chapter 1 introduction - shodhganga : a...

CHAPTER 1 INTRODUCTION

Evaluation of some plant constituents for immunomodulatory effect 1

Mankind has, throughout its existence, used plant material not only as a

source of nutrition but also for numerous other purposes. The plants

provided food, clothing, shelter, and medicine. Plants are considered to

be medicinal if they possess pharmacological activities of possible

therapeutic use. Medicinal plants have been a rich source of medicines

because they produce a host of bioactive molecules, most of which

probably evolved as chemical defenses against predation or infection.

Plant derived drugs are used to cure mental illness, skin diseases,

tuberculosis, diabetes, jaundice, hypertension and cancer. Medicinal

plants play an important role in the development of potent therapeutic

agents. Plant derived drugs came into use in the modern medicine

through the uses of plant material as indigenous cure in folklore or

traditional systems of medicine. Over the past decade, herbal medicines

have been accepted universally, and they have an impact on both world

health and international trade. Hence, medicinal plants continue to play

an important role in the healthcare system of a large number of the

world’s population (Akerele, 1988). Traditional medicine is widely used in

India. Even in USA, use of plants and phytomedicines has increased

dramatically in the last two decades. A National Centre for

Complementary and Alternative Medicine has been established in USA.

The herbal products have been classified under ‘dietary supplements’

and are included with vitamins, minerals, amino acids and ‘other

products intended to supplement the diet’ (Rao et al., 2004). Use of

plants as a medicinal remedy is an integral part of the South African

cultural life (Brandt et al., 1995). It is estimated that 27 million South



Africans use herbal medicines from more than 1020 plant species (Meyer

et al., 1996).

Plants have been the basis of many traditional medicine systems

throughout the world for thousands of years and continue to provide

mankind with new remedies. Plant based medicines initially dispensed

in the form of crude drugs such as tinctures, teas, poultices, powders,

and other herbal formulations (Samuelsson, 2004). The plant based

indigenous knowledge was passed down from generation to generation in

various parts of the world throughout its history and has significantly

contributed to the development of different traditional systems of

medicine. The use of plants as medicines has involved the use of crude

plant drug or the isolation of active compounds, beginning with the

isolation of morphine from opium in the early 19th century (Kinghorn,

2001) and subsequently led to the isolation of early drugs such as

cocaine, codeine, digitoxin and quinine, of which some are still in use.

Numerous molecules have come out of ayurvedic experiential base,

examples include Rauwolfia alkaloids for hypertension, psoralens in

vitiligo, holarrhena alkaloids in amoebiasis, guggulsterones as

hypolipidemic agents, Mucuna pruriens for Parkinson’s disease,

piperidines as bioavailability enhancers, bacosides in mental retention,

picrosides in hepatic protection, phyllanthins as antivirals, curcumin in

inflammation, withanolides, and many other steroidal lactones and

glycosides as immunomodulators (Patwardhan, 2000).

Herbal medicines are currently in demand and their popularity is

increasing day by day. About 500 plants with medicinal use are



mentioned in ancient literature and around 800 plants have been used

in indigenous systems of medicine. India is a vast repository of medicinal

plants that are used in traditional medical treatments (Chopra et al.,

1956). The WHO (world health organization) estimated that about 80% of

the populations living in the developing countries rely almost exclusively

on traditional medicines for their primary health care needs. In almost

all the traditional medicines, the medicinal plants play a major role and

constitute the backbone of the traditional medicines. Indian Materia

Medica includes about 2000 drugs of natural origin almost all of which

are derived from different traditional system and folk fare practices

(Narayana et al., 1998). India has an ancient heritage of traditional

medicine. The various indigenous systems use several plant species to

treat different ailments in which Ayurveda is the important and most

primitive system of medicine. The Ayurvedic system of medicine

developed an extensive use of medicines from plants dating from at least

1000 BC.

1.1. AYURVEDA

Ayurveda is the oldest system of medicine, which came in to existence

during Vedic times (about 900 BC). It is an upveda (part) of Atharva

Veda. The word Ayurveda is derived from ‘Ayur’ meaning life and ‘Veda’

meaning science. Thus, ayurveda literally means science of life.

Ayurveda deals with the physical, as well as spiritual health. It is based

on the concept of strengthening the host defenses against different

diseases (Thatte and Dahanukar, 1986). The medicinal form is governed



by the laws of nature, which suggest that life is a combination of senses,

mind, body and soul. It was the first holistic system of diagnosis and

treatment integrating nutrition, hygiene, rejuvenation, and herbal

medicine. It reveals the potential of various herbs generally in polyherbal

formulations as drugs (Dash and Kashyap, 1980). One of the main

approaches in Ayurvedic medicine is to “increase the body’s natural

resistance to the disease/stress” known as “Rasayana” (Rejuvenation)

(Pallabi et al., 1998).

Ayurvedic medicine considers the human body to be in balance

with nature. The body is believed to be a dynamic and resilient system

that can cope with all stresses from its environment while maintaining

the ability to heal itself.''' The main objectives of Ayurveda are to

maintain and promote health by preventing physical, mental, and

spiritual ailments and to cure disease through natural medicine, diet,

and a regulated lifestyle. According to ayurveda, the structural aspect of

every individual comprises five elements - earth, water, fire, air and

space. According to Ayurvedic theory a harmonious balance between

three humors of the body viz,. 'Vayu', 'Pitta' and 'Kaf' is needed for positive

health; imbalance of these may cause disease. A significant part of

Ayurvedic therapeutics is preventive in nature. It aims to promote positive

health so that individuals do not suffer from disease. This is the concept

of "Vyadhirodhak chamatav", i.e. capacity of the body to resist disease

(Katiyar et al., 1997). The practice of Ayurveda therapeutics consisted of

8 sections divided into 180 chapters and listed 314 plants, which are

used as medicines in India.



In the ayurveda literature, there was much stress on the collection

of plant material during a particular season, from a particular locality

and at a certain time of day. It is now well established that the

concentration and profile of secondary metabolites in a plant depend on

environmental, nutritional and photoperiodic factors. Thus, the correct

type of plant material is essential. Around 1,250 plants are currently

used in various Ayurvedic preparations (Chatterjee and Pakrashi, 1991;

Mills and Kerry, 2000).

Ayurveda remains an important system of medicine and drug

therapy in India. Plant alkaloids are the primary active ingredients of

Ayurvedic drugs. Today the pharmacologically active ingredients of many

Ayurvedic medicines are being identified and their usefulness in drug

therapy being determined. Ayurvedic medicines generally come in the

form of powders, tablets, decoctions, and medicated oils, that are

prepared from natural herbs, plants and minerals. In addition, the

diseases treated and cured by Ayurvedic medicines do not cause any

side effects.

1.2. HERBAL MEDICINE

Medicinal plants play an important role in the development of potent

therapeutic agents. About 500 plants with medicinal use are mentioned

in ancient texts and around 800 plants have been used in indigenous

systems of medicine. Indian subcontinent is a vast repository of

medicinal plants that are used in traditional medical treatments (Chopra

et al., 1956), which also forms a rich source of knowledge. The various



indigenous systems such as Siddha, Ayurveda, Unani and Allopathy use

several plant species to treat different ailments (Rabe and Staden, 1997).

In India around 20,000 medicinal plant species have been recorded

recently (Dev, 1997), but more than 500 traditional communities use

about 800 plant species for curing different diseases (Kamboj, 2000).

Plant derived drugs are used to cure mental illness, skin diseases,

tuberculosis, diabetes, jaundice, hypertension and cancer. These drugs

provide outstanding contribution to modern therapeutics; for example:

serpentine isolated from the root of Indian plant Rauwolfia serpentina in

1953, was a revolutionary event in the treatment of hypertension and

lowering of blood pressure. Vinblastine isolated from the Catharanthus

roseus (Farnsworth et al., 1967) is used for the treatment of Hodgkins,

choriocarcinoma, non-hodgkins lymphomas, leukemia in children,

testicular and neck cancer. Vincristine is recommended for acute

lymphocytic leukemia in childhood advanced stages of hodgkins,

lymphosarcoma, cervical and breast cancer (Farnsworth and Bingel,

1977). Medicinal herbs as potential source of therapeutics aids has

attained a significant role in health system all over the world for both

humans and animals not only in the diseased condition but also as

potential material for maintaining proper health.

1.3. PLANT AS A SOURCE OF MEDICINE

The plants are of great importance in human’s life. They provide food,

clothing and shelter and besides these plants are the major sources of

medicines. Medicinal plants have been a major source of cure of human



diseases since time immemorial. Much of the medicinal use of plants

seems to have been developed through observations of wild animals, and

by trial and error. As time went on, each tribe added the medicinal

power of herbs in their area to its knowledge base. A great variety of

plants are used for medicinal treatment.

The whole plant or from different organs, like leaves, stem, bark,

root, flower, seed, etc may be used as herbal drugs. Some drugs are

prepared from excretory plant product such as gum, resins and latex.

These parts taken as whole or compressed as tablets or made into pills,

used to make infusions (teas), extracts, tinctures, etc., or mixed with

excipients to make lotions, ointments, creams, etc.

Rather than using a whole plant, pharmacologists identify, isolate,

extract, and synthesize individual components, thus capturing the active

properties. The medicinal properties of a plant are due to presence of

active ingredients or active constituents or phytochemicals. The activity

of these phytochemicals depends upon the solvent used and the method

of extraction (Eloff, 1998). In addition to active ingredients, plants

contain minerals, vitamins, volatile oils, glycosides, alkaloids,

bioflavonoids, and other substances that are important in supporting a

particular herb's medicinal properties. These elements also provide an

important natural safeguard. Isolated or synthesized active compounds

can become toxic in relatively small doses; it usually takes a much

greater amount of a whole herb, with all of its components, to reach a

toxic level. Plants used as medicines offer synergistic interactions

between constituents both active and other. The therapeutic potentials



of plant are being used from the ancient times by the simple process

without the isolation of the pure compounds i.e. in the form of crude

drug and the therapeutic action of crude drug is determined by the

nature of its constituents, these constituents are known as

phytochemicals.

1.4. PHYTOCHEMICALS

Plants are the biosynthetic laboratory of phytochemicals. Phytochemicals

are naturally occurring, biologically active chemical compounds in

plants. The prefix “Phyto” is from a Greek word meaning plant. These are

chemical compounds formed during the plants normal metabolic

processes. These chemicals are often referred to as “secondary

metabolites” of which there are several classes including alkaloids,

flavonoids, coumarins, glycosides, gums, polysaccharides, phenols,

tannins, terpenes and terpenoids (Harborne, 1973; Okwu, 2004). In

addition to compounds that are necessary for the growth and

reproduction of plants, i.e. carbohydrates, proteins and lipids, plant cells

synthesize a tremendous number of secondary metabolites, which do not

appear to be strictly necessary for the survival of the plant. These

secondary metabolites or phytochemicals are produced as a response to

external stimuli, e.g. infection or nutritional or climatic changes, and

they may be accumulated in only certain parts of the plant (Verpoorte,

1999). In plants, phytochemicals act as a natural defense system for

host plants and provide colour, aroma and flavour. More than 4000 of

these compounds have been discovered to date and it is expected that



scientists will discover many more. Phytochemicals are protective and

disease-preventing particularly for some forms of cancer and heart

diseases.

Plants develop these numerous non- nutritive chemicals for their

protection and offence which are known as phytochemicals. Besides,

protection of plants these phytochemicals have various therapeutic

applications such as anti-oxidant, anti-diabetic, memory enhancer,

reducing cholesterol, adaptogen, anticancer, immunomodulator etc. The

thousands of phytochemicals that have been discovered are grouped

based on function and sometimes source.

The various phytochemicals have been grouped in to the main classes of

phytochemicals which are as follows:

§ Alkaloids: Alkaloids are naturally occurring organic substances

which constitute major class of chemical group present in plant.

These have heterocyclic ring containing nitrogenous nucleus. They

are basic in nature and bitter in taste. Alkaloids rank among the most

efficient and therapeutically significant plant substances (Okwu,

2005). Some 5,500 alkaloids are known and they comprise the largest

single class of secondary plant substances which contain one or more

Nitrogen atoms, usually in combination as part of a cyclic structure

(Harborne, 1973). They are usually organic bases and form salts with

acids and when soluble gives alkaline solutions. Examples include

nicotine, cocaine, morphine and codeine (Papaver somniferum),

quinine (Cinchona succirubra), reserpine (Rauwolfia vomitoria), which

has a large demand worldwide. Alkaloid production is a characteristic



of all plant organs. They exhibit marked physiological activity when

administered to animals (Okwu and Okwu, 2004). Alkaloids possess

anti-tumor activity (vinblastine and vincristine), antimicrobial

(cepharanthine), analgesic activity (morphine) etc.

§ Carbohydrates: These are soluble substances present in the cell sap

often associated with the other constituents i.e. glycosides. The

pharmaceutically important carbohydrates are polysaccharides and

polyuronides. Crude drugs mostly consist of carbohydrates either

simple like hexose, pentose etc. or complex like polysaccharides. The

pharmaceutical important ones are polysaccharides (e.g. inulin,

starch etc.) and polyuronides (contains uronic acid e.g. gums like

Acacia, tragacanth etc.).

§ Glycosides: These include a wide range of chemical sub groups

containing a glycan (sugar) and an aglycan (non-sugar). These are

polar compounds which consist of atleast one sugar molecule linked

to another moiety. Pharmaceutically important glycosides include

saponins and anthracin derivatives. Glycosides mainly participate in

the stimulation of cardiac system, CNS stimulation and immune

system, they also possess antimicrobial activity e.g. anthraquinone

glycosides, saponin glycosides, cyanogenic glycosides (yields HCN).

§ Phenolic compounds: These are wide range plant substances

possess an aromatic ring bearing one or more hydroxyl substituents.

They are water soluble as they most frequently occur combined with

sugar as glycosides. They are usually located in the cell vacuole. The

presence of phenols is considered to be potentially toxic to the growth



and development of pathogens (Okwu and Okwu, 2004).This group

includes flavonoids, tannins and other phenols.

v Flavonoids – These are among the most widely distributed natural

products in plants occurring both in the free state and as

glycosides. They are mainly water soluble compounds. There

chemical structure is based upon C6-C3-C6 carbon skeleton.

Flavonoids apotent water-soluble super antioxidants and free

radical scavengers which prevent oxidative cell damage, have

strong anti-cancer activity and protects against all stage of

carcinogens. Flavonoids in the body are known to reduce the risk

of heart diseases (Urquiaga and Leighton, 2000). In terms of anti-

cancer activity, they inhibit the initiation, promotion and

progression of tumors (Urquiaga and Leighton, 2000; Okwu,

2004). In recent times, plant flavonoids have attracted attention

as potentially important dietary cancer chemo-protective agents

(Hertog et al., 1993; Elangevan et al., 1994). Some isoflavones act

as allelochemicals widely used in insecticides (Kandaswami et al.,

1994).

v Tannins – These are amorphous, rarely crystalline substances,

soluble in water and alcohol and having an astringent and bitter

taste. Chemically they are complex phenolic substances classified

on the basis of hydrolysis product. They occur widely in vascular

plants, in angiosperms their occurrence associated with woody

tissues. Hydrolysable tannins are based on gallic acid, usually as

multiple esters with D-glucose, while the numerous condensed



tannins (often proanthocyanides) are derived from flavonoid

monomers (Harborne, 1973; Okwu, 2005). Many physiological

activities such as stimulation of phagocytic cells, host mediated

tumor activity and wide range of anti-infective action have been

assigned to tannins (Okwu and Okwu, 2004).

§ Terpenoids: These are natural products whose structure may be

divided into isoprene units, hence also called as isoprenoids.

Ginkgolide A (helps in blood circulation), taxol (used in carcinoma

treatment), andrographolide (hepatoprotection) are some of its

examples. Triterpenoids are compounds with a carbon skeleton based

on six isoprene units and which are derived biosynthetically from the

cyclic C30 hydrocarbon, squalene. They are colourless, crystalline,

often have high melting points and are optically active substances.

The essential triterpenoids are saponins, steroids and cardiac

glycolsides which occur mainly as glycosides. Triterpenes occur

especially in the waxy coatings of leaves and on fruit such as apple

and pear and they may serve a protective function in repelling insects

and microbial attack (Harborne, 1973).

1.5. PHARMACOLOGICAL STUDIES

The classical way of pharmacological screening involves sequential

testing of new extracts from biological material in isolated organs

followed by tests in whole animals, mostly rats and mice but also higher

animals if indicated. Most drugs used nowadays in therapy have been

found and evaluated with these methods. Medicinal plants have



contributed in the most comprehensive way for the development of

herbal medicines and preparations. For past several decades, the extract

of numerous medicinal plants are experimented for various in vivo and

in vitro pharmacological studies like immunomodulatory,

hepatoprotective, anticancer, anthelmintic, antimicrobial etc.

1.5.1. IMMUNOMODULATORY ACTIVITY

As the name suggests Immunomodulatory activity associated with the

immunomodulator which regulate the immune system. Immune system

is a collection of mechanisms within an organism that protects against

disease by identifying and killing pathogens and tumor cells. It detects a

wide variety of agents, from viruses to parasitic worms, and needs to

distinguish them from the organism's own healthy cells and tissues in

order to function properly. It uses various endogenous molecules for

distinguishing ‘self’ and ‘non self’ and mounting defense reactions in a

very selective and specific manner (Steven et al., 2002).

1.5.1.1. Immune system (Roitt, 1984; Guyton, 1991)

The defense system of our body that prevents the entry of

microorganisms and eliminates the microbes, which infect our body

system, is known as immune system. The immune system is composed

of many interdependent cell types that collectively protect the body from

bacterial, parasitic, fungal, viral infections and from the growth of tumor

cells. The cells of the immune system can engulf bacteria, kill parasites

or tumor cells, or kill viral-infected cells.



Figure 1.1 Different organs of the immune system (See: Modric)

1.5.1.2. The Organs of the Immune System

The immune system is composed of many organs (figure 1.1) which are

given as follow:

Bone Marrow: All the cells of the immune system are initially derived

from the bone marrow through a process called hematopoiesis. The bone

marrow produces B cells, natural killer cells, granulocytes and immature

thymocytes, in addition to red blood cells and platelets.

Thymus: The function of the thymus is to produce mature T cells.

Immature thymocytes, also known as prothymocytes, leave the bone

marrow and migrate into the thymus. The mature T cells are then

released into the bloodstream.

Spleen: The spleen is an immunologic filter of the blood. It is made up of

B cells, T cells, macrophages, dendritic cells, natural killer cells and red



blood cells. In the spleen, B cells become activated and produce large

amounts of antibody. Also, old red blood cells are destroyed in the

spleen.

Lymph Nodes: The lymph nodes function as an immunologic filter for

the bodily fluid known as lymph. Lymph nodes are found throughout the

body. Composed mostly of T cells, B cells, dendritic cells and

macrophages, the nodes drain fluid from most of our tissues.

1.5.1.3. Cells of the Immune System

Various cells which are found in immune system are as follow (Figure

1.2):

Figure 1.2: Cells of the immune system (See: Todar)

T-Cells

T lymphocytes are usually divided into two major subsets that are

functionally and phenotypically different. The T helper subset, also

Lymphocytes Granulocytes

B Cell

Stem Cell

Natural killer cell

T Cell Neutrophil Eosinophil

Basophil

Mast cell Monocyte

Dendritic Cell

Macrophage Plasma Cell

Memory Cell

Th Cell

Tc Cell



called the CD4+ T cell, is a pertinent coordinator of immune regulation.

T helper cell augments or potentiates immune responses by the secretion

of specialized factors that activate other white blood cells to fight off

infection. Another important type of T cell is called the T

killer/suppressor subset or CD8+ T cell. These cells are important in

directly killing certain tumor cells, viral-infected cells and sometimes

parasites. Both types of T cells can be found throughout the body.

Natural Killer Cells

Natural killer cells, often referred to as NK cells, are similar to the killer

T cell subset (CD8+ T cells). They function as effector cells that directly

kill certain tumors such as melanomas, lymphomas and viral-infected

cells, most notably herpes and cytomegalovirus-infected cells.

B Cells

The major function of B lymphocytes is the production of antibodies in

response to foreign proteins of bacteria, viruses, and tumor cells.

Antibodies are specialized proteins that specifically recognize and bind to

one particular protein. Antibody production and binding to a foreign

substance or antigen, often is critical as a means of signaling other cells

to engulf, kill or remove that substance from the body.

Granulocytes or Polymorphonuclear (PMN) Leukocytes

Another group of white blood cells is collectively referred to as

granulocytes or polymorphonuclear leukocytes (PMNs). Granulocytes are

composed of three cell types identified as neutrophils, eosinophils and

basophils, based on their staining characteristics with certain dyes.

These cells are predominantly important in the removal of bacteria and



parasites from the body. They engulf these foreign bodies and degrade

them using their powerful enzymes.

Macrophages

Macrophages are important in the regulation of immune responses. They

are often referred to as scavengers or antigen-presenting cells (APC)

because they pick up and ingest foreign materials and present these

antigens to other cells of the immune system such as T cells and B cells.

This is one of the important first steps in the initiation of an immune

response. Stimulated macrophages exhibit increased levels of

phagocytosis and are also secretory.

Dendritic Cells

Dendritic cells, which also originate in the bone marrow, function as

antigen presenting cells (APC). In fact, the dendritic cells are more

efficient apcs than macrophages. These cells are usually found in the

structural compartment of the lymphoid organs such as the thymus,

lymph nodes and spleen. However, they are also found in the

bloodstream and other tissues of the body. It is believed that they

capture antigen or bring it to the lymphoid organs where an immune

response is initiated.

1.5.1.4. Immunity (Goldsby et al., 2001)

The capacity of immune system to protect the host body from infections,

pathogens and diseases is known as immunity. It is of two types i.e.

Innate immunity and Adaptive immunity.

Innate immunity: It developed early in evolution and present in some or

other form in most multicellular organism.



Adaptive immunity: The resistance that an individual acquires during

life is known as adaptive immunity. The ability to respond specifically to

a particular infection and to generate memory cell is the basis of

acquired immunity. The acquired immunity response is of two types:

Humoral immune response and cell mediated immune response.

Humoral immune response: Host defense that is mediated by antibody

present in the plasma, lymph and tissue fluids is known as humoral

immune response. It protects against extracellular bacteria and foreign

macromolecules.

Cell mediated response: It refers to the specific immune responses that

do not involve antibodies. Induction of cell mediated response consists of

specifically sensitization of T-lymphocytes against the antigen. The

activated lymphocytes release biologically active products (lymphokines),

those are responsible for the manifestation of cell mediated response.

Macrophages and other mononuclear cells under the effect of

lymphokines affect the destruction of microorganism and other

processes involved in cell mediated response.

1.5.1.5. Immunomodulators

An immunomodulator may be defined as a substance, biological or

synthetic, which can stimulate, suppress or modulate any of the

components of the immune system including both innate and adaptive

arms of the immune response (Agarwal and Singh, 1999). The functional

and efficiency of immune system may be influenced by many exogenous

and endogenous factors. Apart from the natural mechanism, there are

compounds that are capable of interacting with immune system, to up



regulate or down regulate specific aspects of the host response can be

classified as immunomodulators and the mechanism involve with these

is known as immunomodulation. The basic strategy underlying

immunomodulation is to identify aspects of the host response that can

be enhanced or suppressed in such a way as to augment or complement

a desired immune response. Immunomodulators are of two types i.e.

Immunostimulants and Immunosuppressant.

Immunostimulant: These agents are envisaged to enhance body's

resistance against infections (and may be against allergy, autoimmunity,

and cancer as well). By this definition these agents are inherently non-

specific in nature, but they can act through both the innate and adaptive

arms of the immune response. In healthy individuals the

immunostimulants are expected to serve as prophylactic or promotive

agent i.e. as immune potentiators by enhancing the basal levels of

immune response, and in individuals with impairment of immune

response as immunotherapeutic agent (Agarwal and Singh, 1999).

Immunosuppressant: These agents could be used for control of

pathological immune response in autoimmune diseases, graft rejection,

graft versus host disease, hypersensitivity immune reaction (immediate or

delayed type), and immune pathology associated with infections. Out of the

list the maximum use of these agents has been for prevention of graft

rejection and treatment of autoimmune diseases (Agarwal and Singh,

1999).

These immunomodulators may be synthetic drugs or may be of

herbal origin. Due to side effect of synthetic drugs, herbal drugs gaining



popularity in the world market.

1.5.2. ANTHELMINTIC ACTIVITY

Helminths are of major public health and economic importance to both

man and livestock throughout the tropics. It is estimated that 60-80% of

the world’s population is affected by helminths with a vast majority of

these in developing countries (Farnsworth, 1988). Ascariasis, affects

about one quarter of the world’s population, especially the growth and

nutritional status of children (Latham, 1977). Control of helminthiasis

has therefore been the centre of focus in biomedical research since time

immemorial. Both the medical and veterinary professions have tried to

control helminthiasis by administration of synthetic drugs (Sebuguzi,

2000). However, these drugs are becoming increasingly expensive with

some having serious side effects (Siddiqui and Hussein, 1992).

Therefore, plant originated drugs have been tested for their efficacy

against helminths. Various plant originated drugs are used traditionally

for anthelmintic treatment and several plants and their extracts have

been widely investigated for their anthelmintic properties.

1.5.3. ANTIMICROBIAL ACTIVITY

The world we live in is one full of microbes. Our body temperature and

wealth of nutrients provide an ideal home for these micro-organisms to

thrive. Some microbes are beneficial for human being and play

important role in providing food, chemicals and medicines etc but there

are many microorganisms which cause diseases in human, animals and



plants. The drugs which are used to kill or resist the growth of microbes

are known as antimicrobial agent.

The increasing prevalence of multidrug resistant strains of

bacteria and the recent appearance of strains with reduced susceptibility

to antibiotics adds urgency to the search for new infection-fighting

strategies. In particular, the search for components with antimicrobial

activity has gained increasing importance in recent times, due to growing

worldwide concern about the alarming increase in the rate of infection by

antibiotic-resistant microorganisms (Davies, 1994). Hence, there is a

constant need for new and effective therapeutic agents. Medicinal plants

represent a rich source of antimicrobial agent (Mahesh and Satish,

2008). Earlier many plants have been investigated for their antimicrobial

property.

1.5.4. ANTIOXIDANT ACTIVITY

Oxygen is essential for the survival of all on this earth. During the

process of oxygen utilization in normal physiological and metabolic

processes approximately 5% of oxygen gets univalently reduced to

oxygen derived free radicals (Yu, 1994; Halliwell and Gutteridge, 1989)

like superoxide, hydrogen peroxide, hydroxyl and nitric oxide radicals.

Free radicals are very detrimental in attacking lipids in cell

membranes and also DNA, inducing oxidations that cause membrane

damage such as membrane lipid peroxidation and a decrease in

membrane fluidity and also cause DNA mutation leading to cancer

(Cerutti, 1994; Piett, 2000). Free radicals and oxidants activate nuclear



factor-ĸB, a nuclear transcription factor, resulting in an upregulation of

pro-inflammatory mediators such as interleukin-1, interleukin-8 and

tumor necrosis factor- (Grimble, 1994). This in turn stimulates the

immune response; increases oxidant production and can lead to further

tissue damage. Free radicals stress leads to tissue injury and

progression of disease conditions, such as arthritis, hemorrhagic shock,

atherosclerosis, diabetes, hepatic injury, aging and ischemia. The

production of free radicals is inextricable linked to the inflammatory

process. Free radicals prime the immune response, recruit inflammatory

cells and are innately bactericidal (Grimble, 1994; Allen, 2003).

Figure 1.3: Antioxidant and free radicals

The scavenger of free radical is known as antioxidant (figure 1.3).

Besides playing an important role in physiological systems, antioxidants

have been used in the food industry to prolong the shelf life of foods,

Stable Chemical Free radicals

Oxidation

Antioxidant

Stable Chemicals



especially those rich in polyunsaturated fats. These components in foods

are readily oxidised by molecular oxygen and is a major cause of quality

deterioration, nutritional losses, off-flavour development and

discolouration. The addition of synthetic antioxidants, such as propyl

gallate, butylated hydroxy anisole (BHA), butylated hydroxy toluene

(BHT) and tertiary butyl hydroquinone has been widely used industrially

to control lipid oxidation in foods. However, the use of these synthetic

antioxidants has been questioned due to their potential health risks and

toxicity (Kahl and Kappus, 1993).

The consumption of fruits and vegetables containing antioxidants

has been found to offer protection against these diseases. Dietary

antioxidants can augment cellular defenses and help to prevent oxidative

damage to cellular components (Halliwell, 1989). The search for

antioxidants from natural sources has received much attention and

efforts have been put into identify compounds that can act as suitable

antioxidants to replace synthetic ones.

On the basis of the above back ground the present investigation

has been under taken to evaluate, the two plants, i.e. Swertia chirata

and Gymnema sylvestre for their immunomodulatory, anthelmintic,

antimicrobial and antioxidant activity.



1.6. AIM AND OBJECTIVES

The aim of the present study is to evaluate the extracts of Gymnema

sylvestre and Swertia chirata for different pharmacological studies.

The present study involves the following steps:

I. Preliminary Study

a) Collection of plant materials

b) Processing of collected plant materials

• Drying of plant materials

• Grinding of plant materials

• Storage of plant materials

c) Extraction of phytochemicals

• Solvent extraction (successive extraction)

II. Phytochemical Analysis

III. Chromatographic study

• Thin Layer Chromatography

IV. In-vitro studies

a) Anthelmintic Studies

b) Antimicrobial Studies

• Antibacterial Activity

• Antifungal Activity

c) Antioxidant Assay

• Determination of total phenolic contents

• Ferric reducing power activity

• Free radical scavenging activity by DPPH method



• Hydroxyl radical scavenging activity

• Nitric oxide scavenging activity

V. In-vivo Studies

a) Immunomodulatory Studies

• Haemagglutination test

• Delayed type Hypersensitivity test

1.6. AUTHENTICATION OF THE PLANT MATERIAL

The identification of the plant materials was done by Taxonomist and

Herbarium incharge, Department of Botany, Dr. H.S. Gour University,

Sagar.

Voucher specimen number:

Swertia chirata: Bot/Her/3116

Gymnema sylvestre: Bot/Her/B/1314

chapter 1 introduction - shodhganga : a...

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