1.

INTRODUCTION

Biological organisms maintain their species-specificity and

identity mainly by the unchanged genomic structure throughout their
life period. In multicellular organisms starting from zygote to the death
of organisms, at each stage, the ratio of cell growth and cell death is
very strictly balanced through development and homeostatic processes
(Bolsover et al., 1997).

Cancer is a disease characterized by disorderly division of cells,

combined with the malignant behaviour of these cells. Malignant cancer cells
tend to spread, either by direct growth into adjacent tissue through invasion, or
by implantation into distant sites by metastasis (the process where by Cancer
Cells can move through the blood stream or lymphatic system to distant
locations). Cancer may affect people at all ages, but risk tends to increase
with age. It is one of the principal causes of death in developed countries
(Cooper, 1993).

Cancer is the common term for all the malignant tumours, the types of
tumours include Benign and Malignant. The tumour causing no danger to
health is called benign. Any tumour that cause danger (Cotran , 2003).

However, it is preferable to speak of different cancers affecting different

organs or structures, like cancer in liver, gall bladder, tongue, breast etc.
Cancer differs not only in tissue origin and morphological appearance but also
in etiology, age and sex, incidence, clinical course, prognosis and many other
factors (Ranajit Sen, 2004).

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 Lymphoma is a type of cancer that originates in lymphocytes or, more
rarely, of histocytes. Collectively, these cell types circulate in the vessels of the
lymphatic system. There are many types of lymphoma. Lymphomas are part of
the broad group of diseases called hematological neoplasms (Sabin, 1902).

The lymphatic system consists of organs, such as the spleen, lymph

nodes and lymphatic vessels that manufacture leukocytes. It is also responsible
for moving lymph fluid to the bloodstream. This fluid is made of plasma and
various types of white blood cells, which fight some types of infection and
other threats, such as cancer (Guillermo Oliver and Michael Detmar, 2002).

Lymphoid tissue is formed by various types of immune cells that work

together to resist infection and other threats, such as cancer. Lymphocytes are
the main type of cell found in the lymphoid tissue. In patients with lymphoma,
the lymphocytes become abnormal and grow in an uncontrolled fashion. As the
cells continue to grow and divide, the lymph glands or other organs in which
the lymphocytes begin to enlarge. The cells form tumours that begin to develop
in the body. Organ function may become affected as the lymphocyte masses
grow larger, making it more difficult for normal cells to function
(http://en.wikipedia.org).

Because lymphoid tissue is present in many parts of the body,

lymphomas can develop almost anywhere. In addition, lymphomas are capable
of spreading to other areas of the body including the liver, bone marrow and
spleen.

There are more than 25 different types of tumours that are considered as
lymphomas. These tumours can be classified into two types

Hodgkin’s lymphoma
Non – Hodgkin lymphoma (www.lymphoma.org).

2
 Hodgkin’s lymphoma category, also known as Hodgkins disease, is
named after the physician who identified it in 1832. All other types of
lymphomas are considered non-Hodgkins lymphoma.

Hodgkins disease is a type of lymphoma or cancer of the lymphoid

tissue. Lymphoid tissue is part of the lymphatic system. A major component of
the immune system, the lymph system consists of organs, lymph nodes and
vessels. The lymphatic system is responsible for manufacturing and
transporting lymph (Fluid made of plasma and white blood cells) from tissues
to the blood stream (http://en.wikipedia.org).

Non-Hodgkin’s lymphoma can start anywhere, it generally develops in

the lymph nodes. In some cases, the disease can develop in patches of
lymphatic tissue in organs such as the spleen, stomach or intestines.

The symptoms and clinical courses of this disease are diverse. Usually,
the first indication is swelling of the lymph gland, enlarged tonsils and
adenoids and painless, rubbery nodes in the cervical preclavicular areas.
Children usually develop enlarged nodes in the cervical region, dyspnea and
coughing with the progression of the disease symptoms specific to the area
involved and systemic complaints of fatigue, malaise, weight loss, fever and
night sweats appear. Diagnosis requires histologic evaluation of biopsied
lymph nodes or tonsils, bone marrow, liver bowel or skin (Ranajit Sen, 2004).

The major risk factors for cancer are tobacco, alcohol consumption,
infectins, dietary habits and behavioural risk factors. This offers the prospect
for initiating primary and secondary prevention measures for control and
prevention of cancers (Murthy and Mathew, 2004).

3
 Cancer is essentially a problem of abnormal cell growth. Under the
influence of chemicals, viruses, and free radicals, normal cells are converted to
tumour masses that divide in uncontrolled manner (Nwafor et al., 2001).

Free radicals have been implicated in more than one hundred disease
conditions in humans, including cardiovascular, brain and ocular dysfunctions,
arthritis, ischemia and reperfusion injury of many tissues, diabetes, tumour
promotion and carcinogenesis, and AIDS. Antioxidants / free radical
scavengers function as inhibitors at both initiation and promotion / propagation
/ transformation stages of tumour promotion / carcinogenesis and protect cells
against oxidative stage. Antioxidants inhibit both initiation and promotion
stages in carcinogenesis and counteract cell immortalization and
transformation. Oxygen in the form of free radicals (molecules, which are
highly reactive, they bind and destroy body components) affect the normal cell
and can cause cancer. The free radicals also have been known to cause many
diseases including heart disease. (Halliwell et al., 1992).

In our body, the activity of free radicals are naturally controlled and
delimited by another group of chemical compounds called “antioxidants”,
which are present in higher levels in our regularly consumed food materials.
The beta-carotene, Vitamin C and Vitamin E (Tocopherol) are some important
antioxidant substances. These are known to be effective against cancer by
scavenging the accumulated free radicals in our body. In normal cells the ratio
of free radicals and antioxidants are strictly balanced (Heinonen, 1998).

Under certain conditions, the group of free radicals is increased above

normal that stimulates the normal cells to become cancerous. The reactive
oxygen species like super oxide anions, hydroxyl radicals and hydrogen

4
peroxide are produced as by-products of oxidative respiration and lipid
metabolism. All these are permanent enemies integrity of DNA within the cell
(Stevenson et al., 1994).

High consumption of fruits and vegetables is associated with reduced

risk of several cancers including lung, oral, pancreas, larynx, Oesophagus,
bladder, stomach and cervical cancers (AICR, 1997).

The consumption of edible plants, fresh fruits and green vegetables has
been demonstrated to prevent the occurrence of a number of diseases in
humans and animals. Fresh vegetables, fruits and their seeds and rich sources
of Vitamins C, E and  carotene, and / or protease inhibitors. Compounds
which may protect the organism against free radical – induced injury and
diseases (Hocman, 1989).

Cancer, Chemoprevention is a new promising strategy for prevention,

inhibition or reversal of carcinogenesis, induced by specific natural or synthetic
chemicals (Hanspeter et al., 2000). For a large number of the world’s rural
population, medicinal plants are the only source for the prevention and
treatment of various pathological diseases, from time immemorial. Several
medicinal plants and their constitutions have been reported, to prevent,
multistage carcinogenesis (Craig, 1999).

Plant products have been a source of medicinal agents since time

immemorial. From the dawn of civilization, men have been utilizing the
important biological properties of various plants for treatment of different
disease. Even today, plants are the most exclusive source of drugs for the
majority of world’s population and plant products constitute about 25% of
prescribed medicines (Fransworth and Bingel, 1977; Principe, 1989).

5
 Tephrosia purpurea has been used for centuries in the Indian traditional
medicine, for the treatment of various inflammatory disorders. It is considered
beneficial for liver, spleen and kidney disorders. Also, it has the property to
cure all types of wounds (Joshi, 2000). Experimental studies suggest that the
roots of Tephrosia purpurea exerts antiulcer and antitumour promoting effect
(Saleem et al., 1993).

Objectives of the present investigation:

Evaluation of the antioxidant and antitumour status of the methanolic

extract of Tephrosia purpurea leaves against DLA tumour bearing mice, are
given below:

 Analysing the Toxic effect

 Evaluation of antitumour effect
 Determination of antioxidant activity

6
 2. REVIEW OF LITERATURE

Free radicals are naturally produced by some systems within the body
and have beneficial effect that cannot be overlooked. The immune system is
the main body system that utilizes free radicals. Foreign invaders or damaged
tissue is marked with free radicals by the immune system (www.exrx.net).

Free radical can be defined chemical species possessing an unpaired

electron, which is formed by hemolytic cleavage of a covalent bond of a
molecule, by the loss of a single electron from a normal molecule or by the
addition of a single electron to a normal molecule. Most of the molecular
oxygen consumed by aerobic cells during metabolism is reduced, it becomes
‘activated’ and reacts readily with a variety of biomolecules. This partial
reduction occurs in one electron step, by addition of one, two and four electron
to O2, which leads to successive formation of ROS (Green and Hill, 1984;
Chessman and Slater, 1993).

Free radicals and other active derivatives of oxygen are inevitable

byproducts of biological redox reactions. The effects of the action of free
radicals on membranes include the induction of lipid peroxidation and fatty
acid de-esterification (Arora et al., 2002). Other important consequences of
free radical damage include peroxidation of lipids, oxidation of proteins and
enzymes, strand breaks and adduct formation in nucleic acids (Evans and
Burdon, 1993; Stadtmen and Berlett, 1997; Ward, 1988).

Detrimental effects caused by free radicals occur as a consequence of

imbalance between formation and inactivation of ROS (Ahmed et al., 2000).

7
Reactive Oxygen Species

The oxidants / free radicals are species with very short half life, high
reactivity and damaging activity towards macromolecules like proteins, DNA
and lipids. These species may be either oxygen derived (ROS, reactive oxygen
species) or nitrogen derived (RNS, reactive nitrogen species) (Evans and
Halliwell, 1999).

A free radical can be defined as any molecular species capable of

independent existence that contains an unpaired electron in an atomic orbital.
Many radicals are unstable and highly reactive. They can either donate an
electron to or accept an electron from other molecules, therefore behaving as
oxidants or reductants (Cheeseman et al., 1993).

The generation of free radicals may be accidental or deliberate and the

major source would be leakage from Electron Transport Chain. Free radical
generation is a chain process. The reaction between H 2O2, Fe3+ and O2 produces
OH radicals and several other radicals. In addition to this reaction, Superoxides
are also converted into singlet oxygen where one of the unpaired electron is
moved to high energy orbital and changes its spin direction. It gets paired and
may also react with the double bonds of all the macromolecules present in the
cell. The output of the chain reaction is different types of free radicals and
peroxy radicals (Tripathi, 1998).

The oxygen derived species include Superoxide (O 2-), Hydroxyl ( OH  ),

Hydroperoxyl ( HO.2 ) peroxyl radical ( ROO. ) and alkoxyl radical ( RO. ) as

free radicals. Non radicals are hydrogen peroxide (H2O2), hypochlorous acid

(HOCl), Ozone (O3) and singlet oxygen (  O2 ) (Evans and Halliwell, 1999).

8
 Fig. 1. ROS Metabolism

Superoxide radical

Superoxide anion radical is known to be generated during several

metabolic processes in the respiring cells (Fridovich, 1995). This besides being
itself toxic, generates more toxic species such as OH. which promote lipid
peroxidation (Fridovich, 1976; Utsumi et al., 1977) denaturation of certain
enzymes (Bartosz et al., 1977).

It regulates metabolites capable of signaling and communicating

important informations to the cellular genetic machinery (McCord, 2000).

Hydroxyl radical

The hydroxyl radical, OH  , is the neutral form of the hydroxide ion.

Hydroxyl radicals are highly reactive and consequently short lived; however,
they form an important part of radical chemistry. It is also an important radical
formed in radiation chemistry, since it leads to the formation of hydrogen
peroxide and oxygen.

9
 The hydroxyl radical has a very short in vivo half – life of approx.
10-9 seconds and a higher reactivity. This makes it a very dangerous compound
to the organism. Unlike superoxide, which can be detoxified by superoxide
dismutase, the hydroxyl radical cannot be eliminated by an enzymatic reaction,
as this would require its diffusion to the enzyme’s active site. As diffusion is
slower than the half – life of the molecule, it will react with any oxidizable
compound in vicinity. It can damage virtually all types of macromolecules:
carbohydrates, nucleic acids (mutations), lipids (lipid peroxidation) and amino
acids (e.g. conversion of phenyl alanine to m-tyrosine and o-tyrosine). The
only means to protect important cellular structure is the use of antioxidants
such as glutathione and of effective repair systems (http://en.wikipedia.org).
Hydroxyl radicals are most damaging radicals within the body. This type of
free radical can be formed from O2 and H2O2 via the Harber – Weiss reaction.

Fe2+, Fe2+,
H 2O2  O.2 OH  OH-  O 2  (Prise et al.1989)
-

Harber – Weiss reaction

Singlet oxygen

Singlet oxygen is not a free radical, but can be formed during radical
reactions and also cause further reactions. Singlet oxygen violates Hund’s rule
of electron filling in that it has eight outer electrons existing in pairs leaving
one orbital of the same energy level empty. When oxygen is energetically
excited one of the electrons can jump to empty orbital creating unpaired
electrons. Singlet oxygen can then transfer the energy to a new molecule and
act as a catalyst for free radical formation. The molecule can also interact
with other molecules leading to the formation of a new free radical
(Karlsson, 1997).

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Hydrogen peroxide

Hydrogen peroxide is the most stable ROMs. This is to say that it is the
least reactive and the most readily detected. H2O2 may be generated directly by

divalent reduction of O2 or indirectly by univalent reduction of H 2O2  O.2 .

-

H2O2 is the primary product of the reduction of O 2 by numerous oxidases, such

as XO, uricase, D-amino acid oxidase and α-hydroxy acid oxidase localized in
peroxysome (Oshino et al., 1973).

Peroxy radical

Protonation of O2 gives rise to ·OOH radical, which can initiate

peroxidation.

e- Dismutation
Reaction catalase
- [2H2O2 2H2O + O2
O2 O2
[2O2- + 2H H2O2 + O2 H2O2 Glutathione peroxidase
Superoxide
Ground Hydrogen [H2O2 + 2GSH GSSG + 2H2O]
State peroxide
Oxygen
(Fe2 + H2O2 Fe3+ + OH + OH -)
NADP+ NAD(P)H
Fenton Reaction
Fe2+
[O2 + H2O2 O2 + OH + OH - ]
Haber - Weiss Reaction

OH Hydroxy Radical

Fig. 2. Generation of ROS in cells. The major forms of ROS and their mechanism in
cell chemical (Chance et al., 1979)

Oxidative stress and Biomolecules

Oxidative stress

When overall generation of ROS and RNS exceeds the total antioxidant
activity in the body, the resulting condition is called oxidative stress. This
stress may be mild or severe (Irshad and Chaudhuri, 2002).

11
 Oxidative stress results in the damage of biopolymers including nucleic
acid, protein, polyunsaturated fatty acids and carbohydrates (Khajuria, 1997).

Severe oxidative stress

(negative effect)

Excess ROS/RNS & Low antioxidant defense

Damage to biomolecules (Lipid, DNA, Protein)

Lipid peroxidation DNA damage Protein damage

(Damage to membrane ion (started breakage (Damage to
receptor,
channel, ion transports). Base Modification) enzyme ion
channel)

Raised intracellular Ca2+ Raised

intracellular
Ca2+

Cellular damage with release of more radicals

Cell death and tissue damage

Carcinogenesis, atherosclerosis, aging etc.

Fig. 3. Severe oxidative stress

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Lipid peroxidation

Primary target of free radicals are unsaturated bonds in the lipids of

membranes. These leads to the loss of membranes fluidity, receptor alignment
and cellular lysis. It also attacks the sulphur containing enzymes resulting in
the inactivation, cross linking and denaturation. In a living system, lipid
peroxydation is induced by free radicals and reactive oxygen species, which
ultimately damage the cells. The free radicals damage the membranes and
thereby change the receptor’s alignment and ion channels. These changes
finally affect the normal functioning of a cell by affecting the process of signal
transduction (Tripathi, 1998).

Fig. 4. Metabolism of LPO

Oxidative DNA damage

ROS can cause oxidative damage to DNA both nuclear and

mitochondrial. The nature of damage includes mainly base modification,
deoxyribose oxidation, strand breakage and DNA protein cross – links. The
damage may be the result of extrinsic and intrinsic process including ionizing
radiation, toxic chemical ingestion, UV light exposure and oxygen derived free

13
radicals (Gotz et al., 1994). Reaction of 1O2 with DNA can lead to
strand breaks and formation of altered bases (Moan and Berg, 1992;
Di Mascio et al., 1990).

Damage to proteins and carbohydrates

Reactions of ROS with protein is more selective yielding specific

products. Among the amino acids histidine, trytophan, methionine and
tyrosine are more reactive towards this ROS. Oxidation of these amino acids
results in sulphoxides and short-lived endoperoxides. They may toxic to other
cells (Moan and Berg, 1992; Sies and Packer, 2000; Bandyopadhyay et al.,
1999 ; Devasagayam et al., 1999).

Radical mediated protein oxidation may lead to oxidative modification

of protein side chain, back bone cleavage and protein – protein dimerization.
Sulphur containing side chains are particularly vulnerable to oxidation at
sulphur. Oxidative damage to carbohydrates can also produce products that are
reactive with protein and can result in damage (Stadtman and Berlett, 1997).
Heart
Kidney Brain

Lung Skin
Liver Joints

Krythro cytes Toxic free

GI Tract
radical damage

Blood Vessels

Eye Multiorgan

Cataractogenesis ROS Degenerative retinal

Fig. 5. Effect of oxidative stress on various organs of the body

14
Oxidative Stress and diseases

Oxidative damage crucial to biomolecules due to excess generation of

reactive oxygen species has been implicated as a major cause of organ damage
and hence compounds capable of negative such damage have potential
benefits. The excess generation of ROS during various pathophysiological
states can lead to alterations of the cellular constituents resulting in diseased
conditions (Sreepriya and Devaki, 2001).

Oxidative damage and free radical mediated process have been

implicated in the pathogenesis of a variety of diseases like atherosclerosis,
cancer, liver damage, rheumatoid arthritis, immunological incompetence,
neurodegenerative disorders, (Roy et al., 1998) cancer, diabetes
(Satyanarayana et al., 2004). Ischemia, reperfusion injury, acute hypertension
(Hemnani and Parihar, 1998). Antioxidants thus play an important role to
protect human body against damage by reactive oxygen species.

Cancer

Today it is almost impossible for anyone to escape a personal

experience with cancer for it strikes in two out of every three families. It is a
ravaging disease which consumed one’s flesh and invades internal organs to
produce death. Cancer is a group of disease characterized by the disregulated
proliferation of abnormal cells that invade and disrupt surrounding tissues.
Being a major cause of death its social and economical impact is
overwhelming (Chatterjee et al., 2002).

Cancer was regarded as a disease that began locally and spread

progressively to the regional lymph nodes and only at a late stage via the blood
stream to distant sites. With the discovery of wayward genes, misbehaving
proteins and lots of other information, cancer is in a new shape. Cancer cells
simulate embryonic cells in many ways and again differ from normal cells.

15
16
 Cancer cells are almost similar with embryonic cells on their way to be
a patch of skin or a bundle of nerves. Both of them divide and form ill-defined
clumps, migrate and populate new areas. But while embryonic cells stop
proliferating and mature into adult tissue, the cancer cells just keep dividing
(Ranajit Sen, 2004).

Fig. 7. Stages of tumour development

Tobacco consumption remains the most important avoidable cancer risk,

between 2.5 and 30% of all cancers in developed countries (Tobacco costs
more than you think, WHO, Geneva, 1995). These estimates are based on
occurrence of cancer of mouth, Pharynx, larynx, Oesophages, Lung, bladder
and pancreas (Murthy et al., 1990).

Epidemiological studies carried out in India and abroad have shown that
increased alcohol consumption is casually associated with cancers at various

17
sites, mainly oral cavity, pharynx, larynx and oesophages (Nandakumar et al.,
1990; Sankaranarayanan et al., 1989; Sankaranarayanan et al., 1989 and 1990;
Monographs on the evaluation of carcinogenic risk to humans; Alcohol
drinking, IARC, Lyon, 1988).

Oxidative damage is one of the major causes of cancer induction. A free

radical is a molecule with an unpaired electron. The molecule, which looses an
electron, becomes free radicals giving rise to a self perpetuating chain system
(Shackelford et al., 2000). The majority of carcinogenic agents are regarded as
powerful generators of free radicals that initiate the chain reaction and damage
the cells and its components.

Cancer are classified by the type of cell, that resembles the tumour and,
therefore, the tissue presumed to be the origin of the tumour. Examples of
general categories include:

Fig. 8. Types of Lymphoma

Carcinoma : Malignant tumours derived from epithelial cells. This group represents
the most common cancers, including the common forms of breast, prostate, lung and
colon cancer.

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Lymphoma and Leukemia : Malignant tumours derived from blood and bone
marrow cells.
Sarcoma : Malignant tumours derived from connective tissue, on mesenchymal cells.

Mesothelioma : Tumours derived from the mesothelial cells lining the peritoneum
and the pleura.

Glioma : Tumours derived from glia, the most common type of brain cell.

Germ cell tumour : Tumours derived from reproductive cells, most commonly found
in the testicle and ovary (http://en.wikipedia.org).

Cancer treatment

For cancer treatment, every physician should have a thorough

knowledge on the principles of diagnosis, investigation and management
process. The type of cancer and its unique pattern of behaviour, opens up a
change for wide range of control methods. In recent years with advancement in
the modern technology of cancer cells, cancer biology has achieved greater
accuracy in diagnosis and staging methods. Cancer is cured or controlled by
different phases of treatment. The major common methods of treatment and
their therapeutic values are briefly described below:

1. Conventional therapies

i) Surgery ii) Chemotherapy iii) Radiation Therapy

iv) Hormone (endocrine) therapy.

2. Modified chemotherapy

Photodynamic therapy (PDI)

3. Immune based cancer therapies

i) Immunotherapy (Biological therapy) ii) Monoclonal antibody therapy

(MCAb) iii) Adaptive immunotherapy or cellular immunotherapy iv) Cancer
Vaccines (Ranajit Sen, 2004)

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1) Surgery

Surgery is a kind of therapy to artificially remove the cancerous portions in

the body. It is the oldest and most clear-cut method of control (Myers, 1989).

Radiation therapy

Radiotherapy is another conventional type of cancer treatment for which

X-rays and gamma rays are used. This technique is adapted particularly in
relatively confirmed areas (Nathan, 2000).

iv) Hormone (endocrine) therapy

The main goal of hormone therapy is to prevent cell division by

manipulating specific hormone and thus preventing tumour formation.
Actually, the hormone therapy does not kill the cancer cells. This kind of
treatment is used to control prostate and breast cancers. Like other therapies, it
also expresses some good result in the combination form. It is combinely used
with both chemotherapy and radiation therapy. It also has some adverse side
effects (Davies 1985; Nathan 2000).

Photo-dynamic therapy (PDT)

In PDT a light-sensitive drug (hematoporphyrin-derivative) is injected into

the body and is absorbed by cancer cells. An endoscope is than used to shine a
laser light on to it produce a chemical reaction which kills the tumours cells,
surrounding tissue is unaffected (Nathan, 2000).

Immunotherapy

Immunotherapy is the recent technique to stimulate our body’s natural

ability (immune system) to fight disease or to protect the body from some of
the side effects of cancer treatment. Immunotherapy is based on the principle
that the cancer cells make an abnormal / excessive amount of protein. The

20
protein content of cancer cell acts as an antigen. The immune response,
which is produced by our immune system correctly identifies and detects the
tumour-derived antigens and fights them (Rosenberg, 1999 and 2001;
Chilunkar, 2001).

Monoclonal antibodies therapy (MCAb)

Monoclonal antibodies coupled with radioisotopes or toxic compounds

are used to treat cancer (Kreuaer and Massey, 2001).

Adoptive immunotherapy

Adoptive immunotherapy takes in to account the cancer fighting

capabilities of T-Lymphocytes. T-Lymphocytes constitute a part of our white
blood cells and play a crucial role in our immune system. They recognize
antigens in an unique way and directly kill cells having the property of antigen.
In adoptive immunotherapy, T-Lymphocytes recognizes tumour cells and
effectively kills them in in vitro condition. This kind of cancer treatment
overcomes the problem of weak tumour response and the ability of cancer to
suppress the immune system. Adoptive immunotherapic treatment was
effective on eradicating the tumour in animals under laboratory condition. It
can also be successfully used against the renal and melanoma cancers (Kreuaer
and Massey, 2001).

Cancer vaccines

Cancer vaccines stimulate the immune system in cancer patients. After

cancer vaccination, our immune system recognizes the tumour as a foreign
component of the body (Rosenberg et al, 1998)

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Gene therapy

Gene therapy is the method to introduce genes into the body. It enables
to reverse or stop the cancerous growth (Rosenberg, 1999; Nathan, 2000;
Mulherkar, 2001; Kreuaer and Massey, 2001).

Bone Marrow Transplantation (BMT) or Peripheral stem cell

Transplantation (PSCT) :

BMT / PSCT are used in Cancer treatment. The BMT may be of three
types.

a. Autologouos

The cells saved from the patient earlier will be used for transplantation.

b. Allogeneic

The cells donated by a healthy person are used for transplantation.

c. Syngeneic

The cells donated by an identical twins are used for transplantation.

Both BMT and PSCT provide the patient with healthy stem cells. This
technique of stem cell treatment replace the cells damaged due to very high
doses of chemotherapy or radiation treatments (Nathan, 2000; Kreuer and
Massey, 2001).

Chemotherapy

Chemotherapy is an important and potential type of treatment, which is

done by drugs both synthetic and plant based medicines are used for this
purpose. The drugs that have ability to kill the cells are called cytotoxic
compounds/substances. There are over 40 proven drugs that are currently used
to localize the cancerous spots and also acts as a potential anticancer drugs

22
even at the metastasis condition. Drugs are also used to boost the effect of
radiotherapy in the form of adjuvant chemotherapy. The chemotherapeutic
compounds are also used as palliative to reduce pain and discomfort caused by
severe, metastasis condition of cancer (Moran Campbell et al., 1974; Pondy
and Rosenberg, 1981; Davis, 1985; Salman and Sartorelli, 1989).

Anticancer drugs also affect rapidly dividing normal cells and are thus
likely to (Rang and Dale, 2003)

 Depress bone marrow

 Impair wound healing
 Depress growth in children
 Causes sterility
 Hair loss
 Teratogenic and
 Common side effects are nausea and vomiting.

Chemotherapy is an effective treatment against cancer either singly or in

combination with surgery and/or radiotherapy. In chemotherapy, drugs
like cisplatin, carboplatin, cyclophosphamide, doxorubicin, melphalan,
mitomycin-c, gemcitabine etc., have been used for the treatment of cancers
(Black et al., 1990; Black et al., 1990).

However, therapeutic efficacy of most of them are limited due to

the development of various side effects in the host and / or the acquired
drug resistance by the cancer cells (Black et al., 1990; Kartalou and
Essigmann 2001).

In an attempt to abate these side effects and better remedy against

various malignancies, many plant derivatives have been used with varying
success (Roja and Rao 2000).

23
 From the dawn of civilization, men have been utilizing the important
biological properties of various plants for the treatment of different diseases.
Even today, plants are the most exclusive source of drugs for the majority of
world’s population and plant products constitute about 25% of prescribed
medicines (Farnsworth and Bingel, 1979).

India is gifted with a large number of plants possessing important

therapeutic values. Such plants are being used by the Indians for centuries in
the traditional system of medicine (Biswanath, 1996).

Cancer causing agents

 Genetic factors
 Chemical carcinogens
 Physical agents
 Biological agents.

Chemical carcinogenesis:

Many chemical substances cause mutation in DNA. They are called

chemical mutagens, some times mutation in DNA may convert a normal cell to
a cancer cell.

Many works have extensively studied the mechanism of action of

chemical carcinogens (Myers 1989; Nathan 2000). Some important chemical
carcinogens are Aflatoxin B1, Dactinomycin, Nitrosamines, and inorganic
compounds such as Arsenic, Asbestos, Beryllium, Cadmium and chromium.

Physical agents / Radiation carcinogenesis

Exposure to radiation may damage DNA. Radiant energy, whether in

the form of the UV rays of sunlight or as ionizing radiation, causes mutation in

24
DNA of skin cells. Mutant DNA mediates carcinogenesis, by activation of
oncogenes; which leads to development of cancer of the skin or multiple
tumours of skin (Ranajit Sen, 2004).

Some DNA and RNA viruses are carcinogenic and hence, they are
termed as oncogenic viruses, are responsible for the development of cancer.
Certain herpes and papilloma group of DNA viruses and RNA viruses have
been shown as causative agents in animals and human cancer. Some important
DNA and RNA viruses which cause cancer in humans are

 Epstein barr virus (DNA virus) causes Burkitt’s lymphoma in children

and Nasopharyngeal carcinoma.
 Papilloma virus (DNA virus) causes human cervical cancer.
 Human T-cell Leukemia virus Type- I (RNA virus) it causes T-cell
Leukemia (http://en.wikipedia.org).

Genetic factors:

 Heredity

Some genes in DNA are associated with development of cancer

in susceptible individuals.
eg. Retinoblastoma, cancer of eye develops in people carrying RBI
gene.

Wilmis tumour, Kidney cancer develops in children having the

gene WT1.

 Mutation:
Mutation in normal DNA may convert a normal gene to
oncogene.
eg: Bladder carcinoma is due to substitution of a single nucleotide in DNA by
another nucleotide. Mutation may be due to an error during replication.

25
 Fig. 9. Carcinogenesis and Mechanisms of Chemoprevention

Defense against ROS

Antioxidant defense against the deleterious effects of free radical known

as antioxidants. May be broadly classified into

 Inhibitors or primary antioxidants

 Chain breakers or secondary antioxidants
 Chain terminators or Tertiary antioxidants.

Primary antioxidants or Inhibitors

The preventive defences include efficiency of electron transfer and

sequestration of transition metal ions. Other form of prevention is the removal
of superoxides and peroxides (H2O2 and lipid hydroperoxides) that react with
transition metal ions to produce reactive free radicals. Superoxide dismutase,
catalase and glutathione peroxidase are examples of this group. Antioxidants
exist in membrane as well as in aqueous compartments (Tripathi, 1998).

26
Secondary Antioxidants or Chain breakers

Some non-enzymatic free radical scavengers are mannitol, DMSO,

Dimethyl thiourea (DMTU) which scavange OH radical. Other examples are
spin traps, bilirubin, urate, glutathione, 17-aminosteroids and albumin.
Ceruloplasmin, transferrin and desferoxamine are other antioxidant which
inhibit the iron redox cycling (Tripathi, 1998).

Tertiary antioxidants (or) Chain termination

Some antioxidants act through augmenting the endogenous antioxidant

activity such as ebselen and acetylcysteine which act by enhancing glutathione
peroxidase activity. Third category of natural antioxidant defence is the repair
process, which removes damaged biomolecules before they can accumulate
and alter cell metabolism (Tripathi, 1998).

The term ‘antioxidant’ refers to the activity of numerous vitamins,

minerals and other phytochemicals to protect against the damage caused by
reactive oxygen species (Khlifi et al., 2006).

The term antioxidant has been defined by Halliwell and Gutteridge

(1990) as “any substance that delays or inhibits oxidative damage to a target
molecule”.

Antioxidants work in several ways: they may stop the free radical from
forming in the first place, or interrupt an oxidizing chain reaction to minimize
the damage caused by free radicals. Reactive oxygen species, especially OH 
play a major role in oxidative damage of gastric mucosa in almost all forms of
gastric ulcer (Phull et al., 1995; Das et al., 1997). Inhibition of the release of
free radicals.

27
 Fig. 10. Mechanism of antioxidant activity

The antioxidant enzymes are classified into two types

i) Enzymic Antioxidants
ii) Non-enzymic Antioxidants

Superoxide dismutase (SOD)

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide

into oxygen and hydrogen peroxide. As such it is an important antioxidant
defense in nearly all cells exposed to oxygen (http://en.wikipedia.org).


SODs are a family of metallo enzymes that convert O2 to H2O2.
2H
O2  + O2  H2O2 + O2.
SOD

SOD is the most important enzyme because it is found virtually in all

aerobic organisms. There are four families of SOD : Cu-SOD, Cu-Zn-SOD,
Mn-SOD and Fe-SOD (Oberley and Oberley, 1984). The transition metal of
 
the enzyme reacts with O2 taking its electron. O2 is the only known
substrate for SOD.

28
Catalase (CAT)

Catalase is an enzyme, which is present in most cells, and catalyses the

decomposition of hydrogen peroxide to water and oxygen. CAT is a heme –
containing protein.
CAT
2H2O2 2H2O2 + O2

CAT is found to act 104 times faster than peroxidase. It is localized

mainly in mitochondria and in subcellular respiratory organelles (Pryor, 1986)

Glutathione peroxidase (GPx)

Glutathione peroxidase enzyme is a well-known first line of defence

against oxidative stress, which in turn requires glutathione as a cofactor, it is
involved in the generation of the nucleotide precursors of DNA via the
reduction of ribonucleotides to deoxyribonucleotides (Meister, 1991). GPx
catalyses the oxidation of GSH to GSSG at the expense of H2O2.

Active site of this enzyme contains selenium. GSHPx exists in two

forms of which one is selenium dependent and other selenium independent.
Later one removes H2O2 and lipid hydroperoxides to form water and stable
hydroxyl lipids respectively.

Se-GSHPx
H2O2 + 2GSH 2H2O2 + GSSG

Se-GSHPx
ROOH + 2GSH ROH + H2O + GSSG

Se-independent GPx named as Glutathione-S-transferase (GST)

removes organic hydroperoxides including free fatty acid hydro peroxides but
not hydrogen peroxide. Therefore it is believed that Se-GPx and GST are

29
involved in glutathione dependent defense against lipid peroxidation (Halliwell
and Gutteridge, 1992).
Glutathione Reductase

Glutathione reductase is an enzyme which reduces glutathione

disulphide (GSSG) to the sulfhydryl form GSH, which is an important cellular
antioxidant (Meister and Anderson, 1998; Mannervik, 1987).

For every mole of GSSG one mole of NADPH is required. The NADPH
is generated through the pentose phosphate pathway.

In cells exposed to high levels of oxidative stress, like red blood cells,
upto 10% of the glucose consumption may be used for production of the
NADPH needed for this reaction.

Glutathione-S-Transferase (GST)

GST Hx + RSG
Rx + GSH

Se-independent GSHPx named as glutathione-S-transferase (Mannervik,

1974) removes organic hydroperoxides including free fatty acid
hydroperoxides but not hydrogen peroxide. Therefore it is believed that Se-
GSHPx and GST are involved in the glutathione dependent defense against
lipid peroxidation.

Glutathione

Glutathione is a tripeptide. It contains an usual peptide linkage between

the amino group of cysteine and the carboxyl group of the glutamate side
chain. Glutathione, an antioxidant, protects cells from toxins such as free
radicals (Struznka et al., 2005).

30
 Thiol groups are kept in a reduced state within a ~5 mmol in animal
cells. In effect, glutathione reduces any disulfite bonds formed within
cytoplasmic proteins to cysteines by acting as an electron donor. Glutathione
is found almost exclusively in its reduced form, since the enzyme which reverts
its oxidized form (GSSG), glutathione reductase, is constitutively active and
inducible upon oxidative stress. In fact, the ratio of reduced to oxidized
glutathione within cells is often used scientifically as a measure of cellular
toxicity. Glutathione is not an essential nutrient since it can be synthesized
from the amino acids L – cysteine, L – glutamate and glycine. (http:// en.
wikipedia.org)

Vitamin C

In living organisms, ascorbate is an antioxidant, as it protects the body

against oxidative stress (Padayatty et al., 2003). Vitamin C readily scavenges
Reactive oxygen species (ROS) Ozone, ONOO -, No2, No and hypochlorous
acid (Noroozi et al., 1998). Vitamin C neutralizes ROMs and reduces
oxidative DNA damage and genetic mutations. (Frei, 1994).

As an antioxidant, Vitamin C’s primary role is to neutralize free

radicals. Since ascorbic acid is water soluble, it can work both inside and
outside the cells to combat free radical damage. Vitamin C is an excellent
source of electrons; therefore, it can donate electrons to free radical such as
hydroxyl and superoxide radicals and quench their reactivity (Adrianne
Bendich, 1990).

31
 PUFA or
Peroxides
PUFA ROS
Peroxides Vit. E Vit. C GSH
ROS
NADP+ PUFA
Alcohols
NADPH
Vit. E

PUFA Vit. E Vit. C GSSG

Alcohols Vit. E

Fig. 11. Central Role of Glutathione in antioxidant network

Vitamin E

Vitamin E is a fat soluble vitamin components of biological membranes,

is located predominantly in cellular and sub-cellular membranes serves as a
potent radical chain breaking antioxidant to protect membrane phospholipids
against peroxidation, presumably by functioning as an electron donor to free
radicals (Urano et al., 1992). Vitamin E as an important preventing factor for
lipid peroxidation. Because of antioxidant properties, Vitamin E neutralises
ROMs and reduces oxidative DNA damage and genetic mutations (Frei, 1994).

Vitamin E is thought to be an important chain-breaking antioxidant,

which plays an important role in various stages of carcinogenesis through its
contribution to immuno competence, membrane and DNA repair, and
decreasing oxidative DNA damage (Kimmick et al., 1997).

Vitamin E can directly scavenge ROMs. It is the major lipid-soluble

antioxidant present in all cellular membranes, which protects against LPO
(Machlin and Bendich, 1987).

The major function of vitamin E is its role as a physiological membrane

bound antioxidant, protecting cell membrane lipids from oxidative damage
initiated by ROMs (Garland et al., 1993). Vitamin E is thought to be important
in protecting the breast from free radical induced damage (London et al.,
1985).

32
Antimetabolites

Antimetabolites are structurally very similar to some of the normal

metabolites that are required for cell functioning and replication. Because they
appear to be essential metabolites, antimetabolites are able to enter the cell,
interfere with the synthesis of essential cell components by competing with
natural enzyme substrates or by contributing to the synthesis of abnormal cell
constituents by blocking or subverting one or more of the metabolic pathways
involved in DNA synthesis. In general, their greatest toxicity occurs in tissues
that are very active metabolically. They are much less uniform in their activity
than are alkylating agents (Ranajit Sen, 2004).

Methotrexate (Amethopterin, oncotrex, biotrex, MTX).

Methotrexate is the main folate antagonist and antimetabolite, most

widely used in cancer therapy. Folates are essential for the synthesis of purine
nucleotides and thymidylate, which in turn are essential for DNA synthesis and
cell division.

Chemically methotrexate is N-[4-[[(2,4-diamino-6-pteridinyl)methyl]

Methylamino]benzoyl]-L-glutamic acid.

Molecular weight : 454.45 C20H22N8O5 and the structural formula is

H2N N N
CH3
N CH2N CONH
N
NH2 HOOCH 2CH2C C COOH (http://en.wikipedia.org.)
H

33
 MTx
FH2(glu)n
MTx

DHFR

FH2(glu)n+
F(glu)n FH2(glu)n One carbon Unit

THYMIDYLATE
SYNTHETASE

DTMP DUMP

Fig. 12. The action of methotrexate on thymidylate synthesis

(Ranajit Sen, 2004)

Medicinal plants

Medicinal plants and it multivarious uses in the health, pharmaceutical

and cosmetic industry is substantially cost heritage which merits rediscovery
and active advocacy.

Plant based remedies have always been an integral part of traditional

medicine throughout the world. The increasing demand for herbal medicine in
recent years due to their fewer side effects in comparison to synthetic drugs
and antibiotics has highlighted the need for conservation and propagation of
medicinal plants (Bantaon et al., 1999).

The revival of interest in natural drugs especially those derived from

plants, stored in the last decades mainly because of the wide spread belief that
“green medicines”, are healthier and safer than the synthetic ones (Sinha,
1996). India has one of the oldest, richest and most diverse cultural traditions
associated with the use of medicinal plants. India is blessed with vast
medicinal plants diversity (Saini, 2000).

34
 Herbal drugs have gained importance in recent years because of their
efficacy and cost effectiveness. These drugs are invariably single plant extracts
or fractions, thereof or mixtures of fractions/extract of different plants which
have been carefully standardized for their safety and efficacy (Dev, 1997).

These herbal medicines by way of decoctins, oils, powders

and ointments will be a sure panacea for many ills (Valiantha and
Chattopadhyay, 1998).

Many species of plants reported to be medicinal have been to produce

beneficial effects in the treatment of various diseases (Ladeji et al., 1996). On
the other hand herbal drugs used in the Indian system of medicine are claimed
to be effective and safe in liver diseases (Senthamarai et al., 2000).

The major problem with the cancer therapies such as allopathic

chemotherapy, radiation therapy and other therapies is their irreversible side
effects. These adverse effects have promoted the researchers for natural source
of drugs. The use of plants as medicine is as old as human civilization. From
the time of Galen (A.D.180), the juice of woody night shade (Solanum
dulcamara) has been used to treat cancer, (Srivastsava et al., 2000).

The national cancer institute of USA began an organized programme to

screen anticancerous agents in 1955 with the establishment of cancer
chemotherapy national service center. In USA under the NCI programme, over
35000 plants were screened for anticancer activity between 1960 to 1986 and
2000 crystalline plant derived compounds were isolated and tested for the
activity against P388 lymphocytic Leukemia and KB Carcinoma in cell cultures
(Irfan A Khan and Atiya Khanum, 2002).

35
 In the indigenous medicine all over the world for over 2000 years,
plants have been used against many kind of cancers. The earliest record of
herbal treatment can be traced to ancient Chinese and Greek texts (Trease and
Evans, 1989) unani and ayurvedic system also used a large number of plants
for the treatment of cancer (Javed Ahmad & Farooqi, 1991).

The use of crude natural products for curing disease resembling cancer
has been reported in ancient Egyptian medical records dating back to
1550 B.C. and such reference are available in the Chinese records as well
(Hastwel, 1967).

Tephrosia purpurea is used as a folk remedy for the treatment of cancer

in parts of the south India. The present study is designed to evaluate the
effectiveness of MeTp leaves in modifying the tumour process, as well as
oxidative alterations in DLA tumour.

Tephrosia purpurea

Botanical name : Tephrosia purpurea

English name : Wild Indigo
Tamil name : Kattukkolinchi
Hindi name : Sarphonk, Sharpunkha
Family : Fabaceae

Tephrosia purpurea (Plate 1) is native to tropical Asia and is found

from India and Sri Lanka to Southern China and through South East Asia to
tropical Australia and the Polynesian Islands. It is now neutralized and
cultivated pantropically (www.motherherbs.com).

36
 Tephrosia purpurea occurs naturally in grassy fields, waste places and
thickets, on ridges, and along roadsides, in Java. In Hawaii, it grows near the
seashore.

Upto 400 m altitude, it generally grows at low altitudes, but may be

found to 1300 m altitudes. It prefers dry, gravelly or rocky and sandy soils, but
in Madras (India) it grows well on loamy soils. It is tolerant of saline – sodic
soil conditions.

Different parts like Roots, Leaves, Seeds and Bark of the plants are used
for various purposes.

Traditional medicinal uses

It is used traditionally as Folk medicine. According to Ayurveda, plant

is digestible, antihelmintic, alexiteric, leprosy, ulcers, antipyretic, alternative,
cures diseases of liver, spleen, heart blood, tumour and asthma.

According to Unani system of medicine, root is diuretic, alloys thirst,

enriches blood, cures diarrhoea, useful in bronchitis, inflammations, boils and
pimples; Leaves are tonic to intestines and a promising appetizer. Good in
piles, syphilis and gonorrhoea (Soni et al., 2006).

Other uses

It also checks the soil erosion. Leaves are used as fodder. Seeds can be
used as substitute to coffee. It also helps in fixing Nitrogen.

37
 3. MATERIALS AND METHODS

The present investigation entitled “Antioxidant and antitumour status of

the methanolic extract of Tephrosia purpurea leaves against Dalton’s
lymphoma ascites tumour induced in mice” was carried out by following the
methodology given below.

1. Collection of plant material and preparation of extracts

2. Selection of Animals
3. Induction of Dalton’s Lymphoma ascites tumour in the experimental
animals.
4. Experiments

PHASE I
 Toxicity studies

PHASE II
 Effect of methanolic extract of Tephrosia purpurea (MeTp) on
survival period
 Effect of methanolic extract of Tephrosia purpurea on body
weight

PHASE III
 Liver function tests
 Effect of MeTp on hematological parameters
 Assay of Enzymatic antioxidants
 Assay of Non-Enzymic antioxidants
 LPO
 Histopathology
5. Phytochemical studies
 Qualitative Analysis

38
1. COLLECTION OF PLANT MATERIAL AND PREPARATION OF
EXTRACTS

The leaves of Tephrosia purpurea were collected from Tanjore District,

were authentificated by Dr. Ramachandran, Reader in Botany, Kongunadu Arts
and Science College, Coimbatore.

Fresh leaves were picked and washed with water. Leaves of Tephrosia
purpurea were air-dried and powdered coarsely. The powder obtained (25 g)
was macerated in methanol (250 ml) in an incubatory shaker (150 rev / min.
25o C) for 72h, by refreshing the solvent at the end of the first day. After
removing the plant residues by filtration, each filterate was concentrated under
reduced pressure and lyophilized to get the extract. The greenish brown residue
was obtained. The extract yielded a greenish brown residue solid weighing (1g)
and was preserved in a refrigerator at 4 o C until further use. The yield of
prepared extract was 4%.

2. SELECTION OF ANIMALS

Inbred Swiss albino mice (6-7 weeks old) of both sexes weighing 20 to
25 g were obtained from Small Animal’s Breeding Centre of Kerala
Agricultural University, Mannuthy, Thrissur. The animals were housed in
polypropylene cages maintained in controlled temperature (27± 2 oC) with 12 h
light and 12 h dark cycle. They were fed with standard pellet diet (Lipton,
Indian Laboratories, Bangalore) and water ad libitum.

3. INDUCTION OF DALTON’S LYMPHOMA ASCITES TUMOUR IN

EXPERIMENTAL ANIMALS

Dalton’s lymphoma ascitic (DLA) cells, originally obtained from Amala

Cancer Research Centre, Thrissur, Kerala. The tumour was maintained in vivo
by serial intraperitoneal (i.p.) transplantation of 1 x 10 6 tumour cells (0.25 ml

39
in phosphate buffered saline, pH 7.4) per animal. Tumour-transplanted mice
usually survived for 19 to 21 days.

4. EXPERIMENTS

PHASE I

TOXICITY STUDIES:

The animals were randomly divided into four groups containing 3

animals

Group I: Served as normal which received only the feed and water.

Group II: The animal of this group treated with the MeTp at 100 mg / kg
body weight for 21 days.

Group III: The animal of this group treated with the MeTp at 200 mg/kg
body weight for 21 days.

Group IV: Treated with MeTp at 300 mg / kg body weight for 21 days.

These animals were treated with MeTp for 21 days by orally on 22 nd

day, the animals were sacrificed by cervical dislocation under mild ether
anesthesia, the liver was removed and grossy observed, sections of these
organs was fixed with 10% formalin, embedded in paraffin sectioned at 5µ
thick and stained with haemotoxylin and eosin for histological analysis.

HISTOPATHOLOGICAL EXAMINATION

-Galigher and Kayloff (1971)

Tissue processing

Liver tissue is placed in 10% formal saline (10 % formalin in 9 % sodium

chloride) for 1hr to rectify shrinkage due to higher concentration of formalin. The

40
tissue is dehydrated by ascending grades of Isopropyl alcohol by immersing in 80 %
Isopropanol overnight, 100 % Isopropyl alcohol for 1 hour. The dehydrated tissues
were cleared in two changes of Xylene, 1 hour each. Then the tissues were
impregnated with histology grade paraffin wax (melting point 58-60°C) at 60°C for 2
changes of 1 hour each. The wax impregnated tissues were embedded in paraffin
blocks using the same grade wax. The paraffin blocks were mounted and cut with
rotary microtome at 3 micron thickness. The sections were floated on a tissue
floatation bath at 40°C and taken on glass slides and smeared with equal parts of egg
albumin and glycerol. The sections were then melted in an incubator at 60°C and after
5 mins the sections were allowed to cool.

Tissue staining

The sections were deparaffinised by immersing in Xylene for 10 mins in

horizontal staining jar. The deparaffinised sections were washed in 100 % Isopropyl
alcohol and stained in Ehrlich’s hematoxylin for 8 mins in horizontal staining jar.
After staining in hematoxylin, the sections were washed in tap water and dipped in
acid alcohol to remove excess stain (8.3% HCl in 70 % Alcohol). The sections were
then placed in running tap water for 10 mins for blueing (show alkalization). The
sections were counter stained in 1 % aqueous eosin (1gm in 100 ml tap water) for one
minute and the excess stain is washed in tap water and the sections were allowed to
dry. Complete dehydration of stained sections was ensured by placing the sections in
the incubator at 60°C for 5 mins. When the sections are cooled, they are mounted in
DPX mount having the optical index of glass (the section were wetted in Xylene and
inverted on to the mountant placed on coverslip).

The architecture was observed at low power objective. The liver cell injury
and other aspects were observed under high power dry objective.

41
PHASE II

EFFECT OF MeTp ON SURVIVAL PERIOD

The animals were randomly divided into four groups, each containing
6 animals.

Group I: Normal

Group II: DLA control

Group III: DLA induced+Treated with MeTp at 300 mg / kg body weight

All the mice were transplanted with one million cells of DLA intra
peritoneally. Group III and Group IV was treated with MeTp once / day orally
for 21 days. The treatment was stopped after 21 days. Survival period of these
animals were monitored up to 60 days and mean survival times (MST) was
found.

Survival period of treated groups were compared with control group and
percent increase in life (T/C %) was calculated by the formula.

% Increase in life span =  T/C x 100  - 100

 MST of treated group 
= x 100  - 100
 MST of DLA control group 

EFFECT OF MeTp ON BODY WEIGHT

All the mice were weighed on the day of tumour transplantation and at 5
days intervals.

PHASE III

42
EFFECT OF MeTp ON HEMATOLOGICAL PARAMETERS

From the results obtained from Phase I and II, the effective dose (300
mg / kg body weight) for the further experimental studies has been selected.

The animals were divided into 5 groups containing 6 animals in each group.

Group I: Normal
Group II: DLA Control
Group III: DLA induced + Treated with MeTp at 300 mg/kg body weight
Group IV : Methotrexate (Standard drug) 3.4 mg / kg body weight.
Group V: Treated with MeTp at 300 mg/kg body weight.

In order to find the influence of MeTp on the hematological status of

DLA bearing mice, the comparison was made among the above mentioned five
groups of mice on the 22nd day after transplantation with normal mice.

In Phase II, it was observed that the untreated DLA bearing mice were
found to be started dying from 18 th day and all the animals were died on 24 th
day.

The treatment was given for 21 days on 22nd day the animals were
sacrificed to analyse various biochemical parameters.

At the end of the experimental period, all the mice were killed after an
over night fast by decapitation. Blood was collected from freely flowing tail
vein and used for the estimation Hemoglobin (Hb) content, Red blood cell
count (RBC) and white blood cell count (WBC) were estimated.

The remaining blood was centrifuged and serum was used for the
estimation of biochemical parameters.

43
 After collecting the blood samples, the mice were killed by cervical
dislocation. The liver was excised rinsed in ice-cold normal saline solution
followed by cold 0.1 M Tris-HCl (pH 7.4), blotted, dried and weighed. A 10%
w/v homogenate was prepared in 0.1 M Tris-HCl buffer and was used for the
Biochemical parameters. The rest of the homogenate was centrifuged at 1500
rpm for 15 min at 4oC.

LIVER FUNCTION TESTS :

Liver function marker enzymes and other lipid profiles have been
analyzed to confirm the non toxic effect of the drug which have been analyzed
in phase I. The standard methods have been followed to analyze the liver
function marker enzymes such as Aspartate transaminase, Alanine
Transaminase, Alkaline phosphatase, Acid phosphatase, Lactate
dehydrogenase and lipid profile like Cholesterol, Phospholipids, Triglycerides.

ESTIMATION OF ASPARTATE TRANSAMINASE (AST)

- Reitman and Frankel (1957)

Principle

The enzyme catalyses the following reaction

AST
L – Aspartate + α-ketoglutarate Oxaloacetate + L-Glutamate

The oxaloacetate is measured by the reaction with 2,4 dinitrophenyl

hydrazine giving a brown colored hydrazone after the addition of sodium
hydroxide. The color developed is read at 520 nm.

44
Reagents

 Phosphate buffer : 0.1M, pH 7.5.

Sol A : 0.1M solution of Monobasic sodium phosphate.
Sol B : 0.1M solution of Dibasic sodium phosphate
Mixed 16ml of A and 84ml of B, diluted to a total of 200ml.
 Substrate : Dissolved 146mg of α-ketoglutarate and 13.3gm of aspartic
acid in 1N NaOH with constant stirring. Adjusted the pH to 7.4 and
made upto 1000ml with phosphate buffer.
 Standard Pyruvate, 2mM : Dissolved 22mg of sodium pyruvate in
100ml of phosphate buffer. 0.2ml of standard contains 0.4mM of
sodium pyruvate.
 Dinitrophenyl hydrazine reagent, 1mmol / L : 200mg/ L in 1mol / L
HCl.
 0.4N NaOH : Dissolved 16gm of NaOH in 1L distilled water.

Procedure

0.2ml of sample and 1.0ml of the buffer substrate was incubated for
60mins at 37°C. To the control tubes, enzyme was added after arresting the
reaction with 1.0ml of DNPH and the tubes were kept at room temperature for
20mins. Then 10ml of 0.4N NaOH was added. A set of standard pyruvate were
also treated in a similar manner. The color developed was read at 520nm.

The enzyme activity in serum was expressed as μmoles of pyruvate

liberated / L and in liver homogenate as µmoles of pyruvate liberated / min /
mg protein.

45
ESTIMATION OF ALANINE TRANSAMINASE (ALT)
-Reitman and Frankel (1957)

Principle

The enzyme catalyses the following reaction:

ALT
L – Alanine + α-ketoglutarate Pyruvate + L-Glutamate

The pyruvate is measured by the reaction with 2,4-

dinitrophenylhydrazine giving a brown colored hydrozone after the addition of
sodium hydroxide. The color developed is read at 520nm.

Reagents

 Phosphate buffer : 0.1m, pH 7.5.

 Substrate : Dissolved 146mg of α-ketoglutarate and 17.8gm of L-alanine
in 1N NaOH with constant stirring. Adjusted the pH to 7.4 and made up
to 1000ml with phosphate buffer.
 Standard Pyruvate, 2mM : Dissolved 22mg of sodium pyruvate in
100ml of phosphate buffer. 0.2ml of standard contains 0.4µM of sodium
pyruvate.
 Dinitrophenyl hydrazine reagent, 1mmol/L : 200mg/L in 1mol / L HCl.
 0.4 N NaOH : Dissolved 16gm of NaOH in 1L distilled water.

Procedure

0.2ml of Sample and 1.0 ml of the buffer substrate were incubated for
30 mins at 37°C. To the control tubes, enzyme was added after arresting the
reaction with 1.0 ml of DNPH and the tubes were kept at room temperature for
20 mins. Then 10 ml of 0.4 N NaOH was added. A set of standard pyruvate
was also treated in a similar manner. The color developed was read at 520nm.

46
 The enzyme activity in serum was expressed as μmoles of pyruvate
liberated / L and in liver homogenate as μmoles of pyruvate liberated/min/mg
protein.

ESTIMATION OF ACID PHOSPHATASE (ACP)

- King (1965a)
Principle

The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolyzed with the liberation of phenol and inorganic
phosphate. The liberated phenol is measured at 700 nm with Folin-Ciocalteau
reagent.

Reagents

 Citrate buffer: 0.1M, pH 5.

A : Citric acid (21.01 g in 1000 ml)
B: Sodium citrate (29.41 g in 1000 ml)
20.5 ml of A and 29.5 ml of B, diluted to a total of 100 ml.
 Disodium phenyl phosphate, 100 mmol/L: Dissolved 2.18 g in water,
heated to boil, cooled and made to a litre. Added 1.0 ml of chloroform
and stored in the refrigerator.
 Buffer - Substrate: Prepared by mixing equal volume of the above two
solutions. This has a pH of 5.0.
 Folin-Ciocalteau reagent: Mixed 1.0 ml of reagent with 2.0 ml of water.
 Sodium Carbonate solution, 15% : Dissolved 15 g of anhydrous sodium
carbonate in 100ml of water.
 Standard Phenol solution, 1g/L : Dissolved 19 pure crystalline phenol in
100 mmol/L HCl and made to a litre with the acid.
 Working standard solution: Diluted 10ml of stock standard to 100ml
with water. This contains 100μg phenol/ml.

47
Procedure

Pipetted out 4.0ml of the buffered substrate into a test tube and
incubated at 37°C for 5 mins. Added 0.2 ml of sample and incubated further
for exact 60 mins. Removed and immediately added 1.8 ml of diluted phenol
reagent. At the same time a control was set up containing 4.0 ml buffered
substrate and 0.2 ml of sample which 1.8 ml of phenol reagent was added
immediately. Mixed well and centrifuged. To 4.0 ml of the supernatant added
2.0 ml of sodium carbonate. Took 4.0 ml of working standard solution and for
blank taken 3.2 ml water and 0.8 ml of phenol reagent. Then added 2.0 ml of
sodium carbonate. Incubated all the tubes at 37°C for 15 mins. Read the color
developed at 700 nm.
The activity of serum acid phosphatase was expressed in μmoles of
phenol liberated / L. The activity in tissue homogenate was expressed as
nmoles of phenol liberated / min / mg protein.

ESTIMATION OF ALKALINE PHOSPHATASE (ALP)

- King and Armstrong (1934)

Principle

The method used was that of King and Armstrong in which disodium
phenyl phosphate is hydrolysed with the liberation of phenol and inorganic
phosphate. The liberated phenol is measured at 700nm with Folin -Ciocalteau
reagent.

Reagents

 Sodium Carbonate - Sodium bicarbonate buffer, 100mmol/L: Dissolved

6.36g anhydrous sodium carbonate and 3.36g sodium bicarbonate in
water and made to a litre.

48
  Disodium phenyl phosphate, 100mmol/L: Dissolved 2.18g in water,
heated to boil, cooled and made to a litre. Added 1.0ml of chloroform
and stored in the refrigerator.
 Buffer-Substrate: Prepared by mixing equal volume of the above two
solution. This has a pH of 10.
 Folin-Ciocalteau reagent: Mixed 1.0ml of reagent with 2.0ml of water.
 Sodium Carbonate solution, 15%: Dissolved 15g of anhydrous sodium
carbonate in 100ml of water.
 Standard Phenol solution, 1g/L: Dissolved 1 g pure crystalline phenol in
100 mmol/L HCl and made to a litre with the acid.
 Working standard solution: Added 100 ml diluted phenol reagent to
5.0 mlof stock standard and diluted to 500 ml with water. This contains
10 μg of phenol / ml.

Procedure

Pipetted out 4.0ml of the buffer substrate into a test tube and incubated
at 37°C for 5 mins. Added 0.2 ml of sample and incubated further for exact 15
mins. Removed and immediately added 1.8 ml of diluted phenol reagent. At
the same time a control was set up containing 4.0 ml buffer substrate and 0.2
ml of sample to which 1.8 ml of phenol reagent was added immediately. Mixed
well and centrifuged. To 4.0 ml of the supernatant added 2.0 ml of sodium
carbonate. Took 4.0 ml of working standard solution and for blank taken 3.2
ml of water and 0.8 ml of phenol reagent. Then added 2.0ml of sodium
carbonate. Incubated all the tubes at 37°C for 15 min. Read the color
developed at 700 nm.
The activity of serum alkaline phosphatase was expressed in μmoles of
phenol liberated/ L. The activity in tissue homogenate was expressed as
μmoles of phenol liberated / min / mg protein.

49
ALT
L – Alanine + α-ketoglutarate Pyruvate + L-Glutamate
ESTIMATION OF LACTATE DEHYDROGENASE (LDH)
- King (1965b)

Principle

The lactate is acted upon by lactate dehydrogenase to form pyruvate in

the presence of NAD. The pyruvate forms pyruvate phenyl hydrazone with
2,4-dinitrophenyl hydrazine. The color developed is read in a
spectrophotometer at 440 nm.

Reagents

 Glycine buffer, 0.1 M, pH 10 : 7.505 g of glycine and 5.85 g of

sodium chloride were dissolved in 1 litre of water.
 Buffered substrate: 125 ml of glycine buffer and 75 ml of 0.1N
NaOH were added to 4 g of lithium lactate and mixed well.
 Nicotinamide Adenine Dinucleotide: 10mg of NAD was
dissolved in 2 ml of water.
 2,4-dinitrophenyl hydrazine : 20 mg of DNPH was dissolved in
100 ml of 1N HCl.
 0.4 N NaOH.
 Standard Pyruvate, 1μmol/ml: 11 mg of sodium pyruvate was
dissolved in 100 ml of buffered substrate (1 μmole of pyruvate /ml).
 NADH solution, 1 μmol/ml: 8.5 mg/10 ml buffered substrate.

Procedure

Placed 1.0 ml buffered substrate and 0.1 ml of sample into each of two
tubes. Added 0.2 ml of water to the blank. Then to the test added 0.2 ml of
NAD. Mixed and incubated at 37°C for 15 mins. Exactly after 15 mins, 1.0ml
of dinitrophenyl hydrazine was added to each (test and control). Left for further
15 mins. Then added 10 ml of 0.4 N Sodium hydroxide and the color

50
developed was read immediately at 440 nm. A standard curve with sodium
pyruvate solution with the concentration range 0.1 -1.0 μmole was taken.

LDH activity in serum was expressed as μmoles of pyruvate liberated /

L and in liver homogenate as nmoles of pyruvate liberated / min / mg protein.

ESTIMATION OF CHOLESTEROL
- Zak (1977)

Principle

Cholesterol reacts with ferric chloride in the presence of concentrated

sulphuric acid to give a pink color. The intensity of color developed is directly
proportional to the amount of cholesterol present and is read at 540nm in a
colorimeter.

Reagents

 Stock Ferric chloride : 840 mg of pure dry ferric chloride was weighed
and dissolved in 100 ml of glacial acetic acid.
 Ferric chloride precipitating reagent : 10 ml of stock ferric chloride
reagent was taken in 100 ml of standard flask and made up to the mark
with pure glacial acetic acid.
 Ferric chloride diluting reagent : 8.5 ml of stock ferric chloride was
diluted to 100ml with pure glacial acetic acid.
 Standard Cholesterol solution : 100 mg of cholesterol was dissolved in
100 ml with glacial acetic acid. The concentration of working standard
is 100 μg /ml.
 Working standard : 10 ml of stock was dissolved in 0.85 ml of stock
ferric chloride reagent and made up to 100 ml with glacial acetic acid.
The concentration of working standard is 100 μg/ml.

51
Procedure

To 0.1 ml of serum added 4.9 ml of ferric chloride precipitating reagent.

Centrifuged and to 2.5 ml of supernatant added 2.5 ml of ferric chloride
diluting reagent. Added 4.0 ml of concentrated sulphuric acid. A blank was
prepared simultaneously by taking 5.0 ml of diluting reagent and 4.0 ml of
concentrated sulphuric acid. A set of standards (0.5 - 2.5 ml) were taken and
made up to 5.0 ml with FeCl2 diluting reagent. Then added 4.0 ml of
con.H2SO4. After 30 mins, the intensity of color developed was read at 450 nm
against reagent blank. The amount of cholesterol in the serum was expressed as
mg/dl and mg / g in tissues.

ESTIMATION OF PHOSPHOLIPIDS
– Rouser et al. (1970)

Principle

The organic phospholipid phosphorus is converted to inorganic

phosphorus, which reacts with ammonium molybdate to form
phosphomolybdic acid, which on reduction and reaction with ANSA forms a
stable blue color and has absorption at 660 nm

Reagent
 TCA 10 %

 70 % Perchloric acid

 3 % Ammonium molybdate : 3 g of Ammonium molybdate was

dissolved in 100 ml of distilled water.

 3 % Ascorbic acid: 3 g of Ascorbic acid was dissolved in 100 ml

of distilled water.

 Standard: 35.1 mg of KH2PO4 was dissolved in 100ml of water.

This contains 80μg of phosphorous/ml.

52
Procedure
0.1 ml of lipid extract was diluted to 2.0 ml with distilled water. 1.0 ml
of perchloric acis was added to the tubes and digested on a sand bath till the
solution became colorless and then it was made up to 5.0 ml with distilled
water. Standard phosphate solution and blank containing distilled water were
mixed with 0.8 ml of perchloric acid and the final volume was made upto 5.0
ml with distilled water. 0.5 ml each of ammonium molybdate and ascorbic acid
were added and the mixture was kept in a boiling water bath for 6 minutes. The
color developed was read at 710 nm.

Phosphorus content was multiplied by a factor 25, which gave the

weight of phospholipids. Phospholipids are expressed as mg/100ml in serum
and mg/g in tissues.

ESTIMATION OF TRIGLYCERDIES
– Rice (1970)

Principle

The glycerol moiety is oxidized to formaldehyde and the later

condensed with ammonia and 2,4-pentanedione (acetyl acetone) to produce
3,5-diacetyl 1,4-dihydrotoludine, which is yellow in color and has absorption at
450 nm.

Reagents
 Chloroform-Methanol mixture (2:1 v/v)

 Supersaturated Sodium chloride : 0.89 g of sodium chloride was

dissolved in 100 ml of distilled water.

 Activated Silicic acid: It was activated by washing silicic acid with 4

N or 2 N HCl and then with distilled water until the pH wash neutral.
After drying, ether was added in sufficient amount, stirred well and

53
 the supernatant was discarded. Silicic acid was then dried at 60˚C
and activated at 100˚C over night prior to use.

 0.2 N H2SO4: 0.56 ml of sulphuric acid was added to 100 ml with

distilled water.

 Alcoholic potassium hydroxide 0.4% : 400 mg of KOH was

dissolved in 100 ml of 5% ethanol.

 Sodium-metaperiodate 0.1 M: 50 mg of sodium metaperiodate was

dissolved in 10 ml of distilled water

 Sodium mettaarscnite 0.5 M: 650 mg of sodium metaarsenite was

dissolved in 10 ml of distilled water.

 Chromotropic acid reagent: 1.14 g of chromotropic acid was

dissolved in 100 ml of distilled water and stored as stock solution in
a brown bottle. Before use, 10 ml of this solution was mixed with
4.5 ml of sulphuric acid- water mixture in the ratio of 2:1 (v/v).

 Thio urea: 7% solution in chloroform

 Standard : 100 mg of tripalmitin was dissolved in 100 ml of

chloroform which gave the concentration of 1.0 mg/ml.

Procedure

Took 0.1 ml of Folch-wash aliquot, 1 ml of chloroform-methanol

mixture was added. 50 mg of activated silicic acid was added to it, shaken
vigorously and allowed to stand for 30 mins. After centrifugation, to a 0.5 ml
aliquot of supernatant (as well as to standard and blank), 0.5 ml of alcoholic
potassium hydroxide solution was added and the mixture was saponified in a
60-70oC water bath for 20 min. To this, 0.5 ml of 0.2 N H 2SO4 was added and
kept in a boiling water bath for 10 mins. After cooling the tubes, 0.1 ml of
sodium metaperriodate was added and allowed to stand for 10 mins.

54
 The excess periodiate was reduced by the addition of 0.1 ml of sodium
metaarsenite. Then, 5.0 ml of chromattropic acid was added, mixed thoroughly
and kept in a boiling water bath for 30 mins. After cooling, 0.5 ml of thiourea
solutioin was added and the color developed was read at 570 nm using a
photochem colorimeter.

Triglycerides are expressed as mg / 100 ml in serum and mg/g in tissue

ESTIMATION OF NA+-K+-ATPase
– Bonting (1970)

Principle

Na+K+ ATPase transports Na, K against concentration gradient at the

cost of ATP molecule liberating inorganic phosphate (Pi). The inorganic
phosphorous liberated is estimated by Fiske and Subbarow method.
Reagents
 184 mM Tris- HCl buffer, pH 7.5
 50mM MgSO4
 50mM KCl
 600mM NaCl
 1mM EDTA
 40mM ATP

Procedure

1.0 ml of tris buffer and 0.2 ml of each of the above reagents were
mixed together. Thus the assay medium in a final volume of 2.0 ml, contained
92 mM tris buffer, 5 mM MgSO4, 60 mM NaCl, 1 mM EDTA and 4 mM ATP.
After 10min, equilibration at 37°C in an incubator, reaction was started by the
addition of 0.1 ml of homogenate. The assay medium was incubated for 15

55
min. after incubation the reaction was arrested by the addition of 1.0 ml of 10%
TCA. The phosphorus content in the supernatant was estimated by Fiske and
Subbarow method.

The enzyme activity is expressed as micromoles of Pi liberated / min /

mg protein.

ESTIMATION OF Mg2+-ATPase
– Ohnishi et al. (1982)
Principle

The activity of enzyme was estimated by the inorganic phosphorus

liberated is estimated by Fiske and Subbarow method.

Reagents
 375 mM Tris- HCl buffer pH 7.6
 25 mM MgCl2
 10 mM ATP

Procedure

The assay was estimated by the addition of 0.1 ml of homogenate to an

incubation medium containing 0.1 ml of water and 0.1 ml of each of the above
reagents. The final concentration of Tris buffer, MgCl 2 and ATP were 75 mM,
5 mM and 2 mM in total incubation volume of 0.5 ml. The reaction was
terminated after 15 min by the addition of 1.0 ml of 10 % TCA. The liberated
Pi was estimated by the method of by Fiske and Subbarow.
The enzyme activity is expressed as micromoles of Pi liberated / min /
mg protein.

56
ESTIMATION OF Ca2+-ATPase
– Ohnishi et al. (1982)

Principle
The activity of enzyme was estimated by The inorganic phosphorus
liberated is estimated by Fiske and Subbarow method.

Reagents

 375 mM Tris- HCl buffer pH 7.6

 25 mM CaCl2
 10 mM ATP

Procedure

The assay was estimated by the addition of 0.1 ml of homogenate to an

incubation medium containing 0.1 ml of water and 0.1 ml of each of the above
reagents. The final concentration of Tris buffer, CaCl 2 and ATP were 75 mM,
5 mM and 2 mM in total incubation volume of 0.5 ml. The reaction was
terminated after 15 min by the addition of 1.0 ml of 10 % TCA. The liberated
Pi was estimated by the method of by Fiske and Subbarow.
The enzyme activity is expressed as micromoles of Pi liberated / min /
mg protein.

HEMATOLOGICAL PARAMETERS

In order to find the influence of MeTp on the hematological status of

DLA bearing mice, parameters such as Hb, RBC and WBC were analyzed.

57
ESTIMATION OF HEMOGLOBIN

Reagent

 Ferricyanide reagent (Diluent): 100 mg of ferrycyanide, 50 mg of

potassium cyanide and 1.0 g of sodium bicarbonate were dissolved in
minimum amount of distilled water and made upto one litre with
distilled water.

 Cynomethaemoglobin standard : Standard was bought commercially

and had concentration of 16 g / dl. It was stored at 4oC.

Procedure

0.02 ml blood was diluted with 5.0 ml of ferricyanide reagent after 10

min. the color was read against the diluent blank at 540 nm together in a
photochem colorimeter.

ESTIMATION OF RED BLOOD CELLS

- Chesbrough and McArthur (1972)

PRINCIPLE

The blood specimen is diluted (usually 200 times) with red cell diluting
fluid which does not remove the white cells but allows the red cells to be
counted under 400X magnification in a known volume of the fluid. Finally the
number of cells in undiluted blood is calculated and reported as the number of
red cells / mm3.

REAGENTS

 RBC diluting fluid – (Trisodium citrate – 3.0 g, distilled water – 99.0 ml

and formalin – 1.0 ml)

58
PROCEDURE

The whole blood was taken into the RBC pipette exactly upto the 0.5
mark (Thoma pipette mark 101) and the diluting fluid (formal citrate solution)
was immediately drawn up to the mark 101. The pipette was rotated between
the thumb and the forefinger. This gave a dilution of 1:200.
The cover glass was placed in position over the ruled area using gentle
pressure. The suspension was mixed thoroughly by rotating the pipette for
about a minute, holding it in a horizontal position, and finally shook at
sidewise. The fluid was expelled from the stem of the pipette and filled the
chamber immediately by holding the pipette at an angle of 45 ° and slightly
touching the tip against the edge of the cover glass. There should not be any
bubbles under the cover glass. Then the red corpuccells were allowed to settle
for 2 to 3 min. The number of RBCs was counted in 180 small squares (4
squares of 16 at each four corners and one of 16 at centre). The cells touching
the lower and right hand lines were not counted, but the cells touching the
upper and left hand lines were counted. The cells counted are expressed as
million cells /mm3 blood.

CALCULATION

Number of RBCs / mm3 = Number of cells counted in 5 squares x 10000

ESTIMATION OF WHITE BLOOD CELLS

-Chesbrough and McArthur (1972)

PRINCIPLE

Blood is diluted with acid solution which removes the red cells by
hemolysis and also accentuates the nuclei of the white cells; counting is done
with a microscope under the low power (100X magnification) and knowing the

59
volume of fluid examined and dilution of the blood, the number of white cells
in undiluted whole blood is calculated and reported as the number of WBCs /
mm3.
REAGENTS

 WBC diluting fluid–(Turk solution) (Acetic acid–3.0ml and distilled

water–97.0ml).

PROCEDURE

The whole blood was taken upto the mark 0.5 in WBC pipette and
diluted upto the mark 11 with WBC fluid as described in RBC counting and
filled the counting chamber in the same manner. Then the cells are allowed to
settle for 3 minutes. The neubaur counting chamber was used to count the cells
in the four corners and each of these 4 sq mm. areas is subdivided into 16
squares by using the low power objective and a medium ocular. While
counting, the cells included were those touching the lines on the left and
bottom. The difference between the two squares millimeter area should not be
more than 10 WBCs. The white blood cells were expressed as thousand cells
/ mm3 blood.

CALCULATION

Number of WBCs / mm3 = Number of cells counted x 50

ASSAY OF ANTIOXIDANT CONTENTS

An antioxidant is a substance that prevents oxidative damage caused by

free radicals. Living organisms possess enzymic and Non-enzymic antioxidant
defense system to protect against these radical mediated disorders.

60
 The enzymic antioxidants like SOD, CAT, GPx, GR and GST were
assayed. The Non-enzymic antioxidants (Denense by enzymes) those which
canot be produced by the human body but may protect against pro-oxidant
forces when administered as supplements (Sen, 1995). The standard methods
have been followed to analyze the Non-Enzymic antioxidants such as Reduced
Glutathione, Vitamin C and Vitamin E were analyzed.

ESTIMATION OF SUPEROXIDE DISMUTASE (SOD)

-Das et al. (2000)

Principle

The method involves generation of superoxide radical of riboflavin and

its detection by nitrite formation from hydroxylamine hydrochloride. The
nitrite reacts with sulphanilic acid to produce a diazonium compound which
subsequently reacts with naphthylamine to produce a red azo compound whose
absorbance is measured at 543 nm.

Reagents

 50 mM Phosphate buffer, pH 7.4

 20 mM L-Methionine
 1 % (v/v) Triton X-100
 10 mM Hydroxylamine hydrochloride
 50 μM EDTA
 50 μM Riboflavin
 Greiss reagent: 1 % Sulphanilamide, 2 % Phosphoric acid and
0.1 % Naphthylethylene diamine dihydrochloride.
Procedure

Pipetted out 1.4 ml aliquot of the reaction mixture in a test tube.100 µl

of the sample was added followed by a preincubation at 37°C for 5 min. 80 µl

61
of riboflavin was added and the tubes were exposed for 10 min to 200 W
Philips fluorescent lamps. The control tube contained equal amount of buffer
instead of sample. The sample and its respective control were run together. At
the end of the exposure time, 1.0 ml of Greiss reagent was added to each tube
and the absorbance of the color formed was measured at 543 nm.
One unit of enzyme activity was defined as the amount of SOD capable
of inhibiting 50 % of nitrite formation under assay condition.

ESTIMATION OF CATALASE (CAT)

- Sinha (1972)

Principle

Catalase causes rapid decomposition of hydrogen peroxide to water.

Catalase
2H2O2 2H2O+O2

The method was based on the fact that dichromate in acetic acid
reduced to chromic acetate when heated in the presence of H 2O2 with the
formation of perchloric acid as an unstable intermediate. The chromic acetate
thus produced was measured colorimetrically at 610 nm. Since dichromate has
to absorbency in this region, the presence of the compound in the assay
mixture did not interfere with the colorimetric determination of chromic
acetate. The catalase preparation was allowed to split H 2O2 for different periods
of time. The reaction was stopped at specific time intervals by the addition of
dichromate / acetic acid mixture and the remaining H 2O2 was determined by
measuring chromic acetate colorimetrically after heating the reaction.

Reagents

62
  0.01 M Phosphate buffer, pH 7.0
A: 0.1M Monobasic sodium phosphate
B: 0.1M Dibasic sodium phosphate
Mixed 39 ml of A and 61 ml of B is diluted to a total of 200 ml. 10 ml
of this solution is further diluted to 100 ml with distilled water.
 0.2 M Hydrogen peroxide
 Stock Dichromate / acetic acid solution: Mixed a 5 % potassium
dichromate with glacial acetic acid (1:3 by volume).
 Working Dichromate/acetic acid solution: The stock was diluted to 1:5
with water to make the working dichromate / acetic acid solution.

Procedure

The assay mixture contained 0.5 ml of H 2O2, 1.0ml of buffer and 0.4 ml
of water. 0.2 ml of the enzyme was added to initiate the reaction. 2.0 ml of the
dichromate / acetic acid reagent was added after 0, 30, 60, 90 seconds of
incubation. To the control tube the enzyme was added after the addition of the
acid reagent. The tubes were then heated for 10 min. and then color developed
was read at 610 nm.
The activity of catalase was expressed as μmoles of H 2O2 decomposed /
min / mg protein.

ESTIMATION OF GLUTATHIONE PEROXIDASE (GPx)

- Rotruk et al., (1979)

Principle

Glutathione (GSH) was measured by its reaction with DTNB to give a

compound that absorbs at 412 nm.

Reagents

63
  0.4 M Sodium Phosphate buffer, pH 7.0
 10 mM Sodium azide
 2.5 mM Hydrogen peroxide
 4 mM Reduced glutathione
 10 % TCA
 0.3 M Phosphate solution
 0.04 % DTNB in 1 % Sodium citrate
 Reduced glutathione standard: 20 mg reduced glutathione was dissolved
in 100 ml of water.

Procedure

0.4 ml of buffer, 0.1 ml of sodium azide, 0.2 ml of reduced glutathione,

0.1 ml of H2O2, 0.2 ml of enzyme and 1.0 ml of water were added to a final
incubation volume of 2.0 ml. The tubes were incubated for 0, 30, 60, 90
seconds. The reaction was then terminated by the addition of 0.5 ml TCA. To
determine the glutathione content, 2.0 ml of the supernatant was removed by
centrifugation and added to 3.0 ml disodium hydrogen phosphate solution and
1.0 ml of DTNB reagent. The color developed was read at 412 nm. Standards
in the range of 200-1000 μg were taken and treated in the similar manner.
The activity was expressed in terms of μg of glutathione utilized / min /
mg protein.

ESTIMATION OF GLUTATHIONE REDUCTASE (GR)

- Goldberg and Spooner (1983)
Principle

Glutathione reductase catalyses the reduction of oxidized glutathione

(GSSG) to reduced glutathione (GSH) and is assayed by measuring the
decrease in absorbance at 340nm.
GR
NADPH (NADH) + GSSG NADP+ (NAD)+ + 2GSH

64
Reagents

 0.3 M Phosphate buffer, pH 6.8.

 25 mM EDTA.
 12.5mM oxidized glutathione.
 3mM NADPH.

Procedure

0.2 ml of sample, 1.5 ml of buffer, 0.5 ml EDTA, 0.2 ml GSSG and 0.1
ml NADPH was added. The decrease in optical density of the enzyme was
measured against that of the blank at 340 nm.
The enzyme activity is calculated in terms of µmoles of NADPH
oxidized / min / mg protein.
ESTIMATION OF GLUTATHIONE-S- TRANSFERASE (GST)
- Habig et al. (1974)
Principle

The enzyme was assayed by its ability to conjugate GSH with CDNB,
the extent of conjugation causing a proportionate change in the absorption at
340nm.

Reagents

 1 mM-1-Chloro, 2,4-dinitrobenzene (CDNB) in ethanol.

 1 mM Glutathione.
 0.1 M Phosphate buffer, pH 6.5.

Procedure

The assay was done at 25°C under conditions giving activities linear
with respect to incubation times and protein concentrations for atleast 3 mins.

65
 The enzyme activity was determined by monitoring the change in
absorbance at 340 nm in a spectrophotometer. 0.1 ml of both substrates (GSH
and CDNB) was taken in 0.1 M phosphate buffer (pH 6.5) at room temperature
to make a volume of 2.9 ml. The reaction was started by adding 0.1 ml of liver
homogenate to this mixture. The readings were recorded against distilled water
blank for a minimum of 3 mins. The complete assay mixture without the
enzyme (liver homogenate) served as the control to monitor non-specific
binding of the substrates. Care was taken to ensure that the final concentration
of ethanol in the mixture was always less than 4 %.

Calculation

GST activity was calculated using the extinction co-efficient of the

product formed (9.6 mm-cm-) and the values have been expressed as mean ±
SD of μmoles of CDNB-GSH conjugated formed / min / mg protein.

ESTIMATION OF TOTAL REDUCED GLUTATHIONE (GSH)

- Moron et al. (1979)

Principle

The method was based on the reaction of reduced glutathione with

DTNB to give a compound that absorbs at 412 nm.
Reagents

 Metaphosphoric acid : 1.67 gm of glacial metaphosphoric acid, 0.2 gm

of EDTA and 30 gm of NaCl in 100 ml of distilled water.
 0.4 M Na2HPO4.
 DTNB reagent : 40 mg of DTNB in 100 ml of 1% trisodium citrate.
 Standard Glutathione: 20 mg of reduced glutathione was dissolved in
100 ml of distilled water.

66
Procedure

1.0 ml of 10 % tissue homogenate was precipitated with 4.0 ml of

metaphosphoric acid. The precipitate was removed by centrifugation. To 2.0
ml of the supernatant, 2.0 ml of disodium hydrogen phosphate and 1.0 ml of
DTNB reagent was added. The absorbance was read within 2 mins at 412 nm
against a reagent blank. A set of standards was also treated in the above
manner.
The amount of glutathione is expressed as μg / mg protein.

ESTIMATION OF ASCORBIC ACID

-Omaye et al. (1979)

Principle

Ascorbic acid was oxidised by copper to form dehydroascorbic acid and

diketoglutaric acid. These products were treated with 2,4-dinitrophenyl
hydrazine to form the derivative of bis 2,4-dinitrophenyl hydrazine. This
compound, in strong sulphuric acid undergoes a rearrangement to form a
product with an absorption band that is measured at 520 nm. The reaction was
run in the presence of thiourea to provide a mildly reducing medium, which
helps to prevent interference from non-ascorbic acid chromogens.

Reagents

 5 % TCA.
 65 % Sulphuric acid.
 DTCS reagent: 3gm of 2,4-dinitrophenyl hydrazine, 0.4 gm of
thiourea and 0.05 gm of copper sulphate were dissolved in 9 N
sulphuric acid and made upto 100 ml with the same.
 Standard solution: standard in the range of 4-20 μg/ml were prepared
in 5% oxalic acid.

67
Procedure

1.0 ml of 10 % homogenate was precipitated with 5 % ice-cold TCA

and centrifuged for 20 mins at 3,500 g. 1.0ml of the supernatant was mixed
with 0.2 ml of DTCS reagent and incubated for 3 hours at 37°C. Then 1.5 ml
of ice-cold 65 % sulphuric acid was added, mixed well and the solutions were
allowed to stand at room temperature for an additional 30 mins. Absorbance
was determined at 520 nm.
The results are expressed as μg / mg protein.

ESTIMATION OF VITAMIN E
– Varle et al. (1980)

Principle
Tocopherol can be estimated using Emmerie-Engel reaction, which is
based on the ferric to ferrous ions by tocopherols, which then forms a red color
with 2,2 dipyridyl. Tocopherols and carotenes are first extracted with Xylene
and the extinction read at 460 nm to measure carotenes. A correlation is made
for these after adding ferric chloride and reading at 520 nm.

Reagents
 Absolute ethanol

 Xylene

 2,2-dipyridyl: 1.2 g/L of n-propanol

 Ferric chloride solution: 1.2 g of FeCl3.6H2O in one litre of ethanol.

 Standard solution D,L-α-tocopherol: 10 mg/L in absolute ethanol 0.91

mg of α-tocopherol is equivalent to 100 mg of tocopherol acetate.

68
  Same extraction: Weighed 1.0 g the tissue and were homogenised in a
blender and transferred to a conical flask. Added 50 ml of 0.1 N
sulphuric acid slowly without shaking. Stoppered and allowed to stand
overnight. The next day, the contents of the flask were shaken
vigorously and filtered through Whatmann No.1 paper, discarding the
initial 10-15 ml of the filtrate. Aliquot of the filtrate was used for the
estimation.

Procedure

Into 3 stoppered centrifuge tubes (test, standard and blank) pipetted out 1.5
ml of each liver tissue extract, 1.5 ml of the standard and 1.5 ml of water
respectively. To the test and blank added 1.5 ml of ethanol and to the standard
added 1.5 ml of water. Added 1.5 ml of xylene to all the tubes, stoppered,
mixed well and centrifuged.

Transferred 1.0ml of xylene layer into another stoppered tube, taking care
not to include any ethanol or protein, added 1.0ml of 2,2 dipyridyl reagent to
each tube, stoppered and mixed. Pipetted out 1.5 ml of the mixtures into
spectrophotometer curettes and read the absorbance of test and standard against
the blank at 460 nm. Then in turn beginning wit the blank, added 0.33 ml of
ferric chloride solution. Mixed well and after exactly 15 min read test and
standard against the blank at 520 nm. The amount of vitamin E can be
calculated using the formula.

(∆A520nm-∆A460 nm × conc [s] × 0.29) × Total volume

Vitamin E (μg/g) =
(∆A520nm × Vol for experiment × wt of sample

69
LIPID PEROXIDATION

Peroxidation of membrane lipids is likely to lead to a disturbance of the

Membrane integrity (Richter, 1987; Vliet and Blast, 1992).

ESTIMATION OF LIPID PEROXIDATION (LPO)

- Buege and Aust (1978)

Principle

Malondialdehyde has been identified as the product of lipid

peroxidation that reacts with thiobarbituric acid to give a red color absorbing at
535 nm.

Reagents

 Stock TCA-TBA-HCl reagent: 15% w/v trichloroacetic acid,

0.375 w/v thiobarbituric acid and 0.25 N HCl. The solution was
heated mildly to assist the dissolution of the TBA.

Procedure

To 1.0 ml of the sample, 2.0 ml of TCA- TBA-HCl reagent was added

and mixed thoroughly. The solution was heated for 15 min in a boiling water
bath. After cooling, the flocculent precipitate was removed by centrifugation at
1,000 g for 10 min. The absorbance was determined at 535nm against a blank
that contains all the reagents minus the sample.
The results were expressed as nmoles of MDA formed/min/mg protein
using an extinction coefficient of the chromophore 1.56 x 105 Mcm and
expressed as nmoles of MDA formed/min/mg protein.

70
ESTIMATION OF PROTEIN
- Lowry et al. (1951)

Principle

The blue color developed by the reduction of the phosphomolybdic

phosphotungstic components in the Folin Ciocalteau reagent by the amino
acids tyrosine and tryptophan present in the protein plus the color developed by
the biuret reaction of the protein with the alkaline cupric tartarate are measured
at 660 nm.

Reagents

 2 % Sodium carbonate in 0.1 N NaOH (Reagent A)

 0.5 % Copper sulphate in 1 % potassium sodium tartarate
(Reagent B)
 Alkaline copper reagent: Mixed 50 ml of A and 1.0 ml of
B prior to use
 Folin-Ciocalteau reagent: Mixed 1 part of reagent with 2
parts of water.
 Stock standard: Weighed 50 mg of bovine serum albumin and
made up to 50 ml in a standard flask with saline.
 Working standard: Diluted 10 ml of the stock of 50 ml with
distilled water. 1.0 ml of this solution contains 200 μg of protein.

Procedure

Pipetted out 0.2 to 1.0 ml working standard solution, 0.1 ml of the

sample was taken. The volume in all the tubes were made up to 1.0ml with
distilled water. Added 5.0 ml of alkaline copper reagent to each tube. Mixed
well and allowed to stand for 10 mins. Then added 0.5 ml of Folin-Ciocalteau
reagent. Mixed well and incubated at room temperature for 30 minutes. A

71
reagent blank was also prepared. After 30 minutes, the blue color developed
was read at 660 nm.
The serum protein was expressed as g / dl and in liver homogenate as
mg / gm tissue.

Histopathological studies

The Histopathological analysis of the liver of the experimental animals

of phase III was carried out by following the methodology given in Phase I.

PHYTOCHEMICAL ANALYSIS
(Paech and Tracey, 1955)

IDENTIFICATION OF ALKALOIDS AND FLAVONOIDS

ALKALOIDS

Dragendroff’s Test

8 g of Bi (NO3)3 5H2 O was dissolved in 20 ml of Nitric acid and 2.72g

of Potassium iodide in 50 ml, of water. They were mixed and allowed to stand
when Potassium nitrate crystals out. The supernatant was decanted off and
made upto 100ml with distilled water. The alkaloids were regenerated from the
precipitate by treating with sodium carbonate followed by extraction of the
liberated base with ether.
To 0.5 ml of alcoholic solution of Tephrosia purpurea was added. 2.0
ml of HCl. To this acidic medium 1 ml of reagent was added. An orange red
precipitate produced immediately indicates the presence of alkaloids.

Wagner’s Test (Iodine-Potassium-Iodide Solution)

1.0g of iodine and 2.0 g of potassium iodide solution was diluted to

100 ml. 10 ml of alcoholic extract of Tephrosia purpurea was acidified by

72
adding 1.5% v/v of HCl and a few drops of Wagner’s reagent. Formation of
yellow or brown precipitate confirmed the presence of alkaloids.

Meyer’s Test (Potassium Mercuric Iodide)

1.36 g of Mercuric chloride was dissolved in 60 ml of distilled water

and 5 g of potassium iodide in 10 ml of water. The two solutions were mixed
and diluted to 100 ml with distilled water.

To 1 ml of acidic aqueous solution of Tephrosia purpurea few drops of

reagent was added. Formation of white or pale precipitate showed the presence
of alkaloids.

FLAVONOIDS

In a test tube containing 0.5 ml of alcoholic extract of the Tephrosia

purpurea, 5-10 drops of dilute hydrochloric acid and small piece of Zn or Mg
were added and the solution was boiled for few minutes. In the presence of
flavonoids, reddish pink or dirty brown colour was produced.

IDENTIFICATION OF TANNINS AND SAPONINS

TANNINS

Ferric chloride test

To 1-2 ml of an aqueous solution of Tephrosia purpurea extract, few

drops of 5% solution of aqueous ferric chloride solutioin was added. A bluish
black color which disappears on addition of few ml of dilute H 2SO4 was
followed by the formation of yellowish brown precipitate.

73
SAPONINS

In a test tube containing about 5.0 ml of an aqueous extract of

Tephrosia purpureaa drop of sodium bicarbonate was added. The mixture was
shaken vigorously and kept for 3 minutes. A honeycomb like froth was formed
and it showed the presence of saponins.

IDENTIFICATION OF PHENOLS
PHENOLS
Ferric Chloride test

1 ml of alcoholic solution of Tephrosia purpurea was diluted with water

followed by a few drops of 10% aqueous ferric chloride solution was added.
Formation of blue or green colour indicates the presence of phenols.

Lead acetate test

1 ml of alcoholic solution of Tephrosia purpurea was diluted to 5 ml
with distilled water and to this few drops of 1% aqueous solution of lead
acetate was added. A yellow precipitate was formed to indicate the presence of
phenols.

Libermann’s test

A small quantity of alcoholic extract of the Tephrosia purpurea was

dissolved in 0.5 ml of 20% sulphuric acid solution, followed by the addition of
a few drops of aqueous sodium nitrate solution. A red color was obtained on
dilution and it turned blue when made alkaline with aqueous sodium hydroxide
solution.

74
IDENTIFICATION OF GLYCOSIDES AND RESIN

GLYCOSIDES

A small amount of alcoholic extract of Tephrosia purpurea was

dissolved in 1.0 ml of water and then aqueous sodium hydroxide solution was
added. Formation of a yellow color indicates the presence of glycosides.

RESINS
To 2.0 ml of chloroform or ethanolic extract of Tephrosia purpurea 5-
10 ml of acetic anhydride was added, dissolved by gently heating, cooling and
then 0.5 ml of sulphuric acid was added. Bright purple color was produced. It
indicates the presence of resins.

IDENTIFICATION OF STEROIDS
Libermann-Burchard’s test

To 1.0 ml of Tephrosia purpurea petroleum ether extract in chloroform,

1ml of concentrated sulphuric acid was added followed by the addition of 2.0
ml of acetic anhydride solution. A greenish color developed and it turned blue.
It indicated the presence of steroids.

Salkowski reaction

To 2.0 ml of chloroform extract of Tephrosia purpurea 1.0 ml of

concentrated sulphuric acid was added carefully along the sides of the test tube.
A red color was produced in the chloroform layer.
Statistical analysis

The values were represented as the mean of six values ± S.D. The
results were statistically analyzed using the statistical package (MINITAB,
version 14). One way analysis of variance was employed for comparison
among the six groups followed by Fisher’s test. Statistical significance was set
at p<0.05.

75
 4. RESULTS AND DISCUSSION

Combating cancer is of paramount importance today. Multidisciplinary

scientific investigations are making the best efforts to combat this disease, but
in the sure-shot, perfect cure is yet to be brought into world medicine (Gordon
and Newman, 2001).

Chronic diseases such as cancer, and other non-communicable diseases

are fast replacing communicable diseases in India and other developing
countries.

Many herbs have been evaluated in clinical studies and are currently
being investigated phytochemically to understand their tumricidal actions
against various cancers (Premalatha and Rajgopal, 2005). The rich and diverse
plant sources of India are likely to provide effective anticancer agents. One of
the best approaches in the search for anticancer agents from plant resources in
the selection of plants based on ethnomedical leads (Spiridon, 2006).

The present study was undertaken to investigate the antioxidant and

antitumour status of the methanolic extract of Tephrosia purpurea leaves
against Daltons Lymphoma Ascites tumour induced in mice and the results are
obtained under the following headings.

PHASE – I
TOXICITY STUDIES

Plate 2 exhibits the histological examination of the liver revealed that

there was no potential toxicity or damage done to the liver, which is similar to
that of normal when different doses (100, 200, 300 mg / kg body weight) of

76
MeTp were given to the normal mice for 21 days. Among these doses, highest
concentration of methanolic extract of Tephrosia purpurea (300 mg/kg body
weight) was selected for the experimental studies.

PHASE II

Phase II includes the study of the survival period and observing the
change in the body weight.

SURVIVAL PERIOD

Monitoring the survival period and change in body weight are important
indicators of the effect of the antitumour agents. The effect of methanolic
extract of Tephrosia purpurea on the survival of tumour bearing mice is given
in Table 1.

Table 1. Effect of Tephrosia purpurea on survival period of DLA tumour

bearing mice

Groups Survival in days Life Span (%) % increase in life

Normal > 60 -- --
DLA Control 19.25  6.3 100.0 --
DLA + MeTp 54.80  2.8* 188.42 88.42
Values are expressed as Mean  SD (n = 3)
Statistical comparison (*P <0.05)

Group III compared with Group II

Following the tumour transplantation, the mice survived for 19 to 21

days. The survival time of DLA tumour bearing mice significantly decreased
when compared to normal mice, while the survival period of Tephrosia
purpurea treated DLA tumour bearing mice was found to be increased (54-55
days) significantly.

77
 The present findings are in corroboration with earlier reports of the anti-
tumour activity of many medicinal plants. Badami et al. (2003) reported that
the Solanum pseudocapsicum significantly increased MST of DLA tumour
bearing mice.

Kumar et al. (1998) reported that the life span of DLA tumour bearing
mice, treated with Berberis asiatica was increased. The life span of Mucuna
pruriens treated EAC bearing mice was increased when compared to untreated
EAC bearing mice (Rajeshwar et al., 2005).

Treatment with MeTp increased the life span of tumour bearing mice. It
may be concluded that MeTp by arresting the tumour growth, increases the life
span of DLA tumour bearing mice. Thus the methanolic extract of Tephrosia
purpurea has antitumour activity against DLA tumour bearing mice.

BODY WEIGHT

Following the tumour transplantation, the increase in belly size and

body weight with sluggish movement of the animal was noted from 7 th day.
The change in the body weight was observed on every 5 days interval from the
period of tumour transplantation and the results obtained are presented in
fig. 13 and plate 3.

Body weight of the tumour bearing mice was significantly increased

when compared to normal. The methanolic extract of Tephrosia purpurea
treated tumour bearing mice showed a slight decrease in body weight when
compared to DLA tumour bearing mice.

The present findings are in corroboration with earlier reports of

Rajeshwar et al. (2005) who reported that the Mucuna pruriens treated EAC
bearing mice showed a significant decrease in the body weight.

78
 31

change in body weigt (g)

29
27
25
23
21
19
17
1 5 10 15 20 25 30 35 40 45 50 55 60
Survival period

Normal DLA DLA+MeTp

Fig.13. Effect of Tephrosia purpurea on body weight

Badami et al. (2003) reported a significant decreaes in the body weight

of DLA tumour bearing mice treated with Solanum pseudocapsicum.

The treatment of Tephrosia purpurea caused a significant decrease in

body weight when compared to DLA control. The change in the body weight
of the animals suggests the tumour growth inhibiting activity of MeTp might
be due to the presence of flavonoidal compounds tephrosin, pongaglabol and
semiglabrin present in Tephrosia purpurea may be involved in this action.
Flavonoids have been reported to possess antitumour activity.

PHASE III

It includes analysis of liver function tests, hematological parameters and

Antioxidant contents.

LIVER FUNCTION TESTS

It includes the analysis of liver function marker enzymes in serum and
lipid profiles in liver tissues.

79
Liver function marker enzymes

Table 2 and 3 exhibit the changes in liver function marker enzymes such
as AST, ALT, ALP, ACP and LDH in different experimental groups.

Table 2. Effect of Tephrosia purpurea on liver function marker enzymes

AST and ALT in serum of DLA tumour bearing mice

DLA + DLA +
Parameter Control DLA MeTp
MeTp Methotrexate

AST 10.89  0.69 27.53  1.55a 10.72  0.06b 10.75  0.04 10.49  0.53

ALT 24.46  1.95 36.42  1.24a 24.24  1.40b 23.70  0.04 24.49  1.05
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III

Units: AST, ALT - µ moles of pyruvate liberated/L

Mice induced with Daltons Lymphoma Ascites tumour developed

significant hepatic damage observed from elevated levels of AST and ALT as
compared to the normal mice. Treatment with MeTp caused a significant
reduction in the levels of AST and ALT enzymes indicating the beneficial
effect of Tephrosia purpurea against the DLA tumour. The methotrexate
treated DLA tumour bearing mice showed a better effect than the MeTp treated
group.

Rohini et al. (2004) reported that a significant increase in the levels of

serum AST and ALT in fibrosarcoma bearing rats was observed and it was
decreased by treatment with Bacopa monniera, which indicated the antitumour
activity and correlates with the obtained results.

80
 Increased level of AST and ALT in Fibrosarcoma bearing rat was found
when compared to the normal. The treatment with Muthumarunthu (a herbal
formulation) significantly decreased the AST and ALT level (Palani et al.,
1998).

Table 3. Effect of Tephrosia purpurea on liver function marker enzymes

ACP, ALP and LDH in serum of DLA tumour bearing mice
DLA +
Parameter Control DLA DLA+MeTp MeTp
Methotrexate

ACP 14.14  0.04 30.54  0.02a 13.08  0.04b 14.10  0.05 13.12  0.03

ALP 103.26  5.4 150.55 30.9a 100.922.33b 100.59  1.11 70.71  0.37

LDH 6.19  0.05 11.30  0.07a 5.05  0.04b 6.04  0.04 5.19  0.05
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III

Units : ACP, ALP - moles of phenol liberated / L

LDH moles of pyruvate liberated / L

Daltons lymphoma ascites tumour administration to normal mice

significantly increased the serum ALP, ACP and LDH levels whereas the
MeTp treated DLA tumour bearing mice showed a significant decrease which
is near normal. In the methotrexate treated mice, the serum ALP, ACP and
LDH levels were found to be near normal.

The inference is supported by the findings of Natesan et al., (2007) who

reported the antitumour activity of the methanol extract of Careya arborea
(bark). The inoculation of DLA cells to tumour control animals caused
significant increase in the ALP and LDH levels when compared to normal
animals. The treatment with methanolic extract of Careya arborea
significantly decreased the ALP and LDH level.

81
 Jose Jeena et al. (1999) reported that a significant increase in the levels
of serum ALT and ALP in N-nitrosodiethylamine induced
hepatocarcinogenesis was observed and it was decreased by treatment with
Emblica officinalis, Phyllanthus amarus and Picrorrhiza kurroa, which
indicated the antitumour activity and correlates with the obtained results.

Increased level of ALP in hepatocarcinogenic rat was found when

compared to the normal. The treatment with Cacao liquor extract significantly
decreased the ALP level (Amin et al., 2004). It was reported that the presence
of tumours in the human body or in experimental animals is known to affect
many functions of the liver.

Damage to the structural integrity of liver is reflected by an increase in

the level of AST (Schimdt et al., 1975) because these are cytoplasmic in
location and are released into the circulation after cellular damage (Sallie et al.,
1991). The elevated levels of serum ALP and LDH in tumour inoculated
animals indicated the liver damage and loss of functional integrity of cell
membranes (Gupta et al., 2004).

The significant reversal of these changes towards the normal by

methanolic extract of Tephrosia purpurea treatment in most of the cases
demonstrated the potent hepatoprotective activity of methanolic extract of
Tephrosia purpurea.

Increased levels of AST, ALT, LDH, ACP and ALP following DLA
administration could be attributed to the damaged structural integrity of the
liver cell membrane thus causing leakage of the cellular enzymes in to the
blood.

82
 MeTp used in the present study seems to offer protection and maintain
the structural integrity of the hepatocellular membrane and thus preventing the
leakage of cellular enzymes. This was evident from the significant reduction in
serum AST, ALT, LDH and thus offering protection against DLA tumour in
mice.

LDH, a cytosolic enzyme is a regulator of biochemical reactions in the

body tissues and fluid. Disturbances in cell membranes, were estimated by
measuring the leakage of LDH (Germano et al., 1999).

Our results indicate that the administration of methanolic extract of

Tephrosia purpurea has stabilized the membrane and attenuating the loss of
activity.

LIPID PROFILE

In this study, the effect of Tephrosia purpurea on lipid profile levels in

liver tissues of DLA tumour has been assessed.

Table 4. Effect of Tephrosia purpurea on hepatic lipid profiles in DLA

tumour bearing mice
DLA + DLA +
Parameter Control DLA MeTp
MeTp Methotrexate
Cholesterol 8.40  0.40 5.04  0.55a 8.05  0.11b 8.01  0.18 8.410.08
12.62 
Phospholipids 8.55  0.9a 11.42  0.06b 11.47  0.05 12.55  0.1
1.06
Triglycerides 6.74  0.5 3.85  0.23a 7.07  0.19b 6.95  0.09 7.02  0.18
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III
Units : Cholesterol, phospholipids, Triglycerides – mg/g in tissue

83
 Daltons lymphoma ascites tumour administration to normal mice
significantly decreased the cholesterol, phospholipids and triglycerides levels
as compared to control mice. Treatment with methanolic extract of Tephrosia
purpurea was significantly increased to near normal level. The standard drug,
methotraxate treated DLA tumour bearing mice significantly increased to near
normal mice.

The present work was supported by the findings of Chitra et al. (2003)
who reported the antitumour activity of the extracts of Embelin (berries). The
various parameters in liver such as cholesterol, phospholipids and triglycerides
were significantly decreased in tumour bearing rat when compared to control.
Treatment with Embelin significantly increased the lipid profile level to near
normal.

Natesan et al. (2007) reported that the activity of cholesterol and

triglycerides were significantly decreased in the tumour condition when
compared to normal and treatment with Careya arborea (bark) significantly
increased to near normal.

In the present study a significant depletion of triglycerides, cholesterol

and phospholipids from liver of the animals was observed indicating the
increased lipolytic activity in tumour bearing animals (Ekman et al., 1982).

Lipids are the structural components of the membrane, involved in

maintaining the cell integrity of the cells and in maintaining the fluidity of the
cellular membrane components.

Cholesterol is required for normal growth and cell functions. The cell
maintains a specific distribution of this sterol among its different membranes
(Kumar – Jain, 1975; Yeagle, 1985). The plasma membrane has the highest

84
cholesterol concentration, whereas in liver and kidney mitochondria, the levels
were lower (Colbeau et al., 1971; Jain, 1981; Lange and Ramos, 1983).

The normalisation of lipid levels in tissues of MeTp treated DLA

tumour bearing mice might be due to the decreased lipolytic activity. It might
also be due to the stabilization of the membrane by the action of the
components of MeTp.

Membrane bound ATPases

ATPase enzymes were found in the plasma membrane of virtually every

human cell and is common to all cellular life. It helps to maintain the cell
potential and regulate the cellular volume. In the present study various
enzymes like Na-+K+, Mg2+ and Ca++ ATPases were analysed.

Table 5. Effect of Tephrosia purpurea on membrane bound ATPase

enzymes in DLA tumour bearing mice
DLA + DLA +
Parameter Control DLA MeTp
MeTp Methotrexate
Na+-K+
2.05  0.05 0.73  0.17 a 1.86  0.30b 2.03  0.05 1.790.50
ATPase
Mg2+ ATPase 23.92  2.0 17.38 1.13a 23.26 1.15b 23.31  1.12 24.25  1.4
Ca++
6.07  0.08 2.89  0.36a 5.93  0.74b 6.07  0.10 6.08  0.07
ATPase
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III
Units : Na+-K+, Mg2+ and Ca++ . moles of pi liberated / min / mg protein

Daltons lymphoma ascites tumour administration to normal mice

significantly decreased the liver Na +-K+ ATPase, Ca++ATPase and Mg++
ATPase enzyme levels as compared to control mice. Treatment with MeTp
and Methotrexate significantly increased when compared to normal.

85
 The results are in accordance with the earlier report of Senthilnathan et
al. (2005) who reported the decreased levels of the ATPase enzymic activities
of Na+-K+ ATPases, Mg2+ ATPase and Ca++- ATPase in lung cancer. Treatment
with withaniya somnifera elevated the levels of this enzymes.

The activities of Na+-K+ ATPase, Ca2+ ATPase and Mg2+ ATPase were
decreased significantly in the liver due to DLA tumour bearing mice. It is
restoration of all three ATPases to near normal levels, the drug treated animals
can be attributed to the potent membrane stabilizing effect of MeTp extract.
Tephrosia purpurea contains flavonoids and other constituents like phenolic
compounds, sterols, glycosides etc. Flavonoids are known to influence the
permeability of biomembranes. These compounds are known to interact with
Na+-K+-ATPase pump in animal cells and may have the ability to catalyse
electron transport. In the presence of flavonoids, the Na + K+ pump regained its
normal efficiency and the cell assumed normal properties (Havsteen, 1983).
These compounds possesses antioxidant and antiradical properties (Cotelle et
al., 1996). Antioxidant activity of flavonoids may also be due to their structural
features and its action in the membrane. Inhibition of LPO and membrane
stabilizing action of Tephrosia purpurea may be responsible for the restoration
of ATPases activity in MeTp treated group.

Based on the data obtained it was observed that the Tephrosia purpurea
provide stabilization of membrane bound enzyme profiles against the DLA
tumour in mice.

Hematological parameter

To evaluate the antitumour effect of Tephrosia purpurea the change in

hematological parameters were analysed and the results obtained are presented
in Table 6.

86
Table – 6. Effect of Tephrosia purpurea on Hematological parameters in
DLA tumour bearing mice

Groups RBC Hb WBC

Normal 4.06  0.25 13.50  0.77 8.53  0.04
DLA Control 2.61  0.17a 7.50  0.70a 22.31 0.02a
DLA + MeTp 4.02  0.07b 13.99  0.31b 7.55  0.03b
DLA + 4.2  0.07 14.20  0.43 8.56  0.03
Methotrexate

MeTp 3.89  0.48 13.66  0.34 7.53  0.03

Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III

The results of the present investigation are in accordance with the study
of Rajeshwar et al., (2005) who reported that the Hb and RBC levels were
decreased in EAC bearing mice and WBC level was increased in EAC bearing
mice. In the Mucuna pruriens treated EAC bearing mice, the Hb, RBC and
WBC levels were found to be near normal.

Natesan et al. (2007) reported that the Hb and RBC levels were
decreased and WBC level was increased in DLA tumour bearing mice. In
Careya arborea treated DLA tumour bearing mice, the Hb, RBC and WBC
levels were reversed to near normal.

Our findings are similar to the study of Badami et al. (2003) who
showed that the treatment with Solanum pseudocapsicum decreased the WBC
level and increased the RBC and Hb level in DLA tumour bearing mice.

Usually, in cancer chemotherapy the major problems that are being

encountered are of myelosuppression and anemia (Price et al., 1958; Hongland

87
et al., 1982). The anemia encountered in tumour bearing mice is mainly due to
reduction in RBC or hemoglobin percentage, and this may occur either due to
iron deficiency or due to hemolytic or myelopathic conditions (Fenninger and
Mider 1954). Treatment with MeTp brought back the hemoglobin (Hb)
content, RBC and WBC count more or less to normal levels. This clearly
indicates that MeTp possess protective action on the hemopoietic system.

ANTIOXIDANTS
ENZYMIC ANTIOXIDANTS
The enzymatic antioxidants that prevent the cellular and molecular
damage caused by the Reactive oxygen species like SOD, CAT, GPx, GR and
GST, were analysed and the results obtained are presented in Table 7 and 8.
Table 7. Effect of Tephrosia purpurea on enzymic antioxidants SOD, CAT
and GPx in DLA tumour bearing mice
DLA +
Parameter Control DLA DLA + MeTp MeTp
Methotrexate
SOD 11.51  1.5 3.27  0.6a 7.75  1.4b 7.80  0.37 10.75  2.20
CAT 207.05  2.07 108.431.82a 183.28  2.98b 183.732.70 206.07  2.16
GPx 58.72  3.4 24.30  2.20a 46.19  1.93b 47.20  3.13 56.72  2.23
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III

Units: SOD – 50% inhibition of nitrite formation / min / mg protein

CAT - moles of H2O2 decomposed / min / mg protein
GPx - g of glutathione utilized / min / mg protein

Daltons lymphoma ascites tumour administration to normal mice

significantly decreased the liver SOD, CAT and GPx level as compared to
control mice. Treatment with methanolic extract of Tephrosia purpurea and

88
Methotrexate significantly increased when compared to DLA tumour bearing
mice (Fig. 14).

Gupta et al. (2004a) who reported that the SOD and CAT enzyme levels
were lowered significantly in the tumour state. Administration of Caesalpinia
bonducella increased the SOD and CAT activity. Gupta et al. (2004b) showed
decreased activities of SOD and CAT in the Bauhinia recemosa treated Ehrlich
ascites carcinoma mice which correlates with the obtained inference.

The present study was supported by Natesan et al. (2007), who reported
that the antitumour activity of methanol extract of Careaya arborea (bark)
showed a significant increase in SOD and CAT activities.

Cells are equipped with enzymatic antioxidant mechanisms that play an

important role in the elimination of free radicals. SOD, CAT and GPx are
involved in the clearance of superoxide and hydrogen peroxide (H 2O2). SOD
catalyses the deminition of superoxide into H 2O2, which has to be eliminated
by glutathione peroxidase and / or catalase (Rushmore et al., 1999). It was
found that from the table, a decrease in SOD activity in DLA – bearing mice
may be due to loss of Mn2+- containing SOD activity in DLA tumour and the
loss of mitochondria, leading to a decrease in total SOD activity in the liver.

SOD is an important defense enzyme which catalyses the dismutation of

superoxide radicals to oxygen and hydrogen peroxide. Catalase is a heme
protein, CAT and peroxidase are considered biologically essential in the
reduction of hydrogen peroxide (Jose Jena et al., 1999).
Fig. 14. Effect of Tephrosia purpurea on
enzymic antioxidants in
DLA tumour bearing mice

89
 SOD

12

formation / min/mg protein

10

50% inhibition of nitrate

8

0
Control DLA DLA + MeTp DLA + MeTp
Methotrexate

CAT

250
decomposed/min/mg protein

200
µmoles of H2O2

150

100

50

0
Control DLA DLA + MeTp DLA + MeTp
Methotrexate

GPx

60
µg of glutathione utilized / min /

50

40
mg protein

30

20

10

0
Control DLA DLA + MeTp DLA + MeTp
Methotrexate

90
 On the other hand the free radical scavenging system, SOD and catalase
are present in all oxygen metabolizing cells and their function is to provide a
defense against the potentially damaging reactivities of superoxide and
hydrogen peroxide (Sun et al., 1989)

In the Daltons lymphoma ascites tumour induced mice, GPx level was
significantly decreased when compared to normal mice. The GPx level was
significantly increased in the methanolic extract of Tephrosia purpurea treated
mice. The methotrexate treated GPx level was found to be near normal. Singh
et al. (2005) reported the GPx level was significantly decreased in
Carcinogenic mice and treatment with Tinospora cordifolia increased the GPx
level.

GPx is a selenoenzyme, catalyses the reaction of hydroperoxides with

reduced glutathione to form glutathione disulfide (GSSG) and the reduction
product of hydroperoxide (Venukumar and Latha, 2002). GPx activity was
elevated in liver may be due to adaptive response to remove the hydrogen
peroxide (Anuradha and Balakrishnan, 1998).

The activity of GPx, an enzyme that reduces the levels of peroxides in

cell and thus protects the cell from peroxidative damage, was inhibited by DLA
exposure.

In the present investigation with DLA bearing mice, the administration

of methanolic extract of Tephrosia purpurea increased the SOD, CAT and GPx
levels, which may indicate the antioxidant and free radical scavenging property
of MeTp. The increase in the SOD, CAT and GPx in MeTp treated group
indicates its potential as an inhibitor of DLA induced intracellular oxidative
stress.

91
Table. 8. Effect of Tephrosia purpurea on enzymic antioxidants GR and
GST in DLA tumour bearing mice
DLA +
Parameter Control DLA DLA + MeTp MeTp
Methotrexate
GR 35.37  2.75 44.120.99a 36.45  0.92b 37.00  1.31 37.32  0.58
GST 8.58  0.35 18.08 0.83a 8.46  0.07b 8.42  0.05 8.38  0.07
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III

Units : GR -  moles NADPH oxidized / min / mg protein

GST -  moles of CDNB conjugated / min / mg protein

GST plays an essential role in liver by eliminating toxic compounds by

conjugating them with glutathione (Venukumar and Latha, 2002). Increased
activity of GST in liver occurs as an adaptive mechanism due to increased
uptake from plasma (Anuradha and Balakrishnan, 1998). The decreased
activity of GST in MeTp administered mice can hasten the decomposition of
lipid hydroperoxides, and there by account for the antitumour effects.

The GR and GST levels were significantly increased in the DLA

induced mice when compared to the control. The methanolic extract of
Tephrosia purpurea and methotraxate treated mice showed significant decrease
in GR and GST activity.

Similar findings are also shown by Amin et al. (2004) who reported that
Cacao liquor treated hepatocarcinogenic animals showed a decrease in the
levels of GR and GST.
The results obtained suggest that the hepatoprotective activities claimed
for Tephrosia purpurea, may be due to the antioxidant activity of MeTp. This
activity could be due to the presence of compounds that inhibit free radical
mediated damage in hepatic tissues. Studies are in progress in order to

92
determine presence of compounds such as flavonoids and polyphenols in
Tephrosia purpurea, which could be responsible for the antioxidant activity
described.
GST activities increased in colorectal tumours (Kanbagli, 2000). It has
been suggested that oxidative stress dependent DNA base lesions were
responsible for the low anti-oxidant enzyme activities in acute lymphoblastic
leukemia and prostate hyperplasia (Senturker et al., 1997; Gierek et al., 2000)
and pro-oxidant enzyme activities found in the present study could indicate an
increased oxidative stress.

NON - ENZYMIC ANTIOXIDANTS:

The non enzymatic antioxidants like GSH, Vitamin C and Vitamin E
also have a number of biological activities such as immune stimulation,
inhibition of nitrosamine formation and alteration of metabolic activations of
carcinogens (Ray and Husain 2002). The results obtained are presented in
Table 9 and Fig. 15.

Table 9. Effect of Tephrosia purpurea on hepatic non-enzymic antioxidants

in DLA tumour bearing mice
DLA + DLA +
Parameter Control DLA MeTp
MeTp Methotrexate
0.122
GSH 2.20  0.21 1.45  0.32b 1.92  0.03 2.05  0.04
a
1.43
Vit. C 7.67  0.52 1.380.25a 7.40  0.27b 7.49  0.28 7.65 0.22
Vit. E. 8.08  1.69 3.43 3.00a 7.53  0.07b 7.46  0.04 8.00 0.05
Values are expressed as  SD (n = 6)
Statistical comparison: (P<0.05)
a – Group II is compared with Group I
b – Group III is compared with Group II
c – Group IV is compared with Group III
Units : GSH, Vit. C  g / mg protein
Vit. E. -  g / g tissue

Daltons lymphoma ascites tumour administration to normal mice

significantly decreased the GSH, Vitamin C and Vitamin E levels when

93
compared to normal mice. Treatment with MeTp was significantly increased to
near normal. Methotrexate treated mice showed a significant increase in GSH,
Vitamin C and Vitamin E level.

GSH level was significantly decreased in EAC bearing mice compared

with normal. In the Caesalpinia bonducella treated mice, GSH level was
significantly increased near to normal mice (Gupta et al. 2004a).

Gupta et al. (2004b) reported that a significant decrease in EAC bearing

mice and the level was significantly increased when treated with Bauhinia
racemosa. Ahmed et al. (1999) reported the remarkable reduction in the
content of GSH, Vitamin E and Vitamin C in cervical carcinoma when
compared to control.

Glutathione, a potent inhibitor of the neoplastic process, play an

important role in the endogenous antioxidant system. It is found to be
particularly high concentration in the liver and is known to have a key function
in the protective process. Excessive production of free radicals resulted in
oxidative stress, which leads to damage of macromolecules (Sinclair et al.
1990). It was also reported that the presence of tumours in the human body or
in experimental animal is known to affect many functions of the vital organs,
especially in the liver when the site of the tumour does not interfere directly
with organ function (DeWys, 1982).

MeTp significantly increased the glutathione content in DLA-bearing

mice. The antitumour effect of methanolic extract of Tephrosia purpurea may
be due to the antioxidant and the free radical quenching property of the
phytoconstituents of MeTp.

94
 Fig. 15. Effect of Tephrosia purpurea on
Non-enzymic antioxidants in
DLA tumour bearing mice
GSH

2.5

2
µg/ / mg protein
1.5

0.5

0
Control DLA DLA + MeTP DLA + MeTP
Methotrexate

Vit. C

8
7
µg / mg protein

6
5
4
3

2
1
0
Control DLA DLA + MeTP DLA + MeTP
Methotrexate

Vit. E.

7
µg / mg protein

5
4

2
1

0
Control DLA DLA + MeTP DLA + MeTP
Methotrexate

95
 Vitamin C, a water – soluble antioxidant and also a pro-oxidant, it is
potential and one of the most effective scavengers of oxygen free radicals and
other O2 derived species. It reacts with a variety of free radicals, fits into the
physiology of cellular transport and metabolism suitable regeneration. It
imparts its protection by undergoing oxidation to dehydroascorbate (Sunitha et
al., 2001).

Vitamin C can protect cell membrane and lipoprotein particles from

oxidative damage by regenerating the antioxidant form of Vitamin E (Buettner,
1993; Beyer, 1994). It imparts its protection by undergoing oxidation to
dehydro ascorbate, for the back conversion to ascorbate GSH is required.
Consequently when GSH is reduced, there is a fall in the level of Vitamin C.

Increased levels of GSH, Vitamin C and Vitamin E in DLA tumour

bearing mice treated with herbal extracts confirm their protective effect against
the oxidative stress and radical scavenging ability. This might be due to the
presence of considerable quantitites of antioxidative components in Tephrosia
purpurea.

Lipid peroxidation
Lipid peroxidation, an autocatalytic free radical chain propagating
reaction, is known to be associated with pathological conditions of a cell.
Malondialdehyde, the end product of LPO was reported to be higher in cancer
tissues than in non diseased organ. The results obtained are presented in Fiure
15.

96
 Fig.16. Effect of Tephrosia purpurea on Liver LPO
Activities in DLA tumour bearing mice

n moles of MDA formed/mg protein 7

0
Control DLA DLA + MeTP DLA MeTP
+Methotrexate

LPO level of DLA bearing mice was significantly increased when

compared to normal mice. MeTp treated DLA bearing mice showed a
significant decrease in LPO level. The treatment with methrotraxate also
showed a significant decrease in LPO activity.

The present study is supported by the findings of Jose Jeena et al.

(1999) who reported that the antitumour effect of Emblica officinalis,
Phyllanthus amarus and Picrorrhiza kurroa against hepatocarcinogenesis. In
EAC bearing mice, the LPO level was increased when compared to
normal. Treatment with Bauhinia racemosa decreased the LPO level
(Gupta et al. 2004b).

Oxidative damage induced by DLA in the formation of highly reactive

oxygen species, which stimulates LPO and cause destruction of the cell
membrane. Treatment with MeTp resulted in the significant fall might be due
to the presence of the antioxidant content of the plant extract.

97
Histological Studies

The hepatoprotective action was also studied by the histological

examination (Plate 4).

Group I – Normal

The liver section of normal mice shows normal histological appearance.

Group II – DLA Control

The DLA tumour bearing mice liver showed slight enlargement of

hepatocytes, dilated sinusoidal spaces containing lymphocytes and portal triads
showing collections of lymphocytes.

Group III – DLA + MeTp

The DLA tumour bearing mice treated with MeTp almost normal
histological appearance of liver cells.

Group IV – DLA + Methotrexate

The DLA tumour bearing mice treated with methotrexate showed

almost normal histological appearance of liver cells.

98
PHYTOCHEMICAL ANALYSIS:

Qualitative analysis of phytochemicals in Tephrosia purpurea:

Many phytochemicals are now studied extensively for their potential

role in reducing the risk and even preventing and treating some diseases.
Herbal preparations are effectively and extensively used for their medicinal
properties and have become increasingly popular worldwide(Kainthla et al.,
2006). Phytochemicals constitute a wide family of natural components with a
considerable range of bioactive properties with potential clinical applications
as anticancer drugs.

A large number of phytochemicals, not recognized as essential nutrients,

apparently play an important antioxidant role in the body; practically all diet
contain atleast some phytochemicals. Fruits, vegetables and herbs are
particularly rich sources (Lin 1995).

Phytochemical constituents in Tephrosia purpurea

Secondary metabolites Observation

Alkaloid +

Flavonoid +

Steroid +

Phenolics +

Tannins +

Saponins -

Resins -

Glycosides +

99
 Major classes of phytochemicals with potential for antioxidant,
phytosterols, tannins, chlorophylls, terpenoids, allytic compounds and indoles
(Cooper et al., 1997).

Phytochemical analysis of Tephrosia purpurea showed the presence of

Secondary metabolites like alkaloids, Flavanoids, glycosides etc., might be
responsible for antioxidative or antitumour properties.

These phytochemical might be responsible for the antitumours and

antioxidant effects of methanolic extract of Tephrosia purpurea.

100
 5. SUMMARY AND CONCLUSION

The incidence and mortality rates of cancer vary widely across the
world, but the highest rates were reported every year from developing
countries, particularly from India. Lymphoma cancer is one among the few
human cancers with the vast potential for prevention. In cancer, enormous
production of free radicals in the system has been reported. A major target of
reactive oxygen species is cell membrane, due to the high content of
polyunsaturated fatty acids. Reactive oxygen species mediated lipid
peroxidation causes damage to cellular DNA, membrane structure and
inhibition of functions of several enzymes and alterations in the immune
system.

In our country, since ancient time leaves, bark, seed and various other
parts of plants have been conventionally practiced in traditional medicine. The
utility of such leaves are considered to be valuable in terms of the medicinal
properties, ready availability and practicability even at house hold leaves. The
medicinal properties may vary from plant to plant and there is awareness
among the people to realize the need for the naturopathy treatment for
permanent relief from physiological upsets and ailments and even from some
chronic diseases like asthma, nervous debility, cancer etc.

The present study was carried out to evaluate the of antitumour and
antioxidant effects of Tephrosia purpurea against Dalton’s lymphoma ascites
carcinoma in Swiss albino mice and it was carried out in three different phases.

In Phase I, the toxic effect of the methanolic extract of Tephrosia

purpurea was analysed. In short term toxicity study, the histological
examination of the MeTp treated normal mice for 21 days at the doses of 100,

101
200, 300 mg / kg body weight, showed the nontoxic effect for all the doses.
Among the three doses, the highest dose was selected for the further
experimental studies.

In the next phase of the investigation the treatment was given for 21
days, then the survival period and the change in body weight were observed
upto 60 days. The life span of DLA tumour bearing mice was found to be
decreased whereas MeTp treated mice showed a significant increase in the life
span.

DLA tumour bearing mice showed a significant increase in their body

weight. Treatment with MeTp, the body weight was found to be significantly
decreased. Administration of drug reduced the tumour incidence and growth
resulting in decreased mortality rates when compared to DLA tumour bearing
mice.

The experiment was followed by analyzing all the required biochemical

parameters to confirm the antioxidant and antitumour effects (Phase III). Liver
cells participate in a variety of metabolic activities due to the presence of
different enzymes. Hence the evaluation has been carried out relating to liver
marker enzymes in serum, Lipid profiles, ATPase enzymes, Enzymic and non-
enzymic antioxidants and lipid peroxidation in liver tissues, and supported by
histopathological studies. Hematological parameters were also analyzed.

Cytosolic marker enzymes such as AST, ALT and LDH, lysosomal

marker enzymes ALP and ACP in serum. The liver marker enzymes were
increased in DLA tumour bearing mice. Administration of MeTp caused a
significant decrease in the levels of liver function marker enzymes.

102
 The lipid components such as total cholesterol, phospholipids and
triglycerides were decreased and administration of MeTp restored these levels
to near normal in DLA tumour bearing mice.

Membrane damage is a basic feature of malignant cells. The membrane

bound ATPase activities namely sodium potassium ATPase, Magnesium
ATPase and calcium ATPase were decreased indicating the severity of the
disease. The activities were reverted to near normal levels upon MeTp
treatment, indicating its membrane stabilizing action.

The Hematological parameters such as RBCs and Hb levels were

decreased in DLA tumour bearing mice and treatment with MeTp increased the
RBC and Hb levels to near normal. The WBC level was increased in DLA
tumour bearing mice and treatment with MeTp reverted to near normal.

The levels of enzymic antioxidants such as SOD, CAT and GPx were
significantly decreased and a increase in the levels of GR and GST were
observed in DLA tumour bearing mice. Administration of MeTp helped in
maintaining the cellular redox status of the animals by increasing the efficiency
of antioxidant defence systems of components.

The decrease in the activity of non-enzymic antioxidants such as

Glutathione, Vitamin C and Vitamin E were speculated to be due to the
damaging effects of free radicals produced following DLA tumour.
Administration of the extract reverted the non-enzymic levels to near normal,
confirming the antioxidant effect of the MeTp.

Animals with DLA tumour had a significant increase in the level of

lipid peroxides as compared to normal. The increase in malondialdehyde levels
in the liver suggests the enhanced lipid peroxidation leading to tissue damage

103
and failure of antioxidant defense mechanisms to prevent formation of
excessive free radicals. Treatment with Tephrosia purpurea significantly
reversed these changes. Hence, it is possible that the mechanism of antitumour
of Tephrosia purpurea is due to its antioxidant effect also.

The histological study showed the recovery of the damaged liver cells in
the MeTp treated mice, the cytoarchitecture was restored to the same as normal
level. Histological examination also confirmed the anti-tumour potential of the
drug.

Plant derived extracts containing antioxidant contents and other active

principles exhibited antitumour activity in experimental animals. It was
proposed that the additive and synergistic activity of phytochemicals present in
methanolic extract of Tephrosia purpurea might be responsible for its potent
antitumour activity which can be inferred from the increased the life span of
DLA tumour bearing mice.

The qualitative analysis of phytochemicals indicates the presence of

secondary metabolites such as alkaloids, flavonoids and tannins, which might
be responsible for the antioxidant and antitumour activity of MeTp.

Scope for further research

 A systemic approach is needed for identifying the active

constituents from Tephrosia purpurea.

 More human clinical trails are recommended in near future to

authentically project the Tephrosia purpurea as safe antitumour
agent.

104