CHAPTER: 1

Introduction

1.0 INTRODUCTION
1.1 General:

The health of human being has adversely affected by the diseases and also their well
being. Differemt infectious agents are responsible for causing these ailments since many
years. Non communicable diseases have become the major health related problem in the
21st century (Zimmet P et al.,2001). Since time immemorial, human has been
experimenting, analyzing, utilizing the unexhaustive,and exploring bounties of nature-
relentlessly. It is by the help of these experiments many human being and animal
ailments have been successfully treated and cured like hepatitis, malaria, depression,
jaundice, inflammation, and skin disorders. Many other diseases like, cancer, diabetes,
and hepatitis etc on which extensive research is continued to find a „cure‟ which is
effective in complete irradiation of disease, safe and economical as well. On the
globalization and liberalization, the price of drugs and treatment measures are increasing
every day, hindering the health care budgets of not only third world countries but of
individuals too. Keeping in view the patient compliance, investigations have been taken
to choose the possibilities of using plants, to treat human. To conquer these issues, the
synthetic drugs which are used concomitantly in few individuals may cause side effects
or drug interactions when used to cure such ailments for example, sulfonamides prohibit
metabolism or excretion of antidiabetic drug sulfonylureas hence producing
hypoglycemia, whereas metabolism of rifampicin increases to reduce their hypoglycemic
impact. Hence, taking in consideration the synthetic- drug interactions and side effects
there is a new expectation to look in for plants and herbal preparations for their
therapeutic uses and values.
1.2 Herbal Medicine :
Traditional and herbal remedies are currently being encouraged, recommended and
promoted by WHO in national health care systems due to their ease of availability at low
cost as well as because they are safer and most people have their faith in these
remedies(Handa et al., 1995) . Total health defined by WHO, is not just about the
Chapter 1: Introduction 2

absence of any disease, but also a state of physical, mental, social and spiritual well
being. Diseases today such as cancer, depression, heart troubles etc are arising due to the
faulty nutritions and stress. Allopathy medication are largly not able to cure these
diseases and just offers temporary relief of the symptoms because these diseases have
compenents that are mental or emotional. Need of alternative therapy is required greatly,
to cover a good health for all of us. To overcome the illness(Gupta et al., 2000) Herbal
therapy may be best to perform Since ancient times, in India different parts of the several
medicinal plants are used to cure specific ailment. Since many centuries indigenous
systems of medicine like Ayurvedic, Unani and Siddha have been in existed. Nearly 70%
of village population were served by these systems of medicine.Diseases that are
obstinate and are not curable in other systems medicine can be treated by herbal
medicines. The backbone of the traditional medicine that are the Medicinal plants, have
been the subjected to intense pharmacological studies in last couple of decades, In the
development of drug the lead compond source and therapeutic value of new compound's
potential source has been brought about in acceptance by the value of medicinal plants. A
revival of intrest has come up in the world to use herbal medicines in past few years
beacuse of these facts. The WHO has developed guidelines to formulate traditional
remedies that supports the member states and has also appreciated that the fact is that
most of our world population is depending upon the traditional medicine and hence their
potential uses, safety and efficacy (Mukherjee et al. , 1998) studies were conducted.
various experts in the field managed the importance and the challenges of conducting
herbal drug's clinical research, the pharmacological and toxicological evaluation, toxic
herbal drugs in use, animal models for toxicity and safety evaluation and biological
standardization by simple bioassay.( Padh et al ., 2000)
The quality of the medicinal plants for pharmaceutical proposes should be high as that of
medicinal preparations. However, it is not possible to perform assay when the bioactive
ingredient is not known for a specific chemical entity. In practice, those medicinal plant
materials who have known active ingredient their assay procedures are not conducted.
The complex composition of drugs which are been used in the form of whole plant, or
parts of plant and of plant extract in them problem comes during Standardization.
Generally, the standardization of the expected drug's active compounds do not reflect the
Chapter 1: Introduction 3

reality. In few cases only the activity of drug depends on the single component. It is the
result of concerted activity of many of the compounds that are active and of
accompanying substances that are inert. Though the pathological mechanism is not
affected directly by these inert accompanying components and because these inert
components may be influencing the bioavailability as well as the active components
excretion hence it might become reasonable to use the complex mixtures of components
that is provided by a medicinal plant. Increase in the stability of active component with
reduction in rate of side effect by the component of inert plant. There might be effects
like additive or potentiating if the plant drug contains different active compounds.
To ensure the therapeutic efficacy of traditional remedies, standardization is done. The
assurance of quality of traditional remedies depends on the following categories that are;
batch analysis along with good manufacturing practice and by doing standardization
methods of preparation

1.2.1 Draw backs of synthetic or Allopathic drugs : (Rao et al. , 2000)

* In the development of new drug, high cost and long time is taken.
* Toxicity: Iatrogenic diseases is a new branch of medicine.
* Non renewable source of the basic raw materials. synthetic drugs utilizes fossil
resources such as petrochemicals
* Chemical industries produces environmental pollution.
1.2.2 Positive side of Ayurvedic or plant drug : (Rao et al. , 2000)
* Long history of use, public acceptance, better patient tolerance as well.
* Renewable source.
* Environmental friendly Cultivation & processing.
* Local availability, in developing countries especially.
For traditional medicines many plant species have been utilized but it is very necessary to
establish the scientific basis of traditional plant medicine's therapeutic actions because
they serve as the source for the effective drugs development. To ensure the safty of the
product standarization as well as quality control is done by scientific examination. In this
research work, therapeutic uses of traditional medicinal plants for their antidiabetic,
Chapter 1: Introduction 4

antioxidant, and hepatoprotective properties. Example- Platy cladus Orientalis and

Occimum canum was examined.

1.3 Diabetes :
Diabetes mellitus is a clinical syndrome characterized by hyperglycaemia due to absolute
or relative deficiency of insulin . This can arise in many different ways, but is most
commonly due to autoimmune type 1 diabetes or to adult-onset type 2 diabetes. Lack of
insulin affects the metabolism of carbohydrates, protein , fat and can cause a significant
disturbance of water and electrolyte homeostasis. Death may result from acute metabolic
decomposition, while long standing metabolic derangement is frequently associated with
functional and structural changes in the cells of the body, with those of the vascular
system being particular susceptible. These changes lead to the development of clinical
„complications „ of diabetes which is characteristically affect the eye, the kidney and the
nervous system.( Dorland‟s Illustrated Medical dictionary,31st edition)

Diabetes occurs world-wide and the incidences of both type 1 and type 2 diabetes are
rising; it is estimated that in 2000, 171 million people had diabetes, and this is expected
to double by 2030. This globle pandemic principally involves type 2 diabetes, to which
several factors contribute, including greater longevity, obesity, unsatisfactory diet,
sedentary life style and increasing urbanization. Many cases of type 2 diabetes remain
undetected.

The prevalence of known diabetes in Britain is around 2-3%, but is higher in the middle
and far East ( e.g. 12% in the Indian subcontinent ). A pronounced rise in the prevalence
of type 2 diabeyespccurs in migrant population to industrialized countries, as in Asian
and Afro- Caribbean immigrantsto the UK. Type 2 diabetes is now being observed in
children and adolescents, particularly in some ethnic groups, such as Hispanic and Afro-
Americans

1.3.1 Aetiological Classification of Diabetes Mellitus : .( Dorland‟s Illustrated Medical

dictionary,31st edition)
 Chapter 1: Introduction 5

Type 1 diabetes:-

 Immune-mediated
 Idiopathic
Type 2 diabetes:-

Other specific type

 Genetic defect of β-cell function

 Genetic defect of insulin action
 Pancreatic disease (e.g. pancreatitis,pancreatectomy,neoplastic disease, cystic fibrosis,
haemochromatosis, fibrocalculous pancreatopathy)
 Excess endogenous production of hormonal antagonists to insulin (e.g. growth hormone-
acromegaly; glucocorticoids-Cushing‟s syndrome; glucagon-glucagonoma)
 Drug induced (e.g. corticosteroids,thiazide diuretics, phenytoin )
 Viral infections (e.g. congenital rubella, mumps, coxsackie virus B )
 Uncommon forms of immune-mediated diabetes.
Associated with genetic syndrome; (e.g. Down‟s syndrome, Klinefelter‟s syndrome,
Turner syndrome )

1.3.1.1 Type 1 diabetic mellitus :

type 1 d. mellitus is one of the two major type of diabetes mellitus, characterized by
abrupt onset of symptoms, insulinopenia, and dependence on exogenous insulin to sustain
life; peak age of onset is 12 years, although onset can be at any age. It is due to lack of
insulin production by the beta cells of the pancreas, which may result from viral
infaction, autoimmune reactions, and probably genetic factors; islet cell antibodies are
usually detectable at diagnosis. When it is inadequately controlled, lack of insulin causes
hyperglycemia leads to overflow glycosuria, osmotic diurasis, hyperosmolarty,
dehydration and diabetic ketoacidosis.

1.3.1.2 Type 2 diabetic mellitus :

Chapter 1: Introduction 6

Type 2 d. mellitus one of the two major types of diabetes mellitus, characterized by peak
age of onset between 50 and 60 years, gradual onset with few symptoms of metabolic
disturbance (glycosuria and its consequences ), and no need for exogenous insulin;
dietary control with or without oral hypoglycemic is usually effective. Obesity and
genetic factors may also be present. Diagnosis is based on laboratory tests indicating
glucose intolerance. Basal insulin secretion is maintained at normal or reduced levels, but
insulin release in response to a glucose load is delayed or reduced.

Table - 1.1 : Comprative Clinical Features of Type 1 and Type 2 Diabetes

COMPRATIVE CLINICAL FEATURES OF TYPE 1 AND TYPE 2 DIABETES

TYPE 1 TYPE 2

Typical age at onset <40 year >50 year

Duration of symptoms weeks months to year

Body weight normal or low obese

Ketonuria yes no

Rapid death without yes no

Treatment of insulin
Autoantibodies yes no

Dibeteic complication no 25%

At diagnosis
Family history of dibetes uncommon common

Other autoimmuno disease common uncommon

1.3.1.3 Other forms of Diabetes:-

A number of unusual genetic diseases are associated with diabetes. In families, diabetes
is caused by single gene defects with autosomal dominant inheritance.
Chapter 1: Introduction 7

These uncommon subtype typically present as „maturity-onset diabetes of the young

(MODY) and constitute less than 5% off all cases of diabetes.

1.4 Investigations Treatment and Management of Diabetes mellitus (Davidson‟s

Principle & Pratice of Medicine ,20th edition)

1.4.1 Urine Testing :

Glucose-testing the urine for glucose is a common procedure for detecting diabetes ,
using sensitive glucose specific dipsticks.if possible, testing should be performed on
urine passed 1-2 hours after a meal since this will detected more cases of diabetes than a
fasting specimen . glycosuria always warrants further assessment by blood testing .the
most common cause of glycosuria is a low renal threshold, which is common during
pregnancy and in young people. Renal glycosuria is a benign condition unrelated to
diabetes.

1.4.1.1 Ketones:

ketone bodies can be identified by the nitroprusside reaction, which is primarily specific
for acetoacetate, The test is conveniently carried out using tablets or dipsticks for
ketones. Ketonuria may be found in normal people who have been fasting or exercising
strenuously for long periods, who have been vomiting repeatedly, or who have been
eating a diet high in fat and low in carbohydrates, Ketonuria with glycosuria, the
diagnosis of dibetes is highly likely.

1.4.1.2 Protein Test :

Dipstick testing for albumin is a standard procedure to identify the presence of renal
disease ( or urinary infaction )in people with diabetes . this will detect urinary albumin
greater than 300 mg/l. smaller amounts of urinary albumin (microalbuminuria) can be
measured and these provide indicators of the risk of developing dibeteic nepropathy
and/or macrovascular disease.

1.4.2 Blood Testing :

Chapter 1: Introduction 8

1.4.2.1 Glucose:

Laboratory glucose testing in blood relies upon enzymatic reaction (glucose oxidase )
and is cheap, usually automated and highly reliable . however variation in blood glucose
depends on wether the patients has eaten recently, so it is important to consider the
circumstances in which the blood sample was taken.blood glucose can also be measured
with colorimetric or other testig sticks, which is often read with apotable electronic
meter. These are used for capillary (fingerprick) testing to monitor diabetes treatment.

1.4.2.2 Blood Lipids:

The concentration of serum lipids-total cholesterol, low density and high density
lipoprotein (LDL and HDL) cholesterol and triglyceride- is another important index of
overall metabolic control in diabeteic patients and should measured at diagnosis and
regularly therafter,ideally the triglyceride concentration should be measured in the fasting
state.

1.4.3 Treatment and Management of Diabetes mellitus:

These are exciting development in the research for better methods of treating diabetes,
Transplantation of isolated pancreatic islets (usually into the liver via the portal vein) has
been safely achieved in a small number of humens. Progress is being made towards
meeting the seeds of supply,purification and storage of islets but the problem of
bioincompatibility, rejection and autoimmuno destruction remain. Nevertheless the
development of methods of inducing tolerance to transplanted islets and the use of stem
cells or transformation of hepatocytesfor make insulin by genetic engineering mean that
this may still prove most promising approach in the long term.

1.4.3.1 Primary Prevention of Diabetes:

Treatment of type I diabetes mellitus is to administer enough insulin so that the patient
will have carbohydrate, fat, and protein metabolism that is as normal as possible. Insulin
is available in several forms. “Regular” insulin has a duration of action that lasts from 3
to 8 hours, whereas other forms of insulin (precipitated with zinc or with various protein
Chapter 1: Introduction 9

derivatives) are absorbed slowly from the injection site and therefore have effects that
last as long as 10 to 48 hours. Ordinarily, a patient with severe type I diabetes is given a
single dose of one of the longer-acting insulin every day to increase overall carbohydrate
metabolism throughout the day. Then additional quantities of regular insulin are given
during the day at those times when the blood glucose level tends to rise too high, such as
at mealtimes. Thus, every patient is provided with an individualized pattern of treatment.
In persons with type II diabetes, dieting and exercise are usually recommended in an
attempt to induce weight loss and to reverse the insulin resistance. If this fails, drugs may
be administered to increase insulin sensitivity or to stimulate increased production of
insulin by the pancreas. In many persons, however, exogenous insulin must be used to
regulate blood glucose. In the past, the insulin used for treatment was derived from
animal pancreata . . However, human insulin produced by the recombinant DNA process
has become more widely used because some patients develop immunity and sensitization
against animal insulin, thus limiting its effectiveness
Chapter 1: Introduction 10

Fig 1.1: Management of diabetic patients undergoing surgery

Establish adequate diabetic control at least 2-3 days pre-operative

Stop metformin 24-48 hrs before surgery

Contact anaesthetist well in advance

perform opration as early as possible in the morning

On the morning of surgery omit usual insulin or oral anti-diabetic

drug and cheak blood glucose,electrolytes,urea and creatinine

Type 1 dibetes Type 2 dibetes

Major surgery
Minor surgery

At 0800-0900 hrs establish intravenous

infusion of 500 ml 10% dextrose +10-20
unit short acting insulin+20mmol K+ Simple observe measure blood glucose
given at arate of 100ml/hrs. frequentlyGlucose /insulin/potassium iv if
necessary post-operatively
Cheak blood glucose using blood glucose
meter or strip 2-4 hrs and adjust insulin
content of infusion to maintain values
within the range 5-11 mmol/l
 Chapter 1: Introduction 11

Fig 1.2:The role of hyperglycemia-induced accumulation of reactive oxygen species and

oxidative stress in causation of diabetic vascular complications

Hyperglycemia

Endothelial Activation of Formation of advanced Activation of

nitric oxide protein kinase C glycation end products polyol
synthase (AGE) pathway
uncopuling

Activation of reactive oxygen species

Induction of oxidative stress

Induction of Reduction of Activation of Increased AGE Altered gene

DNA damage nitric oxide protein formation expression
kinase C
Chapter 1: Introduction 12

Table 1.2: Some of the plants reported to possess antidibetic activity

Botanical Name Family Common name Reference

Acacia arabica Fabaceae Babul Singh, 1975

Aegle marmelos Rutaceae Wood apple Kamalakkanan,2003

Allium cepa L. Liliaceae onion Augusti, 1973

Allium sativum Alliaceae garlic Rabinkov, 1998

Aloe vera (L.) Aloaceae Aloe Ajabnoor, 1990

Artemisia pallens Compositae Davana Subramaniam, 1996

Annona squamosa L . Annonaceae Sugar apple Shirwaikar, 2004

Azadirachta indica Meliaceae Neem Chattopadhyay, 1987

Biophytum sensitivum (L) Oxalidaceae Life plant Puri, 1998

Beta vulgaris L. Chenopodiaceae Garden beet Yoshikawa,1996

Boerhavia diffusa L Nyctaginaceae Tar vine Pari, 2004

Cassia auriculata L. Leguminosae Tanner‟s Cassia Pari, 2002

Cajanus cajan Fabaceae Pigeon pea Amalraj, 1998

Coccinia indica Cucurbitaceae Ivy gourd Shibib, 1993

Casearia esculenta Flacourtiaceae Carilla fruit Prakasam, 2002

Catharanthus roseus Apocynaceae Madagascar periwinkle Nammi, 2003

Chapter 1: Introduction 13

Camellia sinensis Theaceae Green tea Anderson, 2002

Enicostemma littorale Blume Gentiaceae Nahi Maroo,2002

Hibiscus rosa sinensis L . Malvaceae China Rose Sachdewa 2003

Helicteres isora L Sterculiaceae, Screw tree Chakrabarti, 2002

Ipomoea batatas (L.) Convolvulaceae Sweet patato Matsui, 2002

Mangifera indica L. Anacardiaceae Mango Muruganandan, 2005

Mucuna pruriens Leguminosae Velvet bean Akhtar,1990

Murraya koenigii (L.) Rutaceae curry-leaf tree Khan, 1995

Ocimum sanctum Lamiaceae Holy Basil Gholap

Salacia reticulate Celastaceae Salacia Karunanayake, 1984

Swertia chirayita Gentianaceae Indian Gentian Saxena, 1993

1.5 Hepatotoxicity :

The liver, which is a versatile organ present in the vertebrates, is center to the metabolic
disposition of virtually all endogenous and exogenous chemicals. It has a vast
arrangement of effects including biochemical regulation, metabolism and excretion.
Plasma proteins like fibrinogen, clotting factors and albumin‟s synthesis are carried out
by liver. It involves the metabolism of proteins, carbohydrates and fat. It acts also as
storage for proteins, and glycogen, vitamins and various metals. It plays important role in
the regulation of blood pressure by transferring blood from portal to systemic circulation
Chapter 1: Introduction 14

and its reticuloendothelial system take part in immune mechanism. Liver injury induced
by drug is an unresolved problem, which has impact that is well beyond the number of
actual cases that occur annually. Despite of a rigorous pre-clinical and clinical review
process (Bissell et al. ,2001), it has emerged as the most common cause of post-
marketing withdrawal of medications. Indiscriminate use of drugs like
Paracetamol(Davidsons et al . ,1996), Anti-malarial drugs(Selby et al., 1985), Anti-
tubercular drugs(Woo J et al. , 1992), Oral contraceptives of hormonal origin,
Antidepressants, Anti-arrhythmic drugs(Lee et al .,1995), and Analgesics etc. are
threatening the integrity of the liver. The drug induced injuries of liver ranges from
increase in asymptomatic liver function tests to fulminant hepatic failure. It may occur in
a number of different forms which may cause liver failure including acute drug-induced
hepatitis, cholestasis, chronic hepatitis, and steatohepatitis. Many drugs may cause
hepatic injury pattern in more than one type. In addition, Alcohol consumption in the
society is increasing. One of the other complication of alcoholism, that is the third most
frequent and common cause of death among those 25 to 64 years of age(Lieber et al .,
1988) is Cirrhosis of liver. Inspite of the fact that there is plethora of hepatotoxins leading
to pandemic of hepatic injuries, we still lack in a specific hepatoprotective agent.
Presently used agents like, multivitamins and few polyherbal preparations and folic acid
could not give assurance of an effective hepatic protection but could only provide a
supportive therapy.
There are hardly any proven remedies for the treatment of the prevalent liver disorders.
No drug has been developed yet in the modern system of medication which could
stimulate the liver function, and protect it from damage or help in the regeneration of
hepatic cells. The only available drugs for the treatment and cure of liver disorders are
corticosteroids and immuno-suppressive agents but the use of these drugs are followed by
various serious side effects. Use of indigenous drugs clinicaly has been an upsurge in the
recent years, like herbal medicinal plants, originally used in the traditional system of
medicine. They are effectively being tried now in variety of pathophysiological states.
There is rapid increase in the need of agent that can protect the liver from damages.
Though varities of such plants like, Picrorrizia kurrooa, Swertia chirota, Piper logum and
Acacia catechu etc. have already been investigated to know their hepatoprotective nature,
Chapter 1: Introduction 15

some Ayurvedic drugs for example, Kumariasav and Tamra bhasma also shows the same
property(Katewa et al., 2001) as above, but information regarding the correlation in-
between pharmacological and the phytoconstituents present is still lacking.

1.5.1 Treatment of Liver Diseases by HerbalMedicines:

It is very interesting to note that there is no drug available in the modem system of
medicine for treating hepatic disorders; only certain herbal preparations are available to
treat this quite vulnerable disease. Medicinal systems conceptualize a general imbalance
of the dichotomous energies leads to the disease and they focus on medicine that balance
these energies and maintain good health. In spite of the phenomenal advances in cellular,
biochemical and therapeutic approaches to many diseases, liver diseases remain
enigmatic today. Though liver diseases are among the important diseases affecting
humans, there is a dearth of effective remedies to treat them satisfactorily. None of the
available preparations are specific for liver disorders. The indigenous system of medicine
in India has abundant data on drugs available for the treatment of various liver disorders.
These drugs have been used for centuries and have been claimed to offer significant
relief. Besides, the folklore remedies, many plant products are also commonly used to
treat liver disorders throughout India.

When we compare many ayurvedic drug with silymarin, the hepatoprotective effect was
found to be similar, or in many cases, superior to the effect of silymarin (Ajith et al.,
2007, Amresh et al., 2004).
Chapter 1: Introduction 16

Table1.3: Some of the plants reported to possess hepatoprotective activity

Botanical Name Family Extract Reference

Acacia catechu Leguminosae Ethyl acetate Jayasekhar et al 997

Adhatoda vasica Acanthaceae Ethanolic extract of Bhattacharyya et al

leaves 2005

Aerva lanata Amaranthaceae Alcoholic extract of entire Majmudar et al 1999

herb

Bombax ceiba Bombacaceae Methanolic extract of Ahsana et al 2005

leaves

Cassia tora Leguminosae Ethanolic extract of Kumud et al 2000

leaves

Elephantus scaber Asteraceae Root extract Rajesh et al 2001

Emblica officinalis Euphorbiaceae Aqueous fruits extract Jeena et al 2000

Ficus hispida Moraceae Methanolic extract of Mandal et al 2000

leaves

Garcinia kola Guttiferae Light pertroleum ether Farombi et al

extract of seeds 2000

Helminthostachys Ophioglossaceae Methanolic extract of Suja et al 2004

zeylanica rhizomes

Hygrophila auriculata Acanthaceae Aqueous extract of Shanmugasundara

roots m et al 2006
Chapter 1: Introduction 17

Plumbago Aqueous and alcoholic

Plumbagoginaceae Tilak et al 2004
zeylanica extracts of roots

Phyllanthus Aqueous and alcoholic

Euphorbiaceae Jalalpure et al 2006
distichus extracts of fruit pulp

Saroostemma Ethyl acetate extracts of stem

Ascepiadaceae Sethuraman 2003
brevistigma bark

Kalanchoe pinnata Crassularaceae Ethanolic extract of Yadav et al 2003

juice of leaves

1.6 Free Radicals :

Oxygen is very important for our life , however, oxidative damage may occur in case of
an excess of oxygen and which may also lead to death. The damage occurs due to the
oxidation of certain products to toxic free radicals and not due to the oxygen‟s presence.
These toxic free radicals are produced within living cells and are part of the normal
metabolic processes of cell which includes detoxification processes and immune system
defenses.The excessive generation of these toxic free radicals and reactive oxygen
species (ROS), such as hydroxyl radicals and hydrogen peroxide and superoxide anions
contribute to the development of different diseases like cancer, rheumatoid arthritis,
certain neurodegenerative diseases, ageing, and tissue damage, mainly when the
production of free radical exceeds the tissues's capacity to remove them(Larkins et al.,
1999). Free radicals play another important role in the followings, inflammation,
atherosclerosis, ischemia of the heart, brain, small intestine, kidney, liver, diabetes
mellitus,disorders of prematurity and radiation injury. Free radicals seem to be one of the
final common pathways of cell damage, they may affect the cell membrane and also the
nuclear DNA. The damage of cell membrane is by protein's cross-linking, by the lipids
(Bhendes et al., 2002) critical alterations. In aerobic organisms, the free radicals
scavengers which act like anti-oxidant are responsible for the defense system against
these free radicals. The functioning of Free radical scavengers is by donating an electron
to the free radical and stabilising it by pairing with the unpaired electron. The mechanism
of Anti-oxidant defense involves both non enzymatic and enzymatic mechanisms, which
Chapter 1: Introduction 18

specific enzymes like superoxide dismutase, catalase, glutathione peroxidase and non
enzymatic mechanisms, which use nutrients and minerals(Aggarwal et al., 2005). The
term ROS is collective term which include both oxygen radical-centered free radicals and
non-radical oxidants. Free radicals may be defined as atomic or molecular species which
has at least one unpaired electron on its outer shell(Roberfroid et al ., 1995). ROS include
the following superoxide (.O2), hydroxyl (.OH), alkoxyl radicals (RO'), peroxyl radicals
(ROO') and hydroperoxyl (HOO') . Non radical oxidants, that can easily shift to be
oxidized into radicals,examples are singlet oxygen (.O2)( Hu C et al., 2005) and
hydrogen peroxide (H2O2) .

1.6.1Oxygen species production Sources:

Through the effect of either endogenous or exogenous production, ROS comes in the
contact with the metabolizing organism. Few main source of endogenous ROS generation
are Oxygen metabolism, mitochondrial energy generation and liver‟s detoxification
reactions, certain examples of exogenous sources(Roberfroid et al.,1995) are exposure to
environmental pollutants, cigarette smoke, alcohol consumption and fungal/viral
infections and ionizing radiation For controlling antimicrobial activity and regulating
proliferation of cell in the body ROS may be beneficial, however, oxidative stress occurs
when generation of free radicals and the other oxidative molecules are much beyond the
capacity of defense network in body, they are not detoxified effectively.
1.6.2 Stress of oxygen :
several pathological situations are caused by free radicals and ROS for example
atherosclerosis, cardiovascular disease, cancer, arthritis, Alzheimer's disease, age-related
disorders and stroke they also generate oxidative stress(Frank et al., 2002). To avoid
oxidative stress an equilibrium between oxidants and antioxidants is required.
Antioxidants decreased level may force oxidative stress on the cell. This may be caused
intrinsically for example, by alterating the activity of cellular antioxidant defence system
by mutation of DNA or extrinsically by dietary mineral deficiency (cofactors) and by
depleting the antioxidant defence by toxins and other factors.Oxidative stress may arise
from increase in level of oxidants in cell. Oxidative stress by activating regulation of the
immune defense system may cause adaptation of the cell or organism. however, cell
Chapter 1: Introduction 19

injury or cell death may also be caused. DNA molecules are damaged by the cellular
interaction with ROS, which indicates that oxidative stress is likely to play an vital role in
increasing the risk of cancer by enhancing, carcinogenesis, and mutagenesis and
aging(Acworth et al., 1997 and Halliwell et., 2004). Alteration of structure or
composition of enzymes are generated by oxidative stress and receptors and transport
proteins affect their functions. These faulty proteins are degraded and than removed from
the cell(Acworth et al., 1997).
1.7 Antioxidants :
Antioxidants are those compounds which, when present in low concentrations as
compared to oxidizable substrates, can fulfill free radicals and protect the biological
systems against free radicals(Arnao et al., 2000 and Diplock et al., 1998) by significantly
delaying or inhibiting oxidation of the substrate. Categration of Antioxidants are
synthetic or natural. Synthetic antioxidants compounds are with phenolic structures of
alkyl substitution with varying degrees and such as butylated hydroxyanisole (BHA) and
butylated hydroxytoluene (BHT). These are suspected that they may cause negative
effects on health like carcinogenicity(Barlow et al., 1990 and Ito N et al., 1983) hence
their use is being restricted and synthetic antioxidant is increasingly been replaced by
naturally occuring antioxidants(Chang et al., 2000 and Koleva et al., 2002). Another
categration of Antioxidants can be either free radical scavengers (nonenzymatic) which
traps and decompose free radicals or cellular or extracellular enzymes (enzymatic) which
may inhibit peroxidase reactions that are involved in the free radical's production.The
free radical scavengers or non enzymatic antioxidants include the followings, ascorbate
(Vit. C)( Kojo et al., 2004 and Suh et al., 2003), polyphenols(Aviram et al., 2005),
tocopherols (Vit. E)( Pryor et al., 2000), carotenoids(. Niles et al., 2004), α-lipoic
acid(Holmquist et al., 2007), glutathione(Giustarini et al., 2008), and flavonoids. The
antioxidant enzymes include, superoxide dismutase, catalase and glutathione peroxidase.
For intracellular defence enzymatic antioxidants are of great importance, whereas for
defence against extracellular oxidants non-enzymatic antioxidants mechanism are been
used. Natural antioxidants are phenolic compounds (tochopherols, anthocyanins,phenolic
acids, and flavonoids), carotenoids, nitrogen compounds (chlorophyll
derivatives,alkaloids amino acids and amines), and vitamin C and E,
 Chapter 1: Introduction 20

phospholipids(Morello et al., 2002).Dietary antioxidants(Siddhuraju et al., 2002) are the

antioxidant compounds that are endogenous constituents present in the food. Dietary
antioxidants are those substrate in food that decreases significantly the free radical's
adverse effect like reactive oxygen species (ROS) and reactive nitrogen species (RNS) or
both on normal physioligical function in human being as described by The Food and
Nutrition Board of the National Academy of Sciences (National Academy of Science,
1998). Free radicals are the molecules or the molecular fragments that contains one or
more than one unpaired electrons. The unpaired electrons presence may confers a
considerable degree of reactivity to free radicals (Valko et al., 2004). Free radicals are
present everywhere in our body and by normal physiological processes it can be
generated and including the aerobic metabolism and inflammatory responses, that are
used to eliminate the invading pathogenic microorganisms(Hussain et al., 2003). From
endogenous sources like mitochondria, cytochrome P450 metabolism, inflammatory cell
activation(Inoue et al., 2003) and peroxisomes production of reactive oxygen species are
made. Modification of biologically relevant macromolecules such as DNA,
carbohydrates, lipids(Troszynska et al., 2002), and proteins etc occurs due to the
imbalance between ROS/RNS and in antioxidant defense systems. Inhibition of oxidation
of these molecules by antioxidants and initiation of oxidizing chain reactions(Klein et
al.,2000) is prevented to avoid these modifications. Either by donating the electron, or
through the singlet oxygen, or by hydrogen atom, and prooxidant metal ions deactivation
they search for free radicals.( Morello et al., 2002)
These antioxidants act at different and several stages, by -
 Replacing damaged, target molecules
 Repairing damaged, target molecules
 Restricting reactive species formation
 Binding metal ions, required for highly reactive species formation like OH
 By the help of antioxidant enzymes or by reaction directly where the antioxidant itself
would be used up scavenging ROS is done
 Quenching and scavenging singlet oxygen (electrons rearrangement that may produces
rapid oxygen)( Gutteridge et al 1994).
Chapter 1: Introduction 21

Defense that are provided by the antioxidant systems for the survival is crucial and that
can also operate at different levels through radical formation prevention within the cells,
intercepting formed radicals, damaged molecules increase, excessively damaged
molecules recognition, which are rather eliminated during replicationare to prevent
mutations from occurring and not being repaired, and repairing oxidative damage.
Classification of non-enzymatic antioxidants are either water soluble or lipid soluble,
which depends upon whether they are primarily acting on aqueous phase or in lipophilic
region of cell membranes. Hydrophilic antioxidants include the following, Vitamin C
and certain polyphenol flavonoid groups, where as lipophilic antioxidants are those that
includes ubiquinols, procyanidins carotenoids, apocynin, and few polyphenol flavonoid
groups tochopherols(Middleton et al., 2000), and retinoids. Non enzymatic antioxidants
include the antioxidant , oxidative enzyme inhibitors and transition metal chelators like
ethylene diamine tetra-acetic acid (EDTA) and enzyme cofactors. Synthetic antioxidants
are toxic and hence they are used limited, such as BHA and BHT.The studies upon the
radical scavenging activity(Molyneux et al., 2004) of medicinal plants are extensively
being carried out over last few years to search the low toxic sources of novel
antioxidants. Plants represent the new source of compounds with antioxidant
activity(Scartezzini et al., 2000) because they produce antioxidant in a large number to
control oxidative stress that are caused by sunbeams and oxygen.

Table 1.4: Some of the plants reported to possess antioxidant activity

S.No Botanical Common/ Part used Reference

English name

1. Curcuma domestica Turmeric Leaf Reddy et al., 2005

2. Cuscuta reflexa Roxb. Akashabela Stem Yadav et al.,, 2001

3. Daucus carota Linn. Carrot Root Bhishayee et al.,1995

4. Emblica officinalis Amla Fruit Bhattacharya et.,1999

Chapter 1: Introduction 22

5. Foeniculum vulgare Mill Fennel Fruit oil Ruberto et l.,2000

6. Glycyrrhiza glabra Linn Mulethi Root Mortez et al.,2003

7. Mangifera indica Linn. Mango Root Martinez etal. 2000,

8 Ocimum sanctum Linn. Tulsi Leaf Devi et al., 1999.

9. Psoralea corylifolia Babchi Seed Haraguchiet al., 2000.

10. Santalum album Linn. chandan Heartood Banerjee et al.,1993

11. Solanum nigrum Linn Makoi Leaf Sultana et al.,1995

12. Withania somnifera Dunal Ashwagandha Root, Leaf Vande et al.,1982

13. Asparagus racemosus Liliaceae Shoot Kamat et al.,2000.

14. Baccharis coridifolia DC. Asteraceae Aerial parts Mongelli et al.,1997.

15. Bryonia alba Linn. Cucurbitaceae Root K aragezyan et .,1981

16. Cichorium intybus Linn. Asteraceae Leaf Sultanaet al.,, 1995

17. Crithmum maritimum Apiaceae Essen- oil Ruberto et al.,2000.

18. Cynara scolymus Linn. Asteraceae Leaf Gebhardt et al.,1997.

19. Emilia sonchifolia DC. Asteraceae Leaf Shylesh et al.,1999.

20. Eucalyptus camaldulensis Myrtaceae Leaf Okamura et al.,1993,.

21. Eucommia ulmoides Oliver Eucommiacee Leaf Hseieh et al.,2000

22. Ginkgo biloba Linn. Ginkgoaceae Leaf Kose et al., 1987.

23. Lavandula angustifolia Lamiaceae Aerial parts Hohmann et a.,1999.

24. Lycium barbarium Linn. Solanaceae Fruit Ren et al.,1995.

Chapter 1: Introduction 23

25. Melissa officinalis Linn. Lamiaceae Aerial parts Hohmann et al.,1999

26. Murraya koenigii (Linn.) Rutaceae Leaf Patel et al.,1979.

27. Myrica gale Linn. Myricaceae Fruit Mathiesenet al.,1995.

28. Picrorrhiza kurroa Royle Scrophulariace Rhizome Anadan et al.,1999,

ae

29. Piper nigrum Linn. Piperaceae Fruit Manosroi et al.,1999

30. Plantago asiatica Linn. Plantaginaceae Seed Toda et al.,1995.

31. Prunus domestica Linn. Rosaceae Fruit Donovan et al.,1998