention is a promising field in phytotherapy (Bala and

Grover, 1989).

Antimutagenic properties elicited by plant species have a full range of prospective

applications in human health. Herbal remedies and phytotherapy drugs, containing active

principles are currently developed to protect against electrophile (e.g, free radical) attack

on DNA and its widespread outcomes such as ageing and cancer. The occurrence rate of

cancer is increasing worldwide and the determination of chemopreventive or

chemoprophylaxis compound is important in the effort to reduce the risk of cancer. A plant

extract indicating antimutagenicity is not necessarily an anticarcinogen; however, it is an

indication of possible candidates for such purposes (Ghazali et al., 2011).

Gene mutations are readily measured in bacteria and other cell systems when they

cause a change in the growth requirements of the cell, whereas chromosome damage in


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mammalian cells is typically measured by observing the cell’s chromosomes under

magnification for breaks or rearrangements. The Salmonella typhimurium microsome

assay (Salmonella test; Ames test) is a widely accepted short-term bacterial assay for

identifying substances that can produce genetic damage that leads to gene mutations. The

test uses a number of Salmonella strains with pre-existing mutations that leave the bacteria

unable to synthesize the required amino acid histidine, and therefore unable to grow and

form colonies in its absence. New mutations at the site of these pre-existing mutations, or

nearby in the genes, can restore the gene’s function and allow the cells to synthesize

histidine. These newly mutated cells can grow in the absence of histidine and form

colonies. For this reason, the test is often referred to as a reversion assay

(Kristien et al., 2000). Natural antimutagens from edible and medicinal plants are of

particular importance because they may be useful for human cancer prevention and have

no undesirable xenobiotic effects on living organisms. Natural antioxidants may reduce or

inhibit the mutagenic potential of mutagens and carcinogens. The cellular mutability

control by natural antimutagens can provide ways for preventing mutations that

conceivably result in cancer as well as diseases caused by genotoxic agents (Negi, 2003;

Zahin, 2010). Mutagenicity can also be useful as an anticancer tool, as most anticancer

drugs are mutagenic (e.g., the spindle-disturbing substances taxol and vinblastine).

2.8.1. Plants as antimutagenic agents

Determination of antimutagenicity of plant extracts is important in the discovery of

new effective anticarcinogenic treatments. A plant extract indicating antimutagenicity is

not necessarily an anticarcinogen, however, it is an indication of a possible anticarcinogen.

Bauhinia galpinii, Buddleja saligna, Clerodendrum myricoides, Millettia sutherlandii and

Sutherlandia frutescens were reported as antimutagenic plants. The toxicity of

Datura stramonium is well documented and had been linked to deaths and poisonings for


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centuries (Friedman, 2004; Steenkamp et al., 2004). Surprisingly, the leaf and seed pod

material indicated antimutagenicity. This species has many uses in traditional medicine

(Van Wyk et al., 1997), but is administered at low concentrations. Hypoxis

hemerocallidea, Sutherlandia frutescens, Catharanthus roseus and Tulbaghia violaceae

were screened for antimutagenic activity (Hutchings et al., 1996; Van Wyk et al., 1997).

Of the extracts screened, only the extract of S. frutescens indicated activity at the highest

concentration. S. frutescens is commonly known as the ‘kankerbos’ ‘cancer bush’ and

antimutagenicity in the leaf and stem mixture was anticipated.

2.9. Genotoxicity

The screening of new drugs for potential genotoxicity is an important step during

research and development. For this purpose, short-term in vitro assays such as the alkaline

Comet assay and the Cytokinesis Block Micronucleus (CBMN) test are used to identify

genotoxic compounds and to select non-genotoxic ones for further therapeutic utilization

(Fenech, 2000; Kiskinis et al., 2002; Hartmann et al., 2003).

Crucial early event in carcinogenesis is the induction of the genomic instability

which enables an initiated cell to evolve into a cancer cell by achieving a greater

proliferative capacity (Fenech and Crott, 2002). It is well known that cancer results from

an accumulation of multiple genetic changes that can be mediated through chromosomal

changes that have the potential to be cytogenetically detectable (Solomon et al., 1991). It

has been hypothesized that the level of genetic damage in peripheral blood lymphocytes

reflects the amount of damage in the precursor cells that lead to the carcinogenic process in

target tissues (Hagmar et al., 1998).

The cytokinesis-block micronucleus (CBMN) assay in human lymphocytes is one

of the most commonly used methods for measuring DNA damage because it is relatively


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easier to score micronuclei than chromosome aberrations. Micronuclei originate from

chromosome fragments or whole chromosomes that fail to engage with the mitotic spindle

and therefore lag behind when the cell divides. Compared with other cytogenetic assays,

quantification of micronuclei confers several advantages, including speed and ease of

analysis, no requirement for metaphase cells, and reliable identification of cells that have

completed only one nuclear division. This prevents confounding effects caused by

differences in cell division kinetics because expression of micronuclei, nucleoplasmic

bridges, or nuclear buds is dependent on completion of nuclear division (Umegaki and

Fenech, 2000). Because cells are blocked in the binucleated stage, it is also possible to

measure nucleoplasmic bridges originating from asymmetrical chromosome

rearrangements and/ or telomere end fusions (Stewenius et al., 2005). Nucleoplasmic

bridges occur when the centromeres of dicentric chromosomes or chromatids are pulled to

the opposite poles of the cell at anaphase.

In the CBMN assay, binucleated cells with nucleoplasmic bridges is easily

observed because cytokinesis is inhibited, preventing breakage of the anaphase bridges

from which nucleoplasmic bridges are derived. Thus, the nuclear membrane forms around

the nucleoplasmic bridge. Both micronuclei and nucleoplasmic bridges occur in cells

exposed to DNA-breaking agents (Fenech et al., 2002). In addition to micronuclei and

nucleoplasmic bridges, the CBMN assay allows for the detection of nuclear buds, which

represent a mechanism by which cells remove amplified DNA and are therefore

considered a marker of possible gene amplification. The CBMN test is gradually replacing

the analysis of chromosome aberrations in lymphocytes because micronuclei,

nucleoplasmic bridges, and nuclear buds are easy to recognize and score and the results

can be obtained in a shorter time (Serrano, 2001).


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2.9.1. Plants as antigenotoxic agents

Investigation of the mutagenic and antimutagenic potentials of herbal plants used in

traditional medicines is generating great interest with the growing evidence for their safe

consumption and or low genotoxic effects over the long term (Elgorashi et al., 2003;

Verschaeve et al., 2004; Deciga Campos et al., 2006). However, some medicinal plants

exhibited mutagenic activity (Cordoso et al., 2006). The frequency of the presence of

micronuclei serves as a measure of in vitro or in vivo exposure to mutagens and

carcinogens. The micronucleus assay has been used in the toxigenetic evaluation of

Ambelania occidentalis, a plant rich in alkaloids for cancer treatment (Castro et al., 2009)

and was found to be nongenotoxic. Similarly, the micronucleus test showed that

Memecylon umbellatum (used for the treatment of gonorrhea) was not genotoxic

(Shetty et al., 2010). Inula viscosa has been evaluated for its genotoxic properties. The

aqueous leaf extracts of I. viscosa induced significant amounts of chromosomal aberrations

and micronucleus formation (Askin and Aslanturk, 2010).

2.10. Antimicrobials

Infectious diseases are the world’s leading cause of premature deaths, killing

almost 50,000 people every day. In recent years, drug resistance to human pathogenic

bacteria, fungi and viruses has been commonly reported from all over the world (Piddock

and Wise, 1989; Singh et al., 1992; Mulligen et al., 1993; Davis, 1994; Robin et al., 1998).

However, the situation is alarming in developing as well as developed countries due to

indiscriminate use of antibiotics. The drug-resistant bacteria, fungal pathogens and viruses

have further complicated the treatment of infectious diseases in immune compromised,

AIDS and cancer patients (Rinaldi, 1991; Diamond, 1993). In the present scenario of

emergence of multiple drug resistance to human pathogenic organisms, this has

necessitated a search for new antimicrobial substances from other sources including plants.


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2.10.1. Plants as antibacterial agents

Plants are used to treat common infectious diseases and some of the traditional

medicines are still included as part of the habitual treatment of various maladies.

For example, the use of bearberry (Arctostaphylos uva-ursi) and cranberry (Vaccinium

macrocarpon) juice to treat urinary tract infections is reported in different manuals of

phytotherapy, while species such as lemon balm (Melissa officinalis), garlic (Allium

sativum) and tee tree (Melaleuca alternifolia) are described as broad-spectrum

antimicrobial agents (Heinrich et al., 2004). Plants such as Allium sativam, Zingiber

officinale, Cinnamomum cassia, Cinnamomum verum, Thymus vulgaris, Salvia

officinalis, Origanum vulgare, Rosmarinus officinalis, Ocimum basilicum,

Lavandula sp., Mentha piperita, Monarda fistulosa, Monarda didyma, Hydrastis

canadensis, Berberis aquifolium, Berberis vulgaris and Coptis chinensis have antibacterial

activity against Staphylococcus sp., Streptococcus sp., Proteus sp., Pseudomonas sp.,

Mycobacterium sp., Escherichia coli, Salmonella sp., Clostridium sp., Klebsiella sp., and

Bacillus subtilis.

2.10.2. Plants as antifungal agents

Fungal diseases represent a critical problem to health and they are one of the main

causes of morbidity and mortality worldwide (CSIR, 1998). Human infections, particularly

those involving the skin and mucosal surfaces, constitute a serious problem, especially in

tropical and subtropical developing countries. In humans, fungal infections range from

superficial to deeply invasive or disseminated, and have increased dramatically in recent

years. The treatment of mycoses has lagged behind bacterial chemotherapy and fewer

antifungal than antibacterial substances are available. Therefore, a search for new

antifungal drugs is extremely necessary (Portillo et al., 2001).


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Acalypha fruticosa, Bauhinia tomentosa, Caesalpinia pulcherrima, Cassia alata,

Cinnomomum verum, Costus speciosus, Diospyros ebenum, Elephantopus scaber,

Hydnocarpus alpine, Hyptis suaveolens, Ichnocarpus frutescens, Mundulea sericea,

Ocimum basilicum, Osbeckia chinensis, Peltophorum pterocarpum, Punica granatum,

Sphaeranthus indicus, Tinospora cordifolia and Toddalia asiatica have activity against

dermatophytes and opportunistic pathogenic fungi (Fortes, 2008).

2.10.3. Plants as antiviral agents

Genital herpes has been a public health concern worldwide (Nahmias et al., 1973).

The infections are caused mostly by herpes simplex virus type 2 (HSV-2) although a

proportion of cases is attributable to HSV-1 (Kalinyak et al., 1977; Corey et al., 1983;

Yoosook et al., 1989). The manifestation of disease may not be so severe in normal or

immune competent hosts, however, a number of patients always encounter recurrent

attacks (Reeves et al., 1981; Whitley and Roizman, 1997). Effective antiherpes drugs are

now available. However, they are very expensive and most patients with frequent attacks

may not be able to afford the cost of long-term treatment. Moreover, the number of cases

will probably increase with time, especially those who have also been infected with human

immunodeficiency virus (Hammer and Inouye, 1997). For this reason, search for new,

effective and inexpensive drugs from natural sources is necessary.

The phytochemical, podophyllotoxin, isolated from the aqueous extract of

Podophyllum peltatum and Phyllanthus urinaria inhibited HSV type 1 (HSV-1). The roots

of the medicinal plant Carissa edulis, have remarkable anti-HSV activity in vitro and in

vivo for both wild type and resistant strains of HSV. Barleria lupulina and Clinacanthus

nutans extracts could inactivate HSV-2 directly. The virucidal activities appeared to be

much higher for B. lupulina than for C. nutans extracts (Tolo et al., 2006).


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2.11. The family Ancistrocladaceae

Ancistrocladaceae is a small palaeotropical family of flowering plants. By rbcL

(ribulose1, 5-bisphosphate carboxylase) gene sequence comparison (Albert, 1992), they

have been found to belong to an ‘extended caryophyllid’ clade of families (including

Droseraceae, Nepenthaceae, Plumbaginaceae, Polygonaceae, Tamaricaceae, and

Frankeniaceae), which had formerly been placed at widely separated positions in the plant

kingdom (Schlauer, 1997). Further studies, that also considered anatomical and

phytochemical data, support the grouping of these families within one common clade

(Hegnauer, 1989).

Species of the Dioncophyllaceae and the Ancistrocladaceae, the two small tropical

plant families known in the traditional medicine of several tropical countries, were shown

to contain naphthylisoquinoline alkaloids (Ruangrungsi et al., 1985; Pokorny, 1995).

These compounds display, amongst a wide range of other biological effects, activities

against Plasmodium falciparum and Plasmodium berghei in erythrocytic forms

(Boyd et al., 1994).

2.11.1. Pharmacological and phytochemical properties of the genus Ancistrocladus

Belonging to the genus Ancistrocladus due to the presence of naphthylisoquinoline

alkaloids, the tropical lianas show remarkable antitrypanosomal (Bringmann and

Feineis, 2000; Bringmann, 2003), antileishmanial (Bringmann et al., 2000), fungicidal

(Bringmann et al., 1992), and antimalarial activities. The crude extract of A. tectorius is

used in traditional medicine to treat dysentery and malaria (Ruangrungsi et al., 1985) and

has additionally been found to exhibit antiviral (Said et al., 2001) and antitumoral

activities (Chen et al., 1981). A. cochinchinensis, a large hooking climber growing as an

endemic species in the south of Vietnam (Hoang, 1991), is used as diuretic, antifebrile and


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antiphlogistic agent. Bioactive constituents of A. robertsoniorum, a tropical liana

indigenous to Kenya has a rich source of secondary metabolites (LeÂonard, 1984; Isahakia

and Robertson, 1994).

The antiviral alkaloids from crude extracts of the tropical liana A. Korupensis are

reported to have HIV inhibitory michellamines as well as the antimalarial korupensamines.

These alkaloids were used by the U.S. National Cancer Institute (NCI) for anti-HIV drug

development and clinical trials. Extracts of this tropical plant material tested in vitro were

active against HIV, and bioassay guided fractionation led to the discovery of

michellamine B. The tested compound showed essentially equivalent activity against

HIV-1 and HIV-2, and toxicity in test animals (Boyd, 1988).

Some compounds of A. korupensis have high antimalarial activities against

Plasmodium falciparum both in vitro and in vivo and showed remarkable activities against

the pathogens of Leishmaniasis, Chagas disease and African sleeping sickness

(Bringmann et al., 2000).

2.11.2. Ancistrocladus heyneanus

Ancistrocladus heyneanus Wall. ex J. Graham a woody climber from tropical

forests of Western Ghats of India is the only species representing the monogeneric family

Ancistrocladaceae in India (Pai et al., 2008). The first representatives of the interesting

class of alkaloids of the family have first been isolated from A. heyneanus (Govindachari

and Parthasarathy, 1970; Govindachari et al., 1972, 1973).

A. heyneanus is used by the traditional medicine practisers in Tamilnadu and

Kerala for the treatment of various types of cancers. However, due to lack of scientific data

for this plant, there is an immense need of a high throughput screening of this medicinal

plant A. heyneanus. This can be achieved by analysing their characters by employing

pharmacological tools.


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