PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING PROPERTIES AND ANTIDIABETIC POTENTIALS OF METHANOL EXTRACT OF THE STEM (BARK) OF ALSTONIA BOONEI ON ALLOXAN-INDUCED DIABETIC ALBINO RATS

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ABSTRACT
This study was carried out to investigate the phytochemical constituents of the methanol extract of the stem (bark) of Alstonia boonei (MEAB), in vitro antioxidant activity of the extract and its possible antidiabetic and antioxidant potential using alloxan- induced diabetic rats as model. The qualitative analysis of the extract showed a wide range of phytochemicals, which could be physiologically potent in ameliorating several diseases. Quantitative phytochemical analysis revealed that the extract contains relatively high concentration on tannin (7.375±0.005mg/100g), flavonoid (6.176±0.003 mg/100g) and phenols (5.867±0.003 mg/100g). The quantitative result of antioxidant vitamins shows that vitamin C was highest (24.91±0.005mg/100g) compared to vitamin A (1.314±0.002µg/g) and vitamin E (0.886±0.002mg/100g). The methanol extract of Alstonia boonei scavenged 1, 1- diphenyl-2-picrylhydrazyl radical (DPPH.) in a concentration dependent manner with a correlation coefficient (R2) of 0.7066, indicating antioxidant activity with effective concentration that inhibits 50 percent of the radicals (EC50) of 12.33 ± 0.2µg/ml compared to ascorbic acid standard EC50 of 98 ± 0.02µg/ml. The superoxide radical scavenging activity was concentration-dependent with an EC50 of 7.03±0.42µg/ml compared with ascorbic acid and rutin standards with EC50 of 812.97±0.97µg/ml and 3.47 ± 0.6µg/ml respectively. The extract also showed hydroxyl radical scavenging activity with an EC50 of 42.75±0.02µg/ml compared to α- tocophenol standard with EC50 of 232.31 ± 6.97µg/ml. The nitric oxide radical scavenging activity shows that the extract scavenged nitric oxide radical in a concentration dependent manner with 500µg/ml being more effective than 500µg/ml of ascorbic acid standard. There was a significant increase (P > 0.05) in the serum glucose level in group 2 (diabetic untreated) compared to group 1 (normal control). Significant decrease (P < 0.05) in glucose serum concentration was recorded in all groups treated with the extract and the standard drug compared to group 2 (diabetic untreated) . There was a significant increase (P < 0.05) in urea, creatinine, sodium ion and chloride ion concentrations of group 2 (diabetic untreated) compared to group 1 (normal control). Significant decrease (P < 0.05) in urea, creatinine, sodium ion and chloride ion concentrations was recorded in all groups treated with the methanol extract and the standard drug(group 3 to 6) compared to group 2 (diabetic untreated). There was no significant increase (P > 0.05) in potassium ion concentration of group 2 when compared with group 1. There was a significant increase (P < 0.05) in the serum concentration of Aspartate aminotransferase, (AST), Alanine aminotransferase, (ALT) and Alkaline phosphatase, (ALP) of group 2 (diabetic untreated) when compared with group 1 and significantly reduced (P < 0.05) in all groups treated with the extract and standard drugs when compared to group 2. Serum superoxide and catalase activities were significantly (P < 0.5) reduced in group 2 (diabetic untreated) when compared to the normal control. Serum superoxide and catalase activities increased significantly (P < 0.05) in all groups treated with the extract and standard drugs when compared to group 2 (diabetic untreated). There was a significant increase (P < 0.05) in serum malondialdehyde (MDA) concentration of group 2 (diabetic untreated) and a significant reduction (P < 0.05) in all groups treated with the extract and the standard drug compare to group 2. These results suggest that methanol extract of the stem (bark) of Alstonia boonei (MEAB), possesses and antidiadetic and antioxidant potentials.

TABLE OF CONTENTS

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Title Page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of Contents vi
List of Tables xii
List of Figures xiii
List of Plates xv
List of Abbreviations xvi

CHAPTER ONE: INTRODUCTION
1.1 Alstonia boonei 2
1.1.1 Morphology of Alstonia boonei 2
1.1.2 Classification of Alstonia boonei 2
1.1.3 Uses of Alstonia boonei 6
1.2 Reactive Oxygen Species (ROS) 6
1.2.1 Superoxide Radical 7
1.2.2 Hydroxyl Radical 8
1.2.3 Nitric Oxide 8
1.3 Diabetes: Definition and Types 9
1.3.1 Complications of Diabetes 9
1.3.2 Oxidative Stress and Diabetic Complications 10
1.4 Phytochemicals 14
1.5 Aim and Objectives 15
1.5.1 Aim of the Study 15
1.5.2 Specific Objective of the Study 15
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials 16
2.1.1 Plant Material 16
2.1.2 Equipment 16
2.1.3 Chemicals/ Reagents 16
2.1.4 Animals 17
2.2 Methods 17
2.2.1 Experimental Design 17
2.2.2 Collection of Plant Material 17
2.2.3 Preparation of Plant Material 18
2.2.4 Induction of Diabetes 18
2.2.5 Determination of Median Lethal Dose (LD50) 18
2.2.6 Determination of Hypoglycemic Potentials of the Extract 18
2.2.7 Serum Glucose Concentration 19
2.2.8 Blood Collection 19
2.2.9 Qualitative Phytochemical Screening 19
2.2.9.1 Test for Carbohydrate 20
2.2.9.2 Test for Reducing Sugar 20
2.2.9.3 Test for Terpenoids 20
2.2.9.4 Test for Saponin 20
2.2.9.5 Test for Flavonoids 20
2.2.9.6 Test for Alkaloids 20
2.2.9.7 Test for Glycosides 21
2.2.9.8 Test for Tannins 21
2.2.9.9 Test for Steroids 21
2.2.9.10 Test for Hydrogen Cyanide 21
2.2.9.11 Test for Phenols 22
2.2.10 Quantitative Determination of Phytochemical Constituents 22
2.2.10.1 Determination of Carbohydrate Content 22
2.2.10.2 Determination of Hydrogen Cyanide Content 22
2.2.10.3 Determination of Saponin Content 22
2.2.10.4 Determination of Alkaloid Content 22
2.2.10.5 Determination of Tannin Content 23
2.2.10.6 Determination of Glycoside Content 23
2.2.10.7 Determination of Phenol Content 23
2.2.10.8 Determination of Flavonoid Content 23
2.2.10.9 Determination of Steroid Content 23
2.2.11 Quantitative Determination of Anti-Oxidant Vitamins 24
2.2.11.1 Determination of Vitamin C 24
2.2.11.2 Determination of Vitamin A 24
2.2.11.3 Determination of Vitamin E 24
2.2.12 Determination of Free Radical Scavenging Activity 25
2.2.12.1 Quantitative DPPH Radical-Scavenging Assay 25
2.2.12.2 Hydroxyl Radical (.OH) Scavenging Assay 25
2.2.12.3 Superoxide (O2¬.-) Scavenging Assay 26
2.2.12.4 Nitric Oxide Radical (NO.) Scavenging Assay 26
2.2.13 Biochemical Parameters 27
2.2.13.1 Determination of Urea Concentration 27
2.2.13.2 Determination of Creatinine Concentration 27
2.2.13.3 Determination of Sodium Ion (Na+) Concentration 28
2.2.13.4 Determination of Potassium Ion (K+) Concentration 29
2.2.13.5 Determination of Chloride Ion (Cl-) Concentration 30
2.2.13.6 Assay for Serum Alanine Aminotransferases (ALT) Activity 31
2.2.13.7 Assay for Serum Aspartate Aminotransferase (AST) Activity 32
2.2.13.8 Assay for Serum Alkaline Phosphatase (ALP) Activity 34
2.2.13.9 Determination of Lipid Peroxidation (Malondialdehyde) level 35
2.2.13.10 Assay for Superoxide dismutase (SOD) activity 36
2.2.13.11Assay for Catalase activity 37
2.2.14 Histopathology 38
2.3 Statistical Analysis 38

CHAPTER THREE: RESULTS
3.1 The Acute Toxicity Test of the Methanol Extract of Alstonia boonei 39
3.2 Qualitative Phytochemical Composition of Methanol Extract of Alstonia boonei 41
3.3 Quantitative Phytochemical Composition of the Methanol Extract
of Alstonia boonei 43
3.4 Quantitative Antioxidant Vitamins Composition of the Methanol
Extract of Alstonia boonei 45
3.5 Quantitative DPPH. Radical Scavenging Activity of the Methanol
Extract of Alstonia boonei 47
3.6 Hydroxyl Radical Scavenging Activity of the Methanol
Extract of Alstonia boonei 49
3.7 Superoxide Radical Scavenging Activity of the Methanol
Extract of Alstonia boonei 51
3.8 Nitric Oxide Radical Scavenging Activity of the Methanol
Extract of Alstonia boonei 53
3.9 Mean Fasting Blood Glucose Concentration of Diabetic Rats
Treated with Methanol Extract of Alstonia boonei 55
3.10 Mean Serum Urea Concentration of Diabetic Rats Treated
with Methanol Extract of Alstonia boonei 57
3.11 Effect of Methanol Extract of Alstonia boonei on the Serum
Creatinine Concentration of Diabetic Rats 59
3.12 Mean Serum Sodium ion Concentration of Diabetic Rats
Treated with Methanol Extract of Alstonia boonei 61
3.13 The Mean Serum Potassium ion Concentration of Diabetic
Rats Treated with Methanol Extract of Alstonia boonei 63
3.14 The Mean Serum Chloride ion Concentration of Diabetic Rats
Treated with Methanol Extract of Alstonia boonei 65
3.15 The Mean Serum Alanine Aminotranferase Activity of Diabetic
Rats Treated with Methanol Extract of Alstonia boonei 67
3.16 The Mean Serum Aspartate Aminotranferase Activity of Diabetic
Rats Treated with Methanol Extract of Alstonia boonei 69
3.17 The Mean Serum Alkaline Phosphatase Activity of Diabetic
Rats Treated with Methanol Extract of Alstonia boonei 71
3.18 The Mean Serum Malondialdehyde Concentration of Diabetic
Rats Treated with Methanol Extract of Alstonia boonei 73
3.19 The Mean Serum Superoxide Dismutase Activity of Diabetic Rats
Treated with Methanol Extract of Alstonia boonei 75
3.20 The Mean Serum Catalase Activity of Diabetic Rats Treated with
Methanol Extract of Alstonia boonei 77
3.21 Histological Photomicrograph of the Pancreas from the Experimental Rats 79
3.22 Histological Photomicrograph of the Kidney from the Experimental Rats 81

CHAPTER FOUR: DISCUSSION
4.1 Discussion 83
4.2 Conclusion 87
4.3 Suggestions for Further Study 87
REFERENCES 88
APPENDICES 99

LIST OF TABLES

Table 1: Standard ALT readings 32
Table 2: Standard AST readings 34
Table 3: Acute Toxicity Test of the Methanol extract of Alstonia boonei 40
Table 4: Qualitative Phytochemical Composition of Alstonia boonei 42
Table 5: Quantitative Phytochemical Composition of the Methanol extract of Alstonia boonei 44
Table 6: Quantitative Antioxidant Vitamins Composition of the Methanol extract of Alstonia boonei 46
Table 7: Quantitative DPPH. Radical Scavenging Activity of Alstonia boonei 48
Table 8: Hydroxyl Radical Scavenging Activity of Alstonia boonei 50
Table 9: Superoxide Radical Scavenging Activity of Alstonia boonei 52

LIST OF FIGURES
Figure 1: Stem of Alstonia boonei 4
Figure 2: Leaves of Alstonia boonei 5
Figure 3: schematic representation of protein kinase C and polyol pathways in the pathogenesis of diabetic complications 11
Figure 4: Increased production of AGE precursors and their pathologic consequences 12
Figure 5: Increased flux by hyperglycemia through hexosamine pathway 13
Figure 6: Activation of PARP and modification of GAPDH by ROS-induced DNA Damage 14
Figure 7: Nitric oxide radical scavenging activity of Alstonia boonei 54
Figure 8: The mean serum fasting glucose concentration of diabetic rats
in different group 56
Figure 9: The Mean Serum Urea Concentration of Diabetic Rats 58
Figure 10: Showing the mean serum creatinine concentration of diabetic rats 60
Figure 11: Bar chart showing the mean serum sodium ion concentration of diabetic rats 62
Figure 12: Mean serum potassium ion concentration of diabetic rats treated with different concentration of the extract 64
Figure 13: Bar chart showing mean serum chloride ion concentration of diabetic rats 66
Figure 14: Mean Serum Alanine Aminotranferase Activity of Diabetic Rats 68
Figure 15: Showing the mean serum Aspartate Aminotransferase of diabetic rats treated with different concentration of the extract 70
Figure 16: The mean serum Alkaline Phosphatase activity of diabetic rats and normal rats 72
Figure 17: Showing the mean serum malondialdehyde concentration of diabetic rats 74
Figure 18: The mean serum Superoxide Dismutase activity of diabetic rats 76
Figure 19: Showing the mean serum Catalase activity of diabetic 78

PLATES
Plate 1: Photomicrograph of the pancreas from the experimental rats showing the
pancreatic islets (PI) 80
Plate 2: Photomicrograph of the kidney from the experimental rats showing the
glomerulus (G) and the renal tubules 82

LIST OF ABBREVIATIONS
ANOVA: Analysis of variance
CAT: Catalase
EDTA: Ethylene diamine tetraacetic acid
ROS: Reactive Oxygen Species
GPX: Glutathione peroxidase
OH.: Hydroxyl Radical
H2O2: Hydrogen Peroxide
SOD: Superoxide Dismutase
MDA: Malondialdehyde
NaOH: Sodium hydroxide
SPSS: Statistical package for social science
WHO: World health organization
GLUT: Glucose transporter
IDDM: Insulin Dependent Diabetic Mellitus
NO: Nitric Oxide
ADA: American Diabetes Association
LADA: Latent Autoimmune Diabetes in Adult
AGEs: Advanced Glycation End Products
PKC: Protein Kinase C
NAD+: Nicotinamide Adenine Dinucleotide (oxidized)
NADH: Nicotinamide Adenine Dinucleotide (reduced)
GAPDH: Glyceraldehyde 3-Phosphate Dehydrogenase
DPPH: 2,2-Diphenyl-1-Picrylhydrazyl
ALT: Alanine Aminotransferase
AST: Aspartate Aminotransferase
ALP: Alkaline Phosphatase

CHAPTER ONE
INTRODUCTION
Diabetes is one of the most challenging health problems in the twenty first century (Rahman et al., 2009). Diabetes currently afflicts 171 million people worldwide (Boden and Taggart, 2009). Normal non-diabetic patients maintain plasma glucose <100 mg/dl in the fasting and <135 mg/dl in the post prandial period (Rossetti et al., 2008). Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both (American Diabetes Association, 2009a). Diabetes mellitus is classified as type 1, type 2, with other specific examples and gestational diabetes (American Diabetes Association, 2008). Type 1 diabetes is known as insulin dependent diabetes or Juvenile-onset diabetes and type 2 diabetes is known as non-insulin dependent or adult onset diabetes (American Diabetes Association, 2009b). A slowly progressive form of type 1 diabetes was acknowledged as latent autoimmune diabetes in adults (LADA) by the World Health Organization (WHO) and American Diabetes Association (ADA) (Van Deutekom et al., 2008). Classification schemes define type 1 diabetes as a state of absolute insulin deficiency and type 2 as a state of insulin resistance combined with inadequate insulin secretion (Greenbaum et al., 2009). Type 1 diabetes is an autoimmune disease where auto reactive immune cells attack insulin producing β-cells, destroying insulin reserve leading to hyperglycemia (Eldor et al., 2009). The rate of loss of β-cell function is affected by factors like age at diagnosis, degree of metabolic control, immune status, genetics and marked inter-individual variation (Palmer, 2009). The symptoms of type 1 diabetes are significant weight loss and ketoacidosis (Ludvigsson et al., 2008). Diabetes Type 2 diabetes is a progressive disease characterized by declining β-cell function that in concert with insulin resistance, leads to loss of glycemic control and eventual diabetes complications (Nauck et al., 2009). Type 1.5 diabetes also known as latent autoimmune diabetes in adults (LADA) is an important form of diabetes although it is frequently under estimated (Mayer et al., 2007). Type 1.5 diabetes is also known as slowly progressive type I diabetes, autoimmune diabetes in adults with slowly progressive β-cell failure, autoimmune diabetes not requiring insulin at diagnosis, autoimmune diabetes in adults and type 1.5 diabetes (Dunn et al., 2008). Type 1.5 diabetes has a later onset and slower progression towards an absolute insulin requirement (Cernea et al., 2009). Type 1.5 diabetes occurs in about 10% of patients classified as type 2 diabetes and not initially requiring insulin (Agardh et al., 2009). Diagnosis of type 1.5 diabetes is difficult due to lack of defining features (Jasem et al., 2010).
1.1 Alstonia boonei

PHYTOCHEMICAL CONSTITUENTS, FREE RADICAL SCAVENGING PROPERTIES AND ANTIDIABETIC POTENTIALS OF METHANOL EXTRACT OF THE STEM (BARK) OF ALSTONIA BOONEI ON ALLOXAN-INDUCED DIABETIC ALBINO RATS