UHUO, EMMANUEL NNAEMEKA (PG/Ph.D /10 /57831 ) · UHUO, EMMANUEL NNAEMEKA (PG/Ph.D /10 /57831 ) EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF ... Abakpa, Enugu, Congratulation for

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UHUO, EMMANUEL NNAEMEKA

(PG/Ph.D/10/57831)

EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF AND FRUIT EXTRACTS OF Kigelia africana ON SOME OXIDATIVE AND BIOCHEMICAL PARAMETERS IN

ALLOXAN-INDUCED DIABETIC RATS

FACULTY OF BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY

Paul Okeke

Digitally Signed by: Content manager’s Name DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre

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EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF AND FRUIT EXTRACTS OF Kigelia africana ON SOME

OXIDATIVE AND BIOCHEMICAL PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

BY

UHUO, EMMANUEL NNAEMEKA

(PG/Ph.D/10/57831)

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA

NSUKKA

JANUARY, 2015

75

TITLE

EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF AND FRUIT EXTRACTS OF Kigelia africana ON SOME

OXIDATIVE AND BIOCHEMICAL PARAMETERS IN ALLOXAN-INDUCED DIABETIC RATS

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY (Ph.D) IN MEDICAL BIOCHEMISTRY, UNIVERSITY OF NIGERIA,

NSUKKA

BY

UHUO, EMMANUEL NNAEMEKA (PG/Ph.D/10/57831)

DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA

NSUKKA

SUPERVISORS: PROF. L.U.S. EZEANYIKA DR V.N. OGUGUA

JANUARY, 2015

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CERTIFICATION

UHUO, Emmanuel Nnaemeka, a postgraduate student with Registration Number PG/Ph.D/10/57831 in the Department of Biochemistry has satisfactorily completed the requirements for the research work for the degree of Doctor of Philosophy (Ph.D) 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.

PROF L.U.S. EZEANYIKA DR V. N. OGUGUA (Supervisor) (Supervisor) PROF O.F.C. NWODO EXTERNAL EXAMINER (Head of Department)

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DEDICATION

This Thesis is dedicated wholly to God Almighty who, out of His infinite mercy granted me

good health, wisdom and ability to produce this work and the entire members of Uhuo‘s

family.

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ACKNOWLEDGEMENTS

I sincerely wish to express my profound gratitude to my supervisors, Prof. L.U.S. Ezeanyika

and Dr V.N Ogugua, for their immense contributions, guidance, tolerance and fatherly role

throughout the course of this study. My appreciation goes to the Head of Department, Prof.

OFC.Nwodo and all the lecturers of the Department of Biochemistry- Prof. O.U. Njoku, Prof.

I.N.E. Onwurah, Prof. F.C. Chilaka and Dr Parker Elijah Joshua, for their wonderful

interactions with me all these years of my academic sojourn in the department. I happily

remember my fellow students, and colleagues whose interaction and love also made this

programme successful. I, with due respect appreciate the sacrifices of all the members of

Uhuo’s family. They have contributed immensely and May God reward them.

My sincere appreciation also goes to all the staff of Shalon Medical Laboratory for their help

in my laboratory work, Department of Botany and Crop Science University of Nigeria,

Nsukka for their help during phytochemical and proximate analyses. Included in the list of

appreciation are the staff of the Department of Home Science, Nutrition Dietetics, University

of Nigeria, Nsukka and Ogugua‘s family at Ayamelum, Anambra State for their numerous

contributions and assistance.

Calvary greetings to all members of Assemblies of God Church, Upper Housing Estate,

Abakpa, Enugu, Congratulation for prayers all these years. I must humbly appreciate the

moral support of Rev. Dr. Onyemaech Oti even when the going was so tough. Finally, may I

most humbly submit to God Almighty who has been everything good and possible to me. I

praise His Holy Name in the name of our Lord and Saviour Jesus Christ, Amen.

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ABSTRACT

Globally, the estimated incidence of diabetes and projection for the year 2030 as given by the International Diabetes Federation (IDF) is 350 million. Kigelia africana is highly used for ethnomedicinal purposes although there is paucity of scientific information on its use. This work was therefore, aimed at evaluating the anti-diabetic and antioxidative potential of the plant. Ethanol, methanol and n- hexane extracts of the leaves of Kigelia africana were used for the study. Alloxan diabetes was induced in a total of 60 adult male albino rats weighing between 90 and 160 g. The alloxan was dissolved in cold normal saline. After 72 hr, diabetes was confirmed and the rats were divided into twelve (12) groups of five (5) rats each. Group 1 served as the normal control, group 2 was the diabetic untreated, group 3 received 2.5 mg /kg b.wt of glibenclamide, groups 4, 6 and 8 received ethanol, methanol and n-hexane leaves extract while group 5, 7 and 9 received ethanol, methanol and n-hexane fruit extract respectively of 500 mg/kg b.wt of the extracts. Groups 10-12 were administered equal combination of the leaves and fruits extracts. The rats were fed orally for 21 days after which some biochemical and oxidative parameters were statistically analysed. Phytochemical screening for different bioactive compounds was done using standard methods and indicated the presence of flavoniods, alkaloids, saponins, soluble carbohydrates, tannin, steroids, glycosides and reducing sugars. Proximate analysis revealed the presence of proteins (13.9%), carbohydrates (63.5%), fats and oil (11.4%) and crude fibre (2.2%). LD50 showed that the extracts were safe. The glucose level decreased while body weight increased in all the treated groups compared with the diabetic rats untreated. Oral administration of 500mg/kg b.w of K. africana extract significantly reduced (p<0.05), the sorbitol, glycohaemoglobin (HbA1c), total protein, and vitamin C concentrations in diabetic rats (groups 4-12) in comparison with the positive control. There were significant differences in glycohaemogolin, sobitol, total protein and vitamin C concentration in diabetic rats fed with a combination of the two parts of the plant extracts (groups 10-12) as against groups 4-9 administered single extracts. Malondiadehyde (MDA) concentration significantly decreased (p < 0.05) in all the test groups compared with the diabetic untreated rats. Low density lipoprotein, total cholesterol, and triacylgycerol levels decreased significantly (p < 0.05) in the treated groups in comparison with the positive control animals (group 3). However, administration of 500 mg/kg b.w of K. africana increased significantly (p<0.05) the high density lipoprotein (HDL) across the test groups as against the diabetic untreated group. Significant decreased (p<0.05) in the lipid profiles (except HDL) was recorded in groups 10, 11 and 12 treated with a combination of two parts (leaf and fruit) of K. africana in comparison with groups 4-9 orally fed with a single plant extract. Furthermore, the data recorded significantly increased (p < 0.05) antioxidant enzymes (SOD, CAT GPX) activities in diabetic treated groups (both combination and single) with reference to the positive control group. Similarly, significant increase (p > 0.05) of SOD and CAT activities and SOD percentage inhibition was observed in group 3 treated with 2.5 mg/kg b.wt of glibenclamide (standard) compared with all the test groups. Significant reduction (p < 0.05) in the activities of ALT, ALT and total bilirubin concentration were observed in the test groups treated with the extracts compared with the diabetic untreated rats. ALT activity and total bilirubin level decreased significantly (p < 0.05) in groups 10, 11 and 12 administered a combination of leaf and fruit extracts as against groups 4-9 treated with either leaf or fruits only. The results suggest that management and prevention of diabetic complications can be achieved by the use of K. africana.

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TABLE OF CONTENTS

PAGE Title Page .. .. .. .. .. .. .. .. .. .. i Certification .. .. .. .. .. .. .. .. .. .. ii Dedication .. .. .. .. .. .. .. .. .. .. iii Acknowledgements .. .. .. .. .. .. .. .. .. iv Abstract .. .. .. .. .. .. .. .. .. .. vi Table of Contents .. .. .. .. .. .. .. .. .. vii List of Figures .. .. .. .. .. .. .. .. .. .. xiii List of Tables .. .. .. .. .. .. .. .. .. .. xv List of Abbreviations .. .. .. .. .. .. .. .. .. xvi CHAPTER ONE: INTRODUCTION 1.1 Kigelia africana … … … … … … … … 3

1.1.1 Description of Kigelia africana … … … … … … 3

1.1.2 Taxonomy of Kigelia africana … … … … … … 4

1.1.3 Traditional uses of Kigelia africana … … … … … … 4

1.1.4 Chemical constituents of Kigelia africana … … … … … 5

1.1.5 Antibacteria and antifungi … … … … … … … 6

1.2 Diabetes ... ... … … … … … … … 7

1.2.1 Diabetes mellitus ... ... ... ... ... ... ... ... 7

1.2.2 Diabetes Type 1 and 2 … … … … … … … 8

1.2.3 Insulin resistance … … … … … … … … 9

1.2.4 Diabetic complications … … … … … … … 10

1.3 Hyperglycemia and diabetic complication … … … … … 10

1.4 Mechanism of tissue damage mediated by hyperglycemia … … … 11

1.4.1 Aldose reductase pathway … … … … … … … 11

1.4.2 Non-enzymatic glycation … … … … … … … 13

1.4.3 Carbonyl stress in diabetes … … … … … … … 14

1.4.4 Activation of protein kinase C isoforms … … … … … 15

1.5 Oxidative stress … … … … … … … … 17

1.5.1 Mechanism of increased oxidative stress in diabetes mellitus … … 17

1.5.2 Glucose autoxidation … … … … … … … … 19

1.5.3 Free radicals … … … … … … … … 20

1.5.4 Reactive oxygen species and oxidative stress … … … … 20

1.6 Antioxidant system … … … … … … … … 22

1.6.1 Scavenging properties of antioxidants … … … … … … 23

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1.6.2 Positive and negative effects of free radicals … … … … … 25

1.7 Lipid peroxidation … … … … … … … … 26

1.8 Antioxidant supplementation in diabetes mellitus … … … … 28

1.9 Alloxan … … … … … … … … … 29

1.9.1 Alloxan diabetes and streptozotocin diabetes … … … … … 29

1.9.2 Alloxan: Mechanism of action … … … … … … 31

1.9.3 Beta cell toxicity and diabetogenicity of alloxan … … … … 32

1.9.4 Streptozotocin: Mechanism of action and beta cell selectivity … … 35

1.9.5 Beta cell toxicity of streptozotocin … … … … … … 35

1.9 Rationale for the study … … … … … … … 36

1.10 Aim and objectives of the study … … … … … … 37

1.10.1 Aim of the study … … … … … … … … 37

1.10.2 Specific objectives of the study … … … … … … 37

CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials … … … … … … … … … 38

2.1.1 Chemicals … … … … … … … … … 38

2.1.2 Instrument/Equipment … … … … … … … 38

2.1.3 Drug … … … … … … … … … … 38

2.1.4 Plant material … … … … … … … … … 38

2.2 Methods … … … … … … … … … 39

2.2.1 Animal management … … … … … … … … 39

2.2.2 Preparation of plant extracts … … … … … … … 39

2.2.3 Design of the experiment … … … … … … … 39

2.2.4 Yield of extracts … … … … … … … … 40

2.2.5 Phytochemical analysis of the crude extracts … … … … … 40

2.2.5.1 Test for the presence of alkaloids … … … … … … 40

2.2.5.2 Test for carbohydrates … … … … … … … 40

2.2.5.3 Test for reducing sugar … … … … … … … 40

2.2.5.4 Test for protein … … … … … … … … 41

2.2.5.5 Test for fats and oil … … … … … … … … 41

2.2.5.6 Test for glycosides … … … … … … … … 41

2.2.5.7 Test for acidic substances … … … … … … … 41

2.2.5.8 Test for the presence of flavonoids … … … … … … 41

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2.2.5.9 Test for the presence of steroids … … … … … … 41

2.2.5.10 Test for tannins … … … … … … … … 42

2.2.5.11 Test for resins … … … … … … … … 42

2.2.5.12 Test for saponins … … … … … … … … 42

2.2.3.13 Test for terpenoids and steroids … … … … … … 43

2.2.6 Proximate Analysis … … … … … … … … 43

2.2.6.1 Crude protein … … … … … … … … … 43

2.2.6.2 Crude fat … … … … … … … … … 45

2.2.6.3 Moisture … … … … … … … … … 45

2.2.6.4 Ash /Mineral matter … … … … … … … … 46

2.2.6.5 Crude fibre … … … … … … … … … 46

2.2.6.6 Carbohydrate or nitrogen free extract (NFE) … … … … 47

2.2.7 Acute toxicity test … … … … … … … … 47

2.2.7.1 Determination of LD50 of the extract … … … … 47

2.2.8 Induction of diabetes … … … … … … … … 48

2.2.9 Determination of fasting and random glucose concentrations … … 48

2.2.10 Determination of sorbitol concentration … … … … … 49

2.2.11 Determination of total protein concentration … … … … … 50

2.2.12 Determination of haemoglobin glycosylation … … … … 51

2.2.13 Determination of malondialdehyde concentration … … … … 53

2.2.14 Determination of vitamin C concentration … … … … … 55

2.2.15 Assay of catalase activity … … … … … … … 57

2.2.16 Assay of superoxide dismutase (SOD) activity … … … … 58

2.2.17 Assay of glutathione peroxidase activity … … … … … 62

2.2.18 Determination of total cholesterol concentration … … … … 63

2.2.19 Determination of high density lipoprotein (HDL) cholesterol concentration … 64

2.2.20 Determination of low density lipoprotein (LDL) cholesterol concentration … 65

2.2.21 Determination of triacylglycerol concentration … … … … 66

2.2.22 Assay of aspartate aminotransferase (AST) activity … … … … 68

2.2.23 Assay of alanine aminotranferase (ALT) activity … … … … 70

2.2.24 Determination of total bilirubin concentration … … … … 71

2.3 Statistical analysis … … … … … … … … 72

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CHAPTER THREE: RESULTS

3.1 Qualitative phytochemical composition of ethanol, methanol

and n-hexane leaf and fruit extracts of Kigelia africana … … … 73

3.2 Quantitative phytochemical composition of ethanol, methanol


3.3 Percentage proximate compositions of ethanol, methanol


3.4 Percentage yield of leaf and fruit samples of Kigelia Africana … … 79

3.5 Acute toxicity studies … … … … … … … … 81

3.6 Effect of ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana on sugar level of diabetic rats … … … … 83

3.7 Body weights of diabetic rats treated with ethanol, n-hexane

and methanol extracts of leaves and fruits of Kigelia africana before and after experiment … … … … … … 85

3.8 Effect of ethanol, methanol and n-hexane leaf and fruit

extract of Kigelia africana on sorbitol concentration in alloxan-induced diabetic rats … … … … … … 87

3.9 Effect of ethanol, methanol and n-hexane leaf and fruit extract of

Kigeria africana on total protein in alloxan-induced diabetic rats … … 89 3.10 Effect of ethanol, methanol and n-hexane leaf and fruit

extract of Kigelia africana on glycosylated haemoglobin concentration in alloxin-induced diabetic rats … … … … 91

3.11 Effect of ethanol, methanol and n-hexane extracts of leaf

and fruit of Kigelia africana on malondialdehyde (MDA) concentration in alloxan-induced diabetic rats … … … … 93

3.12 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on vitamin C concentration in alloxan-induced diabetic rats … … … … … … 95

3.13 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on catalase activity in alloxan-induced diabetic rats … … … … … … 97

3.14 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on superoxide dismutase (SOD) activity in alloxan- induced diabetic rats … … … … 99

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3.15 Effect of ethanol methanol and n-hexane leaf and fruit

extracts of Kigelia africana on percentage inhibition of superoxide dismutase in alloxan-induced diabetic rats … … … 101


extract of Kigelia africana on glutathione peroxidase activity in alloxan-induced diabetic rats … … … … … 103

3.17 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on cholesterol concentration in alloxan-induced diabetic rat … … … … … … 105


extract of Kigelia africana high density lipoprotein in alloxan-induced diabetic rats … … … … … … 107


extracts of Kigelia africana on low density lipoprotein concentration in alloxan-induced diabetic rats … … … … 109


extracts of Kigelia africana on triacylglycerol (TAG) concentration in alloxan-induced diabetic rats … … … … 111

3.21 Effect of ethanol, methanol and n-hexane leaf and fruit extract

of Kigelia africana on aspartate aminotranferase (AST) in alloxan-induced diabetic rats … … … … … … 113


extract of Kigelia africana on alanine aminotransferase (ALT) in alloxan-induced diabetic rats … … … … … 115

3.23 Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on total bilirubin concentration in alloxan-induced diabetic rats … … … … … … 117

CHAPTER FOUR: DISCUSSION 4.1 Discussion … … … … … … … … 119

4.2 Conclusion … … … … … … … … 125

REFERENCES … … … … … … … … … 126 APPENDICES … … … … … … … … … 140

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LIST OF FIGURES Fig.1: Flower, leaves and fruit of Kigelia africana … … … … 4 Fig. 2: Pathway leading to AGE formation … … … … 12 Fig. 3: Formation of advanced glycation end- products (AGEs)

by combination of glycation and oxidation … … … … … 16 Fig. 4: Pathways that contribute to oxidative stress in response

to increased glucose flux … … … … … … … 19 Fig. 5. Endogenous stimuli leading to ROS generation … … … … 21 Fig. 6: Interactions between endogenous antioxidants

in the process of detoxifying lipid peroxides … … … … … 24 Fig. 7: Phasic glucose response to a diabetogenic dose of alloxan or streptozotocin 30 Fig. 8: Redox cycling reaction between alloxan and dialuric acid … … … 34 Fig. 9a: Structure of streptozotocin … … … … … … … 35 Fig. 9b: Structure of methylnitroso urea … … … … … … 35 Fig. 10: Reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) … 53 Fig. 11: Effect of ethanol, n-hexane and methanol extracts of leaf

and fruit of Kigelia africana on sorbitol concentration in alloxan-induced diabetic rats … … … … … … 88

Fig. 12: Effect of ethanol, n-hexane and methanol extracts of leaf and fruit of Kigelia africana on protein concentration in alloxan-induced diabetic rats … … … … … … 90

Fig. 13: Effect of ethanol, n-hexane and methanol extracts of leaf

and fruit of Kigelia africana on glycosylated haemoglobin concentration in alloxan-induced diabetic rats … … … … 92

Fig. 14: Effect of ethanol, n-hexane and methanol extracts of leaf and

fruit of Kigelia africana on malondialdehyde concentration in alloxan-induced diabetic rats … … … … … … 94


and fruit of Kigelia africana on vitamin C concentration in alloxan-induced diabetic rats … … … … … … 96

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Fig. 16: Effect of ethanol, n-hexane and methanol extracts of

leaf and fruit of Kigelia africana on catalase activity in alloxan-induced diabetic rats … … … … … … 98


fruit of Kigelia africana on superoxide dismutase activity in alloxan-induced diabetic rats … … … … … … … 100


fruit of Kigelia africana on percentage of superoxide dismutase inhibition in alloxan-induced diabetic rats … … … … … 102


and fruit of Kigelia africana on glutathione peroxidase activity in alloxan-induced diabetic rats … … … … … 104

Fig. 20: Effect of ethanol, n-hexane and methanol extracts of leaf and fruit of Kigelia africana on percentage of total cholesterol concentration in alloxan-induced diabetic rats … … … … … … 106


fruit of Kigelia africana on percentage of high density lipoprotein concentration in alloxan-induced diabetic rats … … … … 108


fruit of Kigelia africana on percentage of low density lipoprotein concentration in alloxan-induced diabetic rats … … … … 110


and fruit of Kigelia africana on percentage of triacylglycerol concentration in alloxan-induced diabetic rats … … … … 112


and fruit of Kigelia africana on aspartate aminotransferase activity in alloxan-induced diabetic rats … … … … … 114


and fruit of Kigelia africana on alanine aminotransferase activity in alloxan-induced diabetic rats … … … … … 116


and fruit of Kigelia africana on total bilirubin concentration in alloxan-induced diabetic rats … … … … … … 118

LIST OF TABLES Table 1: Vascular complications of diabetes mellitus … … … … 8

Table 2: Comparison of the chemical properties of alloxan and streptozotocin … 32

Table 3: Volumes of the various materials/reagents put into the sample and blank … 55

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Table 4: Volumes of the various materials/reagents put into the sample and blank … 59

Table 5: Absorbance of AST in the serum … … … … … 69

Table 6: Absorbance of ALT in the serum … … … … … 71

Table 7: Qualitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana … … … 74

Table 8: Quantitative phytochemical composition of ethanol, methanol

and n-hexane leaf and fruit extracts of Kigelia afrcana … … … 76 Table 9: Percentage proximate composition of ethanol, methanol and

n-hexane leaf and fruit extracts of Kigelia afrcana … … … … 78 Table 10: Percentage yield of leaf and fruit samples of Kigelia afrcana … … 80 Table 11a: First acute toxicity test (LD50) of Kigelia africana extracts … … 82 Table 11b Second acute toxicity test (LD50) of Kigelia africana extracts … … 83 Table 12: Effect of ethanol, n-hexane and methanol extracts of

leaves and fruits of Kigelia africana on glucose level of diabetic and non-diabetic rats … … … … … … 84

Table 13: Body weights of diabetic and non-diabetic rats treated

with ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana before and after experiment … … … 86

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LIST OF ABBREVIATIONS

A Alloxan

AAS Atomic abosorption spectrophotometer

AST Aspartate aminotransferase

ALT Alanine aminotransferase

ADP Adinine diphosphate

AGEs Advanced glycation end- product

AVP Arginine vasopressin

AQP-2 Aquaporin-2

AVPR2 Arginine vasopressin 2 receptor

AQP-2 Aquaporin-2

ATP Adenosine triphosphate

BH4 Tetrahydrobiopterin

CAT Catalase

Ca2+ Calmodulin.

Cu2+ Copper (ii) Iron

CUZnSOD Copper zinc superoxide dismutase

DAG Diacylglycerol

DHLA Dihydrolipoic acid

DMSO Dimethyl sulphuroxide

DNA Deoxyribo neucleic acid

3-DG 3-Deoxy glucosone

DM Diabetes mellitus

DI Diabetes inspidus

EDTA Ethylene diamine tetraacetate

EDRF Endothelium derived relaxing factor

eNOS Endothelia nitric oxide synthase

FAD Flavin adenine dinucleotide

Fe2+ Iron

FMN Flavin mononucleotide

GAPD Glyceraldehydes -3-Phosphate dehydrogenase

GLO Glyoxal

GR Glutathione reductase

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GSH Glutathione

GSSG Oxidized glutathione

GPx Glutathione peroxidase

GLUT2 Glucose transporter

HDL High density lipoprotein

HOCL Hydrochlorous acid

H2O2 Hydrogen peroxide

HO. Hydroxyl radical

HNE 4-Hydroxyl-2-nonenal

HK Hexokinase

IDDM Insulin -dependent diabetes mellitus

I.N.T 2-(4-iodophenyl)-3-(4-nitrophenol)-5-Phenyltetrazolium chloride

1L-1 Interleukin -1

LA Lipoic acid

LD50 Lethal dose

LDL Low density lipoprotein

MNU N- methyl-N-nitrosourea

MDA Malondialdehyde

Mn-SOD Manganese superoxide dismutase

MGD Methylglyoxal

NADPH Nicotinamide adenine dinucleotide phosphate (Reduced form)

NAD+ Nicotinamide adenine dinucleotide (oxidized form)

NIDDM Non- insulin dependent diabetes mellitus

NOS Nitrogen oxygen species

NO Nitric oxide

ONOO- Peroxylnitrite 1O2 Singlet oxygen

O2-. Superoxide anion

PDGF Platelet- derived growth factor

PKC Protein kinase C

PS Phosphotidyl serine

PPP Pentose phosphate pathway.

PVS Polyvinyl sulphate

PUFA Polyunsaturated fatty acid

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PVN Paraventricular nuclei

RAGE Advanced glycation end- product

RNS Reactive nitrogen species

RO. Alkoxyl radicals

ROS Reactive oxygen species

STZ Streptozotocin

SON Supraoptic nuclei

SOD Superoxide dismutase

TG Triacylglycerol

TLC Thin layer chromatography

TNF Tumor necrosis factor

TBA 2-Thiobarbituric acid

TCA Trichloloroacetic acid

UKPDS United kingdom prospective diabetes study

VEGF Vascular endothelial growth factor

XOD Xanthine oxidase

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CHAPTER ONE

INTRODUCTION

Diabetes mellitus is a metabolic disorder resulting from a defect in insulin secretion, insulin

action or both. Insulin deficiency in turn leads to chronic hyperglycemia with disturbances of

carbohydrate, fat and protein metabolism (Kumar et al., 2011).

During diabetes, failure of insulin-stimulated glucose uptake by fat and muscle cause glucose

concentration in the blood to remain high, consequently glucose uptake by insulin-

independent tissue increases. Increased glucose flux both enhances oxidant production and

impairs antioxidant defenses by multiple interacting non-enzymatic, enzymatic and

mitochondrial pathways (Klotz 2002; Mehta et al., 2006). These include activation of protein

kinase C isoforms (Inoguchi et al., 2000), increased hexosamine pathway (Kaneto et al.,

2001), glucose autoxidation (Brownlee, 2001), increased methylglyoxal and advanced

glycation end-product (AGEs) formation (Thornalley, 1998) as well as increased polyol

pathway flux ( Lee and Chung, 1999). These seemingly different mechanisms are the results

of a single process-that is, overproduction of superoxide by the mitochondrial electron

transport system (Tushuizen et al., 2005). This hyperglycaemia-induced oxidative stress

ultimately results in modification of intracellular proteins resulting in an altered function and

DNA damage, activation of the cellular transcription (NFK B), causing abnormal changes in

gene expression, decreased production of nitric oxide, and increased expression of cytokines,

growth factors and pro-coagulant and pro-inflammatory molecules (Palmer et al., 1988;

Evans et al., 2002; Klotz, 2002; Taniyama and Griendling, 2003). Oxidative stress is

responsible for molecular and cellular tissue damage in a wide spectrum of human diseases

(Halliwell, 1994), amongst which is diabetes mellitus. Diabetes produces disturbances of

lipid profiles, especially an increased susceptibility to lipid peroxidation (Lyons, 1991),

which is responsible for increased incidence of atherosclerosis (Guigliano et al., 1996), a

major complication of diabetes mellitus . An enhanced oxidative stress has been observed in

these patients as indicated by increased free radical production, lipid peroxidation and

diminished antioxidant status (Baynes, 1991).

Globally, the estimated incidence of diabetes and projection for year 2030, as given by

International Diabetes Federation is 350 million (Ananda et al., 2012). Currently available

pharmacotherapies for the treatment of diabetes mellitus include oral hypoglycemic agents

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and insulin. However these drugs do not restore normal glucose homeostasis and they are not

free from side effects (Bandawane et al., 2011). In view of the adverse effects associated with

the synthetic drugs and as plants are safer, affordable and effective, conventional antidiabetic

plants can be explored (Kumar et al., 2010). Over 400 traditional plants have been reported

for the treatment of diabetes (Ramachandran et al., 2011). Furthermore, following World

Health Organization recommendations, investigation of hypoglycemic agents from medicinal

plants has become more important (Kumar et al., 2010). Also diabetes has been treated orally

with several medicinal plants based on folklore medicine since ancient times.

Kigelia africana (Lam.) Benth (Family: Bignoniaceae) plant has many medicinal properties

due to the presence of numerous secondary metabolites. These compounds include iridiods,

flavonoids, naphthoquinones and volatile constituents (Houghton, 2002; Gorman, 2004;

Asekun et al., 2006). Experimentally, the plant has shown antibacterial, antifungal,

antineoplastic, analgesic, anti-inflammatory and antioxidant properties (Saini et al., 2009).

Crude extracts of herbs and spices and other materials rich in phenolics are of increasing

interest in the food industry because they retard oxidative degradation of lipids and thereby

improving the quality and nutritional value of food. Flavonoids, are groups of polyphenolic

compounds with known properties, which include free radical-scavenging and anti-

inflammatory activities (Frankel, 1995).

An enhanced oxidative stress has been observed in diabetic patients as indicated by increased

free radical production, lipid peroxidation and diminished antioxidant status (Baynes, 1991).

In diabetes mellitus, alterations in the endogenous free radical scavenging defense

mechanisms may lead to ineffective scavenging of reactive oxygen species, resulting in

oxidative damage and tissue injury. Oxidative stress is currently suggested as mechanism

underlying diabetes and diabetic complications. Oxidative stress may cause oxidative damage

of cellular membranes and changes in the structural and functional integrity of sub- cellular

organelles and many produce effects that result in the various complications in diabetic

disease (West, 2000). Recently, there has been an upsurge of interest in the therapeutic

potentials of plants, as antioxidants in reducing free radical induced tissue injury. Although

several synthetic antioxidants, such as ascorbic acid, butylated hydroxyanisole and butylated

hydroxytoluene are commercially available, they are quite unsafe and toxic (Viny, et

al.,2010). A survey of literature revealed that there is no experimental evidence of the

antidiabetic effect of Kigelia africana. Therefore, the present work explores this and will in

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addition study its potential effect on the oxidative and biochemical parameters of alloxan-

induced diabetic rats.

1.1 Kigelia africana

1.1.1 Description of Kigelia africana

Nature has been a source of medicinal agents including modern drugs for the thousands of

years (Cordell, 2000). Plant based traditional medicine system continues to play an essential

role in health care with about 80% of the world’s inhabitants relying mainly on traditional

medicines for their primary health care .

Kigelia africana (Lam) Benth of Bignoniaceae family is widely distributed in South central

and West Africa. It is known as the cucumber or sausage tree because of its huge fruits

(average 0.6m in length and 4kg in weight) which hang from long fibrous stalks. It is also

known as Balm Khene in Hindi and is distributed all over India but found in abundance in

West Bengal. It is found mostly in wetter areas and spread abundantly across wet Savannah

and riverine areas (Sofowora et al., 1980). The plant grows approximately 10m high, with

odd pinnate, composite opposite leaves; leaflets are ovate- to- oblong in shape and 4-18 cm

long. The flowers are found in the spring or summer season, hanging ancillary panicles up to

2 m long. It has a corolla with fused petals, irregularly bell shaped 9-13 cm long, yellowish

outside and purple on inside . Fruits are oblong, 30-50 cm long, hanging on stalk for several

months.

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(a) Flower (b) Leaves and fruit

Fig. 1: Flower, leaves and fruit of Kigelia africana

1.1.2 Taxonomy of Kigelia africana

Kingdom: Plantae

Order: Scrophulariales

Family: Bignoniaceae

Genus: Kigelia Dc-Sausage family

Species: Kigelia africana (Lam) Benth

Scientific name: Kigelia africana

1.1.3 Traditional Uses of Kigelia africana

The Kigelia plant has medicinal properties with characteristics of bitterness and astringent. It

has long history of use by African countries particularly for medicinal properties. Several

parts of the plant are employed for medicinal purposes by certain aboriginal people. In Mali

during famine the seeds are roasted and eaten. The baked fruits of K.africana are used for

fermentation of beer. Most commonly traditional healers use it to treat a wide range of skin

ailments like fungal infections, boils, psorasis, and eczema.

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It has also internal applications including treatment of dysentery, ringworm, tapeworm,

postpartum haemorrhage, malaria, diabetes, pneumonia and tooth care (Gill, 1992). The

Tonga women of Zambezi valley regularly apply cosmetic preparations of kigelia fruits to

their faces to ensure a blemish- free complexion (Pooleg, 1993). In folk medicine, the fruits

of the plant are used as dressing for ulcers, purgative and for increasing the flow of milk in

lactating women (Oliver-Bever, 1986). The roots are said to yield a bright yellow dye. The

Shona people from India tend to use the bark or root as powder or infusion for application to

ulcers, or in the treatment of pneumonia, as a gargle for toothache and backache (Maisiri and

Gundidza, 1999).

In West Africa, the root and unripe fruit is used as vermifuge and as a treatment for

haemorrhoids and rheumatism. The bark is traditionally used as remedy for syphilis and

gonorrhea. The fruits and bark ground and boiled in water are taken orally or used as an

enema in treating children’s stomach ailments – usually worms. The unripe fruit is used in

Central Africa as a dressing for wounds, haemorrhoids and rheumatism. Palm wine exracts of

leaf, bark and fruit extracts of the tree are used for treatment of venereal diseases (Walt el

al., 1962).

1.1.4 Chemical Constituents of Kigelia africana

K. africana plant has many medicinal properties due to the presence of numerous secondary

metabolites. These compounds include irridiods, flavonoids and naphthoquinones and

volatile constituents (Houghton, 2002; Gorman et al., 2004; Asekun et al., 2006). Pinnatal

and isopinatal were isolated from tropical trees that belong to the plant family of

Bignoniaceae. Pinnatal was found in a root bark extract of the plant. Thin layer

chromatography (TLC) examination of the most active fractions of both stem bark and fruits

showed the presence of some major components which were found to be norviburtinal and B-

sitosterol. Gouda et al.(2006) isolated a furanone derivative, 3- (21-hydroxyethyl)-5-(2”-

hydroxypropyl)- dihydrofuran -2(3H)- one and four irridoids, 7 hydroxy viteoid II, 7 hydroxy

eucommic acid, 1- hydroxyl – 10- deoxyecuommiol and 10-deoxy eucommoil together with

seven known irridoids, jiofuran, jioglutolide, 1-dehydroxy-3, 4- dehydroaucubigenin, des-p-

hydroxybenzoyl kisasagenol B, ajugol, verminoside and 6-trans-caffeoyl ajugol from the fruit

(Gouda et al., 2003). They also isolated a phenyl propanoid derivatives identified as 6-p-

coumaroyl-sucrose together with ten known phenylpropanoid and phenylethanoid derivatives

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and a flavonoid glycoside from fruits of K.africana (Gouda et al., 2006). The structures of the

isolated compounds were characterized by different spectroscopic methods. Govodachari et

al. (1971) isolated kigelin as the major constituent of the plant from the root heartwood.

1.1.5 Antibacterial and Antifungal

A biologically monitored fractionation of the methanolic extracts of the root and fruits led to

the isolation of the naphthoquinones, kigelinone, iso-pinnatal, dehydro-α- Lapachone, and

lapachol and the phenyl propanoids, p-coumaric acid and ferulic acid as the compounds

responsible for the observed antibacterial and antifugal activity (Binutu et al., 1996). The

compounds isolated were tested for their activities against Staphylococcus aureus, Bacillus

Subtilis, Corynebacterium diphetheriae, Aspergillus niger, A. flavus, Candida albicans and

Pullularia pullularis (Aureobasiduim sp). The steroids and flavonids are hygroscopic and

have fungicidal properties.

Chemical investigation showed that the aqueous extracts of the stem bark of the plant contain

irridoids as major components. In the light of traditional uses of this plant, antimicrobial

activities of the aqueous extracts and two major irridoids were tested against Bacillus subtilis,

Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albican.

The crude aqueous extracts showed significant antimicrobial activity, which could be

partially explained by the activity of the irridoids present (Akunyili et al., 1991). The fruits

are popular sources of traditional medicine throughout Africa. The stem bark has been widely

analyzed for pharmacological activity but fruit is limited despite more extensive use in

traditional remedies.

In the microtitre plate bioassay, stem bark and fruit extracts of K.africana showed similar

antibacterial activity against Gram negative and Gram positive bacteria. A mixture of free

fatty acids exhibiting antibacterial effect was isolated from the ethyl acetate extract of the

fruits using bioassay-guided fractionation. Palmitic acid, already known to possess

antibacterial activity, was the major compound in this mixture. These results confirm

antibacterial activity of K. africana fruits and stem bark, and support the traditional use of the

plant in therapy of bacterial infections (Grace et al., 2002). A disc diffusion susceptibility

test was used to screen concentrated extracts from the bark of 3 medicinal plants (Aistonia

boonei de wild, Morinda lucida Benth and K. africana) for antimicrobial activity (Kwo and

Craker, 1996). Solvents with different polarities were used for the extraction (methylene

Chloride, ethyl acetate, 95% ethanol and acetonitrite), and the extracts were tested against

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Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and

Pseudomonas aeruginosa. The patterns of inhibition varied with the plant extract, the solvent

used for extraction and the organism tested. The largest zone of inhibition was observed for

ethanol extracts of K. africana against S. aureus and P. aeruginosa. S. aureus was the most

inhibited new organism. No inhibitory effects were observed against C. albican. The extent

of the inhibition of the bacteria was related to the concentration of the plant extract (Kwo and

Craker, 1996).

1.2 Diabetes

1.2.1 Diabetes Mellitus

Diabetes mellitus (DM) in all its heterogeneity has taken the centre stage as one of the

ultimate medical challenges. Diabetes complications are the major cause of morbidity and

mortality in patients with Diabetes mellitus (Wolf, 1993). Diabetes mellitus is considered to

be one of a rank free radical diseases which propagates complications with increased free

radical formation (Baynes, 1991; Varvarovska et al., 2004).

One of the major hypotheses proposed to explain the hyperglycemia-induced onset of

diabetic complications is an increase in oxidative stress (Brownlee, 2001; Sheetz and King,

2002, Creager et al., 2003). Similar to their proposed role in the onset of diabetic

complications, reactive oxygen species (ROS) such as superoxide, (02.-), hydroxyl radical,

(OH.) and hydrogen peroxide H2O2 have been linked to other diseases and injury states,

including Alzheimer’s disease, (Yamagishi et al., 2001), Parkinson’s disease (Practico, 1999;

Hyun et al., 2002), Chronic obstructive pulmonary disease (Practico et al; 1998), and

Ischemia (Roberts and Morrow, 2000). Evidence suggests that ROS function not only as

mediators of destruction, but also as intracellular second messengers that regulate signal

transduction cascades and gene expression (Varvarovska, et al., 2004).

1.2.2 Diabetes Type 1 and 2

Diabetes is associated with a variety of metabolic abnormalities. The so-called metabolic

syndromes include hyperglycaemia characterized by hypertriaglyceridemia, reduced High

Density lipoprotein cholesterol (HDL) and abnormal postprandial lipidemia, atherosclerosis

and pro-coagulant state. The metabolic syndrome represents a cycle whereby insulin

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resistance leads to compensatory hyperinsulinemia which maintain normal plasma glucose

and may exacerbate insulin resistance.

Type 1 diabetes or insulin dependent diabetes mellitus (IDDM) is a complex multifactorial

disease involving severe destruction of the insulin-producing pancreatic β-cells. Type 1

diabetes is generally associated with young juvenile onset. Type 2 diabetes or non-insulin

dependent diabetes mellitus (NIDDM) typically occurs with older age and obesity. Although

glycemic control, insulin treatment and other chemical therapies can control many aspects of

diabetes, numerous complications are common and diverse. Diabetic patients have an

increased risk of developing various clinical complications that are largely irreversible and

due to microvascular or macro -vascular disease (Table 1)

Table 1: Vascular complications of diabetes mellitus

Microvascular Macrovascular

Nephropathy Ischemic heart disease

Retinopathy Stroke

Neuropathy Peripheral vascular disease

Source: (Jakus, 2000).

The impact of micro-vascular disease in diabetes includes nephropathy, retinopathy and

neuropathy (Table 1). Macrovascular disease is associated with the 2-4 fold increased risk for

atherosclerosis and ischemic heart disease that occur in diabetic individuals. The

complications of macrovascular disease are important causes of morbidity, mortality and

disability in people with Type 2 diabetes mellitus. Although the increased death rate is

mainly due to cardiovascular disease, deaths from non-cardiovascular causes are also

increased. In the pathogenesis of diabetic complications, important risk factors include not

only duration of diabetes, but also dyslipidemia, hypertension and cigarette smoking. The

results of the diabetic control and complications trails clearly establish hyperglycemia as the

major causal factor for the development of diabetic micro vascular complication

(Jakus,2000).

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The role of hyperglycaemia as an independent risk factor (not the major) for the development

of cardiovascular disease is supported by United Kingdom prospective Diabetes Study.

Improving glycaemic control delays the onset and reduces the severity of diabetic

complication. However, even with intensive current antidiabetic agents, more than 50% of

diabetic patient with type 2 suffer poor glycaemic control and 18% develop serious

complications within six years of diagnosis (Jakus, 2000). Thus,there is need for new

antidiabetic agents.

1.2.3 Insulin Resistance and Oxidative Stress

Several studies show that acute hyperglycemia can impair the physiological homeostasis of

many systems in living organisms. Excessive hyperglycemia may impair insulin activity and

sensitivity by the mechanism of “glucose toxicity” (Mooradian, 1999). Insulin stimulates the

uptake and utilization of glucose in muscle and adipose tissue, inhibits glycogenolysis and

gluconeogenesis in the liver and lipolysis in adipose tissue. Deficient action of insulin

reverses the metabolism of carbohydrates. Thus, with increased lipolysis are enhanced level

of free fatty acids and their oxidation in liver. In animal models, hyperglycemia increases

fatty acid availability in muscle. Thus, both “glucotoxicity” and “lipotoxicity” could lead to

insulin resistance and hyperinsulinemia (McGarry and Dobbis, 1999). It appears that insulin

resistance must occur in both muscle and liver for Type 2 diabetes. Both hyperglycemia and

insulin resistance are accompanied by reduced insulin action. Hyperglycemia and insulin

resistance may also be accompanied by oxidative stress.

Ceriello (2001) hypothesized a model that oxidative stress represents the common pathway

through which hyperglycaemia and insulin induce a depressed insulin action. This point of

view is supported by studies with antioxidants, which are able to improve the activity of

insulin.

1.2.4 Diabetic Complications

Diabetes is both a metabolic and vascular disease associated with numerous long-term

clinical complications that contribute to increased morbidity of the disease(Schalkwijk and

Stehouwer 2005). Vascular complications of diabetes can be divided into micro-and macro-

vascular. Retinal and renal microangiopathy cause diabetic retinopathy and nephropathy

respectively while microangiopathy of the vasa nervorum is important in the pathogenesis of

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neuropathy. Clinically, the complications are manifested as blindness, end stage renal failure,

defective nerve conduction and impaired wound healing. Macroangiopathy in diabetes refers

to a disease of larger vessels consisting mainly of an accelerated form of atherosclerosis that

affects the coronary, carotid and peripheral arteries, thus increasing the risk of myocardial

infarction, angina, congestive heart failure and stroke (Fantus, 2002).

Hypoglycemia has been identified as a risk factor for the development of diabetic

complications. A number of equally tenable hypotheses have been put forward to account for

the association of complications with this small molecule, glucose. These include but are not

limited to increased aldose reductase pathway, advanced glycation end- products (AGES)

formation, oxidative stress and increased protein kinase C (PKC) pathway. All these

mechanisms have been extensively studied and reviewed over a number of years (Wolff et

al., 1991; Guigliano et al., 1996; Brownlee, 2005). An abnormal activity of aldose reductase

pathway by sustained hyperglycemia seems to trigger a number of cellular and molecular

changes that are responsible for the micro- and macrovascular complications.

1.3 Hyperglycemia and Diabetic Complications

The factors that strongly affect the risk of diabetic complications are disease duration and

degree of glycemic control (Nathan, 1998 ; Semenkovich and Heinecke,1997). These

observations have given rise to the “glucose hypothesis” which suggests that glucose

mediates many of the deleterious effects of diabetes. Although, this appears to be an over-

simplification of a complex process, it has gained strong support from clinical trials in Type 1

and Type 2 diabetes. Both the Diabetes Control and Complication Trial and United Kingdom

Prospective Diabetes Study found that strict glycemic control dramatically lowered the

incidence of retinopathy, nephropathy and neuropathy (Nathan, 1998). This salutary finding

suggests that hyperglycemia promotes or even initiates these complications. Therefore,

glucose itself may be toxic to the micro vasculature. However, strict glycemic control alone

does not prevent diabetic complications, suggesting the involvement of additional factors.

Thus, factors other than glucose, such as abnormalities in lipoproteins, metabolic

derangements (insulin resistance, elevated free fatty acid levels) and variations in gene

controlling lipid metabolism might be important in macrovascular as well as microvascular

disease (Semenkovich and Heinecke, 1997).

1.4 Mechanism of Tissue Damage Mediated by Hyperglycemia

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Proposed mechanisms for the pathogenesis of diabetic complications include formation of

advanced glycosylation end-products (AGES) (Baynes and Thorpe, 2000; Stem, et al.,2002

), oxidative stress ( Monnier, 2001), carbonyl stress (Baynes and Thorpe, 2000; Monnier,

2001), increased protein Kinase C activity (Ishie et al., 1998), altered growth factor or

cytokine activities ( Sharma and Ziyadeh, 1997), reductive stress or pseudohypoxia (Ido et

al., 1997), and mitochondrial dysfunction (Nishikawa et al., 2000; Brownlee, 2001). Some of

these hypothesis overlap. For example, AGES might promote growth factor expression and

oxidative stress, and oxidative stress might promote AGES formation ( Baynes and Thorpe,

2000; Monnier, 2001). All these hypotheses are supported by extensive data, but a unifying

hypothesis remains elusive. The existence of several credible hypotheses might mean that

different tissues are differentially vulnerable to various oxidative pathways.

1.4.1 Aldose Reductase Pathway

An increased flux of glucose via the polyol pathway leads to the intracellular accumulation of

sorbitol. Accumulation of this non-permeable sugar in cells especially the lens and nerves

results in osmotic stress, cellular edema, redox imbalance, depletion of water soluble

antioxidants and susceptibility to oxidative insult (Cameron et al., 1999). Under

normoglycemia, most of the cellular glucose is phosphorylated to glucose 6-phosphate by

hexokinase. A minute part of non-phosphorylated glucose (approximately 8%) enters the so-

called polyol pathway, the alternative route of glucose metabolism (Maria et al., 2007),

implicating the enzyme aldose reductase. Aldose redcutase normally reduces toxic aldehydes

in the cell to inactive alcohols, but when the glucose concentration in the cell becomes too

high, glucose reduces to sorbitol in the presence of aldose redctase and NADPH which is

later oxidized to fructose by the sorbitol delydrogenase at the cost of NAD+ (Fig. 2)

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Fig. 2: Pathway leading to AGE formation. AGE formations arise from decomposition of

Amadori products, fragmentation products of polyol pathway lipid peroxidation products

which react with amino group of protein. HK = hexokinase; Glo = glyoxal; MGO =

methylglyoxal; 3-DG = 3-deoxy glucosone; PPP = Pentose Phosphate Pathway.

Source: (Maria et al., 2007)

Under hyperglycemia, there is an increase in the use of glucose through the pentose

phosphate pathway together with increased conversion of glucose via the polyol pathway

(more than 30% of glucose.

The sorbitol pathway increases in activity in diabetes in those tissues that do not require

insulin for cellular glucose uptake, such as the retina, kidney, peripheral nerves and blood

vessels. This pathway may impair endothelial function through some mechanisms. First,

sorbitol does not diffuse through cell membranes easily and accumulates, causing osmotic

damages. Sorbitol accumulation decreases other osmolytes such as myo-inositol and taurine .

However, the relatively low expressions of aldose reductase in the endothelial cells may not

be sufficient to cause significant sorbitol accumulation. Secondly, hyperglycemia leads to

over flow of the products of the polyol pathway along with depletion in the reduced

nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is an essential reducing

equivalent for the regeneration of reduced glutathione (GSH) by glutathione reductase (Fig 2)

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and for the activity of the NADPH- dependent thioredoxin system, two important cell

antioxidants against oxidative damage. Cells have several sources of NADPH, including the

two dehydrogenases of the pentose–phosphate pathway (glucose 6-phosphate dehydrogenase

and 6-phosphogluconate dehydrogenase; both insulin- induced enzymes), the malic enzyme

and the NADPH-dependent isocitrate dehydrogenase. The impairment of the hexose

monophosphate shunt leads to a reduced NADPH availability, and negatively influences

other enzymes and systems involved in defensive process against oxidative agents, such as

the catalase and glutathione systems. Several papers have been published that underline the

role of glucose 6- phosphate dehydrogenase deficiency in the pathogenesis of diabetes (West,

2002,)

NADPH is a cofactor of important enzymes of reactive nitrogen species (RNS) and reactive

oxygen species (ROS) metabolism, nitrogen oxygen species (NOS) (Nishikawa et al., 2000)

and NADPH- Oxidase respectively. Intracellular depletion of NADPH leads to a decreased

NO synthesis since NADPH is cofactor of NO synthesis, which synthesizes NO from L-

arginine. All isoforms of NOS contain a reductase domain and an oxygenase domain

separated by a calmodulin-binding region. NOS requires five cofactors/ prosthetic groups

such as flavin adenine dinucleotide, FAD, flavin mononucleotide (FMN), heme,

tetrahydrobiopterin (BH4) and Ca2+- calmodulin.

1.4.2 Non-Enzymatic Glycation

The non-enzymatic reaction of glucose with free amino groups, a variety of the long -lived

proteins represents another important mechanism of diabetic pathology that ultimately leads

to accumulation of advanced alycation end products (AGES). These products can form

covalent cross-linkages with proteins such as collagen and laminin and increase the stiffness

of the extracellular matrix (Tsilibary et al., 1988). Apart from this direct protein modification,

circulating advanced glycation end-products (AGEs) have been found to bind receptors for

AGEs, called receptor for advanced glycation end-products (RAGE) on various cells

(Endothelia cells, macrophages and mesangial cells) ( Li at al., 1996).

R – NH2 + O H – H2O R – N = CH R – NH-CH2

H-C-OH

Protein D- glucose Schiff base oxo-amine R

(Amadori product )

H2O H -C- OH C =O

R

R

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Binding of these receptors, stimulate the expression of pro-inflammatory cytokines such as

tumor necrosis factor α (TNF -α) and interleukin - 1 (1L-1), growth factor such as vascular

endothelial growth factor (VEGF) and platelet – derived growth factor (PDGF) and

transcription factor (Yamagishi et al ., 1998). RAGE activation increases the vascular

permeability that generates ischemia and microaneurysm in the eye and hemorrhage in the

brain (Paolino and Garner, 2005).

Cataract occurs as a result of the precipitation of glycated lens crystalline proteins and

glycation of retinal vessels leading to inflammation and microhemorrhage. Accumulation of

AGE products such as carboxy methyllysine, pentosidine, and imidozolene in the aqueous

humor has been implicated in the diabetic retinopathy (Franke et al, 2003)

1.4.3 Carbonyl Stress in Diabetes

Carbonyl stress explains increased modification of protein in diabetes, uremia and other

diseases by a generalized increase in the concentration of reactive carbonyl precursors of

AGEs, glycoxidation and lipoxidation products (Lyons and Jenkins, 1997). These carbonyls

may damage not only proteins but phospholipids and nucleotide base DNA. Carbonyl stress

may result from an increase in the deficiency of detoxification of carbonyl compounds.

Carbonyl stress refers to the intracellular generation and accumulation of reactive compounds

with carbonyl groups. These include both six- carbon derivative of glucose such as 3-

deoxyglucosone, and 3–carbon fragmentation products of glycolytic intermediate,

glyceraldehyde–3-phosphate called glyoxal and methyl glyoxal (Begenbardt et al., 1998).

These reactive species form covalent linkages with the amino groups of proteins both intra –

and extracellularly resulting in altered structure and function.

1.4.4 Activation of Protein Kinase C isoforms

Excess glucose may activate protein kinase C (PKC) directly by several mechanisms,

including through de novo synthesis of diacylglycerol (DAG), by activation of phospholipase

C, and by inhibition of DAG kinase (Keogh et al., 1997) or indirectly (via ligation of AGE

receptors or increased activity of the polyol pathway). Increased activity of protein kinase C

results in functional changes to vascular cells via activation of phospholipase A2 (the

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enzyme supplying the substrates arachidonic acid for prostaglandin production), the

expression of growth factors e.g. transforming growth factor–β, endothelia, and vascular

endothelial growth factor) and alterations in the expression of certain basement membrane

proteins (Koya, et al., 1997). Glycation may be responsible for the increased deformability of

granulocytes observed in diabetes (Sulochana et al., 2001).

Glycated products can be oxidized by several ROS, including HO. and ONOO- to give AGEs

shown in the Fig.3 below (Yim et al., 2002; Ahmed et al., 2005). Glycoxidation products

such as pentosidine and NE carboxyl methyl lysine are the best chemically characterized

AGEs compounds found in humans. The non- enzymatic glycation reaction proceeds slowly

through different stages leading to alteration of protein structure and molecular surface

topology that profoundly change the biochemical properties of the affected molecule. The

major biological effects of excessive glycation include inhibition of regulatory molecule

binding , cross linking of glycated proteins, trapping of soluble protein by glycated

extracellular matrix, decreased susceptibility to proteolysis, inactivation of enzymes and

transformation factors and abnormalities in relation to immune complex formation (Turk,

2001;Yim et al., 2002; Ahmed et al., 2005; Halliwell and Gutteridge, 2007). Glycation is

faster at elevated glucose in diabetic patients. Some tissues such as the liver, kidneys, and

erythrocytes are more susceptible to AGEs formation than others (Bohlender et al., 2005).

Glycated haemoglobin (HbA1c) contains a glucose amadori product attached to the N–

terminal valine of the β-chain. Glucose also glycates CuZnSOD in the erythrocyte, decreasing

its activity; this may account for the lower SOD activity reported in the blood of some

diabetics (Aral et al., 1987). Both CuZnSOD and ceruloplasmin can fragment after glycation

to release pro- oxidant copper ions (Islam et al., 1995). Fig. 3 shows the formation of

advanced glycation end-products (AGEs) by combination of glycation and oxidation.

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Fig. 3: Formation of advanced glycation end products (AGEs) by combination of glycation

and oxidation.

Source: (Maria et al., 2007).

Fig. 3 depicts another way of making AGEs by first oxidizing the glucose and then allowing

the oxidation product to react with protein. In the presence of transition metals, glucose can

be oxidized to produce O-2, H2O2, HO. and toxic dicarbonyls which can damage proteins

(Halliwell and Gutteridge, 2007).The above reactions occur under hyperglycemic condition

with production of radicals which can attack biomelecules such as lipids, proteins and DNA.

Under these conditions depletion of antioxidant enzymes such as SOD, CAT GPx could be

attributed to increased levels of the ROS produced.The disequilibrium between free radical

and antioxidant in favour of the former contributes to AGE formation and the word

glycoxidation is often used to describe the pathway involved. Once formed, AGE – modified

protein causes more oxidative stress. Glycation of protein in the electron transport chain can

impair normal electron flow and promote “leakage” to form O2-. Binding of glucose to amino

group on both Apo B and on lipids in LDL facilities LDL oxidation. Thus AGEs formation is

probably a significant contributor to the onset of diabetes complication mainly atherosclerosis

(Mehta et al., 2006).

1.5 Oxidative Stresses

An alteration in the level of oxidant and antioxidants, called oxidative stress initiated by

hyperglycemia contributes to tissue pathology. Early studies showed that glucose

autoxidation occurs in the presence of metal ions generating superoxide radical (O2-) and

hydrogen peroxide (H2O2) and if the scavenging enzymes superoxide dismutase and catalase

are impaired this may result in the formation of hydroxyl radicals (HO.) that react rapidly

Oxidation

107

with and damage lipids, protein and DNA (Baynes, 1991). So many data (clinical and

experimental) have clearly documented the depletion of extra and intracellular antioxidants in

the diabetic state and the prevention of complications by antioxidant supplementation

(Baynes and Therpe, 2000; Cederberg et al., 2001; Yamagishi et al., 2001). Browlee (2001)

speculates that in the setting of hyperglycemia, overproduction of superoxide by

mitochondrial electron transport chain and the resultant oxidative stress is the unifying

mechanism linking the major biochemical pathways triggered by hyperglycaemia. Oxidative

stress may be important in diabetes not just because of its role in the development of

complication but because persistent hyperglycaemia, secondary to insulin resistance may

induce oxidative stress and contribute to beta cell destruction in Type 2 diabetes (Maria et al.,

2007). Protein kianse C activation induced by glycemia may also represent a common

pathway by which oxidants and glycation products may mediate their advanced effects.

1.5.1 Mechanism of Increased Oxidative Stress in Diabetes Mellitus

Many studies have shown that increased lipid peroxides and/or oxidative stress are present in

diabetic subjects. Oxidative stress can be increased before clinical signs of diabetic

complications. In diabetes, oxidative stress is caused by both increased production of reactive

oxygen species (ROS), sharp reduction in antioxidant defenses and altered cellular redox

status (West, 2000). Hyperglycaemia may lead to increased generation of free radicals via

multiple mechanisms. Patients with diabetes may be prone to acute and chronic oxidative

stress which enhances the development of late diabetic complications. Enhanced oxidative

stress in hyperglycemia is indicated by urinary excretion of 8 -iso –prostaglandin F2 alpha.

Oxidative stress measured as index an of lipid peroxidation and protein oxidation has been

shown to increase in both insulin-dependent diabetes and non-insulin dependent diabetes

(Nishikawa et al., 2000; Cerieillo et al., 2001; Mohora et al., 2006; Stephens et al., 2006).

Although, the source of oxidative stress remains unclear, it has been suggested that the

chronic hyperglycaemia in diabetes enhances the production of ROS from glucose

autoxidation, protein glycation and glycoxidation, which leads to tissue damage. Also,

cumulative episode of acute glycemia can be source of acute oxidative stress.

During diabetes or insulin resistance, increased oxidative glucose metabolism itself increases

mitochondria production of O2-, which will be then converted to OH., and H2O2 (Nishikawa

et al, 2000). Beyond glucose, ROS formation is also increased by free fatty acids, through

direct effects on mitochondria (Evans et al., 2002). Enhanced oxidative stress in diabetes

108

(Type 2) has further a variety of important effects in atherogenesis, including lipoprotein

oxidation, particularly low density lipoprotein (LDL) oxidation. Lipid peroxidation of

polyunsaturated fatty acid (PUFA), one of the radical reactions in vivo, can adequately reflect

increased oxidative stress in diabetes (Slatter et al., 2002). The over-production of O.2, in

particular by mitochondria, causes inhibition of the glyceraldehyde -3-phosphate

dehydrogenase (GAPDH) and of cytochrome enzyme of the electron transport system

responsible for oxidative phosphorylation associated with the Krebs cycle (Nishikawa et al.,

2000). Hyperglycaemia- induced GAPDH inhibition was a consequence of poly (ADP-

ribosylation) of GAPDH by poly (ADP-ribose) polymeras], which was activated by DNA

strand breaks produced by mitochondria superoxide over production (Du et al., 2003).

As a result, glycolytic intermediates upstream of GAPDH accumulate, leading to increased

substrate- directed activity of the diacylglycerol synthetic pathway , which further activates

PKC (protein kinase C isoforms) and NADPH oxidase , as well as the hexosamine and polyol

biosynthetic pathways ( Fig 4).

Oxidative stress in the etiology of diabetic complications is illustrated in the diagram below:

Non-enzymatic

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Fig. 4: Pathways that contribute to oxidative stress in response to increased glucose flux

Source: (Maria et al., 2007).

1.5.2 Glucose Autoxidation

Hyperglycaemia –induced oxidative stress also occur in non-nucleated cells lacking

mitochondria and the NADPH oxidase (i.e. erythrocytes) (Jain, 1989). There must therefore

be another mechanism of ROS formation in these cells. One hypothesis is glucose

autoxidation. Glucose and many of its metabolites can react with hydrogen peroxide in the

presence of transition metals, such as Fe2+ and Cu2+, to form hydroxyl radical (OH.), the most

reactive ROS (Robertson et al., 2003).

1.5.3 Free Radicals

Free radicals are defined as atoms or molecules that contain one or more unpaired electrons,

making them unstable and highly reactive. The most important ROS are superoxide anion

(O2-) hydroxyl radical (OH.) hydrogen peroxide (H2O2), alkoxyl (RO.), peroxyl (ROO.) and

hydrochlorous acid (HOCl). Other non-oxygen species existing as reactive nitrogen species

(RNS), such as nitric oxide (NO) and peroxynitrite have also important bioactivity. ROS are

Poly(ADP-Ribose) Polymerase

Excess Mitochondria

110

continuously generated in physiological conditions and effectively eliminated by several

intracellular and extracellular antioxidant systems (Halliwell and Guttreridge, 1999). Free

radicals may be electrically neutral or either positively or negatively charged. They attack

sites of increased electron density such as the nitrogen atom present in proteins and DNA

predominantly and carbon-carbon double bonds present in polyunsaturated fatty acids and

phospholipids to produce additional free radicals which are often reactive intermediates

(Knight, 1999). Uncontrolled production of ROS often leads to damage of cellular

macromolecules,DNA, lipids and proteins and compromise cell function leading to cell death

by necrosis or apoptosis.

1.5.4 Reactive Oxygen Species and Oxidative Stress

Oxidative stress is defined as a disturbance in the proxidant- antioxidant balance in favor of

the former leading to potential damage and disruption of redox signaling and control. Cellular

metabolism generates reactive oxygen species (ROS). Molecular or ground state oxygen can

be activated to a ROS by means of energy transfer (e.g. under the influence of ultraviolet

radiation), forming singlet oxygen (1O2), or by electron transfer, forming incomplete

reduction products i.e. the superoxide anion radical (O2-). Small amount of oxygen (between

0.4 and 4% of all oxygen consumed) are reduced to O-2 by the mitochondria electron

transport chain during the course of normal respiration which is essential for generating ATP

(Boveris, 1984). Subsequently, O-2 can be converted into other ROS and RNS as shown in

Fig.5.

Under normal conditions, O2 - molecules are quickly converted to H2O2 by the key

mitochondrial enzyme, manganese superoxide dismutase (Mn-SOD) within the mitochondria

and by copper and zinc (CuZn-SOD) in the cytosol (Mendez et al., 2006). H2O2 is then

either detoxified to H2O and O2 by glutathione peroxidase (in the mitochondria) in

conjunction with glutathione reductase, or diffuse into the cytosol and detoxified by catalase

in peroxisomes. H2O2 can also be converted to the highly reactive hydroxyl radicals (HO-) in

the presence of reduced transition metals such as Cu or Fe (Fenton reaction). Further reactive

oxygen species may be derived from H2O2 such as the hypochlorite (OCL-) peroxyl radicals

(ROO.) and alkoxyl radicals (RO.) or from peroxidation of polyunsaturated fatty acids

(PUFA) such as conjugated dienes, lipid hydroperoxides and malondialdehyde (MDA)

(Taniyama and Griendling, 2003).

111

Production of one ROS may lead to the production of the other through radical chain

reaction. As summarized in Fig.5, O-2 is produced by one electron reduction of oxygen by

several different oxidases including NAD(P)H oxidase , xanthine oxidase, cyclooxygenase

and even endothelial nitric oxide synthase (eNOS) under certain conditions (Guzik et al.,

2002; Mehta et al., 2006).

Reactive nitrogen species (RNS) include free radicals like nitric oxide (NO.) and nitrogen

dioxide (NO2), as well as non-radicals such as peroxylnitrite (ONOO-). NO., also known as

endothelium-derived relaxation factor (EDRF) produced from L-arginine by eNOS in the

vasculature is considered vasculo protective. However, NO. can easily react with O2-,

generating the highly reactive molecule ONOO-. Thus, variation in the production of and O-2

by endothelium might provide one mechanism for the regulation of vascular tone and NO.

hence of blood pressure.

Fig. 5: Endogenous stimuli leading to ROS generation. The endogenous antioxidant enzymes

functions to maintain redox equilibrium.

Source: (Maria et al., 2007)

Although these ROS and RNS differ with regard to their stability, reactivity and molecular

targets, a common denominator is that the generation of ROS exceeding the antioxidant

capacity of the cells results in damage and oxidation of biomolecules such as lipids, proteins

and nucleic acids (Maria et al., 2007).

1.6 Antioxidant System

112

The reactive oxygen intermediates produced in mitochondria, peroxisomes, and the cytosol,

are scavenged by cellular defense systems including enzymatic (e.g. super oxide dismutase,

glutathione peroxidase, glutathione reductase, and catalase) and non-enzymatic antioxidants

(e.g. glutathione (GSH), thioredoxin, lipoic acid, ubiquinol, albumin, uric acid, flavonoids,

vitamins A, C and E, ).These antioxidants are located in the cell membranes, the cytosol or

blood plasma (Maritim et al., 2003). A major cellular thiol antioxidant and redox buffer of

the cell is reduced glutathione (GSH) which is regenerated most efficiently from oxidized

form (GSSG) by glutathione reductase and reduced nicotinamide adenine dinucleotide

phosphate (NADPH). Glutathione is highly abundant in cytosol (1 - 11 mM) nuclei (3 –

15mM); and mitochondria (5 – 11mM) and is the major soluble antioxidant in these

compartments (Masella et al., 2005; Valko et al., 2007). GSH in the nucleus maintains the

redox state of critical protein sulfhydryl that are necessary for DNA repair and expression.

Oxidized glutathione is accumulated inside the cell and the ratio of GSH/GSSG is a good

measure of oxidative stress of an organism (Valko et al., 2007). There are many other redox

couples in the cell, examples include NAD+ /NADH, ascorbate/dehydroascorbate,

NADP+/NADPH, and α – lipoic acid (LA)/dihydrolipoic acid (DHLA).

The main protective roles of glutathione against oxidative stress are (i) glutathione is a

cofactor of several detoxifying enzymes against oxidative stress, e.g. glutathione peroxidase,

(GPX), glutathione reductase, glyoxalases and enzymes involved in leucotriene synthesis.

(ii) GSH can react with ONOO- leading to formation of some nitrosothiol (GSNO), which

can decompose to regenerate NO., hence GSH can to some extent, recycle ONOO- to NO.

(iii) GSH scavenges hydroxyl radicals and singlet oxygen directly; detoxifying hydrogen

peroxides by the catalytic action of glutathione peroxidase (iv) Glutathione is able to

regenerate the most important antioxidants lipoic acid, vitamin C and E, back to their active

forms. Glutathione can reduce the tocopherol radicals of vitamin E directly or indirectly via

reduction of semi dehydroascorbate to ascorbate (Masella et al., 2005). The capacity of

glutathione to regenerate the most important antioxidant is linked with the redox state of

glutathione disulphide – glutathione couple (GSSG/2GSH) (Pastore et al., 2003)

1.6.1 Scavenging Properties of Antioxidants

A number of major cellular antioxidant defense mechanisms exist to neutralize the damaging

effects of free radicals. Enzymatic antioxidant system (Cu, Zn and Mn- superoxide dismutase

(SOD), catalase,glutathione (GSH), glutathione peroxidase (GPX), and glutathione reductase

113

(GR) function by indirect or sequential removal of ROS, thereby terminating their activities

(Jakus, 2007). The biological roles of these antioxidants are shown in following equations.

2O2. - + 2H + H2O2 + O2 SOD H2O2 +O2

2H2O2 2H2O2 + O2

2GSH + H2O2 + H2O +GSSG

GSSG + NADPH + H+ 2GSH + NADP+

ROO. + VitE –OH + Vit E – O

. + ROOH

Vit E – O. + Asc AH Vit-OH + AscA

. ....................................(vi)

Source :Jacus, 2007.

These antioxidant enzymes and vitamins catalyze the reactions that neutralize free radicals

and reactive oxygen species. They form the body’s endogenous mechanisms to help protect

against free radical-induced cell damage. The antioxidants enzymes glutathione peroxidase,

catalase, and superoxide dismutase metabolize oxidative toxic intermediates. Vitamin C and

E molecules can interrupt free radical chain reactions by capturing the free radical. The free

hydroxyl group on the aromatic ring is responsible for the antioxidant properties. The

hydrogen from this group is donated to the free radical, resulting in a relatively stable free

radical form of vitamin E.

To minimize transition metal – induced catalysis of Fenton and Haber Weiss reaction which

generate the most reactive hydroxyl radical, Several specific metal binding proteins such as

ceruloplasmin, ferritin, transferrin, hepatoglobin, lactoferrin, and albumin ensures that these

metals (copper and iron) are cryptic (Jacus, 2007).Non-enzymatic antioxidant systems consist

of scavenging molecules that are endogenously produced (GSH,ubiqinol,uric acid) or those

derived from the diet (vitamin C and E, carotenoids,α-lipoic acid and selenium).These

molecules donates electrons, and themselves become free radicals that can either initiate

chain reactions, or conversely be regenerated.

CAT

GPX

GR

114

Haber-Weiss H2O2 + O2-. O2 + OH- +

.O

Fenton H2O2 + Fe2+ OH- +

.OH + Fe3+

Fig. 6: Interactions between endogenous antioxidants in the process of detoxifying lipid

peroxide (Maria et al., 2007).

Regneration of endogenous antioxidants occurs through a cooperative set of reactions. The

hydroxyl radicals as shown in Fig. 6 can abstract an electron from polyunsaturated fatty acid

(LH) to give rise to carbon-centered lipid radicals (L.). The lipid radical (L

.) can further

interact with molecular oxygen to give a lipid peroxyl radical (LOO.), the lipid peroxyl

radical (LOO.) is reduced within the membrane by the reduced form of vitamin E resulting in

the formation of a lipid hydroperoxide and a radical of vitamin E. The vitamin E radical is

reduced back to vitamin E by vitamin C leaving behind the vitamin C radical. The oxidized

vitamin E radical can also be reduced by GSH. The oxidized glutathione (GSSG) and the

vitamin C radicals are reduced back to GSH and vitamin C, respectively by dehydrolipoic

acid (DHLA) which itself is converted to α-lipoic acid (LA). Α –lipoic acid (LA) after

reduction by nicotinamide adenine dinuclotide phosphate (NADPH) to dehydrolipioc acid

(DHLA) is able to facilitate the non-enzymatic regeneration of vitamin C and GSH, both of

which are able to regenerate vitamin E (Maria et al., 2007).

1.6.2 Positive and Negative Effects of Free Radicals

Respiratory burst is a remarkable property of the neutrophils, macrophages, β- cells and other

phagocytic cells (Kerrigan et al., 2009; Sluauch, 2011). These cells after activation increase

their oxygen uptake,which may raise up to 50 folds. This increased oxygen uptake is then

115

followed by the breakdown of the oxygen by respiratory burst oxidase enzyme as shown

below:

O2 + NADPH 2O2-. + NADP+ + H+

O.2 can be converted to H2O2 by SOD. The H2O2 can be transformed to HOCl by

myeloperoxidase.

2O2-. H2O2 HOCl

O2 and HOCl are powerful oxidizing agents that degrade microbes.

The presence of low concentrations of free radicals is important for normal cellular redox

status, immune function and intracellular signaling. Free radicals can serve as second

messengers or modify oxidation- reduction (redox) states. They are involved in some

enzymes activation, drugs detoxification and play an essential role in muscle contraction

(Finaud et al., 2006).

1.7 Lipid Peroxidation

Lipid peroxidation refers to the oxidative deterioration of lipid. It is the process in which free

radicals ‘steal’ electrons from the lipids in cell membranes resulting in cell damage. Lipid

peroxidation proceeds by free radical chain reaction. Polyunsaturated fatty acids are most

often being affected because of the presence of multiple double bonds in between which lie

methylene bridges (-CH2-) that possess reactive hydrogens.When the radical removes

hydrogen atom, it leaves behind an unpaired electron in the lipid (Niki, 2009). This in turn

leads to chain reaction. L-H + OH.→ H2O + L

.

The lipid radicals formed lead to cell damage. Three mechanisms are able to induce lipid

peroxidation: autoxidation (by free radicals reaction), photoxidation and enzyme action.

Autoxidation is a radical-chain process involving three stages: initiation, propagation and

termination. The general process of lipid peroxidation consists of three stages: nitiation,

propagation and termination. Initiation occurs when oxygen is partly reduced by Fe2+ to

NADPH-Oxidase

SOD Myeloperoxidase

116

species able to abstract a hydrogen atom from a methylene carbon .The resulting alkyl radical

reacts with oxygen to form a peroxy radical (LOO.), which itself can liberate LOOH via

hydrogen abstraction from a neighbouring alkyl bonds.

Nfe2++O2 nfe3++reduced O2

I+LH I H + L (initiation) L+O LOO.

In propagation, fatty acid radicals react with molecular oxygen forming a peroxyl-fatty acid

radical. This radical is also an unstable species that reacts with another free radical acid,

producing a different fatty radical and a lipid peroxide or acyclic peroxide if it had reacted

with itself. The cycle continues as the new fatty acid radical react in the same way.

LOO.+ L H LOOH + L (propagation) Termination occurs when new radicals reacts and produce a non-radical species. Anioxidant

vitamin E and antioxidant enzymes play a major role in the termination process (Marnett,

2002).

Photo-oxidation occurs when singlet oxygen of highly electrophilic reacts with unsaturated

lipids. In the presence of sensitizers (chlorophyll, porphrins, myoglobin,riboflavin, bilirubin),

a double bond interacts with singlet oxygen produced from O2 by light. The oxygen is added

at either end carbon of a double bond which takes the trans-configuration. Thus, the possible

reaction of singlet oxygen with double bond produces hydroperoxides (Hossam and

Mohamed, 2013).

F2+ can substantially enhance lipid peroxidation by decomposing LOOH to highly reactive

Lipid alkoxyl radicals (LO) that behave as organic and branch lipid peroxidation

Fe2++ LOOH fe3+ + LOH + LO LO + L H LOH+L Excess Fe2+ can also complete, as election donor, for LOO. and LO inhibiting both

propagation and chain braking reaction and causing the Fe2+depevalent termination of lipid

peroxidation.

Fe2++ LOO. / LO fe3+ + LOOH / LO (Termination).

117

Malondialdehyde (MDA) is a late–stage lipid oxidation by-product that can be formed non

enzymatically as a by-product of cycloxygenase activity (Slatter et al., 2002). MDA is a

highly toxic product formed in part by lipid oxidation-derived free radicals. Many studies

have shown that its concentration is considerably high in diabetes mellitus correlating with

poor glycemic control (Slatter et al., 2002, Hoff et al., 2003). MDA is a volatile molecule that

reacts, via Schiff base formation, with free amine groups of proteins, lipids and DNA. It is

estimated that up to 80% of MDA is protein-bound (Slatter et al., 2002). In addition,

accumulation of MDA affects membrane organization by increasing phosphotidyl serine (PS)

externalization. Accumulation of MDAand MDA adducts were correlated with many disease

state, such as hepatitis C, Down syndrome (Muchova et al., 2001), cancer (Marneth et al.,

2002), liver injury (Tuma, 2002), neurodegenerative disease and diadetes mellitus (Slatter et

al., 2002).

4-Hydroxy–nonenal is another lipid oxidation by-product which can form non-enzymatically

by-product. 4-Hydroxy–nonenal is formed from scission of precursor lipid hydroperoxide and

degradation of cyclic intermediates in lipid oxidation (Hoff et al., 2003). Compared with

MDA, 4-HNE is more reactive with proteins, potentiated by the ability for Michael addition

as well as Schiff based information. 4-HNE reacts with lysine from proteins and forms

pyrrols, cysteine and histidine. Few studies have demonstrated the accumulation of 4-HNE

in diabetes mellitus. 4-HNE is increased in microsomes and mitochondria of the IDDM

model mice (Traverso et al., 2002) and in the kidney of STZ-induced diabetic rats.

Lipid hydroperoxides are intermediates of lipid oxidation and are also formed enzymatically

through the action of lipoxygenases. Both forms of lipid peroxidation by-products are

structurally similar and can be further modified to hydroxyl fatty acid, leukotrienes, and

lipids by lipoxygenases and glutathione peroxidase. These molecules are ultimately important

in regulation of inflammation and atherosclerosis (Funk and Cyrus, 2001 and Natarajan and

Nadle, 2003). They initiate apoptosis in vascular smooth muscle cells (Dandona and Mjada,

2002).There is evidence that both non-enzymatically and enzymcatically formed lipid

hydroperoxides and their derivatives are elevated in diabetic state and are associated with a

few of the diabetic complications.

Non-enzymatically produced lipid hydroperoxide increased in the retina of STZ- induced

diabetic rats (Kowluru, 2003) and in the plasma of NIDDM (Nourooz-zadeh et al., 1997) and

IDDM (Davison et al., 2002) patients.

118

Isoprostanes are nonenzymatic products of arachidonic acid oxidation that are formed in situ

in the cell membrane and are released through the action of phospholipases. Isoprostanes

metabolites, such as epoxyisoprostanes and epoxycyclopentenones, stimulate endothelia cell

protein expression and synthesis (Subbanagounder et al., 2002). In addition isoprostane have

been used extensively as indicators of oxidative stress in cigarette smoking – induced

oxidation, pesticide exposure, chronic obstructive pulmonary disease, athero-thrombotic

disease, heart disease, atherosclerosis and diabetic mellitus (Subbanagounder et al., 2002).

They offer specificity and sensitivity advantages over MDA, the classic oxidant indicator.

1.8 Antioxidant Supplementation in Diabetes Mellitus

Given the involvement of oxidative stress in diabetic complications, supplementation with

antioxidants could be of interest since they delay the appearance or the development of

vascular complications. Some information is available on the effects of treatments with

classical antioxidants such as vitamin E, vitamin C or lipoic acid. Specifically, vitamin E

normalizes retinal blood flow and PKC activity in vascular tissue of diabetic rats (Kunisaki et

al., 1995). Lipoic acid has been suggested but not proven, to decrease the severity of diabetic

neuropathy by maintaining G-SH level and / or by its direct antioxidant properties (Vincent et

al., 2004). Vitamin E decreases the extent of diabetic complications, including renal damage,

and embryo pathway, nerve damage and vascular dysfunction. It equally delays lipid

peroxidation.

1.9 Alloxan

Alloxan and streptozotocin are the most prominent diabetogenic chemicals in diabetes

research. Both are cytotoxic glucose analogues. Although their cytotoxicity is achieved via

different pathways, their mechanisms of beta cell selective action are identical (Lenzen,

2007). In 1938 Wohler and Liebig synthesized a pyrmidine derivative, which they later called

alloxan (Lenzen et al., 1996). In 1943, interest in alloxan increased when Dunn and Mc

letchie reported that it could induce diabetes in animals 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. The reduction product of alloxan,

dialuric acid, has been shown to be diabetogenic in animals and to cause ultrastructural

changes identical to those observed in response to alloxan (Jorns et al., 1997). It was

reported that streptozotocin is diabetogenic and could cause diabetes by specific necrosis of

119

the pancreatic beta cell. Research has provided a unifying explanation for selective toxicity

of these most prominent diabetogenic agents.

1.9.1 Alloxan Diabetes and Streptozotocin Diabetes

Fig. 7 shows a schematic diagram of the tetraphasic and triphasic glucose responses to

alloxan and streptozotocin respectively, when injected (Lenzen, 2007). The responses are

accompanied by corresponding inverse changes in plasma insulin and sequential ultra

structural changes resulting in necrotic beta cell death. A first transient hypoglycemia phase

of up to 30 minutes starts within minutes of alloxan injection. This short lived hypoglycemic

response is the result of a transient stimulation of insulin secretion as documented by an

increase in the plasma insulin concentration.

Fig. 7: Phasic glucose response to a diabetogenic dose of alloxan (tetraphasic, i-iv) or

stretozotocin triphasic, the first phase dose not develop in case of streptozotocin, (11-1V).

Source: (Lenzen 2008)

As demonstrated in the diagram above, the initial transient hypoglycemic phase is not

observed in response to streptozotocin injection, since streptozotocin does not inhibit

glucokinase. Morphological alterations are minimal during this phase. The second phase

120

starts with an increase in the blood glucose concentration 1h after administration of the

toxins, and a decrease in plasma insulin. This first hyperglycemic phase, which usually lasts

2-4h, is caused by inhibition of insulin secretion leading to hypoinsulinemia. The third phase,

again a hypoglycemic phase typically occurs 4-8h after the injection of the toxins and last

several hours. It may be so severe that it causes convulsions, and may even be fatal without

glucose administration, in particular when liver glycogen stores are depleted through

starvation. This severe transitional hypoglycemia is produced by the flooding of the

circulation with insulin as a result of toxin – induced secretory granule and cell membrane

rupture (Lenzen., 2008). The fourth phase is the permanent diabetic hyperglycemic phase.

Morphologically, complete degranulation and loss of beta cell integrity is seen within 12-48h.

Non–beta cells remain intact, demonstrating the beta cell- selective character of the toxin.

Thus, injection of alloxan and streptozotocin principally induce the same blood glucose and

plasma insulin response and cause an insulin-dependent type 1- like diabetic syndrome. All of

the described morphological features of beta cell destruction are characteristic of necrotic cell

death (Lenzen, 2007).

1.9.2 Alloxan: Mechanism of Action

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 causes a state of insulin-dependent diabetes through its ability to induce ROS

formation, resulting in the selective necrosis of beta cells (Lenzen, 2008). Due to its chemical

properties, in particular the greater stability (Table 2), streptozotocin is the agent of choice for

reproducible induction of a diabetic metabolic state in experimental animals (Lenzen et al.,

1996). Alloxan on the other hand, as a model compound of ROS-mediated beta cell toxicity,

is the agent with the greater impact upon the understanding of ROS mediated mechanisms of

beta cell death in type 1 and type 2 diabetes mellitus.

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Table 2: Comparison of the chemical properties of alloxan and streptozotocin

Alloxan Streptozotocin Chemical name 2,4,5,6 – Tetraoxypyrimidine

2,4,5,6-Pyrimidinetetrone 2-Deoxy-2- ([(methylnitrosoamino)carbonyl]amino)-D-glucopyranose

Chemical structure Oxygenated pyrimidine derivative; barbituric acid derivative (5-ketobarbituric acid)

Cytotoxic methylnitrosourea moiety (N-methyl-N-nitrosourea) attached to the glucose (2-deoxyglucose) molecule; glucosamine derivative

Chemical properties Very hydrophilic, beta cell-toxic glucose analogue (partition coefficient – 1.8); weak acid

Hydrophilic, beta cell-toxic glucose analogue

Chemically unstable (half-life of 1.5 min at pH 7.4 and 37oC, decomposing to alloxanic acid); stable at acid pH

Relatively stable at pH 7.4 and 34oC (at least for up to 1h)b

Chemical reactivities Thiol reagent that is reduced to dialuric acid in the presence of GSH and other thriols

DNA alkylating agent

A protoxin; intracellular metabolism of this xenobiotic generates toxic ROS through redox cycling with dialuric acid over a long time period (>1h)

Protein alkylating agent

‘Compound 305’, a non-toxic alloxan-GSH adducts of unknown structure with a characteristic absorbance at a wavelength of 305nm; a small amount is formed during each redox cycle.

NO donor

Mode of toxicity Generation of ROS DNA alkylation

Source: (Lenzen, 2008)

1.9.3 Beta Cell Toxicity and Diabetogenicity of Alloxan

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Alloxan can generate reactive oxygen species (ROS) in a cyclic reaction with its reduction

product, dialuric acid as depicted in the reaction below:

(i) AH2 + O2 AH. + O2

- + H+

(ii) AH. + O2 A + O2- + H+

(iii) AH 2 + O2 + H+ AH.

+ H2O2

(iv) AH. + O2

. - + H+ A + H2O2

(v) H2O2 + e- OH. + OH-

(vi) Fe2+ + H2O2 Fe3+ + OH. + OH-

(vii) Net: O2. - + H2O2 O2 + OH

. + OH-

Metal

(viii) A + 2GSH AH2 + GSSG

(ix) A + GSH AH. + GS

.

(x) AH. + GSH AH2 + GS

.

(xi) 2H+ + 2O2. - H2O2 + O2

(xii) 2 H2O2 O2 + 2H2O

2GSH + H2O2 2H2O + GSSG

(Lenzen, 2008).

Chemical redox cyclic reactions between alloxan and dialuric acid and protective reactions of

cytoprotective enzymes (reactions i-ii). In beta cells the toxic action is initiated by the

radicals formed in this redox reaction (Oberley, 1988). Autoxidation of dialuric acid

generated 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

were generated (v-vii). The autoxidation of dialuric acid involves the intermediate formation

Catalyst

SOD

Catalase

GPx

123

of the alloxan radical (reaction i-v) (Winterbourn and Munday, 1989). The reduction of

alloxan to dialuric acid in the cell requires the presence of a suitable thiols, typically the

tripeptide glutathione (GSH) to generate the redox cycling partner, dialuric acid, and oxidized

glutathione (viii). The triketone structure of alloxan is vitally important for this two – step

reaction with glutathione (Elsner et al., 2007) which generate the alloxan radical as an

intermediate product (ix-x). Other thiols such as cysteine, which are present at lower

concentrations in the cell, dithiol and ascorbic acid are also suitable reducing agents and may

therefore contribute to alloxan reduction (Elsner et al., 2006). Alloxan can also generates

ROS by reacting with thiol groups on proteins such as enzymes and albumin. When kept in

the oxidized form, alloxan does not generate ROS (Elsner et al., 2006). Thus, alloxan is not

cytotoxic in the absence of thiol such as GSH or when restricted to the extracellular space

(Elsner et al., 2006). The reduction product dialuric acid is also not toxic when kept in the

reduced form ( Elsner et al., 2006). However in contrast to alloxan, dialuric acid autoxidises

spontaneously in the presence of O2, fig. 7, thus generating cytotoxic ROS in the absence of

thiol (Winterbourn and Munday, 1989). As a result of subsequent redox cycling it can also

induce diabetes and cause beta cell lesions as alloxan (Jorns et al., 1997)

124

Fig. 8: Redox cycling reaction between alloxan and dialuric acid. A, alloxan; AH2 = dialuric

acid, GS = glutathione radical; GSSG=oxidized glutathione; OH = hydroxyl radical; O-2 =

superoxide radical.

Apparently, the superoxide radical is not the species responsible for the cytotoxicity of

alloxan and dialuric acid. Several lines of evidence point to hydroxyl radicals as the principal

culprit (Lenzen, 2008).

1.9.4 Streptozotocin: Mechanism of Action and Beta Cell Selectivity

Streptozotocin inhibits insulin secretion and causes a state of insulin dependent diabetes

mellitus. Both effects can be attributed to its specific chemical properties, namely its

alkylating potency (Lenzen, 2008). As with alloxan, its beta cell specificity is mainly the

result of selective cellular uptake and accumulation.

Fig. 9a: Streptozotocin Fig. 9b: Methylnitrosourea

(Source: Lenzen, 2008)

Streptozotocin, a nitroso urea analogue in which the N- methyl-N-nitroso urea (MNU) moiety

Fig. 9 is linked to the carbon -2 of a hexose. The toxic action of streptozotocin and

chemically related alkylating compounds requires their uptake into the cells. Nitroso urea is

usually lipophilic and tissue uptake through the plasma membrane is rapid; however, as a

result of the hexones substitution, streptozotocin is less lipophilic. Streptozotocin is

selectively accumulated in the pancreatic beta cells via the low affinity GLUT2 glucose

transporter in the plasma membrane. Thus insulin-producing cells that do not express this

glucose transporter are resistant to streptozotocin (Schnedl et al., 1994). This observation also

explains the greater toxicity of streptozotocin compared with N- methyl–N-nitrosourea in

cells that express GLUT2, even though both substances alkylate DNA to a similar extent

(Elsner et al., 2007). The importance of the GLUT2 glucose transporter in this process is also

125

shown by the observation that streptozotocin damages other organs expressing this

transporter, particularly kidney and liver.

1.9.5 Beta Cell Toxicity of Streptozotocin

It is generally assumed that the toxicity of streptozotocin is dependent upon the DNA

alkylating activity of its methylnitrosourea moiety (Murata et al., 1999), especially at O-6

position of guanine (Lenzen, 2007). The transfer of the methyl group from streptozotocin to

the DNA molecule causes damage, which along a defined chain of events (Pieper et al.,

1999), results in the fragmentation of DNA. Protein glycosylation may be an additional

damaging factor. In the attempt to repair DNA, poly (ADP- ribose) polymerase (PARP) is

over-stimulated. This diminishes cellular NAD+ and subsequently ATP stores. The depletion

of the cellular energy stores ultimately results in beta cell necrosis. Although streptozotocin

also methylates proteins, DNA methylation is ultimately responsible for beta cell death, but it

is likely that protein methylation contributes to the functional defects of the beta cells after

exposure to streptozotocin. Inhibitors of poly ADP – ribosylation suppress the process of

DNA methylation. Thus, injection of nicotinamide and PARP inhibitors parallel with or prior

to the administration of streptozotocin are well known to protect beta cells against toxic

action of streptozotocin and to prevent development of a diabetic state (Schein et al., 1967).

Mice deficient in PARP are resistant to beta cells death mediated by streptozotocin, in spite

of DNA fragmentation. The absence of PARP prevents the depletion of the cofactor NAD+

and the subsequent loss of ATP (Masutani et al., 1999) and thus cell death.

1.10 Rationale for the Study

Most medicinal plants presently employed by traditional herbalists are used without scientific

investigation. It is therefore important to access and document the ethnomedical claims of

medicinal plants. For the fact that diabetes mellitus is a global health crisis, the current

incidence of diabetes is about 380 million and 592 million is estimated by 2035. Diabetes

caused at least USD 540 billion dollars in health care expenditure (International Diabetes

Federation report, 2014). In addition, the conventional antidiabetic drugs are with serious side

effects and cannot completely normalize blood glucose level. This prompted the scientific

investigation of K. africana, which could help to develop new drugs that can be used in the

treatment and management of several ailments, especially diabetes. Therefore, the present

work is undertaken to explore the antidiabetic and antioxidant potentials of ethanol, methanol

and n-hexane leaf and fruit extracts of K. africana on alloxan-induced diabetic rats.

1.10 Aim and Objectives of the Study

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1.10.1 Aim of the Study

In this study, the effects of oral administration of ethanol, methanol and n-hexane extracts of

K. africana leaves and fruits in alloxan-induced diabetic rats were evaluated.

1.10.2 Specific Objectives of the Study

The above-mentioned aim of the study was achieved through the following specific

objectives: Determination of the effect of the extracts on

(i) blood glucose level of both normal and alloxan-induced diabetic rats,

(ii) body weight of both normal and alloxan-induced diabetic rats,

(iii) the concentration of sorbitol of both normal and alloxan-induced diabetic rats,

(iv) the concentration of total protein of both normal and alloxan-induced diabetic rats,

(v) glycosylated haemoglobin level of both normal and alloxan-induced diabetic rats,

(vi) lipid peroxidation index (MDA) of both normal and alloxan-induced diabetic rats,

(vii) vitamin C concentration of both normal and alloxan-induced diabetic rats,

(viii) the activities of some antioxidant enzymest (SOD, CAT, GPx) of both normal and

alloxan-induced diabetic rats,

(ix) lipid profiles of both normal and alloxan-induced diabetic rats,

(x) The activities of liver marker enzymes (ALT, AST), and the concentration of total

bilirubin and comparison of the effectiveness and synergistic effect of the extracts.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

127

2.1.1 Chemicals

These were analytical grade products and include ethanol, methanol, n-hexane,

ethylene diamine tetraacetate (EDTA), hydrochloric acid, sulphuric acid, trichloloroacetic

acid, (TCA), 2-thiobarbituric acid (TBA), alloxan monohydrate (sigma-Aldrich, USA), 1-

chloro-2, 4 dinitrothiobenzene, glutathione peroxidase kit (Randox Laboratories Limited,

UK), protein assay kits (Randox Company, USA), superoxide dismutase kit (Randox

Company, USA), glycosylated haemoglobin kit (Randox company,USA), sorbitol kit, lipid

profile kit, slucose test (Life Scan Inc, California, USA)

2.1.2 Instruments/Equipment

Atomic Absorption Spectrophotometer (AAS) Scientific 210 VGP, Buck, Centrifuge

PIC, England, pH meter Pye, Unican 293, England, Refrigerator, Air Thermocool, UV

Spectrometer,UNICO – UV-2012 , water bath DK-SA,Gallenkamp, England, Rotary

Evaporator, Model Modulyo 4K, England.

2.1.3 Drug

A dose of 2.5 mg/ kg body weight of glibenclamide was used as potent standard

(reference) drug (Nigerian German Chemical Plc).

2.1.4 Plant Material

The leaves and fruits of Kigelia africana were obtained from the plant in its natural

habitat in Omor Village, Ayamelum L.G.A, Anambra State, Nigeria and authenticated in the

Herbarum Unit of Department of Plant Science and Biotechnology, University of Nigeria

Nsukka.

2.2 Methods

2.2.1 Animal Management

Male albino rats (6-8 weeks old) of average weight 65 ±0.50 g were used for this

study. For LD50 determination mice of about 4-6 weeks old with average weight of 28±0.40 g

were used. These animals were obtained from the Department of Zoology, 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.

128

2.2.2 Preparation of Plant Extracts

Fresh leaves and fruits of K. africana were dried in the laboratory at room temperature,

crushed and soaked separately in 200 ml of analytical grade (98 %) methanol, Ethanol and n-

hexane for 72 hours. The mixture was filtered and evaporated with rotary evaporator at room

temperature to obtain the crude extract.

2.2.3 Design of the experiment

The animals were divided into 12 (twelve) groups of 5 (five) rats each. Treatment was carried

out orally using intubation tube. Groups 4-9 received 500 mg/kg body weight of the extract

while groups 10-12 were given 250 mg/kg body weight each of leaves and fruits extracts. The

groups were as follows:

Group 1: Normal rats administered normal saline

Group 2: Diabetic untreated rats

Group 3: Diabetic rats treated daily with a reference drug (glibenclamide) at a dose of

2.5mg/kg body weight.

Group 4: Diabetic rats treated with n-hexane extract of K. africana leaves

Group 5: Diabetic rats treated with n-hexane extract of K. africana fruits

Group 6: Diabetic rats treated with ethanol extract of K. africana leaves

Group 7: Diabetic rats treated with ethanol extract of K. africana fruits

Group 8: Diabetic rats treated with methanol extract of K. africana leaves

Group 9: Diabetic rats treated with methanol extract of K. africana fruits

Group 10: Diabetic rats treated with n-hexane extract of K. africana leaves and fruits

Group 11: Diabetic rats treated with methanol extract of K. africana leaves and fruits

Group 12: Diabetic rats treated with methanol extract of K. africana leaves and fruits. At the

end of 21 days post- treatment, the animals were bled through ocular puncture and blood

samples were received into clean dry test tubes for estimation of some biochemical and

oxidative parameters.

2.2.4 Percentage Yield of Extract

% Yield = 100pulp sample ground of (g)Weight

evaporatedextract dry of (g)Weight ×

2.2.5 Phytochemical Analysis of the Crude Extract

129

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

plant constituents: alkaloids, flavonoids, glycosides, proteins, carbohydrates, reducing sugars,

saponins, tannins, oil, resins and terpenoids.

2.2.5.1 Test for the presence of alkaloids

Exactly 0.2 g of leaf /fruit extract of K. africana was boiled with 20% hydrochloric

acid (5 ml) on a steam bath allowed to cool and then filtered. One millilitre of the solution

was treated with 2 drops of Dragendorff reagent (Bismuth potassium iodide solution). A red

precipitate indicated the presence of alkaloids

2.2.5.2 Test for carbohydrates

The leaf/fruit extract (0.1 g) was shaken vigorously in 5 ml of distilled water and then

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

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

ring layer at the inter-phase indicates the presence of carbohydrates.

2.2.5.3 Test for reducing sugar

A quantity (0.1 g) of the crude extract was mixed with 5 ml of equal parts of Fehling’s

solution A and B and heated on a water bath 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 0.1 g of the sample and the mixture left to stand

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

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

presence of proteins.

2.2.5.5 Test for fats and oil

A quantity (0.1 g) of the sample was pressed on the fliter 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.

2.2.5.6 Test for glycoside

130

Two grammes (2 g) of the extracts were mixed with 30 ml of water. The mixture was heated

on water bath 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 (0.2 ml each) until it turned alkaline (tested with

litmus paper). It was then boiled in waterbath for 3 minutes. A brick red precipitate indicated

presence of glycoside

2.2.5.7 Test for acidic substances

The sample (0.1 g) was placed in a clean dry test tube and sufficient distilled

water added. This was kept in a water bath 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 (0.2 g) of the extract was heated with 10 ml of ethyl acetate in boiling water for 3

minutes. The mixture was filtered and the filtrate used for the test. Four millilitres (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 10 ml of lead acetate solution (90% w/v) and 20 ml of aqueous ethanol (50%)

were added 1 g of the sample in a 200 ml 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 M sodium

hydroxide solution were added. A reddish brown interphase showed the presence of steroids.

2.2.5.10 Test for tannins

Two grammes of the sample 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

131

i) Precipitate test: A weighed quantity of ( 0.2 g) of the sample was extracted with 15 ml

of 96% ethanol. The alcoholic extract was then poured into 20 ml of distilled water in a

beaker. A precipitate indicated the presence of resins.

ii) Colour test: A weighed quantity of (0.2 g) of the 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 water bath 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 (0.1 g) was weighed and boiled with 5 ml of distilled water on a hot water bath

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

following tests.

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

mixture shaken vigorously. The formation of an emulsion indicated the presence

of saponins.

ii) Frothing test: One millitre (1 ml) of the filtrate was diluted with 4 ml of distilled

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

2.2.3.13 Test for terpenoids and steroids

Exactly 9 ml of ethanol (96%) was added to 9 g of the sample and refluxed for a few

minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath and then

5 ml 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 2.5 ml 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 0.5 ml of chloroform extract was evaporated to dryness on a waterbath

and heated with 3 ml of concentrated sulphuric acid for 10 minutes on a waterbath. A gray

colour indicated the presence of terpernoids.

2.2.6 Proximate Analysis

132

Percentage concentrations of protein, carbohydrate, crude fiber, moisture and ash in the

leaves and fruits of K.africana extract were determined using the AOAC method (1990).

2.2.6.1 Crude protein

Principle

The crude protein content was determined using the micro Kjeldahl method. The method is

generally used to determine nitrogen (N) in substances which contain N as ammonium salts,

nitrates or organic N compounds. Since it measures the total amount of N in a compound

only a rough indication of the total content can be obtained hence the term crude protein. The

quantity of N measured was then multiplied by 6.25 to obtain the protein content of the

compound. The multiplication factor can vary with some materials (AOAC, 1990).

The N of protein and other compounds were converted to ammonium sulphate by acid

digestion with boiling H2SO4.The acid digest was cooked, diluted with water and made

strongly basic with NaOH. Ammonium was released and distilled into a 4% boric acid

solution. The amount of ammonium borate formed was determined with standard H2SO4 or

HCl.

The indicator used, bromocresol green, gave a pink colour end point at a hydrogen ion

concentration corresponding to a solution of NH4CI. Boric acid is so weak that it has no

appreciable influence on the pH. The reactions are represented as:

NH3 +HBO3 NH4 +BO3-

H+ +BO2 HBO2 (AOAC, 1990)

The method involved three major steps:

A. Digestion of the sample

B. Distillation of the ammonia into a trapping solution.

C. Quantification of the ammonia by titration.

(a) Digestion: A small quantity of sample of K. africana leaf or fruit extract (0.1 g) was

weighed in kjeldhal flask containing 2.0 g sodium sulphate/copper sulphate as catalyst.

Concentrated H2SO4 (20 ml) was introduced into the flask and the content gently digested to

yield a clear solution.

133

(b) Distillation: The content of the flask was washed with 220 ml distilled water into a

distillation flask and cooled under ice blocks. To the flask, 100 ml of 4% boric acid was first

added; later, 3 drops of screened methyl red was also added to the distillate and titrated

against 0.5 N Na2SO4 solution.

(c) Back titration: After cooling, 40% NaOH (50 ml) was added and the distillate was

titrated against 0.5 N Na2SO4 solution

The percentage nitrogen was calculated using the relationship

% Nitrogen = 100sample ofWeight

×××× MWNDfNT

Where

T = Titre volume

N = Normality of acid

Df = Dilution factor

MWN = Molecular weighted of nitrogen

% protein = % Nitrogen x 6.25

Where 6.25 =conversion factor of Nitrogen to protein

2.2.6.2 Crude fat

Principle

The sample was continuously extracted with ether. After extraction, the ether extract was

evaporated to dryness and the residue designated the ether extract also contained organic

acids, oil, pigments, alcohols and fat-soluble vitamins and was referred to as crude fat. Many

of the complex lipids, such as phospholipids are not completely extracted in this procedure

(Ensimger and Olentine, 1978).

Procedure

A washed, dried and cooled quick-fit flask was weighed. Extracts of K. africana leaves and

fruits were individually weighed into the extraction thimble and placed in the quick-fit

soxhlet apparatus. The solvent flask containing 250 ml of diethyl ether was connected to a

condenser. The set-up was heated for 16 hrs for complete extraction. The extract was

134

evaporated at 700C to remove any remaining solvent present. The apparatus was re-weighed

and percentage fat calculated as follows:

% Crude fat = 100sample ofWeight

oil ofWeight ×

2.2.6.3 Moisture

Procedure

Two grammes (2 g) of freshly collected samples of leaves and fruits of K. africana were

respectively weighed and dried in the oven at 1100C to a constant weight. The dishes and

samples were cooled and re-weighed and percentage moisture content calculated using. The

relationship is as follows:

W1 =Weight of sample

W2 = Initial weight of sample and dish

W3 = Final weight of dry sample

2.2.6.4 Ash /Mineral matter

Principle

Ash is defined as the mineral matter of a feed or material since it includes for the most part

the organic or mineral components of the feed or material (Ensiminger et al., 1978; Cullisoin,

1982). The sample was heated at 6000C to burn off all organic materials. The inorganic

material which did not volatilize at this temperature was designated ash.

Procedure

Into previously weighed porcelain dishes were put 2 g samples of leaves and fruits of K.

africana and reweighed. The crucible and samples were placed in a muffle furnace at 6000C

for 3hrs. The ashes and crucible were cooled in a desecrator and re-weighed. The percentage

ash content was calculated using.

100WW

WW

12

13 ×−−

% Ash = 100WW

WW

12

13 ×−−

Where W1 = Weight of crucible W2 = Weight of crucible and sample

100W

WW

1

32 ×−

135

W3 = Weight of crucible and ash

2.2.6.5 Crude fibre

Principle

This fraction was designed to include those materials in food which were of low digestibility

namely cellulose, certain hemicelluloses and some of the lignin, if present. Some of the

lignin, however, may be included in the nitrogen free extract. A moisture- free, ether extract

was digested first with weak acid solution (1.25% H2SO4) and then with a weak base solution

(1.25% NaOH). The organic residue left digestion is collected.

The loss of weight on ignition was called crude fibre.

Procedure

Samples of the leaf or fruit of K. africana extracts (2 g) each was weighed into 500 ml

beakers containing pre-heated concentrated H2S04 (40 ml). The content was boiled for 30

mins and filtered. The residue was washed three times with hot water, and then 150 ml of

pre-heated KOH and drops antifoam agent (loctanol) were added to the sample in the beaker

and heated to boiling. The mixture was boiled slowly for more 30 minutes, filtered and

washed three times with hot water. Acetone was then used in washing it three times in cold

extraction unit and the content dried at 1300C for one hour.

After ashing the content at 5000C, the ash was weighed and the percentage fibre calculated as

follow:

% Crude fibre = 100sample ofWeight

fibre ofWeight ×

2.2.6.6 Carbohydrate or Nitrogen-Free Extract (NFE)

Principle

Proximate analysis of carbohydrate is also known as Nitrogen-Free Extract (NFE)

determination. It includes mostly sugars and starches and also some of the more soluble

hemicelluloses and some of the more soluble lignin according to the method of Cullison,

(1982). Since this fraction was designed to include the more soluble carbohydrates, it is

sometimes refereed to as the carbohydrate portion of the material being analyzed.

136

Procedure

NFE was calculated by subtracting the sum of the fractions from 100 as follows:

100 – (% moisture + % crude protein +%crude fat +crude fibre +%ash) = NFE.

2.2.7 Acute Toxicity Test

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

Median lethal dose (LD50) is the 1og 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 of Lorke (1983).Thirteen experimental mice were distributed in

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

administered differently 10 mg/kg, 100 mg/kg and 1000 mg/kg body weight of the K.

africana fruit and leaf extracts. The extracts were injected intraperitoneally.

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

conditions. Based on the percentage survival, further increased dose of 1,600, 2000 and 2900

mg/kg body weight were administered respectively and the fourth mice received only normal

saline. The mice were observed for another 24 hours. The LD50 was calculated as the

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

2.2.8 Induction of Experimental Diabetes

The animals were fasted for 24 hours before induction. A total of eighty-four (84) rats with

blood glucose concentration 60 – 90 mg/dl were injected intraperitonally with alloxan

monohydrate (Sigma, USA) dissolved at cold normal saline in a dose of 160 mg/kg body

weight. The animals were fed with Bendel feed and Flour Mill Limited pelleted Guinea

Grower Mash. After seventy-two (72) hours blood glucose concentrations were determined

using glucometer (Acu-chek). Rats with blood glucose concentrations of 200 mg/dl and

above were used for determination of antidiabetic and antioxidative effects of K. africana leaf

and fruit extracts. The diabetic rats were divided into nine groups of five rats each. The

extract was dissolved in dimethyl sulphoxide (DMSO) and normal saline (1:10 v : v ) and

was given orally with oro-gastric tube throughout the duration of the experiment.

2.2.9 Determination of Fasting blood Glucose Concentration

Fasting blood sugar was determined using Accu-check Active glucometer by Roche

diagnostics Turkey, A.S according to the method of Mark and Dawson (1965).

Principle

Glucose oxidase

137

Glucose Gluconic acid + H2O2 H2O2 H2O + O

O+ Acceptor Coloured Complex +H2O

The method is based on the reaction of glucose and oxygen in the presence of glucose

oxidase to yield gluconic acid and hydrogen peroxide. The hydrogen peroxide subsequently

oxidizes the dye in a reaction mediated by peroxides producing a blue coloured form of the

dyes. The intensity of the blue colour is proportional to the glucose concentration in the

sample and it is measured and read by the ONE TOUCH meter.

The one-touch glucometer was essentially a reflectance meter. The amount of light reflected

in reagent area of the dextrostix measured in a readout meter scale was a measure of the

concentration of glucose in the blood. Snips were made on the tail of the animal to release

blood on the sensitive spot on the glucometer.

Reagents

ONE TOUCH glucometer (lifescan inc. Johnson- Johnson Company, USA) and test strips

were used.

The composition of the test strips is:

Glucose oxidase (14/U)

Peroxidase (11/U)

3-methyl-2-benzothiazolinonehydrazone hydrochloride (0.06 mg)

3-dimethylaminobenzoic acid (0.12 mg)

Procedure

Code key was inserted into the glucometer code key opening and ensured that the code on the

glucometer matches the code on the test strip. A fresh new strip was inserted with the orange

pad facing up until it went no further into the glucometer opening for test strips. The result

was displayed after 5 seconds in mg/dl.

2.2.10 Determination of Sorbitol Concentration

Sorbitol concentration was determined according to the method of Bergmeyer (1974)

Principle

Peroxidase

O-toluidine

138

D-Glucose + ß-NADH D-Sorbitol +ß-NAD+

Procedure:

Phosphate buffer (2 ml) solution was pipetted into a test tube. One milllitre (1 ml) of sample

was followed by 0.1 ml of nicotinamide adanine dinucleotide solution.The tube was mixed

and extinction E1 at 340 nm was measured. Sorbitol dehydrogenase (0.5 ml) was added and

mixed by inversion, allowed to stand for 60 mins after which the extinction E2 was read

again.

Sorbitol Conc. = E1/E2 × Concentration of std.

Whrere E1 and E2 = absorbancies

2.2.11 Determination of Total Protein Concentration

Total protein concentration was determined using protein assay kits according to the method

of Lowry et al. (1951)

Principle

This method is based on the principle that cupric ions, in an alkaline medium, interact with

peptide bonds of proteins resulting in the formation of a coloured complex.

Reagents contained in assay kits (Randox company, USA)

Contents

1. Biuret Reagent

Sodium hydroxide

Na – K – tartrate

Potassium iodide

Cupric sulphate

Concentration

100 mmol/L

16 mmol/L

15 mmol/L

6 mmol/L

2. Blank Reagent

Sodium hydroxide

Na – K – tartrate

100 mmol/L

16 mmol/L

3. Standard

Protein

60 g/L (6.0 g/dl)

139

Procedure

Distilled water (0.02 ml) was pipetted into reagent blank (B) test tube only. Standard solution

(0.02 ml) was added to another test tube labeled ST (standard) only. After which 0.02 ml of

the sera from the different rats were added to different test tubes labeled SA (sample) only.

Biuret reagent (1.0 ml) was added to all the three sets of test tubes. The content was mixed

thoroughly and incubated for 30 minutes at 25OC (room temperature). Absorbance of the

Sample (Asample) and of the Standard (Astandard) against the reagent blank was read at a

wavelength of 530 nm.

The total Protein concentration was calculated as follows:

Total Protein Conc. = Conc. StandardA

A

Standard

Sample ×

Where:

A sample = Absorbance of the Sample

A standard = Absorbance of the Standard

2.2.12 Determination of Haemoglobin Glycosylation

Principle

A haemolyzed preparation of the whole blood is mixed continuously for 5 minutes with a

weakly binding cation–exchange resin. During this time HbAo binds to the resin. After the

mixing period, a filter is used to separate the supernatant containing the glycohaemoglobin

from the resin.

The percentage glycohaemoglobin is determined by measuring the absorbance at 415 mm for

the haemoglobin and the total haemoglobin fraction. The ratio of the two absorbencies gives

the present haemoglobin

Reagent: Commercial kits obtain from TECO Diagnostics, USA

140

Content Concentration

Resin Reagent 8 mg/ml cation – exchange

Resin buffered at pH 6.9

Lysing Reagent 10 mm potassium cyanide,

surfactant added

Glycohaemoglobin standard 10% glycohaemoglobin

Serum separator -

Preparation of Reagents

1. Glycohaemoglobin lysing reagent: contents were brought to room temperature.

2. Glycohaemoglobin cation–exchange Resin: contents were brought to room

temperature. Each of the 5 tubes was swirl and gently inverted for a minimum of 10

times after addition.

Procedure

A. hemolysate preparation:

The lysing reagent (500 µl) was dispersed into tubes labelled standard, control and sample.

Well- mixed blood sample (100 µl) was placed into the appropriately labeled tube, mixed

well and allowed to stand for 5 minutes.

B. Glycohaemoglobin Preparation.

Exactly 3 ml of glycohaemoglobin cation-exchange resin was dispensed into 13 x 100 mm

tube labeled standard, control and sample 1. The haemolysate sample (100 µl) was added

into the sample test tubes. Filter separators were positioned in the test tube so that the rubber

sleeve is approximately 1cm above the liquid level. The tubes were placed on rotator and

mixed continuously for 5 minutes. Filter separator was pushed into the tubes until the resin

was firmly packed. The supernatant was poured into another tube for absorbance

measurement. The instrument was adjusted to zero absorbance at 415 mm with deionized

water as the blank.

Absorbance was read and recorded for standard, control and sample. These readings were for

glycohaemoglobin.

C Total Haemoglobin

Deionized water ( 5 µl ) was put into tubes labeled standard, control and sample 1.

141

The haemolysate (20 µl) was added to the appropriate tubes, adjusted to zero at 415 mm with

deionized water as the blank. The absorbance value of the standard, control, and sample were

recorded. These readings were for total haemoglobin.

Calculation

Result of the glycosylated HG was determined in the following manner:

% Glyco (unknown) = (standard) R

Conc Standard (unknown) R ×

Where

R (unknown) = Ratio (unknown) = (unknown) Hb AbTotal

(unknown) Glyco Ab

R (standard =Ratio (standard = (Standard) Hb Total Ab

(Standard) Glyco Ab

= 1

Conc Std

(Std) Glyco Abs

Hb Total Abs

(Unknown) Hb Total Abs

(Unknown) Glyco Ab ××

2.2.13 Determination of Malondialdehyde Concentration

Malondialdehyde concentration was determined by the method of Wallin et al.

(1993).

Principle

The principle of the estimation is based on the fact that thiobarbituric acid (TBA)

reacts with malondialdehyde (MDA) to give a red or pink colour, which absorbs maximally

at 532 nm (Fig. 10).

MDA + 2TBA MDA: TBA adduct + H2O

N N

OH CHO CH2 CHO

HS

+

S OH

CH2-CH=HC

N N

N SH N HO

142

Reagent Preparation

(a) Thiobarbituric acid was prepared by dissolving 1.0 g of the compound in 83 ml of

distilled water on warming. After complete dissolution the volume was made up to 100 ml

with distilled water.

(b) 25% Trichloroacetic acid (TCA): Trichloroacetic acid (12.5 g) was dissolved is distilled

water and made up to 50 ml in a volumetric flask with distilled water.

(c) Normal saline: Sodium chloride (0.9 g) was dissolved in 10 ml of distilled water and

make up to 100 ml with distilled water.

Procedure

To 0.1 ml plasma in the test tube was added 0.45 ml of normal saline and mixed

thoroughly before adding 0.5 ml of 25% trichloroacetic acid (TCA) and 0.5 ml of 1%

thiobarbituric acid. To the blank was added the same volume of trichloroacetic acid,

thiobarbituric acid and saline and 0.10 ml of distilled water instead of plasma. The mixture

was placed in the waterbath and heated at 95oC for 40 minutes. The turbidity was removed by

centrifuging the mixture. It was allowed to cool before reading the absorbance of the clear

supernatant against reagent blank at 532 nm. Thiobarbituric acid reacting substances were

quantified as lipid peroxidation product by referring to a standard curve of malondialdehyde

(MDA) concentration equivalent generated by acid hydrolysis of 1,1,3,3–tetraethoxypropane

(TEP) prepared by serial dilution of a stock solution.

Table 3: Volumes of the various materials/reagents put into the sample and blank tubes

Blank Test

Plasma (ml) --- 0.10

2

TBA MDA Pink colour complex

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

OH

H

OH OH

143

Distilled water (ml) 0.10 ---

Normal saline (ml) 0.45 0.45

25% TCA (ml) 0.50 0.50

1% TBA (ml) 0.50 0.50

Preparation of Lipid Peroxidation Standard Curve

Lipid peroxidation standard curve was prepared using 1,1,3,3, tetraethoxypropane

(TEP) according to the method of Albro et al. (1986).

Principle

Tetraethoxypropane reacts with HCl in the required proportion to release MDA which

then reacts with TBA to produce TBA-MDA adduct that absorbs maximally at 532 nm.

Procedure

TEP (24.6 mg) was dissolved in 80 ml of deionised water and the volume made up to

100ml. This served as the stock solution. Working standards were prepared by diluting the

stock 1:50, 1:75, 1:100, 1:150, 1:200, 1:250, 1:400, 1:500, 1:800, and 1:1200 with 0.01

NH4Cl.

Five tubes were set up and 0.5 ml of the serial dilutions added accordingly. A quantity

(2.5 ml) of the 20% TCA was added to each tube and the mixture allowed to stay for 10

minutes. Then 2.5 ml of 0.05 M H2SO4 and 3 ml of TBA (0.67%) were added. The set up was

incubated for one hour in boiling water. After the one hour, the tubes were cooled in a

running tap and 4 ml of butan-1-ol added. The tube contents were mixed with a vortex. This

was then centrifuged for 30 minutes at low speed of whisperfuge centrifuge model B84. The

butanol layer on top containing the TBA reactive substance was carefully collected and its

absorbance read at 532 nm against a butanol blank on a spectrophotometer (Pye Unicam SP

500). The standard curve of OD against MDA concentration of the stock solution contained

15 nmol (2.46 µg) of MDA per ml.

2.2.14 Determination of Vitamin C Concentration

Vitamin C concentration was determined using the method Goodhart and Shils

(1973)

Principle

144

This method involves the oxidation and conversion of ascorbic acid to diketogluconic

acid in strong acid solution. A diphenylhydrazine is formed by the reaction of sulphuric acid

with 2, 4-dinitrophenylhydrazine. Cupric ions act as the oxidizing agent, followed by

hydrazone formation. The hydrazone dissolves in strong sulphuric acid solution to produce a

light red colouration, whose intensity gives a measure of the concentration of ascorbic acid.

The addition of thiourea as a reducing agent adds specificity by avoiding interference from

non-ascorbate chromogens.

Reagents for Vitamin C

i) Trichloroacetic acid (TCA) (10% w/v): Trichloroacetic acid (10 g) was dissolved

in 20 ml distilled water and the volume made up to 100 ml.

ii) 2, 4-Dinitrophenylhydrazine Reagent: Concentrated sulphuric acid (25 ml) was

added to 75 ml of chilled water to give 9.0 N H2SO4. Crystalline 2, 4-

dinitrophenylhydrazine (2 g) was dissolved in 100 ml of 9.0 N H2SO4 and the

resultant solution was filtered and stored in a brown bottle in the refrigerator.

iii) Thiourea Solution: Thiourea (10 g) was dissolved in 100 ml of 50% ethanol and

the solution stored in the refrigerator.

iv) Cupric Sulphate (1.5% w/v): Cupric sulphate (1.5 g) was dissolved in 10 ml of

distilled water and made up to 100 ml.

v) Combined Colour Reagent: This was prepared fresh each day by mixing:

2, 4-dinitrophenylhydrazine reagent (5 ml)

Cupric sulphate solution (0.1 ml) and

Thiourea solution (0.1 ml).

vi) Sulphuric acid (85%): Concentrated H2SO4 (180 ml) was added to 20 ml of

distilled water. The solution was thoroughly mixed, cooled and stored in a glass-

stoppered bottle in the refrigerator.

vii) Ascorbic Acid Standard: Ascorbic acid (1 g) was dissolved and diluted to 100 ml.

This was further diluted with distilled water just before use to give a working

standard of 2 mg/100 ml (1:50 dilution ratio).

Procedure

To serum (0.1 ml) in a test tube was added 10% trichloroacetic acid (1 ml) and

chloroform (0.5 ml). The test tube was stoppered, shaken vigorously for 15 seconds and

centrifuged at 10,000 rpm for 5 minutes. The clear supernatant (1 ml) was pipetted into

another sample test tube. Five hundred microllitres (500 µl) of trichloroacetic (10%) was

145

added to 0.5 ml distilled water and to 0.5 ml of freshly prepared ascorbic acid standard to

give the blank and the working standard respectively.

Freshly prepared colour reagent (0.4 ml) was added to the blank, working standard

and test sample. The resulting solution was thoroughly mixed, stoppered and placed in a

waterbath for 1 hour at 56OC. The test tubes were cooled in an ice bath for 5 minutes.

Ice-cold 85% sulphuric acid (2 ml) was slowly added to each test tube with mixing

and allowed to stand at room temperature for 30 minutes. Absorbance of test sample and

standard was measured against blank at 500 nm

Ascorbic acid concentration was calculated thus:

2)(

)( ×SA

TAmg Ascorbic acid/100 ml

Where A (T) = Absorbance of test sample

A (S) = Absorbance of standard

2.2.15 Assay of Catalase Activity

Catalase was assayed by the method described by Aebi (1983)

Principle

The ultraviolet absorption of hydrogen peroxide can be easily measured at 240 nm.

On the decomposition of hydrogen peroxide with catalase, the absorption decreases with time

and from this decrease catalase activity can be measured.

Reagent

Phosphate buffer, pH 7

3.522 g KH2 PO4 and 7.268 g Na2H PO4.2H20 in 1000 ml water.

Hydrogen peroxide solution

0.085ml of 30% hydrogen per oxide in 25 ml phosphate buffer.

Procedure

Immediately following the addition of 1ml phosphate buffer and 2 ml diluted

haemolysate into the blank test tube (B) and 1 ml hydrogen peroxide and 2 ml haemolysate

146

into the sample test tube (T), the change of absorbance of test sample against blank at 240 nm

was recorded every 15 seconds for 1 minute on a spectrophotometer.

Table 4: Volumes of the various materials/reagents put into the sample and blank tubes

Blank Test Sample

RBC lysate (Haemolysate) (ml) 2 2

Phosphate buffer (ml) 1 ---

Hydrogen peroxide solution (ml) --- 1

Calculation

The activity of catalase is calculated by using the following formula:

Catalytic concentration (unit/l) = 00693.0

23.02

1

×

A

ALog

Where Al is absorbance at t = 0 second

A2 is absorbance at t = 15 seconds

0.23 and 0.00693 = constant

2.2.16 Superoxide Dismutase Activity

Superoxide dismutase activity was assayed using the method described by Fridovich

(1989) as contained in the commercial kit.

Principle

The role of superoxide dismutase (SOD) is to accelerate the dismutation of the toxic

superoxide radical (O2-.), produced during oxidative energy process, to hydrogen peroxide

and molecular oxygen. This method employs xanthine and xanthine oxidase (XOD) to

generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-

phenyltetrazolium chloride (I.N.T) to form a red formaxan dye.

The superoxide dismutase activity is then measured by the degree of inhibition of this

reaction. One unit of SOD is that which causes a 50% inhibition of the rate of reduction of

INT under the condition of assay.

Xanthine uric acid + O-2 Xanthine

Oxidase

147

INT O-2 Formazan dye

OR

O2: + O2

: + 2H+ O2 + H2O2

Reagent


RIa Mixed substrate

Xanthine 0.05 mmol/L

INT 0.025 mmol/L

RIb Buffer

CAPS 40 mml/L, pH10.2

EDTA 0.94 mmol/L

R2 Xanthine Oxidase 80 U/L

CAL Standard 5.30 u/ml (per vial)

Reconstitution of solution

i. Mixed substrate:

The content of one vial of mixed substrate was reconstituted with 20 ml of buffer.

ii. Xanthine oxidase: One vial of mixed oxidase was reconstituted with 10 ml of distilled

water.

iii. Standards: One vial of standard was reconstituted with 10 ml of distilled water.

Subsequent dilutions of the standard were prepared with 0.01 M phosphate buffer (pH

7.0). Serial dilutions were prepared with the standard for the preparation of standard

curve.

Procedure

Washed erythrocytes in the various centrifuge tubes set up for the assay, were made

up to 2 ml with cold distilled water, mixed and left to stand in refrigerator at 4oC for 15

minutes. A twenty-five (25) fold dilution of the rat haemolysate was done using 0.01 M

phosphate buffer, pH 7.0, so that the percentage (%) inhibition fell between 30% and 60%.

Not all samples were within this range. Fifty microlitres (50 µl) of the haemolysate (diluted

sample) and 50 µl of the standard were added separately to 1.7 ml of the mixed substrate,

mixed properly and then incubated at 37oC in a water bath. Two hundred and fifty microlitres

(250 µl) of xanthine Oxidase (XOD) was added to the solution, mixed well and transferred

into a cuvette. The initial absorbance (A1) was read after 30 seconds and the final absorbance

148

(A2) read after 3 minutes. Measurement was done against air at 505 nm. The procedure is

summarized as follows:

Sample Standard Diluted

Diluent S2-S6 Sample

Diluted sample (ml) - - 0.05

Standard (ml) - 0.05 -

Ransod sample (ml) 0.05 - -

Diluents (ml) 1.7 1.7 1.7

The tube contents were mixed well, then

Xanthine oxidase (ml) 0.25 0.25 0.25

The content of the different tubes were mixed well and the initial absorbance A1 was

read after 30 second. The final absorbance A2 was read after 3 minutes.

Calculation

3

AA 12 − = ∆A/min of standard or sample

Where

A1 = Initial absorbance

A2 = final absorbance after 3 min

All standard rates and diluted sample rates were converted into percentages of the sample

diluents rate and subtracted from 100% to give a percentage inhibition.

100min/S

sample/min

i∆Α∆Α

= % inhibition.

Where Asample = Absorbance of sample

Asi = Absorbance of sample diluent rate

A plot of percentage inhibition for each standard against log10 (standard conc. in U /ml)

was made. The percentage inhibition of sample was used to obtain units of SOD from the

standard curve. SOD U/ml of whole blood= SOD U/ml from standard curve x dilution factor.

149

SOD Standard Curve Preparation

This was calibrated using the standard supplied with the Kit. The following dilutions

were made of the standard CAL (or S6) to produce the standard curve.

Standard Undiluted solution Sample

S6 Undiluted standard diluent

S5 5 ml of S6 5 ml

S4 5 ml of S5 5 ml

S3 5 ml of S4 5 ml

S2 3ml of S3 6 ml

Absorbance was read at 505 nm

2.2.17 Assay of Glutathione Peroxidase Activity

Glutathione peroxidase activities of the rats were assayed using the method of Paglia

and Valentine (1967).

Principle

Glutahtione peroxidase (GPx) catalyses the oxidation of glutathione (GSH) by

cumene hydroperoxide in the presence of glutathione reductase (GR) and NADPH. The

oxidized glutathione (GSSG) is immediately converted to the reduced form with a

concomitant oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is

measured.

Reaction Principle

2GSH + ROOH ROH + GSSG + H2O

GSSG + NADPH + H+ NADP+ + 2GSH

Reagent

Contents Concentration in the Test

RIa Glutathione 4mmol/L

Glutathione Reductase ≥0.5µ/L

NADPH 0.34mmol/L

RIb Buffer

Phosphate buffer 0.05mol/L; pH 7.2

GP

GR

150

EDTA 4.3mmol/L

R2 Cumene Hydroperoxide 0.18mmol/L

R3 Diluting Agent

Procedure

Heparinized whole blood was used. The blood sample (0.05 ml) was diluted with 2 ml

of diluting agent and mixed thoroughly. Diluted sample (0.02 ml) was pipetted into a sample

test (T) tube only. Distilled water (0.02 ml) was pipetted into the reagent blank (B) test tube

only. One milliliter (1.0 ml) of the reagent (R1) was pipetted into the sample (T) and reagent

blank (B), both were placed in water bath at 37oC. Exactly 0.04 ml of cumene hydroperoxide

was pipetted into both the sample test and reagent blank. Immediately the initial absorbance

of sample test (T) and reagent blank (B) were read after one minute and again after 1 and 2

minutes at a wavelength of 340 nm.The blank value was subtracted from the sample value.

The procedure is represented below:

Substance added Sample (T) Blank (B)

Diluted sample (ml) 0.02 -

Distilled H2O (ml) - 0.02

Reagent R1 (ml) 1.00 1.00

Cumene hydroperoxide (ml) 0.04 0.04

These reagents were mixed immediately and absorbance read at 340 nm

Manual Calculation

Glutathione Peroxidase concentration was calculated as follows:

U/L of Haemolysate = 8412 x ∆ A340 nm/minute.

The result obtained using the RANSEL kit is in unit/litre of haemolysate and must be

multiplied by the appropriate dilution factor to obtain the result in unit/litre of whole blood,

thus

R x 41 = Unit/litre (U/L) or whole blood

where R= result and 41 = dilution factor.

2.2.18 Determination of Total Cholesterol Concentration

Principle

The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator

quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of

phenol and peroxidase.

151

Reaction Principle

Cholesterol ester +H2O Cholesterol + fatty acid.

Cholesterol + O2 Cholestene-3-one + H2O2

2 H2O2 + Phenol + 4 Aminoantipyrine Quinoneimine +4H20

Reagent

Reagents used are commercial kit obtained from Randox Lab Limited, UK.

Content Concentration.

Reagent 4-Aminoantipyrine 0.30 mmol/L

Phenol 6 mmol/L

Peroxidase > 0.5 u/ml

Cholesterol esterase > 0.15 u/ml

Cholesterol oxidase >0.1u/ml

Pipes buffer 80 mmol, PH 6.8

CAL Standard

Procedure

Three test tubes were labeled blank, standard and sample respectively. Into the blank

were added 10 ml of distilled water and 10 µl of standard to the standard labeled test tubes.

Sample serum (10 µl) was added to the appropriately labeled test tube. Reagent (1000 µl) was

added to the three sets of the tubes, mixed and incubated at 370c for 5 minutes. The

absorbance of the sample (A sample) was measured against the reagent blank within 60

minutes at 500 nm.

Calculation

Conc. of cholesterol in sample = mg/dlin standard of Conc.AStandard

ASample ×∆∆

2.2.19 Determination of High Density Lipoprotein (HDL) Cholesterol

Concentration

High density lipoprotein cholesterol (HDL) level was determined by the method of

Albers (1978) as contained in the QCA commercial kit used.

Principle

Cholesterol esterase

Peroxidase

Cholesterol oxidase

152

LDL and very low density lipoprotein (VLDL) are precipitated from the serum by the

action of a polysaccharide in the presence of divalent cations. Then high density lipoprotein –

cholesterol (HDL –Cholesterol) present in the supernatant is determined.

Cholesterol– ester +H2O Cholesterol + Fatty acid

Cholesterol +1/2 O2+H2O Cholestene-3-one + H2O2

2H2O2 + 4–Aminoantipyrine + DCFs Quinoneimine +H2O

Procedure

The procedure involved two steps.

(A) Precipitation step.

The serum sample (0.3 ml) was pipetted into labeled centrifuge tubes.

A drop of the precipitant solution or reagent (10g/c of dextran sulphate, IM of magnesium

acetate and stabilizers) was added to each of the centrifuge tubes.

The contents in the various tubes were thoroughly mixed and allowed to stand for 15

minutes at room temperature (20-250C) and then centrifuged at 2,000 rpm. The concentration

of cholesterol in the supernatant was determined

Calculation

HDL–Cholesterol concentration in the sample was calculated using the following general

formula:

5.52standardA

sampleA × = mg/dl HDL–Cholesterol

OR

36.1standardA

sampleA × mmol/dl HDL-Cholesterol

where 52.5 and 1.36 are constants.

2.2.20 Determination of Low Density Lipoprotein (LDL) Cholesterol Concentration

Chol. Oxidase

Chol.esterase

Peroxidase

153

LDL-Cholesterol can be determined as the difference between total cholesterol and

the cholesterol content of the supernatant after precipitation of the LDL fraction by polyvinyl

sulphate (PVS) in the presence of polyethylene glycol monomethyl ether.

LDL-Cholesterol = Total Cholesterol –Cholesterol in the supernatant

Reagent


Precipitation Reagent

Polyvinyl Sulphate 0.7g/L

EDTA (Na2 salt) 5.0 mM

Polyethylene glycol monomethyl ether 170.g/L

Stabilizers

Procedure

1. Precipitation Reaction

The precipitation solution (3 drops or 0.1 ml) was carefully measured into test tubes

labelled accordingly. The serum (0.2 ml) was added to the labeled test tubes. The contents

were thoroughly mixed and left to stand for 15 minutes at room temperature (20-250C).Then,

the mixture was centrifuged at 2,000 rpm for 15 minutes and the cholesterol concentration in

the supernatant was determined.

2. Cholesterol Determination

The concentration of serum total cholesterol was determined according to the

QCACHOD –PAP method.

Calculations

The LDL-Cholesterol concentration in the sample was calculated using the following

general formula.

LDL-Cholesterol (mg/dl) =Total Cholesterol (mg/dl) - 1.5 × Supernatant cholesterol

(mg/dl).

2.2.21 Determination of Triacylglycerol Concentration

Triacylglycerol concentration was determined using the method of Albers et al.,

(1978).

154

Principle

The triacylglycerol concentration was determined after enzymatic hydrolysis with

lipases. The indicator is a quinoneimine formed from hydrogen peroxide, 4-aminophenazone

and 4-chlorophenol under the catalytic influence of peroxidase .

Reaction

Triacylglycerol + H2O Glycerol +fatty acids

Glycerol +ATP Glycerol -3- phosphate + ADP.

Glycerol -3- Phosphate + O2 Dihydroxyacetone +Phosphate+H2O2

2H2O2 +4- aminophenaxone + 4 – Chorophenol Quinoneimine+HCl +H2O

Reagent


Reagent1

Buffer 40 mmol/L,pH 7.6

4-Chlorophenol 5.5 mmol/L

Magnesium ions 17.5 mmol/L

Reagent 2 Enzyme reagent

4 – aminophenazone 0.5 mmol/L

ATP 1.0 mmol/L

Lipases > 150 u/ml

Glycerol –Kinases > 0.4 u/ml.

Glycerol -3- phosphoperoxidase > 15 u/ml.

Cal. Standard 743 mg/dl

Procedure

One hundred microllitre (100 µl) of the reagent 1 was pipetted into the reagent blank

tube, standard tube and the sample tubes. In the standard test tube was added 10 µl of the

standard (CAL) while 10 µl of the sample was pipetted into the sample tube mixed

Lipases

Glycero-kinase

Glycerol peroxidase

Peroxidase

155

thoroughly and incubated for 10 minutes at 20-250c. Absorbance of the sample and the

standard were measured against the reagent blank within 60 minutes at 546 nm

Triacyglycerol concentration in

mmol/L = 29.2A

A

Standard

Sample ×∆∆

Asample = Absorbance of sample

Astandard = Absorbance of standard

2.29 = constant

2.2.22 Aspartate Aminotransferase Activity

Principle

AST is measured by monitoring the concentration of oxaloacetate hydrazone formed

with 2, 4-dinitrophenylhydrazine. The source of the enzyme was serum.

α- Oxoglutarate + L-aspartate L-Glutarate+oxoacetate

Reagents

Commercial kits were obtained from Quimica Clinica Aplicada, Spain.


Reagent 1. Phosphate buffer 100 mmol/L, pH7.4

L –Aspartete 100 mmol/L

α - Oxoglutarate 2.0 mmol/L

Reagent 2. 2, 4- dinitrophenyl hydrazine 2.0 mmol/L

Procedure

The blank and the sample test tubes were set up. Into the blank and sample were

added 0.1 ml distilled water and 0.1 ml serum respectively. A quantity (0.5 ml) of reagent 1

was added the test and blank, thoroughly mixed and incubated for exactly 20 minutes at

370C. Reagent 2 (5 ml) was then added in each tube, mixed well and incubated for exactly 20

minutes at 20-250C. Two hundred and fifty microllitre of 1.9 N sodium hydroxide was then

added, the solution was mixed and the absorbance of the sample was read against blank after

5 minutes at 550 nm.

The assay procedure is represented below:

AST in Serum

156

Substance added Blank Test

Serum (ml) - 0.1 Distilled

water (ml) 0.1 -

Reagent 1 (ml) 0.5 0.5

Then mixed well and incubated at 370C for 20 minutes

Reagent 2 (ml) 5 5

The content was mixed well and incubated for 20 minutes at 20-250C

NaOH (ml) 2.5 2.5

The absorbance was read at 550 mm after 5 minutes

Calculation AST activity was obtained from the standard table below (Table 5)

Table 5: Absorbance and corresponding AST activity in serum

Absorbance U/L Absorbance U/L

0.020 7 0.100 36

0.030 10 0.110 41

0.040 13 0.120 47

0.050 16 0.130 52

0.060 19 0.140 59

0.070 23 0.150 67

0.080 27 0.160 76

0.090 31 0.170 89

2.2.23 Assay of Alanine Aminotransferase (ALT) Activity

Principle

ALT in serum is measured by monitoring the concentration of pyruvate hydrazone

formed with 2, 4-dinitrophengl hydrazine.

α-Oxoglutarate + L- alanine L-Glutamate + pyruvate

Reagents

ALT in Serum

157

A commercial kit obtained from Quimica Clinica Aplicada (S.A, Spain) was used.

Contents Concentration

R1. Phosphate buffer 100mmol, pH 7.4

L-alannie 200 mmol/L

α – oxoglutarate 2.0 mmol1/L

R2. 2, 4. Dinitrophenyl hydrazine 2.0 mmo1/L

Procedure

The blank and sample test tubes were set up. Into the blank and sample test tubes were

added 0.1 ml distilled water and serum respectively. The reagent (0.5 ml) was added to both

blank and sample tubes, mixed thoroughly and incubated for 20 minutes at 37oC. Reagent 2

(5 ml) was added to each tube, mixed thoroughly and incubated for exactly 20 minutes at 20-

25oC. Five hundred microlitres (500 µl) 1.9 N NaOH was then added mixed and the

absorbance of the sample was read against blank after 5 minutes at wavelength of 550 nm.

The assay procedure is represented bellow.

Substance added Blank Test

1. Serum (ml) - 0.1

2. Reagent 1 (ml) 0.5 0.5

3. Distilled water (ml) 0.1 0.5

Mixed and incubated at 37oC for 20 minutes

4. Reagent 2 (ml) 5 5

Incubated for 20 minutes at 20-25oC

5. NaOH (ml) 2.5 2.5

The absorbance was read at 550 nm after 5 minutes

Calculation

ALT activity in the serum was obtained from the Table 6 below:

Table 6: Absorbance and corresponding ALT activity in serum

Absorbance U/L Absorbance U/L

0.050 4 0.275 48

0.050 8 0.300 52

0.075 12 0.325 57

0.100 17 0.350 62

0.125 21 0.375 67

158

0.150 25 0.400 72

0.175 29 0.425 77

0.200 34 0.450 83

0.225 39 0.475 88

0.250 43 0.500 94

2.2.24 Determination of Total Bilirubin Concentration

Principle

Bilirubin reacts with diazotized sulphanilic acid (DSA) to form a red azo dye. The

absorbance of this dye at 578 nm is directly proportional to the total bilirubin concentration in

the sample. Water soluble bilirubin glucuronides react directly with DAS whereas the

albumin conjugated indirect bilirubin will only react with DSA in the presence of caffeine (an

accelerator): Total bilirubin = Direct bilirubin + Indirect bilirubin.

Reagents

The reagents used in this assay were constituents of kit obtained from Randox

Laboratories Ltd. USA.

Reagents 1 Concentration

Sulphanilic acid 29 mmol/L

Hydrochloric acid 0.17N

Reagent 2

Sodium nitrite 25 mmol/L

Reagent 3

Caffeine l 0.26 mmol/L

Sodium benzoate 0.52 mmol/L

Reagent 4

Tartrate 0.93 mmo/L

Sodium hydroxide 1.9 N

Procedure

The blank and sample tubes were set up. Into the tubes, 0.2 ml of reagent 1, 1 ml of

reagent 3, and 0.02 ml of sample were added. Into the sample tube were added 0.2 ml of

reagent 1, 0.05 ml of reagent 2, 1 ml of reagent 3 and 0.02 ml of sample. The different tubes

were mixed thoroughly and allowed to stand for 10 minutes at 20-250C. Then 1 ml of reagent

159

4 was added to each of the tubes and allowed to stand for 10 minute at 20-250C. Asorbance

(ATB) was read at 578 nm against blank.

Calculation

Total bilirubin (mg/dl) = 10.8 × ATB (578 nm)

Where 10.8 = constant.

2.3 Statistical Analysis

Data were reported as means ± SEM, where appropriate. One-way analysis of

variance (ANOVA) and One-way analysis of variance with repeated measures were used to

analyze the experimental data and Duncan multiple test range was used to compare the group

means obtained after each treatment with control measurements. Differences were considered

significant when p ≤ 0.05.

CHAPTER THREE

RESULTS

3.1 Qualitative Phytochemical Composition of Ethanol, Methanol and n-Hexane

Leaf and Fruit Extracts of Kigelia africana

Table 7 shows relative trace presence of saponins and terpeniods in all the extract

samples. In the same vein, hyhrogen cyanide and steroids were found to be present in trace

160

concentrations in extracts except methanol leaf extract which exhibited moderate presence of

these bioactive compounds. Relative moderate amount of soluble carbohydrates was found in

all the extracts except ethanol leaf, n-hexane fruit and ethanol fruit extracts which exhibited

trace amount carbohydrates. Interestingly, flavoniods was found in high concentration in the

extracts.

161

Table 7: Qualitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana.

Extract Soluble

carbohydrates Tannins Alkaloids Hydrogen

cyanide Saponins Flavonoids Reducing

sugars Steroids Glycosides Tepenoids

Ethanol leaf + + + + + ++ + + ++ +

Ethanol fruit + ++ ++ + + +++ + + ++ +

Methanol fruit ++ +++ ++ + + +++ +++ + ++ +

Methanol leaf ++ +++ ++ ++ + ++ ++ ++ ++ +

n-hexane leaf ++ ++ + + + ++ + + + +

n-hexane fruit + +++ ++ + + +++ ++ + ++ +

NB + Present in trace concentration ++ Present in moderately high concentration +++ Present in very high concentration

162

3.2 Quantitative Phytochemical Composition of Ethanol, Methanol and n-Hexane

Leaf and Fruit Extracts of Kigelia africana

Table 8 shows the quantitative composition of bioactive compounds present at various

concentrations. Significant increased of flavoniod was recorded in ethanol fruit, methanol

fruit and n-hexane fruit extracts compared with the leaf extracts. Trace concentration of

hydrogen cyanide was found in the extracts. All the extracts contained moderate

concentration of alkaloids. Tannin level was high in all the extracts except the ethanol leaf

extract that was relatively low compared with other extracts as shown in Table 8.

163

Table 8: Quantitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia afrcana.

Extract Soluble

carbohydrates (mg/100g)

Tannins (mg/100g)

Alkaloids mg/100g

Hydrogen cyanide mg/100g

Saponins mg/100g

Flavonoids mg/100g

Reducing sugars (mg/100g)

Steroids (mg/100g)

Glycosides (mg/100g)

n-hexane leaf 3.01±0.13ac 6.14±0.35ab 2.75±0.12ab 0.12±0.01ac 0.52±0.01ab 2.84±0.02ab 25.67±1.03ab 0.52±0.02ab 2.72±0.21ab

Methanol leaf 1.44±0.05ab 10.31±0.42ac 3.12±0.11ab 0.29±0.04ac 0.52±0.01ab 2.84±0.01ab 26.67±1.02ab 0.62±0.01ab 2.57±0.13ab

Ethanol leaf 0.96±0.02ab 4.11±0.65ab 2.67±0.12ab 0.03±0.001ad 0.52±0.02ab 2.32±0.04ab 50.85±3.36ad 0.53±0.03ab 2.39±0.15ab

n-hexane fruit 0.95±0.01ab 10.87±0.36ac 3.22±0.11ab 0.94±0.02ab 0.56±0.02ab 3.32±0.01ab 26.08±1.02ab 0.67±0.03ab 2.69±0.12ab

Methanol fruit 1.92±0.25ab 10.87±0.22ac 3.21±0.13ab 0.91±0.01ab 0.53±0.01ab 3.36±0.02ab 20.35±1.01ac 0.59±0.02ab 2.62±0.11ab

Ethanol fruit 0.96±0.17ab 9.08±0.14ac 3.54±0.11ab 0.95±0.02ab 0.56±0.03ab 3.63±0.02ab 26.95±5.14ab 0.57±0.01ab 2.874±0.14ab

Results are expressed in mean ± SD; n = 3 Mean values with different letters as superscripts across the column are considered significant (p < 0.05) while mean values with different letters as superscripts across the column are considered significant (p > 0.05)

164

3.3 Percentage Proximate Composition of Ethanol, Methanol and n-Hexane Leaf

and Fruit Extracts of Kigelia afrcana

The percentage proximate composition of K. africana was shown on the Table 9. K. africana

leaf demonstrated high percentage protein concentration compared with the fruit. Relative

percentage of fibre content was found. Carbohydrates concentration was high in fruit as

against leaf.

165

Table 9: Percentage proximate composition of ethanol, methanol and n-hexane leaf and fruit

extracts of Kigelia afrcana.

Moisture (%)

Ash (%) Fats (%)

Fibre (%)

Protein (%)

Carbohydrate (%)

Leaf 5.5 2.7 11.4 2.2 13.9 63.5

Fruit 5.1 1.8 3.7 1.3 10.4 77.5

3.4 Percentage Yield of Leaf and Fruit Samples of Kigelia africana

166

The percentage yield of 200 g sample of Kigelia africana leaf and fruit extracted with

ethanol, methanol and n-hexane are depicted in Table 10. Ethanol fruit extract showed

highest yield compared with other samples. In the same vein, methanol leaf gave the lowest

yield.

Table 10: Percentage yield of leaf and fruit extracts of Kigelia africana

167

Extract Percentage (%)

Ethanol leaf extract 5.4

Ethanol fruit extract 10.5

Methanol leaf extract 6.0

Methanol fruit extract 7.7

n-Hexane leaf extract 2.9

n-Hexane fruit extract 3.0

168

3.5 Acute Toxicity Studies

This study showed no mortality up to a dose of 5000 mg/kg body weight. Tables 11 a and b

depict the result of the LD50.

Table 11a: First phase acute toxicity test (LD50) of Kigelia africana extract

Groups and Doses Weight (g) Number of Deaths

Group 1 (10 mg/kg b.wt) 29.8 Zero

29.0

26.6


28.4

29.6


23.8

23.7

169

Table 11b: Second phase of acute toxicity test (LD50) of K. africana extracts

Groups and Doses Weight (g) Number of Deaths




170

3.6 Effects of Ethanol, n-Hexane and Methanol Extracts of Leaves and Fruits of

Kigelia africana on glucose Level of Diabetic Rats

The glucose levels of rats before the experiment in all groups were found to be non-

significant (p>0.05) compared with the glucose level of group 2 rats (diabetic untreated) as

shown in Table 12. Rats in all the groups except groups 7, 9, 10 and 11 exhibited significant

increase in glucose level (p<0.05) compared with that of group 2 rats at 72 hours after

induction. At day 21, a significant increase (p<0.05) was also observed in the glucose level of

rats in all groups compared with the glucose level of rats in group 2 (diabetic untreated).

There was no significant (p>0.05) variation in the glucose level of rats in group 1 (normal

control) at 72 hours after induction and day 21 after treatment compared with the glucose

level before the induction. The glucose level increased significantly (p<0.05) in groups 2, 3,

4, 5, 7, 9, 10, 11 and 12 rats at 72 hours after induction and day 21 after treatment compared

with the glucose level before the induction. On the other hand, the glucose level of rats in

groups 6 and 8 had a significant increase (p<0.05) at 72 hours after induction and non-

significant increase (p>0.05) at day 21 after treatment.

171

Table 12: Effect of ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana on fasting blood glucose level of diabetic rats

Treatment Groups glucose Level (mg/dl) Before

Induction 72 Hours After

Induction After 21 Days

Treatment Group 1 (Normal Control) 76.20±5.02ab 78.80±2.71ab 75.40±4.22ab Group 2 (Diabetic Untreated) 67.40±3.50ab 558.40±14.01ac* 405.40±15.96ac* Group 3 (Standard Control) 66.40±3.91ab 321.00±115.16ab* 241.20±116.79ab* Group 4 (Diabetic + n-Hexane Leaf Extract)

65.20±2.28ab 363.20±136.38ab* 228.80±79.92ab*

Group 5 (Diabetic + n-Hexane Fruit Extract)

74.20±4.96ab 288.40±62.86ab* 193.20±38.32ab*

Group 6 (Diabetic + Ethanol Leaf Extract)

72.20±4.96ab 314.80±159.19ab* 184.40±54.50ab

Group 7 (Diabetic + Ethanol Fruit Extract)

76.40±9.07ab 464.80±159.32ac* 273.20±93.59ab*

Group 8 (Diabetic + Methanol Leaf Extract)

69.80±10.37ab 393.00±150.16ab* 163.80±68.81ab

Group 9 (Diabetic + Methanol Fruit Extract)

64.00±5.33ab 467.60±122.21ac* 185.80±53.60ab*

Group 10 (Diabetic + n-Hexane Leaf and Fruit Extract)

63.80±3.02ab 523.80±80.91ac* 183.80±74.24ab*

Group 11 (Diabetic + Ethanol Leaf and Fruit Extract)

64.60±3.20ab 479.60±142.28ac* 269.60±108.64ab*

Group 12 (Diabetic + Methanol Leaf and Fruit Extract)

66.60±12.73ab 342.40±121.43ab* 219.80±131.40ab*

Results are expressed in mean ± SD; n = 5 Mean values with different letters as superscripts across the column compared with group 2 (diabetic untreated) are considered significant (p<0.05) while mean values with asterisk (*) as superscripts across the row compared with the sugar level before the experiment are considered significant (p>0.05) 3.7 Body Weights of Diabetic Rats Treated With Ethanol, n-Hexane and Methanol

Extracts of Leaves and Fruits of Kigelia africana before and After Experiment

172

Significant (p<0.05) increases in the body weights of group 1 rats (normal control) and

diabetic rat in groups 6, 8 and 9 treated with ethanol leaf, methanol leaf and methanol fruit

extracts of K. africana respectively after experiment compared with that obtained before the

experiment. Conversely, non-significant (p>0.05) decrease was observed in the body weights

of the animals in other groups after the experiment compared with the body weights of the

animals before the experiment (Table 13).

Table 13: Body weights of diabetic rats treated with ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana before and after experiment

173

Treatment Groups Body Weight (g) 0 hr After 21days

Group 1 (Normal Control) 92.59±5.87ab 130.36±17.83ac Group 2 (Diabetic Untreated) 173.66±12.24aa 156.16±13.14aa Group 3 (Standard Control) 94.58±5.80aa 107.34±18.41aa Group 4 (Diabetic + n-Hexane Leaf Extract) 175.97±9.60aa 182.58±8.18aa Group 5 (Diabetic + n-Hexane Fruit Extract) 116.23±12.44aa 131.79±12.46aa Group 6 (Diabetic + Ethanol Leaf Extract) 103.94±8.30ab 125.98±14.59ac Group 7 (Diabetic + Ethanol Fruit Extract) 119.83±40.91aa 127.94±37.44aa Group 8 (Diabetic + Methanol Leaf Extract) 81.40±4.45ab 112.44±13.83ac Group 9 (Diabetic + Methanol Fruit Extract) 75.38±6.05ab 101.38±17.57ac Group 10 (Diabetic + n-Hexane Leaf and Fruit Extract)

87.86±11.53aa 101.01±14.93aa

Group 11 (Diabetic + Ethanol Leaf and Fruit Extract)

86.71±9.77aa 95.12±4.09aa

Group 12 (Diabetic + Methanol Leaf and Fruit Extract)

70.29±4.42ab 90.06±18.75aa

Results are expressed in mean ± SD; n = 5 Mean values with different letters as superscripts across the row are considered significant (p<0.05)

3.8 Effects of Ethanol, Methanol and n-Hexane Leaf and Fruit Extracts of Kigelia

africana on Sorbitol Concentration in Alloxan-Induced Diabetic Rats

174

The sorbitol concentration in all the test groups decreased significantly (p<0.05) compared

with the untreated diabetic animals (Group 2). A significant (p<0.05) reduction of sorbitol

concentration was recorded in groups 10, 11 and 12 treated with a combination of the leaf

and fruit extracts of K. africana compared with the diabetic rats not treated. There was non

significant increase (p>0.05) of sorbitol concentration in all the test rats compared with the

normal control rats (group 1) as depicted in Fig. 11. Similarly, non-significant increase

(p>0.05) of sorbitol concentration was recorded in group 10 (diabetic + n-hexane leaf and

fruit extract) in comparison with group 3 treated with the reference drug, glibenclamide.

175

Fi.g. 22: Effect of ethanol, n-hexane and methanol extracts of leaf and fruit of Kigelia africana on

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Group 11 Group 12

Treatment Group

Mea

n S

orb

ito

l Co

nc

(mg

/ml)

Fig. 11: Effect of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on sorbitol concentration in alloxan-induced diabetic rats

Group 1: Normal Control (normal saline); Group2: diabetic rats (no treatmen) Group 3: Diabetic rats treated with glibenclamide; Group 4: Diabetic + n-hexane leaf extract Group 5: Diabetic + n-hexane fruit extract; Group 6: Diabetic + ethanol leaf extract Group 7: Diabetic + Ethanol fruit extract; Group 8: Diabetic + methanol leaf extract Group 9: Diabetic + methanol fruit extract; Group 10: Diabetic + n-hexane leaf and fruit extracts Group 11: Diabetic + ethanol leaf and fruit extracts; Group 12: Diabetic + methanol leaf and fruit extracts.

176

3.9 Effect of Ethanol, Methanol and n-Hexane Leaf and Fruit Extract of

Kigeria africana on Total Protein Concentration in Alloxan-Induced

Diabetic Rats

Fig. 12 reveals significant increased (p>0.05) of total protein was recorded in all test

groups compared with the positive control rats (group 2). Total protein concentrations

in groups 10, 11 and 12 orally fed with a combination of the leaf and fruit extract

showed significant increase (p<0.05) compared with other test groups except group 6

administered ethanol leaf plant extract. A non-significant increase (p>0.05) of total

protein was noted across all test groups (groups 4-12) compared with the total protein

concentration of group 3 rats fed with the standard drug.

177


0

1

2

3

4

5

6

7


Treatment Group

Mea

n T

ota

l Pro

tein

Co

nc

(g/d

l)

Group 1: Normal Control (normal saline); Group 2: diabetic rats (no treatment) Group 3: Diabetic rats treated with glibenclamide; Group 4: Diabetic + n-hexane leaf extract Group 5: Diabetic + n-hexane fruit extract; Group 6: Diabetic + ethanol leaf extract Group 7: Diabetic + Ethanol fruit extract; Group 8: Diabetic + methanol leaf extract Group 9: Diabetic + methanol fruit extract; Group 10: Diabetic + n-hexane leaf and fruit extracts Group 11: Diabetic + ethanol leaf and fruit extracts; Group 12: Diabetic + methanol leaf and fruit extracts.

Fig. 12: Effect of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on total protein concentration in alloxan-induced diabetic rats

178

3.10 Effect of Ethanol, Methanol and n-Hexane Leaf and Fruit Extract of

Kigelia africana on Glycosylated Haemoglobin Concentration in Alloxin-

Induced Diabetic Rats

The mean HbA1c level decreased significantly (p<0.05) in all the test groups

compared with the HbA1c level of untreated diabetics rats (group 2). No change in

HbA1c level was observed in group 10 rats treated with a combination of n-hexane

leaf and fruit extract in ratio of 1:1 compared with group 3 rats treated with the

standard drug. A significant increase (p<0.05) HbA1c level was recorded in all the

test groups against the normal control rats (negative control). Group 10 (diabetic+ n-

hexane leaf and fruit extract, ratio 1:1) demonstrated a non-significant (p>0.05)

reduction of HA1c concentration compared with groups 4 -9 rats orally fed with

single plant extract (Fig. 13).

179

0

2

4

6

8

10

12


Treatment Group

Mea

n H

bA

ic L

evel

(%

)


Fig. 13: Effect of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on glycosylated haemoglobin concentration in alloxan-induced diabetic rats

Treatment

180

3.11 Effects of Ethanol, Methanol and n-Hexane Extracts of Leaf and Fruit of

Kigelia africana on Malondialdehyde (MDA) Concentration in Alloxan-


Lipid peroxidation measured as malondialdehyde (MDA) observed significantly

increase (p<0.5) in all the test groups compared with untreated control as shown in

Fig. 14. A significant decrease of MDA concentration was recorded in groups 10, 11,

and 12 treated with the combination of the plant extract compared with the groups

administered with single extract (groups 4-9). Similarly, a significant decrease

(p<0.05) of MDA concentration was observed in the diabetic rats treated with n-

hexane leaf and fruit extract (group 10) compared with MDA concentration of other

test groups .The concentration of MDA in diabetic rats treated with n-hexane and

fruit extracts (group 10 ) significantly reduced (p<0.05) as against group 11 and 12.

181

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1


Treatment Group

Fig. 14: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on malondialdehyde concentration in alloxan-induced diabetic rats

Group 1: Normal Control (normal saline); Group 2: diabetic rats (no treatment) Group 3: Diabetic rats treated with glibenclamide; Group 4: Diabetic + n-hexane leaf extract Group 5: Diabetic + n-hexane fruit extract; Group 6: Diabetic + ethanol leaf extract Group 7: Diabetic + Ethanol fruit extract; Group 8: Diabetic + methanol leaf extract Group 9: Diabetic + methanol fruit extract; Group 10: Diabetic + n-hexane leaf and fruit extracts Group 11: Diabetic+ ethanol leaf and fruit extracts; Group 12: Diabetic + methanol leaf and fruit extracts.

Mea

n M

DA

Con

c (m

mol

/L)

182

3.12 Effects of Ethanol, Methanol and n-Hexane Leaf and Fruit Extracts of

Kigelia africana on Vitamin C Concentration in Alloxan-Induced Diabetic

Rats

There was a general decrease in vitamin C concentration in all the test groups and the

untreated diabetic group compared with the vitamin concentration of normal control

rats (group 1). There was statistically significant increase (p<0.05) of vitamin C level

in group 10 rats treated with a combination of n-hexane leaf and fruit extracts

compared with other test groups. There was no change in vitamin C concentration in

group 8 (diabetic + methanol leaf extract) compared with the group 9

(diabetic+methanol fruit extract). The diabetic rats administered 2.5 mg/kg body

weight of glibenclamide demonstrated an increased (p<0.05) vitamin C level

compared with the vitamin C concentration of rats in group 2 (diabetic untreated

rates), see Fig. 15.

183

0

0.5

1

1.5

2

2.5


Treatment Group

Mea

n V

it C

Co

nc

(mg

/ml)

Fig. 15: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on vitamin C concentration in alloxan-induced diabetic rats

Treatment


184


Kigelia africana on Catalase Activity of Alloxan-Induced Diabetic Rats

Across the test groups was recorded a statistically significant increase (p<0.05) of

serum catalase activity (Fig. 16) compared with the untreated diabetic rats (positive

control; group 2). Similarly, a significant increase (p<0.05) of catalase activity was

observed in the diabetic rats treated with reference drug (glibenclamide) in

comparison with the catalase activity of all the test groups. In the same pattern, groups

10 and 12 treated with n-hexane leaf/fruit extract and methanol leaf + fruit extract

respectively demonstrated a non-significant increase (p>0.05) of catalase activity

compared with other test groups (groups 4 to 9) administered with a single plant

extract.

185

0

1

2

3

4

5

6

7


Treatment Group

Mea

n C

atal

ase

Act

ivit

y (I

U/L

)

Fig. 16: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on catalase activity in alloxan-induced diabetic rats

Treatment


186

3.14 Effects of Ethanol, Methanol and n-Hexane Leaf and Fruit Extracts of K.

africana on erythrocyte SOD Activity in Alloxan-Induced Diabetic Rats

The activities of superoxide dismutase (SOD) reduced significantly (p<0.05) in all the

test groups compared with the normal control (group 1).There were statistically

significant (p<0.05) decreases in SOD activities of all test groups compared with the

untreated diabetic rats (group 2) as shown in Fig. 17. Superoxide dismutase activities

of the test groups (groups 10, 11 and 12) administered with the combination of the

extracts were significantly increased (p<0.05) compared with other test groups treated

with the single extracts (groups 4-9). The same observation was noted in the test

treated with the standard drug. However, SOD activities of the test rat in group 6

(diabetic treated with ethanol leaf extract) increased compared with the SOD activities

in groups 4, 7, and 8 treated with single extracts of leaf fruits of K. africana .In the

same vein, the activities of SOD in the diabetic rat administered with 2.5 mg /kg body

weight of glibenclamide increased significantly (p<0.05) as against all the test groups.

187

0

0.5

1

1.5

2

2.5

3


Treatment Group

Mea

n S

OD

Act

ivit

y (I

U/L

)

Fig. 17: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of K. africana on erythrocyte SOD activity in alloxan-induced diabetic rats


188

3.15 Effects of Ethanol Methanol and n-Hexane Leaf and Fruit Extracts of

Kigelia africana on Percentage Inhibition of erythrocyte SOD Activity in

Alloxan-Induced Diabetic Rats

Fig. 18 demonstrates statistically significant decrease (p<0.05) of percentage

inhibition of SOD activity in the test groups compared with the normal control group.

A significant reduction (p<0.05) of percentage inhibition of SOD activity occurred in

the diabetic untreated rats (group 2) compared with the percentage inhibition of SOD

activity in normal control. Diabetic rats in groups 10, 11 and 12 treated with a

combination of leaf and fruit extracts recorded a non significantly (p>0.05) increase

of percentage inhibition of SOD activity compared with groups 4, 5, 6, 7, 8 and 9

administered monotherapically with leaf and fruit extracts of K. africana.

Conversely, group 6 treated with ethanol leaf extracts showed significant increase

(p<0.05) of percentage inhibition of SOD activity as against groups 4, 7 and 8 of the

same treatment pattern. Furthermore, non-significant reduction (p>0.05) of percentage

inhibition was observed in diabetic administered the n-hexane leaf and fruit extracts

compared with the diabetic rats treated with 2.5 mg/kg body weight of glibenclamide

(group 3).

189

0

10

20

30

40

50

60

70

80


Treatment Group

Mea

n S

OD

Inh

ibit

ion

(%

)

Fig. 18: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on % inhibition of SOD in alloxan-induced diabetic rats


190

3.16 Effects of Ethanol, Methanol and n-Hexane Leaf and Fruit Extract of

Kigelia africana on Glutathione Peroxidase Activity in Alloxan-Induced

Diabetic Rats

Fig. 19 shows activity of glutathione peroxidase (GPx) which increased significantly

(p<0.05) in all the test groups treated with both single and combination of the leaf and

fruit of K. africana extract in comparison with the GPx activity of the rats in group 1

(normal control rats). The combination therapy in groups 10, 11 and 12 demonstrated

significant increase (p<0.05) of GPx activity compared with groups 4-9 of the test

groups treated with a single plant extract (monotherapy).The test groups 10, 11 and 12

of diabetic rats treated with combined leaf and fruit extracts increased in GPx activity

significantly relative to group 3 treated with the standard drug.

191

Fi.g. 19: Effect of ethanol, n-hexane and methanol extracts of leaf and fruit of Kigelia africana on glutathione peroxidase activities in alloxan-induced diabetic rats

0

200

400

600

800

1000

1200

1400

1600


Treatment Group

Mea

n G

Px

Act

ivit

y (I

U/L

)


192


Kigelia africana on Total Cholesterol Concentration in Alloxan-Induced

Diabetic Rats

Fig. 20 shows relative increase in total serum cholesterol concentration in diabetic

rats treated in groups 5-12 compared with the total cholesterol concentration of

normal control in group 1, however such increase was found to be non significant

(p>0.05). A significant (p<0.05) decrease was noted in the diabetic rats administered

with the standard drug compared with the untreated diabetic rats (group 2). Similar

trend of result was observed in total cholesterol concentration of groups 10, 11 and 12

treated with a combination of the extracts compared with the total cholesterol

concentration in diabetic untreated rats. However, a non-significant decrease (p>0.05)

was observed in the total cholesterol concentration of the diabetic rats (group10)

administered with a combination of n-hexane leaf and fruit extracts compared with

standard drug. Furthermore, significant decreases (p<0.05) of total cholesterol

concentration in group 10 and 12 were observed in comparison with diabetic

untreated rats.

193

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Treatment Group

Fig. 20: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on total cholesterol concentration in alloxan-induced diabetic rats


Mea

n T

otal

Cho

l Con

c (m

g/dL

)

194


Kigelia africana on High Density Lipoprotein Concentration in Alloxan-


Significant reduction (p<0.05) of serum HDL cholesterol was noted in groups 4, 5, 6

7, 8 and 12 treated with different solvent extracts of leaf and fruit of K. africana as

shown in Fig. 21 compared with the HDL concentration normal control rats in group

1. Conversely, the HDL concentration of rats in test groups 10 and 11 diabetic rats

administered with a combination of n-hexane leaf/fruit and methanol leaf/fruit

extracts respectively increased, though not significant (p>0.05) compared with the

HDL concentration of rats in group 3 treated with the standard drug (2.5 mg/kg body

weight). Similarly, a significant decrease (p<0.05) of HDL concentration was

obtained in groups 10, 11 and 12 treated with combination of the two parts of the

plant compared with the diabetic untreated rats (Fig. 21).

195

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2


Treatment Group

Fig. 21: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on high density lipoprotein concentration in alloxan-induced diabetic rats

Treatment


Mea

n H

DL

Con

c (m

g/dL

)

196


Kigelia africana on serum Low Density Lipoprotein Concentration in

Alloxan-Induced Diabetic Rats

Fig. 22 shows significant increase (p<0.05) in the concentration of low density

lipoprotein (LDL) in test groups 2, 4, 6 and 7 compared with the concentration of low

density lipoprotein of the control rats (group 1). Non-significant (p>0.05) variation of

low density lipoprotein (LDL) concentration across the test groups 4 through 9

(diabetic +single plant extract) compared the LDL concentration of rats in groups 10

and 12 treated with a combination of K. africana leaf and fruit extracts. Invariably,

significant (p<0.05) decrease of LDL concentration was observed in all, except group

4 as against diabetic rats untreated (Group 2).

197

0

0.5

1

1.5

2

2.5


Treatment Group

Fig. 22: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on low density lipoprotein concentration in alloxan-induced diabetic rats


Mea

n L

DL

Con

c (m

g/dL

)

198


Kigelia africana on Triacylglycerol (TAG) Concentration in Alloxan-


Serum triacylglycerol (TAG) concentration decreased significantly (p<0.05) in all the

test groups (group 4 to group 12) orally fed K. africana extracts compared with the

TAG concentration of rats (group 2). Furthermore, a significant increase (P<0.05) of

TAG concentration in group 4 to group 9 was observed in comparison with normal

control rats (group 1). However, a non significant (p>0.05) decrease of TAG

concentration in group 10, 11 and 12 administered with a combination of leaf and

fruit extracts of K. africana was noticed compared with groups 4 - 9 treated with

single extract of the plant. Also as shown in Fig. 23, group 3 (standard treatment)

demonstrated observable significant (p>0.05) changes in comparison with group 10,

11 and 12 treated with n-hexane leaf/fruit, ethanol leaf/fruit and methanol leaf/fruit

extracts in that order.

199

0

0.2

0.4

0.6

0.8

1

1.2

1.4


Treatment Group

Mea

n T

AG

Co

nc

(mg

/dl)

Fig. 23: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on triacylglycerol concentration in alloxan-induced diabetic rats

Treatment


Mea

n T

AG

Con

c (m

g/dL

)

200


Kigelia africana on Aspartate Aminotranferase (AST) Activity in Alloxan-


A significant (p<0.05) reduction of plasma AST activity was recorded in all the test

groups administered with Kigelia leaf and fruit extracts compared with diabetic rats

not treated (group) (Fig. 24). Groups 10,11 and 12 fed orally with n-hexane leaf +

fruits, ethanol leaf + fruit and methanol leaf + fruit K africana extracts respectively,

demonstrated non significant decreased (p > 0.05) of AST activity compared with

AST activity of rats in groups 4 – 9 rats treated with 500 mg/kg body weight of the

leaf or fruit extract alone. There was significant decrease (p < 0.05) of AST activity in

group 10 (diabetic + n-hexane leaf and fruit) compared with the AST activity of rats

in group 3 treated with 2.5mg/kg body weight of glibenclamide.

201

0

10

20

30

40

50

60

70


Treatment Group

Mea

n A

ST

Act

ivit

y (I

U/L

)

Fig. 24: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on aspartate aminotransferase activities in alloxan-induced diabetic rats

Treatment


202


Kigelia africana on Alanine Aminotransferase (ALT) Activity in Alloxan-

Induced Diabetic Rats.

A significant decrease (p<0.05) of plasma ALT activity was recorded across all the

test groups in comparison with the diabetic rats not treated (group 2) (Fig. 25).

Groups 10, 11 and 12 rats that received combination of n-hexane leaf + fruit, ethanol

leaf + fruit and methanol leaf + fruit extract respectively showed a significant

reduction (p<0.05) of ALT activity compared with the ALT activity of rats in other

test groups (4-9) that received a single extract of leaf or fruit of the plant. There was

reduction of ALT activity, though not significant (p>0.05) in groups treated with a

combination of the plant extracts compared with the diabetic rats administered

glibenclamide (reference drug).

203


0

5

10

15

20

25

30

35

40


Treatment Group

Mea

n A

LT

Act

ivit

y (I

U/L

)

Fig. 25: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on alanine aminotransferase activities in alloxan-induced diabetic rats


204


Kigelia africana on Total Serum Bilirubin Concentration in Alloxan-


There was statistically significantlly decrease (p<0.05) of total bilirubin concentration

in all the treated groups when compared with the total bilirubin concentration of

untreated diabetes rats (group 2) (Fig. 26). In the same view non significant decrease

(p>0.05) of the total bilirubin concentration was noted in group 10 (diabetic + hexane

leaf and fruit extract) in comparison with group 3 animals treated with the standard

drugs. There was also a significant decrease (p<0.05) of total bilirubin concentration

in test groups 5-9 compared with the diabetic rats treated with standard drug.

205

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Treatment Group Treatment Fig. 26: Effects of ethanol, methanol and n-hexane extracts of leaf and fruit of Kigelia africana on total bilirubin concentration in alloxan-induced diabetic rats


Mea

n T

otal

Bili

rubi

n C

onc

(mg/

dL)

206

206

CHAPTER FOUR

DISCUSSION

In animals, diabetes induced experimentally has provided important approach on the physiologic and biochemical derangement of the diabetic state.

Many of the derangements have been characterized in hyperglycemic animals. Significant changes in lipid metabolism and structure also occur in

diabetes.

This study evaluated the antidiabetic and antioxidative properties of Kigelia africana in alloxan-induced diabetic rats. From the results obtained diabetic

rats had much higher blood glucose level than that of the normal control. Changes in blood glucose levels reflect abnormalities in ß- cells structure and

function. Alloxan causes glucose oxidation and reduction in insulin release by the destruction of ß- cells of the islets of langerhans (Siyem et al., 2002).

In this study, rats with blood sugar level of 200 mg/dl and greater were considered diabetic. Administration of K. africana leaf and fruit extracts restored

glucose level in alloxan- induced diabetic rats near the normal level. Glibenclamide was used as a standard drug to compare the activity of K. africana

extract in reference to blood glucose reduction. The results revealed that the extracts at a dose of 500 mg/kg body weight showed significant effect at 21st

day indicating that the extracts possess hypoglycemic activities. The comparable effect of the extract (500 mg/kg) with glibenclamide (2.5 mg/kg) may

suggest similar modes of action, since the main mechanism of the action of glibenclamide is the stimulation of insulin release and the inhibition of

glucagon secretion. It has been described that glibenclamide is effective in moderate diabetic state and ineffective in severe diabetic animals where

pancreatic ß- cells are totally destroyed (Suba et al., 2004). The hypoglycemic effect of medicinal plant extracts generally depends upon the degree of ß-

cell destruction (Grover et al., 2000). The possible mechanism by which the plant extract brings about its hypoglycemic action may be by potentiating

the insulin effect by increasing pancreatic secretion of insulin from ß- cells (Stanely et al., 2000). The findings also suggest that K. africana leaf and fruit

many generate ß- cells and have protective effect on ß- cells from glucose toxicity. This report was buttressed by the results of liver marker enzymes that

were assayed in this study. As other studies showed, plant extracts might bring about their hypoglycemic effect through insulin secretion from the

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remaining ß-cell and insulin sensitivity (Ko et al., 2005; Leng et al., 2004). Some plants have also been observed to exert hypoglycemic activity through

insulin stimulatory effect (Ravi et al., 2004). In general, there is little biological knowledge on the specific modes of action of plants in the treatment of

diabetics, but most of the plants have been found to contain substances like glycosides, alkaloids, terpernoids, and flavonoids that are frequently

implicated as having antidiabetic effects (Low and Kazkin, 2002). This was also buttressed by the results of the phytochemistry of K. africana which

revealed high percentage of flavonoids, glycosides, alkaloids, terperniods that are frequently implicated as having antidiabetic effects effects (Low and

Kazkin, 2002). These plant constituents can lower blood glucose level.

The alloxan-induced diabetic rats had a marked loss of body weight. This would be expected as one of the effects of diabetes is body weight loss. Free

radicals generated under hyperglycemic condition could attack major biomolecules such as proteins, DNA and lipids and which could lead to the weight

loss recorded in this work. Increased synthesis of ketone bodies coupled with increased lipolysis seen in diabetes leads to a severe body weight loss.

However, the diabetic rats orally fed with Kigelia plant extracts had a remarkable gain in body weight compared with untreated diabetic rats. Significant

increase (p<0.05) in body weight was recorded in the group administered a combination of the plant extracts in comparison with the group treated with a

single extract. In addition, the observed decrease in body weight of diabetic animals agrees with result of Torres et al., 1999, who noticed a marked

reduction in the body weight of animal with significant increase in serum triacylglycerol in STZ- induced diabetic rats.

Sorbitol concentration significantly decreased (p<0.05) across all the test animals in reference to diabetic untreated rats. This reduction is probably due to

the antioxidant contents of the plant extracts. Sorbitol is a product of polyol pathway and is a feature of diabetic complications. It could be suggested that

some of the active constituents of K. africana extracts inhibit the activity of aldose reductase, the major enzyme in the polyol pathway. An increased flux

of glucose via the polyol pathway leads to intracellular accumulation of sorbitol. Accumulation of this non-permeable sugar in cells especially the lenses

and nerves results in osmotic stress, cellular edema, redox imbalance, depletion of water soluble antioxidant and susceptibility to oxidative insult

(Cameron et al., 1996). This is implicated in the pathogenesis of long term complication in diabetes mellitus.

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This study further revealed significant reduction (P<0.05) of sorbitol concentration in groups 10-12 rats treated with the combination of leaf and fruit

extracts relative to animals treated with the reference drug (2.5 mg of glibenclamde). This is in line with the fact that synthetic drugs do not restore

normal glucose homeostasis and are not free from side effect (Bandawane et al., 2011).

Throughout the circulatory life of the red cells, glycohaemoglobin is formed continuously by the addition of glucose to the N- terminal of the hemoglobin

beta chain. This process which is non-enzymatic reflects the average exposure of hemoglobin to glucose over an extended period. In a classical study,

Trivell et al., showed glycohemoglobin in diabetic subjects to be elevated 2-3 folds over the levels found in normal individuals. These support the results

of this study. A significant (p<0.05) increase in glycosylated hemoglobin level in the diabetic rats untreated with reference to the normal control animals

(group 1) was observed in this study. The increase is in accord with the report of several other researchers (Testamarian and Cohen, 1992; Langerstroer

and Pieper et al., 1993; Ting et al., 1996). The increased glycosylation may be as a result of diabetic complications caused by oxidative stress. Generally,

decreased glycoheamoglobin level was observed in diabetic rats treated with K. africana extracts as against diabetic rats not treated. Decrease in

glycoheamoglobin level could be attributed to the extracts’ ability to reduce glucose level in the blood stream with corresponding decrease in glycated

heamoglobin level.

A significantly increase (p<0.05) in serum total protein was recorded in all the test groups treated with the plant extracts in comparison with the diabetic

untreated rats. Decrease in serum total protein was observed in untreated diabetic rats with reference to test groups both single and combination of the

plant extracts. This is in tandem with the proximate composition of the plant that revealed approximate 13% protein. Invariably, the effect of free

radicals on plasma protein has been discussed as diabetic rats undergo oxidative stress.

High concentration of MDA in diabetic untreated established oxidative stress status in the animals. In hyperglyceamic condition, glucose is one of the

major sources of free radicals. Lipid peroxidation measured as malondiadehyde (MDA) significantly (p<0.05) decreased in all the test groups compared

with diabetic rats untreated (group 2). Groups 10, 11 and 12 treated with a combination of leaf and fruit extracts showed significant (p<0.05) decrease in

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MDA concentration as against groups 4-9 treated with single extract. Reduction in the lipid peroxidation index in treatment groups indicates the ability of

the extracts to stem down the oxidative stress by moping up free radical that lead to lipid breakdown. The bioactive constituents of the extracts such as

flavoniods, alkaloids as revealed by phytochemistry results could be implicated in free radical scavenging properties of the extracts.

This study revealed marked increase in serum levels of triacylglycerol, total cholesterol, and low density lipoprotein cholesterol (LDL- Cholesterol) in

alloxan–induced diabetic rats. Diabetes is associated with altered lipid levels. The most commonly observed lipid abnormalities in diabetes are

hypertrigyceridemia and hypercholesterolemia (Shephened, 2005; Shirwaikar et al., 2006) and these contribute to coronary artery disease (Arvind et al.,

2002). This lends credence to the significant (P<0.05) increase of total cholesterol, triacylglycerol and low density lipoprotein in the diabetic rats used in

this study. K. africana treated rats, showed a reduction in these lipids which buttressed the hypolipidemic effect of the plant. The hypolipidemic effect

may be due to inhibition of fatty acid synthesis (Kumar et al., 2011). It could also be attributed to the increase in the reverse cholesterol transport

pathway and decreased cholesterol concentration from the intestine due to α- glucosidase inhibition. In normal metabolism insulin activates the enzymes

lipoprotein lipase and hydrolyses triacylglycerol. A deficiency in insulin results in inactivation of these enzymes thereby causing hypertriglyceridemia

(Shirwaikar et al., 2004; Maruithupandian et al., 2011). Administration of a combination of leaf and fruit extracts of K. africana (groups 10-12) resulted

in a significant (p<0.05) improvement in lipid parameters when compared with the diabetic control animals (group-2). It can be further stated that K.

africana plant extracts have the potential to correct the lipid abnormalities, thus delaying lipid peroxidtion in diabetic condition.

The reports on the status of antioxidants and antioxidant enzymes in diabetic state are very contradictory; both increase and decrease of antioxidant

activity have been reported (Matkovice et al., 1982; Kaji et al., 1985). The report on the SOD activity in diabetic state is controversial with some authors

reporting no change in SOD activity (Kesavulu et al., 2000; Sekero et al., 2000) while others reported increased (Sundaram et al., 1996; Maritin et al.,

2003) and decreased SOD activity (Sundaram et al., 1996). In the present study, significant (p<0.05) decreases in the activities of SOD, CAT and GPx

were recorded in diabetic rats not treated compared with the normal control group. An observed significant (p<0.05) increases of these antioxidant

enzymes were recorded in groups 10-12 treated with a combination of two parts of K.africana extracts as against groups 4-9 with monotherpeutic

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administration of leaf and fruit extracts of the same. Reduction of the antioxidant enzymes was observed in diabetic rats not treated with reference to test

rats treated with the standard drug. This is in line with the report that products of membrane lipid peroxidation and other oxidants like H2O2 may react

with superoxide dismutase resulting in oxidative modification thereby causing loss of enzyme activity in diabetic condition (Sundaram et al., 1996). The

result, also concords with the reports that the relatively low expression of antioxidant enzymes such as catalase and superoxide dismutase, pancreatic ß-

cells may be vulnerable to reactive oxygen species (ROS) attack when the system is under oxidative stress situation (Lenzen et al., 1996; Tiedge et al.,

1997). Similarly, elevated levels of free radicals, due to insufficiency of the antioxidant defense system, may lead to disruption of cellular functions,

oxidative damages to protein, DNA, membranes and enhance their susceptibility to lipid peroxidation (Baynes, 1999) under uncontrolled diabetic

condition. Also hyperglycemia leads to glycation and inactivation of superoxide dismutase thus attributing to its decrease. In the study, the animals

treated with K. africana extracts showed increase in the activity of antioxidant enzymes as against untreated diabetic rats (group 2) and this unveiled the

extracts’ potential in mopping up or scavenging free radicals generated under oxidative stress mediated diabetes. The bioactive compound, favonoid may

be implicated in the scavenging activity of the plant extracts in oxidative condition.

The fact that normal cells are protected against peroxidative damage in vivo can be attributed to efficient antioxidant mechanisms. This antioxidant

protection is in part a function of the integrity of each cellular constituent, and in part a reflection of antioxidant system within the cell. In disease

conditions, where oxidative stress plays causative and/or exacerbating roles, this mechanism is impaired. Antioxidant vitamins, such as vitamin C and E

may be then low in such system. From the above premise, the low plasma vitamin C concentration obtained in group 2 of untreated diabetic rats

compared with the control group 1 and all the test rats (groups 4-12) is a manifestation of oxidative stress. Oxidative stress inactivates antioxidants

(Fantone and Ward, 1982), thus depletion of antioxidant vitamin C is expected. The fact that vitamin C protects against oxidative stress is now generally

accepted (Delatre and Bonnefont-Rousselot, 1998; Fakoya et al., 1998).Thus; decrease in vitamin C concentration in the present study of untreated

diabetic rats is probably a consequence of its protective roles.

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Herperglyceamia induces liver damage through free radicals generated from glucose autoxidation, advanced glycation end products formation (AGEs)

and oxidation of lipid biomelecules. In this study, damage of the hepatocytes was established by increased activity of liver marker as observed in diabetic

rats not treated. The increase in serum activity of ALT, AST and the concentration of total bilirubin measured indicated severe damage to liver

hepatocytes of untreated diabetic rats as against test groups treated with K. africana extracts. Increased serum ALT and AST activity could be associated

with hepatocellular damage and was in line with the report of Lieberman et al., 1991. The statistical reduction (p<0.05) of the liver marker enzymes

observed in groups 10-12 demonstrated the synergistic effect of the combination of the plant extracts in comparison with monothererpy. Significant

decrease (p<0.05) of serum activity of ALT and AST was recorded in group 3 treated with standard drug in reference to the positive control group 2

(diabetic rats untreated). The reduction of the activity of these liver markers enzymes, ALT and AST could be attributed to the ability of the extracts to

repair and regenerate the damaged hepatocytes. The possible mechanism by which this could be achieved is by mopping up or scavenging free radical

generated under stress condition. Flavonoids and other bioactive constituents of K. africana as revealed by photochemical results could be implicated in

free radical scavenging activity of the extracts.

Bilirubin accumulates because its transfer from liver cells to the bile is inhibited. Conjugation of bilirubin in the liver in diabetic condition is impaired

probably because of liver damage. The significant (p<0.05) increase of serum bilirubin concentration was observed in diabetic untreated as against

normal control rats (Group 1). The increase could be due to inability of the liver to metabolize bilirubin formed during the break down of heamoglobin in

red cells of diabetic animals. Free radicals were implicated in these conditions because breakdown of heamoglobin can be facilitated by the oxidant

species. Hence increased bilirubin concentration correlated with extent of liver damage in hyperglyceamic condition. The diabetic groups 10-12 treated

with a combination of leaf and fruit extracts showed significant (p<0.05) decrease in bilirubin concentration compared with groups 4-9 fed with a single

extract. Generally, test groups demonstrated decreased in bilirubin concentration as against untreated diabetic rats. The reduction in the bilirubin

concentration confirmed the potency of the extracts to repair the damaged hepatocytes of the diabetic rats. K. africana extracts reduced the concentrations

of the liver markers ALT, AST and bilirubin in the test groups with reference to the untreated diabetic rats. Thus, supporting that K. africana is relatively

free from side effect as revealed by the median lethal dose (LD50)

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4.2 Conclusion

From the results, it can be concluded that K. africana extracts possess anti-hyperglyceamic effect via α-glucosidase inhibition with corresponding

increase in body weight of diabetic rats treated with the extracts. Significant reduction of glycoheamogloin level and sorbitol concentration was obtained

in all the diabetic treated groups in reference to positive control, supporting glucose reduction ability of the extracts. The extracts were found to have

lipid- lowering effects through reduction of total serum cholesterol, triacyglycerol and low density lipoprotein. High density lipoprotein increased thus

increasing reverse cholesterol transport pathway. K. africana extracts exhibit antioxidant properties by reducing malondiadehyde concentration; hence

retarding lipid peroxidation. Antioxidative potential of the extracts was ascertained through increase in activities of antioxidant enzymes; CAT, SOD,

GPx and antioxidant vitamin (Vitamin C) in test animals as against positive group. The increase in the antioxidant activities was due to the ability of the

extracts to mop-up free radical generated under stress conditions. Total protein was improved in all the treatment groups. The results revealed reduction

in liver marker; ALT and AST and reduction in total bilirubin concentration and therefore confirmed the safety profile of the extracts. Furthermore,

combination of leaf and fruit extracts provided better results compared with the groups treated with single plant extract. In general, the possible

mechanisms by which K. africana brings about antidiabtic activities include: glycosidase (glucosidase) inhibitor mechanism, α-amylase inhibitor

mechanism, inhibition of hepatic glucose metabolizing enzyme mechanism, antioxidant mechanism, inhibition of glycosylation of haemoglobin

mechanism and modulation of glucose absorption from the gut. Therefore, management and prevention of diabetes complications can be achieved by use

of K. africana extracts.

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APPENDICES

Appendix I

Descriptives

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

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Total_Cholesterol Group 1=Normal Control 5 2.7220 .26042 .11646 2.3986 3.0454 2.52 3.17

Group 2=Diabetic Untreated 5 4.0020 .47568 .21273 3.4114 4.5926 3.65 4.68

Group 3=Standard Control 5 2.4220 1.20188 .53750 .9297 3.9143 .86 4.04

Group 4=Diabetic + n-hexane leaf extract 5 3.7000 .73089 .32686 2.7925 4.6075 3.02 4.82

Group 5=Diabetic + n-hexane fruit extract 5 3.1020 .61459 .27485 2.3389 3.8651 2.31 3.75

Group 6=Diabetic + ethanol leaf extract 5 3.3360 .76229 .34090 2.3895 4.2825 2.60 4.48

Group 7=Diabetic + ethanol fruit extract 5 3.1480 .45024 .20136 2.5889 3.7071 2.60 3.62

Group 8=Diabetic + methanol leaf extract 5 3.1960 .52439 .23451 2.5449 3.8471 2.62 3.75

Group 9=Diabetic + methanol fruit extract 5 3.1440 .59252 .26498 2.4083 3.8797 2.31 3.65

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 2.3000 .99091 .44315 1.0696 3.5304 .86 3.25

Group 11=Diabetic + ethanol leaf and fruit extracts 5 2.9740 .47305 .21156 2.3866 3.5614 2.60 3.75

Group 12: Diabetic + methanol leaf and fruit extracts 5 2.8000 1.15588 .51692 1.3648 4.2352 2.02 4.77

Total 60 3.0705 .81628 .10538 2.8596 3.2814 .86 4.82

HDL Group 1=Normal Control 5 1.7140 .12876 .05758 1.5541 1.8739 1.57 1.86

Group 2=Diabetic Untreated 5 .6520 .15770 .07053 .4562 .8478 .49 .90

Group 3=Standard Control 5 1.4460 .37747 .16881 .9773 1.9147 1.00 1.86

Group 4=Diabetic + n-hexane leaf extract 5 1.2200 .45918 .20535 .6498 1.7902 .70 1.88


Group 6=Diabetic + ethanol leaf extract 5 1.3040 .34551 .15452 .8750 1.7330 1.01 1.89

Group 7=Diabetic + ethanol fruit extract 5 1.2720 .23931 .10702 .9749 1.5691 1.02 1.60

Group 8=Diabetic + methanol leaf extract 5 1.0720 .21253 .09505 .8081 1.3359 .89 1.44

Group 9=Diabetic + methanol fruit extract 5 1.3640 .39488 .17660 .8737 1.8543 .86 1.73

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 1.5520 .31539 .14105 1.1604 1.9436 1.15 1.86


Group 12: Diabetic + methanol leaf and fruit extracts 5 .9660 .27208 .12168 .6282 1.3038 .50 1.17

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Total 60 1.2737 .38193 .04931 1.1750 1.3723 .49 1.89

LDL Group 1=Normal Control 5 .7000 .19761 .08837 .4546 .9454 .51 .96


Group 3=Standard Control 5 1.2800 .57814 .25855 .5621 1.9979 .52 2.12


Group 5=Diabetic + n-hexane fruit extract 5 1.3680 .52328 .23402 .7183 2.0177 .45 1.73

Group 6=Diabetic + ethanol leaf extract 5 1.5120 .46170 .20648 .9387 2.0853 .71 1.89

Group 7=Diabetic + ethanol fruit extract 5 1.4720 .82302 .36807 .4501 2.4939 .71 2.70

Group 8=Diabetic + methanol leaf extract 5 1.3560 .37441 .16744 .8911 1.8209 .89 1.71

Group 9=Diabetic + methanol fruit extract 5 1.4660 .99854 .44656 .2262 2.7058 .26 2.86

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 1.2000 .54199 .24238 .5270 1.8730 .48 1.86

Group 11=Diabetic + ethanol leaf and fruit extracts 5 1.4000 .61033 .27295 .6422 2.1578 .63 1.96

Group 12: Diabetic + methanol leaf and fruit extracts 5 1.3660 .59458 .26591 .6277 2.1043 .77 2.07

Total 60 1.4108 .62774 .08104 1.2487 1.5730 .26 2.90

TAG Group 1=Normal Control 5 .4820 .04817 .02154 .4222 .5418 .40 .52

Group 2=Diabetic Untreated 5 1.2140 .45785 .20476 .6455 1.7825 .65 1.80

Group 3=Standard Control 5 .6120 .07190 .03216 .5227 .7013 .51 .70

Group 4=Diabetic + n-hexane leaf extract 5 .8740 .31150 .13931 .4872 1.2608 .52 1.32

Group 5=Diabetic + n-hexane fruit extract 5 .7700 .12000 .05367 .6210 .9190 .68 .98

Group 6=Diabetic + ethanol leaf extract 5 .7360 .09017 .04032 .6240 .8480 .61 .86

Group 7=Diabetic + ethanol fruit extract 5 .7160 .04930 .02205 .6548 .7772 .68 .77

Group 8=Diabetic + methanol leaf extract 5 .7240 .06804 .03043 .6395 .8085 .67 .84

Group 9=Diabetic + methanol fruit extract 5 .7400 .03742 .01673 .6935 .7865 .68 .78

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 .6900 .12550 .05612 .5342 .8458 .50 .83

Group 11=Diabetic + ethanol leaf and fruit extracts 5 .6640 .05459 .02441 .5962 .7318 .60 .75

Group 12: Diabetic + methanol leaf and fruit extracts 5 .6780 .04266 .01908 .6250 .7310 .62 .74

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Total 60 .7417 .23142 .02988 .6819 .8014 .40 1.80

Total_Protein Group 1=Normal Control 5 6.2840 .60842 .27210 5.5285 7.0395 5.35 6.85


Group 3=Standard Control 5 5.2260 .95411 .42669 4.0413 6.4107 4.25 6.68



Group 6=Diabetic + ethanol leaf extract 5 5.0700 1.02696 .45927 3.7949 6.3451 4.00 6.63






Group 12: Diabetic + methanol leaf and fruit extracts 5 4.9880 .38213 .17089 4.5135 5.4625 4.65 5.62

Total 60 4.8982 .83774 .10815 4.6818 5.1146 2.22 6.85

MDA Group 1=Normal Control 5 .3140 .06580 .02943 .2323 .3957 .25 .42



Group 4=Diabetic + n-hexane leaf extract 5 .6600 .03391 .01517 .6179 .7021 .62 .70









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Total 60 .6320 .15707 .02028 .5914 .6726 .25 .99

SOD Group 1=Normal Control 5 2.5876 .24085 .10771 2.2886 2.8866 2.41 3.00












Total 60 1.9825 .32308 .04171 1.8991 2.0660 1.14 3.00

SOD_Inhibition Group 1=Normal Control 5 66.3720 6.17639 2.76217 58.7030 74.0410 61.87 76.87

Group 2=Diabetic Untreated 5 38.3140 4.36617 1.95261 32.8927 43.7353 30.80 41.87

Group 3=Standard Control 5 56.1600 3.49829 1.56448 51.8163 60.5037 50.90 60.10

Group 4=Diabetic + n-hexane leaf extract 5 39.1720 7.13070 3.18895 30.3181 48.0259 29.16 48.10

Group 5=Diabetic + n-hexane fruit extract 5 50.8120 1.50747 .67416 48.9402 52.6838 48.98 52.67

Group 6=Diabetic + ethanol leaf extract 5 50.9020 2.23998 1.00175 48.1207 53.6833 48.30 54.00

Group 7=Diabetic + ethanol fruit extract 5 47.8900 5.54368 2.47921 41.0066 54.7734 40.30 53.75

Group 8=Diabetic + methanol leaf extract 5 48.8180 4.50035 2.01262 43.2301 54.4059 41.66 53.06

Group 9=Diabetic + methanol fruit extract 5 50.6500 3.98732 1.78318 45.6991 55.6009 45.30 56.38

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 55.6800 4.34404 1.94271 50.2862 61.0738 48.30 59.16

Group 11=Diabetic + ethanol leaf and fruit extracts 5 53.1660 3.20759 1.43448 49.1833 57.1487 49.58 57.50

Group 12: Diabetic + methanol leaf and fruit extracts 5 52.4340 5.57843 2.49475 45.5075 59.3605 45.72 58.58

234

234

Total 60 50.8642 8.28818 1.07000 48.7231 53.0052 29.16 76.87

Catalase Group 1=Normal Control 5 6.3332 .35302 .15787 5.8949 6.7715 6.00 6.85












Total 60 5.7189 .64699 .08353 5.5518 5.8860 4.09 6.88

GPx Group 1=Normal Control 5 1375.5560 96.63523 43.21659 1255.5675 1495.5445 1296.79 1529.92

Group 2=Diabetic Untreated 5 540.0900 571.83362 255.73177 -169.9352 1250.1152 91.82 1465.79



Group 5=Diabetic + n-hexane fruit extract 5 877.1640 124.87457 55.84560 722.1117 1032.2163 661.80 982.30








235

235

Total 60 883.5792 307.48676 39.69637 804.1469 963.0114 91.82 1529.92

ALT Group 1=Normal Control 5 18.4000 2.30217 1.02956 15.5415 21.2585 16.00 22.00









Group 10=Diabetic + n-hexane leaf and fruit extracts 5 18.8000 1.92354 .86023 16.4116 21.1884 16.00 21.00



Total 60 23.0167 5.79448 .74806 21.5198 24.5135 11.00 40.00

AST Group 1=Normal Control 5 42.4000 5.68331 2.54165 35.3432 49.4568 38.00 52.00












236

236

Total 60 51.8833 6.83979 .88301 50.1164 53.6502 38.00 68.00

Total_Bilirubin Group 1=Normal Control 5 .8640 .36916 .16509 .4056 1.3224 .66 1.52


Group 3=Standard Control 5 1.4660 .52429 .23447 .8150 2.1170 1.06 2.28



Group 6=Diabetic + ethanol leaf extract 5 1.8120 .53691 .24011 1.1453 2.4787 .99 2.47

Group 7=Diabetic + ethanol fruit extract 5 1.6720 .55639 .24883 .9812 2.3628 1.23 2.28






Total 60 1.6822 .52492 .06777 1.5466 1.8178 .66 2.85

HbAic Group 1=Normal Control 5 4.0200 .47117 .21071 3.4350 4.6050 3.60 4.80



Group 4=Diabetic + n-hexane leaf extract 5 5.9600 1.11490 .49860 4.5757 7.3443 4.80 7.30









237

237

Total 60 5.8367 1.56844 .20248 5.4315 6.2418 3.60 10.60

Sorbitol Group 1=Normal Control 5 .3694 .06985 .03124 .2827 .4561 .30 .48



Group 4=Diabetic + n-hexane leaf extract 5 .5190 .06368 .02848 .4399 .5981 .43 .59









Total 60 .4997 .09225 .01191 .4759 .5235 .30 .74

Vit_C Group 1=Normal Control 5 2.0800 .51186 .22891 1.4444 2.7156 1.50 2.70

Group 2=Diabetic Untreated 5 .7400 .16733 .07483 .5322 .9478 .60 1.00


Group 4=Diabetic + n-hexane leaf extract 5 1.0000 .15811 .07071 .8037 1.1963 .80 1.20

Group 5=Diabetic + n-hexane fruit extract 5 1.0800 .13038 .05831 .9181 1.2419 .90 1.20

Group 6=Diabetic + ethanol leaf extract 5 1.0400 .11402 .05099 .8984 1.1816 .90 1.20

Group 7=Diabetic + ethanol fruit extract 5 1.0200 .19235 .08602 .7812 1.2588 .80 1.30

Group 8=Diabetic + methanol leaf extract 5 .9600 .11402 .05099 .8184 1.1016 .80 1.10

Group 9=Diabetic + methanol fruit extract 5 .9600 .05477 .02449 .8920 1.0280 .90 1.00

Group 10=Diabetic + n-hexane leaf and fruit extracts 5 1.4400 .55946 .25020 .7453 2.1347 1.00 2.10


238

238

Group 12: Diabetic + methanol leaf and fruit extracts 5 1.1000 .10000 .04472 .9758 1.2242 1.00 1.20

Total 60 1.1433 .39504 .05100 1.0413 1.2454 .60 2.70

Post Hoc Tests Multiple Comparisons

Dependent Variable (I) Group (J) Group

Mean Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

Total

Cholesterol

LSD Group 1=Normal Control Group 2=Diabetic Untreated -1.28000* .46912 .009 -2.2232 -.3368

Group 3=Standard Control .30000 .46912 .526 -.6432 1.2432

Group 4=Diabetic + n-hexane leaf extract -.97800* .46912 .042 -1.9212 -.0348

Group 5=Diabetic + n-hexane fruit extract -.38000 .46912 .422 -1.3232 .5632

Group 6=Diabetic + ethanol leaf extract -.61400 .46912 .197 -1.5572 .3292

Group 7=Diabetic + ethanol fruit extract -.42600 .46912 .368 -1.3692 .5172

Group 8=Diabetic + methanol leaf extract -.47400 .46912 .317 -1.4172 .4692

Group 9=Diabetic + methanol fruit extract -.42200 .46912 .373 -1.3652 .5212

Group 10=Diabetic + n-hexane leaf and fruit extracts .42200 .46912 .373 -.5212 1.3652

Group 11=Diabetic + ethanol leaf and fruit extracts -.25200 .46912 .594 -1.1952 .6912

Group 12: Diabetic + methanol leaf and fruit extracts -.07800 .46912 .869 -1.0212 .8652

Group 2=Diabetic Untreated Group 1=Normal Control 1.28000* .46912 .009 .3368 2.2232

Group 3=Standard Control 1.58000* .46912 .001 .6368 2.5232

Group 4=Diabetic + n-hexane leaf extract .30200 .46912 .523 -.6412 1.2452

Group 5=Diabetic + n-hexane fruit extract .90000 .46912 .061 -.0432 1.8432

Group 6=Diabetic + ethanol leaf extract .66600 .46912 .162 -.2772 1.6092

Group 7=Diabetic + ethanol fruit extract .85400 .46912 .075 -.0892 1.7972

Group 8=Diabetic + methanol leaf extract .80600 .46912 .092 -.1372 1.7492

Group 9=Diabetic + methanol fruit extract .85800 .46912 .074 -.0852 1.8012

239

239

Group 10=Diabetic + n-hexane leaf and fruit extracts 1.70200* .46912 .001 .7588 2.6452

Group 11=Diabetic + ethanol leaf and fruit extracts 1.02800* .46912 .033 .0848 1.9712

Group 12: Diabetic + methanol leaf and fruit extracts 1.20200* .46912 .014 .2588 2.1452

Group 3=Standard Control Group 1=Normal Control -.30000 .46912 .526 -1.2432 .6432

Group 2=Diabetic Untreated -1.58000* .46912 .001 -2.5232 -.6368

Group 4=Diabetic + n-hexane leaf extract -1.27800* .46912 .009 -2.2212 -.3348









Group 4=Diabetic + n-hexane

leaf extract

Group 1=Normal Control .97800* .46912 .042 .0348 1.9212

Group 2=Diabetic Untreated -.30200 .46912 .523 -1.2452 .6412








Group 11=Diabetic + ethanol leaf and fruit extracts .72600 .46912 .128 -.2172 1.6692

Group 12: Diabetic + methanol leaf and fruit extracts .90000 .46912 .061 -.0432 1.8432

240

240


fruit extract

Group 1=Normal Control .38000 .46912 .422 -.5632 1.3232



Group 4=Diabetic + n-hexane leaf extract -.59800 .46912 .209 -1.5412 .3452


Group 7=Diabetic + ethanol fruit extract -.04600 .46912 .922 -.9892 .8972


Group 9=Diabetic + methanol fruit extract -.04200 .46912 .929 -.9852 .9012




Group 6=Diabetic + ethanol

leaf extract













fruit extract




241

241


Group 5=Diabetic + n-hexane fruit extract .04600 .46912 .922 -.8972 .9892


Group 8=Diabetic + methanol leaf extract -.04800 .46912 .919 -.9912 .8952

Group 9=Diabetic + methanol fruit extract .00400 .46912 .993 -.9392 .9472




Group 8=Diabetic + methanol

leaf extract







Group 7=Diabetic + ethanol fruit extract .04800 .46912 .919 -.8952 .9912






fruit extract







242

242






Group 10=Diabetic + n-

hexane leaf and fruit extracts

Group 1=Normal Control -.42200 .46912 .373 -1.3652 .5212


Group 3=Standard Control -.12200 .46912 .796 -1.0652 .8212



Group 6=Diabetic + ethanol leaf extract -1.03600* .46912 .032 -1.9792 -.0928







leaf and fruit extracts










243

243



Group 12: Diabetic +

methanol leaf and fruit

extracts












HDL LSD Group 1=Normal Control Group 2=Diabetic Untreated 1.06200* .18751 .000 .6850 1.4390

Group 3=Standard Control .26800 .18751 .159 -.1090 .6450

Group 4=Diabetic + n-hexane leaf extract .49400* .18751 .011 .1170 .8710

Group 5=Diabetic + n-hexane fruit extract .47000* .18751 .016 .0930 .8470

Group 6=Diabetic + ethanol leaf extract .41000* .18751 .034 .0330 .7870

Group 7=Diabetic + ethanol fruit extract .44200* .18751 .023 .0650 .8190

Group 8=Diabetic + methanol leaf extract .64200* .18751 .001 .2650 1.0190


Group 10=Diabetic + n-hexane leaf and fruit extracts .16200 .18751 .392 -.2150 .5390

Group 11=Diabetic + ethanol leaf and fruit extracts .23600 .18751 .214 -.1410 .6130

Group 12: Diabetic + methanol leaf and fruit extracts .74800* .18751 .000 .3710 1.1250

Group 2=Diabetic Untreated Group 1=Normal Control -1.06200* .18751 .000 -1.4390 -.6850

244

244

Group 3=Standard Control -.79400* .18751 .000 -1.1710 -.4170

Group 4=Diabetic + n-hexane leaf extract -.56800* .18751 .004 -.9450 -.1910

Group 5=Diabetic + n-hexane fruit extract -.59200* .18751 .003 -.9690 -.2150

Group 6=Diabetic + ethanol leaf extract -.65200* .18751 .001 -1.0290 -.2750

Group 7=Diabetic + ethanol fruit extract -.62000* .18751 .002 -.9970 -.2430

Group 8=Diabetic + methanol leaf extract -.42000* .18751 .030 -.7970 -.0430

Group 9=Diabetic + methanol fruit extract -.71200* .18751 .000 -1.0890 -.3350

Group 10=Diabetic + n-hexane leaf and fruit extracts -.90000* .18751 .000 -1.2770 -.5230

Group 11=Diabetic + ethanol leaf and fruit extracts -.82600* .18751 .000 -1.2030 -.4490

Group 12: Diabetic + methanol leaf and fruit extracts -.31400 .18751 .101 -.6910 .0630

Group 3=Standard Control Group 1=Normal Control -.26800 .18751 .159 -.6450 .1090

Group 2=Diabetic Untreated .79400* .18751 .000 .4170 1.1710

Group 4=Diabetic + n-hexane leaf extract .22600 .18751 .234 -.1510 .6030


Group 6=Diabetic + ethanol leaf extract .14200 .18751 .453 -.2350 .5190


Group 8=Diabetic + methanol leaf extract .37400 .18751 .052 -.0030 .7510


Group 10=Diabetic + n-hexane leaf and fruit extracts -.10600 .18751 .574 -.4830 .2710

Group 11=Diabetic + ethanol leaf and fruit extracts -.03200 .18751 .865 -.4090 .3450

Group 12: Diabetic + methanol leaf and fruit extracts .48000* .18751 .014 .1030 .8570


leaf extract

Group 1=Normal Control -.49400* .18751 .011 -.8710 -.1170

Group 2=Diabetic Untreated .56800* .18751 .004 .1910 .9450

Group 3=Standard Control -.22600 .18751 .234 -.6030 .1510

Group 5=Diabetic + n-hexane fruit extract -.02400 .18751 .899 -.4010 .3530

Group 6=Diabetic + ethanol leaf extract -.08400 .18751 .656 -.4610 .2930

245

245






Group 12: Diabetic + methanol leaf and fruit extracts .25400 .18751 .182 -.1230 .6310


fruit extract













leaf extract










246

246




fruit extract













leaf extract

Group 1=Normal Control -.64200* .18751 .001 -1.0190 -.2650



Group 4=Diabetic + n-hexane leaf extract -.14800 .18751 .434 -.5250 .2290





Group 10=Diabetic + n-hexane leaf and fruit extracts -.48000* .18751 .014 -.8570 -.1030

Group 11=Diabetic + ethanol leaf and fruit extracts -.40600* .18751 .035 -.7830 -.0290



fruit extract

Group 1=Normal Control -.35000 .18751 .068 -.7270 .0270


247

247



















Group 8=Diabetic + methanol leaf extract .48000* .18751 .014 .1030 .8570












248

248








extracts


Group 2=Diabetic Untreated .31400 .18751 .101 -.0630 .6910

Group 3=Standard Control -.48000* .18751 .014 -.8570 -.1030






Group 9=Diabetic + methanol fruit extract -.39800* .18751 .039 -.7750 -.0210



LDL LSD Group 1=Normal Control Group 2=Diabetic Untreated -1.32200* .38423 .001 -2.0945 -.5495








Group 10=Diabetic + n-hexane leaf and fruit extracts -.50000 .38423 .199 -1.2725 .2725


249

249










Group 10=Diabetic + n-hexane leaf and fruit extracts .82200* .38423 .038 .0495 1.5945



Group 3=Standard Control Group 1=Normal Control .58000 .38423 .138 -.1925 1.3525












leaf extract

Group 1=Normal Control 1.08800* .38423 .007 .3155 1.8605



250

250










fruit extract













leaf extract








251

251






fruit extract













leaf extract












252

252


fruit extract















Group 2=Diabetic Untreated -.82200* .38423 .038 -1.5945 -.0495
















253

253










extracts












TAG LSD Group 1=Normal Control Group 2=Diabetic Untreated -.73200* .11041 .000 -.9540 -.5100




Group 6=Diabetic + ethanol leaf extract -.25400* .11041 .026 -.4760 -.0320




254

254




Group 2=Diabetic Untreated Group 1=Normal Control .73200* .11041 .000 .5100 .9540

Group 3=Standard Control .60200* .11041 .000 .3800 .8240






Group 9=Diabetic + methanol fruit extract .47400* .11041 .000 .2520 .6960

Group 10=Diabetic + n-hexane leaf and fruit extracts .52400* .11041 .000 .3020 .7460

Group 11=Diabetic + ethanol leaf and fruit extracts .55000* .11041 .000 .3280 .7720


Group 3=Standard Control Group 1=Normal Control .13000 .11041 .245 -.0920 .3520

Group 2=Diabetic Untreated -.60200* .11041 .000 -.8240 -.3800










Group 4=Diabetic + n-hexane Group 1=Normal Control .39200* .11041 .001 .1700 .6140

255

255

leaf extract Group 2=Diabetic Untreated -.34000* .11041 .003 -.5620 -.1180











fruit extract

Group 1=Normal Control .28800* .11041 .012 .0660 .5100












leaf extract






256

256








fruit extract













leaf extract










257

257




fruit extract














Group 1=Normal Control .20800 .11041 .066 -.0140 .4300















258

258












extracts












Total_Protei

n

LSD Group 1=Normal Control Group 2=Diabetic Untreated 2.60200* .40453 .000 1.7886 3.4154


Group 4=Diabetic + n-hexane leaf extract 2.03200* .40453 .000 1.2186 2.8454

Group 5=Diabetic + n-hexane fruit extract 1.83400* .40453 .000 1.0206 2.6474

Group 6=Diabetic + ethanol leaf extract 1.21400* .40453 .004 .4006 2.0274

Group 7=Diabetic + ethanol fruit extract 1.34800* .40453 .002 .5346 2.1614

259

259

Group 8=Diabetic + methanol leaf extract 1.46000* .40453 .001 .6466 2.2734

Group 9=Diabetic + methanol fruit extract 1.51400* .40453 .000 .7006 2.3274



Group 12: Diabetic + methanol leaf and fruit extracts 1.29600* .40453 .002 .4826 2.1094

Group 2=Diabetic Untreated Group 1=Normal Control -2.60200* .40453 .000 -3.4154 -1.7886

Group 3=Standard Control -1.54400* .40453 .000 -2.3574 -.7306




Group 7=Diabetic + ethanol fruit extract -1.25400* .40453 .003 -2.0674 -.4406

Group 8=Diabetic + methanol leaf extract -1.14200* .40453 .007 -1.9554 -.3286

Group 9=Diabetic + methanol fruit extract -1.08800* .40453 .010 -1.9014 -.2746

Group 10=Diabetic + n-hexane leaf and fruit extracts -1.61200* .40453 .000 -2.4254 -.7986

Group 11=Diabetic + ethanol leaf and fruit extracts -1.32000* .40453 .002 -2.1334 -.5066

Group 12: Diabetic + methanol leaf and fruit extracts -1.30600* .40453 .002 -2.1194 -.4926

Group 3=Standard Control Group 1=Normal Control -1.05800* .40453 .012 -1.8714 -.2446

Group 2=Diabetic Untreated 1.54400* .40453 .000 .7306 2.3574

Group 4=Diabetic + n-hexane leaf extract .97400* .40453 .020 .1606 1.7874








260

260



leaf extract

Group 1=Normal Control -2.03200* .40453 .000 -2.8454 -1.2186

Group 2=Diabetic Untreated .57000 .40453 .165 -.2434 1.3834











fruit extract

Group 1=Normal Control -1.83400* .40453 .000 -2.6474 -1.0206












leaf extract

Group 1=Normal Control -1.21400* .40453 .004 -2.0274 -.4006



261

261










fruit extract













leaf extract








262

262






fruit extract

















Group 4=Diabetic + n-hexane leaf extract 1.04200* .40453 .013 .2286 1.8554

Group 5=Diabetic + n-hexane fruit extract .84400* .40453 .042 .0306 1.6574







263

263
















extracts












MDA LSD Group 1=Normal Control Group 2=Diabetic Untreated -.52400* .05322 .000 -.6310 -.4170




264

264







Group 12: Diabetic + methanol leaf and fruit extracts -.26000* .05322 .000 -.3670 -.1530












Group 3=Standard Control Group 1=Normal Control .34400* .05322 .000 .2370 .4510








265

265





leaf extract













fruit extract












Group 6=Diabetic + ethanol Group 1=Normal Control .36800* .05322 .000 .2610 .4750

266

266

leaf extract Group 2=Diabetic Untreated -.15600* .05322 .005 -.2630 -.0490











fruit extract













leaf extract






267

267








fruit extract


Group 2=Diabetic Untreated -.10600 .05322 .052 -.2130 .0010





















268

268


















extracts












SOD LSD Group 1=Normal Control Group 2=Diabetic Untreated 1.09400* .11355 .000 .8657 1.3223


269

269





















Group 3=Standard Control Group 1=Normal Control -.39820* .11355 .001 -.6265 -.1699






270

270







leaf extract













fruit extract











271

271



leaf extract













fruit extract













leaf extract




272

272










fruit extract





















273

273




















extracts












274

274

SOD_Inhibiti

on

LSD Group 1=Normal Control Group 2=Diabetic Untreated 28.05800* 2.91404 .000 22.1989 33.9171

Group 3=Standard Control 10.21200* 2.91404 .001 4.3529 16.0711

Group 4=Diabetic + n-hexane leaf extract 27.20000* 2.91404 .000 21.3409 33.0591

Group 5=Diabetic + n-hexane fruit extract 15.56000* 2.91404 .000 9.7009 21.4191

Group 6=Diabetic + ethanol leaf extract 15.47000* 2.91404 .000 9.6109 21.3291

Group 7=Diabetic + ethanol fruit extract 18.48200* 2.91404 .000 12.6229 24.3411

Group 8=Diabetic + methanol leaf extract 17.55400* 2.91404 .000 11.6949 23.4131

Group 9=Diabetic + methanol fruit extract 15.72200* 2.91404 .000 9.8629 21.5811

Group 10=Diabetic + n-hexane leaf and fruit extracts 10.69200* 2.91404 .001 4.8329 16.5511

Group 11=Diabetic + ethanol leaf and fruit extracts 13.20600* 2.91404 .000 7.3469 19.0651

Group 12: Diabetic + methanol leaf and fruit extracts 13.93800* 2.91404 .000 8.0789 19.7971

Group 2=Diabetic Untreated Group 1=Normal Control -28.05800* 2.91404 .000 -33.9171 -22.1989

Group 3=Standard Control -17.84600* 2.91404 .000 -23.7051 -11.9869

Group 4=Diabetic + n-hexane leaf extract -.85800 2.91404 .770 -6.7171 5.0011

Group 5=Diabetic + n-hexane fruit extract -12.49800* 2.91404 .000 -18.3571 -6.6389

Group 6=Diabetic + ethanol leaf extract -12.58800* 2.91404 .000 -18.4471 -6.7289

Group 7=Diabetic + ethanol fruit extract -9.57600* 2.91404 .002 -15.4351 -3.7169

Group 8=Diabetic + methanol leaf extract -10.50400* 2.91404 .001 -16.3631 -4.6449

Group 9=Diabetic + methanol fruit extract -12.33600* 2.91404 .000 -18.1951 -6.4769

Group 10=Diabetic + n-hexane leaf and fruit extracts -17.36600* 2.91404 .000 -23.2251 -11.5069

Group 11=Diabetic + ethanol leaf and fruit extracts -14.85200* 2.91404 .000 -20.7111 -8.9929

Group 12: Diabetic + methanol leaf and fruit extracts -14.12000* 2.91404 .000 -19.9791 -8.2609

Group 3=Standard Control Group 1=Normal Control -10.21200* 2.91404 .001 -16.0711 -4.3529

Group 2=Diabetic Untreated 17.84600* 2.91404 .000 11.9869 23.7051


Group 5=Diabetic + n-hexane fruit extract 5.34800 2.91404 .073 -.5111 11.2071

275

275

Group 6=Diabetic + ethanol leaf extract 5.25800 2.91404 .077 -.6011 11.1171



Group 9=Diabetic + methanol fruit extract 5.51000 2.91404 .065 -.3491 11.3691

Group 10=Diabetic + n-hexane leaf and fruit extracts .48000 2.91404 .870 -5.3791 6.3391

Group 11=Diabetic + ethanol leaf and fruit extracts 2.99400 2.91404 .309 -2.8651 8.8531

Group 12: Diabetic + methanol leaf and fruit extracts 3.72600 2.91404 .207 -2.1331 9.5851


leaf extract

Group 1=Normal Control -27.20000* 2.91404 .000 -33.0591 -21.3409

Group 2=Diabetic Untreated .85800 2.91404 .770 -5.0011 6.7171











fruit extract



Group 3=Standard Control -5.34800 2.91404 .073 -11.2071 .5111


Group 6=Diabetic + ethanol leaf extract -.09000 2.91404 .975 -5.9491 5.7691

Group 7=Diabetic + ethanol fruit extract 2.92200 2.91404 .321 -2.9371 8.7811

Group 8=Diabetic + methanol leaf extract 1.99400 2.91404 .497 -3.8651 7.8531

Group 9=Diabetic + methanol fruit extract .16200 2.91404 .956 -5.6971 6.0211

276

276

Group 10=Diabetic + n-hexane leaf and fruit extracts -4.86800 2.91404 .101 -10.7271 .9911

Group 11=Diabetic + ethanol leaf and fruit extracts -2.35400 2.91404 .423 -8.2131 3.5051

Group 12: Diabetic + methanol leaf and fruit extracts -1.62200 2.91404 .580 -7.4811 4.2371


leaf extract





Group 5=Diabetic + n-hexane fruit extract .09000 2.91404 .975 -5.7691 5.9491




Group 10=Diabetic + n-hexane leaf and fruit extracts -4.77800 2.91404 .108 -10.6371 1.0811




fruit extract





Group 5=Diabetic + n-hexane fruit extract -2.92200 2.91404 .321 -8.7811 2.9371

Group 6=Diabetic + ethanol leaf extract -3.01200 2.91404 .306 -8.8711 2.8471

Group 8=Diabetic + methanol leaf extract -.92800 2.91404 .752 -6.7871 4.9311

Group 9=Diabetic + methanol fruit extract -2.76000 2.91404 .348 -8.6191 3.0991


Group 11=Diabetic + ethanol leaf and fruit extracts -5.27600 2.91404 .076 -11.1351 .5831


Group 8=Diabetic + methanol Group 1=Normal Control -17.55400* 2.91404 .000 -23.4131 -11.6949

277

277

leaf extract Group 2=Diabetic Untreated 10.50400* 2.91404 .001 4.6449 16.3631





Group 7=Diabetic + ethanol fruit extract .92800 2.91404 .752 -4.9311 6.7871






fruit extract





Group 5=Diabetic + n-hexane fruit extract -.16200 2.91404 .956 -6.0211 5.6971




Group 10=Diabetic + n-hexane leaf and fruit extracts -5.03000 2.91404 .091 -10.8891 .8291







Group 3=Standard Control -.48000 2.91404 .870 -6.3391 5.3791


Group 5=Diabetic + n-hexane fruit extract 4.86800 2.91404 .101 -.9911 10.7271

278

278

Group 6=Diabetic + ethanol leaf extract 4.77800 2.91404 .108 -1.0811 10.6371



Group 9=Diabetic + methanol fruit extract 5.03000 2.91404 .091 -.8291 10.8891







Group 3=Standard Control -2.99400 2.91404 .309 -8.8531 2.8651


Group 5=Diabetic + n-hexane fruit extract 2.35400 2.91404 .423 -3.5051 8.2131


Group 7=Diabetic + ethanol fruit extract 5.27600 2.91404 .076 -.5831 11.1351


Group 9=Diabetic + methanol fruit extract 2.51600 2.91404 .392 -3.3431 8.3751


Group 12: Diabetic + methanol leaf and fruit extracts .73200 2.91404 .803 -5.1271 6.5911



extracts










279

279


Group 11=Diabetic + ethanol leaf and fruit extracts -.73200 2.91404 .803 -6.5911 5.1271

Catalase LSD Group 1=Normal Control Group 2=Diabetic Untreated 1.44960* .30321 .000 .8400 2.0592



Group 5=Diabetic + n-hexane fruit extract 1.12580* .30321 .001 .5162 1.7354


Group 7=Diabetic + ethanol fruit extract .99440* .30321 .002 .3848 1.6040




Group 11=Diabetic + ethanol leaf and fruit extracts .67820* .30321 .030 .0686 1.2878








Group 8=Diabetic + methanol leaf extract -.89180* .30321 .005 -1.5014 -.2822







280

280



Group 6=Diabetic + ethanol leaf extract .61940* .30321 .047 .0098 1.2290








leaf extract







Group 8=Diabetic + methanol leaf extract -.68600* .30321 .028 -1.2956 -.0764






fruit extract







281

281





Group 12: Diabetic + methanol leaf and fruit extracts -.92420* .30321 .004 -1.5338 -.3146


leaf extract













fruit extract











282

282



leaf extract













fruit extract

















283

283
























extracts








284

284





GPx LSD Group 1=Normal Control Group 2=Diabetic Untreated 835.46600* 157.29156 .000 519.2101 1151.7219











Group 2=Diabetic Untreated Group 1=Normal Control -835.46600* 157.29156 .000 -1151.7219 -519.2101


Group 4=Diabetic + n-hexane leaf extract -343.82400* 157.29156 .034 -660.0799 -27.5681



Group 7=Diabetic + ethanol fruit extract -62.14200 157.29156 .695 -378.3979 254.1139

Group 8=Diabetic + methanol leaf extract -308.94000 157.29156 .055 -625.1959 7.3159





285

285

Group 3=Standard Control Group 1=Normal Control -657.97400* 157.29156 .000 -974.2299 -341.7181

Group 2=Diabetic Untreated 177.49200 157.29156 .265 -138.7639 493.7479

Group 4=Diabetic + n-hexane leaf extract -166.33200 157.29156 .296 -482.5879 149.9239










leaf extract



Group 3=Standard Control 166.33200 157.29156 .296 -149.9239 482.5879










fruit extract





286

286




Group 9=Diabetic + methanol fruit extract -.29800 157.29156 .998 -316.5539 315.9579





leaf extract













fruit extract









287

287





leaf extract













fruit extract












Group 10=Diabetic + n- Group 1=Normal Control -363.06800* 157.29156 .025 -679.3239 -46.8121

288

288

hexane leaf and fruit extracts Group 2=Diabetic Untreated 472.39800* 157.29156 .004 156.1421 788.6539


Group 4=Diabetic + n-hexane leaf extract 128.57400 157.29156 .418 -187.6819 444.8299



















Group 10=Diabetic + n-hexane leaf and fruit extracts 30.09800 157.29156 .849 -286.1579 346.3539




extracts






289

289







ALT LSD Group 1=Normal Control Group 2=Diabetic Untreated -16.20000* 2.79643 .000 -21.8226 -10.5774





Group 7=Diabetic + ethanol fruit extract -5.20000 2.79643 .069 -10.8226 .4226



Group 10=Diabetic + n-hexane leaf and fruit extracts -.40000 2.79643 .887 -6.0226 5.2226



Group 2=Diabetic Untreated Group 1=Normal Control 16.20000* 2.79643 .000 10.5774 21.8226









290

290



Group 3=Standard Control Group 1=Normal Control 3.20000 2.79643 .258 -2.4226 8.8226

Group 2=Diabetic Untreated -13.00000* 2.79643 .000 -18.6226 -7.3774











leaf extract

Group 1=Normal Control 7.60000* 2.79643 .009 1.9774 13.2226









Group 11=Diabetic + ethanol leaf and fruit extracts 6.20000* 2.79643 .031 .5774 11.8226

Group 12: Diabetic + methanol leaf and fruit extracts 5.60000 2.79643 .051 -.0226 11.2226


fruit extract

Group 1=Normal Control 4.40000 2.79643 .122 -1.2226 10.0226


291

291



Group 6=Diabetic + ethanol leaf extract .80000 2.79643 .776 -4.8226 6.4226

Group 7=Diabetic + ethanol fruit extract -.80000 2.79643 .776 -6.4226 4.8226

Group 8=Diabetic + methanol leaf extract .00000 2.79643 1.000 -5.6226 5.6226






leaf extract



Group 3=Standard Control .40000 2.79643 .887 -5.2226 6.0226


Group 5=Diabetic + n-hexane fruit extract -.80000 2.79643 .776 -6.4226 4.8226








fruit extract

Group 1=Normal Control 5.20000 2.79643 .069 -.4226 10.8226






292

292

Group 8=Diabetic + methanol leaf extract .80000 2.79643 .776 -4.8226 6.4226


Group 10=Diabetic + n-hexane leaf and fruit extracts 4.80000 2.79643 .093 -.8226 10.4226




leaf extract





Group 5=Diabetic + n-hexane fruit extract .00000 2.79643 1.000 -5.6226 5.6226








fruit extract









Group 10=Diabetic + n-hexane leaf and fruit extracts 6.60000* 2.79643 .022 .9774 12.2226

Group 11=Diabetic + ethanol leaf and fruit extracts 5.60000 2.79643 .051 -.0226 11.2226

293

293




Group 1=Normal Control .40000 2.79643 .887 -5.2226 6.0226






Group 7=Diabetic + ethanol fruit extract -4.80000 2.79643 .093 -10.4226 .8226


Group 9=Diabetic + methanol fruit extract -6.60000* 2.79643 .022 -12.2226 -.9774








Group 4=Diabetic + n-hexane leaf extract -6.20000* 2.79643 .031 -11.8226 -.5774





Group 9=Diabetic + methanol fruit extract -5.60000 2.79643 .051 -11.2226 .0226


Group 12: Diabetic + methanol leaf and fruit extracts -.60000 2.79643 .831 -6.2226 5.0226



extracts




294

294

Group 4=Diabetic + n-hexane leaf extract -5.60000 2.79643 .051 -11.2226 .0226





Group 9=Diabetic + methanol fruit extract -5.00000 2.79643 .080 -10.6226 .6226


Group 11=Diabetic + ethanol leaf and fruit extracts .60000 2.79643 .831 -5.0226 6.2226

AST LSD Group 1=Normal Control Group 2=Diabetic Untreated -17.60000* 3.52373 .000 -24.6849 -10.5151









Group 11=Diabetic + ethanol leaf and fruit extracts -7.20000* 3.52373 .047 -14.2849 -.1151

Group 12: Diabetic + methanol leaf and fruit extracts -6.20000 3.52373 .085 -13.2849 .8849

Group 2=Diabetic Untreated Group 1=Normal Control 17.60000* 3.52373 .000 10.5151 24.6849

Group 3=Standard Control 6.20000 3.52373 .085 -.8849 13.2849



Group 6=Diabetic + ethanol leaf extract 6.80000 3.52373 .060 -.2849 13.8849

Group 7=Diabetic + ethanol fruit extract 6.60000 3.52373 .067 -.4849 13.6849


295

295





Group 3=Standard Control Group 1=Normal Control 11.40000* 3.52373 .002 4.3151 18.4849

Group 2=Diabetic Untreated -6.20000 3.52373 .085 -13.2849 .8849











leaf extract


Group 2=Diabetic Untreated -4.20000 3.52373 .239 -11.2849 2.8849








Group 11=Diabetic + ethanol leaf and fruit extracts 6.20000 3.52373 .085 -.8849 13.2849

Group 12: Diabetic + methanol leaf and fruit extracts 7.20000* 3.52373 .047 .1151 14.2849

296

296


fruit extract













leaf extract













fruit extract





297

297









leaf extract



Group 3=Standard Control .40000 3.52373 .910 -6.6849 7.4849










fruit extract









298

298











Group 6=Diabetic + ethanol leaf extract -7.80000* 3.52373 .032 -14.8849 -.7151

Group 7=Diabetic + ethanol fruit extract -8.00000* 3.52373 .028 -15.0849 -.9151







Group 1=Normal Control 7.20000* 3.52373 .047 .1151 14.2849



Group 4=Diabetic + n-hexane leaf extract -6.20000 3.52373 .085 -13.2849 .8849








Group 12: Diabetic + Group 1=Normal Control 6.20000 3.52373 .085 -.8849 13.2849

299

299


extracts



Group 4=Diabetic + n-hexane leaf extract -7.20000* 3.52373 .047 -14.2849 -.1151

Group 5=Diabetic + n-hexane fruit extract -6.60000 3.52373 .067 -13.6849 .4849







Total_Bilirub

in

LSD Group 1=Normal Control Group 2=Diabetic Untreated -1.42800* .29109 .000 -2.0133 -.8427



Group 5=Diabetic + n-hexane fruit extract -.90000* .29109 .003 -1.4853 -.3147


Group 7=Diabetic + ethanol fruit extract -.80800* .29109 .008 -1.3933 -.2227







Group 3=Standard Control .82600* .29109 .007 .2407 1.4113




300

300







Group 3=Standard Control Group 1=Normal Control .60200* .29109 .044 .0167 1.1873












leaf extract










301

301




fruit extract













leaf extract













fruit extract



302

302











leaf extract













fruit extract







303

303































304

304




extracts












HbAic LSD Group 1=Normal Control Group 2=Diabetic Untreated -6.04000* .41396 .000 -6.8723 -5.2077


Group 4=Diabetic + n-hexane leaf extract -1.94000* .41396 .000 -2.7723 -1.1077

Group 5=Diabetic + n-hexane fruit extract -2.14000* .41396 .000 -2.9723 -1.3077


Group 7=Diabetic + ethanol fruit extract -1.94000* .41396 .000 -2.7723 -1.1077

Group 8=Diabetic + methanol leaf extract -2.16000* .41396 .000 -2.9923 -1.3277

Group 9=Diabetic + methanol fruit extract -2.12000* .41396 .000 -2.9523 -1.2877


Group 11=Diabetic + ethanol leaf and fruit extracts -1.00000* .41396 .020 -1.8323 -.1677


Group 2=Diabetic Untreated Group 1=Normal Control 6.04000* .41396 .000 5.2077 6.8723

Group 3=Standard Control 5.08000* .41396 .000 4.2477 5.9123

Group 4=Diabetic + n-hexane leaf extract 4.10000* .41396 .000 3.2677 4.9323

305

305

Group 5=Diabetic + n-hexane fruit extract 3.90000* .41396 .000 3.0677 4.7323

Group 6=Diabetic + ethanol leaf extract 4.36000* .41396 .000 3.5277 5.1923

Group 7=Diabetic + ethanol fruit extract 4.10000* .41396 .000 3.2677 4.9323

Group 8=Diabetic + methanol leaf extract 3.88000* .41396 .000 3.0477 4.7123

Group 9=Diabetic + methanol fruit extract 3.92000* .41396 .000 3.0877 4.7523

Group 10=Diabetic + n-hexane leaf and fruit extracts 5.56000* .41396 .000 4.7277 6.3923

Group 11=Diabetic + ethanol leaf and fruit extracts 5.04000* .41396 .000 4.2077 5.8723

Group 12: Diabetic + methanol leaf and fruit extracts 4.70000* .41396 .000 3.8677 5.5323

Group 3=Standard Control Group 1=Normal Control .96000* .41396 .025 .1277 1.7923

Group 2=Diabetic Untreated -5.08000* .41396 .000 -5.9123 -4.2477


Group 5=Diabetic + n-hexane fruit extract -1.18000* .41396 .006 -2.0123 -.3477









leaf extract

Group 1=Normal Control 1.94000* .41396 .000 1.1077 2.7723





Group 7=Diabetic + ethanol fruit extract .00000 .41396 1.000 -.8323 .8323


306

306






fruit extract













leaf extract












307

307


fruit extract




Group 4=Diabetic + n-hexane leaf extract .00000 .41396 1.000 -.8323 .8323









leaf extract













fruit extract





308

308
















Group 7=Diabetic + ethanol fruit extract -1.46000* .41396 .001 -2.2923 -.6277















309

309






extracts












Sorbitol LSD Group 1=Normal Control Group 2=Diabetic Untreated -.31660* .03906 .000 -.3951 -.2381












310

310























leaf extract






311

311








fruit extract













leaf extract










312

312




fruit extract













leaf extract













fruit extract



313

313































314

314








extracts












Vit_C LSD Group 1=Normal Control Group 2=Diabetic Untreated 1.34000* .15706 .000 1.0242 1.6558




Group 6=Diabetic + ethanol leaf extract 1.04000* .15706 .000 .7242 1.3558


Group 8=Diabetic + methanol leaf extract 1.12000* .15706 .000 .8042 1.4358

Group 9=Diabetic + methanol fruit extract 1.12000* .15706 .000 .8042 1.4358


315

315



Group 2=Diabetic Untreated Group 1=Normal Control -1.34000* .15706 .000 -1.6558 -1.0242











Group 3=Standard Control Group 1=Normal Control -.96000* .15706 .000 -1.2758 -.6442











Group 4=Diabetic + n-hexane Group 1=Normal Control -1.08000* .15706 .000 -1.3958 -.7642

316

316

leaf extract Group 2=Diabetic Untreated .26000 .15706 .104 -.0558 .5758











fruit extract













leaf extract





317

317









fruit extract













leaf extract








318

318

Group 9=Diabetic + methanol fruit extract .00000 .15706 1.000 -.3158 .3158





fruit extract








Group 8=Diabetic + methanol leaf extract .00000 .15706 1.000 -.3158 .3158
















319

319

















extracts












*. The mean difference is significant at the 0.05 level.

320

320

Documents

UHUO, EMMANUEL NNAEMEKA (PG/Ph.D /10 /57831 ) · UHUO, EMMANUEL NNAEMEKA (PG/Ph.D /10 /57831 ) EFFECTS OF ETHANOL, METHANOL AND N-HEXANE LEAF ... Abakpa, Enugu, Congratulation for