90

TITLE

THE EFFECT OF SOME SOLVENT EXTRACTS OF Colatropis gigantea LEAF ON SOME OXIDATIVE

PARAMETERS IN DIABETIC RABBITS

BY

UHUO, EMMANUEL NNAEMEKA (PG/M.Sc/07/43734)

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA

NSUKKA

A DISSERTATION SUBMITTED TO SCHOOL OF POSTGRADUATE STUDIES IN PARTIAL FULFILMENT

OF THE REQUIREMENTS FOR AWARD OF DEGREE OF MASTER OF SCIENCE (M.Sc) IN MEDICAL

BIOCHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA

SUPERVISOR: DR. V. N. OGUGUA

OCTOBER, 2009

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CERTIFICATION

UHUO, Emmanuel Nnaemeka, a postgraduate student with Registration Number PG/M.Sc/07/43734 in the Department of Biochemistry has satisfactorily completed the requirements for coursework and research for the degree of Master of Science (M.Sc) in Medical Biochemistry. The work embodied in this report is original and has not been submitted in part or full for any other diploma or degree of this or any other university.

DR. V. N. OGUGUA PROF. I. N. E. ONWURAH (Supervisor) (Head of Department)

EXTERNAL EXAMINER

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DEDICATION

This project work is wholly dedicated to God Almighty who mercifully

granted me health, strength, ability and wisdom to produce it and also to my

beloved family members.

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ACKNOWLEDGEMENTS

Foremost, I thank God Almighty for guiding me throughout this project work. I

most sincerely express my appreciation and gratitude to my project supervisors: Dr. V. N.

Ogugua, who encouraged me in bringing this study to a successful completion. My

unreserved gratitude also goes to Prof. O. U. Njoku, for his moral support.

I very much appreciate the goodwill and encouragement of all the lecturers and

technical staff of the Department of Biochemistry. I thank the Head of Department, Prof.

I. N. E. Onwurah and and other lecturers: Prof. P. N. Uzoegwu, Prof. O. F. C. Nwodo,

Mr. P. A. C. Egbuna, Prof F. C. Chilaka, Dr. S. O. Eze, Mr. O. C. Enechi and Mr. O. E.

Ikwuagwu for their valuable support towards this work .

I appreciate the work of Dr. V. O. Shoyinka of the Department of Microbiology

and Parasitology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka (UNN),

for the pains he took for the detailed analysis of my histopathological slides. The

assistance given to me by Mr. Uche Nwachi, Dr. Parker Elijah Joshua, Mr. Felix Eze and

Mr. Austin Eze, are highly appreciated. More to that, I appreciate the help of my fellow

postgraduate students and colleagues, especially Chinedu Okonkwo.

I also acknowledge the help of the staff of Histology and Histopathology

Laboratory of University of Nigeria Teaching Hospital, Faculty of Biological Sciences’

Animal Research House and Animal House of the Department of Home Science,

Nutrition & Dietetics of University of Nigeria,Nsukka. My family has been supportive

and encouraging. I am indebted to my dear parents: Mr. and Mrs. Uhuo and my younger

brother, Samuel Uhuo for the time, love and resources they put together to see that this

project succeeded. It is not easy to forget. Thank you all.

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ABSTRACT

The effect of Colatropis gigantea leaf extract was studied on alloxan-induced

hyperglycaemic rabbits to evaluate the possible hypoglycaemic and antioxidant

properties of C. gigantea in diabetes mellitus. Screening for the most effective organic

extract revealed that acetone fraction significantly decreased the blood glucose level

(p

95

TABLE OF CONTENTS PAGE

Title Page .. .. .. .. .. .. .. .. .. .. i Certification .. .. .. .. .. .. .. .. .. .. ii Dedication .. .. .. .. .. .. .. .. .. .. iii Acknowledgements .. .. .. .. .. .. .. .. .. iv Abstract .. .. .. .. .. .. .. .. .. .. v Table of Contents .. .. .. .. .. .. .. .. .. vi List of Figures .. .. .. .. .. .. .. .. .. .. x List of Tables .. .. .. .. .. .. .. .. .. .. xi List of Abbreviations .. .. .. .. .. .. .. .. .. xii CHAPTER ONE: INTRODUCTION

1.1 Diabetes mellitus … … … … … … … …

3

1.1.1 Types of diabetes mellitus … … … … … …

3

1.1.1.1 Type 1 diabetes mellitus … … … … … …

4

1.1.1.2 Type 2 diabetes mellitus … … … … … …

6

1.1.1.3 Gestational diabetes … … … … … …

7

1.1.1.4 Other types of diabetes mellitus … … … … …

8

1.1.2 Complications in diabetes mellitus … … … … …

9

1.1.2.1 Microangiopathy … … … … … … … …

12

96

1.1.2.2 Macroangiopathy … … … … … … … …

13

1.1.3 Oxidative stress caused by hyperglycaemia … … …

… 14

1.1.4 Free radicals and their role in diabetes … … … … …

14

1.2 Generation of reactive oxygen species (ROS) in human body …

… 15

1.3 Oxidative stress and tissue injury … … … … …

… 18

1.3.1 Mechanism of tissue injury … … … … …

… 15

1.4 Pancreas … … … … … … … …

21

1.4.1 Anatomy of the pancreas … … … … … … …

21

1.4.2 Structure of the pancreas … … … … … …

17

1.4.3 Types of pancreatic tissues … … … … …

… 23

1.4.4 Functions of pancreas … … … … … …

… 24

1.5 Insulin … … … … … … … …

… 24

1.6 Antioxidants … … … … … … …

… 25

97

1.6.1 Superoxide dismutase (SOD) … … … …

… … … 26

1.6.2 Catalase … … … … … … …

… … 27

1.6.3 The glutathione system … … … … … …

… 28

1.6.4 Vitamin C as Antioxidant … … … … …

… … 24

1.6.4.1 Vitamin E (Alpha t ocopherols … … … … …

… 30

1.6.4.2 Vitamin C (Ascorbic acid) … … … …

… … 31

1.7 Aim of Research … … … … … … … … 32 1.8 Research objective … … … … … … … … 32

CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials … … … … … … … … …

33

2.1.1 Chemicals … … … … … … … … …

33

2.1.2 Drugs … … … … … … … … … …

33

2.1.3 Instruments/Equipment … … … … …

34

2.1.4 Plant material … … … … … … … …

34

2.1.5 Animals … … … … … … … …

34

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2.2 Methods … … … … … … … … …

35

2.2.1 Experimental design … … … … … … … …

35

2.2.2 Plant treatment … … … … … … … …

36

2.2.3 Extraction procedure … … … … … … … …

36

2.2.4 Determination of yield of extract … … … … … …

38

2.2.5 Phytochemical analysis of the crude extract … … … …

38

2.2.5.1 Test for the presence of alkaloids … … … … … …

38

2.2.5.2 Test for carbohydrates … … … … … … …

38

2.2.5.3 Test for reducing sugar … … … … … … …

38

2.2.5.4 Test for protein … … … … … … ..

38

2.2.5.5 Test for fats and oil … … … … … … … 39

2.2.5.6 Test for glycoside … … … … … … …

39

2.2.5.7 Test for acidic substances … … … … … … …

39

2.2.5.8 Test for the presence of flavonoids … … … … … …

39

2.2.5.9 Test for the presence of steroids … … … … … …

39

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2.2.5.10 Test for tannins … … … … … … … …

40

2.2.5.11 Test for resins … … … … … … … …

40 2.2.5.12 Test for saponins … … … … … … …

… 40

2.2.5.13 Test for terpenoid and steroid … … … … … … 40 2.2.6 Acute toxicity test … … … … … … … 41 2.2.6.1 Determination of LD50 of the extract … … … … … 41 2.2.7 Anti-diabetic evaluation … … … … … … 41 2.2.7.1 Induction of diabetes mellitus … … … … … … 41

2.2.8 Assay methods … … … … … … … …

42

2.2.8.1 Blood glucose determination … … … … … …

42

2.2.8.2 Lipid peroxidation determination … … … … … … 43 2.2.9 Glutathione peroxidase assay … … … … … … 45 2.2.10 Catalase assay … … … … … … … … … 45

2.2.11 Superoxide dismutase (SOD) assay … … … … …

48

2.2.12 Total protein determination … … … … … …

49

2.2.13 Determination of vitamin C level … … … … … …

50 2.2.14 Determination of blood pH … … … … … … …

52

2.2.15 Histopathologic techniques … … … … … …

52

100

2.2.15.1 Microscopic observation of slides … … … … …

53

2.3 Statistical analysis … … … … … … …

53

CHAPTER THREE: RESULTS 3.1 Extract yield … … … … … … … …

54

3.2 Phytochemical analysis of Colatropis gigantea … … … …

54

3.3 Micronutrient analysis of Colatropis gigantea … … … …

52

3.4 LD50 of C. gigantea extract … … … … … … …

55

3.5 Effect of the fractionated extracts of C. gigantea on glucose

level in diabetic rabbits … … … … … … …

55

3.6 Effect of acetone fraction of C. gigantean leaf extract on

plasma malondialdehyde (MDA) concentrations … … … …

58

3.7 Effect of acetone fraction of C. gigantea leaf extract on catalase activity …

60

3.8 Effect of acetone fraction of C. gigantea leaf extract on SOD activity …

62

3.9 Effect of acetone fraction of C. gigantea leaf extract on

glutathione peroxidase activities … … … … … …

64

3.10 Effect of C. gigantea leaf extract on vitamin C level … … …

66

3.11 Effect of acetone fraction of C. gigantea leaf extract on protein concentration…

68

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3.12 Effect of acetone fraction of C. gigantea leaf extract on blood pH … …

70

3.13 Histologic changes … … … … … … …

68

CHAPTER FOUR: DISCUSSION 4.1 Discussion … … … … … … … …

74

4.2 Suggestions for Further Research … … … … …

78

REFERENCES … … … … … … … … … 79 APPENDICES … … … … … … … … … 84

102

LIST OF FIGURES PAGE

Fig. 1 Schematic Representation: Hyperglycemia and biochemical processes lead to

oxidative stress and vascular effects … … … … …

… … 10

Fig. 2 Influence of hyperglycemia on plasma protein … … …

… 11

Fig. 3 The free radical mechanism of lipid peroxidation … …

… 17

Fig. 4 Free radical mediated peroxidation of unsaturated fatty acid leading

to

damage and the generation of numerous end products … …

… 20

Fig. 5 Structure of the pancreas is made up of two types of tissues … … 22 Fig. 6 Role of the pentose phosphate pathway in the glutathione peroxidase reaction of erythrocytes (G – S – S – G, oxidized glutathione; Se; G – SH reduced glutathione, selenium cofactor) … … … 29

Fig. 7 Structure of tocopherol (Vit. E) … … … …

… 30

Fig. 8 Structural formula of ascorbic acid and dehydroascorbic acid …

… 31

Fig. 9 Fractionation of C. gigantea leaves … … … … …

… 37

Fig. 10 Reaction of thiobarbituric acid (TBA) and malondialdehyde …

… 43 Fig. 11 Effect of acetone fraction of C. gigantean leaf extract on

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the plasma MDA concentration of experimental rabbits … … …

59

Fig. 12 Effect of acetone fraction of C. gigantean leaf extract on

the catalase activity of experimental rabbits … … … …

61

Fig. 13 Effect of acetone fraction of C. gigantean leaf extract on

the SOD activity of experimental rabbits … … … … …

63

Fig. 14 Effect of acetone fraction of C. gigantean leaf extract on

the glutathione peroxidase activity of experimental rabbits … …

65

Fig. 15 Effect of acetone fraction of C. gigantean leaf extract on

the vitamin C concentration of experimental rabbits … … …

67

Fig. 16 Effect of acetone fraction of C. gigantean leaf extract on

the total protein concentration of experimental rabbits … … …

69

Fig. 17 Effect of acetone fraction of C. gigantean leaf extract on

the blood pH of experimental rabbits … … … … …

71

LIST OF TABLES

Table 1 Procedure for MDA determination … … … … …

44

Table 2 Procedure for catalase assay … … … … … …

47

Table 3 Phytochemical constituents of C. gigantea leaf extract … …

54

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Table 4 Micronutrient constituent of C. gigantea leaf extract … … …

55

Table 5 Effect of solvent fraction of C. gigangia leaf on glucose level (Phase I)…

56

Table 6 Effect of acetone fraction of C. gigantea leaf on glucose level (Phase II)…

57

105

LIST OF ABBREVIATIONS

AGES Advanced glycosylation end products

DNA Deoxyribonucleic acid

EDTA Ethylene diamine tetraceate

EC Enzyme commission

GSH Reduced glutathione

GSSG Oxidized glutathione

FAD Flavin adenine dinucleotide

GPX Glutathione peroxidase

HDL High density lipoprotein

H2O2 Hydrogen peroxide

IDDM Insulin dependent diabetes mellitus

IL-1 Interleukins 1

IL-6 Interleukins 6

LD50 Median lethal dose

LDL Low density lipoprotein

LOO∙ Lipid peroxyl radical

MDA Malondialdehyde

MODY Maturity onset diabetes of the young

NADPH Nicotinamide adenine dinucleotide

NO Nitric oxide

NO2 Peroxynitrite

NIDDM Non insulin dependent diabetes of the young

O2∙ Superoxide radicals

OH∙ Hydroxyl radical

PMN Polymorphonuclear

RO∙ Alkoxyl radical

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ROS Reactive oxygen specie

SE Selenium cofactor

SOD Superoxide dismutase

TXA2 Thromboxane A2

TCA Trichloro acetic acid

TNF-α Tumour necrotic factor

TBARS Thiobarbituric acid reactive substance

TGF-B Transforming growth factor

VEGF Vessel endothelium growth factor

VLDL Very low density lipoprotein

4HDA 4-hydroxyaikenals

CHAPTER ONE

INTRODUCTION

Diabetes mellitus is a widespread disease with great social impact. It is a

syndrome, initially characterized by a loss of glucose homeostasis resulting from defects

in insulin secretion and insulin action, both resulting in impaired metabolism of glucose

and other energy – yielding fuels such as lipids and protein (Scheen, 1997).

The quality of life and the life span of the patients with the disease depend on its

complications. Hence, there is an increased interest in dealing with this disorder.

Convincing evidences of the role of free radicals and oxidative stress in the pathogenesis

and complications of diabetes mellitus have been established over times. It was shown

that the patients were put under increasing oxidative stress in conjunction with different

biochemical defects – the inactivation of nitric oxide, which is key to maintaining

vascular tones. Significant changes in lipid metabolism and structure also occur in

diabetes. In these cases the structural changes are clearly oxidative in nature and are

associated with development of vascular disease in diabetes (Baynes et al., 1999).

Oxidant free radicals play a relevant role in the etiology and pathogenesis of a

variety of diseases such as diabetes mellitus, cancer, hypertension, and cardio vascular

diseases and are considered to be the principle causative agents of aging (Jeon et al.,

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2002). Diabetes mellitus and its sequels, neuropathy and angiopathy, are conditions in

which free radical are involved both in human and in the experimental model. The

increase in oxygen free radicals in diabetes could be primarily due to increase in blood

glucose levels, which upon autoxidation generate free radicals and secondarily due to

effect of diabetogenic agents (alloxan) (Szkudelska et al., 2001).

In diabetes, hypoinsulineamia increases the activity of the enzyme, fatty acyl

coenzymes (coenzyme A oxidase), which initiate β-oxidation of fatty acids resulting in

lipid peroxidation (Baynes, 1995). Increased lipid peroxidation impairs membrane

functions by decreasing membrane fluidity, and changing the activity of membrane-

bound enzymes (Baynes, 1995). Its products (lipid radicals and lipid peroxide) are

harmful to the cells in the body and are associated with atherosclerosis and brain damage.

In diabetic rabbits, increased lipid peroxidation was associated with hyperlipidemia.

Liver, an insulin dependent tissue that plays a pivotal role in glucose and lipid

homeostasis is severely affected during diabetes mellitus.

During diabetes a profound alteration in the concentration and composition of

lipid occurs. Despite the great strides that have been made in the understanding and

management of diabetes, the disease and disease related complications are increasing

unabated (Tiwari et al., 2002). Inspite of the presence of known antidiabetic medicine in

the pharmaceutical market, remedies from medicinal plants are used with success to treat

this disease (Bhattaram et al., 2002). Many traditional plants are used throughout the

world for the treatment of diabetes mellitus. Plant drugs and herbal formulation

(Bhattacharya et al., 1997) are frequently considered to be less toxic and more free from

side effects than synthetic one. Based on World Health Organization (WHO)

recommendations for hypoglycemic agents of plants origin used in traditional medicine

are important. The attributed anti-hyperglycemic effect of these plants is due to their

ability to restore the function of pancreatic tissues by causing an increase in the insulin

output or inhibit the intestinal absorption of glucose or to the facilitation of metabolites in

the insulin dependent process.

Treatment with herbal formulations has an effect on protecting B-cells and

smoothing out fluctuation in glucose levels (Elder, 2004; Jia et al., 2003). There is

limited biological knowledge on the specific modes of action in the treatment of diabetes

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but most of the plants have been found to contain substances like glucosides, alkaloids,

terpenoids, flavonoids etc. that are frequently implicated as having anti-diabetic effect

(Loew et al., 2002).

In addition to the orthodox hypoglycaemic therapies there are also many plants and

plants extracts which possess marked hypoglycaemic activity. From ancient times, such

materials have been used for the treatment of diabetes mellitus and still finds extensive

use in the traditional medicine worldwide. There have been several comprehensive

reviews covering plant hypoglycaemics (Oliver–Bever and Zahnd, 1979). Several reports

have also shown a good number of plants with antidiabetic activity as well as their

constituents, worldwide usage, and the chemical structures of their phytochemicals

(Handa et al., 1989).

Studies by Ugochukwu and Babady (2003), Pushparaj (2000), Grover et al. (2002)

and Steven et al. (2004) described the antidiabetic properties of Gongronema latitolium,

Averrhoa Bilinibi, Brassica Juncea seeds and Vernonia amygdalina leaf extracts

respectively using animal models. There have also been successful clinical trials of some

of these antidiabetic plants. It is generally agreed that medicinal plants and their products

are relatively safer than synthetic drugs and offer a more holistic approach to treatment.

Perhaps the way nature made it, medicinal plant constituents mimic more closely the

natural constitution of the animal (human) system (Evans, 2002). About 80% of the

world’s population relies on herbal medicines and governments of third world countries,

unable to sustain a complete coverage with Western-type drugs, have encouraged the

national development of traditional treatments (Evans, 2002). Presently, the World

Health Organization is taking an official interest in such developments in the bid to make

health care available for all.

1.1 Diabetes Mellitus

In the early years of modern medicine, diabetes was variously thought to be a

disease of the kidney, the stomach, the blood and the liver and as far as 1857, it was

considered a disease of the nervous system. The result of the experiment of Oskar

Minkowski and Joseph Von Mering in Strasbourg in 1889, however showed that diabetes

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is a disorder associated with pancreas (Milton et al., 1996). Diabetes has unusually high

concentration of glucose in the blood, a condition called hyperglycemia (Lehninger

2000). Insulin helps most cells of the body to take up biological fuels, including the sugar

(glucose). As the beta cells of islets of Langerhans are destroyed and the pancreas stop

producing this crucial hormone, glucose accumulates in the blood, giving rise to

abnormal glucose level that is the hallmark of diabetes.

1.1.1 Types of Diabetes Mellitus

There are four major classes of diabetes mellitus based on their aetiology and

clinical manifestation (Field-May, 1998).

1.1.1.1 Type 1 Diabetes Type 1 diabetes, also known as insulin–dependent diabetes mellitus (IDDM),

usually begins in childhood and is thought to be a result of autoimmune destruction of the

pancreatic beta cells (the cells that produce insulin, also called islet cells). Destruction of

beta cells results in a complete or almost complete loss of insulin production.

(a) Aetiology of Type 1 Diabetes Type 1 diabetes is a discrete disorder and its pathogenesis involves environmental

triggers that may activate autoimmune mechanisms in genetically susceptible individuals,

leading to progressive loss of pancreatic islet B-cells (Harrison et al., 1999). In many

respect, attempts to explain the aetiology of Type 1 diabetes have be disappointing

despite decades of research (Harrison et al., 1999; Atkinson and Maclare, 1994).

Predisposition is mediated by a number of genes that interact in a complex manner with

each other and the environment.

The fact that Finland has the world’s highest incidence of Type 1 diabetes, 35

cases per 100,000 women (persons) when compared with Estonica whose population is

linguistically and ethnically very similar (Tuomilochto et al., 1992) but has only a third

of the Finland incidence of Type 1 diabetes is an indication that environmental factors

might have a particular powerful influence on the aetiology of Type 1 diabetes. Despite

these compelling epidemiological findings, the environmental factor(s) that precipitate

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Type 1 diabetes in genetically susceptible individuals have remained speculative,

although diary products and early weaning onto cow’s milk, and increased dietary nitrate

and nitrite have been suggested to be associated factors (Tuomilochto et al., 1997).

The environmental agents that cause this injury are difficult to identify owing to

the long period between exposure and the onset of hyper-glycaemia. Iron could be toxic

to insulin – producing cells by promoting free radical mediated stress. Up to 80% of

people with haemochromatosis develop diabetes. Men with high iron store (low

transferring receptor, ferritin ratio) have a 25 – fold increase in risk of developing

diabetes.

(b) Common symptoms of Type 1 Diabetes Common symptoms of Type 1 diabetes include:

Increase thirst and frequent urination: As excess sugar builds up in the blood stream,

fluid is pulled from tissues, this may result to thirsty. As a result, one could drink and

urinate more than usual.

Extreme hunger: Without enough insulin to move sugar into the cells, muscles and

organs become depleted of energy. This triggers intense hunger that may persist even

after eating.

Weight loss: Despite eating more than usual to relieve constant hunger, weight loss is

experienced. Without the energy sugar supplies, muscle tissue and fat stores will

simply shrink.

Fatigue: If the cells are deprived of sugar, tiredness and irritable could occur.

Blurred vision: If blood sugar level is too high, fluid may be pulled from tissue

including the lenses of the eyes. This may affect the ability to focus. Lastly slow-

healing sore or frequent infection.(Wikipedia,2007)

(c) Risk factors for Type 1 Diabetes

Heredity: If one has close relative (parent or sibling) with Type 1 diabetes then

one is at an increased risk of developing the condition.

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Race: Type 1 diabetes strikes people of every race, but it is more common in

some white populations – especially Northern Europe and less common in, for

example, people of Asian origin (Harrison et al., 1999)

Age: Approximately half of people diagnosed with Type 1 diabetes are under 20.

Peak incidence is at about 11 years of age.

(d) Treatment of Type I Diabetes People with Type 1 diabetes must take exogenous insulin for survival to prevent

the development of ketoacidosis

1.1.1.2 Type 2 Diabetes Type 2 diabetes is characterized by insulin resistance and/or abnormal insulin

secretion, either of which may predominate. Type 2 diabetes has an adult onset. People

with Type 2 diabetes are not dependent on exogenous insulin, but may require it for

control of blood glucose concentration if this is not achieved with diet or with oral

hypoglycaemic agents.

(a) Aetiology of Type 2 Diabetes Diabetes epidemic relates particularly to Type 2 diabetes, and is taking place both

in developed and developing nations (Zimmet, 1992). Type 2 diabetes is strongly

associated with a sedentary lifestyle and obesity. Although, it is numerically more

prevalent in the general population. Type 1 diabetes is the most common chronic disease

of children. But with increasing prevalence of Type 2 diabetes in children and

adolescents, the order may be reversed within one or two decades (Fagot-Gampagna et

al., 2000; Fagot-Gampagna and Narayam, 2001).

(b) Common symptoms of Type 2 Diabetes Subject may develop symptoms such as increase in thirst, increase in urination,

foul odour of urine, which may be dark in colour, breath with fruity or sweet smell,

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stomach cramps, numbness, or tingling in feet or hands, and blurred vision. Wounds may

not heal fast.

(c) Risk factor for Type 2 Diabetes Heredity: Type 2 diabetes often runs in families.

Race: A number of populations are at increased risk of Type 2 diabetes, including;

Native Americans, people of Asian or African. Caribbean descent and Pacific

Islander groups.

Age: Nearly all people diagnosed with Type 2 diabetes are over 30 years old. Half of

all new cases are aged 55 and above.

Obesity: Insulin resistance increases with lack of exercise.

Women who have had gestational diabetes. Previous gestational diabetes increase

risk of Type 2 diabetes developing later on in life.

Women who have given birth or babies weighing 4.0 kg or more (Zimmet, 1992).

(d) Treatment of Type 2 Diabetes The treatment for Type 2 diabetes depends largely on the individual situation.

Obesity causes insulin resistance, so the first line of attack for people who have diabetes

and are over weight is usually dietary control and a weight loss program. Physical

activity also makes the body’s cell more responsive to insulin. Most people are therefore

treated by “Diet and Exercise” when newly diagnosed. If the “Diet and Exercise”

approach is unsuccessful then there are number of oral medications (tablets) that can be

used to help the blood glucose control in Type 2 diabetes. The right combinations of diet,

exercise and adequate medication when needed is the key to effective management of

Type 2 diabetes (Fagot-Gampagna et al., 2000; Fagot-Gampagna and Narayam, 2001).

1.1.1.3 Gestational Diabetes

Gestational diabetes is a form of diabetes that develops during pregnancy. It

occurs in up to 4% of all pregnancies in the general population. Hormones produced by

the placenta during pregnancy cause insulin resistance in the mother. This means that the

113

mother needs to produce extra insulin in order to keep blood glucose concentrations

within normal limits (ADA, 2007).

If the body cannot produce sufficient insulin during pregnancy, then gestational

diabetes set in. Insulin resistance increases drastically after 24th week of pregnancy.

High blood glucose concentrations can be dangerous to the baby and may lead to a

number of possible complications with the pregnancy. So, it is important for the health

of the baby and for the mother that the blood glucose concentration should remain as near

normal as possible. For this reason the diagnosis criteria for gestational diabetes are

slightly stricter than they are for other types of diabetes (Wikipedia, 2007).

(a) Common symptoms of gestational diabetes Gestational diabetes share many characteristics of Type 2 diabetes in common,

but it is not Type 2 and it will usually disappear after delivery. Sometimes either Type 1

or Type 2 may be “unmasked” by the added stresses on the female body during

pregnancy – in which case, the diabetes remain after the baby has been born.

Although the diabetes usually goes away after delivery, there is an increased risk

of developing gestational diabetes again in future pregnancies and of developing Type 2

diabetes in later life (ADA, 2007).

(b) Risk factors for gestational diabetes Obesity: Insulin resistance increases with body weight.

Heredity: If one has a close relative (parent or siblings) with Type 2 diabetes then

one is at an increased risk of gestational diabetes.

Age: Gestational diabetes is more common in pregnant women more than 25 years

old.

Previous gestational diabetes.

Previous birth of a baby weighing 4.0 kg or more.

Pregnant women at high risk of developing gestational diabetes should ideally be

tested as soon as possible after the pregnancy has been confirmed, and then again

between the 24th and 28th weeks of pregnancy (ADA, 2007).

(c) Treatment of gestational diabetes

114

Treatment of Gestational diabetes is usually through diet and exercise.

Sometimes insulin injections are required for the duration of the pregnancy.

1.1.1.4 Other types of diabetes

There are also a number of more specific forms of diabetes, including

Maturity onset diabetes of the young (MODY).

Diabetes induced by certain medications or chemicals.

Diabetes caused by other hormone conditions.

All type of diabetes have the common hallmark of a high blood glucose concentration

which is caused by complete or partial lack of insulin, but the root of the cause is slightly

different in each case (WHO, 1999).

1.1.2 Complications in Diabetes Mellitus

Diabetes is associated with a significant medical problem. Severe hyperglycemia

may result in coma or even death. Milder hyperglycemia, if present for many years,

increases the risk of cardiovascular disease, which can manifest as a heart attack,

congestive heart failure, stroke, gangrene of the extremities (necessitating amputation in

some cases), or kidney failure (Goycheva et al., 2006).

Diabetes mellitus is wide spread disease that affect all nationalities and ages. The

number of patients in 2003 has reached an epidemic proportion totalling a whopping 194

million with patients of 20 to 79 years of age affected (5.1% of the population in this

group). A rise to 50% is expected in 2010, mainly from new cases in Africa, Asia and

South America (Goycheva et al., 2006). A projection of this figure shows that in 2025

diabetes patients will be 333 million or 6.3% of the total population on earth (Goycheva

et al., 2006).

To this alarming trend must be added the fact that chronic complication of

diabetes – micro and macro angiopathy, are the cause of 4 times higher mortality in

patients with diabetes mellitus in comparison with healthy individual.

To understand the essence of aetiopathogenic mechanisms, which are at the root

of diabetic complications development, is an essential challenge to modern medical

115

science and practice. Nowadays diabetic micro and macro angiopathy are considered to

be polyaetiological multifactoral diseases where persistent hyperglycemia plays the

leading part (Baynes, 1991). On the other hand it contributes to origin of oxidative stress.

Along with others, endogenous and exogenous factors take a considerable place in

diabetes pathogenesis. Hence, the patients are exposed to continuously increasing

oxidative stress caused by the prolonged hyperglycemia and conditioned by different

pathophysiological process. The diagram below (Fig. 1 describes the three basic

consequencies of hyperglycaemia: Advanced glycation end product formation, glucose

auto-oxidation and sorbitol increase (Goycheva et al., 2006).

HYPERGLYCEMIA

OXIDATIVE STREES

AGEs formation Glucose Autooxidation

Sorbitol way

Endothelial disfunction

Coagulation

Thrombocyte activity

NO, endothelin – 1 prostacyclin

TXA2

VASSAL

COMPLICATIONS

Lipid peroxidation

Leucocyte adhesion

Formation of “foamy” cells

TNF-α

Fibrinolisis

Hypercoagulation

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Fig. 1: Schematic Representation: Hyperglycaemia and biochemical processes lead to

oxidative stress and vascular effects. (Goycheva et al., 2006)

AGES = Advanced glycosylation end products, TxA2 – Thromboxane A2

TNF- = Tumor necrotising factor -

NO = Nitric Oxide.

It is evident that in state of chronic hyperglycemia non-enzyme glycosylation of

protein set in. Their formation mechanism is presented in Fig. 2

Non-enzyme glycosylation of proteins

In Mallard reaction

Unstable Schiff bases

Amadori products

Long semilife proteins Short semilife proteins

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Fig. 2: Influence of hyperglycemia on plasma protein

(Goycheva et al.., 2006)

A brief period of semi life of most cellular and plasma proteins does not provide

the possibility for Amadori poroducts to transform further. By contrast, protein with

semi life, a part of Amadori products undergo partial oxidative degradation and

carboxymethllysine. The rest are included into series of intermediate and subsequent

mallard reactions until formation of pigmented, fluorescent and containing cross – “links

“advanced mailard products” called also advanced glycosylation end products” (AGEs),

for example pentosidine and others. They can be determined as a class of heterogeneous

compounds of monosaccharides and proteins, obtained by consecutive reactions of

dehydration, condensation (Bowulee, 1994). This produces a combination of glucose

with plasma proteins free radicals. Together with transformed proteins, they contribute to

the intensification of oxidative stress and vessel injury (Goycheva et al., 2006).

1.1.2.1 Microangiopathy

It is a specific complication and is the most clearly expressed and is characteristic

for patients with diabetes mellitus Type 1 but affects also the other forms of the disease.

Important clinical problems are micro vascular lesions in retinal, neuronal and kidney

vessel leading to retinopathy, neuropathy (Parvanora et al., 2002).

No change Carboxmethyllisine Advanced glycosylation end products

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(a) Retinopathy Retina is characterized by high content of lipid and increased consumption of

oxygen. This makes it particularly susceptible to the influence of reactive oxygen types

formed in condition of hyperglycemia. It is established that markers of oxidative stress

(Malondialdehyde, sulfhydril protein) in sub retinal liquid in patients with proliferate

retinopathy are changed significantly in comparison with healthy control and patients

without retinopathy. In addition to this, activation of protein kinase C intensifies

synthesis of vasoactive prostanoids and lead to changes in retinal blood stream

(Parvanora et al., 2002).

(b) Nephropathy

It is proved that “final products of advanced glycosylation” take principle position

in pathogenesis of diabetic nephropathy. Observed initially kidney hyperperfusion and

hyper filtration are connected with activation of phospholipase A2 by protein kinase C.

Advanced diabetes however, leads to kidney vasoconstriction and increased deposition of

extra cellular matrix contributing to systemic hypertension and nephrosclerosis.

(c) Neuropathy The diminished neural perfusion and capillary occlusion due probably to

thrombosis, oedema of endothelial cells or proliferation connect neuropathy with micro

vascular disease. It is considered that reactive oxygen special (ROS) damage neural

fibres (Parvanova et al,2002)

1.1.2.2 Macroangiopathy

It is atheroslerotic process, which because of metabolic troubles in diabetes, set in

earlier age. It occurs more often and shows a faster evolution and heaviness in

comparison with the non-diabetic. Macroangiopathy is more characteristics of diabetes

mellitus Type 2. Artherosclerotic lesions are formed mainly in the big and medium size

arteries and can cause ischaemic changes in the heart, the brain or the extremities. This

can lead to myocardial infarction, brain stroke or necrosis of the foot.

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Unlocking factor for atherogenesis is lesion of endothelial cells in persistant

hyperglycemia and oxidative stress.

Nowadays the concept that reactive oxygen types and lipid peroxidation products are

leading factors in endothelium lesion is being held.

In addition to endothelia dysfunction it is shown that upset lipid metabolism takes

central position in atherosclerosis evolution (Parvanova et al., 2002). Patients with

diabetes Type 2 have usually increased levels of triglycerides (especially of VLDL) and

diminished quantity of HDL – cholesterol – two risk factors for cardio-vascular disease.

Concentrations of LDL-cholesterol do not differ significantly from these in persons

without diabetes, but in constrast to them LDL – particles are smaller, thicker and

oxygenated. They are more strongly atherogenetic than normal, bigger and more motile

LDL – particles.

Oxidation of LDL diminishes their ability to fluctuate between the lumen and

blood vessels wall. As the oxidized LDL enable extracellular release of lipids and

lysosome enzymes and reinforce atherogenesis, they are cytotoxic for endothelia cells.

That is why oxidative modification of lipoproteins appears to play a key role in formation

of “foamy” cells, which is irreversible stage in the atherogenesis chain (Jiala et al., 1996)

The above described relation between lipid oxidation and atherogenesis is an important

fact and possibililty for favourable therapeutic influence on evolution of Ischaemic

disease of the heart and other manifestation of macroangiopathy.

1.1.3 Oxidative Stress Caused by Hyperglycaemia Oxidation acts upon endothelium expression of growth factors from its adjacent

cells. The transforming growth factor B (TGF – B) secreted by mesangealial cells and

growth factor of vessel endothelium (VEGF) stimulate its cells proliferation and suppress

their ability for regenerating after damage.

Hyperglycemia effects are reinforced additionally by reactive oxygen types

formed in the course of non-enzyme glycosylising of proteins and glucose autoxidation

occurring simultaneously (Browlee, 1994). Obtained “end-products of advanced

glycosylation” can activate endothelial cells attaching to specific receptors on their

surface. In these conditions endothelial permeability increases, accumulation of adhesive

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molecules deepens and synthesis of interleukins (1L-1, 1L-6) and tumour necrotizing

factors- (TNF-) is reinforced (Frostegard et al., 1991).

1.1.4 Free Radical and their Role in Diabetes Mellitus

A free radical is defined as any species which contains one or more unpaired

electrons in its outer ring but capable of independent existence (Escobales et al., 2005;

Siore et al., 2005). Free radical also includes atoms or molecules, which contain

unpaired electrons. Since they have very strong tendency to exist in a paired rather than

unpaired state, free radicals indiscriminately pick up electrons from other atoms, which in

turn convert those other atoms into secondary free radicals, thus, setting off a chain

reaction which can cause substantial biological damage (Virag, 2005).

Free radical damage is believed to play a role in atherosclerosis, cataract

formation, and some of the other complications of diabetes. To counteract the

destructiveness of free radicals, the body possesses a complex system of antioxidant

defenses that utilizes various vitamins, minerals and other naturally occurring substances.

Diabetes have been reported to have significantly higher free-radical activity, as well as

significantly lower concentrations of antioxidants compared with healthy controls. These

changes were of greater magnitude in patients with disease complications than in those

without complication. It is possible; therefore, that supplementing with foods, nutrients

and herbs that have antioxidant activity would help prevent diabetic end-organ damage

(Virag, 2005).

1.2 Generation of reactive oxygen species (ROS) Reactive oxygen species is a collective term referring to free radicals and non-

radicals derived from oxygen either by electrons transfer or by energy transfer reactions.

Oxygen free radicals include superoxide radical (O∙2) and hydroxyl radicals (OH) while

non-radicals are hydrogen peroxide (H2O2), singlet oxygen (O2) and Nitric oxide (NO).

Some other free radicals include lipid peroxy radicals (LOO), alkoxy radical (RO),

peroxynitrite (NO2) etc (Aruoma et al., 1991). Free radicals and reactive species are

derived either from normal essential metabolic activities in the human body or from

external sources. Free radicals formation occurs continuously in the cell as a

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consequence of both enzymatic and non-enzymatic reactions. Enzymatic reactions,

which serve as sources of free radicals, include those involved in respiratory chain,

phagocytosis and prostaglandin synthesis and in the cytochrome system (Shanmganathan

et al., 2005).

Some of internally generated sources of free radicals are mitochondria,

phagocytes, Xanthenes oxidase, reaction involving iron and other transition metals

arachidonate pathways, peroxisomes, exercise, inflammation and ischaemia/reperfusion

(Brighenti et al., 2005).

The external generated sources of free radicals include: cigarette smoke, environmental

pollutants, radiations, ultraviolet light, certain drugs, pesticides, anesthetic, industrial

solvents and ozone (Ozgumer et al., 2005).

Polyunsaturated fatty acids (PUFAs) are particularly susceptible to peroxidation

because they contain multiple double bonds in between which lie methylene –CH2–

groups that possess especially reactive hydrogens. Any reactive species capable of

abstracting a hydrogen atom from the –CH2– group of PUFA initiates the process of lipid

peroxidation which like any other radical reaction, is a chain reaction involving three

major steps: initiation, propagation and termination.

Initiation

The initiation of lipid peroxidation is caused by attack of ROS (most commonly

∙OH) which is capable to abstract a hydrogen atom from a methylene group (–CH2–) of

an unsaturated fatty acid producing a lipid radical (fatty acid radical).

HO∙ + RH R∙ + H2O -- -- -- -- (1)

Propagation

The fatty acid radical is unstable, it is stabilized by molecular rearrangement,

followed by reaction with oxygen to give a peroxyl-fatty acid radical (lipid peroxyl

radical). This too is an unstable species that reacts with an adjacent fatty acid producing a

new fatty acid radical and a lipid hydroperoxide or a cyclic peroxide if it had reacted with

itself. This cycle continues as the new fatty acid radical reacts in the same way.

122

R∙ + O2 ROO∙ -- -- -- -- (2)

ROO∙ + RH R∙ + ROOH -- -- -- -- (3)

The lipid hydroperoxide is also unstable and in the presence of a metal catalyst

such as iron forms a reactive alkoxy radical.

ROOH + Fe2+ OH– + RO∙ + Fe3+ -- -- (4)

Termination

When a radical reacts, it always produces another radical, which is why the

process is called a “chain reaction.” The radical reaction stops when two radicals react

and produce a non-radical species. This happens only when the concentration of radical

species is high enough for there to be a high probability of two radicals colliding. Living

organisms have evolved different molecules that speed up termination by donating

electrons to free radicals and therefore protect the cell membrane. One important such

molecule is vitamin E.

R∙ + R∙ R_R -- -- -- -- -- (5)

R∙ + ROO∙ ROOR -- -- -- -- -- (6)

ROO∙ + ROO∙ ROOR + O2 -- -- -- -- (7)

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Fig. 3: The free radical mechanism of lipid peroxidation (Wikipedia, 2009)

1.3 Oxidative stress and tissue injury

Oxidative stress is defined as any disturbance of the pro-oxidant to antioxidant

balance in favour of the pro-oxidant (Ozturk et al., 2005). It is a condition associated

with an increased rate of cellular damage mediated by oxygen and oxygen-derived

oxidants commonly known as reactive oxygen species (Xu et al., 2005).

Reactive oxygen species (ROS) are free radicals with highly reactive oxidizing

potentials. In normal cell, there is an appropriate pro-oxidant: antioxidant balance.

However, this balance under oxidative stress is titled in favour of pro-oxidant due to

124

either increased production of reactive oxygen species or decrease in antioxidants levels.

(Matsu

moto et al., 2005)

Reactive oxygen species have been implicated in many disease states, which

ranges from malaria, sickle cell anaemia (Stern, 1993), infertility, to arthritis, connective

tissue disorder, carcinogenesis, ageing, toxin exposure, physical injury, infection and

acquired immune deficiency syndrome (Furukawa et al., 2004).

Free radical – mediated tissue injury is a final pathway of damaged and integral

component of wide variety of disparate pathophysiological and even physiological

processes (Reilly and Buckley, 1990). It is thought that a significant part of the damage

is related to the effect on cell membrane structures, which are highly vulnerable to radical

disturbances. Evidence abide that the effects of ROS can be as drastic as cells being

completely perforated, so that their contents leak into the blood stream (Sims et al.,

2004). Besides membrane effects, free radicals can damage DNA bases, primarily

guanine via lipid peroxy or alkaoxy radicals or through covalent bonding of MDA

resulting in strand breaks and cross links (Chen et al., 2004). ROS can also induce

oxidation of critical site groups in proteins and DNA, which will alter structure and

functions of species molecules. Free radicals are known to trigger the accumulation of

polymorphonuclear (PMN) leucocytes which can adhere to capillary cell walls or even

plug capillary and venules, thus resulting in Macro-circulatory derangements,

inflammation and tissue damage (Aliyez et al., 2005).

Free radicals can attack the DNA molecule through its deoxyribose sugar moiety

or through its base constituent, purine or pyrimidine to produce a wide range of product

that induces cell cancer. The most important target of OH radical attacks is the

polyunsaturated fatty acids. Abstraction of a hydrogen atom from a PUFA initiates the

process of lipid peroxidation (Yoshimi et al., 2005). Numerous products are formed,

presenting special analytical problems as shown in Fig. 4.

125

∙OH + RH H2O + R -- -- -- -- -- 8

X

__

(1)

__ __ __

oOH

HOHH

L

Y

H O O

O O

+ MDA

O

C

R

X

Y

PUFA

126

Fig 4: Free radical mediated per oxidation of unsaturated fatty acid leading to damage

and the generation of numerous end products

(Yoshimi et al., 2005)

Polyunsaturated fatty acid

(MDA) = Malondialdehyde

(4HDA) = 4 – hydroxyalkenals

LH = Lipid molecule

L = Lipid peroxyl radical

1.4 Pancreas

1.4.1 Anatomy of the pancreas

The pancreas (shown below) is an elongated, flat, hammer shaped organ that is

light tan or pinkish in colour, analogous in its structures to the salivary glands which is

located across the back of the abdomen right behind the stomach and touching the spleen.

Its right extremity, being broad, is called the head, and is connected to the main portion

of the organ, or body, by a slight constriction, the neck. Its left extremity gradually tapers

to form the tail (Wikipedia, 2007).

PUFA Radical

Lipid peroxyl radical

4 HDA

Lipid peroxide

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Fig. 5: Structure of the pancreas is made up of two types of tissues (Wikipedia, 2007)

128

1.4.3 Types of Pancreatic Tissues

Pancreas is made up of two types of tissues:

(i) Exocrine tissue

The exocrine tissue secretes digestive juice to the duodenum. Pancreatic juice is

composed of two products critical to proper digestion, digestive enzymes (e.g.

Trypsinogen, Chymotrypsinogen, Amylases e.t.c) and bicarbonate. As food chyme into

the small intestine from the stomach, two things happen”

Acid must be quickly and efficiently neutralized to prevent damage to the

duodenal mucosa.

Macromolecular nutrients – proteins, fats and starch – must be broken down

much further before their constituents can be absorbed through the micosa

into blood. The pancreas plays a vital role in accomplishing both of the

objectives; so vital in fact that insufficient exocrine secretion by the pancreas

leads to starvation, even if the animal is consuming adequate quantities of

high quality food.(Wikipedia 2007)

(ii) Endocrine tissue

The endocrine tissue (Islets of Langerhans) secretes hormones, which are taken up

by the blood stream and are concerned with sugar metabolism. Glucagon raises the

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concentration of glucose (sugar) in the blood. Insulin stimulates cells to utilize glucose.

Somatostatin may regulate the secretion of glucagons and insulin.

The Islet has no ducts, but rich in capillaries with a fenestrated endothelium.

Pale cells contain granules differing in alcohol-solubility and staining characteristics for

the differentiation of

(a) Alpha cells (20%), located mostly in the periphery of the dorsal

pancreatic bud, secrete glucagons,

(b) Beta calls (75%), located in the center of the Islets, secrete insulin, C

peptide, and proinsulin. Insulin promotes the intracellular movement of

glucose and glycogen storage thereby lowering the glucose concentration

in the blood.

(c) Delta cells, (5%), dispersed. Produce somatostatin, which inhibits insulin

and glucagons release.

(d) F cells: located in the periphery of the ventral pancreatic buds. Secrete

pancreatic polypeptide. Blood drained from the pancreas and bearing the

polypeptide hormones passes via the portal flow, to the liver. (Polonsky

and O Meara, 2001)

1.4.4 Functions of the pancreas

The pancreas has a dual role – It helps digest food and also secretes hormones

that, among other things, affect the concentration of sugar in the blood.

(1) It synthesizes enzymes to aid in digestion and produces the hormone insulin to

regulate blood sugar concentrations in the blood stream.

(2) The digestive juices secreted by the pancreas combine with juices from the

intestines to complete the job of breaking down proteins, carbohydrates, and fats.

(3) The pancreas secretes enzymes that have the capacity to reduce virtually all

digestible macromolecules into forms that are capable of being absorbed. Substance in

the pancreatic juice also helps neutralize stomach acids that pass from the stomach into

the small intestine.(Wikipedia,2007)

Alcoholism, gallstones and viral infections can cause a serious inflammation of

the pancreas called pancreatitis. When gallstones block the bile duct, the flow of

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pancreatic juice is stopped. This may also lead to pancreatitis. Hypoglycaemia (too little

sugar in the blood) can result from over production of insulin by the islets of langerhans.

Tumors on the islets themselves, or large tumour on other organs near the pancreas, can

cause the release of excess insulin.

1.5 Insulin

To generate energy, cells need food in a very simple form. Food taken in is

broken down into simple sugar called glucose. Glucose provides the energy needed for

daily activities. Blood transports glucose to the cells where it will be used, stored, or

converted to fat. When the amount of glucose in blood reaches a certain concentration,

the pancreas releases insulin. As more glucose enters the sells, the concentration of

glucose in the blood stream drops. Without insulin, the glucose cannot be stored which

allows the concentration of glucose in the blood to rise.

Insulin concentrations are normal determined by a feedback control system that is

responsive to the prevailing concentration of plasma glucose (Polonsky and O’ meara,

2001). The overall sensitivity of the pancreatic B-cell to glucose is determined by the

sensitivity of peripheral tissues to the action of insulin with insulin resistant subjects

having higher insulin concentrations and insulin secretion rate than insulin – sensitivity

subjects. Insulin is also secreted in response to amino acids and fatty acids and the

magnitude of this response being modulated by a variety of neural (For example,

sympathetic and parasympathetic autonomic nerves) and hormonal factor (For example,

glucagons, glycogen – like peptide I, gastric inhibitory polypeptide and somatostatin).

Glucose, however, is the overriding influence.

Normal insulin secretion shows a rapid response to glucose and a complete

pulsatile profit. The increase in insulin – secretion that occurs after the intravenous

administration of glucose is virtually instantaneous; even after oral glucose ingestion,

increase in insulin secretion occur within minutes. Early manifestations of disordered B-

cell function include delayed and blunted responses to glucose, temporal irregularities in

the pulses and oscillations of insulin secretion, and loss of the tight coupling between

pulses of insulin secretion and pulses of glucose. Diabetes is also associated with

impaired conversion of pro-insulin to insulin. As a result, circulating pro-insulin

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concentrations comprise a greater proportion of total immune-reactive insulin

concentrations in diabetic than in normal individual (Polonsky and O meara, 2001).

1.6 Antioxidants

Antioxidant is a molecule stable enough to donate an electron to a free rampaging

radical and when present at low concentration compared to those of the oxidizable

substrate, significantly delays or inhibits oxidation of that substrate (Cateri et al., 2005).

Antioxidant defense protect cells against the potential damage induced by oxygen free

radicals and can be categorized as either preventive antioxidant, which include the

enzyme superoxide dismutase, catalase and glutathione peroxidase, among others, or the

chain – breaking antioxidants such as vitamin E, ubiquinone, urate, glutathione. The

preventive antioxidants eliminate the species involved in the initiative of free radical

chain reactions, whereas the chain – breaking antioxidants repair oxidizing radicals

directly (Bonnefont – Rousslet et al., 2000).

Human body has several mechanisms to counteract damage by free radicals and

other reactive species. One mechanism could be endogenous while other could be

enzymatic. The endogenous mechanism could be mediated by a system of antioxidants

such as ascorbic acid (Vitamin C), tocopherol (Vitamin E), caroteniods, glutathione,

ubiquinone, uric acids, phenolic and non-phenolic compounds such as flavonoids and

other polyphenols are also antioxidant (Medany et al., 2005).

Endogenous antioxidants perform their function by removing oxygen or

decreasing oxygen concentration, chelating catalytic metallic ions, removing key reactive

oxygen species (ROS) such as superoxide and hydrogen peroxide. They also scavenge

initiating free radicals such as hydroxyl, alkoxyl and peroxyl species breaking the chain

of an initiated sequence and also by quenching/scavenging singlet oxygen. On the other

hand, cellular antioxidant enzymes operate at different levels by preventing radical

formation, intercepting radicals when formed, repairing oxidative damage caused by

radicals, facilitates the elimination of damage molecule and catalyzing effectively

antioxidant reactions.

1.6.1 Superoxide dismutase (SOD) [EC.1.15.1]

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Superoxide dismutase formally known and identified with such names as

erythrocuprein, indophenol oxidase and tetrazolium oxidase catalyses the dismutation of

superoxide anion to hydrogen peroxide and molecular oxygen (Ukeda et al., 1997).

O∙2 + O∙2 + 2H+ H2O2+O2 -- -- -- -- (9)

Superoxide dismutase is ubiquitous metaloproteins that play a major role in living

cells and have been widely used as pharmacological tools in the study of

pathophysiological mechanism.

Superoxide dismutase (SODs) is present in all aerobic organisms and most

subcellular compartments that generate activated oxygen. It is therefore assumed that

SOD has a control role in the defense against oxidative stress.

There are three isozymes of superoxide dismutase classified on the basis of metal

cofactor the Mn-SOD found in the mitochondria of eukaryotic cells, the Cu/Zn-SOD is

found in the cytosol and chloroplast and the Fe-SOD found in the chloroplast (Bowler et

al., 1992). The Mn-SOD and Fe-SOD are also found in prokaryotes and eukaryote algae.

It was reported that diabetes has multiple effects on the protein levels and activity

of this enzyme which further augment oxidative stress by causing a suppressed defense

response. Hence, modulation of this enzyme in target organs prone to diabetic response.

Hence, modulation of this enzyme in target organs prone to diabetic complication such as

the kidney may prove beneficial in the prevention and management of kidney failure

(Johansen et al., 2005).

1.6.2 Catalase (EC.1.11.6)

Catalase is a haem-containing enzyme that catalyses the dismutation of hydrogen

peroxide into water and oxygen. The enzyme is present in all aerobic eukaryotes. It is

important in the removal of hydrogen peroxide generated in peroxosomes (microbodies)

by oxidases involved in oxidation of fatty acids, the glyoxylate and purine metabolism.

133

Catalase was one of the first enzymes to be isolated in a highly purified state. Multiple

forms of catalase have been described and all forms are tetramers.

Catalase is very sensitive to light and has a rapid turn over rate. This may be as a

result of light absorption by the heam group or perhaps hydrogen peroxide inactivation.

Stress conditions which reduce the rate of protein turnover such as salinity, heat shock or

cold, caused the depletion of catalase activity.

The reaction catalysed by catalase include (Equation 2 and 3)

2H2O2 2H2O + O2 (Catalytic Activity) -- -- -- -- (10)

Decomposition

ROOH + AH2 H2O + ROH + A (Periodic Activity) -- -- (11)

Oxidation

1.6.3 The Glutathione System

Glutathione (GSH), present in plants, animals and bacteria often at high levels,

can be thought of as a redox buffer. It is derived from glycine, glutamate and aspartate.

The oxidized form of glutathione (GSSG), produced in the course of its redox activities,

contains two glutathione molecules linked by a disulfide bond. Glutathione acts as a

direct scavenger as well as a co-substrate for glutathione peroxidase. It is a major

intracellular redox system (Johansen et al., 2005).

Pentose phosphate pathway in erythrocytes provides NADPH for the reduction of

oxidized glutathione to reduced glutathione catalysed by glutathione reductase, a flavo

protin enzyme containing FAD (Fig. 5). In turn, reduced glutathione removes H2O2 from

the erythrocytes in a reaction cataysed by glutathione peroxidases, an enzyme that

contains the trace element selenium. This reaction is important, since accumulation of

H2O2 may decrease the life span of the erythrocytes by increasing the rate of oxidation of

hemoglobin to methemoglobin.

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The activities of glutathione peroxidase (GPX) and glutathione reductase (GSSG–

PX) have been reported to be higher in cellular compartment i.e. mitochondria and

microsomes where excess free radicals are known to occur.

Fig. 6: Role of the pentose phosphate pathway in the glutathione peroxidase reaction of

erythrocytes (G–S–S–G, oxidizing glutathione; G – SH reduced glutathione, Se, selenium

cofactor).

FAD Se PENTOSE PHOSPHATE PATHWAY

NADP+

NADPH+ H +

GLUTATHIONE REDUCTASE

2G - SH

G – S – S - G

H2O2

GLUTATHIONE PEROXIDASE

2H2O

2H

135

\

1.6.4 Vitamins as Antioxidant

Vitamins are essential component of dietary requirement and are important in

both growth and repair of body tissues and organs. Of all naturally existing vitamins A,

B, C, D, E and K, only vitamin E and C as well as B carotenoid have antioxidant

potential.

These antioxidants nutrient have specific activities and they work synergistically

to enhance the overall antioxidant capacity of the body. The balance between the

production of free radicals and the antioxidant defences in the body has important health

implications. If there are too many free radicals produced and too few antioxidants, a

condition known as “oxidative stress” develop which may cause serious cell/organ

damage. Some water-soluble and fat-soluble vitamins can act in vitro as potential

antioxidants by scavenging free radicals (Zhang et al., 2005).

1.6.4.1 Vitamin E (Alpha Tocopherols)

The term vitamin E refers to a fat soluble factor discovered in 1992 to be essential

for reproduction in rats, but now used as a generic term to include all entities that exhibit

the biological activity of - tocopherols (Morlay et al., 2004; Meydani et al., 2005). The

structural formula of α–tocopherol (Vit E) is given in Fig. 6.

CH2(CH2CH2CHCH2)3H

CH3 CH3

CH3

H3C

HO CH H

H

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Fig. 7: Structure of tocopherol (Vit. E)

Alpha tocopherol, the major constituent of the fat-soluble vitamin E is probably

the most important free radicals scavenger within biomembranes. Vitamin E reacts with

free radicals and reduces the risk of disease outcome. It also affords protection against

cancer, ischaemia and reperfusion injury, cataract, arthritis and certain neurological

disordes (Jackson et al., 2004). Vitamin E intake enhances body’s immune response and

also inhibits the conversion of nitrites in the stomach to nitrosamines, which are cancer

promoters. It is also known to reduce the effect of cardiovascular disease by inhibiting

the oxidation of low-density lipoprotein, which usually damages the arterial walls. It

inhibits prostaglandin synthesis, which stimulate platelet aggregation (Meydani, 2004).

1.6.4.2 Vitamin C (Ascorbic acid)

Ascorbic acid, a water-soluble vitamin is a six-carbon compound. It is

structurally related to glucose and other hexoses. It is reversibly oxidized in the body to

dehydroascorbic acid. The structural formulae of ascorbic and dehydroascorbic acid are

given below 7.

O= C

HO = C

HO - C

HO - C

HO - C

O = C

O = C

O = C

O = C

O = C - H

O

O

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Ascorbic acid Dehydroascorbic acid

Fig. 8: Structural formula of ascorbic acid and dehydroascorbic acid

This vitamin is a free radical scavenger in the cells as well as in the fluids between cells. It is considered to be one of the most important antioxidant in extra

cellular fluids. Vitamin C regenerates vitamin E from the tocopheroxyl radicals

(Hathcock et al., 2005). Its protective effect extends to cancer,coronary artery disease,

arthritis and ageing. In cancer, it suppresses the formation of carcinogens such as

nitrosamine and quinines (Pother, 1997). Vitamin C protects the oxidation of low density

lipid (LDL) and increases high density lipid (HDL) levels and may also lower cholesterol

level in the blood thus reducing the risk of cardiovascular disease (Meydani, 2004).

Vitamin C supplement has also been inversely associated with decreased cataract risk.

1.7 Aim of Research

Inspite of the presence of known antidiabetic drug in the pharmaceutical market,

remedies from medicinal plants have been used with success to treat diabetes mellitus

(Bhattanram et al., 2002). Many traditional plant treatments/remedies for diabetes are

used throughout the world especially in Africa. Therefore, this investigation is aimed at

finding the antidiabetic effect of Colatropic gigantea leaf extracts, antioxidant potential

and its effect on some oxidative parameters in diabetic rabbits.

1.8 Research Objectives

Despite the great stride that have been made in the understanding and

management of diabetes, the disease and diseases related complications are increasing

unabated (Twari et al., 2002). It is therefore the aim of this research to:

(1) Determine the effect of this extract on blood glucose and protein levels in both

normal and diabetic rabbits.

CH2 OH

138

(2) Determine the effect of Colatropis gigantea leaf extract on oxidative index;

malondialdelyde (MDA) in alloxan induced – diabetic rabbits.

(3) Evaluate the effect of this extract on the activity of anti-oxidant enzymes,

(catalase, glutathione peroxidase, superoxide dismutase) and antioxidant vitamin

(vitamin C) level in both normal and diabetic rabbits.

(4) Study the effect of the extract on anatomy of the liver and pancreas of normal and

diabetic rabbits

(5) Determine the blood pH in both normal and diabetic rabbits.

It is hoped that the out come of this study may aid in the management of human diabetes.

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CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemicals

The chemicals used for this study are of analytical grades and include ethanol,

methanol, n-hexane, butanol, chloroform, acetone, ethylacetate, ethylene diamine

tetracetate (EDTA), hydrochloric acid, sulphuric acid, sodium hydroxide, trichloroacetic

acid, (TCA) adrenaline, 2-thiobarbituric acid (TBA), alloxan monohydrate (sigma-

Aldrich, USA) and 1 chloro-2,4-dinitrothiobenzene, glutathione peroxidase kit (Randox

Laboratories Limited, United Kingdom), Protein kit (Randox Company, USA). Glucose

test (Life Scan Inc, California, USA)

2.1.2 Drug

The known hypoglycaemic drug used was glibenclamide 10mg/kg body weight.

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2.1.3 Instruments/Equipment Atomic Absorption Spectrophotometer ( AAS) Scientific 210 VGP, Buck

Beakers Pyrex

Centrifuge PIC England

Conical Flasks Pyrex

Filter paper Whatman, England

Glass funnel Kimax

Hot plate Gallenkamp

Measuring Cylinder Pyrex England

Micro Pipette Perfect (p) U.S.A

MK.V Orbital Shaker Gallenkamp

Mortar Life scan (J and J)

Oven, Model 301 Fisher

pH Meter Pye,unicam 293, England

Pipette Pyrex

Refrigerator Haier Thermocool

Spatula Pyrex

Syringe (1 ml, 2 ml) DANA JET, Nigeria

Test-tubes Pyrex, England

UV. Spectrophotometer UNICO-UV-2102

Water bath Dk-8A Gallenkamp, England

Rotary Evaporator Gallenkamp, England

Crucible pot Gallenkamp, England

2.1.4 Plant Material The leaves of the plant Colatropis gigantea were obtained from Ogadimma

Research Farm in Effiom, Ebonyi state. The plant was identified at Department of

Botany, University of Nigeria, Nsukka.

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2.1.5 Animals The rabbits used for this study were male of about 12 – 16 weeks old with average

weight of 700 ± 50g. The mice used for LD50 determination were 4-6 weeks old with

average weight of about 25.50 ± 0.50g. They were obtained from the Faculty of

Veterinary Medicine, University of Nigeria, Nsukka. The animals were kept under

optimum temperature of 25oC for 14 days with free access to water and food before the

experiment commenced.

2.2 METHODS 2.2.1 Experimental design

The study was made up of two phases.

Phase I

Phase I was the screening phase at which the most potent fractionated extract

amongst the fractions was noted and used in the second phase.

Diabetes was induced by slow intraperitoneal injection of 1% solution of alloxan

(200mg/kg body weight) in normal saline and administered within few minutes of

preparation. Diabetic state was confirmed after three days using glucometer. The

animals were administered with the extract twice daily intraperitoneally at a dose of

300mg/kg body weight for five days. Screening was done by estimating the fraction that

reduced the elevated blood glucose level close to normal using glucometer.

A total of eighteen (18) rabbits were used and was divided into nine groups:

Group 1: Normal rabbits fed on normal rabbit chow and water ad libitum.

Group 2: Diabetic rabbits not treated and given water ad libitum.

Group 3: Diabetic rabbits treated with glibenclamide (standard) and water ad

libitum

Group 4: Diabetic rabbits treated with n-hexane fraction and water ad

libitum.

Group 5: Diabetic rabbits treated with chloroform fraction and water ad

libitum.

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Group 6: Diabetic rabbits treated with ethylacetate fraction and water ad

libitum.

Group 7: Diabetic rabbits treated with butanol fraction and water ad libitum.

Group 8: Diabetic rabbits treated with acetone fraction and water ad libitum.

Group 9: Diabetic rabbits treated with methanol fraction and water ad

libitum.

Phase II

This Phase of the study was made up of four (4) groups of three (3) rabbits each.

Diabetic induction was done as described in the phase I. Treatment was administered

twice daily intraperitoneally at a dosage of 300mg/kg body weight for five (5) days.

Blood samples were collected through ear vein for biochemical analysis. Liver and

pancreatic tissues were dissected out, washed in normal saline and kept in formal calcium

removed for histopathological study.

A total of twelve rabbits were used and divided into groups.

Group 1: Normal rabbit fed on normal rabbit chow and water ad libitum.

Group 2: Diabetic rabbits not treated and water ad libitum.

Group 3: Diabetic rabbits treated with Glibenclamide (Standard) and water ad

libitum.

Group 4: Diabetic rabbits treated with most active fraction (acetone fraction) and

water ad libitum

2.2.2 Plant treatment

Fresh leaves of C. gigantea were dried under room temperature, crushed and

soaked in 200ml of analytical grade of methanol for 48 hours then filtered and was

evaporated with rotary evaporator at room temperature to obtain the crude extract.

2.2.3 Extraction procedure

The crude extract was subjected to fractionation using different organic solvents

(Fig. 7). It was first extracted using n-hexane. The n-hexane soluble fraction was obtained

and concentrated using rotary evaporator at an optimum temperature of 25oC. The

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resulting residue (residue – A) was dried and then fractionated using chloroform. The

soluble chloroform fraction was concentrated using rotary evaporator and obtained

chloroform extract while the resulting residue (residue B) was subsequently fractionated

using ethyl acetate. The ethyl acetate soluble fraction was concentrated using rotary

evaporator and an insoluble residue (residue C). The insoluble residue obtained was re-

suspended in acetone, filtered and the filtrate concentrated. The residue obtained (residue

D) was further fractionated using methanol. The methanol soluble fraction was

concentrated and the methanol concentrate was obtained while the insoluble portion

obtained was dried; this was soluble in water.

Fractionation with n - hexane

Extraction with methanol, dried

Dried and blended C. gigantea

Whole extract

Residue A

n – hexan fraction (concentrate)

Residue B

Residue C

Fractionation with Chloroform Chloroform fraction (concentrate)

Fractionation with ethyl acetate

Ethyl acetate fraction (concentrate)

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Fig. 9: Fractionation of C. gigantea leaves

2.2.6 Determination of Yield of Extract

% Yield = 100)(

)(

PulpSampleGroundgWeightEvaporatedExtractofgWeight

2.2.7 Phytochemical Analysis of the Crude Extract

The preliminary analysis involved testing for the presence or absence of the

following plant constituents: alkaloids, flavonoids, glycosides, protein, carbohydrate,

reducing sugars, saponins, tannins, oil, resins and terpenoids.

2.2.5.1 Test for the Presence of Alkaloids

Exactly 0.2g of the concisely grounded leaves of C. gigantea was boiled with 2%

hydrochloric acid (5ml) on a steam bath, it was allowed to cool and then filtered. One

milliliter of the solution was treated with 2 drops of drahendroff reagent (Bismuth

Fractionation with acetone Residue D

Acetone fraction (concentrate)

Fractionation with methanol Residue E

Methanol fraction (concentrate)

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potassium iodide solution). A red precipitate indicated the presence of alkaloids

(Harborne, 1984).

2.2.5.2 Test for Carbohydrates

The ground sample (0.1g) was shaken vigorously in 5ml of distilled water and

then filtered. To the aqueous filtrate was added few drops of molisch reagent followed by

vigorous shaking. Concentrated sulphuric acid (1ml) was carefully added to the solution.

A browning layer at the inter-phase indicate the presence of carbohydrate.

2.2.5 .3 Test for Reducing Sugar

A quantity, 0.1g of the ground sample was mixed with 5ml of equal parts of

feeling’s solution I and II and heated on a waterbath for 5 minutes. A brick red precipitate

showed the presence of reducing sugar.

2.2.5.4 Test for Protein

To 5 ml of distilled water was added 0.1g of the ground sample and left to stand

for 3 hours and then filtered. To 2ml portion of the filtrate was added 0.1ml of million’s

reagent, shaken and kept for observation. Formation of yellow precipitate showed the

presence of proteins.

2.2.5.6 Test for Fats and Oil A quantity, 0.1g of the ground sample was pressed between filter paper and the

paper was observed. A control was also prepared by placing 2 drops of olive oil on filter

paper.

Translucency of the filter paper indicated the presence of fats and oil.

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2.2.5.6 Test for Glycoside

Two grammes (2g) of the ground sample were mixed with 30ml of water. The

mixture was heated on waterbath for 5 minutes and filtered. To 5 ml of the filtrate was

added a mixture of Fehling’s solution A and B of equal volumes (0.2ml each) until it

turned alkaline (tested with litmus paper). It was then boiled in waterbath for 3 minutes.

Brick red precipitate indicated presence of glycoside.

2.2.5.7 Test for Acidic Substances

Ground sample (0.1g) was placed in a clear dry test tube and sufficient distilled

water added. This was kept in a waterbath and then cooled. A strip of neutral litmus paper

was dipped with the filtrate. Red colour of the litmus paper indicated acidity.

2.2.5.8 Test for the Presence of Flavonoids A quantity, (0.2g) of ground sample was heated with 10ml of ethylacetate in

boiling water for 3 minutes. The mixture was filtered and the filtrate used for the test.

Four millilitre (4 ml) of the filtrate were shaken with 1% aluminum chloride solution (1

ml) and observed. A yellowish colouration in the ethylacetate layer indicated the

presence of flavonoids.

2.2.5.9 Test for the Presence of Steroids

To a mixture of 10ml of lead acetate solution (90% w/v) and 20 ml of aqueous

ethanol (50%) were added to 1 g of the ground sample in a 200ml conical flask. The

mixture was placed on a boiling waterbath for 3 minutes, cooled and filtered.

The filtrate was extracted twice with 15 ml of chloroform. Five ml of the

chloroform extract was evaporated to dryness on a waterbath. To the residue, 2 ml of 3,5-

dinitrobenzoic acid solution (2% in ethanol) and 1ml of 1 N sodium hydroxide solution

were added. A redish brown interphase showed the presence of steroids.

2.2.5.10 Test for Tannins

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Two grammes of the ground plant material were boiled in 5ml of 45% ethanol for

5 minutes. The mixture was cooled and filtered. To one ml of the filtrate were added 3

drops of lead acetate solution.

A gelatinous precipitate indicated the presence of tannins.

2.2.5.11 Test for Resins

i) Precipitate test: A weighed quantity of 0.2g of the ground sample was extracted

with 15 ml of 96% ethanol. The alcoholic extract was then poured into 20ml of distilled

water in a beaker. A precipitate occurring indicated the presence of resins.

ii) Colour test: A weighed quantity of 0.2g of the ground sample was extracted with

chloroform and the extract concentrated to dryness. The residue was dissolved in 3 ml of

acetone and another 3ml of concentrated hydrochloric acid was added. The mixture was

heated in a waterbath for 30 minutes.

A pink colour, which changes to magenta red, indicated the presence of resins.

2.2.5.12 Test for Saponins

The sample (0.1g) was weighed and boiled with 5ml of distilled water on a hot

waterbath for 5 minutes. The mixture was filtered hot, allowed to cool and the filtrate

used for the following test.

ii) Emulsion test: To 1ml of the filtrate, 2 drops of olive oil was added and the

mixture shaken vigorously. The formation of emulsion indicated the presence

of saponins.

iii) Frosting test: A given volume of 1 ml of the filtrate was diluted with 4 ml of

distilled water, shaken vigorously and then observed on standing for a stable

froth.

2.2.5.13 Test for Terpenoid and Steroid

A volume of 9 ml of ethanol was added to 9g of the ground sample and refluxed

for a few minutes and filtered. The filtrate was concentrated to 2.5ml on a boiling

waterbath and then 5ml of hot water was added. The mixture was allowed to stand for 1

hour and the waxy matter was filtered off. The filtrate was extracted with 2.5ml

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chloroform using separating funnel. To 0.5 ml of the chloroform extract in a test tube was

carefully added 1 ml of concentrated sulphuric acid to form a layer. A reddish brown

interphase showed the presence of steroids. Another 0.5ml of chloroform extract was

evaporated to dryness on a waterbath and heated with 3 ml of concentrated sulphuric acid

for 10 minute on a waterbath. A gray colour indicated the presence of terpernoids.

2.2.6 Acute Toxicity Test

2.2.6.1 Determination of LD50 of the Extract (Lorke, 1983)

Median lethal dose (LD50) is the log dose of a drug that kills 50% of the

population to which the drug is administered. Investigation on the acute toxicity study

LD50 of the extract was determined using the method described by Lorke (1983). Thirteen

experimental mice were distributed in 3 groups.

These studies were conducted in two stages; 3 groups of mice were administered

10mg/kg, 100mg/kg, and 1000mg/kg body weight of the extract of C. gigantea. The

extracts were injected intraperitoneally.

The mice were observed for 24hours reactions, abnormal behaviour and general

body conditions). Base on the percentage survival rates further increase dose of 1,600mg,

2000mg/kg, 2900mg/kg body weight were administered to 3 mice respectively and the

fourth mice received only normal saline which served as the control. The mice were

observed for another 24hours and the number of death was recorded. The LD50 was

calculated as the geometric mean of highest non-lethal and the lowest lethal doses.

2.2.7 Anti-diabetic Evaluation

2.2.7.1 Induction of Diabetes

Phase I

Twenty four rabbits with sugar concentrations of 60-90mg/dl after 12 hours of

fasting were injected intraperitoneally (i.p) with 200mg/kg body weight of freshly

prepared alloxan monohydrate (Sigma, USA) in normal saline. The animals were fed

with Bendel Feed and Flour Mill Limited Pelletized Guinea Growers mash.

After three days, blood sugar concentrations were determined using glucometer.

Blood sugar concentrations found to be 150mg/dl and above were used for determination

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of hypoglycemic effects of C. gigantea leaves extract. The diabetic rabbits were divided

into eight groups of two animals each. The extract dissolved in the normal saline and was

injected intraperitoneally.

Group 1 represented the control group, while group 2 represented diabetic not

treated as described in the experimental design. Group 3 received glibenclamide as

standard hypoglycaemic agent. Groups 4 – 9 received different fraction of the extract at

the dose of 300mg/kg body weight twice daily for five days.

After five days treatment, Groups 4 – 9 were screened down to one group using

blood sugar level as a marker that determined the most active fraction.

Phase II

Twelve rabbits having sugar concentration of 60 – 90 mg/dl after 12 hours of

fasting were injected intraperitoneally (i.p.) with 200 mg/kg body weight of freshly

prepared alloxan monohydrate (Sigma, USA) in normal saline. After 3 days, blood sugar

was determined using glucometer. Blood sugar