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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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