Results and Discussion

I N T R O D U C T IO N

Nature has bestowed on us a very rich botanical wealth and a large

number of diverse types of plants in different parts of the Country (Chitravadivu
et al., 2009). India has rich medicinal plants flora of more than 7500 species.
Over 50% of all modern clinical drugs are natural product origin and natural
products play important roles in drug development in the pharmaceutical
industry (Preethi et al., 2010). Medicinal plants and herbs have been used for
the eradication of disease and human suffering since antiquity (Shahid-UdDaula and Mohammad-Anwarul, 2009).
Natural products such as herbs, fruits and vegetables become popular in
recent years due to public awareness and increasing interest among consumers
and scientific community (Thaipong et al., 2006). Natural products which
contain antioxidant properties such as phenolics, include flavonoids and
phenolic acids (Klimczak et al., 2007), carotenoids and vitamins (Rupasinghe
and Clegg, 2007). Herbal medicines have recently attracted much attention as
alternative medicines useful for treating or preventing life style related disorders
and relatively very little knowledge is available about their mode of action. There
has been a growing interest in the analysis of plant products which has
stimulated intense research on their potential health benefits (Anbarasu
et al., 2011). The majority of the worlds population in developing countries still
relies on herbal medicines to meet their health needs in cases when synthetic
medicine could not relieve patients who suffer from painful illnesses like cancer.
In the modern system of medicine, several chemotherapeutic agents have been
developed as a result of screening of the medicinal plants in various parts of the
world (Radha et al., 2008).
Cancer is the second leading cause of death worldwide next to
cardiovascular diseases

and

can

be

treated

with

surgery,

radiation,

chemotherapy, hormone therapy (Gupta et al., 2004) and biological therapy

(Dashora et al., 2010). Emerging evidence suggests that a number of plants
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

are known to be the source of useful drugs in modern medicine and have been
accepted currently as one of the main source of cancer chemoprevention,
drug discovery and development (Gonzales and Valerio, 2006; Kathiriya
et al., 2010) due to their diverse pharmacological properties including cytotoxic
and cancer chemopreventive effects (Dahiru et al., 2005). Plant derived agents
are being used for the treatment of cancer. Several anticancer agents including
taxol,

vinblastine,

vincristine,

the

camptothecin

derivatives,

topotecan,

irinotecan and etoposide derived from epipodophyllotoxin are in clinical use

all over the world (Shoeb, 2006).
Tumor is a mass of tissues which proliferate rapidly, spread throughout
the body and may eventually cause death of the host. Chemotherapy is an
effective treatment against various types of cancer either singly or in
combination with surgery and/or radiotherapy. However, chemotherapeutic
effects of most of the drugs showed limited efficacies due to the development of
various side effects. This fostered our attempts to evaluate some plant products
against cancer as they are less likely to cause serious

side effects. Many

Indian spices (Unnikrishnan and Kuttan, 1990) and plants (Babu et al., 1995)
are quoted to be useful in different types of cancer.
In the human body the free radicals are continuously produced due to
the oxygen utilization by the cells of the body (Vilasrao et al., 2010). This
generates a series of reactive oxygen species (ROS) like super oxide anion
(O2-) and hydroxyl (HO) radicals and non free radical species such as H2O2,
singlet oxygen (O2) and nitric oxide (NO). Reactive oxygen species
and reactive nitrogen species are associated with many pathological
conditions such as atherosclerosis, ischemia, and reperfusion injury of many
tissues, central nervous system injury, gastritis and cancer (Costa et al., 2009;
Manokaran et al., 2005). Recently there has been an upsurge of interest
in

the

therapeutic

potentials

of

medicinal

plants

as

antioxidants

in

reducing such free radical induced tissue injury (Pourmorad et al., 2006).
Phytocompounds like flavonoids and phenolic acids, commonly found in
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

plants (Pawlowska et al., 2008) have been reported to have multiple biological
and pharmacological

activities including antioxidative, cytotoxic, anticancer

(Cesquini et al., 2003; Conforti et al., 2008; Rajkumar et al., 2011) antimicrobial,
antiviral (Dash and Murthy, 2011) and antiinflammatory activities (Middleton,
2000; Kumar et al., 2011).
Antioxidants cause protective effect by neutralizing free radicals, which
are toxic byproducts of natural cell metabolism. The human body has several
mechanisms to counteract oxidative stress by producing antioxidants, which
are either naturally produced in situ, or externally supplied through foods and/or
supplements. These antioxidants act as free radical scavengers by preventing
and repairing damages caused by ROS, and therefore can enhance the
immune defense and lower the risk of cancer and degenerative diseases
(Pham-Huy et al., 2008).
Antioxidants are any substance that delay or inhibit oxidative damage to
a target molecule. At a time one antioxidant molecule can react with single free
radicals and are capable to neutralize free radicals by donating one of their own
electrons, ending the carbon-stealing reaction. Antioxidants prevent cell and
tissue damage as they act as scavenger. Cell produce defense against
excessive free radicals by their preventive mechanisms, repair mechanisms,
physical defenses and antioxidant defenses. The body has developed several
endogenous antioxidant catalase (CAT), glutathione peroxidase (GPx),
glutathione reductase (GR) and non-enzymatic defense system included
vitamin E, vitamin C and reduced glutathione (GSH). Oxidative stress is a
harmful condition that occurs when there is an excess of ROS and/or a
decrease in antioxidant levels, this may cause tissue damage by physical,
chemical, psychological factors that lead to tissue injury in human and causes
different diseases (Tian et al., 2007; Saikat et al., 2010).
The Ehrlich Lymphpma Ascite (ELA) tumor was initially described as a
spontaneous murine mammary adenocarcinoma. It is a rapidly growing
carcinoma with very aggressive behavior and is able to grow in almost all
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

strains of mice. In ascetic form it has been used as a transplantable tumor

model to investigate the antitumor effects of several substances (Segura et al.,
2000). Ascite fluid is the direct nutritional source for tumor cells. So, a rapid
increase in ascite fluid with tumor growth would be a mean to meet the
nutritional requirement of tumor cells (Rajeshwar et al., 2005). Intraperitoneally
propagated ELA tumor cells in Swiss albino mice are an important experimental
approach to study the biology of human cancer. The methanolic extract of aerial
parts of Momordica cymbalaria hook F, crude extract and diethylether fractions
of Alangium salvifolium flower and ethanolic extract of Tragia Plukenetii
R. Smith were found to retard the tumor development and increased the life
span of ELA tumor

bearing mice (Jeevanantham et al., 2011; Meenakshi

Sundaram et al., 2008 ; Zahan, et al., 2011).

Modulation of immune response, as a possible therapeutic measure,
by using medicinal plant products has become a subject of scientific
investigation. Indian medicinal plants are commonly used for the treatment of
various ailments and are considered by many to have advantages over the
conventionally used drugs, which are expensive and known to have harmful
side effects (Khare, 2004). Modulation of the immune response through
stimulation or suppression may help in maintaining a disease-free state. Agents
that activate host defense mechanisms in the presence of an impaired
immune responsiveness can provide supportive therapy to conventional
chemotherapy. Immunostimulation in a drug induced immunosuppression and
immunosuppression in an experimental hyper reactivity model by the same
preparation can be to the true immunomodulation (Kalpesh Gaur et al., 2009).
The immune system is vulnerable to the free radical induced oxidative
stress. The cellular and humoral components of the immune system are
particularly sensitive to increased levels of reactive oxygen species, which may
cause premature immunosenescence (Joharapurkar et al., 2004). It is essential
to counteract this oxidative stress and there by enhance the immunity of body
system. Allopathic drugs are available for counteracting the oxidative stress
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

and hence improve immunity, but the side effects and prohibitive cost of
these allopathic drugs makes it necessary to search for an alternative
(Meera et al., 2008).
There are many plants, which are having immunostimulatory activity
whereas other has immunosuppressant activity. Apart from being specifically
stimulatory or suppressive action, certain agents have been shown to possess
activity to normalize or modulate pathophysiological processes and are hence
called immunomodulatory agents (Bafna and Mishra, 2005). The plant products
have long been used as immunomodulators by the traditional healers. Scientific
literature is continuously reporting plant drugs having immunostimulatory
activity. Immunomodulation using plant extracts such as Eclipta alba, Centella
asiatica, T. Chebula, T. bellerica and E. officinalis can provide an alternative
to conventional chemotherapy for a variety of diseases, especially when the
host defense mechanism has to be activated under the condition of impaired
immune response (Tan and Vanitha, 2004; Srikumar et al., 2006).
The

active

principle

of

plants

acting

as

antioxidants

and

immunomodulators can reduce the toxicity of specific chemotherapeutic agents

used in the treatment of cancer. The immunomodulators have a biphasic
effect on the immune response either by stimulating or inhibiting the immune
response and other host defense mechanisms. Immunomodulators not only
have immunorestorating function but also can be used to produce an
immunocompetent state so as to ward off many diseases. Therefore, great
attention is given for the plant materials that can stimulate immune system.
Plant extracts with antioxidant activity are traditionally used to strengthen
the natural immune defenses. Immunomodulatory agents of plant origin
enhance the immune responsiveness of an organism against a pathogen
by nonspecifically activating the immune system (Razdan and Roy, 2008;
Jayathirtha and Mishra, 2004).
Docking is frequently used to predict the binding orientation of small
molecule drug candidates to their protein targets in order to predict the affinity
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

and activity of the small molecule. Hence docking plays an important role in the
rational design of drugs (Kitchen et al., 2004). Docking analysis by Grid Based
Ligand Docking with Energetics (GLIDE) is used to predict the binding
properties of disease related target proteins. The software searches for
favourable interactions between one or more typically small ligand molecules
and a larger receptor molecule usually a protein (Gane and Dean, 2000).
The plants selected for the present research work were Cynodon
dactylon (Cd) and Terminalia catappa (Tc). Cd (Family-Poaceae), commonly
known as doob (Hindi), aroogumpillo (Tamil), is called creeper in India. The
Cd has been shown to possess variety of medicinal properties. The aqueous
fluid extract of Cd leaf was used to study the antidiabetic, antioxidant and
hypolipidemic efficacy in diabetic rats (Karthik and Ravikumar, 2011; Rai et al.,
2010). Antiulcer activity (Patil et al., 2005), hepatoprotective activity (Surendra
et al., 2008) and cardioprotective effect (Garjani et al., 2009) of alcoholic extract
of Cd have been reported. Immunomodulatory and antioxidant activity of the Cd
protein fractions were evaluated in Swiss albino mice (Santhi and Annapoorani,
2010; Santhi et al., 2010).
Terminalia catappa (Tc) is (family - combretaceae) also known as badam
widely grown in tropical regions of the world as an ornamental tree. The
aqueous and cold extracts of leaves of the Tc have been reported to be
antioxidant, hepatoprotective, and antidiabetic (Nagappa et al., 2003 ; Ahmed
et al., 2005). More and more pharmacological studies have reported that the
extract of Tc leaves and fruits have antibacterial, anticancer, antioxidant,
anti-HIV reverse transcriptase, antiinflammatory, antidiabetic effects and
hepatoprotective activities (Mohale et al., 2009). Tc leaf protein has antioxidant
activity against ELA implanted Swiss albino mice (Santhi and Annapoorani,
2009).
In particular Cynodon dactylon and Terminalia catappa received a lot of
attention in the research worldwide because of their potent antioxidant activity.
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals

Results and Discussion

So far, no studies were undertaken to explore the antioxidative, antitumor

and immunomodulatory activity of flavonoid fractions of Cd (CdFf) and Tc (TcFf)
leaves. The present unique research was undertaken to investigate the
Antioxidative,

antitumorigenic

and

immunomodulatory

efficacy

of

flavonoid fractions of Cynodon dactylon and Terminalia catappa leaves in

experimentally implanted ELA cells in Swiss albino mice.
The present study was carried out in five phases with the following
objectives:
Phase I
1. To extract the flavonoids of Cynodon dactylon (Cd) and Terminalia catappa
(Tc) leaves using different organic solvents
2. To quantify the total flavonoid content of flavonoid rich fractions of Cynodon
dactylon and Terminalia catappa leaves and selection of the flavonoid
fractions (CdFf and TcFf).
Phase II
3. To evaluate the in vitro antioxidative and antitumorigenic potential of CdFf
and TcFf.
Phase III
4. To evaluate the in vivo antioxidative and antitumorigenic activity of CdFf and
TcFf against standard antioxidant silymarin and in ELA tumor induced Swiss
albino mice.
Phase IV
5. To evaluate the immunomodulatory role of CdFf and TcFf in SRBC
immunized mice against pyrogallol, ELA and carbon ink induced Swiss
albino mice.
Phase V
6. To characterize CdFf and TcFf by color reactions of flavonoids,
chromatographic, spectral analysis and in silico studies.
Compatibility of Pongamia pinnata biofuel / diesel blends with few industrial metals