ANTI-DIABETIC PROPERTIES OF N-HEXANE FRUIT EXTRACT OF DACRYODES EDULIS ON ALLOXAN-INDUCED DIABETIC RATS (RATTUS NOVERGICUS)

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ABSTRACT
The present study was undertaken to investigate the anti-diabetic properties of n-hexane extract of Dacryodes edulis on alloxan-induced diabetic rats. A total of 24 and 72 adult albino rats were used for the acute (24 hours) and sub-chronic (15 days) studies respectively. In the acute study, the rats were divided into 6 groups, (A- F) comprising of 4 rats each without replicates. For the sub-chronic study, the rats were divided into 6 groups (A – F) comprising of 12 rats each and the groups were further replicated three times, with 4 rats each. Diabetes was induced in rats of groups B to F, by a single intraperitoneal injection of alloxan, at a dose of 150mg/kg. The extract which was obtained by standard methods was screened phytochemically. The rats in the normal control (group A) and diabetic control (group B) were given water and food only, while the rats in the standard control (group C) received 2.5mg/kg of Glibenclamide (GB) by oral inturbation. Furthermore, rats in groups D, E and F were administered graded doses (400mg/kg, 800mg/kg and 1600mg/kg) of the extract respectively. The behavioural activities of the rats were observed on an hourly basis during the acute studies. The fasting blood glucose levels (FBGLs) of the rats used for the acute anti-diabetic testing were ascertained an hour after the treatments and subsequently on the 3rd, 6th, 12th, and 24th hours respectively. The body weights of the rats in the sub-chronic study were determined before the commencement of treatment (Day 0) and subsequently on a 5 days interval. Blood samples were obtained from the orbital sinus of each rat for the various analyses. Blood samples from the rats in the sub-chronic anti-diabetic testing group were used to ascertain such biochemical parameters as; Total cholesterol level (TCL), High Density Lipoprotein Cholesterol (HDL-C), Low Density Lipoprotein Cholesterol (LDL-C), Total Triglyceride (TG), Creatinine, Blood Urea (BU), Alkaline Phosphatase (ALP), Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) respectively. In addition, post sub-chronic histopathological analysis of the pancreas was carried out in the control and treated rats respectively. The acute fasting blood glucose levels obtained over a 24 hour period showed a significant decrease (P < 0.05) in the animals treated with Glibenclamide drug, while, in the extract treatment groups (D – F), FBGLs decreased insignificantly (P > 0.05). Also, during the acute study, rats in the normal control group displayed agility and lacked the symptoms of diabetes displayed in the other rats induced with diabetes. In group D however, agility was restored in the rats from the 12th to 24th hour. During the sub-chronic treatment, the body weights of the rats didn’t change significantly in groups A, C, D, E and F, while, that of group B increased significantly (P < 0.05). The sub-chronic treatment with D. edulis decreased FBGLs, TCL, TG, LDL-C, AST, ALT and ALP levels significantly (P < 0.05), while the HDL-C, creatinine and BU serum levels increased significantly (P < 0.05). The post sub-chronic histopathological examination of the pancreatic tissues showed that Glibenclamide drug and 1600mg/kg of the extract almost normalised damaged pancreatic architecture, while other diabetes-induced groups showed abnormal pancreatic architecture. This study therefore demonstrates that D. edulis n-hexane fruit extract possesses hypoglycaemic, hypolipidaemic and hepatoprotective effects mediated through restoration of the functions of the pancreatic tissues and even insulinotropic effects.


TABLE OF CONTENTS

Title Page
Table of Contents
List of Figures
List of Plates
List of Tables
Abstract

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1. 1      Introduction
1.2       Justification of Study
1.3       Objectives of Study
1.4       Literature Review
1.4.1    Glibenclamide
1.4.2    Dacryodes edulis
1.4.3    Proximate composition of African pear
1.4.4    Antimicrobial activity of Dacryodes edulis
1.4.5    Antioxidant property of Dacryodes edulis
1.4.6    Antidrepanocytary (anti-sickle cell anaemia) activity of Dacryodes edulis
1.4.7    Toxicity of African pear
1.4.8    Alloxan

CHAPTER TWO: MATERIALS AND METHODS
2.1       Procurement of Dacryodes edulis
2.2       Procurement of Experimental Drugs
2.3       Experimental Animals
2.4       Preparation of n-Hexane Extract
2.5       Phytochemical Analysis
2.6       Lethal Dose (LD50) Determination
2.7       Diabetes Induction in Rats
2.8       Acute Fasting Blood Glucose Monitor of Anti-diabetic Effect of D. edulis
2.9       Experimental Design for Sub-chronic Anti-diabetic Study
2.10     Blood Sample Collection
2.11     Determination of Fasting Blood Glucose (FBG) Level
2.12     Liver Enzyme Assay
2.13     Blood Creatinine Assay
2.14     Blood Urea Assay
2.15     Blood Lipid Profile
2.15.1  Total cholesterol (TC)
2.15.2 High density lipoprotein cholesterol (HDL-C)
2.15.3  Total triglyceride (TG)
2.15.4 Low density lipoprotein cholesterol (LDL-C)
2.16     Post Sub-Chronic Glucose Monitor and Histopathological Studies
Statistical Analysis

CHAPTER THREE: RESULTS
3.1       Qualitative and Quantitative Phytochemical Composition of N-Hexane Extract of Dacryodes edulis Fruit
3.2       Acute Toxicity Studies of N-Hexane Fruit Extract of Dacryodes edulis
3.3       Effects of Single Dose Administration of Dacryodes edulis Fruit Extract
3.3.1    24 hour behavioural monitoring of experimental and control rats
3.3.2    Acute fasting blood glucose levels (FBGLs) monitor of the anti-diabetic effect of D. edulis n-hexane fruit extract
3.4       Effects of N-hexane Extract of Dacryodes edulis Fruit on the Body Weights (BWs) of Diabetic Rats
3.5       Effects of N-hexane Extract of Dacryodes edulis Fruit on the Fasting Blood Glucose Levels (FBGLs) of Diabetic Rats
3.6       Effects  of  N-hexane  Extract  of  Dacryodes  edulis  Fruit  on  Aspartate
            Aminotransferase (AST) Levels of Diabetic Rats
3.7       Effects  of  N-hexane  Extract  of  Dacryodes  edulis  Fruit  on  Alanine Aminotransferase (ALT) Levels of Diabetic Rats
3.8       Effects of N-hexane Extract of Dacryodes edulis Fruit on Alkaline Phosphatase (ALP) Levels of Diabetic Rats
3.9       Effects of N-hexane Extract of Dacryodes edulis Fruit on Creatinine Levels of Diabetic Rats
3.10     Effects of N-hexane Extract of Dacryodes edulis Fruit on Blood Urea (BU) Levels of Diabetic Rats
3.11     Effects of N-hexane Extract of Dacryodes edulis Fruit on the Total Cholesterol (TC) Levels of Diabetic Rats
3.12     Effects of N-hexane Extract of Dacryodes edulis Fruit on High Density Lipoprotein Cholesterol (HDL-C) Levels of Diabetic Rats
3.13     Effects of N-hexane Extract of Dacryodes edulis Fruit on the Total Triglyceride (TG) Levels of Diabetic Rats
3.14     Effects of N-hexane Extract of Dacryodes edulis Fruit on Low Density Lipoprotein Cholesterol (LDL-C) Levels of Diabetic Rats
3.15     Post Sub-Chronic Fasting Blood Glucose Levels of Alloxan-Induced Diabetic and Control Rats
3.16     Post Sub-Chronic Histopathological Features of the Pancreases in Alloxan-induced Diabetic and Control Rats
3.16.1 Post sub-chronic histopathological features of the pancreas of rat from the normal control group
3.16.2 Post sub-chronic histopathological features of the pancreas of rat from the diabetic control group
3.16.3 Post sub-chronic histopathological features of the pancreas of rat from the standard control group
3.16.4  Post sub-chronic histopathological features of the pancreas of rat from 400mg/kg extract treatment group
3.16.5 Post sub-chronic histopathology of the pancreas of rat from 800mg/kg extract treatment group
3.16.6  Post sub-chronic histopathological features of the pancreas of rat from 1600mg/kg extract treatment group
3.16.7 Comparison of the post sub-chronic photomicrograps of the pancreatic tissues of the extract treatment groups with the control groups

CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1       Discussion
4.2       Conclusion
RECOMMENDATIONS
REFERENCES
APPENDICES


CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION
Nutrition is geared towards creating awareness among people about the health implications of their diets. Recently, diet exploration has diverted consumers towards nature (Butt and Sultan, 2009). Diabetes mellitus is a group of metabolic diseases in which a person has high blood sugar (hyperglycaemia), either because the pancreas does not produce enough insulin (type 1 diabetes), or because cells do not respond to the insulin that is produced (type 2 diabetes) (Gardner and Shoback, 2011). This high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger). Once there is an onset of diabetes, it has to be managed henceforth. The goal of management for diabetes is to maintain blood glucose levels as close to normal as possible. There are three basic managements procedures already established,they include; diet, exercise and medication. It is widely recognised that the prevalence of diabetes is rising rapidly (Mokadad et al., 2001). There are more than 12 million people with diabetes in the world as at 1997 and approached 20 million in 2010 (Amos et al., 1997).This metabolic disorder can be induced chemically in animal models like rats using either alloxan monohydrate or streptozotocin. Alloxan-induced diabetes mellitus in rats arises as a result of selective pancreatic beta cells toxicity (Matsuhisa et al., 1997).Hence, insulin-producing cells are destroyed by alloxan.
Dacryodes edulis (G. Don), the African plum or safou, is an evergreen tree indigenous to the central Africa and Gulf of guinea region (Lam, 1985). The tree has a relatively short trunk and a deep, dense crown. The bark is pale gray and rough with droplets of resin. The preferential habitat of Dacryodes edulis is a shady, humid tropical forest. However, it adapts well to variations in soil type, humidity, temperature and day length. The edible fruit is a rich source of nutrients such as lipids, vitamins and proteins. The plant has been long used in traditional medicine of some African countries to treat several ailments such as wounds, skin diseases, dysentery and fever. The extracts and secondary metabolites have been found to show biological activities such as antimicrobial, antioxidant and anti-sickle cell anaemia (Obame et al., 2008; Nguefack, 2009). The production and commercialization of African pear has been on the increase in the last few years. So important has been the trade that transactions are now known to cut across some national and international boundaries (Awono etal., 2002). The prime importance though has been because the fruit is rich in lipids. Infact, research investigations into the chemical composition of safou has focused on the oil content (Omoti & Okiy, 1987; Kinkela & Bezard, 1993).

1.2 JUSTIFICATION OF STUDY
It was the aim of this work to comparatively study the anti-diabetic properties of Dacryodes edulis fruits harvested in Nsukka with the standard drug; Glibenclamide, using albino rat as the animal model. Once there is an onset of diabetes, patients need to use a synthetic drug to control their blood glucose level and to improve blood glucose tolerance throughout their lives. Recently, the use of natural products has gained more interest for remedy of diabetes and other ailments. Hence, with an increase in occurrence of type 1....

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