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

TABLE OF CONTENTS
Title Page
Certification
Dedication
Acknowledgements
Abstract
Table of Contents
List of Figures
List of Tables
List of Abbreviations

CHAPTER ONE: INTRODUCTION
1.1       Kigelia africana
1.1.1    Description of Kigelia africana
1.1.2    Taxonomy of Kigelia africana
1.1.3    Traditional uses of Kigelia africana
1.1.4    Chemical constituents of Kigelia africana
1.1.5    Antibacteria and antifungi
1.2       Diabetes
1.2.1    Diabetes mellitus
1.2.2    Diabetes Type 1 and 2
1.2.3    Insulin resistance
1.2.4    Diabetic complications
1.3       Hyperglycemia and diabetic complication
1.4       Mechanism of tissue damage mediated by hyperglycemia
1.4.1    Aldose reductase pathway
1.4.2    Non-enzymatic glycation
1.4.3    Carbonyl stress in diabetes
1.4.4    Activation of protein kinase C isoforms
1.5       Oxidative stress
1.5.1    Mechanism of increased oxidative stress in diabetes mellitus
1.5.2    Glucose autoxidation
1.5.3    Free radicals
1.5.4    Reactive oxygen species and oxidative stress
1.6       Antioxidant system
1.6.1    Scavenging properties of antioxidants
1.6.2    Positive and negative effects of free radicals
1.7       Lipid peroxidation
1.8       Antioxidant supplementation in diabetes mellitus
1.9       Alloxan
1.9.1    Alloxan diabetes and streptozotocin diabetes
1.9.2    Alloxan: Mechanism of action
1.9.3    Beta cell toxicity and diabetogenicity of alloxan
1.9.4    Streptozotocin: Mechanism of action and beta cell selectivity
1.9.5    Beta cell toxicity of streptozotocin
1.9       Rationale for the study
1.10     Aim and objectives of the study
1.10.1  Aim of the study
1.10.2  Specific objectives of the study

CHAPTER TWO: MATERIALS AND METHODS
2.1       Materials
2.1.1    Chemicals
2.1.2    Instrument/Equipment
2.1.3    Drug
2.1.4    Plant material
2.2       Methods
2.2.1    Animal management
2.2.2    Preparation of plant extracts
2.2.3    Design of the experiment
2.2.4    Yield of extracts
2.2.5    Phytochemical analysis of the crude extracts
2.2.5.1 Test for the presence of alkaloids
2.2.5.2 Test for carbohydrates
2.2.5.3 Test for reducing sugar
2.2.5.4 Test for protein
2.2.5.5 Test for fats and oil
2.2.5.6 Test for glycosides
2.2.5.7 Test for acidic substances
2.2.5.8 Test for the presence of flavonoids
2.2.5.9 Test for the presence of steroids
2.2.5.10  Test for tannins
2.2.5.11 Test for resins
2.2.5.12 Test for saponins
2.2.3.13  Test for terpenoids and steroids
2.2.6    Proximate Analysis
2.2.6.1 Crude protein
2.2.6.2 Crude fat
2.2.6.3 Moisture
2.2.6.4 Ash /Mineral matter
2.2.6.5 Crude fibre
2.2.6.6 Carbohydrate or nitrogen free extract (NFE)
2.2.7    Acute toxicity test
2.2.7.1 Determination of LD50 of the extract
2.2.8    Induction of diabetes
2.2.9    Determination of fasting and random glucose concentrations
2.2.10  Determination of sorbitol concentration
2.2.11  Determination of total protein concentration
2.2.12  Determination of haemoglobin glycosylation
2.2.13  Determination of malondialdehyde concentration
2.2.14  Determination of vitamin C concentration
2.2.15  Assay of catalase activity
2.2.16  Assay of superoxide dismutase (SOD) activity
2.2.17  Assay of glutathione peroxidase activity
2.2.18  Determination of total cholesterol concentration
2.2.19  Determination of high density lipoprotein (HDL) cholesterol concentration
2.2.20  Determination of low density lipoprotein (LDL) cholesterol concentration
2.2.21  Determination of triacylglycerol concentration
2.2.22  Assay of aspartate aminotransferase (AST) activity
2.2.23  Assay of alanine aminotranferase (ALT) activity
2.2.24  Determination of total bilirubin concentration
2.3       Statistical analysis

CHAPTER THREE: RESULTS
3.1       Qualitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.2       Quantitative phytochemical composition of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.3       Percentage proximate compositions of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana
3.4       Percentage yield of leaf and fruit samples of Kigelia Africana
3.5       Acute toxicity studies
3.6       Effect of ethanol, n-hexane and methanol extracts of leaves and fruits of Kigelia africana on sugar level of diabetic rats
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
3.8       Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on sorbitol concentration in alloxan-induced diabetic rats
3.9       Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigeria africana on total protein in alloxan-induced diabetic rats
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
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
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
3.13     Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on catalase activity in alloxan-induced diabetic rats
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
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
3.16     Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on glutathione peroxidase activity in alloxan-induced diabetic rats
3.17     Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on cholesterol concentration in alloxan-induced diabetic rat
3.18     Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana high density lipoprotein in alloxan-induced diabetic rats
3.19     Effect of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana on low density lipoprotein concentration in alloxan-induced diabetic rats
3.20     Effect of ethanol, methanol and n-hexane leaf and fruit extracts of Kigelia africana on triacylglycerol (TAG) concentration in alloxan-induced diabetic rats
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
3.22     Effect of ethanol, methanol and n-hexane leaf and fruit extract of Kigelia africana on alanine aminotransferase (ALT) in alloxan-induced diabetic rats
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

CHAPTER FOUR: DISCUSSION

4.1       Discussion
4.2       Conclusion
REFERENCES
APPENDICES


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="" 0.05="" 10-12="" 3="" 4-12="" 4-9="" 500="" a="" administered="" administration="" against="" all="" and="" animals="" as="" b.w="" ba1c="" c="" cholesterol="" combination="" compared="" comparison="" concentration="" concentrations="" control.="" control="" decreased="" density="" diabetic="" differences="" extracts.="" extracts="" fed="" glycohaemoglobin="" glycohaemogolin="" group="" groups="" however="" i="" in="" kg="" levels="" lipoprotein="" low="" malondiadehyde="" mg="" of="" p="" parts="" plant="" positive="" protein="" rats.="" rats="" significant="" significantly="" single="" sobitol="" sorbitol="" test="" the="" there="" total="" treated="" triacylgycerol="" two="" untreated="" vitamin="" were="" with="">K. africana
increased significantly (p<0 .05="" 10="" 11="" 12="" a="" across="" against="" and="" as="" combination="" decreased="" density="" diabetic="" except="" fruit="" group.="" groups="" hdl="" high="" i="" in="" leaf="" lipid="" lipoprotein="" of="" p="" parts="" profiles="" recorded="" significant="" test="" the="" treated="" two="" untreated="" was="" with="">) 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.


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.....

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