by Shalini Srivastava - Shodhgangashodhganga.inflibnet.ac.in/.../shalini_biochem_phd... · Shalini Srivastava under my supervision. The work presented here is original, carried out

Biochemical studies to evaluate the anti-diabetic, anti-oxidative and immunomodulatory potentials

of Syzygium cumini (Jamun) & Pterocarpus marsupium (Vijaysaar)

Thesis Submitted to

University of Lucknow for the degree of

Doctor of Philosophy

in

Biochemistry

by

Shalini Srivastava M. Sc. (Biochemistry)

Department of Biochemistry University of Lucknow, Lucknow

2014

Dedicated To

My parents

UNIVERSITY OF LUCKNOW LUCKNOW-226007

DR. DEEPAK CHANDRA DEPARTMENT OF BIOCHEMISTRY Associate Professor 0522-2740069(Office), 0522-2740132 (Fax) 94151-64388 (Mobile), e-mail:[email protected]

CERTIFICATE

This is to certify that the work embodied in this thesis entitled

“Biochemical studies to evaluate the anti-diabetic, anti-oxidative and

immunomodulatory potentials of Syzygium cumini (Jamun) &

Pterocarpus marsupium (Vijayasaar)” has been carried out by Ms.

Shalini Srivastava under my supervision. The work presented here is

original, carried out by the candidate herself and has not been submitted

so far, in part or full, for any other degree or diploma of any other

University/Institute. It is further certified that the candidate has fulfilled

the conditions laid down under the ordinance for the degree of Doctor of

Philosophy (Ph.D.) in Biochemistry by the University of Lucknow.

(Deepak Chandra)

Contents SN. Topic Page No.

1. Acknowledgment i-ii

2. Preface iii-iv

3. Abbreviations v-vii

4. Introduction 1-6

5. Review of Literature 7-56

6. Materials and Methods 57-71

7. Objectives 72

8. Chapter I:

Anti-diabetic and anti-oxidative potentials of crude Syzygium cumini and Pterocarpus marsupium extracts

73-107

9. Chapter II:

Anti-diabetic and anti-oxidative potentials of purified Syzygium cumini and Pterocarpus marsupium extracts

108-135

10. Chapter III:

Immunosuppression in diabetes and immunomodulatory properties of Syzygium cumini and Pterocarpus marsupium

136-151

11. Summary 152-165

12. Reference 166-203

13. List of Publications 204

i

ACKNOWLEDGEMENT

“To the Supreme, whose eternal blessings, divine love and spiritual guidance helps us to fulfil all our aspirations.”

Social beings in all conditions, achieve their goals with the assistance, co-operation and support of all the concerned people. I take this moment to express my most sincere appreciation, for the people who have knowingly or unknowingly played a role in my life as a teacher, friend, counsellor and supporter.

I am immensely grateful to my supervisor, Dr. Deepak Chandra, Associate Professor, Department of Biochemistry, University of Lucknow, Lucknow, for his meticulous efforts, inspiring guidance, unstinted co-operation and sentimental support which helped me to complete my thesis with ease. His great sense of humour with intellect, versatile personal qualities and critical appreciation influenced me to enjoy my work a lot. It was a wonderful experience to work under his guidance.

I express my gratitude to Prof. U. N. Dwivedi and Prof. S. K. Agarwal, former Heads, Department of Biochemistry, University of Lucknow, Lucknow, for providing me the necessary infrastructure and resources to accomplish my research work in the department and for their encouragement, moral support and keen interest in my progress.

I sincerely thank Prof. R. K. Mishra, Head, Department of Biochemistry, University of Lucknow, Lucknow, for his indispensable suggestions, constructive criticism, admirable teaching and encouragement. He will always be an inspiration and his passion for teaching will be cherished.

Few words cannot be sufficient to express my gratitude towards a few people. In this view I thank my teachers Prof. P.C. Mishra, Prof. M. K. Misra, Prof. Raj Khanna, Dr. Sudhir Mehrotra, Dr. Samir Sharma, Dr. Kusum Yadav and Dr. Minal Garg for their whole hearted support, consistent help and motivation.

I am grateful to Dr. Desh Deepak, Associate Professor, Department of Chemistry, University of Lucknow, Lucknow, for his help.

I am immensely thankful to my seniors: Dr. Umanath Tripathi, Dr. Ashita Gupta, Dr. Pratima Tripathi, Dr. Ankita Srivastava, Dr. Giti Verma, Mr. Veda Prakash Pandey and Mr. Sanjay Yadav for their wholesome support and substantial help. I thank my juniors: Mrs. Priyanka, Ms. Mandipika, Ms. Padminee, Ms. Nandini, Ms. Ambika and Mr. Bhanu for their constant cooperation and maintaining a healthy environment all along. The time shared with them during this period shall be an everlasting memory.

I take this great opportunity to owe the success in my life to my loving parents. I sincerely dedicate this piece of art & science to the efforts of my father,

Introduction

ii

Dr. D. C. Srivastava and my mother, Dr. (Mrs.) V. K. Srivastava and for their sacrifices which have enabled me to stand where I am and their constant encouragement.

I would do injustice if I do not thank my brothers Dr. Shekhar Srivastava and Mr. Sharad Srivastava, and sister-in-law, Dr. Swati Srivastava, for being my strength and support.

It’s my fortune to gratefully acknowledge my husband Dr. Prashant Kumar Saxena for his concern and support during the writing period of my thesis, which helped me to complete my work with ease. I also thank him for helping me out in statistics.

I thank the staff members of the Department of Biochemistry for their help and cooperation rendered at various stages of this work.

I would be ungrateful if I do not acknowledge all the animals sacrificed during the course of this study.

Shalini Srivastava

iii

PREFACE

Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in insulin

secretion, insulin action, or both. Insulin deficiency in turn leads to chronic

hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism. It is

estimated that 25% of the world population is affected by this disease. Oxidative

stress due to prolonged hyperglycaemia causes tissue damage leading to severe

diabetic complications such as retinopathy, neuropathy, nephropathy, cardiovascular

complications and ulceration. Hyperglycaemia is also one of the potential mediators

of altered defence mechanism and immunosuppression in diabetes. Thus, diabetes

covers a wide range of heterogeneous diseases. Diabetes mellitus may be categorized

into several types but the two major types are type 1, insulin dependent diabetes

mellitus (IDDM) and type 2, non-insulin dependent diabetes mellitus (NIDDM).

Sushruta (6th century BCE) identified diabetes and classified it as

Medhumeha. .He further identified it with obesity and sedentary lifestyle, advising

exercises to help cure it. The word diabetes comes from the Greek word diabenein

which means to pass through, in reference to the excessive urine passed as a symptom

of this disease.

Common symptoms of diabetes are hyperglycemia, polydipsia, polyurea,

weight loss, blurred vision and susceptibility to acquire infections. Drugs are used

primarily to save life and alleviate symptoms. Secondary aims are to prevent long-

term diabetic complications by eliminating various risk factors, to increase longevity.

Insulin replacement therapy is the mainstay for patients with type 1 DM while diet

and lifestyle modifications are considered the cornerstone for the treatment and

management of type 2 DM. Insulin is also important in type 2 DM when blood

glucose levels cannot be controlled by diet, weight loss, exercise and oral

medications. Oral hypoglycaemic agents are also useful in the treatment of type 2

DM. Oral hypoglycaemic agents include sulphonylureas, biguanides, alpha

glucosidase inhibitors, meglitinide analogues, and thiazolidenediones. The main

objective of these drugs is to correct the underlying metabolic disorder, such as

insulin resistance and inadequate insulin secretion.

Preface

iv

Despite considerable progress in the treatment of diabetes by oral

hypoglycemic agents, search for newer drugs continues because the existing synthetic

drugs have several limitations. The herbal drugs with anti-diabetic activity are yet to

be commercially formulated as modern medicines, even though they have been

acclaimed for their therapeutic properties in the traditional systems of medicine. The

plants provide a potential source of hypoglycemic drugs because many plants and

plant derived compounds have been used in the treatment of diabetes. Ayurveda and

other traditional medicinal system for the treatment of diabetes describe a number of

plants used as herbal drugs. Hence, they play an important role as alternative

medicine due to less side effects and low cost.

In the present study the anti-hypergylcemic, anti-oxidative and

immunomodulatory potentials of Syzygium cumini seed and Pterocarpus marsupium

bark have been studied. The crude extracts of these plant parts show anti-

hypergylcemic, anti-oxidative and immunomodulatory potentials. Attempts were

made to purify the active principles responsible for anti-diabetic activity, present in

these plant extracts and elucidate their mechanism of action. The active constituent(s)

present in these medicinal plants might cause pancreatic beta cells re-generation,

insulin release, increase peripheral glucose utilization and may also fight the problem

of insulin resistance. The outcomes of the study will not only be useful in isolating

newer and effective anti-hyperglycemic constituent(s) from these plant extracts but

the isolation of constituents with anti-oxidative potential will also be useful in

managing the pathogenesis of various oxidative stress mediated disorders including

diabetes. Modulation of the immune responses through the stimulatory or suppressive

activity of phyto-extracts may help to maintain a disease-free state in normal or

unhealthy people.

v

ABBRIVIATIONS

ADH Anti-diuretic hormone

ADP Adenosine diphosphate

AGE Advanced glycation end products

ALT Alanine aminotransferase

AMP Adenosine monophosphate

AP Alkaline phosphatase

AST Aspartate aminotransferase

ATP Adenosine triphosphate

α Alpha

β Beta

BSA Bovine serum albumin

b.w. Body weight

CAT Catalase

CDNB 1-chloro 2,4-dinitrobenzene

dl Deciliter

DNPH 2,4-dinitrophenylhydrazine

DPPH Diphenylpicrylhydrazyl

DTNB 5,5-Dithiobis-2-nitrobenzoic acid

EDTA Ethylenediamine tetracetate

ELISA Enzyme linked immune sorbent assay

FBG Fasting blood glucose

FCA Freund’s complete adjuvant

FIA Freund’s incomplete adjuvant

Fig. Figure

g Gram

G6PD Glucose 6-phosphate dehydrogenase

GPx Glutathione peroxidase

GSH Glutathione (reduced)

GSSG Glutathione (oxidized)

GST Glutathione S-transferase

h Hour

Abbreviations

vi

H2O2 Hydrogen peroxide

HK Hexokinase

HRP Horse radish peroxidase

IgG Immunoglobulin G

IgM Immunoglobulin M

IL Interleukins

1U International unit

KDa Kilo Dalton

Kg Kilogram

LDH Lactate dehydrogenase

LDL Low density lipoprotein

LMWA Low molecular weight antioxidants

LPO Lipid peroxidation

LPS Lipopolysaccharide

MDA Malondialdehyde

mg Mili gram

min Minute

ml Mili liter

µM Micromole

mM Milimole

NaCl Sodium chloride

NAD Nicotinamide adenine dinucleotide

NADH Nicotinamide adenine dinucleotide (reduced)

nm Nano meter

NO Nitric oxide

O2·¯ Superoxide radical

OH• Hydroxyl radical

PBS Phosphate buffer saline

P value Degree of significance

PEP Phosphoenol pyruvate

PFK Phosphofructokinase

PK Pyruvate kinase

PKC Protein kinase C

Abbreviations

vii

PM Pterocarpus marsupium

% Percentage

RBC Red blood cells

ROS Reactive oxygen species

rpm Rotation per minute

SC Syzygium cumini

SD Standard deviation

-SH Sulfhydryl group

SOD Superoxide dismutase

TBA Thiobarbituric acid

TBARS Thiobarbituric acid reactive substances

TD Thymus dependent

TEP 1,1,3,3-tetraethoxy propane

TI Thymus independent

TMB Tetra methyl benzidene

Tris Tris (hydroxymethyl) aminomethane

TT Tetanus toxoid

Wt Weight

Introduction

1

Introduction

The human population worldwide appears to be in the midst of an epidemic of diabetes. An estimated 285 million people, corresponding to 6.4% of the world's adult population suffer from diabetes. The number is expected to grow to 438 million by 2030, corresponding to 7.8% of the adult population. Despite the great strides that have been made in the understanding and management of diabetes, the disease and disease-related complications are increasing unabated. Parallel to this, recent developments in understanding the pathophysiology of the disease process have opened several new avenues to identify and develop novel therapies to combat the diabetic plague. Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in combating diabetes and diabetes-related complications. Therefore, as the disease is progressing unabated, there is an urgent need of identifying indigenous natural resources in order to procure them, and study in detail, their potential on different newly identified targets in order to develop them as new therapeutics.

Diabetes is a chronic disorder in which homeostasis of carbohydrate, protein and lipid metabolism is improperly regulated by insulin. It is characterized by elevated fasting and post prandial blood sugar levels. Diabetes mellitus is a complex metabolic disorder resulting from either insulin insufficiency or insulin dysfunction. Type I diabetes (insulin dependent) is caused due to insulin insufficiency because of lack of functional β cells. Patients suffering from this are therefore totally dependent on exogenous source of insulin while patients suffering from Type II diabetes (insulin independent) are unable to respond to insulin and can be treated with dietary changes, exercise and medication. Type II diabetes is the more common form of diabetes constituting 90% of the diabetic population (Modak et al., 2007). Tissues where glucose uptake is insulin independent (cardiac tissue, blood vessels, peripheral nerves, renal medulla and ocular lens) face severe and sustained hyperglycemia (Chandra et al., 2002a). The major complications of diabetes include atherosclerosis, retinopathy, nephropathy and neuropathy etc.

Introduction

2

Diabetes related complications

The major risks of the diabetes disorder are chronic complication affecting

multiple organ systems which eventually arise in patients with poor glycemic control.

Different symptoms, complications and possible risk factors of diabetes mellitus are

summarized in Table 1. Microvascular complications are peripheral neuropathy which

can lead to foot ulcer and possibly progressing to necrosis, infection, gangrene,

sometimes requiring limb amputation. Nephropathy is a progressive kidney disease

caused by angiopathy of capillaries in the glomeruli of kidney. Retinopathy (damage

to retina) is an ocular manifestation of systemic disease which can lead to blindness.

Macrovascular complications include atherosclerosis and myocardial infarction or

ischemic heart disease. Possible mechanisms implicated in hyperglycemia induced

damage are: (1) increased polyol pathway flux, (2) increased advanced glycation end

products formation, (3) activation of protein kinase C (PKC) isoform and (4)

increased hexosamine pathways flux.

Table 1: Symptoms, complications and possible risk factors of diabetes mellitus

Symptoms Complications Possible Risk Factors

Polyurea Microvascular Obesity

Polydipsia Neuropathy High cholesterol

Weight loss Nephropathy Hypertension

Blurred vision Retinopathy Smoking

Tiredness Macrovascular Hereditary

Skin infection Myocardial infarction Sedentary lifestyle

Muscular cramps Atherosclerosis Ketoacidosis

Nausea Vomiting Diabetic coma

Oxidative stress in diabetes

Oxidative stress plays an important role in chronic complications of diabetes

(Elangovan et. al, 2000). Oxidative stress in cells and tissues, results from the

increased generation of reactive oxygen species and from decrease in antioxidant

defense potential (Gumieniczek et al., 2002). Several hypotheses have been put forth

to explain the genesis of free radicals in diabetes. These include autoxidation

Introduction

3

processes of glucose, the non-enzymatic and progressive glycation of proteins with

the consequently increased formation of glucose-derived advanced glycosylation end

products (AGEs), and enhanced glucose flux through the polyol pathway (Oberley,

1988; Tiwari and Rao, 2002; Brownlee et al., 1984). Elevated generation of free

radicals resulting in the consumption of antioxidant defense components may lead to

disruption of cellular functions and oxidative damage to membranes and may enhance

susceptibility to lipid peroxidation (Baynes, 1991).

Reactive oxygen species (ROS) such as O2–, H2O2 and .OH are highly toxic to

cells. Cellular antioxidant enzymes (superoxide dismutase, catalase, glutathione S-

transferase), and the free-radical scavengers (GSH) normally protect a cell from toxic

effects of the ROS. However, when generation of the ROS overtakes the antioxidant

defense of the cells, oxidative damage of the cellular macromolecules (lipids,

proteins, and nucleic acids) occurs, leading finally to various pathological conditions.

ROS-mediated lipid peroxidation, oxidation of proteins, and DNA damage are well-

known outcomes of oxygen-derived free radicals, leading to cellular pathology and

ultimately to cell death (Bandyopadhyay et al., 1999)

The management of diabetes is mainly aimed at alleviating the symptoms and

minimizing the micro and macrovascular complications. Oral hypoglycemic drugs,

viz, biguanide (metformin), thiazolindinediones, sulphanylureas, meglitinides etc,

play an important role in the management of non insulin-dependent diabetes mellitus,

but none have been unequivocally successful in maintaining euglycaemia and in

avoiding late complications of diabetes. Inspite of several advances in therapeutics

and detailed understanding of the disease, diabetes still remains a major cause of

morbidity and mortality in the world (Bastaki, 2005). Plants have been the major

source of drugs in Indian system of medicine and other ancient systems of the world.

Earliest description of curative properties of medicinal plants is found in Rigveda.

Traditional medicines derived from medicinal plants are used by about 60% of the

world’s population (Tag et al., 2012; Gupta, 1994).

Several natural products such as Syzygium cumini, Momordica charantia,

Azadirachta indica, Gymnema sylvestre, Pterocarpus marsupim, Coccinia indica,

Trigonella foenum graecum, Allium sativum and Ocimum sanctum, etc. are being used

Introduction

4

in India as well as other parts of the world for the management of diabetes and to

overcome its complications. These plants are found to be effective and their low cost

and minimal side effects have increased the interest of scientists to develop plant

based drugs for managing diabetes (Modak et al., 2007).

Diabetes has also been associated to immunosuppression (Muller et al., 2011;

Luo et al., 2007), thus the plant based therapies, which attenuate the complications of

diabetes, were also tested for their immunomodulatory activity.

The impact of the immune system in human disease is enormous. Immune

system dysfunction is responsible for various diseases like arthritis, ulcerative colitis,

asthma, allergy, parasitic diseases, cancer and infectious diseases (Patwardhan et al.,

1990). Immune system is a remarkably sophisticated defence system within

vertebrates, to protect them from invading agents. It is able to generate varieties of

cells and molecules capable of recognizing and eliminating limitless varieties of

foreign and undesirable agents. Modulation of the immune system denotes to any

change in the immune response that can involve induction, expression, amplification

or inhibition of any part or phase of the immune response. Thus, immunomodulator is

a substance used for its effect on the immune system. There are generally of two types

immunomodulators based on their effects: immunosuppressants and

immunostimulators. They have the ability to mount an immune response or defend

against pathogens or tumors. Immunopharmacology is a comparatively new and

developing branch of pharmacology aims at searching for immunomodulators. The

potential uses of immunodulators in clinical medicine include the reconstitution of

immune deficiency (e.g. the treatment of AIDS) and the suppression of normal or

excessive immune function (e.g. the treatment of graft rejection or autoimmune

disease) (Saroj et al., 2012).

Chemotherapeutic agents available today have mainly immunosuppressive

activity. Most of them are cytotoxic and exert a variety of side effects. This has given

rise to stimulation in the search for investigating natural resources showing

immunomodulatory activity. Many medicinal plants are known to have

immunomodulatory properties and maintain organic resistance against infection by re-

establishing the body’s immune system such as Azadirachta indica (van der Nat et al.,

Introduction

5

1987), Terminalia chebula (Sohni and Bhat, 1996) and Murraya koenigii (Shah et al.,

2008). The phytochemical constituents like terpenoids, steroids, proteins and tannins

(Biswas et al., 2002) are considered to exhibit this immunomodulatory property.

Flavonoids from Plantago species (Chiang et al., 2003) and Syzygium samarangense

(Kuo et al., 2004) have also shown immunomodulatory activity. In our study the two

common Indian trees tested for immunomodulatory effects are Syzygium cumini

(seeds) and Pterocarpus marsupium (bark).

Syzygium cumini (SC), a member of family Myrtaceae is commonly known as

Jamun or Jambul in Hindi and Black Plum or Black Berry in English. Various parts of

this plant have been recognized to possess several medicinal properties in the

traditional system of medicine. The bark of the plant is astringent, refrigerant,

carminative, diuretic, digestive, anti-helminthic, febrifuge, constipating, stomachic

and antibacterial. The fruits and seeds are used to treat diabetes, pharyngitis,

spleenopathy, urethrorrhea and ringworm infection. The leaves have antibacterial

properties and are used to strengthen the teeth and gums. The leaves have also been

extensively used to treat diabetes, constipation, leucorrhoea, fever, gastropathy,

dermopathy and to inhibit blood discharges in the feces (Jagetia and Baliga, 2002;

Pepato et al., 2001; Pari and Saravanan, 2002; Mitra et al., 1995). In addition,

pharmacological evaluation of this plant reveals its anti-diabetic, hypolipidemic,

antioxidant, anti-HIV, anti diarrhoeal, anti-inflammatory, antibacterial, antipyretic,

radioprotective and neuropsychopharmacological activity (Srivastava and Chandra,

2013; Jadhav et al. 2009). Pterocarpus marsupium (PM) belongs to the family

fabaceae and is commonly known as Indian Kino in English and Vijaysar in Hindi.

Pterocarpus marsupium shows anti-diabetic, hepatoprotective and cardiotonic

activity. Studies have also reported its ability as a COX-2 inhibitor (Devgun et al.,

2009).

Several reports are available regarding the anti-hyperglycemic effects of SC

and PM but the data about their active constituents, their mechanism of action is not

conclusive. Oxidative stress greatly contributes to the progression of diabetic

complications. The medicinal plants mentioned above are reported to exhibit

antioxidant potential but it is inconclusive that their anti-oxidative property is due to

Introduction

6

an independent activity associated to their constituent (s) or simply because of

normoglycemic condition achieved due to their consumption. Therefore, the present

study is planned to systematically evaluate the anti-hyperglycemic and anti-oxidative

potentials of SC and PM. The study is also aimed to isolate and characterize the active

constituents present in these plants and responsible for anti-hyperglycemic and anti-

oxidative potential, using alloxan induced diabetic rats as model. The effects of these

plant extracts on immune system in normal and diabetic rats were also tested in order

to evaluate their immunomodulatory potential.

Review of Literature

7


DIABETES

Diabetes is characterized as an abnormal pathological condition resulting due to imbalance in secretion of hormone(s), primarily insulin secreted from β cells of pancreas. Diabetes is one of the major health problems not only in developing countries like India but throughout the world (Al Ali et al., 2013). Two types of diabetes are known:

Diabetes insipidus: Diabetes insipidus is often called as water diabetes. The symptoms of diabetes

insipidus are, excessive thirst, passing large volume of diluted urine and a general feeling of weakness, which are much similar to the other type of diabetes i.e. diabetes mellitus (sugar diabetes). Diabetes insipidus can be caused either by a defect in pituitary gland or a defect in kidney (nephrogenic diabetes insipidus). These defects cause either under secretion of the anti-diuretic hormones (ADH) or vasopressin. ADH is responsible for re-absorption of water in kidney but due to its under secretion in diabetes insipidus the body’s water retention capacity is reduced and the patient passes large volume of urine. Thus the symptoms observed include profound thirst, dehydration and low blood pressure.

Diabetes mellitus: Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterized

by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both. Banting and Best isolated insulin in 1922 and treated a diabetic patient with it. Insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. In mammals, insulin is synthesized in the pancreas within the beta cells (β-cells) of the islets of Langerhans. In β cells, insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the center portion of the molecule (i.e., C-peptide). The remaining polypeptides (51 amino acids in total), the B- and A- chains, are linked together by disulfide bonds to form insulin.


8

Insulin is a hormone that is central to regulating carbohydrate and fat

metabolism in the body. The stimuli for insulin secretion include ingested protein and

glucose in the blood produced from digested food. Insulin binds to the extracellular

portion of the alpha subunits of the insulin receptor which in turn causes a

conformational change in the insulin receptor that activates the kinase domain that

resides on the intracellular portion of the beta subunits. This further activates a

cascade of protein kinases and a whole series of enzyme

phosphorylation/dephosphorylation reactions which account for the effects of

insulin. Insulin causes cells in the liver, muscle, and fat tissues to take up glucose and

amino acids from the blood, activate protein synthesis from amino acids and glycogen

and triglyceride synthesis from glucose. Insulin stops the use of fat as an energy

source by inhibiting the release of glucagon. In the absence of insulin, glucose is not

taken up by body cells and the body begins to use fat as an energy source

or gluconeogenesis. Thus failure in the control of insulin levels, results in diabetes

mellitus.

Two types of diabetes mellitus have been reported, a) Type I or insulin

dependent diabetes mellitus (IDDM), found among 10% of the total diabetes mellitus

patients (Ejaz and Wilson, 2013), b) Type II or non insulin dependent diabetes

mellitus (NIDDM) and represents about 90% of the total diabetes mellitus patients

(Booth et al., 2013).

Type I DM also known as juvenile diabetes is the classical threatening form of

diabetes characterized by the autoimmune destruction of insulin producing β-cells of

islets of langerhans leading to absolute insulin deficiency. Type I DM is the result of

an unfavorable interaction between environmental factors and an inherited

predisposition of the disease. The environmental factors that might lead to type I

diabetes includes viral infections specially those caused by the coxsackie and other

enterovirus , mycobacterium, chemical toxins in the food and neonates exposure to

cow’s milk constituents, which may cross react with specific β-cell antigens (Virtanen

et al., 1993; Tisch and McDevitt, 1996). Type I diabetes is managed with insulin

injection, life style adjustments and careful monitoring.


9

Type II diabetes mellitus is also known as adult-onset diabetes. Insulin

resistance and disorders of insulin secretion represent core elements in the

pathogenesis of type II diabetes mellitus (Giorgino et al., 2005). The natural history of

type II diabetes usually begins with obesity due to insulin resistance which ultimately

led to under secretion of insulin which may be attributed to several metabolic effects

including increased hepatic glucose production. The transition from normal to

impaired glucose tolerance is associated with a decline in insulin stimulated glucose

disposal and a decline in the acute insulin secretory response. This decrease in the

first phase insulin response is responsible for post-prandial hyperglycemia (Lin and

Sun, 2010; Panunti et al., 2004). Fasting hyperglycemia, on the other hand, is caused

by unrestrained basal hepatic glucose output, primarily a consequence of hepatic

resistance to insulin action. Chronic hyperglycemia further impairs β-cell secretory

kinetics and tissue sensitivity to insulin, a phenomenon known as glucotoxicity

(Dailey, 2004).

Biochemistry of diabetes

The plasma glucose levels rarely exceed 120 mg/dl in normal humans, but

higher levels are routinely found in patients with deficient insulin action. When the

plasma glucose level is higher than 180 mg/dl (the maximum limit of renal tubular

absorption of glucose), sugar is excreted in urine (glycosuria). The urine volume is

increased owing to osmotic diuresis and coincident obligatory water loss (polyurea)

and this in turn leads to dehydration (hyperosmolarity), increased thirst and excessive

drinking of water (polydipsia). Protein synthesis decreases in the absence of insulin

partly because the transport of amino acids into muscles is diminished (O’Neill et al.,

2010). Thus the insulin deficient persons are in negative nitrogen balance. The

deficiency of insulin leads to lipolysis resulting in increased plasma fatty acid levels.

When the capacity of the liver to oxidize fatty acids to CO2 is exceeded, β-hydroxy

butyric acid accumulates (ketosis) (Fex et al., 2004). The organism initially

compensates for the accumulation of these organic acids by increasing respiratory

losses of CO2 but if unchecked by the administration of insulin, severe metabolic

acidosis supervenes and the patient dies in diabetic coma.


10

Effect on glucose metabolism

Insulin influences the intracellular utilization of glucose in a number of ways.

In normal person about half of the glucose ingested in converted to energy through

glycolytic pathway and about half is stored as fat or glycogen. In the absence of

insulin, glycolysis is decreased and the anabolic process of gluconeogenesis is

decreased (Magnusson et al., 1992). Only 5% of an ingested glucose load is converted

to fat in insulin deficient diabetics. Insulin increases hepatic glycolysis by increasing

the activity and amount of several key enzymes including glucokinase,

phosphofructokinase and pyruvate kinase (Wu et al., 2005). The flux through the

glycolytic pathway is adjusted in response to conditions both inside and outside the

cell. The rate in liver is regulated to meet major cellular needs: (1) the production of

ATP, (2) the provision of building blocks for biosynthetic reactions, and (3) to lower

blood glucose, one of the major functions of the liver. When blood sugar falls,

glycolysis is halted in the liver to allow the reverse process, gluconeogenesis. In

glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate

kinase are effectively irreversible in most organisms. In metabolic pathways,

such enzymes are potential sites of control, and all three enzymes serve this purpose

in glycolysis. A balance between glucose production and its utilization is necessary to

maintain normal blood glucose levels. Diabetes is characterized by elevated

production and low utilization of glucose (Taylor and Agius, 1998). A number of

changes in several enzymes present in the liver and other tissues are known to occur

in diabetes mellitus e.g. activity of hepatic glucokinase is markedly decreased and

activity of glucose-6-phosphatase is almost doubled (Hinder et al., 2013). This

imbalance results in constant hyperglycemia in the diabetic state. In skeletal muscles

insulin promotes glucose entry through the transporter and also increases Hexokinase

II activity, which phosphorylates glucose and initiates glucose metabolism. Insulin

stimulates lipogenesis in adipose tissue (Polakof et al., 2011) 1- by providing the

acetyl CoA and NADPH required for fatty acid synthesis. 2- by measuring the normal

levels of the enzyme acetyle CoA-carboxylase, which catalyzes the conversion of

acetyl CoA to malonyl CoA. 3- by providing the glycerol involved in triglycerol

synthesis.


11

The action of insulin on glucose transport, glycolysis and glucogenesis occur

within seconds or minutes since they primarily involve the activation or inactivation

of enzyme involved by phosphorylation or dephosphorylation (Denton et al., 1981).

The formation of glucose from non-carbohydrate precursor involves a series of

enzymatic steps, many of which are stimulated by glucagon (through C-AMP), by

glucocarticoid hormones and to a lesser extent by α and β adrenergic agents,

angiotensin II and vasopressin (Roth & Beaudoin, 1987; Kuchler et al., 2010). The

key gluconeogenic enzyme in the liver is phosphoenolpyruate carboxykinase

(PEPCK). Insulin decreases the amount of this enzyme by selectively inhibiting of the

gene that codes for the m-RNA for PEPCK (Scott at al., 1998). The net action of all

the above effects of insulin is to decrease the blood sugar level. In this action, insulin

stands alone against an array of hormones that attempt to counteract this effect.

Effect on lipid metabolism

In patient with insulin deficiency, lipase activity increases, resulting in

enhanced lipolysis and increased concentration of free fatty acids in plasma and liver

(Costabile et al., 2011). Glucagon levels also increases in these patients and this

enhances the release of free fatty acids. Free fatty acids are metabolized to acetyl CoA

and finally to CO2 and H2O via citric acid cycle. In patient with insulin deficiency the

capacity of this process is rapidly exceeded and the acetyl CoA is converted to

acetoacetyl CoA and then to acetoacetic and hyroxy-butyric acids (Bickerton et al.,

2008). Insulin apparently affects either formation or clearance of VLDL and LDL,

since levels of these particles and consequently the levels of cholesterol are often

elevated in poorly controlled hyperglycemia.

Effect on protein metabolism

Insulin generally has an anabolic effect on protein metabolism by stimulating

protein synthesis (Bonadonna et al., 1993; Kimball et al., 1994). Insulin stimulates the

uptake of neutral amino acids in muscles, an effect that is not linked to glucose uptake

or to a subsequent incorporation of amino acids into protein. The effect of insulin on

general protein synthesis in skeletal and cardiac muscles and in liver are exerted at the

level of m-RNA translation (Scheper et al., 2007). In recent years insulin has been


12

shown to influence the synthesis of specific proteins by affecting changes in the

corresponding m-RNA.

OXIDATIVE STRESS

Oxidative stress results from an imbalance between radical-generating and

radical scavenging systems, i.e. increased free radical production or reduced activity

of antioxidant defenses or both. Oxygen is vital for aerobic life processes. However,

about 5% or more of the inhaled O2 is converted to reactive oxygen species (ROS)

such as O2¯, H2O2, and ·OH by univalent reduction of O2 (Harman, 1993). Therefore

under aerobic conditions the cells are always endangered by ROS, which are

efficiently taken care of by the highly powerful antioxidant systems of the cell. When

the balance between ROS production and antioxidant defenses is lost, ‘oxidative

stress’ results which through a series of events deregulates the cellular functions

leading to various pathological conditions including cardiovascular dysfunction,

neurodegenerative diseases, gastroduodenal pathogenesis, metasbolic dysfunction of

almost all the vital organs, cancer, and premature aging (Thomas and Kalyanaraman,

1997).

1) THE MECHANISM OF ROS FORMATION

Although O2 can behave like a radical (a diradical) owing to the presence of

two unpaired electrons of parallel spin, it does not exhibit extreme reactivity due to

quantum-mechanical restrictions. Its electronic structure results in formation of water

by reduction with four electrons, i.e:

O2 + 4H++ 4e- 2H2 O

In the sequential univalent process by which O2 undergoes reduction, several

reactive intermediates are formed, such as superoxide (O2¯), hydrogen peroxide

(H2O2), and the extremely reactive hydroxyl radical (·OH), which are collectively

termed as the reactive oxygen species (ROS). The process can be represented as:

O2 e-

O2¯ e- H2O2 e- ·OH e- H2O

These oxygen-derived pro-oxidants, can cause damage to biological targets

such as lipids, DNA, and proteins, and on the defending systems of the cell, which are


13

composed of enzymes and reducing equivalents, or antioxidants. In general these pro-

oxidants are referred to as reactive oxygen species (ROS) that can be classified into 2

groups of compounds, radicals and nonradicals. The radical group, contains

compounds such as nitric oxide radical (NO·), superoxide ion radical (O2·¯), hydroxyl

radical (·OH), peroxyl (ROO·) and alkoxyl radicals (RO·), and one form of singlet

oxygen (Halliwell et al., 2000). These species are radicals, because they contain at

least 1 unpaired electron in the shells around the atomic nucleus and are capable of

independent existence (Halliwell and Gutteridge, 1999). The occurrence of one

unpaired electron results in high reactivity of these species by their affinity to donate

or obtain another electron to attain stability. The group of nonradical compounds

contains a large variety of substances, some of which are extremely reactive although

not radical by definition. Among these compounds produced in high concentrations in

the living cell are hypochlorous acid (HClO), hydrogen peroxide (H2O2), organic

peroxides, aldehydes, ozone (O3), and O2 as shown in table 1.

i) Some examples of ROS

Superoxide Ion Radical (O2·¯ /HO·

2)

This species possess different properties depending on the environment and

pH. Due to its pKa of 4.8, superoxide can exist in the form of either O2·¯or, at low pH,

hydroperoxyl (HO·2) (Sohal et al., 1989). The latter can more easily penetrate


14

biological membranes than the charged form. Hydroperoxyl can therefore be

considered an important species, although under physiological pH most of the

superoxide is in the charged form. In a hydrophilic environment both the O2·¯ and

HO·2 can act as reducing agents capable, for example, of reducing ferric (Fe +3) ions to

ferrous (Fe +2) ions; however, the reducing capacity of HO·2 is higher. The most

important reaction of superoxide radicals is dismutation (reaction 1), in which a

superoxide radical reacts with another superoxide radical. One is oxidized to oxygen,

and the other is reduced to hydrogen peroxide (Fridovich, 1997).

HO·2 /O2

·¯ + HO·2 /O2

·¯ + H+ H2O2 + O2 (1)

Hydroxyl Radical (·OH)

The reactivity of hydroxyl radicals is extremely high (Halliwell and

Gutteridge, 1999). In contrast to superoxide radicals that are considered relatively

stable and have constant, relatively low reaction rates with biological components,

hydroxyl radicals are short-lived species possessing high affinity toward other

molecules. ·OH is a powerful oxidizing agent that can react at a high rate with most

organic and inorganic molecules in the cell, including DNA, proteins, lipids, amino

acids, sugars, and metals. ·OH is considered the most reactive radical in biological

systems and due to its high reactivity it interacts at the site of its production with the

molecules closely surrounding it.

Hydrogen Peroxide (H2O2)

The result of dismutation of superoxide radicals is the production of H2O2.

There are some enzymes that can produce H2O2 directly or indirectly. Although H2O2

molecules are considered reactive oxygen metabolites, they are not radical by

definition. They can, however, cause damage to the cell at a relatively low

concentration (10 µM). They are freely dissolved in aqueous solution and can easily

penetrate biological membranes. Their deleterious chemical effects can be divided

into the categories of direct activity, originating from their oxidizing properties, and

indirect activity in which they serve as a source for more deleterious species, such as

OH· or HClO. Direct activities of H2O2 include degradation of haem proteins; release


15

of iron; inactivation of enzymes; and oxidation of DNA, lipids, SH groups, and keto

acids (Halliwell and Gutteridge, 1999).

Nitric Oxide (NO·), Peroxynitrite (ONOO¯), and Other Members of the

Family

The nitric oxide, or nitrogen monoxide, radical (NO·) is produced by the

oxidation of one of the terminal guanido nitrogen atoms of L-arginine. In this

reaction, catalyzed by the group of enzymes called nitric oxide synthase (NOS), L-

arginine is converted to nitric oxide and L-citrulline. Three types of the enzymes

exist: neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). One-

electron oxidation results in the production of nitrosonium cation (NO+), while one-

electron reduction leads to nitroxyl anion (NO¯), which can undergo further reactions,

such as interacting with NO· to yield N2O and OH· (Beckman and Koppenol, 1996).

NO· can react with a variety of radicals and substances like H2O2 and HClO to yield a

line of derivatives such as N2O3, NO2¯, and NO3¯. One of the most important

reactions under physiological conditions is that of superoxide and nitric oxide radicals

resulting in peroxynitrite (reaction 2). This reaction helps to maintain the balance of

superoxide radicals and other ROS and is also important in redox regulation (Czapski

and Goldstein, 1995).

NO· + O2·¯ ONOO¯ (2)

The protonated form of peroxynitrite (ONOOH) is a powerful oxidizing agent

that might cause depletion of sulfhydryl (-SH) groups and oxidation of many

molecules causing damage similar to that observed when OH· is involved. It can also

cause DNA damage such as breaks, protein oxidation, and nitration of aromatic amino

acid residues in proteins (Murphy et al., 1998).

The Role of Transition Metals

Most of the transition metals—those in the first row of the D block in the

periodic table contain unpaired electrons and can, therefore, with the exception of

zinc, be considered radicals by definition (Halliwell and Gutteridge, 1999). They can

participate in the chemistry of radicals and convert relatively stable oxidants into

powerful radicals. Among the various transition metals, copper and especially iron are


16

most abundant, present in relatively high concentrations, and are major players in the

Fenton reaction (Fenton, 1894) and the metal-mediated Haber-Weiss reaction (Haber

and Weiss, 1934).

For the production of ·OH, except during abnormal exposure to ionizing

radiation, generation of ·OH in vivo requires the presence of trace amount of transition

metals like iron or copper. A simple mixture of H2O2 and Fe2+ salt forms ·OH, as

given by the following Fenton reaction:

Fe2+ + H2O2 Fe3+ + ·OH + OH¯

Traces of Fe3+ can react further with H2O2 to form the following products:

Fe3+ + H2O2 Fe2+ + O2¯ + H+

Thus, a free-radical mechanism for the generation of .OH may be deduced as

follows:

O2¯ + H2O2 OH¯ + ·OH + O2

Unfortunately, the rate constant for the above reaction is very low but can be

accounted for if the reaction is catalyzed by traces of transition metal ions – the metal-

catalyzed Haber–Weiss reaction. The various steps of this reaction are:

Fe3+ + O2¯ Fe2+ + O2

Fe2+ + H2O2 Fe3+ + ·OH + OH¯

and the net result is:

O2¯ + H2O2 O2 + ·OH + OH¯

However, redox-active free iron or copper do not exist in biological systems, as these

transition metal ions remain bound to proteins, membranes, nucleic acids or low-

molecular weight chelating agents like citrate, histidine, or ATP (Halliwell and

Gutteridge, 1984). However during ischemic condition, and cellular acidosis,

transition-metal ions may be released from some metalloproteins (Chevion et al.,

1993), resulting in generation of ·OH.

ii) Sources of ROS

The cell is exposed to a large variety of ROS and RNS from both exogenous

and endogenous sources (Figure 1). The former include, first, exposure to di-oxygen,


17

which, although a nonreactive biradical, can independently cause oxidation and

damage to proteins and enzymes, exemplified by inhibition of aconitase and fumerase

in the Krebs cycle and glutamate decarboxylase, which results in decreased γ-

aminobutyric acid in the brain (Halliwell and Gutteridge, 1999). Ozone (O3) present

in the upper atmosphere acts as a damaging species to biological tissues. It can

damage lungs, and can serve as a powerful oxidizing agent that can oxidize biological

components directly (Rao and Davis, 2001). Exposure of living organisms to ionizing

and non ionizing irradiation constitutes another major exogenous source of ROS

(Shadyro et al., 2002). Air pollutants such as car exhaust, cigarette smoke, and

industrial contaminants encompassing many types of NO derivatives constitute major

sources of ROS that attack and damage the organism either by direct interaction with

skin or following inhalation into the lung (Koren, 1995). Drugs are also a major

source of ROS (Rav et al., 2001). There are drugs, such as belomycinem and

adreamicine, whose mechanism of activity is mediated via production of ROS, those

like nitroglycerine that are NO· donors, and those that produce ROS indirectly.

Narcotic drugs and anesthetizing gases are considered major contributors to the

production of ROS (Chinev et al., 1998). A large variety of xenobiotics (eg. toxins,

pesticides, and herbicides such as paraquat) and chemicals (eg,mustard gas, alcohol)

produce ROS as a by-product of their metabolism in vivo (Elsayed et al., 1992). One

of the major sources of oxidants is food for a large portion of the food we consume is

oxidized to a large degree and contains different kinds of oxidants such as peroxides,

aldehydes, oxidized fatty acids, and transition metals (Ames, 1986). Food debris that

reaches the intestinal tract places an enormous oxidative pressure on the intestinal-

tract mucosa (Srigirdhar et al., 2001).

Although the exposure of the organism to ROS is extremely high from

exogenous sources, the exposure to endogenous sources is much more important and

extensive, because it is a continuous process during the life span of every cell in the

organism.

For the production of O2¯, normally the tendency of univalent reduction of O2

in respiring cells is restricted by cytochrome oxidase of the mitochondrial electron

transport chain, which reduces O2 by four electrons to H2O without releasing either O2


18

– or H2O2. However, O2 – is invariably produced in respiring cells (Fridovich, 1983).

This is due to the probable ‘leak’ of single electron at the specific site of the

mitochondrial electron transport chain, resulting in inappropriate single electron

reduction of oxygen to O2¯ (Loschen et al.,1974). When the electron transport chain

is highly reduced, and the respiratory rate is dependent on ADP availability; ‘leakage’

of electrons at the ubisemiquinone and ubiquinone sites increases so as to result in

production of O2¯ and H2O2 (Turrens and Boveris, 1980).

For the production of H2O2, peroxisomal oxidases and flavoproteins, as well

as D-amino acid oxidase, L-hydroxy acid oxidase, and fatty acyl oxidase participate.

Cytochrome P-450, P-450 reductase and cytochrome b-5 reductase in the endoplasmic

reticulum under certain conditions generate O2¯ and H2O2 during their catalytic cycles

(Bast et al., 1991). Likewise, the catalytic cycle of xanthine oxidase has emerged as

an important source of O2¯ and H2O2 in a number of different tissue injuries.

Xanthine oxidase, produced by proteolytic cleavage of xanthine dehydrogenase

during ischemia, upon reperfusion in presence of O2, acts on xanthine or

hypoxanthine to generate O2¯ and H2O2 (McCord, 1987).

The phagocytic cells, such as neutrophils, when activated during phagocytosis,

generate O2¯ and H2O2 through activation of NADPH oxidase. Neutrophil

accumulation in inflammated tissue is one of the major reasons of oxidative damage

due to generation of ROS. In addition, spontaneous dismutation of O2 at neutral pH or

dismutation by superoxide dismutase, results in H2O2 production (Morel et al., 1991).

Figure 1: Exogenous and endogenous sources of reactive oxygen species (ROS).


19

2) CONSEQUENCES OF FREE RADICALS GENERATION

Reactive oxygen species can attack vital cell components like polyunsaturated

fatty acids, proteins, and nucleic acids. To a lesser extent, carbohydrates are also the

targets of ROS. These reactions can alter intrinsic membrane properties like fluidity,

ion transport, loss of enzyme activity, protein cross-linking, inhibition of protein

synthesis, DNA damage: ultimately resulting in cell death (Halliwell and Gutteridge,

1990). Some of the well-known consequences of generation of the free radicals in

vivo are: DNA strand scission (Brawn and Fridovich, 1981), nucleic acid base

modification (Moody and Hussan, 1982), protein oxidation (Pacifici et al., 1993) and

lipid peroxidation (Halliwell and Gutteridge, 1990).

Lipid peroxidation

Oxygen radicals catalyse the oxidative modification of lipids (Gardner, 1989).

This peroxidation chain reaction is illustrated in figure 2. The presence of double

bond adjacent to a methylene group makes the methylene C–H bonds of

polyunsaturated fatty acid (PUFA) weaker and therefore the hydrogen becomes more

prone to abstraction. While lipid peroxidation is not initiated by O2¯ and H2O2, ·OH,

alkoxy radicals (RO·), and peroxy radicals (ROO·) result in initiating the lipid

peroxidation (Turrens and Boveris, 1980). This can lead to a self perpetuating process

since peroxy radicals are both reaction initiators as well as the products of lipid

peroxidation. Lipid peroxy radicals react with other lipids, proteins, and nucleic acids;

propagating thereby the transfer of electrons and bringing about the oxidation of

substrates. Cell membranes, which are structurally made up of large amounts of

PUFA, are highly susceptible to oxidative attack and, consequently, changes in

membrane fluidity, permeability, and cellular metabolic functions result.

Figure 2: Mechanism of lipid peroxidation by ROS.


20

DNA damage

ROS can cause oxidative damages to DNA: both nuclear and mitochondrial.

The nature of damages includes mainly base modification, deoxyribose oxidation,

strand breakage, and DNA– protein cross-links. Among the various ROS, ·OH

generates various products from the DNA bases which mainly include C-8

hydroxylation of guanine to form 8-oxo-7,8 dehydro-2’- deoxyguanosine, a ring-

opened product; 2,6-diamino-4-hydroxy-5- formamimodipyrimidine, 8-OH-adenine,

2-OH-adenine, thymine glycol, cytosine glycol, etc. (Wiseman and Halliwell, 1996).

ROS-induced DNA damages include various mutagenic alterations as well. For

example, mutation arising from selective modification of G : C sites specially

indicates oxidative attack on DNA by ROS. The action of 8-oxodeoxy- guanosine as a

promutagen, as well as in altering the binding of methylase to the oligomer so as to

inhibit methylation of adjacent cytosine has been reported in cases of cancer

development (Weitzman et al., 1994). ROS have also been shown to activate

mutations in human C-Ha-ras-1 protooncogene, and to induce mutation in the p53

tumour-suppressor gene (Hussain et al., 1994). Besides, ROS may interfere with

normal cell signalling, resulting thereby in alteration of the gene expression, and

development of cancer by redox regulation of transcriptional factors/activator and/or

by oxidatively modulating the protein kinase cascades. The oxidative damage of

mitochondrial DNA also involves base modification and strand breaks, which leads to

formation of abnormal components of the electron transport chain. This results in the

generation of more ROS through increased leakage of electrons, and therefore further

cell damage. Oxidative damage to mitochondrial DNA may promote cancer and

aging, eventually (Richter, 1988).

Oxidative damage of proteins

During mitochondrial electron transport chain, free radicals are produced

which can stimulate protein degradation. Oxidative protein damage may be brought

about by metabolic processes which degrade a damaged protein to promote synthesis

of a new protein. In the process of cataractogenesis, oxidative modification plays a

significant role in cross-linking of crystalline lens protein, leading to high-molecular-

weight aggregates, loss of solubility, and lens opacity (Guptasarma et al., 1992).


21

Lipofuscin – an aggregate of peroxidized lipid and proteins – accumulates in

lysosomes of aged cells, Alzheimer’s disease brain cells, and ironoverloaded

hepatocytes (Wolf et al., 1986). On the basis of extensive studies on aging processes,

it has been established that catalytically inactive or less active, more thermolabile

forms of enzyme accumulate in cells during aging, and show a dramatic increase in

the level of protein carbonyl content: an index of metal-catalysed oxidation of

proteins (Stadtman and Oliver, 1991). In human erythrocytes, levels of

glyceraldehyde-3-P-dehydrogenase, aspartate aminotransferase, and phosphoglycerate

kinase decline with age together with an increase in protein carbonyl content. The

carbonyl content of protein in rat hepatocytes also increases with age along with

decrease in the activities of glutamine synthetase and glucose-6-P-dehydrogenase,

without any loss in the total enzyme protein (Oliver et al., 1987). An oxidative

inactivation of glutamine synthetase occurs during ischemic-reperfusion injury of

gerbil brain (Oliver et al., 1990) (Figure 3).

Figure 3: An overall picture of the metabolism of ROS and the mechanism of oxidative tissue damage leading to pathological conditions.


22

3) ANTIOXIDANT SYSTEMS INVOLVED IN THE

SCAVENGING PROCESS

a) Primary defense against ROS: Catalytic removal of ROS by

antioxidant enzymes

Superoxide dismutase (SOD), catalase, and peroxidases constitute a mutually

supportive team of defense against ROS. While SOD lowers the steady-state level of

O2¯, catalase and peroxidases do the same for H2O2.

Superoxide dismutase

The first enzyme involved in the antioxidant defense is the superoxide

dismutase: a metalloprotein found in both prokaryotic and eukaryotic cells (Fridovich,

1983). The iron-containing (Fe-SOD) and the manganese-containing (Mn-SOD)

enzymes are characteristic of prokaryotes. In eukaryotic cells, the predominant forms

are the copper-containing enzyme and the zinc-containing enzyme, located in the

cytosol. The second type is the manganese containing SOD found in the

mitochondrial matrix. The biosynthesis of SOD is mainly controlled by its substrate,

the O2– (Fridovich, 1986). Induction of SOD by increased intracellular fluxes of O2

–

has been observed in numerous microorganisms, as well as in higher organisms

(Crapo and McCord, 1976).

Glutathione peroxidase and Glutathione reductase

Glutathione peroxidase catalyses the reaction of hydroperoxides with reduced

glutathione (GSH) to form glutathione disulphide (GSSG) and the reduction product

of the hydroperoxide (Figure 4). This enzyme is specific for its hydrogen donor, GSH,

and nonspecific for the hydroperoxides ranging from H2O2 to organic hydroperoxides

(Meister and Anderson, 1983). It is a seleno-enzyme; two-third of which (in liver) is

present in the cytosol and one-third in the mitochondria. Glutathione disulfide is

recycled back to glutathione by glutathione reductase, using the cofactor NADPH

generated by glucose 6- phosphate dehydrogenase (Freeman and Crapo, 1982).


23

Heme peroxidase

Heme peroxidases such as horseradish peroxidase, lactoperoxidase, and other

mammalian peroxidases have been studied most extensively. The enzyme catalyses

the oxidation of a wide variety of electron donors with the help of H2O2 and thereby

scavenges the endogenous H2O2 (Dawson, 1988).

Catalase

Catalase present in almost all the mammalian cells is localized in the

peroxisomes or the microperoxisomes. It is a hemoprotein and catalyses the

decomposition of H2O2 to water and oxygen and thus protects the cell from oxidative

damage by H2O2 and .OH (Deisseroth and Dounce, 1970).

Figure 4: Catalytic removal of ROS by antioxidant enzymes.

b) Secondary defense against ROS: Free-radical scavengers

In addition to the primary defense against ROS by antioxidant enzymes,

secondary defense against ROS is also offered by small molecules which react with

radicals to produce another radical compound, the ‘scavengers’. When these

scavengers produce a lesser harmful radical species, they are called ‘antioxidants’.

For example, α-tocopherol, ascorbate, and reduced glutathione (GSH) may act in

combination to act as cellular antioxidants (Figure 5). α-tocopherol, present in the cell

membrane and plasma lipoproteins, functions as a chain-breaking antioxidant. Once

the tocopherol radical is formed, it can migrate to the membrane surface and is

reconverted to α-tocopherol by reaction with ascorbate or GSH. The resulting

ascorbate radical can regenerate ascorbate by reduction with GSH, which can also


24

directly scavenge ROS, and the resulting GSSG can regenerate GSH through

NADPH-glutathione reductase system.

Figure 5: Mechanism of free-radical scavenging action of cellular low-molecular weight antioxidants- α-tocopherol, ascorbate, and reduced glutathione (GSH), through NADPH-glutathione reductase (GR) system.

This cooperative activity may explain the synergism obtained when several

scavengers are involved and the beneficial use of large combinations of low molecular

weight antioxidants (LMWA) in antioxidant therapy (figure 6).

Examples of LMWA

1) Glutathione

It is a low-molecular-mass, thiol-containing tripeptide, glutamic acid-cysteine-

glycine (GSH) in its reduced form and GSSG in its oxidized form, in which 2 GSH

molecules join via the oxidation of the -SH groups of the cysteine residue to form a

disulphide bridge. It acts as a cofactor for the enzyme peroxidase, thus serving as an

indirect antioxidant donating the electrons necessary for its decomposition of H2O2. It

is also involved in many other biochemical pathways and cellular functions like

metabolism of ascorbic acid, maintenance of communication between cells,

prevention of oxidation in -SH groups of protein, and copper transport (Chance et al.,

1979). Glutathione can act as a chelating agent for copper ions and prevent them from

participating in the Haber-Weiss reaction, it serve as a cofactor for several enzymes,

such as glyoxylase and those involved in leukotriene biosynthesis, and play a role in

protein folding, degradation, and cross-linking. In addition to its biochemical

functions, it can scavenge ROS directly. GSH can interact with OH·, ROO·, and RO·

radicals as well as with HCLO and ‘O2 Upon reaction with ROS, it becomes a

glutathione radical, which can be regenerated to its reduced form (Gul et al., 2000).


25

2) Melatonin

It is the hormone synthesized by the pineal gland, that helps to regulate

circadian rhythms that also possesses a powerful antioxidant capacity in vitro, as it

scavenges a variety of ROS, mainly through donation of the hydrogen atom by the (-

NH) group. Indirectly it alters the antioxidant activity of the cell, for example, by

induction of synthesis of antioxidant enzymes or modulating other cellular responses

leading to secretion and accumulation of other antioxidants. High local concentrations

of melatonin in the brain may explain its great protective effect against head injury in

rats (Reiter et al., 2002).

3) Histidine dipeptides

It includes the compounds (carnosine, homocarnosine, and anserine) that are

synthesized in the brain and skeletal muscles that have anti-oxidative potentials. They

are considered multifunctional antioxidants, because they can act in many ways to

destroy and remove ROS. They can scavenge directly ·OH, ROO·, and RO· radicals;

bind H2O2; quench efficiently ‘O2; and bind transition metals and prevent them from

participating in the metal-mediated Haber- Weiss reaction. In vivo they diminish

oxidative damage in many systems, including the ischemic process. These compounds

do not exert pro-oxidant effects as other reducing antioxidants sometimes do. They

also act as endogenous buffers and thus prevents protein glycosylation (Boldyrev,

1993).

4) Uric acid

Uric acid provides an excellent example of the adaptation of the organism to

oxidative stress. It is a cellular waste product originating from the oxidation of

hypoxanthine and xanthine by xanthine oxidase and dehydrogenase. Urate, the

physiological state of uric acid, reacts with hydroxyl radicals producing a stable urate

radical that can be regenerated by ascorbate to its prior state, urate. This compound

can act with peroxyl radicals, 1O2, O3, NO·, and other nitrogen oxygen radicals. Urate

also protects protein from nitration; it can chelate metal ions, such as copper and iron,

and prevent them from participating in redox reactions (Ames et al., 1981).


26

5) Dietary source antioxidants

Antioxidant molecules like ascorbic acid, tocopherols, polyphenols and

carotenoids present in green vegetables, fruits, fish and other food items. Both

lipophilic and hydrophilic compounds possess different oxidation potentials reflecting

their ability to donate electrons and act as antioxidants.

a) Ascorbic acid (ascorbate, vitamin C)

It is an example of a water-soluble antioxidant. Like other antioxidants,

ascorbate, which at physiological pH exists as a mono anion, possesses many

biochemical functions in addition to its activity as a scavenger. It is required as a

cofactor for many enzymes, such as proline hydroxylase and dopamine β-

hydroxylase. As an antioxidant, ascorbate is an efficient scavenger, or reducing

antioxidant, capable of donating its electrons to ROS and eliminating them. It can

donate 2 electrons; following donation of 1 electron, it produces the ascorbyl

(semidehydroascorbate or ascorbate) radical, which can be further oxidized to

produce dehydroascorbate. Because the ascorbyl radical is relatively stable, it makes

ascorbate a powerful, important antioxidant. This radical can lose its electron and be

transformed to dehydroascorbic acid or regenerated to the reduced form by obtaining

an electron from another reducing agent, such as GSH or NADH, via the mediation of

an enzyme like NADH-semidehydroascorbate reductase (Carr and Frei, 1999).

The oxidation product, dehydroascorbic acid, can also be regenerated by the

enzyme dehydroascorbate reductase at the expense of 2 molecules of GSH. The

compound dehydroascorbate is not stable and is decomposed to di-keto- L-gulonic

acid and then to oxalic and L-threonic acids, which can be further decomposed to

oxalic acid. In vitro, ascorbate can act as an efficient antioxidant and scavenge a

variety of ROS including hydroxyl, peroxyl, thyil, and oxosulphuric radicals.

Ascorbate is also a powerful scavenger of HClO and peroxynitrous acid and can

inhibit the peroxidation process. It can react with 1O2 and act synergistically with

other antioxidants to regenerate, for example, the tocopherol radical to its reduced

form. Indirect evidence has shown that, in vivo, ascorbate acts directly as an

antioxidant (Arrigoni and De Tullio, 2002).


27

b) Vitamin E (Tocopherol)

It is a chain breaking antioxidant that scavenges ROO. to inhibit the lipid

peroxidation process in biological membranes . There are eight naturally occurring

substances that are known to be members of the vitamin E family. These compounds

have 3 asymmetric carbon atoms, giving 8 optical isomers. Although all possess

antioxidant activity, the RRR-a -tocopherol or d-a -tocopherol is considered the most

effective one, as the others are not retained and absorbed well in body tissues. Other

members of the family consist of d-β, d-γ, and d-δ-tocopherols and d-α-, d-β-, d-γ-,

and d-δ-tocotrienols. These compounds can scavenge other ROS, such as 1O2.

Following interaction, tocopherol is converted to tocopherolquinone and subsequently

to tocopherylquinone. As with other scavengers, a -tocopheryl radical can be recycled

to its active form. Other roles for tocopherol also exist, such as those of a membrane-

stabilizing agent and a potential pro-oxidant compound in some systems when

transition metals are present (Herrera and Barbas, 2001).

Figure 6: The sources and cellular responses to reactive oxygen species (ROS).


28

DIABETES INDUCED OXIDATIVE STRESS AND ITS

COMPLICATIONS

Glucose in chronic excess causes toxic effects on structure and function of

organs. There are many potential mechanisms whereby excess glucose metabolites

traveling along these pathways might cause cell damage. Multiple biochemical

pathways and mechanisms of action for glucose toxicity have been suggested (Figure

7). These include glucose autoxidation, protein kinase C activation, methylglyoxal

formation and glycation, hexosamine metabolism, sorbitol formation, and oxidative

phosphorylation. However, all these pathways have in common the formation of

reactive oxygen species that, in excess and over time, cause chronic oxidative stress,

which has been suggested to be involved in the pathogenesis and progression of

diabetic tissue damage. This is particularly relevant and dangerous for the islet, which

is among those tissues that have the lowest levels of intrinsic antioxidant defenses

(Robertson, 2004).

1. Increased polyol pathway flux

The first enzyme in the polyol pathway is aldose reductase (alditol: NAD(P)+

1-oxidoreductase, EC 1.1.1.21). It is a cytosolic, monomeric oxidoreductase that

Figure 7: Biochemical pathways along which glucose metabolism can form ROS


29

catalyses the NADPH-dependent reduction of a wide variety of carbonyl compounds,

including glucose. Its crystal structure has a single domain folded into an eight-

stranded parallel a/b-barrel motif, with the substrate-binding site located in a cleft at

the carboxy-terminal end of the b-barrel (Wilson et al., 1992). Aldose reductase has a

low affinity (high Km) for glucose, and at the normal glucose concentrations found in

non-diabetics, metabolism of glucose by this pathway is a very small percentage of

total glucose use. But in hyperglycaemic conditions, increased intracellular glucose

results in its increased enzymatic conversion to the polyalcohol sorbitol, with

concomitant decreases in NADPH. In the polyol pathway, sorbitol is oxidized to

fructose by the enzyme sorbitol dehydrogenase, with NAD+ reduced to NADH. Flux

through this pathway during hyperglycaemia varies from 33% of total glucose use in

the rabbit lens to 11% in human erythrocytes. A number of mechanisms have been

proposed to explain the potential detrimental effects of hyperglycaemia-induced

increases in polyol pathway flux. These include sorbitol-induced osmotic stress,

decreased (Na+&K+) ATPase activity, an increase in cytosolic NADH/NAD+ and a

decrease in cytosolic NADPH. Sorbitol does not diffuse easily across cell membranes,

and it was originally suggested that this resulted in osmotic damage to microvascular

cells.Thus, the contribution of this pathway to diabetic complications may be very

much species, site and tissue dependent (Figure 8) (Lee and Chung, 1999).

Figure 8: Involvement of polyol pathway in diabetic complications.


30

2. Increased intracellular formation of advanced glycation end-

products

AGEs are found in increased amounts in diabetic retinal vessels (Stitt et al.,

1997) and renal glomeruli (Horie et al., 1997). They were originally thought to arise

from non-enzymatic reactions between extracellular proteins and glucose. But the rate

of AGE formation from glucose is orders of magnitude slower than the rate of AGE

formation from glucose-derived dicarbonyl precursors generated intracellularly, and it

now seems likely that intracellular hyperglycaemia is the primary initiating event in

the formation of both intracellular and extracellular AGEs (Degenhardt et al., 1998).

AGEs can arise from intracellular auto-oxidation of glucose to glyoxal (Wells-Knecht

et al., 1995), decomposition of the Amadori product (glucose-derived 1-amino-1-

deoxyfructose lysine adducts) to 3-deoxyglucosone (perhaps accelerated by an

amadoriase), and fragmentation of glyceraldehyde- 3-phosphate and

dihydroxyacetone phosphate to methylglyoxal. These reactive intracellular

dicarbonyls — glyoxal, methylglyoxal and 3-deoxyglucosone — react with amino

groups of intracellular and extracellular proteins to form AGEs. Methylglyoxal and

glyoxal are detoxified by the glyoxalase system (Thornalley, 1990). All three AGE

precursors are also substrates for other reductases (Suzuki et al., 1998).

Production of intracellular AGE precursors damages target cells by three

general mechanisms (Figure 9). First, intracellular proteins modified by AGEs have

altered function. Second, extracellular matrix components modified by AGE

precursors interact abnormally with other matrix components and with the receptors

for matrix proteins (integrins) on cells. Third, plasma proteins modified by AGE

precursors bind to AGE receptors on endothelial cells, mesangial cells and

macrophages, inducing receptor-mediated production of reactive oxygen species. This

AGE receptor ligation activates the pleiotropic transcription factor NF-kB, causing

pathological changes in gene expression.


31

Figure 9: Mechanisms by which intracellular production of advanced glycation end-product (AGE) precursors damages vascular cells.

3. Activation of protein kinase C

The PKC family comprises at least eleven isoforms, nine of which are

activated by the lipid second messenger DAG. Intracellular hyperglycaemia increases

the amount of DAG in cultured microvascular cells and in the retina and renal

glomeruli of diabetic animals. It seems to achieve this primarily by increasing de novo

DAG synthesis from the glycolytic intermediate dihydroxyacetone phosphate, through

reduction of the latter to glycerol-3-phosphate and stepwise acylation. Increased de

novo synthesis of DAG activates PKC both in cultured vascular cells and in retina and

glomeruli of diabetic animals (Koya and King, 1998). The β- and δ-isoforms of PKC

are activated primarily, but increases in other isoforms have also been found, such as

PKC-α and - ε isoforms in the retina and PKC- α and - β in glomeruli of diabetic rats.

Activation of PKC has a number of pathogenic consequences by affecting expression

of endothelial nitric oxide synthetase (eNOS), endothelin-1 (ET-1), vascular

endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and

plasminogen activator inhibitor-1 (PAI-1), and by activating NF-kB and NAD(P)H

oxidases (Koya et al., 1997) (Figure 10).


32

Figure 10: Consequences of hyperglycaemia-induced activation of protein kinase C (PKC).

4. Increased flux through the hexosamine pathway

Shunting of excess intracellular glucose into the hexosamine pathway might

also cause several manifestations of diabetic complication. In this pathway, fructose-

6-phosphate is diverted from glycolysis to provide substrates for reactions that require

UDP-N-acetylglucosamine, such as proteoglycan synthesis and the formation of O-

linked glycoproteins (Figure 11). The glycolytic intermediate fructose-6-phosphate

(Fruc-6-P) is converted to glucosamine-6-phosphate by the enzyme glutamine:

fructose-6-phosphate amidotransferase (GFAT). Intracellular glycosylation by the

addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalysed by the

enzyme O-GlcNAc transferase (OGT). Increased donation of GlcNAc moieties to

serine and threonine residues of transcription factors such as Sp1, often at

phosphorylation sites, increases the production of factors as PAI-1 (Du et al., 2000)

and TGF-β1(Kolm-Litty et al., 1998).This pathway is also important role in

hyperglycaemia induced and fat-induced insulin resistance (Hawkins et al., 1997).

Thus, activation of the hexosamine pathway by hyperglycaemia may result in many

changes in both gene expression and protein function, which together contribute to the

pathogenesis of diabetic complications.


33

Figure 11: The hexosamine pathway.

5. Oxidative phosphorylation

Electron flow through the mitochondrial electron-transport chain (Figure 12)

is carried out by four inner membrane-associated enzyme complexes, plus

cytochrome c and the mobile electron carrier ubiquinone. NADH derived from both

cytosolic glucose oxidation and mitochondrial TCA cycle activity donates electrons to

NADH: ubiquinone oxidoreductase (complex I). Complex I ultimately transfers its

electrons to ubiquinone. Ubiquinone can also be reduced by electrons donated from

several FADH2-containing dehydrogenases, including succinate: ubiquinone

oxidoreductase (complex II) and glycerol-3-phosphate dehydrogenase. Electrons from

reduced ubiquinone are then transferred to ubiquinol: cytochrome c oxidoreductase

(complex III) by the ubisemiquinone radical-generating Q cycle. Electron transport

then proceeds through cytochrome c, cytochrome c oxidase (complex IV) and, finally,

molecular oxygen (O2). Electron transfer through complexes I, III and IV generates a

proton gradient that drives ATP synthase (complex V). When the electrochemical

potential difference generated by the proton gradient across the inner mitochondrial

membrane is high, the lifetime of superoxide-generating electron-transport

intermediates such as ubisemiquinone is prolonged. There seems to be a threshold

value above which superoxide production is markedly increased (Korshunov et al.,


34

1997). Thus hyperglycaemia increases the proton gradient above this threshold value

as a result of overproduction of electron donors by the TCA cycle. This, in turn,

causes a marked increase in the production of superoxide.

Figure 12: Production of superoxide by the mitochondrial electron-transport chain.

6. Glyceraldehyde Autoxidation

Glyceraldehyde 3-phosphate is a phosphorylation product formed from

glucose during anaerobic glycolysis. The partner product, dihydroxyacetone

phosphate, also contributes to intracellular glyceraldehyde concentrations via

enzymatic conversion by triose-phosphate isomerase. Thereafter, glyceraldehyde 3-

phosphate is oxidized by glyceraldehyde-phosphate dehydrogenase (GAPDH).

Continuance of glycolysis yields pyruvate, which enters the mitochondria where it is

oxidized to acetyl-CoA, and the processes of the tricarboxylic acid cycle and

oxidative phosphorylation begin. One alternative to this classic pathway of glucose

metabolism is the less familiar route of glyceraldehyde autoxidation (Figure 7,

pathway 1). The potential relevance of this pathway to diabetes mellitus was pointed

out by Wolff and Dean. (Wolff and Dean, 1987), who emphasized that autoxidation of

α-hydroxyaldehydes generates hydrogen peroxide (H2O2) and α-ketoaldehydes. In the

presence of redox active metals, H2O2 can form the highly toxic hydroxyl radical.

This pathway, therefore, forms two potentially toxic substances, α-ketoaldehydes,


35

which contribute to glycosylation-related protein chromophore development, and the

hydroxyl radical, a reactive oxygen species that can cause mutagenic alterations in

DNA.

PHARMACOLOGICAL INTERVENTIONS IN THE

TREATMENT OF DIABETES MELLITUS

Oral hypoglycaemic agents

There are five classes of oral pharmacological agents available to treat

diabetes: Sulfonylureas, Biguanides, Thiazolidinediones, α-glucosidase inhibitors,

meglitinides (Loh and Leow, 2002)

Sulfonylureas

These are derived from sulfonic acid and urea, have remained a keystone of

therapy for type 2 diabetes since 1950’s. Sulfonylureas are insulin secretogogues,

since they control blood glucose levels by stimulating insulin secretion in the

pancreatic β-cells. Insulin secretion is initiated when the drug binds to a cell surface

receptor associated with adenosine triphasphate (ATP)- sensitive potassium channels

on the pancreatic β-cells (Gerich, 1989; Lebovitz, 1990) leading to closure of the KATP

channels and opening of voltage-sensitive calcium channels which in turn causes

rapid influx of calcium and stimulates translocation of insulin-containing secretory

granules to the plasma membrane and the exocytotic release of insulin. Sulfonylureas

are generally well tolerated but the most common and also the most serious adverse

effect associated with these agents is hypoglycemia. (Gerich, 1989; Lebovitz, 1990)

Other side effects with sulfonylurea therapy include dermatological hypersensitivity,

gastrointestinal discomfort and weight gain. (Turner et al., 1996).

Dimethylbiguanide

These are derivative of metformin was introduced in the late 1950s for the

treatment of NIDDM. It is an extract of goat’s rue (Galega officinalis) which contains

guanidine, whose history as a treatment for diabetes can be traced to medieval times

(Bailey and Day, 1989). Metformin is considered as anti-hyperglycemic rather than a

hypoglycemic agent since it does not lower blood glucose level below normal.


36

Various mechanisms have been proposed which account for the anti-hyperglycemic

action of metformin like suppression of basal hepatic glucose production (Cusi and

DeFronzo, 1998), increase in peripheral glucose uptake (Widen and Groop, 1994) and

increase in non-oxidative glucose metabolism (e.g. glycogen formation in skeletal

muscle) but molecular target of metformin action still awaits identification.

Metformin does not stimulate insulin secretion instead it improves insulin sensitivity

and thus decreases the insulin resistance that is prevalent in NIDDM. (Cusi an

DeFronzo, 1998) When used as a monotherapy, it decreases HbA1C and also it does

not promote weight gain. However, the major concern with the biguanide therapy has

been the risk of lactic acidoisis (Sirtori and Pasik, 1994) and gastrointestinal

disturbances like abdominal discomfort and diarrhea, which occur in approximately

20-30% of patients. Biguanites are contraindicated in patients with renal impairment,

hepatic dysfunction and cardiac failure.

Thiazolidinedione

It is a novel drug class of insulin sensitizers (Goldstein, 2000) characterized

for their glucose and lipid-lowering activity. (Sohda et al., 1995) These drugs have

been shown to decrease insulin resistance by improving insulin sensitivity (Mudaliar

and Henry, 2001) without stimulating insulin secretion. TZDs are agonists for the

nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) (Schoonjans

and Auwerx, 2000), which improves insulin sensitivity by increasing the transcription

of insulin-sensitive genes involved in modulating the metabolism of glucose and

lipids. In addition, they control adipocytes differentiation, lower circulating

triglycerides and nonesterified fatty acids levels, increase glycogenesis and glycolysis

in muscle, stimulate glucose oxidation and lipogenesis in adipose tissue, and reduce

gluconeogenesis in liver (Hoffman et al., 1992). They are also reported to stimulate

the tyrosine kinase activity of insulin receptor (Iwamoto et al., 1991). But TZDs are

also associated with some major side effects like edema, weight gain, decreased

hemoglobin, and elevated alanine aminotransferase activity. Triglitazone of this class

was found to cause idiosyncratic hepatic reaction leading to hepatic failure and death

in some patients although pioglitazone and resiglitazone treatment have not shown

liver toxicity (Lebovitz et al., 2002; Chilcott et al., 2001). Mild to moderate


37

hypoglycemia is also reported during combination therapy with sulfonylureas or

insulin (Mudaliar and Henry, 2001).

α-glucosidase inhibitors

These are another class of oral anti-hyperglycemic agents that complitively

inhibit α-glucosidase enzymes found at the brush border of the small intestine

responsible for terminal carbohydrate digestion (Bischoff, 1995). Thus these drugs act

by delaying glucose digestion and absorption. They lower post-prandial blood glucose

level without risk of hypoglycemia and are also not associated with risk of weight

gain. Ascarbose, a nitrogen containing pseudotetrasaccharide, was the first α-

glucosidase inhibitor discovered. Acarbose therapy has also shown significant

reduction in glucose and insulin incremental as well as reduction in relative insulin

resistance (Josse et al., 2003). The major side effect of α-glucosidase inhibitors are

gastrointestinal disturbance including flatulence, diarrhoea, bloating and abdominal

discomfort. It is contradicted in patients with inflammatory bowel disease, cerrhosis

or elevated plasma creatinine. This class of drugs is also associated with dose-

dependent hepatotoxicity.

Meglitinides

These are a novel class of non-sulfonylurea insulin secretogogues

characterized by a very rapid onset and abbreviated duration of action. Repaglinide, a

benzoic acid derivative introduced in 1998, was the first member of the meglitinide

class. Neteglinide is a derivative of the amino acid D-phenylalanine and was

introduced to the market in 2001. Similar to sulfonylureas, meglitinides are insulin

secretogogues, since they control blood glucose levels by directly stimulating insulin

secretion in pancreatic β-cells. Nateglinide initiates a more rapid release of insulin

that is shorter in duration compared to repaglinide (Dunning and Foley, 2000), despite

having an in vivo pharmacokinetic profile that is similar (Weaver et al, 2001). Unlike

sulfonylureas, repaglinide does not stimulate insulin secretion in vitro in the absence

of glucose; rather, it enhances glucose stimulated insulin secretion especially at 180

mg/dl (or 10 nmol/l) glucose. The efficacy of meglitinides is similar to sulfonylurea

(Culy and Jarvis, 2001) indeed, netaglinide produced a more rapid post-parandial


38

increase in insulin secretion, and its duration of response is shorter than that of

glyburide (Hollander et al., 2001). Netaglinide is reported to be a highly effective

insulin secretagogue and anti-hyperglycemic agent when given prior to an oral

glucose load (Keilson et al., 2000). Although the drug is found to be safe and well

tolerated, the most common side effects are nausea, diarrhea, dizziness and

lightheadedness. The incidence of mild hypoglycemia is less with netaglinide than for

repaglinide. There is no report of severe hypoglycemia and body weight gain obtained

yet (Fuchtenbusch et al., 2000).

Insulin treatment in diabetes mellitus

The introduction of insulin to treat diabetes has saved an estimated 5 million

years of life for patients with type 1diabetes during the year 2000 (Owens et al.,

2001). Considerable progress has been made, in recent years, in the production,

formulation and delivery of insulin preparations, as well as the development of insulin

treatment regimens which maintains long-term-normoglycaemia, with a low risk of

hypoglycaemia (Boli, 1999). The importance of the aim of preventing or slowing the

progression of chronic microvascular complications has been conclusively proven

during the last decade, in both type 1 and type II diabetes. Unfortunately, patients

treated with insulin have uniformly poorer glycaemic control compared to those

treated with other therapies. It is an accepted fact that insulin is the most potent

glucose-lowering agent, with hypoglycaemia being the only major dose-limiting

factor. Unlike all oral agents that have limited maximum action, insulin has

progressively more side effects as the dose is increased (Bastaki, 2005).

Herbal treatment of diabetes

The Indian flora has a vast variety of medicinal plants, which are used

traditionally for their anti-diabetic property. However, careful assessment including

sustainability of such herbs, ecological and seasonal variation in activity of phyto-

constituents, metal contents of crude herbal anti-diabetic drugs, thorough toxicity

study and cost effectiveness is required for their popularity.

Literally hundreds of extracts of higher plants used for the management in

diabetes have been screened for their biological activity in both in vitro and in vivo


39

assays to validate the claimed therapeutic effects. The medicinal plants contain a

variety of active constituents that act on an array of targets by various modes and

mechanisms. There are a number of herbal formulations available in the market for

the treatment of diabetes. Gymnema sylvestre seems to be plant that is most

commonly used in most of these formulations (Pushpangadan et al., 2005). A review

of some of the commonly used medicinal plants with well documented anti-

hyperglycemic activity has been done.

Allium cepa

Linn. (Family: Liliaceae) Hindi name: Pyaj; Common name: Onion

Onion bulb and leaves are the important part of diet in Indian community. In a

clinical study treatment of diabetic patients with juice of Allium cepa bulb, controlled

the blood sugar level (Mathew and Augusti, 1975). Ether soluble fraction and

petroleum ether extract of onion has been observed to lower blood sugar level in

normal rabbits and exhibited potent antioxidant activity (Gupta et al., 1977). Dipropyl

disulphide oxide and onion oil produced significant hypoglycemic effect (Augusti,

1976). A sulphur containing amino acid product, S-methyl cystein sulphoxide (at a

dose of 200mg/kg for 45 days) isolated from onion showed potent hypoglycemic

activity in alloxan induced diabetic rats (Kumari et al., 1995; Sheela et al., 1995).

Prolonged administration of freeze dried onion powder (3%) with a diet produced

anti-hyperglycemic, hypolipidemic and antioxidant activities in STZ-diabetic rats

(Babu and Srinivasan, 1997). Onion callus cultures showed greater hypoglycemic

potential over natural onion bulb (Campos et al., 2003). Allium cepa juice (0.4g/100g

b.w. for 4 weeks) exhibited anti-hyperglycemic and antioxidant effects in alloxan

induced diabetic rats and it also repaired hepatic and renal damage caused due to

diabetes (El-Demerdash et al., 2005).

Allium sativum

Linn. (Family: Liliaceae) Hindi name: Lahsun; Common name: Garlic

Allium sativum is an important part of dietary ingredients. Allicin (0.25mg/kg,

orally) from garlic exhibited hypoglycemia in mild diabetic rabbits (Mathew and

Augusti, 1973). In alloxan induced diabetic rabbits ethanol, ethyl acetate and


40

petroleum ether extract (0.25mg/kg, orally) produced anti-hyperglycemic activity

(Jain and Vyas, 1975). Treatment of alloxan diabetic rats with the antioxidant s-allyl

cysteine sulfoxide isolated from garlic ameliorated the diabetic condition almost to

the same extent as did glibenclamide and insulin. It could also stimulate in vitro

insulin secretion from β-cells isolated from pancreas of normal rats (Augusti and

Sheela, 1996). Allium sativum bulb extract (500mg/kg/day) proved to be effective for

treatment of L-thyroxine (L-T4) induced hyperglycemia in rats (Tahiliani and Kar,

2003). A diet containing Allium sativum (12.5%) fed to alloxan induced diabetic rats

for 15 days resulted in reduced blood glucose level as compare to control group

(Jelodar et al., 2005). Herbal extract of garlic (20mg/100 g body weight, orally, daily

for 5 weeks) was reported to produce hypoglycemia, probably by interfering with

food intake of both normal and STZ-diabetic rats (Musabayane et al., 2006). Bis

(allixinato) oxovanadium (IV) from garlic was found to be a potent anti-diabetic agent

(Adachi et al., 2006).

Aloe vera

(Linn.) Burm. f. (Family: Liliaceae) Hindi and Common name: Aloe

Dry sap of plant produced prominent anti-hyperglycemic response in alloxan

induced diabetic Swiss albino mice (500mg/kg, twice daily for 5 days) (Ghannam et

al., 1986). Aloe vera leaf and pulp extract showed hypoglycemic activity in type 1 and

type 2 diabetic rats, the effect was more pronounced in type 2 diabetes as compared

with glibenclamide (Okyar et al., 2001). Ethanolic extract of Aloe vera gel (200 and

300mg/kg b.w., orally) produced hypoglycemic activity along with controlled

carbohydrate metabolizing enzymes in normal fasted, oral glucose fed and STZ-

diabetic rats (Rajsekaran et al., 2004). Oral administration of ethanolic extract

(300mg/kg b.w.) and gel extract (300mg/kg b.w. per day for 21 days) to STZ-diabetic

rats resulted in a significant reduction of fasting blood glucose and improved plasma

insulin level (Rajsekaran et al., 2005; Rajsekaran et al., 2006).


41

Azadirachta indica

A. Juss. (Family: Meliaceae) Hindi name: Neem; Common name: Indian lilac tree

Aqueous extract of Azadirachta indica is known to produce anti-

hyperglycemic and hypoglycemic activities in diabetic dogs (Satyanarayan et al.,

1978). Fresh leaves decoction possessed anti-hyperglycemic activity and increased the

peripheral glucose utilization in normal rats (Chattopadhyay et al., 1987). Crude

ethanol extract of leaves (250mg/kg, for 2 weeks) potentially lowered the blood sugar

level of alloxan induced diabetic rats (Kar et al., 2003). Petroleum ether extract of

seed kernel (2gm/kg b.w.) & seed husk (0.9gm/kg b.w.) has been reported to reduce

oxidative stress in heart tissue and erythrocytes of STZ induced diabetic rats (Gupta et

al., 2004). Beta-sitosterol, a steroid obtained from Azadirachta indica, has been

reported to possess hypoglycemic property (Mukherjee et al., 2006).

Gymnema sylvestre

(Willd) R. Br. (Family: Ascelpiadaceae) Hindi name: Gudmar; Common name:

Periploca of the wood

Plants are grown in tropical regions of India and used as household remedy for

diabetes (Kar et al., 2003). Oral administration of a water soluble fraction G-54

isolated from Gymnema sylvestre administered to 27 type 2 diabetic patients reduced

their insulin requirement, lowered the fasting blood sugar and glycosylated

haemoglobin content. Two water soluble fractions (GS-3 and GS-4) obtained from

leaves were found to double the pancreatic islets and β-cell numbers in diabetic rats

(Shanmugasundaram et al., 1990). Alcoholic leaf extract (500mg/kg, orally) lowered

maximum blood sugar in fasted, glucose fed and diabetic rats along with insulin

released from pancreatic β-cells (Chatopadhyay et al., 1993). Gymnemic acid IV,

isolated from leaves produced potent hypoglycemic effect in STZ-diabetic mice

(Sugihara et al., 2000). A polyherbal formulation containing aqueous extracts of

Gymnema sylvestre produced prominent hypoglycemic activity in normal and diabetic

rats at a dose of 100-500mg/kg/day, orally for acute (6 hours) and for long-term (6

weeks) studies (Mutalik et al., 2005). Gymnemic acid IV isolated from the leaves has


42

been observed to produce anti-hyperglycemic, glucose uptake inhibitory and gut

glycosidase inhibitory effects (Kimura, 2006).

Momordica charantia

Linn. (Family: Cucurbitaceae) Hindi name: Karela; Common name: Bitter gourd

The plant is an annual climber grown mostly in tropical India and commonly

used as vegetable (Saxena et al., 2006). Charantin (50mg/kg, orally) isolated from

Momordica charantia resembled insulin activity and lowered blood sugar levels

(maximum 42% after 4 hours of administration) in rabbits (Lolitkar and Rao, 1966).

Charantin obtained from Momordica charantia caused hypoglycemic effect,

stimulated the insulin release and inhibited gluconeogenesis (Ng et al., 1986).

Hypoglycemic effect and delayed cataract development was reported in alloxan

diabetic rats treated with fruit extract (4g/kg/Day orally for 2 months) (Srivastava et

al., 1988). Ethanolic extract (200mg/kg) of Momordica charantia showed

hypoglycemic activity in normal and streptozotocin diabetic rats; this was explained

to be possibly due to inhibition of glucose-6-phosphatase and fructose-1,6-

biphosphatase in liver, and stimulating hepatic glucose-6- phosphate dehydrogenase

activities (Shibib et al., 1993). Fruit aqueous extract (200mg/kg, orally for 6 weeks),

and exercise potentially lowered blood sugar of type 2 diabetic and hyperinsulinemic

(insulin resistance) rats (Miura et al., 2004). Seed aqueous extract produced

prominent reduction in blood glucose, glycosylated hemoglobin, lactate

dehydrogenase, glucose-6-phosphatase, fructose-1,6- bisphosphatase and glycogen

phosphorylase along with increased hemoglobin, glycogen content and hexokinase,

glycogen synthase activity (Sekar et al., 2005). Plant constituents such as charantin,

vicine and polypeptide-p have been reported to have anti diabetic potential and may

be included as a part of dietary supplement for patients of diabetes (Krawinkel and

Keding, 2006). The plant extract of Momordica charantia has been shown to possess

anti-oxidant and anti-hyperglycemic properties and exerts beneficial effects against

diabetes and associated free radical complications in heart tissue (Tripathi and

Chandra, 2009b). The antihyperglycemic and antioxidant properties of M. charantia

have been found to be comparable with metformin, a standard hypoglycemic drug

(Tripathi and Chandra, 2009a). M. charantia is not only useful in controlling the


43

blood glucose levels, but also has antioxidant potential to protect vital organs such as

heart and kidney against damage caused due to diabetes induced oxidative stress

(Tripathi and Chandra, 2010).

Ocimum sanctum

Linn. (Family: Lamiaceae) Hindi name: Tulasi; Common name: Holy Basil

It is a tropical annual herb grown all over India and use for household

remediation (Mukherjee et al., 2006). Oral administration of alcoholic extract of

leaves of Ocimum sanctum lowered blood sugar level in normal; glucose fed

hyperglycemic and STZ-diabetic rats, along with increased insulin release

(Chattopadhyay, 1993). Ocimum sanctum leaf powder was reported to produce potent

hypoglycemic and hypolipidemic effects in normal and diabetic rats (Rai et al., 1997).

Alcoholic extract of leaves significantly lowered the blood glucose in normal and

alloxan diabetic rats (Vats et al., 2002). Administration of leaves extract 200mg/kg in

STZ-diabetic rats for 30 days led to decreased plasma glucose level by 26.4% (Vats et

al., 2004a).

Pterocarpus marsupium

Roxb. (Family: Fabaceae) Hindi name: Vijayasar; Common name: Indian Malabar

Plants grow throughout India and are use as hypoglycemic plant in folklore

medicine. Aqueous bark extract lowered blood sugar and improved glucose tolerance

of diabetics with no side effects observed (Pandey and Sharma, 1976). From alcoholic

extract of bark the ethyl acetate soluble fraction caused blood sugar lowering effect

and repaired the alloxan induced pancreatic β-cells damage in albino rats

(Chakroborty et al., 1980). (-)-Epicatechin isolated from plant at a dose of 30mg/kg,

i.p. produced anti-hyperglycemic effect in alloxan induced diabetic rats (Sheehan et

al., 1983). The (-) epicatechin from bark increased the cAMP content of the

pancreatic islets associated with increased insulin release, conversion of proinsulin to

insulin and cathepsin B activity in rats (Ahmad et al., 1991). Marsupin and

pterostilbene two phenolic constituents of plant potentially lowered blood glucose at

same level as compared to metformin in STZ-diabetic rats (Manickam et al., 1997).

Aqueous extract (1g/kg, orally) of bark has been observed to produce anti-cataract


44

activity in alloxan diabetic rats (Vats et al., 2004b). Plant extract was prevented the

hyper-triglyceridaemia and hyper-insulinaemia (insulin resistance) in type 2 diabetic

patients (Grover et al., 2005). Aqueous extract (250mg/kg, orally) of dried wood has

been reportd to produce hypoglycemic effect in acute and sub-acute study (Mukhtar et

al., 2005). Butanol subfraction of the alcohol extract of P.marsupium exhibited

significant antidiabetic activity and corrected the metabolic alterations in diabetic rats

(Dhanabal et al., 2006). Aqueous extract of P.marsupium Roxb bark was found to

have ameliorative effect on diabetes associated metabolic alterations (Gayathri and

Kannabiran, 2008). Oral administration of P.marsupium had the ability to improve

streptozotocin-induced chronic diabetic stress (Gupta and Gupta, 2009). Pterocarpus

marsupium extract reveals strong in vitro antioxidant activity and may serve as a

potential source of natural antioxidant for treatment of diabetes (Mohammadi et al.,

2009). The ethanol extract of P.marsupium has been shown to possess significant

antidiabetic, antihyperlipidaemic and antioxidant effects in alloxan induced diabetic

rats. (Maruthupandian and Mohan, 2011). Therapy with methanolic extract of

Pterocarpus marsupium Roxb and Ocimum sanctum Linn has been shown to reverse

dyslipidemia and oxidative stress in alloxan induced type I diabetic rat model (Singh

et al., 2012).

Syzygium cumini

Linn. (Family: Myrtaceae) Syn. Eugenia jambolana (Linn.); Hindi name: Jamun;

Common name: Black Berry

Oral administration of fruit pulp induced hypoglycemic activity in normal and

STZ-diabetic rats along with insulin released from β-cells (Achrekar et al., 1991).

Seed powder provided good symptomatic relief to 30 patients of diabetes (type 2) and

regulated blood sugar levels (Kohli and Singh, 1993). Oral administration of aqueous

seed extract (2.5g/kg, b.w. for one month) to alloxan diabetic rats produced blood

sugar lowering effect which was explained due to increased activity of hexokinase

and decreased activity of glucose-6-phosphatase in liver (Prince et al., 1997).

Alcoholic seed extract (20mg, i.p.) reduced the blood sugar level to 37.17% at 3 hour

and 46.68% at 6 hour of administration in alloxan diabetic mice along with enhanced

insulin secretion (Purohit and Daradka, 2000). Decreased plasma glucose


45

concentrations in STZ-induced diabetic mice was observed at oral administration of

fruit extract (200mg/kg, for 50 days) (Grover et al., 2002). Blood sugar lowering,

hypolipidemic activity, increased serum insulin, increased glycogen content of liver

and muscles and a fall in glycosylated haemoglobin level produced by ethanolic

extract (100mg/kg b.w. orally) of seed (Sharma et al., 2003). Ethanolic seed kernels

extract (100gm/kg b.w.) has been observed to improve glucose tolerance (Ravi et al.,

2004), and produced hypoglycemic and hypolipidemic effects (Ravi et al., 2005) in

STZ-diabetic rats. Aqueous and ethanolic extracts of the fruit-pulp has been reported

to produce antihyperglycemic effect in alloxan diabetic rats, and 24.4% raise in

plasma insulin level in mild diabetic and 26.3% in severely diabetic rabbits (Sharma

et al., 2006). S.cumini seed kernel extracts were found to be potent inhibitors of α-

glucosidase, which might be a possible mechanism for its anti-diabetic activity.

(Shinde et al., 2008). Antioxidant and antiproliferative activities of

anthocyanin/ellagitannin-enriched extracts from S.cumini have also been evaluated

(Aqil et al., 2012).

Trigonella foenum-graecum

Linn. (Family: Fabaceae) Hindi name: Methi; Common name: Fenugreek

Major alkaloid trigonelline from fenugreek seeds produced hypoglycemic

activity (Shani et al., 1974). Ethanol extract (0.8g/kg, i.p.) of leaves has been

observed to reduce blood glucose concentration in alloxan induced diabetic rats.

Lethal doses (LD50) of aqueous leaf extract were 1.9g/kg at intra-peritoneal and

10g/kg at oral dose (Abdel Barry et al., 1997). 4-Hydroxyisoleucine, an insulinotropic

compound isolated from seeds increased the insulin release in glucose fed

hyperglycemic rats (Sauvaire et al., 1998). Seed powder treatment normalized the

enhanced lipid peroxidation and reduced the susceptibility to oxidative stress

associated with depletion of antioxidants in liver of diabetic rats (Anuradha and

Ravikumar, 2001). Maximum 46.64% decrease in blood sugar level of diabetic rats

was observed at oral administration of seed extract (1g/kg, for one month) (Vats et al.,

2003). From fenugreek seeds, the soluble dietary fibre (SDF) fraction at (0.5g/kg,

orally administered twice daily, for 28 days) inhibited platelets aggregation in type 2

diabetic rats and produced beneficial effect in dyslipidemia (Hannan et al., 2003).


46

Restored activity of glutamate dehydrogenase, NAD linked isocitrate dehydrogenase

and D-b-hydroxybutyrate dehydrogenase reported in alloxan induced diabetic rats

upon oral administration of seed powder (5%, for 3weeks). 4-hydroxyisoleucine:5, an

amino acid, isolated from seeds, produced anti-hyperglycemic effect and decreased

the 33% plasma triglyceride, 22% total cholesterol (22%) and 14% free fatty acids

(Narender et al., 2006). The plant extract of Trigonella foenum graecum has been

shown to possess anti-oxidant and anti-hyperglycemic properties and exerts beneficial

effects against diabetes and associated free radical complications in heart tissue

(Tripathi and Chandra, 2009b). Trigonella foenum graecum is not only useful in

controlling the blood glucose levels, but also has antioxidant potential to protect vital

organs such as heart and kidney against damage caused due to diabetes induced

oxidative stress (Tripathi and Chandra, 2010).

IMMUNOMODULATION

Immunomodulation is the adjustment of the immune response to a desired

level, as in immunopotentiation, immunosuppression, or induction of immunologic

tolerance. Immunomodulators are substances that have been shown to modify the

immune system. These are biological or synthetic substances that can stimulate,

suppress or modulate any aspect of the immune system including both adaptive and

innate arms of the immune system.

Classification of immunomodulators

Clinically, immunomodulators can be classified into the following three

categories:

Immunoadjuvants

These are used to enhance the efficacy of vaccines and therefore could be

considered specific immune stimulants. Immunoadjuvants hold the promise of being

the true modulators of the immune response. It has been proposed that they be

exploited as selectors between cellular and humoral helper T1 (Th1) and helper T2

cells (Th2), immunoprotective, immunodestructive, and reagenic [immunoglobulin E

(IgE)] versus IgG type immune responses-posing a real challenge to vaccine designers

(Billiau and Matthys, 2001).


47

Immunostimulants

These are inherently non-specific as they are envisaged as enhancements to a

body’s resistance to infection. They can act through innate as well as adaptive

immune responses. In healthy individuals, the immunostimulants are expected to

serve as prophylactic and promoter agents, i.e., as immunopotentiators, by enhancing

the basic level of immune response. In the individual with impairment of immune

response, they are expected to act as immunotherapeutic agents (Agarwal and Singh,

1999).

Immunosuppressants

These are a structurally and functionally heterogeneous group of drugs, which

are often concomitantly administered in combination regimens to treat various types

of organ transplant rejection and autoimmune diseases (Agarwal and Singh, 1999).

IMMUNOMODULATION IN DIABETES

Diabetes is widely believed to predispose to serious infections. For example,

tuberculosis was a major cause of death among patients with diabetes mellitus before

the advent of insulin therapy (Johnson, 1970). Thus, depression of the natural

defenses against infection in diabetics has long been suspected and investigated. This

altered susceptibility to infection has been ascribed to a depression in the function of

polymorphonuclear leukocytes (Badgdade et al., 1974). Impairment of chemotaxis or

mobilization of polymorphonuclear leukocytes has also been described (Mowat and

Baum, 1971). With respect to immunological mechanisms, the results are conflicting

(Johnson,1970). Bates and Weiss reported that children with poorly controlled

diabetes showed a diminished production of staphylococcal antibody (Bates and

Weiss, 1941). Similarly, low antibody titers against typhoid vaccine were observed in

certain diabetic patients (Richardson, 1933). Depressed reactivity of diabetic human

lymphocytes to phytohemagglutinin stimulation has also been reported (MacCuish et

al, 1974). Mahmoud et al. demonstrated depressed cellular immunological reactivity

against murine Schistosoma mansoni and allograft skin rejection in experimentally

induced diabetic mice (Mahmoud et al, 1976).


48

Immunomodifying effects of diabetogenic drugs such as streptozotocin (STZ)

or alloxan have also been observed, which occur promptly after drug administration,

suggesting the possibility that the acute diabetic state may, in addition to its chronic

effects, rapidly lead to a degree of immunosuppression. However, the current view is

that immunosuppression post-diabetogenic drug administration is a result of direct

immunosuppressive properties of the drugs rather than a consequence of the diabetes

they induce (Cattan et al., 2003; Pericin et al., 2002). This conclusion has appeared

sound because it is based on observations from a number of approaches. For example,

STZ is toxic to lymphocytes in vitro (Gaulton et al., 1985); insulin injections only

have a partial impact on STZ-induced immunosuppression (Nichols et al., 1979); and

islet transplant rejection in NOD mice that are already diabetic can be blocked by

injection of STZ (Takayama et al., 1993; Koulmanda et al., 2003). However, each of

these approaches has considerable limitations as compounds that are toxic in vitro are

not necessarily toxic in vivo; insulin injections do not fully reverse the diabetic state;

and STZ affects presentation of autoantigens targeted by NOD T cells. Furthermore,

increased rates of infection occur in diabetic patients and reduced immunity has been

noted in animal models of chronic diabetes (Moutschen et al., 1992; Joshi et al.,

1999). Hyperglycaemia is a common finding in critically ill patients and intensive

insulin therapy for such patients reduces infections and mortality (van den Berghe et

al., 2001). A link between hyperglycaemia and suppressed adaptive responses has

received little attention; reduced innate rather than adaptive immunity was speculated

to be the basis for increased infections in these hyperglycaemic patients (van den

Berghe et al., 2001).

The mechanisms linking diabetes and immunosuppression are not well

defined. One potential mediator of the altered defence mechanisms is hyperglycaemia.

It has been identified as the main factor contributing to the development of diseases

associated with diabetes mellitus. The immune response in diabetes and the direct

effect of hyperglycaemia on T and B lymphocyte reactivity were analysed. Diabetes

induced an early decrease in IgG levels in the secondary response. However, both

primary responses against a T-cell dependent or independent antigen were affected

after 6 months of diabetes induction. Pre-incubation of lymph node and spleen cells in

a high glucose (HG) containing medium led to a significant time- and dose-dependent


49

decrease in T- and B-cell proliferation. Still viable cells after HG exposition were able

to improve their proliferative response when cultured with the mitogen in a fresh

standard medium. HG diminished cell viability, increased apoptosis and induced

oxidative stress in lymphocytes. These results indicate that HG concentrations can

directly affect lymphoid cell growth. An increase in oxidative stress would be

implicated in this deleterious effect. Prolonged exposure to pathologically HG

concentrations result in the immunosuppressive state observed in diabetes (Rubinstein

et al., 2008).

IMMUNOMODULATORS

Different synthetic immunomodulatory agents have been reported in literature.

These agents can be classified as immunosuppressants and immunostimulants (Saroj

et al. 2012).

Immunosuppressant drugs

These drugs have major role in organ transplantation and auto immune

diseases.

1. Calcineurin inhibitors (Specific T-cell inhibitors): Cyclosporine

(Ciclosporin) is a lipophillic cyclic polypeptide composed of 11 amino acids.

It is a calcineurin inhibitor and selectively inhibits T lymphocyte proliferation,

IL2 and other cytokine production and responce to inducer T-cells.

Lymphocytes are arrested in G0 to G1 phase. Tacrolimus is

an immunosuppressive drug that is mainly used after allogeneic organ

transplant to reduce the activity of the patient's immune system and so lower

the risk of organ rejection. It is also used in a topical preparation in the

treatment of atopic dermatitis(eczema), severe refractory uveitis after bone

marrow transplants, exacerbations of minimal change disease, and the skin

condition vitiligo.

2. Antiproliferative drugs (Cytotoxic drugs): Azathioprine is a purine

antimetabolite. Its selective uptake into immune cells and intracellular

conversion to the active metabolite 6-mercaptopurine, inhibits de novo purine


50

synthesis and causes DNA damage. It is approved for prevention of renal and

other graft rejection. The drug cyclophosphamide has more effect on B cells

and humoral immunity compared to that on T cells and cell- mediated

immunity. It is used in bone marrow transplant. Other drugs of this category

are Methotrexate, Chlorambucil, Mycophenolate mofetil (MMF).

3. Glucocorticoids: Prednisolone is a steroid used to suppress acute rejection

of solid allograft and in chronic graft versus host disease. The steroids are able

to rapidly reduce lymphocyte populations by lysis or redistribution. On

entering cells, they bind to the glucocorticoid receptor and the complex passes

into the nucleus and regulates the translation of DNA.

4. Antibodies: Muromonab CD3 is a murine monoclonal antibody against the

CD3 glcoprotein located near to the T cell receptor on helper T cells. It is used

for treatment of acute rejection of renal allografts as well as cardiac and

hepatic transplantation. Antithymocyte globin (ATG) is a polyclonal antibody

purified from horse or rats immunized with human thymice lymphocytes. It

binds to T-lympocytes and depletes them. It is a potent immunosuppressant

used for suppress acute allograft reject episodes. Other drugs of this category

are Rho (D) immuneglobin, Efalizumab.

Immunostimulant drugs

They stimulate the immune system to fight against immunodeficiencies (like

AIDS), infections and cancers. Examples of these drugs are levamisole which is an

antihelmintic drug that also restores functions of B lymphocytes, T lymphocytes,

monocytes and macrophages. Hence it has been used in colon cancer along with 5-

Fluorouracil. Thalidomide has been utilized in conditions such as, erythema nodosum

leprosum (anti-inflammatory effect), multiple myeloma (anti-angiogenesis) and

rheumatoid arthritis (anti TNF effect). BCG is used in bladder carcinoma.

Recombinant cytokines such as interferons a have been used in tumors and chronic

hepatitis B and C. Interleukin 2 (aldeslukin) has been used in renal cell carcinoma and

melanoma


51


activity. Most of them are cytotoxic and exerts a variety of side effects. This has given


immunomodulatory activity.

IMMUNOMODULATORY POTENTIAL OF PLANTS

According to the World Health Organization (WHO), about three-quarters of

the world population relies upon traditional remedies (mainly herbs) for the health

care of its people. Various plants identified in the Indian Ayurvedic system of

medicine display a wealth of pharmacological properties. The ayurvedic system of

medicine is one of the oldest systems of medicine and includes various

ethnopharmacological activities such as immunostimulation, tonic, neurostimulation,

anti-ageing, antibacterial, antiviral, antirheumatic, anticancer, adaptogenic,

etc.(Agarwal and Singh, 1999) An entire section of the Materia Medica of Ayurveda

is devoted to “Rasayana”, drugs reputed to enhance body resistance (Gulati et al.,

2002). Listed as a class in the texts of traditional Indian Medicine literature, Rasayana

consists of a number of plants reputed to promote physical and mental health, improve

defense mechanisms of the body and enhance longevity. These attributes are similar

to the modern concept of adaptogenic agents, which are known to afford protection of

the human physiological system against diverse stressors (Bhattacharya et al., 2000).

A number of medicinal plants as Rasayanas have been claimed to possess

immunomodulatory activity, e.g., Withania somnifera, Tinospora cordifolia, and

Mangifera indica (Davis and Kuttan, 2000; Singh et al., 2004; Makare et al., 2001).

They can depress or potentiate the host’s capacity to resist infection and tumors non-

specifically, or react specifically to a foreign substance. Immunomodulators act on the

complex network of mechanisms of the immune system in a way not yet fully

elucidated.

Fruits of Emblica officinalis (family: Euphorbiaceae) and whole plant of

Evolvulus alsinoides (family: Convolvulaceae) has been extensively used in Indian

Ayurvedic medicine for varieties of medical disorders. The immunomodulatory

properties of Emblica offýcinalis and Evolvulus alsinoides were evaluated in adjuvant

induced arthritic rat model. The crude aqueous extracts of both the herbs were


52

administered intraperitonially following a repeated treatment profile. There was a

significant reduction in swelling and redness of inflamed areas in treated animals than

in untreated controls. The anti-inflammatory response of both extracts was determined

by lymphocyte proliferation activity and histopathological severity of synovial

hyperplasia. Both extracts showed a marked reduction in inflammation and edema. At

cellular level, immunosuppression occurred during the early phase of the disease.

There was mild synovial hyperplasia and infiltration of few mononuclear cells in

treated animals. The induction of nitric oxide synthase was significantly decreased in

treated animals as compared to controls. These observations suggest that both the

herbal extracts caused immunosuppression. Both are as potent as dexamethasone, a

traditionally used immunosuppressant for arthritis (Ganju et al., 2003).

Mehrota described in vitro immunosuppressive potential of ethanolic extract

of Acorus calamus rhizome. Ethanolic extract of A. calamus inhibited proliferation of

mitogen (phytohaemagglutinin) and antigen (purified protein stimulated human

peripheral blood mononuclear cells (PBMCs). In addition, A. calamus extract

inhibited growth of several cell lines of mouse and human origin. It also inhibited

production of nitric oxide (NO), interleukin-2 (IL-2) and tumor necrosis factor-α

(TNF-α). Intracytoplasmic interferon-γ (IFN-γ) and expression of cell surface

markers, CD16 and HLA-DR, on human PBMC, were not affected on treatment with

A. calamus extract but CD25 expression was down regulated (Mehrotra et al., 2003).

Crude extract of Tinospora cordifolia contained a polyclonal B cell mitogen

which enhanced immune response in mice. An arabinogalactan polysaccharide, G1-

4A from the stem of Tinospora cordifolia examined to modulate induced

immunosuppression. Mice pre-treated with G1-4A exhibited protection against

lipopolysaccharide (LPS) induced mortality (Desai et al., 2007).

Partially purified immunomodulator, G1-4A prevented lipid peroxidation and

restored the activities of superoxide dismutase and catalase enzymes. Likewise,

oxidative damage, induced by peroxynitrite, was also inhibited by partially purified

immunomodulator similar to selective inhibitors of reactive oxygen species (ROS)

like mannitol, superoxide dismutase, sodium azide and antioxidants, GSH and vitamin

C (Kumar et al., 2012).. In further studies, intraperitoneal administration of alcoholic


53

extract of Tinospora cordifolia in Dalton's lymphoma bearing mice not only

augmented the basic function of macrophages such as phagocytosis, but also their

antigen presenting ability and secretion of IL-1, TNF and RNI. It was also indicated

that the extract slow down the tumor growth and increases the life span of tumor

bearing host, thus showing its anti tumor effect through destabilizing the membrane

integrity of Dalton's lymphoma cells directly or indirectly. Thus, the study

demonstrated alcoholic extract of Tinospora cordifolia activated tumor associated

macrophages and showed antitumor effect on the spontaneous T-cell lymphoma and

may have some clinical implications (Singh et al., 2004).

Ethanolic extract of Boerhaavia diffusa, a plant used in Indian traditional

system of medicine, significantly inhibited the cell proliferation (Mungantiwar et al.,

1999). Extracts of B. diffusa roots inhibited human NK cell cytotoxicity in vitro,

production of nitric oxide in mouse macrophage cells, interleukin-2 and tumor

necrosis factor-α (TNF-α), in human PBMCs. Whereas, intracytoplasmic interferon-γ

(IFN-γ) and cell surface markers such as CD16, CD25, and HLA-DR did not get

affected on treatment with B. diffusa extract and demonstrates immunosuppressive

potential of B. diffusa (Mehrotra et al., 2002).

Aqueous leaves extract of biopesticidal plant Nyctanthes arbor-tristis has been

found as a potent immunomodulator (Puri et al., 1994). The extract has been

evaluated as immunorestorative or anti-immunosuppressive agent in the malathion

exposed immunosuppressed mice by studying various immunological parameters

(humoral, cell mediated immune, numerical values of immunocytes and functions of

phagocytes) in treated or untreated malathion-exposed mice. The results revealed that

the immunological parameters which were suppressed with malathion either reverted

back to normal or showed a trend towards normalcy, when treated with aqueous

leaves extract of Nyctanthes arbor-tristis (Puri et al., 1994).

Methanol extract of Eclipta alba and Centella asiatica whole plant showed

phagocytic index and antibody titer has been increased significantly. The F ratios of

the phagocytic index and WBC count were also significant with linearity in the dose-

response relationship (Jayathirtha and Mishra, 2004). The ethanol extract of the root


54

of the plant Cryptolepis buchanani caused significant stimulation of the delayed type

hypersensitivity reaction and humoral antibody production in mice (Kaul et al., 2003).

An aqueous extract of Rhodiola imbricata rhizome stimulated production of

interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in human PBMCs as well as

RAW 264.7 cell line. It also increased production of nitric oxide synergistically in

combination with lipopolysaccharide in RAW 264.7. Furthermore, it increased the

phosphorylated-IκB expression and activated the nuclear translocation of NF-κB in

human PBMCs. Thus, Rhodiola most likely activated proinflammatory mediators via

phosphorylated inhibitory κB and transcription factor NF-κB (Mishra et al., 2006).

PHARMACOLOGY OF IMMUNOMODULATORY ACTIVITIES

FROM PUTATIVE MEDICINAL PLANTS

It has been reported that the “Rasayanas” are rejuvenators, nutritional

supplements and possess strong antioxidant activities. They also exert antagonistic

action on oxidative stressors, giving rise to the formation of different free radicals.

They are used mainly to combat the effects of ageing, atherosclerosis, cancer,

diabetes, rheumatoid arthritis, autoimmune disease and Parkinson’s disease. The

Rasayana herbs seem to operate through immunostimulant, immunoadjuvant, and

immunosuppressant activities or by affecting the effector arm of the immune response

(Chulet and Pradhan, 2010) Mechanisms of immunomodulation activity occur mainly

via phagocytosis stimulation, macrophages activation, immunostimulatory effect on

peritoneal macrophages, lymphoid cells stimulation, cellular immune function

enhancement and nonspecific cellular immune system effect, antigen-specific

immunoglobulin production increase, increased nonspecific immunity mediators and

natural killer cell numbers, reducing chemotherapy-induced leukopenia, and

increasing circulating total white cell counts and interleukin-2 levels (Vaghasiya et

al., 2010; Malik et al., 2009).

A class of herbal medicines alter the activity of immune function through the

dynamic regulation of informational molecules such as cytokines. This may offer an

explanation of the effects of herbs on the immune system and other tissues (Spelman

et al., 2006). Cytokines, a large group of soluble extracellular proteins or


55

glycoproteins, are key intercellular regulators and mobilizers of cells engaged in

immune responses. Classified into family groups (e.g., interleukins, interferons, and

chemokines) based on the structural homologies of their receptors. They are now seen

to be crucial to innate and adaptive inflammatory responses, cell growth and

differentiation, cell death, angiogenesis, and developmental as well as repair

processes aimed at the restoration of homeostasis (Oppenheim, 2001). Their secretion,

by virtually every nucleated cell type, is usually an inducible response to injurious

stimuli (Oppenheim, 2001). In addition, cytokines provide a link between organ

systems, providing molecular cues for maintaining physiological stability (O’Sullivan

et al., 1998).The various cytokines involved in the immune system are shown in the

figure 13.

Figure 13: The various cytokines involved in immune response.

Modulation of the immune responses through the stimulatory or suppressive

activity of a phyto-extract may help maintain a disease-free state in normal or

unhealthy people. Agents that activate host defense mechanisms in the presence of an

impaired immune response can provide supportive therapy to conventional

chemotherapy (Kumar et al., 2012). A high degree of cell proliferation renders bone

marrow a sensitive target, especially to various cytotoxic drugs. In fact, bone marrow

is the organ most affected during any immunosuppression therapy with this class of

drugs. Loss of stem cells and the inability of the bone marrow to regenerate new


56

blood cells results in thrombocytopenia and leucopenia (Bafna and Mishra, 2006)

Saponins are either triterpenoid or steroidal glycosides proven to be essential

phytoconstituents with various pharmacological activities, such as antiallergic,

antiphlogostic, cytotoxic antitumour, antiviral, immunomodulating, antihepatotoxic,

molluscicidal, and antifungal activity. Recently, three diosgenyl saponins isolated

from Paris polyphylla have been reported to have immunostimulant properties (Xiu-

feng et al., 2007). Lymphocyte stimulation tests were performed on eight cycloartane-

type saponins isolated from Astragalus melanophrurius (Calis et al., 1997) to

determine the role of saponins in the immunomodulating effect of the plant. Higher

concentrations of tested compounds have exhibited inhibitory effects.

Cycloartane and oleanane-type triterpenes from these species have

unmistakably induced interleukin-2 activity (Erdem et al., 2005). Immunomodulatory

activities of terpenoid compounds such as glycyrrhizinic acid, ursolic acid, oleanolic

acid, and nomilin have been reported (Raphael and Kuttan, 2003). A novel

triterpenoid has been isolated from the root bark of Ailanthus excelsa Roxb. (Tree of

Heaven), AECHL-1, and has potential as an anticancer agent (Kumar et al., 2010).

Many studies have reported the identification of immunomodulatory

compounds with pharmacological activity and a limited toxicity. In this context,

ethnopharmacology represents the most important way possible to uncover interesting

and therapeutically helpful molecules. The phytochemical analysis of Rasayana plants

has revealed a large number of compounds including tannic acid, flavonoids,

tocopherol, curcumin, ascorbate, carotenoids, polyphenols, etc., which have been

shown to have potent immunomodulatory properties. The herbal mixture preparations

of Indian traditional medicine may stimulate immunomodulation due to their content

of plants with immunomodulatory properties that probably act synergistically. This

hypothesis along with the lack of toxicity can be important to understand their use in

the past as well as currently. Some medicinal plants may stimulate the immune

system, (e.g., Panax ginseng, Ocimum sanctum, Tinospora cordifolia, and Terminalia

arjuna), and some may suppress the immune response (Alternanthera tenella). Also,

various secondary metabolites (e.g., alkaloids, glycosides, saponins, flavonoids,

coumarins, and sterols) exhibit a wide range of immunomodulating activity.

Materials & Methods

57

Materials and Methods

Chemicals: Alloxan monohydrate, 5,5’-Dithio-bis 2-nitrobenzeoic acid (DTNB), 1-chloro-

2,4 dinitrobenzene (CDNB), thiobarbituric acid (TBA), Epinephrine, metformin,

bovine serum albumin, glacial metaphosphoric acid, glutathione, DPPH, ATP, ADP,

NADH, NAD+, pyruvate, PEP, fructose 6-phosphate, glucose 6-phosphate, HRP

labelled rabbit anti-rat secondary antibodies, TMB, H2O2, PFK, PK, HK, LDH,

G6PD, adjuvants (FCA and FIA) and LPS were purchased from Sigma chemical

company Inc., St Louis, Mo, USA. 1,1,3,3 tetraethoxypropane (TEP), aspartate,

alanine α-ketoglutarate, AMP (2-amino 2- methyl 1- propanol), creatinine, glucose,

diacetyl-monoxime, thiosemicarbazide, 2,4-dinitrophenylhydrazine (DNPH), EDTA,

ascorbic acid, potassium dichromate, Coomassie Brilliant Blue G-250 were obtained

from SRL (India), ethanol, HPLC grade methanol, ethyl acetate, benzene, HPLC

grade acetic acid, butanol, pyridine, Folin ciocalataeu’s reagent were purchased from

Qualigens fine chemicals (India). All other chemicals used were of analytical grade.

Animals and their management: Male albino wistar rats weighing 150 to 200g were purchased from Central

Drug Research Institute (CDRI), Lucknow, India, for study and housed at 22±5°C in

the animal room in the department. They were provided a standard pellet diet

(Hindustan Lever Ltd, Mumbai, India) and had free access to water. Prior permission

for animal use and approval of the protocol were obtained from the Institutional

Animal Ethical Committee.

Plant materials: Syzygium cumini (SC) seed powder and Pterocarpus marsupium (PM) bark

powder were purchased from a local market in Lucknow.

Preparation of extracts:

Aqueous extract of SC seed powder

100 g of commercially available seed powder was suspended in 350 ml DDW

and magnetically stirred overnight. The contents were filtered through four layers of

sterile muslin cloth. The residue was re-extracted with 200ml DDW and again


58

filtrated. The filtrates were pooled and residue was discarded. The pooled filtrates

were centrifuged at 5000 rpm for 5 min and the supernatant was collected (400 ml).

This was concentrated by rotavapour (Buchi, R-210, Germany) at 50°C and reduced

pressure, to one-tenth volume (40 ml). The aqueous extract prepared was stored at

4ºC.

Ethanolic extract of SC seed powder

100g of commercially available seed powder was suspended in 300ml of 95%

ethanol, magnetically stirred overnight and filtered through four layers of muslin. The

residue was re-extracted in 200ml of 95% ethanol and filtered. The filtrates were

pooled and centrifuged at 5000 rpm for 5min. The supernatant obtained (400ml) was

concentrated by rotavapour (Buchi, R-210, Germany) at 50°C and reduced pressure to

20 ml. It was further lyophilized and the yield was 2.83%. The lyophilized powder

was stored at -20ºC and re-suspended in water whenever required for dosing.

Purification of ethanolic SC seed extract

200g of commercially available seed powder was suspended in 500 ml DDW,

magnetically stirred overnight, filtered through four layers of muslin and the filtrate

was discarded. The residue was re-suspended and extracted in 400ml of 80% ethanol,

magnetically stirred for 48 hrs and filtered through four layers of muslin. The residue

was re-extracted with 300ml of 95% ethanol for 48 hrs and filtered. The filtrates were

pooled (residue discarded) and centrifuged at 5000 rpm for 10 min. The supernatant

collected (550ml) was concentrated by rotavapour (Buchi, R-210, Germany) at 50°C

and reduced pressure to 35ml. The ethanolic extract prepared was stored at 4ºC.

10ml of this concentrated ethanolic extract (showing better anti-hyperglycemic

activity) was subjected to purification via silica gel (60-120 mesh) chromatography.

The gel was swollen in hexane and fractions were batch eluted with 100% methanol.

The fractions were pooled and lyophilized to 1.33g of powder, which was further

suspended in 10ml of DDW.

This was further purified by sephadex LH 20 beads. The matrix was swollen

in water and 5ml of the above fraction was loaded. Batch elution was carried out with

different ratios of water and methanol (100% water, 70:30, 30:70, 100% methanol),


59

thus four fractions were collected. The four fractions were lyophilized and re-

suspended in DDW. These were tested for their anti-hyperglycemic and anti-oxidative

activities in vivo and in vitro.

HPLC analysis of fraction IV and standard caffeic acid

Fraction IV (lyophilized and re-suspended in water) was 5 times diluted and

subjected to HPLC analysis using C18 column (4.6 x 250 mm, Waters). Gradient

elution was carried out using acetic acid and methanol as mobile phase for 30 min.

The column was eluted with acetic acid and methanol. A gradient of these two was

formed; initially the column was equilibrated with acetic acid and sample was loaded.

The concentration of methanol was increased gradually, 1-10% for 20 min, 10-50%

for next 3 min, 50-100 % for next 3 min. After this the column was again equilibrated

with acetic acid (100%) for next 4 min Standard caffeic acid (100µg/ml) was run

using the same method.

Aqueous extract of PM bark powder

5gm of PM bark powder was suspended in 50ml DDW and boiled for 15 min.

It was then filtered through four layers of muslin and final volume was made upto

50ml. The aqueous extract prepared was stored at 4ºC.

Alcoholic extract of PM bark powder

10g PM bark powder was extracted in 50ml 95% ethanol. It was then filtered

and lyophilized. The lyophilized powder was stored at -20ºC and re-suspended in

water whenever required for dosing.

Purification of PM bark extract

100g of PM bark powder was extracted with 250ml of hot ethyl acetate for 24

hrs after initial de-fatting with n-hexane. The contents were filtered through four

layers of muslin and centrifuged at 5000rpm for 10min. The supernatant (150ml) was

collected and concentrated by rotavapour (Buchi, R-210, Germany) at 50°C and

reduced pressure to 15ml. It was further lyophilized and re-suspended in 6ml of 100%

methanol.


60

4ml of the above methanolic suspension was further chromatographed on

silica gel (60-120 mesh) swollen in hexane. Fractions were batch eluted with benzene

and pooled. The pooled fractions were further lyophilized to 0.22g powder which was

re-suspended in 95% ethanol and stored at -20ºC.

Alloxan induced diabetic rat model:

Diabetic was induced in rats by a single injection of alloxan at a dose 150

mg/kg body weight. Alloxan was dissolved in 0.15 M sterile normal saline and

injected through intraperitoneal route. Diabetes was confirmed by the determination

of fasting blood glucose level (<270 mg/dl).

Body weight determination and blood collection

The body weight of rats was determined prior to blood collection on a

weighing machine. The blood glucose sample was taken by piercing the tail vein.

Blood Glucose

Fasting blood glucose (FBG) level was estimated by one touch ultra

glucometer (Accu check). FBG levels were measured on the fifth day (considered as

day zero) after administration of alloxan and monitored on fifteen days interval (0,15th

and 30th day).

Serum preparation and tissue collection

Rats were fasted overnight and sacrificed by cervical dislocation. Rats were

dissected and blood was taken from the heart and kept at room temperature for 30

min, for serum separation. Blood was centrifuged at 5000 rpm for 10 min and serum

was collected as supernatant and stored at -20oC. Different body tissues (heart, liver

and kidney) were dissected out, washed with sterile normal saline and stored at -20°C.

Preparation of homogenate

10% (w/v) homogenate was prepared using Potter Elvehjem type homogenizer

in ice-cold 50mM phosphate buffer, pH 7.4 containing mammalian protease inhibitor

cocktail from Sigma chemicals (containing AEBSF, pepstatin A, E-64, bestatin,

leupeptin and aprotinin cocktail). The homogenate was centrifuged at 10,000 rpm for


61

30 min at 4oC. The supernatant was used for the assay of antioxidant activities/levels

and lipid peroxidation.

Measurement of lipid peroxidation

Lipid peroxidation was estimated in terms of MDA (malondialdehyde)

formed, according to the method of Ohkawa, et al. (1979) using thiobarbituric acid

(TBA) reagent. To 0.2 ml of the homogenate, added 0.2 ml sodium dodecyl sulphate

[8.1% (w/v)], 1.5 ml glacial acetic acid (20%, pH 3.5) and 1.5 ml thiobarbituric acid

[0.8% (w/v)]. The final volume was made upto 4.0 ml. The contents of the tubes were

vortexed vigorously, heated in a water bath at 90°C for 1 hr and then immediately

cooled under running tap water. To each tube, 1.0 ml of water and 5.0 ml of a mixture

of n-butanol and pyridine (15:1, v/v) were added and the tubes were vortexed and

centrifuged at 800xg for 20 min. The upper layer was aspirated out to measure the

color intensity at 532 nm. The reference used was 1, 1, 3, 3 tetraethoxypropane (TEP).

MDA contents were expressed in nM/gram tissue.

In the thiobarbituric acid (TBA) test reaction one molecule of

malonaldialdehyde reacts with two molecules of TBA with the production of pink

color having an absorption maximum at 530-535 nm.

Superoxide dismutase activity

Superoxide dismutase (SOD) activity was assayed by the method of Misra and

Fridovich, (1972). 3.0 ml reaction mixture contains of 1.5 ml of 0.1 M carbonate-

bicarbonate buffer, pH 10.3; 0.1 ml of 30 mM EDTA, suitable aliquot of enzyme

preparation and water to make up the volume to 2.94 ml. The reaction was started by

addition of 0.06 ml of 15 mM epinephrine. Change in absorbance was recorded at 480


62

nm for one min at 15 sec interval. Control consisting of all the ingredients, except

enzyme preparation, was run simultaneously. One unit of enzyme activity has been

defined as the amount of enzyme required to cause 50% inhibition of auto-oxidation

of epinephrine under experimental condition. Specific activity has been expressed as

the unit of enzyme activity per mg protein.

The auto-oxidation of epinephrine in solution at pH 10.3 produces O2-. O2

-

once formed participated in the oxidation of further molecules of epinephrine in a

chain reaction to give rise to adrenochrome. Adrenochrome exhibits an absorption

maximum at 480 nm. The addition of SOD greatly slows down the rate of oxidation

of epinephrine because of simultaneous and rapid utilization of O2- by the enzyme.

The rate of oxidation of epinephrine to adrenochrome and its inhibition by SOD is

determined by the change in absorbance at 480 nm.

Catalase activity

The activity of catalase was determined by the method of Sinha (1972).

Catalase was assayed colorimetrically at 570 nm. The reaction mixture in a total

volume 1.6 ml contains 1.0 ml of 0.01 M, phosphate buffer, pH 7.0, 0.1 ml of tissue

homogenate-supernatant and 0.5 ml of 2.0 M H2O2. The reaction was stopped by the

addition of 2.0 ml dichromate acetic acid reagent (5% potassium dichromate and

glacial acetic acid mixed in a 1:3 ratio). One unit of enzyme activity has been defined

as the amount of enzyme required to decomposed µmoles H2O2 per min under

experimental condition. Specific activity has been expressed as the unit of enzyme

activity per mg protein.

The dichromate / acetic acid reagent can be thought of as a "stop bath" for

catalase activity. As soon as the enzyme reaction mixture hits the acetic acid, its

activity is destroyed; any hydrogen peroxide which hasn't been split by the catalase

will react with the dichromate to give a blue precipitate of perchromic acid. This

unstable precipitate is then decomposed by heating to give the green solution.

Glutathione S-transferase activity

The activity of GST was determined by the method of Habig at al. (1974).

GST was determined using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The


63

assay mixture contained 1mM GSH, 1mM CDNB and 100mM phosphate buffer, pH

6.5. The reaction was started by the addition of enzyme in linearity range, the rate of

increase in absorbance due to formation of CDNB conjugate of GSH was monitored

at 340 nm for 3 mins. One unit of enzyme activity has been defined as the amount of

enzyme required to form GSH-CDNB conjugate per min. Specific activity has been

expressed as the unit of enzyme activity per mg protein.

Reduced glutathione content

GSH content was determined by the method of Chandra et al. (2002b). 0.2 ml

homogenate were mixed in 0.3 ml precipitating regent (0.2 M glacial meta-phosphoric

acid, 5.1 M NaCl and 5.9 mM EDTA). After centrifugation at 10,000xg for 15

minutes, 0.2 ml supernatant was added to 0.8 ml 0.3 M Na2HPO4, followed by the

addition of 0.1 ml 5,5 dithiobis (2- nitrobenzoic acid); DTNB; 0.04% in 1% sodium

citrate). GSH reduces DTNB to form the colored product 2-dinitro-5-thiobenzoic acid,

absorbance of which is measured at 412 nm.

Protein estimation

Protein was estimated by the method of Lowry et al. (1951) using Folin phenol

reagent. 0.05 ml of homogenate was taken and made up the volume to 1 ml with

distilled water; 5.0 ml of freshly prepared copper reagent was added and mixed

immediately and left for 10 min. at room temperature. 0.5 ml of Folin and Ciocalteau

(1:2 in TDW) reagent was then added and mixed well. The absorbance was taken

after 30 min. at 660 nm. Bovine serum albumin was used as standard.

Protein was also estimated by the method of Bradford, 1976. 0.05 ml of

sample was taken and volume was made upto 1 ml with distilled water; 5.0 ml of

Bradford reagent (contains Coomassie Brilliant Blue G-250, ethanol and

orthophosphoric acid) was added and vortexed. The dye, Coomasie Blue G binds to

the protein and shifts the absorption maximum of the dye from red to blue. The

absorbance was measured at 595 nm. Bovine serum albumin was used as standard.


64

Alanine transaminase and aspartate transaminase estimation

The principle reaction of the colorimetric determination of AST or ALT

activity is based on the reaction of aspartate or alanine with α-ketoglutarate to form

oxaloacetate or pyruvate, respectively. The oxaloacetate or pyruvate formed is

measured by monitoring the concentration of oxaloacetate or pyruvate hydrazone

formed with 2,4-dinitrophenylhydrazine (DNPH). Absorbance was measured at 546

nm (Godkar, 1994). 0.1 ml serum was added to 0.5 ml of substrate and incubated for

30 min at 37°C. 0.5 ml of DNPH was added and the reaction mixture was mixed

thoroughly and kept at room temperature for 30 min. 5 ml of 0.4 N NaOH was added,

mixed and kept at room temperature for 10 min. Absorbance was measured at 540

nm.

Alkaline phosphatase estimation

Paranitrophenyl phosphate is a colorless compound. The enzyme splits off the

phosphate group from it to form P-nitrophenol, which is also colorless in the acidic

solution. Under alkaline condition it is converted to P-nitrophenoxide ions, which

exhibits yellow color. The intensity of yellow color is directly proportional to the

enzyme present in the sample and can be measured at 405 nm. 0.2 ml of substrate was

added to 2.7 ml of AMP (2-amino 2- methyl 1- propanol) buffer, pH 10.3 and

incubated for 5 min at 37°C. 0.1 ml of serum was added and incubated at 37°C for 15

min. 3 ml of 0.25 N NaOH was added and absorbance was measured at 540 nm

(Godkar, 1994).

Creatinine estimation

Creatinine reacts with picric acid in alkaline medium to form a reddish yellow

complex intensity of which is directly proportional to the concentration of creatinine

in the specimen and can be measured at 520 nm. 1.0 ml serum, 3.0 ml of distilled

water, 0.5 ml of 0.66 N sulphuric acid and 0.5 ml of sodium tungstate were mixed.

The contents were centrifuged at 5000 rpm for 15 min. 3.0 ml distilled water was

added to 2.0 ml of the supernatant followed by 1.0 ml of alkaline picrate reagent.

Reaction mixture was mixed and kept at room temperature for 20 min. Absorbance

was measured at 520 nm. Creatinine was used as standard (Godkar, 1994).


65

Urea estimation

Urea reacts with diacetyl-monoxime in hot acidic medium and in the presence

of thiosemicarbazide and ferric ions forms a pink coloured compound which can be

measured. 0.05 ml of serum was added to 5.0 ml of working reagent consisting of

diacetyle momoxine, thisemicarbazide and acid reagent (which contains sulphuric

acid, othrophosphoric acid and ferric chloride). Contents were mixed thoroughly and

tubes were placed on boiling water bath for 15 min. Tubes were cooled immediately

and absorbance was measured at 520 nm after 5 min (Godkar, 1994).

DPPH radical scavenging assay

This assay was done by the method of Gulluce et al. (2006). The hydrogen

atom or electron donation ability of the extracts was measured from the bleaching of

purple colored methanol solution of DPPH. This spectrophotometric assay uses stable

radical diphenylpicrylhydrazyl (DPPH) as a reagent. 50µL of extracts were added to 5

ml of a 0.004% methanol solution of DPPH. After a 30 min incubation period at room

temperature absorbance was read against a blank at 517 nm. Synthetic antioxidant,

butylated hydroxytoluene (BHT) was used as positive control and all tests were

carried out in triplicate.

Serum insulin estimation

The insulin concentration in test serum was measured using ultra sensitive rat

insulin ELISA kit from Crystal Chem, Incorporation, Downers Grove, IL 60515 USA

(catalog no. 90060). 5µl test sample (in total volume of 100µl) was added per well in

the ELISA plate pre-coated with anti-rat insulin antibody. Plate was incubated for 2

hrs at 4ºC and then washed extensively. 100µl of anti-insulin HRP conjugated

antibody (used against a distinct epitope on rat insulin) was added to each well. Plate

was incubated at room temperature for 30min and then washed extensively. 100µl of

substrate solution [tetramethylbenzidene (TMB) – H202] was added to each well and

incubated for 40 min. Reaction was stopped by adding 2N HCL and absorbance was

measured at 450nm. Insulin concentration was calculated using the standard curve.


66

Hexokinase activity

Hexokinase (HK) activity was estimated by the method of Porter and Chassy

(1982) with suitable modifications. The assay is based upon reduction of NAD+

through a coupled reaction with glucose-6-phosphate dehydrogenase (G6PD) and is

determined spectrophotometrically by measuring the increase in absorbance at 340

nm.

D-glucose + ATP HK Glucose-6-phosphate + ADP

Glucose-6-phosphate + NAD+ G6PD Gluconate-6-phosphate + NADH+ H+

One unit of enzyme activity reduces 1µmole of NAD+ per min at room

temperature at pH 8.0 under experimental conditions. 3.0 ml reaction mixture contains

2.28 ml of 50 mM tris-HCL buffer pH 8.0 containing 13.3 mM MgCl2, 0.5 ml of 0.67

M glucose, 0.10 ml 16.5 mM ATP, 0.1 ml of 6.8 mM NAD+ and 0.01 ml (3I.U.) of

G6PD. At zero time add 0.1 ml of approximately diluted hexokinase source.

Phosphofructokinase activity

PFK activity was monitored by the method of Storey (1982) with suitable

modifications. PFK catalyzes formation of fructose 1,6-bisphosphate. In the assay

system formation of fructose 1,6-bisphosphate is coupled to the oxidation of NADH

using pyruvate kinase and lactate dehydrogenase with decrease in absorbance at 340

nm monitored spectrophotometrically.

Fructose-6-phosphate + ATP PFK Fructose 1,6-bisphosphate + ADP

ADP + PEP PK ATP + Pyruvate

Pyruvate + NADH + H+ LDH Lactate + NAD+

One unit of enzyme activity is defined as the amount of PFK that forms 1µmol

of fructose 1,6-bisphosphate per min at room temperature under experimental

conditions. 3.0 ml of reaction mixture contains 2.733 ml of 100 mM tris-HCl buffer

pH 9.0, 30µl of 100mM ATP solution, 40 µl of 56 mM PEP solution, 60 µl of 13.1

mM NADH, 60 µl of 500mM fructose-6-phosphate, 6 µl of 2.5 M KCl, 60 µl of 100


67

mM MgSO4, 6 µl (12U) pyruvate kinase, 5 µl (5U) LDH and 10 µl of appropriately

diluted enzyme (homogenate).

Pyruvate Kinase activity

Pyruvate kinase activity was measured by the method of Imamura and Tanaka

(1982) with suitable modifications. PK catalyzes the formation of pyruvate from PEP.

The spectrophotometric assay involves formation of pyruvate which is coupled to

oxidation of NADH by LDH.

The reaction mixture (3.0 ml) contains 0.80 ml of 50 mM tris-HCl buffer pH

7.5, 0.1 ml of 17 mM PEP, 0.25 ml of 1.3 mM NADH, 0.2 ml of 100 mM MgSO4, 0.1

ml of 44 mM ADP, 2 µl (10U) LDH and finally 0.1 ml of appropriately diluted

enzyme (homogenate) is added and decrease in absorbance was monitored at 340 nm

for 3 min.

One unit of enzyme activity will be defined as the amount that catalyzes the

formation of 1 µmol of pyruvate or oxidizing 1 µmol of NADH in the coupled system

in 1 min under assay conditions.

PEP + ADP PK Pyruvate + ATP

Pyruvate + NADH + H+ LDH L- Lactate + NAD+

Antigen preparation and immunization

Antigenic doses of E.coli lipopolysaccharide (LPS) and tetanus toxoid (TT) were

100µg/rat. LPS was prepared for immunization by mixing equal volume of LPS and

Freund′s adjuvant. Rats were immunized with 0.1 ml of antigenic preparation

intraperitoneally. Each immunization was done after 15 days interval. Antigenic

preparations for 4 consequent immunizations were:

1. First injection was given in Freund′s Complete adjuvant (FCA). (0.5ml FCA +

0.5ml LPS)

2. Second injection was given in Freund′s Complete adjuvant (FCA). (0.5ml

FCA + 0.5ml LPS).


68

3. Third injection was given in Freund′s Complete adjuvant (FCA) and Freund′s

Incomplete Adjuvant (FIA). (0.25ml FCA + 0.25ml FIA+ 0.5ml LPS)

4. Fourth injection was given in Freund′s Incomplete Adjuvant (FIA). (0.5ml

FIA + 0.5ml LPS)

The adjuvant and antigen were mixed properly to form a water in oil emulsion

before injecting.

TT injection were procured from Serum Institute of India Ltd., Pune, and

protein content was estimated. Four immunizations were done at an interval of 15

days.

Blood sample collection

Seven days after each immunization (LPS and TT) blood samples were

collected from rat’s eye. Blood samples were kept at room temperature for 30 minutes

for serum separation and then centrifuged at 5000 rpm for 10 minutes. Serum was

collected and stored at -20�C.

Serum dilution

Sera collected after all four immunizations were serially diluted with 1% BSA

in PBS for ELISA testing.

Testing of antibody titer

Sera of rats were tested for the presence of IgM and IgG antibodies by ELISA.

Levels of IL-4 were also tested for all groups of rats.

Protocol for ELISA to detect IgG and IgM:

Coating Antigen to microtiter plate

The antigens (TT and LPS) were diluted to a final concentration of 1µg/100µl

in phosphate buffer saline (PBS; 50mM pH=7.4) and wells were coated with 100µl

antigen preparation i.e. 1µg antigen. The plate was covered and incubated overnight at

4�C. The coating solution was removed and the plate was washed 4 times with PBS.


69

Blocking

The remaining protein-binding sites in the coated wells were blocked by

adding 400µl blocking solution (1% BSA in PBS) per well. The plate was covered

and incubated for four hours at 4°C. The plate was washed 4 times with PBS.

Incubation with the primary and secondary antibodies

100µl of primary antibody (appropriately diluted rat serum) was added to the

well. The plate was covered and incubated for 2½ hrs at room temperature. The plate

was washed four times with PBS. The 100µl of appropriately diluted secondary

antibodies (anti-rat IgG or IgM, HRP labelled) were added.

Detection

100µl of the substrate solution [Tetramethylbenzidine (TMB) + hydrogen

peroxide] was added. Then 100µl of stop solution (10N H2SO4) was added to the

wells. The absorbance of each well was recorded at 450nm.

Detection of IL-2 levels:

The levels of IL-2 in normal, diabetic and treated rat serum were estimated

using Invitrogen Rat IL-2 ELISA kit. The invitrogen rat IL-2 kit is supplied with rat

IL-2 antibodies coated onto the wells of the ELISA plate. The estimation was

performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-2

solution at appropriate dilutions, 50 µl of unknown serum were added and incubated

for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl of

biotinylated rat anti IL-2 antibody was added to each well and incubated for 2 hrs at

room temperature. After that the plate was washed thoroughly and 50 µl of

streptavidin-HRP conjugate was added to each well. The plate was incubated for 1 hr

at room temperature. After washing, the chromogen (50 µl) solution supplied with the

kit was added to each well and incubated for 20-30 min followed by addition of stop

solution (50 µl). The blue colour turned to yellow which was read at 450nm. Standard

curve was plot and concentration of IL-2 was calculated.


70






solution at appropriate dilutions, 50 µl of unknown serum were added and incubated














soltion at appropriate dilutions, 50 µl of unknown serum were added and incubated










71





performed as per manufacturer’s protocol. Briefly, to each well 50 µl of standard IL-

10 solution at appropriate dilutions, 50 µl of unknown serum were added and

incubated for 2 hrs at room temperature. The plate was washed thoroughly and 50 µl

of biotinylated rat anti IL-10 antibody was added to each well and incubated for 2 hrs

at room temperature. After that the plate was washed thoroughly and 50 µl of




solution (50 µl). The blue color turned to yellow which was read at 450nm. Standard


Objectives

72

OBJECTIVES

1) To evaluate anti-hyperglycemic activity of Syzygium cumini & Pterocarpus

marsupium.

2) To evaluate the effect of Syzygium cumini & Pterocarpus marsupium extracts

on activities of key glycolytic enzymes.

3) To isolate and identify the active component(s) present in the plant extracts of

Syzygium cumini & Pterocarpus marsupium having anti diabetic potential.

4) To evaluate the role of these plant extracts in management of diabetes

associated oxidative stress.

5) To evaluate the immunomodulatory effect of these plant extracts.

Chapter-I

Anti-diabetic and anti-oxidative potentials of crude Syzygium cumini and

Pterocarpus marsupium extracts

Chapter 1: Anti-diabetic and anti-oxidative potentials of crude SC and PM extracts

73

Diabetes mellitus is a common and very prevalent disease affecting the

citizens of both developed and developing countries. It is estimated that 25% of the

world population is affected by this disease. Diabetes mellitus is caused by the

abnormality of carbohydrate metabolism which is linked to low blood insulin level or

insensitivity of target organs to insulin (Maiti et al., 2004). Despite considerable

progress in the treatment of diabetes by oral hypoglycemic agents, search for newer

drugs continues because the existing synthetic drugs have several limitations. The

herbal drugs with anti-diabetic activity are yet to be commercially formulated as

modern medicines, even though they have been acclaimed for their therapeutic

properties in the traditional systems of medicine (Chan et al., 2012). The plants

provide a potential source of anti-hyperglycemic drugs because many plants and plant

derived compounds have been used in the treatment of diabetes. Many Indian plants

have been investigated for their beneficial use in different types of diabetes. Ayurveda

and other traditional medicinal system for the treatment of diabetes describe a number

of plants used as herbal drugs. Hence, they play an important role as alternative

medicine due to less side effects and low cost. The active principles present in

medicinal plants have been reported to stimulate pancreatic beta cells re-generation

and insulin release (Kavishankar et al., 2011). Hyperglycemia is involved in the

etiology of development of diabetic complications. Anti-hyperglycemic herbs

increase insulin secretion, enhance glucose uptake by adipose or muscle tissues and

inhibit glucose absorption from intestine and glucose production from liver (Hui et

al., 2009). Insulin and oral hypoglycemic agents like sulphonylureas and biguanides

are still the major players in the management but there is quest for the development of

more effective anti-diabetic agents.

Oxidative stress plays a pivotal role in the development of diabetes

complications. Oxidative stress and oxidative damage to the tissues are common end

points of chronic diseases, such as atherosclerosis, diabetes and rheumatoid arthritis

(Baynes and Thorpe, 1999). Oxidative stress is currently suggested as mechanism

underlying diabetes and diabetic complications (Kangralkar et al., 2010). During

diabetes, persistent hyperglycemia causes increased production of free radicals,

especially reactive oxygen species (ROS), in all tissues due to glucose auto-oxidation


74

and protein glycosylation. The increase in the level of ROS in diabetes could be due

to their increased production and/ or decreased destruction by nonenzymic

antioxidants, eg. reduced glutathione (GSH) and enzymic antioxidants like catalase

(CAT), glutathione S-transferase (GST), , and superoxide dismutase (SOD). The level

of these antioxidant enzymes critically influences the susceptibility of various tissues

to oxidative stress and is associated with the development of complications in diabetes

(Lipinski, 2001).

EXPERIMENTAL DESIGN

1- To evaluate anti-hyperglycemic effect of aqueous and alcoholic extracts of

SC seed powder at different doses.

Rats were divided into following groups, each group contains five rats

Group I Normal control

Group II Normal + aq. SC treated (1.5 g/kg b.w./day)

Group III Normal + aq. SC treated (3 g/kg b.w./day)

Group IV Normal + aq. SC treated (5 g/kg b.w./day)

Group V Normal + alc. SC treated (50 mg/kg b.w./day)

Group VI Normal + alc. SC treated (100 mg/kg b.w./day)

Group VII Normal + alc. SC treated (200 mg/kg b.w./day)

Group VIII Diabetic control

Group IX Diabetic + aq. SC treated (1.5 g/kg b.w./day)

Group X Diabetic + aq. SC treated (3 g/kg b.w./day)

Group XI Diabetic + aq. SC treated (5 g/kg b.w./day)

Group XII Diabetic + alc. SC treated (50 mg/kg b.w./day)

Group XIII Diabetic + alc. SC treated (100 mg/kg b.w./day)

Group XIV Diabetic + alc. SC treated (200 mg/kg b.w./day)

Group XV Diabetic + Metformin (100 mg/kg b.w./day)


75

2- To evaluate anti-hyperglycemic effect of aqueous and alcoholic extracts of

PM bark powder at different doses.



Group II Normal + aq. PM treated (100 mg/kg b.w./day)

Group III Normal + aq. PM treated (200 mg/kg b.w./day)

Group IV Normal + aq. PM treated (400 mg/kg b.w./day)

Group V Normal + alc. PM treated (150 mg/kg b.w./day)

Group VI Normal + alc. PM treated (300 mg/kg b.w./day)

Group VII Normal + alc. PM treated (500 mg/kg b.w./day)

Group VIII Diabetic control

Group IX Diabetic + aq. PM treated (100 mg/kg b.w./day)

Group X Diabetic + aq. PM treated (200 mg/kg b.w./day)

Group XI Diabetic + aq. PM treated (400 mg/kg b.w./day)

Group XII Diabetic + alc. PM treated (150 mg/kg b.w./day)

Group XIII Diabetic + alc. PM treated (300 mg/kg b.w./day)

Group XIV Diabetic + alc. PM treated (500 mg/kg b.w./day)

Group XV Diabetic + Metformin (100 mg/kg body b.w./day)

3- Duration dependent effect of alcoholic extract of SC seed and aqueous

extract of PM bark on fasting blood glucose levels.



Group II Normal + alc. SC treated (100 mg/kg b.w./day)

Group III Normal + aq. PM treated (200 mg/kg b.w./day)

Group IV Diabetic control


76

Group V Diabetic + alc. SC treated (100 mg/kg b.w./day)

Group VI Diabetic + aq. PM treated (200 mg/kg b.w./day)

Group VII Diabetic + Metformin (100 mg/kg b.w./day)

4- Effect of aqueous and alcoholic extracts of SC seed on body weight.



Group II Diabetic

Group III Normal + aq. SC treated (3 g/kg b.w./day)

Group VI Normal + alc. SC treated (100mg/kg b.w./day)

Group V Diabetic + aq. SC treated (3 g/kg b.w./day)

Group VI Diabetic + alc. SC treated (100mg/kg b.w./day)


5- Effect of aqueous and alcoholic extracts of PM bark on body weight.



Group II Diabetic

Group III Normal + aq. PM treated (200mg/kg b.w./day)

Group IV Normal + alc. PM treated (300mg/kg b.w./day)

Group V Diabetic + aq. PM treated (200mg/kg b.w./day)

Group VI Diabetic + alc. PM treated (300mg/kg b.w./day)

Group VIII Diabetic +Metformin (100 mg/kg b.w./day)

6- Evaluation of toxic effects of SC seed alcoholic extract and PM bark

aqueous extract doses in rats after 2 month exposure.




77

Group II Normal + alc.SC treated (3 g/kg b.w./day)

Group III Normal + alc.SC treated (15 g/kg b.w./day)

Group IV Normal + aq. PM treated (200 mg/kg b.w./day)

Group V Normal + aq. PM treated (1 g/kg b.w./day)

7- To evaluate anti-oxidative effect of aqueous and alcoholic extract of SC in

alloxan induced diabetic rats.



Group II Normal + aq. SC treated (3 g/kg b.w./day)

Group III Normal + alc. SC treated (100 mg/kg b.w./day)


Group V Diabetic + aq. SC treated (3 g/kg b.w./day)

Group VI Diabetic + alc. SC treated (100 mg/kg b.w./day)


Group VIII Diabetic + Vitamin C (150 mg/kg b.w./day)

8- To evaluate anti-oxidative effect of aqueous and alcoholic extract of PM

in alloxan induced diabetic rats.



Group II Normal + aq. PM treated (200 mg/kg b.w./day)

Group III Normal + alc. PM treated (300 mg/kg b.w./day)


Group V Diabetic + aq. PM treated (200 mg/kg b.w./day)

Group VI Diabetic + alc. PM treated (300 mg/kg b.w./day)



78


RESULTS

A- To evaluate the anti-hyperglycemic effect of aqueous and alcoholic

extracts of Syzygium cumini at different doses.

Experiments were conducted to evaluate anti-hyperglycemic effect of aqueous

and alcoholic extracts of SC seeds. Rats were divided into 15 groups according to the

experimental design. The rats were fed with 3 different doses of aq. SC extract 1.5,

3.0 and 5.0 g/kg body weight/day and 3 different doses of alc. SC extract 50, 100 and

200 mg/kg body weight/day. An intraperitonial dose (150 mg/kg body weight) of

alloxan increased FBG levels in groups VIII-XV after 4-5 days of injection. FBG

levels were monitored on day 0 (when rats were confirmed for diabetes) and day 30

(end of experiments). In diabetic control group (VIII), higher FBG level (>270 mg/dl)

was maintained throughout the period of study. On the other hand the oral dose of aq.

extract of SC resulted in significant decrease in FBG levels in diabetic rats. The doses

1.5, 3.0 and 5.0 g/kg body weight/day resulted in decrease in FBG levels from

445.9±18.7 to 317.1±14.4 mg/dl, 460.4±23.6 to 160.6±6.7 mg/dl and 476.8±21.9 to

158.1±4.9 mg/dl respectively, in 30 days treatment (Table 1). Better effects were

observed when diabetic rats were administered orally different doses of alc. SC seed

extracts viz. 50, 100 and 200 mg/kg body weight/day. The FBG levels were decreased

from 455.9±12.9 to 300.8±18.3 mg/dl, 427.6±18.8 to 100.0±10.1 mg/dl and

437.7±19.2 to 103.8±9.8 mg/dl, respectively, in 30 days treatment (Table 1). Normal

rats treated with different doses of aq. and alc. extract of SC seeds did not show any

significant changes in FBG levels as compared to normal control (group I). The 3.0

and 5.0 g/kg body weight doses of aq. SC extract and 100 and 200mg /kg body weight

doses of alc. SC extract showed significant decrease in FBG levels which was better

than metformin (group XV), standard anti-diabetic agent (Table 1).

B- To evaluate the anti-hyperglycemic effect of aqueous and alcoholic

extracts of Pterocarpus marsupium at different doses.

Experiments were conducted to evaluate anti-hyperglycemic effect of aqueous


79

and alcoholic extracts of PM bark. Rats were divided into 15 groups according to the

experimental design. The rats were fed with 3 different doses of aq. PM extract 100,

200 and 400 mg/kg body weight/day and 3 different doses of alc. PM extract 150, 300

and 500 mg/kg body weight/day. An intraperitonial dose (150 mg/kg body weight) of

alloxan increased FBG levels in groups VIII-XV after 4-5 days of injection. FBG

levels were monitored on day 0 (when rats were confirmed for diabetes) and day 30

(end of experiments). In diabetic control group (VIII), higher FBG level (>270 mg/dl)

was maintained throughout the period of study. On the other hand the oral dose of aq.

extract of PM resulted in significant decrease in FBG levels in diabetic rats. The doses

100, 200 and 400 mg/kg body weight/day resulted in decrease in FBG levels from

403.1±16.2 to 335.6±7.7 mg/dl, 373.4±13.6 to 175.6±4.7 mg/dl and 388.3±16.1 to

170.5±5.2 mg/dl respectively, in 30 days treatment (Table 2). Treatment of diabetic

rats with different doses of alc. PM bark extracts viz. 150, 300 and 500 mg/kg body

weight/day decreased the FBG levels from 398.5±13.8 to 350.8±6.1 mg/dl,

355.5±10.8 to 300.3±6.1 mg/dl and 378.9±13.8 to 310.3±7.3 mg/dl, respectively, in

30 days treatment (Table 2). Normal rats treated with different doses of aq. and alc.

extract of PM bark did not show any significant changes in FBG levels as compared

to normal control (group I). The 200 and 400 mg/kg body weight doses of aq. PM

extract showed significant decrease in FBG levels which was better than metformin

(group XV), standard anti-diabetic agent (Table 2).

C- Duration dependent effect of alcoholic extract of SC seed and aqueous

extract of PM bark on fasting blood glucose levels.

The rats were divided into 7 groups according to experimental design. FBG

levels were monitored at day 0 (when rats were confirmed for diabetes), 15th and day

30th. The FBG levels of normal rats groups (group I-III) remained unchanged during

experimental period. Treatment of diabetic rats with alcoholic SC extract (group V)

showed decrease in FBG levels from 427.6±18.8 to 297.6±11.5 and 100.0±10.1 mg/dl

on the 15th and 30th day, respectively. Diabetic rats treated with aqueous PM extract

(group VI) showed decrease in FBG levels from 373.4±13.6 to 223.5±12.4 and

175.6±4.7 mg/dl on the 15th and 30th day, respectively. The anti-hyperglycemic effect


80

shown by alc. SC seed extract and aq. PM bark extract was better than metformin

(group VII) (Table 3).

D- Effect of Syzygium cumini and Pterocarpus marsupium extracts (aq. and

alc.) on body weight.

The change in body weight in the rats during the experimental period was

observed. For each plant extract, the rats were divided into 7 groups. The rats in all

the 7 groups were having almost similar body weight. Gain in body weight was

monitored on the 0 day (rats were confirmed for diabetes), and 30th day. Normal rats

treated with aqueous and alcoholic extracts of SC showed 17.9% and 20.4% increase

(Table 4), whereas those treated with aqueous and alcoholic extracts of PM showed

18.4% and 16.3% increase (Table 5) in body weight respectively. Diabetic (group II)

rats showed 10.4% decrease in body weight on the 30th day (table 4 and 5). Diabetic

rats treated with aqueous and alcoholic extracts of SC showed a total 14.2% and

17.6% increase in body weight respectively at the end of study (Table 4), whereas

those treated with aqueous and alcoholic extracts PM showed a total 15.8% and

13.2% increase in body weight, respectively (Table 5). Diabetic rats treated with anti-

hyperglycemic drug, metformin (group VII) showed 16.5% increase in body weight

on the 30th day.

E- Evaluation of toxic effects of alcoholic extract of Syzygium cumini and

aqueous extract of Pterocarpus marsupium.

Experiments were conducted to establish non-toxic nature of alcoholic SC

seeds extract and aqueous PM bark powder extract doses chosen for the study. Rats

were divided into 5 groups according to the experimental design. The rats were fed

with the dose of alcoholic extract of SC seeds and aqueous extract of PM bark via oral

route for 60 days. The minimum dose of SC extract was 100 mg/kg body weight/day

and maximum dose chosen was 500 mg/kg body weight/day. The minimum dose of

PM extract was 200 mg/kg body weight/day and maximum dose chosen was 1 g/kg

body weight/day. There was no morbidity and the rats of all the groups showed

normal growth as observed by similar gain in body weight (~32-37%) at the end of

experiment, in groups II to V, comparable to that of group I. Effect of these plants

extracts at two doses were observed on liver and kidney function. No significant


81

deviations from control values were observed in serum urea and serum creatinine

values in all the groups treated with SC (seeds) and PM (bark) extracts. No significant

changes from normal control rats were observed in serum transaminases (ALT and

AST) and alkaline phosphatase levels (Table 6).

F- To evaluate anti-oxidative effect of SC and PM extracts (aq. and alc.) in


Experiments were conducted to evaluate the effect of aqueous and alcoholic

extracts of SC (seeds) and PM (bark) on antioxidant (SOD, catalase and GST)

activities, reduced glutathione content and malondialdehyde levels in heart, liver and

kidney issues. Rats were divided into eight groups according to the experimental

design. The rats were fed with aqueous and alcoholic extracts of SC and PM at doses

of 3g and 100 mg/kg body weight/day, and 200 and 300 mg/kg body weight/day

respectively for 30 days.

Cardiac MDA levels

MDA levels were significantly increased from 39.44±1.68 to 105.3±10.93

(p<0.001) in cardiac tissue of diabetic rats. However, the treatment of SC and PM

extracts did not show any significant change in normal rats. Treatment of diabetic rats

with aq. and alc. SC seed extracts resulted in significant decrease (p<0.001) in

elevated MDA levels from 105.30±10.93 to 72.67±7.13 nM/g tissue, and from

105.30±10.93 to 51.08±5.99 nM/g tissue, respectively. Similarly, treatment of

diabetic rats with aq. extract of PM bark resulted in decrease in MDA levels from

105.30±10.93 to 82.87±7.81 nM/g tissue, whereas treatment with alc. PM bark extract

showed significant decrease (p<0.001) in MDA levels from 105.30±10.93 to

65.15±6.63 nM/g tissue. However, there was no significant change observed in

normal rats treated with SC and PM. The beneficial effects of SC and PM on MDA

levels were comparable to metformin (a standard anti-diabetic agent) and vitamin C

(Table 7, 8).

Cardiac GSH Content

GSH content was significantly decreased from 0.93±0.06 to 0.49±0.08 µg/mg

protein (p<0.001) in cardiac tissue of diabetic rats when compared with normal rats.


82

Treatment of diabetic rats with aq. and alc. SC seed extracts resulted in significant

increase (p<0.001) in GSH content from 0.49±0.08 to 0.77±0.07 µg/mg protein, and

from 0.49±0.08 to 1.10±0.11 µg/mg protein, respectively. Similarly, treatment of

diabetic rats with aq. extract of PM bark resulted in increase in GSH content from

0.49±0.08 to 0.71±0.09 µg/mg protein, whereas treatment with alc. PM bark extract

showed significant increase (p<0.001) in GSH content from 0.49±0.08 to 0.89±0.15

µg/mg protein. However, there was no significant change observed in normal rats

treated with SC and PM. The beneficial effects of SC and PM on GSH content were

comparable to metformin (a standard anti-diabetic agent) and vitamin C (Table 9, 10).

Activities of cardiac antioxidant enzymes

Cardiac antioxidant enzyme activities are shown in fig. 1-6. The activities of

antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,

p<0.001 and p<0.001) in cardiac tissue of diabetic rats (group IV) when compared

with normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC

showed a significant (p < 0.001) increase from 8.52±1.82 (diabetic control) to

19.55±1.33 and 23.15±1.88 U/mg protein, respectively, in SOD activity (fig. 1).

Treatment of diabetic rats with aq. and alc. PM bark extract increased the SOD

activity from 8.52±1.82 (diabetic control) to 15.67±1.34 and 20.12±1.23 U/mg protein

(fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a significant (p

< 0.001, group V and VI) reversal of decreased catalase activity from 74.09±5.5

(diabetic control) to 100.7±4.8 and 119.54±5.5 U/mg protein respectively (fig. 3).

Treatment of diabetic rats with aq. and alc. PM bark extract increased the catalase


(fig. 4). The GST activity in diabetic rats was significantly (p<0.001) decreased to

1.27±0.09 from 2.39±0.15 U/mg protein observed in normal rats. The treatment of

diabetic rats with aq. and alc. SC extracts could result in significant improvement in

decreased GST activity, as it was increased from 1.27±0.09 to 1.59±0.12 and

2.25±0.07 U/mg protein when treated aq. and alc. SC extracts, respectively (fig. 5).

Treatment of diabetic rats with aq. and alc. PM bark extract increased the GST


(fig. 6). Normal rats treated with aq. and alc. extracts of SC and PM did not show any


83

significant effect on antioxidant enzyme activities when compared to normal control

(group I). On the other hand administration of metformin and vitamin C to diabetic

rats showed significant (p<0.001) increase in antioxidant activities (SOD, catalase and

GST) as compared with diabetic control rats.

Hepatic MDA levels


(p<0.001) in hepatic tissue of diabetic rats. However the treatment of SC and PM




418.07±27.48 to 227.59±25.31 nM/g tissue, respectively. Similarly, treatment of

diabetic rats with aq. extract of PM bark resulted in decrease in MDA levels from

418.07±27.48 to 335.76±28.22 nM/g tissue, whereas treatment with alc. PM bark

extract showed significant decrease (p<0.001) in MDA levels from 418.07±27.48 to

259.87±18.49 nM/g tissue. However, there was no significant change observed in

normal rats treated with SC and PM. The beneficial effects of SC and PM on MDA

levels were comparable to metformin (a standard anti-diabetic agent) and vitamin C

(Table 7, 8).

Hepatic GSH Content


protein (p<0.001) in hepatic tissue of diabetic rats when compared with normal rats.











84

Activities of hepatic antioxidant enzymes

Hepatic antioxidant enzyme activities are shown in fig. 1-6. The activities of


p<0.001 and p<0.001) in hepatic tissue of diabetic rats (group IV) when compared

with normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC




activity from 23.13±1.47 (diabetic control) to 30.89±1.22 and 38.99±1.47 U/mg

protein (fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a

significant (p < 0.001, group V and VI) reversal of decreased catalase activity from

41.99±1.5 (diabetic control) to 62.61±3.8 and 67.25±2.6 U/mg protein respectively

(fig. 3). Treatment of diabetic rats with aq. and alc. PM bark extract increased the

catalase activity from 41.99±1.5 (diabetic control) to 55.64±3.3 and 65.25±3.1 U/mg

protein (fig. 4). The GST activity in diabetic rats was significantly (p<0.001)

decreased to 3.44±0.19 from 6.6±0.35 U/mg protein observed in normal rats. The

treatment of diabetic rats with aq. and alc. SC extracts could result in significant

improvement in decreased GST activity, as it was increased from 3.44±0.19 to

4.9±0.32 and 6.29±0.47 U/mg protein when treated aq. and alc. SC extracts,

respectively (fig. 5). Treatment of diabetic rats with aq. and alc. PM bark extract

increased the GST activity from 3.44±0.19 (diabetic control) to 4.37±0.21 and

4.81±0.58 U/mg protein (fig. 6). Normal rats treated with aq. and alc. extracts of SC

and PM did not show any significant effect on antioxidant enzyme activities when

compared to normal control (group I). On the other hand administration of metformin

and vitamin C to diabetic rats showed significant (p<0.001) increase in antioxidant

activities (SOD, catalase and GST) as compared with diabetic control rats.

Renal MDA levels


(p<0.001) in renal tissue of diabetic rats. However the treatment of SC and PM




85


80.32±7.63 to 56.71±5.49 nM/g tissue, respectively. Similarly, treatment of diabetic

rats with aq. extract of PM bark resulted in decrease in MDA levels from 80.32±7.63

to 66.83±4.11 nM/g tissue, whereas treatment with alc. PM bark extract showed

significant decrease (p<0.001) in MDA levels from 80.32±7.63 to 58.87±5.23 nM/g

tissue. However, there was no significant change observed in normal rats treated with

SC and PM. The beneficial effects of SC and PM on MDA levels were comparable to

metformin (a standard anti-diabetic agent) and vitamin C (Table 7, 8).

Renal GSH Content


protein (p<0.001) in renal tissue of diabetic rats when compared with normal rats.










Activities of renal antioxidant enzymes

Renal antioxidant enzyme activities are shown in fig. 1-6. The activities of


p<0.001 and p<0.001) in renal tissue of diabetic rats (group IV) when compared with

normal rats (group I). The diabetic rats that received aq. and alc. extracts of SC





(fig. 2). The diabetic rats treated with aq. and alc. SC extracts showed a significant (p

< 0.001, group V and VI) reversal of decreased catalase activity from 45.76±1.91


86

(diabetic control) to 50.98±1.2 and 65.79±3.01 U/mg protein respectively (fig. 3).

Treatment of diabetic rats with aq. and alc. PM bark extract increased the catalase

activity from 45.76±1.91 (diabetic control) to 56.77±2.33 and 57.85±2.31 U/mg

protein (fig. 4). The GST activity in diabetic rats was significantly (p<0.001)

decreased to 9.21±0.77 from 18.44±0.85 U/mg protein observed in normal rats. The

treatment of diabetic rats with aq. and alc. SC extracts could result in significant

improvement in decreased GST activity, as it was increased from 9.21±0.77 to

14.76±0.69 and 18.14±0.73 U/mg protein when treated aq. and alc. SC extracts,

respectively (fig. 5). Treatment of diabetic rats with aq. and alc. PM bark extract

increased the GST activity from 9.21±0.77 (diabetic control) to 15.11±0.87 and

15.89±0.88 U/mg protein (fig. 6). Normal rats treated with aq. and alc. extracts of SC

and PM did not show any significant effect on antioxidant enzyme activities when

compared to normal control (group I). On the other hand administration of metformin

and vitamin C to diabetic rats showed significant (p<0.001) increase in antioxidant

activities (SOD, catalase and GST) as compared with diabetic control rats.


87

Table 1 Anti-hyperglycemic effect of aqueous and alcoholic extracts of SC

seed at different doses

Fasting blood glucose levels (mg/dl) Groups Treatments

Day 0 Day 30

I Normal control 98.8±3.7 100.6±6.1

II Normal+Aq SC (1.5g/kg b.w.) 81.2±4.5 93.2±3.8

III Normal+Aq SC (3g/kg b.w.) 83.1±4.6 95.2±3.9

IV Normal+Aq SC (5g/kg b.w.) 87.2±4.2 94.7±4.2

V Normal+Alc SC (50mg/kg b.w.) 96.2±4.1 92.2±3.1

VI Normal+Alc SC (100mg/kg b.w.) 92.8±3.8 98.4±3.5

VII Normal+Alc SC (200mg/kg b.w.) 97.2±4.5 99.8±3.8

VIII Diabetic control 453.6±22.9* 437.1±19.7*

IX Diabetic+Aq SC (1.5g/kg b.w.) 445.9±18.7* 317.1±14.4*

X Diabetic+Aq SC (3g/kg b.w.) 460.4±23.6* 160.6±6.7*

XI Diabetic+Aq SC (5g/kg b.w.) 476.8±21.9* 158.1±4.9*

XII Diabetic+Alc SC (50mg/kg b.w.) 455.9±12.9* 300.8±18.3*

XIII Diabetic+Alc SC (100mg/kg b.w.) 427.6±18.8* 100.0±10.1*

XIV Diabetic+Alc SC (200mg/kg b.w.) 437.7±19.2* 103.8±9.8*

XV Diabetic+Metformin (100mg/kgb.w.)

389.9±4.6* 230.6±3.2*

SC= Syzygium cumini; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group II - VII were compared to group I; group VIII was compared to group I; group IX - XV were compared to group VIII.


88

Table 2 Anti-hyperglycemic effect of aqueous and alcoholic extracts of PM

bark at different doses


Day 0 Day 30

I Normal control 98.8±3.7 100.6±6.1

II Normal+Aq PM (100mg/kg b.w.) 100.3±2.5 102.1±6.2

III Normal+Aq PM (200mg/kg b.w.) 110.3±3.6 112.2±5.3

IV Normal+Aq PM (400mg/kg b.w.) 115.8±3.7 114.9±5.9

V Normal+Alc PM (150mg/kg b.w.) 99.1±2.6 99.8±3.5

VI Normal+Alc PM (300mg/kg b.w.) 109.5±3.7 106.3±3.9

VII Normal+Alc PM (500mg/kg b.w.) 105.9±2.5 106.7±4.7

VIII Diabetic control 453.6±22.9* 437.1±19.7*

IX Diabetic+Aq PM (100mg/kg b.w.) 403.1±16.2* 335.6±7.7*

X Diabetic+Aq PM (200mg/kg b.w.) 373.4±13.6* 175.6±4.7*

XI Diabetic+Aq PM (400mg/kg b.w.) 388.3±16.1* 170.5±5.2*

XII Diabetic+Alc PM (150mg/kg b.w.) 398.5±13.8* 350.8±6.1*

XIII Diabetic+Alc PM (300mg/kg b.w.) 355.5±10.8* 300.3±6.1*

XIV Diabetic+Alc PM (500mg/kg b.w.) 378.9±13.8* 310.3±7.3*

XV Diabetic+Metformin (100mg/kg b.w.)

389.9±4.6* 230.6±3.2*

PM= Pterocarpus marsupium; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group II - VII were compared to group I; group VIII was compared to group I; group IX - XV were compared to group VIII.


89

Table 3 Duration dependent effect of alcoholic extract of SC seed and aqueous extract of

PM bark on fasting blood glucose levels


Day 0 Day 15 Day 30

I Normal control 98.8±3.7 99.8±3.9 100.6±6.1

II Normal+SC (100mg/kg b.w.) 96.2±4.1 95.2±5.6 92.2±3.1

III Normal+PM (200mg/kg b.w.) 100.3±2.5 98.3±4.4 102.1±6.2

IV Diabetic control 453.6±22.9* 445.7±18.9* 437.1±19.7*

V Diabetic+SC (100mg/kg b.w.) 427.6±18.8* 297.6±11.5* 100.0±10.1*

VI Diabetic+PM (200mg/kg b.w.) 373.4±13.6* 223.5±12.4* 175.6±4.7*

VII Diabetic + Metformin (100mg/kg b.w.)

389.9±4.6* 318.3±7.3* 230.6±3.2*

SC= Syzygium cumini; PM= Pterocarpus marsupium; *p<0.001. All values are expressed as mean ± SD. Group II and III were compared to group I; group IV was compared to group I; group V-VII were compared to group IV.

Table 4 Effect of aqueous and alcoholic extracts of SC seed on body weight

Body weight (g) Groups Treatments

Day 0 Day 30 % Gain

I Normal control 98.4±3.1 118.5±2.5 20.4

II Diabetic 95.3±2.3 85.4±3.8* -10.4

III Normal+Aq SC (3 g/kg b.w.) 95.8±2.8 112.9±4.6 17.9

IV Normal+Alc SC (100mg/kg b.w.) 120.4±5.8 144.9±6.5 20.4

V Diabetic+Aq SC (3 g/kg b.w.) 85.2±2.1 97.3±3.5* 14.2

VI Diabetic+Alc SC (100mg/kg b.w.) 82.9±4.2 97.5±4.5* 17.6

VII Diabetic+Metformin (100mg/kg b.w.) 124.4±6.5 144.9±5.4* 16.5

SC= Syzygium cumini; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group III and IV were compared to group I; group II was compared to group I; group V-VII were compared to group II.


90

Table 5 Effect of aqueous and alcoholic extracts of PM bark on body weight

Body weight (g) Groups Treatments

Day 0 Day 30 % Gain

I Normal control 98.4±3.1 118.5±2.5 20.4

II Diabetic 95.3±2.3 85.4±3.8* -10.4

III Normal+Aq PM (200mg/kg b.w.) 94.5±4.4 111.9±5.5 18.4

IV Normal+Alc PM (300mg/kg b.w.) 115.7±6.7 134.6±5.6 16.3

V Diabetic+Aq PM (200mg/kg b.w.) 88.5±4.2 102.5±5.2* 15.8

VI Diabetic+Alc PM (300mg/kg b.w.) 108.3±5.8 122.6±6.9* 13.2

VII Diabetic +Metformin (100mg/kg b.w.)

124.4±6.5 144.9±5.4* 16.5

PM= Pterocarpus marsupium; b.w.=body weight; *p<0.001. All values are expressed as mean ± SD. Group III and IV were compared to group I; group II was compared to group I; group V-VII were compared to group II.

Table 6 Evaluation of toxic effects of SC seed (alcoholic extract) and PM bark (aqueous

extract) doses in rats after 2 month exposure

Gps Treatments Serum

I II III VI V

Normal control Normal+Alc SC (100 mg/kg b.w.) Normal+Alc SC (500 mg/kg b.w. ) Normal+Aq PM (200mg/kg b.w.) Normal+Aq PM (1 g/kg b.w.)

ALT AST AP Creatinine Urea B.W. (U/l) (U/l) (µM/min/l) (mg/dl) (mg/dl) (% inc) 45.3±3.1 137.5±6.7 131.2±3.4 0.56±0.09 90.1±2.1 35.3 46.6±2.9 148.5±5.4 129.6±4.5 0.61±0.08 93.2±3.2 32.1 50.9±3.6 149.6±4.9 132.5±5.1 0.62±0.1 95.4±4.1 34.2 47.9±3.2 144.6±5.1 132.3±3.1 0.62±0.11 92.9±5.3 37.0 49.2±5.2 151.3±8.5 135.1±6.2 0.61±0.15 94.2±5.2 34.3

Aq=Aqueous; Alc=alcoholic; SC= Syzygium cumini; PM= Pterocarpus marsupium; b.w.=body weight; ALT=Alanine aminotransferase; AST= aspartate aminotransferase; AP= Alkaline phosphatase. All values are expressed as mean ± SD


91

Table 7 Effect of aqueous and alcoholic extracts of SC on MDA levels (nM/g tissue) in

alloxan induced diabetic rats

Gps Treatments Tissues

I II III IV V VI VII VIII

Normal control Normal+Aq SC treated Normal+Alc SC treated Diabetic control Diabetic+Aq SC treated Diabetic+Alc SC treatedDiabetic +Metformin Diabetic+Vitamin C

Heart Liver Kidney

39.44±1.68 194.30±9.96 35.81±6.87 41.14±1.22 199.87±8.70 42.11±7.65 38.74±1.65 182.85±6.78 38.34±5.66 105.30±10.93* 418.07±27.48* 80.32±7.63* 72.67±7.13* 299.66±19.50* 60.05±4.12* 51.08±5.99* 227.59±25.31* 56.71±5.49* 78.90±4.11* 302.48±26.30* 59.83±4.13* 50.11±4.19* 210.19±15.11* 51.83±4.83*

All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001.

Table 8 The effect of aqueous and alcoholic extracts of PM on MDA (nM/g tissue)

content in alloxan induced diabetic rats



Normal control Normal+Aq PM treated Normal+Alc PM treated Diabetic control Diabetic+Aq PM treated Diabetic+Alc PM treated Diabetic +Metformin Diabetic+Vitamin C

Heart Liver Kidney 39.44±1.68 194.30±9.96 35.81±6.87 42.35±1.79 186.42±7.87 44.65±6.95 35.88±1.91 198.50±8.44 39.59±5.19 105.30±10.93* 418.07±27.48* 80.32±7.63* 82.87±7.81** 335.76±28.22** 66.83±4.11** 65.15±6.63* 259.87±18.49* 58.87±5.23* 78.90±4.11* 302.48±26.30* 59.83±4.13* 50.11±4.19* 210.19±15.11* 51.83±4.83*

All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001, **p<0.01


92

Table 9 The effect of aqueous and alcoholic extracts of SC on GSH (µg/mg protein)




Normal control Normal+Aq SC treated Normal+Alc SC treated Diabetic control Diabetic+Aq SC treated Diabetic+Alc SC treated Diabetic +Metformin Diabetic+Vitamin C

Heart Liver Kidney

0.93±0.06 1.05±0.06 0.79±0.05 0.98±0.07 1.15±0.07 0.85±0.06 0.89±0.08 1.09±0.08 0.82±0.07 0.49±0.08* 0.64±0.03* 0.46±0.04* 0.77±0.07* 0.79±0.05* 0.57±0.02* 1.10±0.11* 1.03±0.09* 0.78±0.07* 0.76±0.08* 0.83±0.07* 0.65±0.06* 1.15±0.11* 1.08±0.07* 0.78±0.09*

All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001

Table 10 The effect of aqueous and alcoholic extracts of PM on GSH (µg/mg protein)




Normal control Normal+Aq PM treated Normal+Alc PM treated Diabetic control Diabetic+Aq PM treated Diabetic+Alc PM treated Diabetic +Metformin Diabetic+Vitamin C

Heart Liver Kidney

0.93±0.06 1.05±0.06 0.79±0.05 0.85±0.06 1.09±0.07 0.82±0.07 0.95±0.07 0.99±0.05 0.85±0.06 0.49±0.08* 0.64±0.03* 0.46±0.04* 0.71±0.09** 0.83±0.08** 0.55±0.04** 0.89±0.15* 0.97±0.04* 0.76±0.07* 0.76±0.08* 0.83±0.07* 0.65±0.06* 1.15±0.11* 1.08±0.07* 0.78±0.06*

All values are expressed as mean ± SD; Normal treated and diabetic rats were compared with normal rats; diabetic treated rats were compared with diabetic rats; *p<0.001, **p<0.01


93

Figure 1: The effect of aqueous and alcoholic extracts of SC on SOD activity (U/mg protein) in alloxan induced diabetic rats.

Figure 2: The effect of aqueous and alcoholic extracts of PM on SOD activity (U/mg protein) in alloxan induced diabetic rats.

SOD=Superoxide dismutase; 1U of SOD= 50% inhibition of auto-oxidation of

epinephrine/min; Normal treated and diabetic rats were compared with normal rats;

Diabetic treated rats were compared with diabetic rats.


94

Figure 3: The effect of aqueous and alcoholic extracts of SC on Catalase activity (µM H2O2 decomposed/min/mg protein) in alloxan induced diabetic rats.

Figure 4: The effect of aqueous and alcoholic extracts of PM on catalase activity (µM H2O2 decomposed/min/mg protein) in alloxan induced diabetic rats.

1U of Catalase= µmoles H2O2 decomposed/min; Normal treated and diabetic

rats were compared with normal rats; Diabetic treated rats were compared with

diabetic rats.


95

Figure 5: The effect of aqueous and alcoholic extracts of SC on GST (U/mg protein) activity in alloxan induced diabetic rats.

N=Normal, NSC(Aq)=Normal+SC aqueous extract treated, NSC(Alc)=Normal+SC alcoholic extract treated, D=Diabetic, DSC(Aq)=Diabetic+SC aqueous extract treated, DSC(Alc)=Diabetic+SC alcoholic extract, DMet=Diabetic+Metformin treated, DVitC=Diabetic+Vitamin C treated.

Figure 6: The effect of aqueous and alcoholic extracts of PM on GST activity (U/mg protein) in alloxan induced diabetic rats.

N=Normal, NPM(Aq)=Normal+PM aqueous extract treated, NPM(Alc)=Normal+PM alcoholic extract treated, D=Diabetic, DPM(Aq)=Diabetic+PM aqueous extract treated, DPM(Alc) = Diabetic+PM alcoholic extract, DMet=Diabetic+Metformin treated, DVitC=Diabetic+Vitamin C treated.

GST= Glutathione-s-transferase; 1 U of GST= µM GSH-CDNB complex formed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.


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DISCUSSION

Alloxan and streptozotocin are the most prominent diabetogenic chemicals in

diabetes research. In 1838, Wöhler and Liebig (Wöhler and Liebig, 1838) synthesised

a pyrimidine derivative, which was later called alloxan (Lenzen et al., 1996). In 1943,

alloxan became of interest in diabetes research when it was reported that it could

induce diabetes in animals (Dunn and McLetchie, 1943) as a result of the specific

necrosis of the pancreatic beta cells (Peschke et al., 2000). The resulting insulinopenia

causes a state of experimental diabetes mellitus called ‘alloxan diabetes’ (McLetchie,

1982).

Alloxan has two distinct pathological effects: it selectively inhibits glucose-

induced insulin secretion through specific inhibition of glucokinase, the glucose

sensor of the beta cell, and it causes a state of insulin-dependent diabetes through its

ability to induce ROS formation, resulting in the selective necrosis of beta cells.

These two effects can be assigned to the specific chemical properties of alloxan, the

common denominator being selective cellular uptake and accumulation of alloxan by

the beta cell.

Alloxan is a very unstable chemical compound with a molecular shape

resembling glucose (Lenzen and Munday, 1991). Both alloxan and glucose are

hydrophilic and do not penetrate the lipid bilayer of the plasma membrane. The

alloxan molecule is structurally so similar to glucose that the GLUT2 glucose

transporter in the beta cell plasma membrane accepts this glucomimetic and transports

it into the cytosol (Gorus et al., 1982). Alloxan does not inhibit the function of the

transporter (Elsner et al., 2002), and can therefore selectively enter beta cells in an

unrestricted manner (Malaisse et al., 2001).

Selective inhibition of glucose-induced insulin secretion is the major

pathophysiological effect of the thiol group reactivity of alloxan (Lenzen et al., 1987).

Alloxan has a central 5-carbonyl group that reacts very avidly with thiol groups.

Glucokinase (hexokinase IV) is the most sensitive thiol enzyme in the beta cell

(Tiedge et al., 2000), with a half maximal inhibitory concentration in the 1–10 µmol/l

range. At higher concentrations, alloxan can inhibit many functionally important


97

enzymes, as well as other proteins and cellular functions (Konrad and Kudlow, 2002).

Inhibition of glucokinase reduces glucose oxidation and ATP generation, thereby

suppressing the ATP signal that triggers insulin secretion (Gunnarsson and

Hellerström, 1973). Inhibition of glucokinase is achieved within 1 min of exposure to

alloxan. The inhibition of insulin secretion after exposure to alloxan (Weaver et al.,

1978) is restricted to that induced by glucose and its epimer, mannose, both of which

induce insulin secretion through interaction with glucokinase (Lenzen and Panten,

1988). Insulin biosynthesis is also inhibited by alloxan (Niki et al., 1976), most likely

through the same mechanism.

Thiols such as the tripeptide glutathione (GSH), cysteine and dithiothreitol

protect glucokinase against alloxan inhibition because they reduce alloxan to dialuric

acid, which is not thiol reactive (Lenzen et al., 1988). However, only dithiols such as

dithiothreitol (Lenzen and Mirzaie, 1991) can readily reverse alloxan-induced

glucokinase inhibition. They achieve this by reducing functionally essential cysteine

residues of the glucokinase enzyme, which are oxidised through alloxan action

(Lenzen et al., 1988; Lenzen and Mirzaie, 1991). Likewise, glucose protects against

alloxan-induced inhibition of glucose-induced insulin secretion because its binding to

the sugar-binding site of glucokinase prevents the oxidation of the functionally

essential thiol groups.

Alloxan can generate reactive oxygen species (ROS) in a cyclic reaction with

its reduction product, dialuric acid (Munday, 1988), as depicted in the text box

‘Chemical redox cycling reactions between alloxan and dialuric acid, and protective

actions of cytoprotective enzymes’ (reactions i–ii). In the beta cells the toxic action of

alloxan is initiated by free radicals formed in this redox reaction (Winterbourn et al.,

1989). Autoxidation of dialuric acid generates superoxide radicals (iii–iv) and

hydrogen peroxide (iii–iv), and in the Fenton reaction (v), in the presence of a suitable

metal catalyst (typically iron) (vi), hydroxyl radicals (v–vii). The autoxidation of

dialuric acid involves the intermediate formation of the alloxan radical (i–iv)

(Winterbourn and Muday, 1989).


98

The reduction of alloxan to dialuric acid in the cell requires the presence of a

suitable thiol, typically the tripeptide glutathione (GSH) to generate the redox cycling

partner, dialuric acid, and oxidised glutathione (viii) (Brömme et al., 2000). The

triketone structure of alloxan is vitally important for this two-step reaction with

glutathione (Elsner et al., 2008), which generates the alloxan radical as an

intermediate product (ix–x). Other thiols such as cysteine, which are present at lower

concentrations in the cell, dithiols and ascorbic acid are also suitable reducing agents

and may therefore contribute to alloxan reduction (Elsner et al., 2006). Alloxan can


99

also generate ROS by reacting with thiol groups on enzymes (Lenzen and Mirzaie,

1992) and albumin (Sakurai and Miura, 1989). During each typical redox cycle a

small amount of ‘Compound 305’, an alloxan–GSH adduct (Brömme et al., 2002), is

formed. The intracellular concentration of Compound 305 increases in a time-

dependent manner, which gradually decreases the amount of reduced GSH available

in the cell for redox cycling, thus producing a lower pro-oxidative ratio between

alloxan and GSH, rather than a higher antioxidative ratio (Munday, 1988).

Paradoxically the thiols cysteine and GSH have long been reported to protect

rats against the development of alloxan diabetes when injected together with alloxan

(Sen and Bhattacharya, 1952). This observation can now be explained in light of the

established molecular mechanism of alloxan action. When concentrations of reducing

agents in the blood stream or in the extracellular space are significantly increased

through injection of a thiol, more alloxan is reduced extracellularly so that less is

available for intracellular accumulation. Normally the capacity for alloxan reduction,

redox cycling and the generation of ROS in the circulation (Sakurai and Miura, 1989)

is not sufficient to prevent the alloxan molecule from reaching and entering the beta

cell.

The major oxidation pathway of dialuric acid, a chain reaction dependent upon

superoxide radicals, is inhibited by superoxide dismutase (SOD; xi). In the presence

of SOD, an autocatalytic process involving the interaction between dialuric acid and

alloxan becomes important (Munday, 1988), while in the presence of a transition

metal, a third oxidation mechanism, dependent upon hydrogen peroxide, has been

identified (Munday, 1988). This latter step is inhibited by the hydrogen peroxide

inactivating enzyme catalase (Munday, 1988) (xii; text box: Chemical redox cycling

reactions between alloxan and dialuric acid, and protective actions of cytoprotective

enzymes). The other hydrogen peroxide inactivating enzyme, glutathione peroxidase,

can principally act in a similar manner. But this enzyme requires GSH, which is

oxidised in this reaction (xiii). When kept in the oxidised form, alloxan does not

generate ROS (Elsner et al., 2006). Thus, alloxan is not cytotoxic in the absence of

thiols such as GSH or when restricted to the extracellular space (Elsner et al., 2006).

Thiols in the plasma membrane, with which alloxan could interact and generate ROS


100

in a redox cycle, are apparently not present or not accessible to a sufficient extent to

allow the generation of ROS and damage the cells (Elsner et al., 2006).

Thus, it can be concluded that the pancreatic beta cell toxicity and the resultant

diabetogenicity of alloxan are due to redox cycling and the generation of toxic ROS

within the β cells. Different doses of alloxan have been reported in literature to induce

diabetes in different animal models. 70 mg/kg body weight, i.v. dose and 200 mg/kg

body weight i.p. doses have been reported (Zhou et al., 2009). The most widely used

dose to make rats diabetic was found to be 150 mg/kg/body weight in literature

(Khushk et al., 2010). In present study, 150 mg/kg body weight resulted in

hyperglycemic rats. FBG levels were in the range 250 mg/dl to 450 mg/dl, rats

showing glucose levels >270 mg/dl were included in the present study.

The present study was designed to evaluate beneficial effect of two commonly

used natural products i.e. SC and PM in Indian population, on anti-oxidant status and

anti-hyperglycemic activity in alloxan induced diabetic rats. The anti-hyperglycemic

effect of aqueous and alcoholic extracts of SC was evaluated at 3 different doses. For

aqueous extract the 3 doses were 1.5, 3, 5 g/kg b.w./day and for alcoholic extract the 3

doses were 50, 100, 200 mg/kg b.w./day. Administration of aqueous SC extract at a

dose of 1.5 g/kg b.w./day by oral route resulted in 27.5% decrease in FBG levels in

diabetic rats after 30 days, however 3 g/ kg/b.w./day dose resulted in better

management of FBG, as the decreased levels (by 63.3%) observed were close to

normal value (Table 1). With 5 g/kg b.w./day dose the results were comparable to the

results obtained with 3 g/kg b.w./day dose. These doses (3 and 5 g/kg b.w./day) of SC

extracts have not shown any hypoglycemic effect in normal rats. On the other hand

administration of alcoholic SC extract at a dose of 50 mg/kg b.w./day by oral route

resulted in 31.2% decrease in FBG levels in diabetic rats after 30 days, however 100

mg/ kg/b.w./day dose resulted in better management of FBG, as the decreased levels

(by 77%) observed were close to normal value (Table 1). With 200 mg/kg b.w./day

dose the results were comparable to the results obtained with 100 mg/kg b.w./day

dose. These doses (100 and 200 mg/kg b.w./day) of SC extracts have not shown any

hypoglycemic effect in normal rats. The effect of different doses of SC extract on


101

FBG levels has been previously evaluated in various studies (Singh and Gupta, 2007;

Prince et al., 1998).

Similarly, the anti-hyperglycemic effect of aqueous and alcoholic extracts of

PM was evaluated at 3 different doses. For aqueous extract the 3 doses were 100, 200,

400 mg/kg b.w./day and for alcoholic extract the 3 doses were 150, 300, 500 mg/kg

b.w./day. Administration of aqueous PM extract at a dose of 100 mg/kg b.w./day by

oral route resulted in 23.2% decrease in FBG levels in diabetic rats after 30 days,

however 200 mg/ kg/b.w./day dose resulted in better management of FBG, as the

decreased levels (by 59.8%) observed were close to normal value (Table 2). With 400

mg/kg b.w./day dose the results were comparable to the results obtained with 200

mg/kg b.w./day dose. These doses (200 and 400 g/kg b.w./day) of PM extracts have

not shown any hypoglycemic effect in normal rats. On the other hand administration

of alcoholic PM extract at a dose of 150 mg/kg b.w./day by oral route resulted in

19.7% decrease in FBG levels in diabetic rats after 30 days, however 300 mg/

kg/b.w./day dose resulted in 31.3% decrease in FBG levels (Table 2). With 500 mg/kg

b.w./day dose the results were comparable to the results obtained with 300 mg/kg

b.w./day dose. These doses (300 and 500 mg/kg b.w./day) of SC extracts have not

shown any hypoglycemic effect in normal rats. The effect of different doses of PM

extract on FBG levels has been previously evaluated in various studies (Gayathri and

Kannabiran, 2008; Gupta and Gupta, 2009).

Metformin, is a biguanide that affects the intestinal glucose absorption, insulin

secretion and hepatic glucose production to manage the diabetes. In vivo and in vitro

studies have demonstrated that metformin stimulates the insulin-induced component

of glucose uptake into skeletal muscle and adipocytes in both diabetic individuals and

animal models (Klip and Leiter, 1990). In the present study, anti-hyperglycemic

effects resulted due to metformin, used as positive control, were comparable to the

results obtained with these plant extracts (Table 1 and 2). The possible mechanism of

anti-hyperglycemic potential of these plant extract could be due to any of following

reasons viz, increase in the release of insulin from pancreas or an increased uptake of

glucose by the peripheral tissues.


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Duration dependent anti-hyperglycemic effect of alcoholic extract of SC (100

mg/kg b.w./day) and aqueous extract of PM (200 mg/kg b.w./day) was evaluated on

the 15th and 30th day. SC extract showed a significant decrease in FBG, 33.22% and

77.56% on day 15th and 30th, respectively. PM extract showed in 49.85% and 60.60%

decrease in FBG levels on the 15th and 30th day, respectively (table 3). The doses of

SC and PM extracts did not show any hypoglycemic effect in normal rats. The pattern

of duration dependent anti-hyperglycemic effect shown by of SC and PM were

comparable with positive control metformin.

Administration of alloxan is reported to be associated with loss in body weight

(Siddiqui et al., 2005). In spite of the increased food consumption, loss of body

weight may be due to defect in glucose metabolism and excessive breakdown of

protein in tissues is a characteristic of diabetes (Sikarwar and Patil, 2010). In present

study alloxan induced diabetic rats showed a significant decrease in body weight from

118.5±2.5 to 85.4±3.8 g. As shown in table 4 treatment with aqueous (3 g/kg

b.w./day) and alcoholic (100 mg/kg b.w./day) extracts of SC resulted in 14.2% and

17.6% gain in body weight respectively, on 30th day. Similarly aqueous (200 mg/kg

b.w./day) and alcoholic (300mg/kg b.w./day) extracts of PM showed in 15.8% and

13.2% gain in body weight on 30th day (table 5). Treatment with SC and PM extracts

improved body weight of diabetic rats, indicating control over polyphagia and muscle

wasting resulted due to hyperglycemic condition. The restoration of decreased body

weight in diabetic rats after plant extract treatment has been previously reported

(Singh and Gupta, 2007; Prince et al., 1998). Normal rats when treated with these

plant extracts of SC and PM showed gain in body weight which was comparable to

normal control rats. The result obtained with anti-diabetic drug metformin with

respect to gain in body weight was comparable to the effects observed with SC and

PM extracts in diabetic rats.

The present study includes experiments which were conducted to establish the

toxicity of alcoholic SC seed extract and aqueous PM bark extract when administered

for two months. Two doses of these extracts, SC seed (100 and 500 mg/kg b.w./day)

and PM bark extract (200 mg and 1 g/kg b.w./day), were given to normal rats. There

was no morbidity and all the rats showed normal growth (gain in body weight),


103

similar to that of normal rats. Effects of these plant extracts were observed on liver

and kidney function. No significant deviation from control values were observed in

serum urea and creatinine values in all the groups. No significant changes from

control rats were observed in serum transaminases ALT and AST and alkaline

phosphatase levels (Table 6). The data suggest that prolong use of these extracts is

safe. This non-toxic nature of plant extract has been reported previously (Gayathri and

Kannabiran, 2008).

Alloxan induced diabetic rats exhibit most of the diabetic complication

mediated by oxidative stress (Ozturia et al., 1996). The mechanism of diabetes

induction due to alloxan involves free radical mediated destruction of pancreatic β-

cells (Tomlinson et al., 1992). Aqueous and alcoholic extracts of SC and PM have

been reported to possess anti-hyperglycemic activity and are non-toxic. In the present

study, anti-peroxidative and anti-oxidative effects of aqueous and alcoholic extracts of

SC seeds and PM bark were evaluated in diabetic rats in order to establish their anti-

oxidative potential.

Oxidative stress plays an important role in development of complications of

diabetes and it is postulated to be associated with increased lipid peroxidation

(Elangovan et al., 2000). Oxidative stress in cells and tissues results from the

increased generation of reactive oxygen species and from decrease in antioxidant

defense potential (Gumieniczek et al., 2002). Elevated generation of free radicals

resulting in the consumption of antioxidant defense components may lead to

disruption of cellular functions and oxidative damage to membranes and may enhance

susceptibility to lipid peroxidation (Baynes, 1991). The present study showed a

significant (p<0.001) increase in MDA levels in heart, liver and kidney tissues of

diabetic rats (Table 7) suggesting that peroxidative injury may be involved in alloxan

induced diabetes and may lead to other secondary complications. Treatment of

diabetic rats with aqueous and alcoholic extracts of SC could significantly lower the

elevated MDA levels by 30.9% and 51.4% in heart, 28.3% and 45.6% in liver, 25.2%

and 29.4% in kidney, respectively (Table 7). These results are in accordance with the

previously reported anti-peroxidative effect of SC extracts (Prince et al., 2003;

Ahmed et al., 2010). Similarly aqueous and alcoholic extracts of PM could lower the


104

elevated MDA levels by 21.3% and 38.1% in heart, 19.7% and 37.8% in liver, 16.8%

and 27.7% in kidney tissue, respectively (Table 8). These results are in accordance

with the previous studies showing the anti-peroxidative effect PM extract

(Mohammadi et al., 2009; Maruthupandian and Mohan, 2011; Singh et al., 2012).

Reduced glutathione (GSH) is known to protect the cellular system against the

toxic effects of lipid peroxidation (Nicotera and Orrenius, 1986). GSH functions as

direct free radical scavenger, as a co-substrate for glutathione peroxidase (GPx), as a

cofactor for many other enzymes and forms conjugates in endo and xenobiotic

reactions (Gregus et al., 1996). Several studies support the hypothesis that prolonged

hyperglycemia up-regulate the polyol pathway as well as advanced glycation end

products formation and free radical generation rates. A relative depletion of NADPH

occurs due to aldose reductase (AR) activation in different tissues of diabetic rats.

Role of AR has also been implicated in detoxification of lipid peroxidation products

such as 4-hydroxynonenal (4- HNE) and malondialdehyde (MDA) as GSH-aldehyde

adducts (Srivastava et al., 1998). This led to depleted GSH levels at cellular level. Our

results are also in accordance to these findings as depleted GSH levels and elevated

MDA levels were observed in heart, liver and kidney tissues of diabetic rats. Diabetic

rats treated with aqueous and alcoholic extracts of SC and PM resulted in significant

(p<0.001) increase in GSH content when compared with diabetic controls (Table 9).

Diabetic rats treated with aqueous and alcoholic SC extracts resulted in increase in

GSH content by 57.14% and 124.5 % in heart, 23.4% and 60.9% in liver, 23.9% and

69.6% in kidney, respectively (Table 9). Similarly aqueous and alcoholic extracts of

PM resulted in increase in GSH content by 44.9% and 81.6% in heart, 29.7% and

51.6% in liver, 19.6% and 65.2% in kidney, respectively (Table 10). Thus, the

treatment of SC and PM extracts to the alloxan induced diabetic rats resulted in

attenuation in elevated levels of TBARS in different tissues and also increased the

depleted GSH content. These findings suggest that the administration of SC and PM

extracts to the diabetic rats could overcome the oxidative stress status in different

tissues of diabetics. The significant recovery of GSH content by treatment with SC

extracts indicated their protective effect on antioxidants (Prince et al., 2003; Ahmed et

al., 2010). Previously reported antioxidant activity of SC extracts has been attributed

to anthocyanin/ellagitannin present in the extract (Aqil et al., 2012). Pterocarpus


105

marsupium extract has been reported to possess strong in vitro antioxidant activity

and may serve as a potential source of natural antioxidant for treatment of diabetes

(Mohammadi et al., 2009). The ethanol extract of P.marsupium has been shown to

possess significant antidiabetic, antihyperlipidaemic and antioxidant effects in alloxan

induced diabetic rats. (Maruthupandian and Mohan, 2011; Singh et al., 2012).

The initiation and propagation of oxidative stress by overproduction of O2 –

and H2O2 and their conversion to potent toxic oxidants led to cause change in tissue

pathology (Muzykantov, 2001). Therefore, interception and detoxification of O2 – and

H2O2 appear to represent an important therapeutic goal. Superoxide dismutase (SOD),

catalase, and peroxidases constitute a mutually supportive team of defense against

ROS. While SOD lowers the steady-state level of O2¯, catalase and peroxidases do the

same for H2O2.

Reduced activities of SOD and catalase in heart, liver and kidney tissues of

diabetic rats has been observed in the present study (Fig 1, 2, 3 and 4) which may be

due to increased production of reactive oxygen species that can themselves reduce the

activities of these enzymes (Mc-cord et al, 1976). The reduced activities of SOD and

catalase have been reported in poor glycemic control and the inactivation of these

enzymes may be due to glycation of these proteins (Sozmen et al., 2001). The reduced

activities of these enzymes in tissues may lead to number of deleterious effects

(Wohaieb and Godin, 1987). The treatment of diabetic rats with aqueous and

alcoholic extracts of SC resulted in significant (p<0.001) increase in the activities of

anti-oxidant enzymes (SOD, CAT, GST) in heart liver and kidney tissues (figures 1,

3, 5). Similarly treatment of diabetic rats with alcoholic extract of PM resulted in

significant (p<0.001) increase in the activities of anti-oxidant enzymes (SOD, CAT,

GST) in heart liver and kidney tissues, whereas the results obtained with aqueous

extract were not significant (figures 2, 4, 6).

On basis of these findings a possible mechanism to explain the anti-oxidative

protection by SC and PM could be proposed. The diabetics have decreased SOD and

GPx activities due to inactivation by reactive oxygen species or by glycation of

enzymes. The elevated superoxide anions have been reported to inactivate catalase

(Chance et al., 1952). Thus, the altered SOD activity plays an important role on the


106

activity of catalase. Reduced activity of GST observed in diabetic state may also be

due to the inactivation caused by ROS. GST works together with glutathione and GPx

in decomposition of H2O2 or other organic hydroperoxides to non-toxic products

(Freeman and Crapo, 1982). A significant increase/recovery in SOD, catalase and

GST activities in SC and PM extract (aq./alc.) treated diabetic subjects may be due to

following reasons: the anti-hyperglycemic effect observed by these extracts resulted

in decreased glycation of these enzyme proteins (less inactivation of enzymes) which

in turn potentiate their reduction capacity by improving their antioxidant activities.

Achievement of near normoglycemic conditions in diabetic rats treated with SC and

PM extracts resulted in decreased free radical/ROS formation in diabetic tissues

which led to regain the GSH levels and reverse the ROS mediated inactivation of

GST, SOD and CAT activities. The significant recovery of anti-oxidant enzyme

activities by treatment with SC extracts indicated their protective effect on oxidative

stress (Prince et al., 2003; Ahmed et al., 2010). Previously reported antioxidant

activity of SC extracts has been attributed to anthocyanin/ellagitannin present in the

extract (Aqil et al., 2012). Pterocarpus marsupium extract has been reported to

possess strong in vitro antioxidant activity and may serve as a potential source of

natural antioxidant for treatment of diabetes (Mohammadi et al., 2009). The ethanol

extract of P.marsupium has been shown to possess significant antidiabetic, anti-

hyperlipidaemic and antioxidant effects in alloxan induced diabetic rats.

(Maruthupandian and Mohan, 2011; Singh et al., 2012).

Metformin (a biguanide derivative) is considered as an anti-hyperglycemic

rather than a hypoglycemic agent which makes the metformin a drug of choice.

Various mechanisms have been proposed to account for anti-hyperglycemic action of

metformin like suppression of basal hepatic glucose production, increased peripheral

glucose up take, and increased non-oxidative glucose metabolism (Cusi and

DeFronzo, 1998). All these lead to oxidative protection in diabetics. In the present

study the anti-hyperglycemic and anti-oxidative effects observed with SC and PM

extracts (aq. and alc.) treatments were comparable or even better than that of

metformin. Long term use of metformin has been reported to be associated with

several toxic effects on renal and hepatic systems (Cusi and DeFronzo, 1998). The

present study demonstrates that prolonged use of SC and PM extracts did not result in


107

any such toxic effect and also had better or comparable anti-hyperglycemic and anti-

oxidative effects. Thus these findings suggest that developing newer anti-diabetic

agent from these plant extracts may be safe and preferred.

Chapter-II

Anti-diabetic and anti-oxidative potentials of purified Syzygium cumini and

Pterocarpus marsupium extracts

Chapter II: Anti-diabetic and anti-oxidative potentials of purified SC and PM extracts

108

Diabetes mellitus is a fast growing medical problem which is characterised by

disordered metabolism and abnormally high blood sugar levels resulting from the

body’s inability to produce or properly use insulin. A balance between glucose

production and its utilization is necessary to maintain normal blood glucose levels.

Diabetes is characterized by elevated production and low utilization of glucose

(Taylor and Agius, 1998). A number of changes in several enzymes involved in

glucose metabolism present in the liver and other tissues are known to occur in

diabetes mellitus e.g. activity of hepatic glucokinase, phosphofrucokinase and

pyruvate kinase is markedly decreased and activity of glucose-6-phosphatase is

almost doubled (Cahill et al., 1959; Prince et al., 1997; Sharma et al., 2011). The

chronic hyperglycemia of diabetes is associated with long-term dysfunction and

damage to various organs. Hence, there is a need to search for a medication for

lowering glucose as well as modify the alteration of key enzymes involved in

carbohydrate metabolism.

Hyperglycemia is closely associated with increased production of free radical

species and increased oxidative stress (Matsunami et al., 2010). Persistant

hyperglycemic status in diabetes and increased oxidative stress is associated with

altered glucose and lipid metabolism. Hyperglycemia causes oxidative stress and

tissue damage through 5 major mechanisms: (1) increased flux of glucose and other

sugars through the polyol pathway; (2) increased intracellular formation of AGEs

(advanced glycation end products); (3) increased expression of the receptor for AGEs

and its activating ligands; (4) activation of protein kinase C (PKC) isoforms; and (5)

overactivity of the hexosamine pathway. Oxidative stress plays a pivotal role in the

development of diabetes complications, both microvascular and macroovascular

(Baynes, 1991). Lipid peroxide mediated tissue damage has been observed in the

development of both the types of diabetes. A sophisticated enzymatic and

nonenzymatic antioxidant defense system including catalase (CAT), superoxide

dismutase (SOD) and reduced glutathione (GSH) counteracts and regulates overall

ROS levels to maintain physiological homeostasis. Increased concentration of

TBARS and the simultaneous decline in antioxidative defence mechanisms observed


109

in diabetic patients promotes the development of late complications (Karunakaran

and Park, 2013).

The pathogenesis of diabetes and its management by oral hypoglycemic agents

has stimulated great interest in recent years. Despite considerable progress in the

management of diabetes mellitus by synthetic drugs, the search for indigenous natural

anti-diabetic agents is still going on (Kania et al., 2013). Before the development of

modern pharmaceutical treatments, therapeutic capacity was completely dependent on

the use of medicinal herbs for prevention and treatment of diseases (Patel et al.,

2012a). Ethnobotanical information also indicates that more than 800 plants are used

as traditional remedies for treatment of diabetes throughout the world (Patel et al.,

2012b). There is still an unmet need for scientific proof of the antidiabetic activity of

medicinal plants and phytopharmaceuticals with fewer side effects. In view of this,

present study was taken up to explore antidiabetic potential of Syzygium cumini seeds

and Pterocarpus marsupium bark, and also to reduce the risk of late complications

and negative outcomes of diabetes which requires not only to control blood glucose

level but also to control oxidative stress.

Purification of alcoholic SC seed extract The alcoholic extract was subjected to silica gel chromatography and the

adsorbed compounds (mostly phenolics) were batch eluted with 100% methanol. The

eluted fraction was further purified on sephadex LH 20 beads and batch eluted with

different ratios of water and methanol (100% water, 70:30, 30:70,100% methanol).

Four fractions were obtained and tested for anti-hyperglycemic and anti-oxidative

activities. The fraction IV showing best results was further subjected to HPLC

column. Elution profile is given as figure 10. The eluted fraction was characterized as

caffeic acid. The anti-diabetic potential of caffeic acid was evaluated and results are

given below.

Purification of PM extract

The bark powder of PM was extracted with ethyl acetate and subjected to

silica gel beads. The fractions (mostly polyphenols) batch eluted with benzene were


110

pooled and lyophilized. The lyophilized material was re-suspended in minimum

volume of ethanol. The ethanol was evaporated and material was suspended in water

and dosed.

EXPERIMENTAL DESIGN

1- To evaluate the effect of sephadex LH 20 purified fractions of alcoholic

extract of SC on FBG levels.



Group II Normal + SC LH 20 purified fraction I (1.1mg/kg b.w./day)

Group III Normal + SC LH 20 purified fraction II (1.1mg/kg b.w./day)

Group IV Normal + SC LH 20 purified fraction III (1.1mg/kg b.w./day)

Group V Normal + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group VI Diabetic control

Group VII Diabetic + SC LH 20 purified fraction I (1.1mg/kg b.w./day)

Group VIII Diabetic + SC LH 20 purified fraction II (1.1mg/kg b.w./day)

Group IX Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)

Group X Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group XI Diabetic + Metformin (100 mg/kg b.w./day)

2- To evaluate the effect of purified PM extract on FBG levels in diabetic

rats.



Group II Normal + purified PM extract (1mg/kg b.w./day)

Group III Diabetic control

Group IV Diabetic + purified PM extract (1mg/kg b.w./day)


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Group V Diabetic + Metformin (100 mg/kg b.w./day)

3- To evaluate the in vitro anti-oxidant activity of the crude and LH 20

purified extracts of SC by DPPH radical scavenging assay.

I Radical scavenging activity of metformin

II Radical scavenging activity of crude aq. SC extract

III Radical scavenging activity of crude alc. SC extract

IV Radical scavenging activity of SC LH 20 purified fraction I

V Radical scavenging activity of SC LH 20 purified fraction II

VI Radical scavenging activity of SC LH 20 purified fraction III

VII Radical scavenging activity of SC LH 20 purified fraction IV

VIII Radical scavenging activity of BHT

4- To evaluate the in vitro anti-oxidant activity of the crude and purified

extracts of PM by DPPH radical scavenging assay.

I Radical scavenging activity of metformin

II Radical scavenging activity of crude aq. PM extract

III Radical scavenging activity of crude alc. PM extract

IV Radical scavenging activity of purified PM extract

V Radical scavenging activity of BHT

5- To evaluate the anti-oxidative effect of purified SC and PM extracts on




Group II Normal + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group III Normal + purified PM extract (1mg/kg b.w./day)



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Group V Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group VI Diabetic + purified PM extract (1mg/kg b.w./day)\



6- To evaluate the effect of SC and PM purified extracts on serum insulin

levels.



Group II Diabetic control

Group III Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)

Group IV Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group V Diabetic + purified PM extract (1mg/kg b.w./day)

7- To evaluate the effect of purified SC and PM extracts on the activities of

glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate

kinase) in heart, liver and kidney tissues of diabetic rats.



Group II Diabetic control

Group III Diabetic + SC LH 20 purified fraction III (1.1mg/kg b.w./day)

Group IV Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group V Diabetic + purified PM extract (1mg/kg b.w./day)

RESULTS

A- To evaluate the effect of sephadex LH 20 purified fractions of alcoholic

extract of SC on FBG levels.

Experiments were conducted to evaluate anti-hyperglycemic effect of


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sephadex LH 20 purified fractions of alcoholic extract of SC seeds. Rats were divided

into 11 groups according to the experimental design. The rats were fed with the four

fractions purified from sephadex LH 20 at a dose of 1.1mg/kg b.w./day. An

intraperitonial dose (150 mg/kg body weight) of alloxan increased FBG levels in

groups VI-XI after 4-5 days of injection. FBG levels were monitored on day 0 (when

rats were confirmed for diabetes) and day 30 (end of experiments). In diabetic control

group (VI), higher FBG level (>270 mg/dl) was maintained throughout the period of

study. On the other hand the oral dose of SC purified fractions resulted in decrease in

FBG levels in diabetic rats. The decrease in FBG levels on administration of fractions

I, II and III was from 435.4±16.3 to 406.8±15.7 md/dl, 444.2±20.3 to 389.6±12.9

mg/dl and 436.5±20.1 to 352±14.2 mg/dl, respectively. Better results were obtained

with fraction IV, which significantly (p<0.001) decreased the FBG level from

445.2±11.9 to 102.9±4.8 mg/dl (Table 1). Normal rats treated with the four fractions

did not show any significant changes in FBG levels as compared to normal control

(group I). Fraction IV showed significant decrease in FBG levels which was better

than metformin (group XI), standard anti-diabetic agent (Table 1).

B- To evaluate the effect of purified PM extract on FBG levels.

Experiments were conducted to evaluate anti-hyperglycemic effect of purified

extract of PM bark. Rats were divided into 5 groups according to the experimental

design. The rats were fed with the purified fraction of PM at a dose of 1mg/kg

b.w./day. An intraperitonial dose (150 mg/kg body weight) of alloxan increased FBG

levels in groups III-V after 4-5 days of injection. FBG levels were monitored on day 0

(when rats were confirmed for diabetes) and day 30 (end of experiments). In diabetic

control group (III), higher FBG level (>270 mg/dl) was maintained throughout the

period of study. On the other hand the oral dose of PM purified fraction resulted in

decrease in FBG levels in diabetic rats. The FBG level was significantly (p<0.001)

decreased from 413.4±16.8 to 161.7±11.3 mg/dl after treatment (Table 2). Normal rat

treated with the purified fraction of PM did not show any significant change in FBG

level as compared to normal control (group I). PM purified fraction showed

significant decrease in FBG levels which was better than metformin (group XI),

standard anti-diabetic agent (Table 2).


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C- To evaluate the in vitro anti-oxidant activity of the crude and purified

extracts of SC and PM by DPPH radical scavenging assay.

Alcoholic extract of SC seeds exhibited a strong antioxidant property which

was 2-3 fold better than aqueous extract. Better radical scavenging activity shown by

alcoholic SC extract as compared to the aqueous extracts, suggests that the anti-

oxidative component(s) are better extracted in alcoholic extract (fig. 1). All the four

LH-20 purified fractions showed strong antioxidant activity.

The aqueous and alcoholic extracts of PM did not show significant radical

scavenging activity, whereas the purified fraction caused 91% bleaching of purple

color of DPPH radical which confirms its radical scavenging and anti-oxidant activity

(fig. 2).

D- To evaluate anti-oxidative effect of purified SC and PM extracts in


Experiments were conducted to evaluate the effect of purified extracts of SC

(seeds) and PM (bark) on antioxidant (SOD, catalase and GST) activities, reduced

glutathione content and malondialdehyde levels in heart, liver and kidney issues. Rats

were divided into eight groups according to the experimental design. The rats were

fed with SC LH 20 purified fraction IV and PM purified fraction at doses of 1.1mg/kg

b.w./day and 1mg/kg b.w./day respectively for 30 days.

Cardiac MDA levels


(p<0.001) in cardiac tissue of diabetic rats. However the treatment of purified SC and

PM extracts did not show any significant change in normal rats. Treatment of diabetic

rats with SC LH 20 purified fraction IV resulted in significant decrease (p<0.001) in

elevated MDA levels from 105.30±10.93 to 51.09±4.13 nM/g tissue. Similarly,

treatment of diabetic rats with purified PM extract resulted in decrease in MDA levels

from 105.30±10.93 to 57.12±5.53 nM/g tissue. The beneficial effects of purified SC

and PM extracts on MDA levels were comparable to metformin (a standard anti-

diabetic agent) and vitamin C (Table 3).


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Cardiac GSH Content


protein (p<0.001) in cardiac tissue of diabetic rats when compared with normal rats.

Treatment of diabetic rats with SC LH 20 purified fraction IV and purified PM extract

resulted in significant increase (p<0.001) in GSH content from 0.49±0.08 to

1.11±0.12 µg/mg protein, and from 0.49±0.08 to 0.99±0.16 µg/mg protein,

respectively. However, there was no significant change observed in normal rats

treated with purified SC and PM extracts. The beneficial effects of purified SC and

PM extracts on GSH content were comparable to metformin (a standard anti-diabetic

agent) and vitamin C (Table 4).

Activities of cardiac antioxidant enzymes

Cardiac antioxidant enzyme activities are shown in figures 3-5. The activities

of antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,

p<0.001 and p<0.001) in cardiac tissue of diabetic rats (group IV) when compared

with normal rats (group I). The diabetic rats that received SC LH 20 purified fraction

IV showed a significant (p < 0.001) increase from 8.52±1.82 (diabetic control) to

24.11±1.85 U/mg protein in SOD activity. Treatment of diabetic rats with purified

PM bark extract increased the SOD activity from 8.52±1.82 (diabetic control) to

22.31±1.19 U/mg protein (fig. 3). The diabetic rats treated with SC LH 20 purified

fraction IV showed a significant (p < 0.001) reversal of decreased catalase activity

from 74.09±5.5 (diabetic control) to 119.91±5.4 U/mg protein. Treatment of diabetic

rats with purified PM bark extract increased the catalase activity from 74.09±5.5

(diabetic control) to 118.98±5.2 U/mg protein (fig. 4). The GST activity in diabetic

rats was significantly (p<0.001) decreased to 1.27±0.09 from 2.39±0.15 U/mg protein

observed in normal rats. The treatment of diabetic rats with SC LH 20 purified

fraction IV could result in significant improvement in decreased GST activity, as it

was increased from 1.27±0.09 to 2.26±0.08 U/mg protein after treatment. Treatment

of diabetic rats with purified PM bark extract increased the GST activity from

1.27±0.09 (diabetic control) to 2.05±0.07 U/mg protein (fig. 5). Normal rats treated

with purified extracts of SC and PM did not show any significant effect on antioxidant

enzyme activities when compared to normal control (group I). On the other hand


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administration of metformin and vitamin C to diabetic rats showed significant

(p<0.001) increase in antioxidant activities (SOD, catalase and GST) as compared

with diabetic control rats.

Hepatic MDA levels


(p<0.001) in hepatic tissue of diabetic rats. However the treatment of purified SC and





from 418.07±27.48 to 241.48±16.39 nM/g tissue. The beneficial effects of purified

SC and PM extracts on MDA levels were comparable to metformin (a standard anti-

diabetic agent) and vitamin C (Table 3).

Hepatic GSH Content


protein (p<0.001) in hepatic tissue of diabetic rats when compared with normal rats.








Activities of hepatic antioxidant enzymes

Hepatic antioxidant enzyme activities are shown in figures 3-5. The activities

of antioxidants (SOD, Catalase and GST) were significantly decreased (p<0.001,

p<0.001 and p<0.001) in hepatic tissue of diabetic rats (group IV) when compared

with normal rats (group I). The diabetic rats that received SC LH 20 purified fraction

IV showed a significant (p < 0.001) increase from 23.13±1.47 (diabetic control) to

48.81±2.46 U/mg protein in SOD activity. Treatment of diabetic rats with purified


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PM bark extract increased the SOD activity from 23.13±1.47 (diabetic control) to





(diabetic control) to 66.75±3.9 U/mg protein (fig. 4). The GST activity in diabetic rats

was significantly (p<0.001) decreased to 3.44±0.19 from 6.6±0.65 U/mg protein

observed in normal rats. The treatment of diabetic rats with SC LH 20 purified










Renal MDA levels


(p<0.001) in renal tissue of diabetic rats. However the treatment of purified SC and





from 80.32±7.63 to 55.61±4.98 nM/g tissue. The beneficial effects of purified SC and

PM extracts on MDA levels were comparable to metformin (a standard anti-diabetic


Renal GSH Content


protein (p<0.001) in renal tissue of diabetic rats when compared with normal rats.



118







Activities of renal antioxidant enzymes

Renal antioxidant enzyme activities are shown in figures 3-5. The activities of


p<0.001 and p<0.001) in renal tissue of diabetic rats (group IV) when compared with

normal rats (group I). The diabetic rats that received SC LH 20 purified fraction IV

showed a significant (p<0.001) increase from 8.51±1.16 (diabetic control) to

24.8±1.66 U/mg protein in SOD activity. Treatment of diabetic rats with purified PM

bark extract increased the SOD activity from 8.51±1.16 (diabetic control) to





(diabetic control) to 60.27±3.34 U/mg protein (fig. 4). The GST activity in diabetic

rats was significantly (p<0.001) decreased to 9.21±0.77 from 18.44±0.85 U/mg

protein observed in normal rats. The treatment of diabetic rats with SC LH 20 purified











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E- To evaluate the effect of purified SC and PM extracts on serum insulin

levels.

The serum insulin levels were significantly decreased in diabetic rats (0.068

ng/ml) as compared to normal rats (0.32 ng/ml), which were brought back to near

normal levels after treatment with SC sephadex LH-20 purified fractions III and IV.

Treatment of diabetic rats with fraction III restored the serum insulin levels from

0.068 to 0.152 ng/ml, whereas treatment with fraction IV restored the insulin levels

from 0.068 to 0.244 ng/ml. Thus fraction IV showed much better improvement in

serum insulin levels as compared to fraction III (fig. 6). The purified PM fraction

restored the serum insulin levels from 0.068 to 0.22 ng/ml.

F- To evaluate the effect of purified SC and PM extracts on the activities of

glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate

kinase) in heart, liver and kidney tissues of diabetic rats.

Activities of cardiac glycolytic enzymes

Cardiac glycolytic enzyme activities are shown in figures 7-9. The activities of

glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were

significantly decreased (p<0.001 in all three cases) in cardiac tissue of diabetic rats

(group II) when compared with normal rats (group I). The diabetic rats that received

SC LH 20 purified fractions III and IV showed a significant (p<0.001) increase in

hexokinase activity from 0.82±0.05 (diabetic control) to 0.91±0.03 and 1.15±0.07

mU/mg protein, respectively. Treatment of diabetic rats with purified PM bark extract

increased the hexokinase activity from 0.82±0.05 (diabetic control) to 1.2±0.05

mU/mg protein (fig. 7). The diabetic rats treated with SC LH 20 purified fractions III

and IV showed a significant (p < 0.001) reversal of decreased phosphofructokinase

activity from 0.73±0.09 (diabetic control) to 1.05±0.1 and 1.72±0.17 mU/mg protein

respectively. Treatment of diabetic rats with purified PM bark extract increased the

phosphofructokinase activity from 0.73±0.09 (diabetic control) to 1.51±0.12 mU/mg

protein (fig. 8). The pyruvate kinase activity in diabetic rats was significantly

(p<0.001) decreased to 0.47±0.04 from 1.2±0.15 mU/mg protein observed in normal

rats. The treatment of diabetic rats with SC LH 20 purified fractions III and IV could


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result in significant (p<0.001) improvement in decreased pyruvate kinase activity, as

it was increased from 0.47±0.04 to 0.69±0.1 and 0.91±0.07 mU/mg protein

respectively after treatment. Treatment of diabetic rats with purified PM bark extract

increased the pyruvate kinase activity from 0.47±0.04 (diabetic control) to 0.85±0.08

mU/mg protein (fig. 9).

Activities of hepatic glycolytic enzymes

Hepatic glycolytic enzyme activities are shown in figures 7-9. The activities of


significantly decreased (p<0.001 in all three cases) in hepatic tissue of diabetic rats




mU/mg protein respectively. Treatment of diabetic rats with purified PM bark extract















Activities of renal glycolytic enzymes

Renal glycolytic enzyme activities are shown in figures 7-9. The activities of



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significantly decreased (p<0.001 in all three cases) in renal tissue of diabetic rats




mU/mg protein respectively. Treatment of diabetic rats with purified PM bark extract















G- HPLC analyses of SC LH 20 purified fractions III and IV.

Fraction IV (lyophilized and re-suspended in water) was 5 times diluted and

subjected to HPLC analysis using C18 column. Gradient elution was carried out using

acetic acid and methanol as mobile phase for 30 min. A single peak was observed

which corresponded to caffeic acid (Fig. 10). Standard caffeic acid (100µg/ml) was

run using the same method (Fig. 11).


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Table 1 Effect of sephadex LH 20 purified fractions of alcoholic extract of SC

on FBG levels

Groups Treatments Fasting blood glucose levels (mg/dl)

I II III IV V VI VII VIII IX X XI

Normal Normal + SCLI Normal + SCLII Normal + SCLIII Normal + SCLIV Diabetic Diabetic + SCLI Diabetic + SCLII Diabetic + SCLIII Diabetic + SCLIV Diabetic + Metformin

Day 0 Day 30 98.8±3.7 100.6±6.1 92.3±5.5 95.3±4.1 94.1±4.7 97.5±3.4 97.1±5.1 89.5±4.7 99.7±5.2 95.1±3.4 453.6±22.9* 437.1±19.7* 435.4±16.3* 406.8±15.7 444.2±20.3* 389.6±12.9 436.5±20.1* 352.5±14.2* 445.2±11.9* 102.9±4.8* 389.9±4.6* 230.6±3.2*

SC LI, LII, LIII, LIV = Syzygium cumini LH 20 column purified fractions I-IV. *p<0.001.All values are expressed as mean ± SD. Group II - V were compared to group I; group VI was compared to group I; group VII - XI were compared to group VI.

Table 2 Effect of purified PM extract on FBG levels in diabetic rats

Groups Treatments Fasting blood glucose levels (mg/dl)

I II III IV V

Normal Normal + PM Pur. Diabetic. Diabetic + PM Pur. Diabetic+Metformin

Day 0 Day 30 98.8±3.7 100.6±6.1 100.2±3.9 103.1±5.9

453.6±22.9* 437.1±19.7* 413.4±16.8* 161.7±11.3* 389.9±4.6* 230.6±3.2*

PM Pur. = Purified Pterocarpus marsupium extract. *p<0.001. All values are expressed as mean ± SD. Group II was compared to group I; group III was compared to group I; group IV and V were compared to group III.


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Figure 1: In vitro anti-oxidant activity of the crude and purified extracts of SC by DPPH radical scavenging assay.

Figure 2: In vitro anti-oxidant activity of the crude and purified extracts of PM

by DPPH radical scavenging assay.


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Table 3 Effect of purified SC and PM extracts on MDA (nM/g tissue) levels in alloxan

induced diabetic rats



Normal Normal + SCLIV Normal + PM Pur. Diabetic Diabetic + SCLIV Diabetic + PM Pur. Diabetic + Metformin Diabetic + Vit C

Heart Liver Kidney 39.44±1.68 194.30±9.96 35.81±6.87 40.14±1.22 191.25±6.39 39.98±5.99 37.71±1.88 195.61±7.99 38.19±5.11 105.30±10.93* 418.07±27.48* 80.32±7.63* 51.09±4.13* 215.67±19.11* 53.98±4.12* 57.12±5.53* 241.48±16.39* 55.61±4.98* 78.90±4.11* 302.48±26.30* 59.83±4.13* 50.11±4.19* 210.19±15.11* 51.83±4.83*

All values are expressed as mean ± SD; Normal treated and diabetic rats were

compared with normal rats; diabetic treated rats were compared with diabetic rats;

*p<0.001.

Table 4: Effect of purified SC and PM extracts on GSH (µg/mg protein) content in alloxan induced diabetic rats



Normal Normal + SCLIV Normal + PM Pur. Diabetic Diabetic + SCLIV Diabetic + PM Pur. Diabetic + Metformin Diabetic + Vit C

Heart Liver Kidney 0.93±0.06 1.05±0.06 0.79±0.05 0.94±0.03 1.01±0.02 0.81±0.05 0.97±0.08 0.98±0.04 0.91±0.07 0.49±0.08* 0.64±0.03* 0.46±0.04* 1.11±0.12* 1.05±0.07* 0.78±0.06* 0.99±0.16* 1.00±0.05* 0.77±0.06* 0.76±0.09* 0.83±0.07* 0.65±0.06* 1.15±0.21* 1.08±0.07* 0.78±0.09*

All values are expressed as mean ± SD; Normal treated and diabetic rats were

compared with normal rats; diabetic treated rats were compared with diabetic rats;

*p<0.001.


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Figure 3: Effect of purified SC and PM extracts on SOD activity (U/mg protein) in alloxan induced diabetic rats.

SOD=Superoxide dismutase; 1U of SOD= 50% inhibition of auto-oxidation of

epinephrine/min; Normal treated and diabetic rats were compared with normal rats;

Diabetic treated rats were compared with diabetic rats.

Figure 4: Effect of purified SC and PM extracts on CAT activity (U/mg protein) in alloxan induced diabetic rats.

1U of Catalase= µmoles H2O2 decomposed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.


126

Figure 5: Effect of purified SC and PM extracts on GST activity (U/mg protein) in alloxan induced diabetic rats.

N=Normal, NSCLIV=Normal+SC LH 20 column purified fraction IV treated, NPMP=Normal+PM purified extract treated, D=Diabetic, DSCLIV=Diabetic+SC LH 20 column purified fraction IV treated, DPMP=Diabetic+PM purified extract treated, DMet=Diabetic+Metformin treated, DVitC=Diabetic+Vitamin C treated.

GST= Glutathione-s-transferase; 1 U of GST= µM GSH-CDNB complex formed/min; Normal treated and diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.

Figure 6: Effect of purified SC and PM extracts on serum insulin levels.

Diabetic rats were compared with normal rats; Diabetic treated rats were compared

with diabetic rats.


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Figure 7: Effect of purified SC and PM extracts on the activity of hexokinase (mU/mg protein) in heart, liver and kidney tissues of diabetic rats.


with diabetic rats.

Figure 8: Effect of purified SC and PM extracts on the activity of phosphofructokinase (mU/mg protein) in heart, liver and kidney tissues of

diabetic rats.


with diabetic rats.


128

Figure 9: Effect of purified SC and PM extracts on the activity of pyruvate kinase (mU/mg protein) in heart, liver and kidney tissues of diabetic rats.

N=Normal, D=Diabetic, DSCLIII=Diabetic+SC LH 20 purified fraction III treated, DSCLIV=Diabetic+SC LH 20 purified fraction IV treated, DPMP=Diabetic+PM purified extract treated. Diabetic rats were compared with normal rats; Diabetic treated rats were compared with diabetic rats.

Figure 10: HPLC analysis of sephadex LH 20 purified fraction 4.

AU

0.00

1.00

2.00

Minutes0.00 10.00 20.00 30.00


129

Figure 11: HPLC analysis of standard caffeic acid.

AU

0.00

0.50

1.00

Minutes0.00 10.00 20.00

DISCUSSION

Our results with crude extracts (aqueous and alcoholic) of SC showed that the

alcoholic extract has better anti-diabetic properties therefore, we further attempted to

purify alcoholic extract of SC using a combination of silica gel, sephadex LH 20 and

HPLC column chromatography. At LH 20 chromatography step, four fractions were

obtained and tested for their anti-diabetic potential. Among these four fractions, best

results were observed with fraction IV as the FBG level, 445.2±11.9 mg/dl in diabetic

group of rats was decreased to 102.9±4.8 mg/dl (Table 1). Fraction IV was further

subjected to HPLC column and a peak eluted as caffeic acid showed anti-diabetic

effect (figure 10). The studies carried out by Kumar et al., 2008 and Farzwan et al.,

2009 with purified SC compounds (eg. Mycaminose and Cuminoside) have been

reported to possess anti-hyperglycemic effect. Mycaminose, an anti-diabetic

compound isolated from methanolic extract of SC seeds, at a dose of 0.05 g kg/b.w.

resulted in a 28% decrease in elevated FBG levels after 15 days (Kumar et al., 2008).

Another compound, Cuminoside, a phenolic glycoside isolated from methanolic

extract of SC seeds, at a dose of 0.05 g kg/b.w. to diabetic rats led to a 61% reduction

in FBG (Farzwan et al., 2009). In another study, LH II fraction purified from

ethanolic seed extract of E. jambolana, using a combination of silica gel (60–120

mesh) and sephadex LH 20 chromatography, showed a significant anti-

hyperglycaemic effect in alloxan-induced diabetic rabbits. A 0.01 g kg/b.w. dose of


130

LH II fraction led to decreased glycosylated haemoglobin and elevated plasma insulin

levels (Sharma et al., 2011). The purified SC fraction IV in our study showed 77%

decrease in FBG levels which is better than the results obtained in previous studies.

The possible mechanism of decrease in blood glucose level may be by potentiation of

the insulin effect by increasing either the pancreatic secretion of insulin from β-cells

of the islets of Langerhans or its release from the bound form. A number of other

plants have been reported to exert hypoglycemic activity through insulin release-

stimulatory effects (Gupta, 1994). Inhibition of pancreatic α-amylase activity by

aqueous SC seed extract has been reported to be a possible mechanism for its anti-

diabetic action (Karthik et al., 2008).

We further evaluated the effect of SC purified fraction III and IV on serum

insulin levels (figure 6). The serum insulin levels were decreased by 78% (from 0.32-

0.068 ng/ml) in diabetic rats as compared to normal rats. Treatment of diabetic rats

with SC fraction III and IV restored the insulin levels from 0.068 to 0.152 and 0.244

ng/ml respectively. The present study demonstrated a 3.5 fold increase in insulin

levels after SC fraction IV treatment which was in accordance with the previous

results. Ravi et al. showed 3.2 fold increase in plasma insulin levels after treatment of

diabetic rats with crude alcoholic SC extract (Ravi et al., 2004). Sharma et al. showed

a 3 fold increase in insulin levels in diabetic rats treated with SC purified LH II

fraction (Sharma et al., 2011). The anti-hyperglycaemic activity of SC might be

possibly due to either stimulation of insulin release from β cells or insulin mimetic

activity of some components, resulting in direct peripheral glucose uptake, or due to a

combination of the two (Lolitkar and Rao. 1966).

Insulin acts is an activator of key glycolytic enzymes and a suppressor of

gluconeogenic enzymes (Sharma et al., 2011), therefore, we further evaluated the

effect of SC purified fraction III and IV on the activities of glycolytic enzymes,

hexokinase, phosphofructokinase and pyruvate kinase in heart liver and kidney tissues

of diabetic rats (figure 7,8,9). Better results were obtained with fraction IV. The

activity of hexokinase was decreased by 48%, 66% and 65% in heart liver and kidney

tissues of diabetic rats, which was recovered after treatment with SC fraction IV. The

activity of phosphofructokinase was decreased by 68%, 66% and 65% in heart liver


131

and kidney tissues of diabetic rats, which was recovered after treatment with SC

fraction IV. The activity of pyruvate kinase was decreased by 60%, 72% and 74% in

heart liver and kidney tissues of diabetic rats, which was recovered after treatment

with SC fraction IV. In previous studies, aqueous extract of SC resulted in 1.6 and 1.4

fold increase in hepatic hexokinase and phosphofructokinase activity respectively

(Prince et al., 1997; Grover et al., 2000). In the study carried out by Sharma et al., the

LH II fraction purified from ethanolic seed extract of SC restored the decreased

activities of glycolytic enzymes (glucokinase and phosphofructokinase) and increased

the activities of gluconeogenic enzymes (glucose-6- phosphatase and fructose-1,6-

bisphosphatase) to their normal levels in alloxan induced diabetic rabbits. Thus LH II-

induced activation of glycolysis resulted in increased consumption of glucose by

restoring insulin secretion in treated rabbits (Sharma et al., 2011). The anti-

hyperglycemic activity of caffeic acid has been attributed to supressed hepatic glucose

output due to enhanced hepatic glucose utilization and inhibited glucose over-

production (Jung et al., 2006). Kasetti et al. identified the anti-diabetic fraction C

purified from aqueous extract of Syzygium alternifolium as cinnamic acid which

increased the activity of hexokinase in liver and kidney tissues by 1.6 and 2.7 fold

respectively (Kasetti et al., 2012). These results were comparable with the present

study, as fraction IV purified as caffeic acid resulted in increase in hexokinase activity

by 2.6 and 2.3 fold in liver and kidney tissues respectively, as compared to diabetic

rats. Therefore the possible mechanism for decreased FBG levels of diabetic rats after

treatment of purified SC fraction IV might be due to regeneration of pancreartic β

cells and stimulation of insulin secretion, which in turn activates the enzymes of

glycolytic pathway and increases peripheral glucose utilization, thus lowers blood

glucose levels.

The in vivo antioxidant activity of crude aqueous and alcoholic extracts of SC

has been discussed in chapter I. The crude extracts and the four fractions obtained

after sephadex LH 20 chromatography were tested for their in vitro antioxidant

activity by DPPH radical scavenging assay (figure 1). Fraction IV showed maximum

radical scavenging activity and was further selected for in vivo study. Ahmed et al.,

2010 have reported in vitro radical scavenging and anti-lipidperoxidative effects of


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Eugenia jambolana aqueous extracts.Tannins extracted from SC fruit showed good

DPPH radical scavenging activity thus indicating the utilization of the fruit of SC as a

significant source of natural antioxidants (Zhang and Lin, 2009).

The effect of SC purified fraction IV (caffeic acid) was studied on antioxidant

parameters (MDA levels, GSH content and SOD, CAT, GST activities) in heart liver

and kidney tissues of diabetic rats (figure 3,4,5). The MDA levels were significantly

increased in diabetic rats as compared to normal rats which were brought back to near

normal levels after SC fraction IV treatment. The depleted GSH content and

decreased antioxidant enzymes activities in diabetic rats were also restored after SC

fraction IV treatment. Caffeic acid has been reported to decrease oxytetracyclin

induced oxidative stress in liver tissue, where it increased the activities of antioxidant

enzymes SOD and CAT by 1.5 and 1.3 fold respectively. The GSH content was

increased by 2.4 fold and TBARS levels were decreased by 1.8 fold (Jayanthi and

Subash, 2010). Yucel et al., 2012 reported that caffeic acid could reduce the elevated

MDA levels in sciatic nerve tissues of diabetic rats by 1.14 fold. The compound

cuminoside isolated from methanolic extract of SC increased the GSH content and

activities of SOD and CAT by 1.14, 1.06 and 1.09 fold respectively, whereas lipid

peroxidation was decreased by 1.4 fold in liver tissue of diabetic rats (Farzwan et al.,

2009). The antioxidative effect shown by SC LH 20 purified fraction IV in our study

was slightly better than the previous studies as there was a 2 fold decrease in MDA

levels and the GSH content, SOD and CAT activities were increased by 1.64, 2.1 and

1.61 fold respectively in the liver tissues of diabetic rats. Similar results were

observed in the heart and kidney tissues.

Diabetes induced oxidative stress has already been discussed in chapter 1.

SOD plays an important role in protecting cells from oxidative damage by converting

superoxide radicals into hydrogen peroxide, which is then further metabolized by

CAT and GSH-Px, where CAT detoxifies hydrogen peroxide and GSH-Px catalyzes

the destruction of hydrogen peroxide and lipid hydroperoxide. If the CAT and GSH-

Px activity is not sufficiently enhanced to metabolize hydrogen peroxide, this can lead

to an increased hydrogen peroxide and TBARS levels (Haron, 1991).


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It has been hypothesized that caffeic acid protects the rats from oxidative

stress by scavenging the free radicals. Clinical complications in oxidative stress

related diseases might be due to the dysfunctioning of key antioxidant enzymes. SOD,

CAT, GST and GPx are among those enzymes which metabolize endogenous reactive

oxygen species and free radicals. In the present study, the decreased levels of SOD,

CAT and GST were observed in diabetic rats. The ability of caffeic acid to increase

the activities of antioxidant enzymes in diabetic rats implies that caffeic acid

reactivates the antioxidant defence system, thereby increasing the capacity of

detoxification through the enhanced scavenging of free radicals. Caffeic acid

supplement resulted in dramatic increase in the antioxidant enzyme activities and

mRNA levels in both erythrocyte and liver compared with the control group (Jung et

al., 2006). Low levels of antioxidants have been implicated as a risk factor for the

development of liver toxic injury (Maddrey, 2005). An antioxidant should be efficient

and protective, preferably by inhibiting the lipid peroxidation process, where most of

the oxidative damage occurs. Normally the phenolic compounds have been reported

to act by scavenging free radicals and quenching the lipid peroxidation (Nardini et al.,

1998). Caffeic acid has the ability to scavenge the free radicals and attenuate the lipid

peroxidation as indicated by increased levels of enzymic antioxidants (Ozyurt et al.,

2006), our results also support these findings. Thus our study suggests that caffeic

acid (Fig. 12) can prevent diabetes induced complications. This may be attributed to

the antiradical activity of caffeic acid which inhibits lipid peroxidation and enhances

the antioxidant defence against diabetes induced oxidative damage in tissues.

Figure 12: Structure of caffeic acid.


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The results for the anti-hyperglycemic activity of crude aqueous and alcoholic

extracts of PM have been discussed in chapter 1. Aqueous extract of PM showed

better anti-diabetic properties. The bark powder of PM was extracted with ethyl

acetate and subjected to silica gel beads. The fractions (mostly polyphenols) batch

eluted with benzene were pooled and lyophilized. The lyophilized material was re-

suspended in minimum volume of ethanol. The ethanol was evaporated and material

was suspended in water and dosed. The FBG levels were elevated from 100.6±6.1 to

437.1±19.7 mg/dl in alloxan induced diabetic rats. After treatment with purified PM

extract the FBG levels were lowered by 63% (Table 2). An active constituent of PM,

epicatechin, has been reported to reverse hyperglycemia in alloxan induced diabetic

rats, when given before or within 24 hrs after the dose of alloxan (Sheehan et al.,

1983). The anti-hyperglycemic effect of phenolics isolated from PM heartwood has

been studied and 42 % decrease in FBG levels was observed after treatment

(Manickam et al., 1997). The possible mechanism of anti-hyperglycemic effect shown

by purified compounds of PM might be due to the regeneration of damaged β cells

and stimulation of insulin secretion (Sheehan et al., 1983).

We also found that, depleted serum insulin levels in diabetic rats were restored

from 0.068 to 0.22 ng/ml after treatment with purified PM extract (figure 6). The

decreased activities of glycolytic enzymes (hexokinase, phosphofructokinase and

pyruvate kinase) in diabetic rats were also significantly increased after treatment

(figure 7,8,9). Pari and Sathesh, 2006 observed a significant decrease in glucose and

significant increase in plasma insulin levels in diabetic rats treated with pterostilbene,

a polyphenolic compound purified from PM. Grover et al., 2002 evaluated the effect

of aqueous extract of PM on the activities of glycolytic enzymes and observed 2.1 and

1.6 fold increase in activities of hepatic hexokinase and phosphofructokinase, whereas

in our study 2.5 and 2.3 fold increase was observed in activities of hepatic hexokinase

and phosphofructokinase in diabetic rats. The activities of the hepatic enzymes such

as hexokinase was significantly increased whereas glucose-6-phosphatase and

fructose-1,6-bisphosphatase were significantly decreased by the administration

of pterostilbene in diabetic rats (Pari and Sathesh, 2006). Therefore, the possible

mechanism for decreased FBG levels of diabetic rats after treatment with purified PM


135

extract might be due to regeneration of pancreartic β cells and stimulation of insulin

secretion. Insulin further activates the enzymes of glycolytic pathway and increases

peripheral glucose utilization, thus lowers blood glucose levels.

The in vivo antioxidant activity of crude aqueous and alcoholic extracts of PM

has been observed in chapter 1. The crude aqueous and alcoholic extracts and the

purified fraction were tested for their in vitro antioxidant activity by DPPH radical

scavenging assay (figure 2). The purified fraction showed the maximum radical

scavenging activity in vitro and was analyzed for its in vivo antioxidant potential.

The effect of purified PM fraction was studied on antioxidant parameters

(MDA levels, GSH content and SOD, CAT, GST activities) in heart liver and kidney

tissues of diabetic rats (figure 3,4,5). The MDA levels were significantly increased in

diabetic rats as compared to normal rats which were brought back to near normal

levels after treatment. The depleted GSH content and decreased antioxidant enzymes

activities in diabetic rats were also restored after treatment with PM purified fraction.

Attempts were made to purify compound(s) from PM extract using silica gel

chromatography (Mallavadhani and Sahu, 2003). The purified fraction showed

antioxidant potential (McCormack and McFadden, 2013). The peroxyl-radical

scavenging activity of pterostilbene was found to be same as that of resveratrol.

Pterostilbene also was shown to be as effective as resveratrol in inhibiting electrolyte

leakage caused by herbicide-induced oxidative damage, and both compounds had the

same activity as α-tocopherol. (Rimando et al., 2002). Pterostilbene exhibited a

concentration-dependent antioxidant capacity measured by the ABTS method, that

relies on the inhibition of the oxidation of ABTS (2,2′-azino-di-[3-ethylbenzthiazoline

sulphonate]) to ABTS·+ by metmyoglobin (Remsberg et al., 2008). In the present

study the compound purified from PM extract showed antioxidative activity which

was confirmed by both in vivo and in vitro data.

The standard anti-diabetic drug, metformin showed antioxidative effect in vivo

but not in vitro (figure 1,2), which suggests that the antioxidative effect shown by

metformin in vivo might be due to its normoglycemic effect. The purified extracts

showed antioxidative acivity both in vivo and in vitro, which suggests the presence of

constituents in these extracts that directly affect oxidative defense system.

Chapter-III

Immunosuppression in diabetes and immunomodulatory properties of Syzygium

cumini and Pterocarpus marsupium

Chapter III: Immunosuppression in diabetes and immunomodulatory properties of SC and PM

136

Diabetes is widely believed to predispose to serious infections. Experimental

clinical literature supports an association between diabetes and infection (Peleg et al.,

2007). Individuals with diabetes might be at a higher risk of moderate or severe

infection-related morbidity caused by altered defence mechanisms. It was shown that

individuals with diabetes have poorer outcomes after infection and increased

incidence of nosocomial infection compared with normal subjects (Bertoni et al.,

2001). However, the mechanisms linking diabetes and immunosuppression are not

well defined.

One potential mediator of the altered defence mechanisms is hyperglycaemia

(McMahon and Bistrian, 1995). Hyperglycaemia has been identified as the main

factor contributing to the development of diseases associated with diabetes mellitus

and intensive treatment of people with insulin-dependent diabetes mellitus can delay

the onset and slow the progression of diabetic retinopathy, nephropathy, neuropathy,

and micro- and macrovascular complications, or prevent diabetic cardiovascular

disease (Joshi et al., 1999). Although it is difficult to prove a similar causal relation

between hyperglycaemia and infection, there are substantial clinical data supporting

this hypothesis (McMahon and Bistrian, 1995). Moreover, uncontrolled

hyperglycaemia may be associated with increased risk of nosocomial infections in

critically affected patients, even in those without a history of diabetes (Butler et al.,

2005).

Various pathophysiological and biochemical mechanisms have been proposed

to explain the adverse effects of hyperglycaemia. In recent years, an increasing

number of reports have shown that enhanced oxidative stress is a key factor in the

development of abnormalities in diabetes (Baynes, 1991). Reactive oxygen species

(ROS) are by-products of normal metabolic processes in the cell. At low

concentrations, ROS are thought to have some physiological roles (Schreck and

Baeuerle, 1991), including the activation and proliferation of lymphocytes (Whitacre

and Cathcart, 1992). However, at high concentrations they lead to oxidative stress and

cause damage to cellular components including immune system (Schreck and

Baeuerle, 1991). ROS can function as signalling molecules to activate a number of

cellular stress-sensitive pathways that cause cellular damage and they are proposed to


137

be responsible for the late complications of diabetes (Evans et al., 2003).

Hyperglycaemia generates ROS, attenuates antioxidant mechanisms, creating a state

of oxidative stress (Vincent et al., 2004). Enhanced oxidative stress is proposed to be

a major cause of diabetic endothelial dysfunctions (Rodríguez-Mañas et al., 2003),

nephropathy (Lee et al., 2003) and neuropathy (Vincent et al., 2004). Concerning

lymphocyte function, it has been suggested that oxidative stress may play an

important pathogenic role in the development of immunodeficiency (Robinson et al.,

1993).

The anti-hyperglycemic and anti-oxidative properties of the medicinal plants,

Syzygium cumini and Pterocarpus marsupium, have already been described (chapter 1

and 2). In this chapter the immunomodulatory property of these plants has been

studied by analysing the effect of SC seed extract and PM bark extract on humoral

immune response in normal and diabetic rats. The modulation in immune response

has been explained by measuring the serum levels of some of the immunomodulatory

cytokines before and after the herbal therapy in normal and diabetic rats.

EXPERIMENTAL DESIGN

1- Evaluation of antibody (IgG) response in normal and treated rats

immunized with tetanus toxoid.


Group I Normal immunized (100µg/rat)

Group II Normal immunized + PM bark extract treated (200mg/kg

b.w./day)

Group III Normal immunized + SC seed extract treated (3g/kg b.w./day)

2- Evaluation of antibody (IgG) response in normal and treated rats

immunized with E.coli LPS.





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b.w./day)


3- Evaluation of antibody (IgM) response in normal and treated rats





b.w./day)


4- Evaluation of serum levels of IL-4 (pg/ml) in normal and treated rats

immunized with tetanus toxoid and E.coli LPS.


Group I Normal un-immunized

Group II Normal immunized (100µg/rat)

Group III Normal immunized + PM bark extract treated (200mg/kg

b.w./day)

Group IV Normal immunized + SC seed extract treated (3g/kg b.w./day)

5- Evaluation of the effect of purified SC and PM extracts on serum IL-2,

IL-4, IL-6 and IL-10 levels (pg/ml) in diabetic rats.


Group I Normal

Group II Diabetic

Group III Diabetic + SC LH 20 purified fraction IV (1.1mg/kg b.w./day)

Group IV Diabetic + PM purified fraction (1mg/kg b.w./day)


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RESULTS

A. Evaluation of antibody (IgG) response in normal and treated rats

immunized with tetanus toxoid (TT).

Experiments were conducted to evaluate the effect of SC seed and PM bark

extracts on antibody (IgG) levels in rats immunized with thymus dependent antigen,

TT at a dose of 100µg/ml. Four immunizations were performed and sera were

collected after 7 days of each immunization. Rats were divided into three groups.

Normal immunized (NI) rats were not given any treatment whereas the treated rats

were fed with SC and PM extracts at a dose of 3g/kg b.w./day and 200mg/kg b.w./day

respectively. The ELISA values (absorbance at 450nm) are directly proportional to

serum antibody levels at constant antigenic concentration. The anti-TT IgG levels

showed 2-4 fold increase after III immunization in different groups of rats, which

became constant after III immunization. This effect was more pronounced in groups

of rats treated with plant extracts of PM and SC (group II and III) suggesting that the

treatment of rats with these plant extracts resulted in immunostimulation (fig. 1A-D).

B. Evaluation of antibody (IgG) response in normal and treated rats



extracts on antibody (IgG) levels in rats immunized with thymus independent antigen,

LPS at a dose of 100µg/ml. Four immunizations were performed and sera were





serum antibody levels at constant antigenic concentration. Normal rats immunized

(NI) with E.coli LPS did not show significant increase in anti-LPS IgG after II, III and

IV immunizations (fig 2A-D), although, there was slightly better immune response

(Anti-LPS IgG) in groups of immunized rats which received plant extracts of SC and

PM in comparison to NI rats (Fig. 2A-D). But the ELISA values of anti-LPS IgG


140

measured against LPS were 3-4 fold lower than what has been observed in case of

anti-TT IgG (Fig.1A-D).

C. Evaluation of antibody (IgM) response in normal and treated rats



extracts on antibody (IgM) levels in rats immunized with thymus independent antigen,

LPS at a dose of 100µg/ml. Four immunizations were performed and sera were





serum antibody levels at constant antigenic concentration. The sera obtained from rats

immunized with E.coli LPS were tested for the presence of IgM levels and almost

constant IgM levels were observed in group I rats (NI). The groups immunized with

E.coli LPS and treated with the plant extracts of PM and SC (group II and III) showed

slight but insignificant decrease in anti-LPS IgM (Fig.3A-D).

D. Evaluation of serum levels of IL-4 in normal and treated rats immunized

with tetanus toxoid and E.coli LPS.


extracts on serum IL-4 levels in rats immunized with thymus dependent (TT) and

independent antigens (LPS). The rats were immunized with TT and LPS at doses of

100µg/ml. Rats were divided into four groups according to the experimental design.



respectively. The group II rats (NI) immunized with TT showed increase in IL-4

levels from 12.23±2.1 to 18.43±2.3 pg/ml when compared with serum IL-4 levels of

normal unimmunized rats (group I). On the other hand, immunized rats treated with

plant extracts of PM and SC (group III and IV) increased serum IL-4 levels from

18.43±2.3 to 22.23±3.7 and 20.98±3.1 respectively. The group II rats (NI) immunized

with E.coli LPS did not show any significant increase in serum IL-4 levels whereas

group III and IV rats immunized with LPS and treated with plant extracts of PM and


141

SC showed only about 1.2 fold increase (from 12.92±3.1 to 15.63±3.2 and 15.98±2.4

pg/ml respectively) in serum IL-4 levels which was lesser than IL-4 level increase in

rats immunized with TT (Fig. 4).

E. Evaluation of the effect of purified SC and PM extracts on serum IL-2,

IL-4, IL-6 and IL-10 levels (pg/ml) in diabetic rats.

Experiments were conducted to evaluate the effect of purified extracts of SC

seed and PM bark on serum cytokine levels of normal and diabetic rats. Rats were

divided into four groups according to the experimental design. An intraperitonial dose

(150 mg/kg body weight) of alloxan increased FBG levels (>270 mg/dl) in groups II-

IV after 4-5 days of injection. The rats were fed with the purified fractions of SC and

PM at doses of 1.1 and 1mg/kg b.w./day respectively. The level of IL-2 was

significantly decreased in diabetic rats from 10.3±0.5 to 2.3±0.4 pg/ml as compared to

normal rats. After treatment of diabetic rats with purified SC and PM extracts the IL-2

levels were increased from 2.3±0.4 to 7.0±0.33 and 5.8±0.22 pg/ml respectively (Fig.

5A). The level of IL-4 was significantly decreased in diabetic rats from 12.3±0.6 to

4.67±0.35 pg/ml as compared to normal rats. The treatment of diabetic rats with

purified SC and PM extracts resulted in increased IL-4 levels from 4.67±0.35 to

9.2±0.7 and 8.1±0.45 pg/ml respectively (Fig. 5B). The level of IL-6 was significantly

decreased in diabetic rats from 22.3±1.45 to 5.93±0.8 pg/ml as compared to normal

rats. After treatment of diabetic rats with purified SC and PM extracts the IL-6 levels

were increased from 5.93±0.8 to 16.3±0.95 and 14.3±0.67 pg/ml respectively (Fig.

5C). The level of IL-10 was significantly increased in diabetic rats from 4.25±0.25 to

11.3±0.61 pg/ml as compared to normal rats. After treatment of diabetic rats with

purified SC and PM extracts the IL-10 levels were decreased from 11.3±0.61 to

6.3±0.34 and 7.7±0.56 pg/ml respectively (Fig. 5D).


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Figure 1: Antibody (IgG) response in normal and treated rats immunized with tetanus toxoid

A B

C D


143

Figure 2: Antibody (IgG) response in normal and treated rats immunized with E.coli LPS.

A B

C D


144

Figure 3: Antibody (IgM) response in normal and treated rats immunized with E.coli LPS.

A B

C D


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Figure 4: Serum levels of IL-4 in normal and treated rats immunized with Tetanus toxoid and E.coli LPS.

Figure 5: Effect of purified SC and PM extracts on serum IL-2, IL-4, IL-6 levels and IL-10 (pg/ml) in diabetic rats.

A

B


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C

D

DISCUSSION

Various plants identified in the Indian Ayurvedic system of medicine display a

wealth of pharmacological properties. The ayurvedic system of medicine is one of the

oldest systems of medicine and includes various ethnopharmacological activities such

as immunostimulation, tonic, neurostimulation, anti-ageing, antibacterial, antiviral,

antirheumatic, anticancer, adaptogenic, etc.(Agarwal and Singh, 1999) An entire

section of the Materia Medica of Ayurveda is devoted to “Rasayana”, drugs reputed to

enhance body resistance (Gulati et al., 2002). Listed as a class in the texts of

traditional Indian Medicine literature, Rasayana consists of a number of plants reputed

to promote physical and mental health, improve defense mechanisms of the body and

enhance longevity. These attributes are similar to the modern concept of adaptogenic

agents, which are known to afford protection of the human physiological system

against diverse stresses (Bhattacharya et al., 2000). A number of medicinal plants as


147

Rasayanas have been claimed to possess immunomodulatory activity, e.g., Withania

somnifera, Tinospora cordifolia, and Mangifera indica (Davis and Kuttan, 2000;

Singh et al., 2004; Makare et al., 2001). They can depress or potentiate the host’s

capacity to resist infection and tumors non-specifically, or react specifically to a

foreign substance. Immunomodulators act on the complex network of mechanisms of

the immune system in a way not yet fully elucidated.

In the present study the effect of plant extracts of SC and PM was observed on

immune system. Rats were immunized with tetanus toxoid (TT), a thymus dependent

antigen and E.coli LPS, a thymus independent antigen. Four immunizations were

performed with each antigen and the sera were tested for the presence of antibodies

(IgM and IgG) and IL-4 levels by ELISA. Rats immunized with TT showed typical

immune response i.e. increase in IgG levels were observed in different groups of rats

after each immunization and became constant after III immunization. Typical immune

response means that there was activation and proliferation of antigen specific B

lymphocytes and their consequent differentiation to plasma cells and memory cells.

The anti-TT IgG levels (ELISA values) showed 2-4 fold increase after III

immunization in different groups of rats, which became constant after III

immunization. This suggested the formation of sufficient memory cells at this stage.

The effect was more pronounced in groups of rats treated with plant extracts of PM

and SC (group II and III) suggesting that the treatment of rats with these plant extracts

resulted in immunostimulation (Fig. 1A-D).

In the next experiment, rats were immunized with thymus independent antigen

i.e. E.coli LPS. Four immunizations were performed and sera were collected after 7

days of each immunization. Rats were divided into different groups. Normal rats

immunized (NI) with E.coli LPS show less significant increase in anti-LPS IgG

(ELISA values) after II, III and IV immunizations (Fig. 2A-D) as compared to the

results obtained from TT immunization. Although, there was slightly better immune

response (anti-LPS IgG) in groups of immunized rats which received plant extracts of

SC and PM in comparison to NI rats (Fig. 2A-D). But the ELISA values of anti-LPS

IgG tested against LPS were 3-4 fold lower than what has been observed in case of

testing of anti-TT IgG against TT antigen (Fig.1A-D). The animals taken for


148

immunization studies of TT and E.coli LPS were having almost similar level of serum

protein before treatment.

When the sera obtained from rats immunized with E.coli LPS were tested for

the presence of IgM levels, almost constant IgM levels were observed in group I rats

(NI). The groups immunized with E.coli LPS and treated with the plant extracts of

PM and SC (group II and III) showed slight but insignificant decrease in anti-LPS

IgM (Fig. 3A-D).

Depending on nature of antigen, B cell activation proceeds by two different

routes, one dependent on TH cells, the other not. The B cell response to thymus

dependent (TD) antigens requires direct contact with TH cells, not simply exposure to

TH derived cytokines. Antigens that can activate B cells in absence of this kind of

direct participation of TH cells are known as thymus independent (TI) antigens. E.coli

LPS which is a TI-1 antigen is a polyclonal B cell activator (mitogen), it can activate

B cells regardless of its antigenic specificity. The response to TI antigens are

generally weaker and IgM is the predominant antibody secreted, reflecting a low level

of class switching. This highlights the important role played by TH cells in generating

memory B cells, affinity maturation and class switching to other isotypes. An

effective signal for B cell activation involves two distinct signals induced by

membrane events. Binding of a TI-1 antigen to B cell provides both the signals. A TD

antigen provides signal 1 by cross linking B cells but a separate interaction between

CD 40 on the B cell and CD 40L on activated TH is required to generate signal 2. The

expression of CD 40 on B cells has been reported to be stimulated by IL-2 (Goldsby

et al., 2003).

The data of above three experiments suggests that the rats immunized with

thymus dependent antigen TT showed activation, proliferation and differentiation of

B lymphocytes, followed by class switching (IgM→IgG) whereas the rats immunized

with E.coli LPS showed activation and proliferation of B lymphocytes without proper

class switching (IgM→IgG).

These findings were further confirmed by measuring the serum levels of IL-4

in different groups of rats immunized with TT and E.coli LPS. The group II rats (NI)

immunized with TT showed 1.5 fold increase in IL-4 when compared with serum IL-4


149

levels of normal unimmunized rats (group I). On the other hand, immunized rats

treated with plant extracts (group III and IV) showed about 1.7-1.8 fold higher serum

IL-4 levels. This confirmed the interaction of T cell component of immune system

with B cell component as IL-4 secreted by TH2 lymphocytes is involved in class

switching from IgM to IgG in rats immunized with thymus dependent antigen TT.

The group II rats (NI) immunized with E.coli LPS did not show any significant

increase in serum IL-4 levels whereas group III and IV rats immunized and treated

with plant extracts of PM and SC showed only about 1.2 fold increase in serum IL-4

levels which was lesser than IL-4 level increase in rats immunized with TT (Fig. 4).

The data suggests that E.coli LPS could not result in sufficient increase in

serum IL-4 levels and resulting in no or insignificant enhancement and class

switching in group II rats (NI). The treatment of rats (immunized with LPS) with

plant extracts of SC and PM resulted in some increase (1.2 fold) in serum IL-4 levels

and better immune response. It can be concluded that thymus dependent (TT) and

thymus independent (LPS) interact and activate immune system differently which

may be dependent on different serum cytokine levels, such effect of IL-4 has been

demonstrated in our study.

Diabetes has been reported to be associated with immunosuppression leading

to serious infections in previous studies. For example, tuberculosis was a major cause

of death among patients with diabetes mellitus before the advent of insulin therapy

(Johnson,1970). Thus, depression of the natural defenses against infection in diabetics

has long been suspected and investigated. This altered susceptibility to infection has

been ascribed to a depression in the function of polymorphonuclear leukocytes

(Badgdade, 1974). Impairment of chemotaxis or mobilization of polymorphonuclear

leukocytes has also been described (Mowat and Baum, 1971). With respect to

immunological mechanisms, the results are conflicting (Johnson,1970). Bates and

Weiss reported that children with poorly controlled diabetes showed a diminished

production of staphylococcal antibody (Bates and Weiss, 1941). Similarly, low

antibody titers against typhoid vaccine were observed in certain diabetic patients

(Richardson, 1933). Depressed reactivity of diabetic human lymphocytes to

phytohemagglutinin stimulation has also been reported (MacCuish et al, 1974).


150

Mahmoud et al. demonstrated depressed cellular immunological reactivity against

murine Schistosoma mansoni and allograft skin rejection in experimentally induced

diabetic mice (Mahmoud et al, 1976).

Activated TH cells secrete different cytokines of adaptive immunity including

IL-4, IL-2 having role in B cell proliferation and class switching, IL-6 plays key role

in linking innate and adaptive immunity and B cell maturation, and is a pro-

inflammatory cytokine. In the present study the effect of purified SC and PM extracts

on serum cytokine levels (IL-2, IL-4, IL-6, IL-10) of diabetic rats was evaluated. T

Lymphocytes regulate the growth and differentiation of T cells and certain B cells

through the release of secreted protein factors. These factors include interleukin 2 (IL-

2), which are secreted by lectin- or antigen-stimulated T cells and have various

physiological effects. IL-2 is a lymphokine that induces the proliferation of responsive

T cells. In addition, it acts on some B cells, via receptor-specific binding as a growth

factor and antibody production stimulant. The level of IL-2 was significantly

decreased in diabetic rats from 10.3±0.5 to 2.3±0.4 pg/ml as compared to normal rats.

After treatment of diabetic rats with purified SC and PM extracts the IL-2 levels were

increased by 3 and 2.5 folds respectively (Fig. 5A).

Interleukin 4 (IL-4) is produced by CD4 T cells specialized in providing help

to B cells to proliferate and to undergo class switch recombination and somatic

hypermutation. TH2 cells, through production of IL-4, have an important function in

B-cell responses that involve class switch recombination to the IgG1 and IgE

isotypes. The level of IL-4 was significantly decreased in diabetic rats from 12.3±0.6

to 4.67±0.35 pg/ml as compared to normal rats. After treatment of diabetic rats with

purified SC and PM extracts the IL-4 levels were increased by 1.97 and 1.73 folds

respectively (Fig. 5B).

Interleukin 6 (IL-6), also referred to as B-cell stimulatory factor-2 (BSF-2) and

interferon beta-2, is a cytokine involved in a wide variety of biological functions. It

plays an essential role in the final differentiation of B cells into IG-secreting cells, as

well as inducing myeloma/plasmacytoma growth, nerve cell differentiation and in

hepatocytes, acute-phase reactants. The level of IL-6 was significantly decreased in

diabetic rats from 22.3±1.45 to 5.93±0.8 pg/ml as compared to normal rats. After


151

treatment of diabetic rats with purified SC and PM extracts the IL-6 levels were

increased by 2.75 and 2.41 folds respectively (Fig. 5C).

Interleukin 10 (IL-10) is a protein that inhibits the synthesis of a number of

cytokines, including IFN-gamma, IL-2, IL-3, TNF, and GM-CSF produced by

activated macrophages and by helper T cells. IL-10 exhibits immunosuppressive

activity and also serves as a potent inhibitor of proinflammatory cytokine production

eg. IL-6 and IL-2. The level of IL-10 was significantly increased in diabetic rats from

4.25±0.25 to 11.3±0.61 pg/ml as compared to normal rats. After treatment of diabetic

rats with purified SC and PM extracts the IL-10 levels were decreased by 1.8 and 1.5

folds respectively (Fig. 5D).

It has been reported that herbs seem to operate through immunostimulant,

immunoadjuvant and immunosuppressant activities or by affecting the effector arm of

the immune response (Chulet and Pradhan, 2010). Mechanisms of immunomodulation

activity occur mainly via phagocytosis stimulation, macrophages activation,

immunostimulatory effect on peritoneal macrophages, lymphoid cells stimulation,

cellular immune function enhancement and nonspecific cellular immune system

effect, antigen-specific immunoglobulin production increase, increased nonspecific

immunity mediators and natural killer cell numbers, reducing chemotherapy-induced

leukopenia, and increasing circulating total white cell counts and interleukin-2 levels

(Vaghasiya et al., 2010; Malik et al., 2009). Herbal medicines have been reported to

alter the activity of immune function through the dynamic regulation of informational

molecules such as cytokines (Spelman et al., 2006). The present study demonstrates

that purified SC and PM extracts have immunostimulatory potential. The antibody

production by B cells was increased in the treated group of rats and the levels of

serum immunostimulatory cytokines (IL-2, IL-4, IL-6) were increased and

immunosuppressive cytokine (IL-10) was decreased in diabetic rats.

Summary

Summary

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Diabetes is a chronic disorder in which homeostasis of carbohydrate, protein

and lipid metabolism is improperly regulated by insulin. It is characterized by

elevated fasting and post prandial blood sugar levels. Diabetes mellitus is a complex

metabolic disorder resulting from either insulin insufficiency or insulin dysfunction.

Type I diabetes (insulin dependent) is caused due to insulin insufficiency because of

lack of functional β cells. Patients suffering from this are therefore totally dependent

on exogenous source of insulin while patients suffering from Type II diabetes (insulin

independent) are unable to respond to insulin and can be treated with dietary changes,

exercise and medication. Type II diabetes is the more common form of diabetes

constituting 90% of the diabetic population (Modak et al., 2007). Tissues where

glucose uptake is insulin independent (cardiac tissue, blood vessels, peripheral nerves,

renal medulla and ocular lens) face severe and sustained hyperglycemia (Chandra et

al., 2002). The major complications of diabetes include atherosclerosis, retinopathy,

nephropathy and neuropathy etc.

The human population worldwide appears to be in the midst of an epidemic of

diabetes. An estimated 285 million people, corresponding to 6.4% of the world's adult

population suffer from diabetes. The number is expected to grow to 438 million by

2030, corresponding to 7.8% of the adult population. Despite the great strides that

have been made in the understanding and management of diabetes, the disease and

disease-related complications are increasing unabated. Parallel to this, recent

developments in understanding the pathophysiology of the disease process have

opened several new avenues to identify and develop novel therapies to combat the

diabetic plague. Oral hypoglycemic drugs, viz, biguanide (metformin),

thiazolindinediones, sulphanylureas, meglitinides etc, and insulin therapy play an

important role in the management of diabetes mellitus, but have their own limitations

(Bastaki, 2005). Concurrently, phytochemicals identified from traditional medicinal

plants are presenting an exciting opportunity for the development of new types of

therapeutics because of lesser side effects and are cost effective. This has accelerated

the global effort to harness and harvest those medicinal plants that bear substantial

amount of potential phytochemicals showing multiple beneficial effects in combating

diabetes and diabetes-related complications. Therefore, as the disease is progressing

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unabated, there is an urgent need of identifying indigenous natural resources in order

to procure them, and study in detail, their potential on different newly identified

targets in order to develop them as new therapeutics (Tag et al., 2012).

Syzygium cumini (SC), a member of family Myrtaceae is commonly known as

Jamun or Jambul in Hindi and Black Plum or Black Berry in English. Various parts of

this plant have been recognized to possess several medicinal properties in the

traditional system of medicine. Pharmacological evaluation of this plant reveals its

anti-diabetic, hypolipidemic, antioxidant, anti-HIV, anti diarrhoeal, anti-

inflammatory, antibacterial, antipyretic, radioprotective and

neuropsychopharmacological activity (Srivastava and Chandra, 2013). Pterocarpus

marsupium (PM) belongs to the family fabaceae and is commonly known as Indian

Kino in English and Vijaysar in Hindi. Pterocarpus marsupium shows anti-diabetic,

hepatoprotective and cardiotonic activity. Studies have also reported its ability as a

COX-2 inhibitor (Devgun et al., 2009).

Several reports are available regarding the anti-hyperglycemic effects of SC

and PM but the data about their active constituents, their mechanism of action is not

conclusive. Oxidative stress greatly contributes to the progression of diabetic

complications. The medicinal plants mentioned above are reported to exhibit

antioxidant potential but it is inconclusive that their anti-oxidative property is due to

an independent activity associated to their constituent (s) or simply because of

normoglycemic condition achieved due to their consumption. Therefore, the present

study is planned to systematically evaluate the anti-hyperglycemic and anti-oxidative

potentials of SC and PM. The study is also aimed to isolate and characterize the active

constituents present in these plants and responsible for anti-hyperglycemic and anti-

oxidative potential, using alloxan induced diabetic rats as model. The effects of these

plant extracts on immune system in normal and diabetic rats were also tested in order

to evaluate their immunomodulatory potential.

The present study was designed to evaluate anti-hyperglycemic effect of

aqueous and alcoholic extracts of SC at three different doses. An intraperitonial dose

(150 mg/kg body weight) of alloxan induced diabetes in rats and the increased FBG

levels (>270 mg/dl) were monitored on day 0 (when rats were confirmed for diabetes)

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and day 30 (end of experiments). For aqueous extract the three doses were 1.5, 3, 5

g/kg b.w/day and for alcoholic extract the three doses were 50, 100, 200 mg/kg

b.w/day. Administration of aqueous SC extract at a dose of 1.5 g/kg b.w/day by oral

route resulted in 27.5% decrease in FBG levels in diabetic rats after 30 days, however

3 g/ kg/b.w/day dose resulted in better management of FBG, as the decreased levels

(by 63.3%) observed were maximum and close to normal value. With 5 g/kg b.w/day

dose the results were comparable to the results obtained with 3 g/kg b.w/day dose.

These doses (3 and 5 g/kg b.w/day) of SC extracts have not shown any hypoglycemic

effect in normal rats. On the other hand administration of alcoholic SC extract at a

dose of 50 mg/kg b.w/day by oral route resulted in 31.2% decrease in FBG levels in

diabetic rats after 30 days, however 100 mg/ kg/b.w/day dose resulted in better

management of FBG, as the maximally decreased levels (by 77%) observed were

close to normal value. With 200 mg/kg b.w/day dose the results were comparable to

the results obtained with 100 mg/kg b.w/day dose. These doses (100 and 200 mg/kg

b.w/day) of SC extracts have not shown any hypoglycemic effect in normal rats.

Similarly, the anti-hyperglycemic effect of aqueous and alcoholic extracts of

PM was evaluated at three different doses. For aqueous extract the 3 doses were 100,

200, 400 mg/kg b.w/day and for alcoholic extract the 3 doses were 150, 300, 500

mg/kg b.w/day. Administration of aqueous PM extract at a dose of 100 mg/kg

b.w/day by oral route resulted in 23.2% decrease in FBG levels in diabetic rats after

30 days, however 200 mg/ kg/b.w/day dose resulted in better management of FBG, as

the decreased levels (by 59.8%) observed were close to normal value. With 400

mg/kg b.w/day dose the results were comparable to the results obtained with 200

mg/kg b.w/day dose. These doses (200 and 400 g/kg b.w/day) of PM extracts have not

shown any hypoglycemic effect in normal rats. On the other hand administration of

alcoholic PM extract at a dose of 150 mg/kg b.w/day by oral route resulted in 19.7%

decrease in FBG levels in diabetic rats after 30 days, however 300 mg/ kg/b.w/day

dose resulted in 31.3% decrease in FBG levels. With 500 mg/kg b.w/day dose the

results were comparable to the results obtained with 300 mg/kg b.w/day dose. These

doses (300 and 500 mg/kg b.w/day) of SC extracts have not shown any hypoglycemic

effect in normal rats.

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Metformin, is a biguanide that affects the intestinal glucose absorption, insulin

secretion and hepatic glucose production to manage the diabetes. In vivo and in vitro

studies have demonstrated that metformin stimulates the insulin-induced component

of glucose uptake into skeletal muscle and adipocytes in both diabetic individuals and

animal models. In the present study, anti-hyperglycemic effects resulted due to

metformin, used as positive control, were comparable to the results obtained with

these plant extracts.

Duration dependent anti-hyperglycemic effect of alcoholic extract of SC (100

mg/kg b.w/day) and aqueous extract of PM (200 mg/kg b.w/day) was evaluated on the

15th and 30th day. SC extract showed a significant decrease in FBG, 33.22% and

77.56% on day 15th and 30th respectively. PM extract showed in 49.85% and 60.60%

decrease in FBG levels on the 15th and 30th day, respectively. The doses of SC and

PM extracts did not show any hypoglycemic effect in normal rats. The pattern of

duration dependent anti-hyperglycemic effect shown by of SC and PM were

comparable with positive control metformin.

Administration of alloxan is reported to be associated with loss in body

weight. In spite of the increased food consumption, loss of body weight may be due to

defect in glucose metabolism and excessive breakdown of protein in tissues is a

characteristic of diabetes. In present study alloxan induced diabetic rats showed a

significant decrease in body weight from 118.5±2.5 to 85.4±3.8 g. Treatment with

aqueous (3 g/kg b.w/day) and alcoholic (100 mg/kg b.w/day) extracts of SC resulted

in 14.2% and 17.6% gain in body weight respectively, on 30th day. Similarly aqueous

(200 mg/kg b.w/day) and alcoholic (300mg/kg b.w/day) extracts of PM showed in

15.8% and 13.2% gain in body weight on 30th day. Treatment with SC and PM

extracts improved body weight of diabetic rats, indicating control over polyphagia and

muscle wasting resulted due to hyperglycemic condition. Normal rats when treated

with these plant extracts of SC and PM showed gain in body weight which was

comparable to normal control rats. The result obtained with anti-diabetic drug

metformin with respect to gain in body weight was comparable to the effects observed

with SC and PM extracts in diabetic rats.

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The present study includes experiments which were conducted to establish the

toxicity of alcoholic SC seed extract and aqueous PM bark extract when administered

for two months. Two doses of these extracts, SC seed (100 and 500 mg/kg b.w/day)

and PM bark extract (200 mg and 1 g/kg b.w/day), were given to normal rats. There

was no morbidity and all the rats showed normal growth (gain in body weight),

similar to that of normal rats. Effects of these plant extracts were observed on liver

and kidney function. No significant deviation from control values were observed in

serum urea and creatinine values in all the groups. No significant changes from

control rats were observed in serum transaminases ALT and AST and alkaline

phosphatase levels. The data suggest that prolong use of these extracts is safe.

Further, the crude extracts of SC and PM were further purified and evaluated

for their anti-hyperglycemic activity. The alcoholic extract of SC was subjected to

silica gel chromatography and the adsorbed compounds (mostly phenolics) were batch

eluted with 100% methanol. The eluted fraction was further purified on sephadex LH

20 beads and batch eluted with different ratios of water and methanol (100% water,

70:30, 30:70,100% methanol). Four fractions were obtained and tested for anti-

hyperglycemic and anti-oxidative activities. The fraction IV showing best results was

further subjected to HPLC column. The eluted fraction was characterized as caffeic

acid. The bark powder of PM was extracted with ethyl acetate and subjected to silica

gel beads. The fractions (mostly polyphenols) batch eluted with benzene were pooled

and lyophilized. The lyophilized material was re-suspended in minimum volume of

ethanol. The ethanol was evaporated and material was suspended in water and dosed.

The rats were fed with the four fractions purified from sephadex LH 20 at a

dose of 1.1mg/kg b.w/day. FBG levels were monitored on day 0 (when rats were

confirmed for diabetes) and day 30 (end of experiments). In diabetic control group,

higher FBG level (>270 mg/dl) was maintained throughout the period of study. On the

other hand the oral dose of SC purified fractions resulted in decrease in FBG levels in

diabetic rats. The decrease in FBG levels on administration of fractions I, II and III

was from 435.4±16.3 to 406.8±15.7 md/dl, 444.2±20.3 to 389.6±12.9 mg/dl and

436.5±20.1 to 352±14.2 mg/dl, respectively. Better results were obtained with fraction

IV, which significantly (p<0.001) decreased the FBG level from 445.2±11.9 to

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102.9±4.8 mg/dl. Normal rats treated with the four fractions did not show any

significant changes in FBG levels. The rats were fed with the purified fraction of PM

at a dose of 1mg/kg b.w/day. The oral dose of PM purified fraction resulted in

significant (p<0.001) decrease in FBG levels from 413.4±16.8 to 161.7±11.3 mg/dl in

diabetic rats. Normal rats treated with the purified fraction of PM did not show any

significant change in FBG level. SC purified fraction IV and PM purified fraction

showed significant decrease in FBG levels which was better than metformin, standard

hypoglycemic agents.

The effect of these purified extracts of SC and PM were studied on serum

insulin levels. The serum insulin levels were significantly decreased in diabetic rats

(0.068 ng/ml) as compared to normal rats (0.32 ng/ml), which were brought back to

near normal levels after treatment with SC sephadex LH-20 purified fractions III and

IV. Treatment of diabetic rats with fraction III restored the serum insulin levels from

0.068 to 0.152 ng/ml, whereas treatment with fraction IV restored the insulin levels

from 0.068 to 0.244 ng/ml. Thus fraction IV showed much better improvement in

serum insulin levels as compared to fraction III. The purified PM fraction restored the

serum insulin levels from 0.068 to 0.22 ng/ml. Insulin acts is an activator of key

glycolytic enzymes and a suppressor of gluconeogenic enzymes, therefore we further

evaluated the effect of purified SC and PM extracts on the activities of glycolytic

enzymes, hexokinase, phosphofructokinase and pyruvate kinase in heart liver and

kidney tissues of diabetic rats. The decreased activities of glycolytic enzymes

(hexokinase, phosphofructokinase and pyruvate kinase) in diabetic rats were

significantly (p<0.001) increased after treatment.

The anti-hyperglycaemic activity of SC and PM might be possibly due to

regeneration of pancreartic β cells and stimulation of insulin release or insulin

mimetic activity of some components. Insulin further activates the enzymes of

glycolytic pathway and increases peripheral glucose utilization, thus lowers blood

glucose levels.

Oxidative stress plays a pivotal role in the development of diabetes

complications. Oxidative stress and oxidative damage to the tissues are common end

points of chronic diseases, such as atherosclerosis, diabetes and rheumatoid arthritis.

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Oxidative stress is currently suggested as mechanism underlying diabetes and diabetic

complications (Kangralkar et al., 2010). During diabetes, persistent hyperglycemia

causes increased production of free radicals, especially reactive oxygen species

(ROS), in all tissues due to glucose auto-oxidation and protein glycosylation. The

increase in the level of ROS in diabetes could be due to their increased production

and/ or decreased destruction by nonenzymic antioxidants, eg. reduced glutathione

(GSH) and enzymic antioxidants like catalase (CAT), glutathione S-transferase

(GST), , and superoxide dismutase (SOD). The level of these antioxidant enzymes

critically influences the susceptibility of various tissues to oxidative stress and is

associated with the development of complications in diabetes (Lipinski, 2001).

The present study showed a significant (p<0.001) increase in MDA levels in

heart, liver and kidney tissues of diabetic rats suggesting that peroxidative injury may

be involved in alloxan induced diabetes and may lead to other secondary

complications. Treatment of diabetic rats with crude aqueous and alcoholic extracts of

SC could significantly lower the elevated MDA levels by 30.9% and 51.4% in heart,

28.3% and 45.6% in liver, 25.2% and 29.4% in kidney, respectively. Similarly crude

aqueous and alcoholic extracts of PM could lower the elevated MDA levels by 21.3%

and 38.1% in heart, 19.7% and 37.8% in liver, 16.8% and 27.7% in kidney tissue,

respectively Reduced glutathione (GSH) is known to protect the cellular system

against the toxic effects of lipid peroxidation. GSH functions as direct free radical

scavenger, as a co-substrate for glutathione peroxidase (GPx), as a cofactor for many

other enzymes and forms conjugates in endo and xenobiotic reactions. Diabetic rats

treated with aqueous and alcoholic extracts of SC and PM resulted in significant

(p<0.001) increase in depleted GSH content when compared with diabetic controls.

Diabetic rats treated with aqueous and alcoholic SC extracts resulted in increase in

GSH content by 57.14% and 124.5 % in heart, 23.4% and 60.9% in liver, 23.9% and

69.6% in kidney, respectively. Similarly aqueous and alcoholic extracts of PM

resulted in increase in GSH content by 44.9% and 81.6% in heart, 29.7% and 51.6%

in liver, 19.6% and 65.2% in kidney, respectively. Thus, the treatment of SC and PM

extracts to the alloxan induced diabetic rats resulted in attenuation in elevated levels

of TBARS in different tissues and also increased the depleted GSH content. These

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159

findings suggest that the administration of SC and PM extracts to the diabetic rats

could overcome the oxidative stress status in different tissues of diabetics.

Superoxide dismutase (SOD), catalase, and peroxidases constitute a mutually

supportive team of defense against ROS. While SOD lowers the steady-state level of

O2¯, catalase and peroxidases do the same for H2O2. Reduced activities of SOD and

catalase in heart, liver and kidney tissues of diabetic rats has been observed in the

present study which may be due to increased production of reactive oxygen species

that can themselves reduce the activities of these enzymes. The treatment of diabetic

rats with aqueous and alcoholic extracts of SC resulted in significant (p<0.001)

increase in the activities of anti-oxidant enzymes (SOD, CAT, GST) in heart liver and

kidney tissues. Similarly treatment of diabetic rats with alcoholic extract of PM

resulted in significant (p<0.001) increase in the activities of anti-oxidant enzymes

(SOD, CAT, GST) in heart liver and kidney tissues, whereas the results obtained with

aqueous extract were not significant.

The crude (aqueous and alcoholic) extracts and the purified fractions of SC

and PM were tested for their in vitro antioxidant activity by DPPH radical scavenging

assay. SC purified fraction IV and the PM purified fraction showed maximum radical

scavenging activity and were further selected for in vivo study.

The effect of SC purified fraction IV (caffeic acid) was studied on antioxidant

parameters (MDA levels, GSH content and SOD, CAT, GST activities) in heart liver

and kidney tissues of diabetic rats. The MDA levels were significantly increased in

diabetic rats as compared to normal rats which were brought back to near normal

levels after SC fraction IV treatment. The depleted GSH content and decreased

antioxidant enzymes activities in diabetic rats were also restored after SC fraction IV

treatment.

It has been hypothesized that caffeic acid protects the rats from oxidative

stress by scavenging the free radicals. Clinical complications in oxidative stress

related diseases might be due to the dysfunctioning of key antioxidant enzymes. SOD,

CAT, GST and GPx are among those enzymes which metabolize endogenous reactive

oxygen species and free radicals. In the present study, the decreased levels of SOD,

CAT and GST were observed in diabetic rats. The ability of caffeic acid to increase

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the activities of antioxidant enzymes in diabetic rats implies that caffeic acid

reactivates the antioxidant defence system, thereby increasing the capacity of

detoxification through the enhanced scavenging of free radicals. Thus our study

suggests that caffeic acid can prevent diabetes induced complications. This may be

attributed to the antiradical activity of caffeic acid which inhibits lipid peroxidation

and enhances the antioxidant defence against diabetes induced oxidative damage in

tissues. The effect of purified PM fraction was studied on antioxidant parameters

(MDA levels, GSH content and SOD, CAT, GST activities) in heart liver and kidney

tissues of diabetic rats. The MDA levels were significantly increased in diabetic rats

as compared to normal rats which were brought back to near normal levels after

treatment. The depleted GSH content and decreased antioxidant enzymes activities in

diabetic rats were also restored after treatment with PM purified fraction.

On basis of these findings a possible mechanism to explain the anti-oxidative

protection by SC and PM could be proposed. The diabetics have decreased SOD and

GST activities due to inactivation by reactive oxygen species or by glycation of

enzymes. GST works together with glutathione and GPx in decomposition of H2O2 or

other organic hydroperoxides to non-toxic products. A significant increase/recovery in

SOD, catalase, GST and GPx activities in SC and PM extract (aq./alc.) treated

diabetic subjects may be due to following reasons: the anti-hyperglycemic effect

observed by these extracts resulted in decreased glycation of these enzyme proteins

(less inactivation of enzymes) which in turn potentiate their reduction capacity by

improving their antioxidant activities. Achievement of near normoglycemic

conditions in diabetic rats treated with SC and PM extracts resulted in decreased free

radical/ROS formation in diabetic tissues which led to regain the GSH levels and

reverse the ROS mediated inactivation of GST, SOD and CAT activities. The

significant recovery of anti-oxidant enzyme activities by treatment with SC and PM

extracts indicated their protective effect on oxidative stress.

Diabetes has also been associated to immunosuppression (Muller et al., 2011),

thus the plant based therapies, which attenuate the complications of diabetes, were

also tested for their immunomodulatory activity. Modulation of the immune system

denotes to any change in the immune response that can involve induction, expression,

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161

amplification or inhibition of any part or phase of the immune response. Thus,

immunomodulator is a substance used for its effect on the immune system. There are

generally of two types immunomodulators based on their effects:

immunosuppressants and immunostimulators. They have the ability to mount an

immune response or defend against pathogens or tumors. The potential uses of

immunodulators in clinical medicine include the reconstitution of immune deficiency

(e.g. the treatment of AIDS) and the suppression of normal or excessive immune

function (e.g. the treatment of graft rejection or autoimmune disease) (Saroj et al.,

2012).


activity. Most of them are cytotoxic and exert a variety of side effects. This has given


immunomodulatory activity. Many medicinal plants are known to have

immunomodulatory properties and maintain organic resistance against infection by re-

establishing the body’s immune system such as Azadirachta indica, Terminalia

chebula and Murraya koenigii (Shah et al., 2008). The phytochemical constituents

like terpenoids, steroids, proteins and tannins are considered to exhibit this

immunomodulatory property. The immunomodulatory property of Syzygium cumini

(seeds) and Pterocarpus marsupium (bark) has been studied by analysing the effect of

SC seed extract and PM bark extract on humoral immune response in normal and

diabetic rats. The modulation in immune response has been explained by measuring

the serum levels of some of the immunomodulatory cytokines before and after the

herbal therapy in normal and diabetic rats.

Rats were immunized with tetanus toxoid (TT), a thymus dependent antigen

and E.coli LPS, a thymus independent antigen at doses of 100µg/rat each. Four

immunizations were performed with each antigen and the sera were tested for the

presence of antibodies (IgM and IgG) and IL-4 levels by ELISA. Rats immunized

with TT showed typical immune response i.e. increase in IgG levels were observed in

different groups of rats after each immunization and became constant after III

immunization. Typical immune response means that there was activation and

proliferation of antigen specific B lymphocytes and their consequent differentiation to

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162

plasma cells and memory cells. The anti-TT IgG levels (ELISA values) showed 2-4

fold increase after III immunization in different groups of rats, which became constant

after III immunization. This suggested the formation of sufficient memory cells at this

stage. The effect was more pronounced in groups of rats treated with plant extracts of

SC and PM (3g/kg b.w./day and 200mg/kg b.w./day respectively) suggesting that the

treatment of rats with these plant extracts resulted in immunostimulation.

In the next experiment, rats were immunized with thymus independent antigen

i.e. E.coli LPS. Four immunizations were performed and sera were collected after 7

days of each immunization. Rats were divided into different groups. Normal rats

immunized (NI) with E.coli LPS did not show significant increase in anti-LPS IgG

(ELISA values) after II, III and IV immunizations as compared to the results obtained

from TT immunization. Although, there was slightly better immune response (anti-

LPS IgG) in groups of immunized rats which received plant extracts of SC and PM in

comparison to NI rats. But the ELISA values of anti-LPS IgG tested against LPS were

3-4 fold lower than what has been observed in case of testing of anti-TT IgG against

TT antigen.

When the sera obtained from rats immunized with E.coli LPS were tested for

the presence of IgM levels, almost constant IgM levels were observed in normal

immunized rats. The groups immunized with E.coli LPS and treated with the plant

extracts of PM and SC showed slight but insignificant decrease in anti-LPS IgM.

Depending on nature of antigen, B cell activation proceeds by two different

routes, one dependent on TH cells, the other not. The B cell response to thymus

dependent (TD) antigens requires direct contact with TH cells, not simply exposure to

TH derived cytokines. Antigens that can activate B cells in absence of this kind of

direct participation of TH cells are known as thymus independent (TI) antigens. E.coli

LPS which is a TI-1 antigen is a polyclonal B cell activator (mitogen), it can activate

B cells regardless of its antigenic specificity. The response to TI antigens are

generally weaker and IgM is the predominant antibody secreted, reflecting a low level

of class switching. This highlights the important role played by TH cells in generating

memory B cells, affinity maturation and class switching to other isotypes. An

effective signal for B cell activation involves two distinct signals induced by

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163

membrane events. Binding of a TI-1 antigen to B cell provides both the signals. A TD

antigen provides signal 1 by cross linking B cells but a separate interaction between

CD 40 on the B cell and CD 40L on activated TH cells is required to generate signal 2.

The expression of CD 40 on B cells has been reported to be stimulated by IL-2.

The data of above three experiments suggests that the rats immunized with

thymus dependent antigen TT showed activation, proliferation and differentiation of

B lymphocytes, followed by class switching (IgM→IgG) whereas the rats immunized

with E.coli LPS showed activation and proliferation of B lymphocytes without proper

class switching (IgM→IgG).

These findings were further confirmed by measuring the serum levels of IL-4

in different groups of rats immunized with TT and E.coli LPS. The normal rats

immunized with TT showed 1.5 fold increase in IL-4 when compared with serum IL-4

levels of normal unimmunized rats. On the other hand, immunized rats treated with

plant extracts showed about 1.7-1.8 fold higher serum IL-4 levels. This confirmed the

interaction of T cell component of immune system with B cell component as IL-4

secreted by TH2 lymphocytes is involved in class switching from IgM to IgG in rats

immunized with thymus dependent antigen TT. The normal rats immunized with

E.coli LPS did not show any significant increase in serum IL-4 levels whereas group

III and IV rats immunized and treated with plant extracts of PM and SC showed only

about 1.2 fold increase in serum IL-4 levels which was lesser than IL-4 level increase

in rats immunized with TT.

The data suggests that immunization with E.coli LPS could not result in

sufficient increase in serum IL-4 levels and resulting in no or insignificant

enhancement and class switching in normal immunized rats. The treatment of rats

(immunized with LPS) with plant extracts of SC and PM resulted in some increase

(1.2 fold) in serum IL-4 levels and better immune response. It can be concluded that

thymus dependent (TT) and thymus independent (LPS) interact and activate immune

system differently which may be dependent on different serum cytokine levels, such

effect of IL-4 has been demonstrated in our study.

Activated TH cells secrete different cytokines of adaptive immunity including

IL-4, IL-2 having role in B cell proliferation and class switching, IL-6 plays key role

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164

in linking innate and adaptive immunity and B cell maturation, and is a pro-

inflammatory cytokine. In the present study the effect of purified SC and PM extracts

on serum cytokine levels (IL-2, IL-4, IL-6, IL-10) of diabetic rats was evaluated. T

Lymphocytes regulate the growth and differentiation of T cells and certain B cells

through the release of secreted protein factors. These factors include interleukin 2 (IL-

2), which are secreted by lectin- or antigen-stimulated T cells and have various

physiological effects. IL-2 is a lymphokine that induces the proliferation of responsive

T cells. In addition, it acts on some B cells, via receptor-specific binding as a growth

factor and antibody production stimulant. The level of IL-2 was significantly

decreased in diabetic rats from 10.3±0.5 to 2.3±0.4 pg/ml as compared to normal rats.

After treatment of diabetic rats with purified SC and PM extracts the IL-2 levels were

increased by 3 and 2.5 folds respectively.

Interleukin 4 (IL-4) is produced by CD4 T cells specialized in providing help

to B cells to proliferate and to undergo class switch recombination and somatic

hypermutation. TH2 cells, through production of IL-4, have an important function in

B-cell responses that involve class switch recombination to the IgG1 and IgE

isotypes. The level of IL-4 was significantly decreased in diabetic rats from 12.3±0.6

to 4.67±0.35 pg/ml as compared to normal rats. After treatment of diabetic rats with

purified SC and PM extracts the IL-4 levels were increased by 1.97 and 1.73 folds

respectively.

Interleukin 6 (IL-6), also referred to as B-cell stimulatory factor-2 (BSF-2) and

interferon beta-2, is a cytokine involved in a wide variety of biological functions. It

plays an essential role in the final differentiation of B cells into IG-secreting cells, as

well as inducing myeloma/plasmacytoma growth, nerve cell differentiation and in

hepatocytes, acute-phase reactants. The level of IL-6 was significantly decreased in

diabetic rats from 22.3±1.45 to 5.93±0.8 pg/ml as compared to normal rats. After

treatment of diabetic rats with purified SC and PM extracts the IL-6 levels were

increased by 2.75 and 2.41 folds respectively.

Interleukin 10 (IL-10) is a protein that inhibits the synthesis of a number of

cytokines, including IFN-gamma, IL-2, IL-3, TNF, and GM-CSF produced by

activated macrophages and by helper T cells. IL-10 exhibits immunosuppressive

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165

activity and also serves as a potent inhibitor of proinflammatory cytokine production

eg. IL-6 and IL-2. The level of IL-10 was significantly increased in diabetic rats from

4.25±0.25 to 11.3±0.61 pg/ml as compared to normal rats. After treatment of diabetic

rats with purified SC and PM extracts the IL-10 levels were decreased by 1.8 and 1.5

folds respectively. The present study demonstrates that purified SC and PM extracts

have immunostimulatory potential. The antibody production by B cells was increased

in the treated group of rats and the levels of serum immunostimulatory cytokines (IL-

2, IL-4, IL-6) were increased and immunosuppressive cytokine (IL-10) was decreased

in diabetic rats.

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Research Papers Published:

1. Deepak Chandra, UN Tripathi, Shalini Srivastava and A. Swaroop. (2011)

Carbofuran induced biochemical toxicity in mice: protective role of Momordica

charantia. Eu J Exp Biol, 1(1): 106-112.

2. Shalini Srivastava and Deepak Chandra. (2013) Pharmacological potentials of

Syzygium cumini: A review. J Sci Food Agr, 93: 2084-2093.

Documents

by Shalini Srivastava - Shodhgangashodhganga.inflibnet.ac.in/.../shalini_biochem_phd... · Shalini Srivastava under my supervision. The work presented here is original, carried out