– Evaluation of the Anti-Diabetic and Anti-Oxidant Potentials of Methanolic Extract of Helianthus Annuus l. LEAVES in Alloxan- Induced Hyperglycemic Rats –
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ABSTRACT
This study evaluated the anti-diabetic and anti-oxidant potential of methanolic extract of Helianthus annuus L. leaves in alloxan induced hyperglycemic rats. Fresh leaves of the plant were collected from their local habitat in the University of Nigeria, Nsukka environment.
The extract was prepared by cold maceration using 80 % methanol at environmental temperature with intermittent sharing for 48 h to obtain a yield of 10.04 % w/w dry Helianthus annuus extract (HAE).
The extract was tolerated by the rats, which showed no overt signs of toxicity at the dose range 300 – 3600 mg/kg) tested. Even the highest does of 3600 mg/kg did not cause mortality in the rats.
The crude extract produced dose- and time-dependent reduction in fasting blood sugar (FBS) level. HAE (600 mg/kg) at 6 hour post treatment, significantly (p < 0.05) decreased FBS when compared with DW treated group.
However this was not significant (p > 0.05) when compared with GLB- treated group. The normoglycemic OGTT showed no significant (p > 0.05) difference in FBS level between all the treated groups.
At 2 hour post glucose load, there was no significant (p > 0.05) difference compared with the 0 hour FBS level. In hyperglycaemic OGTT, HAE (600 mg/kg) at 2 hour was significantly (p < 0.05) lower when compared with the control.
However, this was not significant (p > 0.05) when compared with GLB (2 mg/kg) treated group. The separation of the extract yielded thirteen (13) fractions.
Bioactivity screening of the fractions at 60 mg/kg showed various degrees of reduction in FBS in time-dependent manner. The HAE, fractions 8-10 and 13 caused 66.74, 61.36, 70.63 and 78.03% reductions in FBS, respectively.
Phytochemical spot tests of the HAE showed the presence of saponins, alkaloids, flavonoids, terpenes, glycosides, tannins and carbohydrates while fractions 8 and 10 contain mainly glycosides and sterols and fraction 13 contains mainly tannins.
In vitro anti-oxidant tests at 400 µg/ml showed that HAE, fractions 8-10 and 13 gave 89.00, 30.42, 88.03 and 92.72 % anti-oxidant activity, respectively using DPPH model and 3.69, 0.95,
0.67 and 0.28 anti-oxidant potential, respectively in FRAP model. Ascorbic acid used as control has antioxidant potential of 2.00 using FRAP model.
The study validates the use of the preparations from H. annuus leaves in folklore diabetic therapy.
TABLE OF CONTENTS
Title page I
Declaration II
Certification III
Dedication IV
Acknowledgements V
Table of content VI
List of tables VIII
List of figures IX
Abstract X
CHAPTER ONE: INTRODUCTION 1
1.1 Introduction 1
1.2 Statement of problems 2
1.3 Objectives of the study 3
CHAPTER TWO: LITERATURE REVIEW
2.1 Plant Description 4
2.2 Medicinal and Folkloric Uses of Helianthus annuus L. 5
2.3 Definitions of Diabetes Mellitus (DM) 6
2.4 Classifications of Diabetes Mellitus 7
2.5. Aetiologies and Predisposing Factors of Diabetes Mellitus 7
2.5.1 Type 1 Diabetes Mellitus 8
2.5.2 Type 2 Diabetes Mellitus 9
2.6 Symptoms of Diabetes 10
2.7 Experimental Diabetes Mellitus 11
2.7.1 Pancreatectomy 11
2.7.2 Administration of Anterior Pituitary Extract 11
2.7.3 Administration of Chemical (Alloxan and Streptozotocin) 11
2.7.4 Excessive Administration of Glucose Over Prolong Period of Time 12
2.7.5 Prolong Administration of Excessive Insulin 13
2.8 Diagnosis of Diabetes Mellitus 13
2.9 Pathophysiology of Diabetes Mellitus 16
2.10 Complication of Diabetes Mellitus 16
2.11 Treatment of Diabetes 19
2.12 Anti-diabetic Principles from Plant Origin 25
CHAPTER THREE: MATERIAL AND METHOD 26
3.1 Materials 26
3.1.1 Equipment 26
3.1.2 Chemicals, Reagents and Drugs: 26
3.1.3 Animal 26
3.2 Methods 27
3.2.1 Plant Collection 27
3.2.2 Extraction of Plant Material 27
3.2.3 Determination of the Weight of the HAE 28
3.2.4 Acute Toxicity Studies 28
3.2.5 Method of Blood Glucose Level Determination 28
3.2.6 Induction of Experimental Diabetics 28
3.2.7 Effect of Graded Doses of HAE on Alloxan-induced Hyperglycaemic Rats 29
3.2.8 Effect of HAE on Oral Glucose Tolerance Test (OGTT) on a Normoglycemic Rats 30
3.2.9 Effect of HAE on OGTT on Alloxan-induced Diabetic Rats 30
3.2.10 Bioassay-guided Isolation and Identification of Active Fraction 30
3.2.10.1 Column Chromatography 30
3.2.10.2 Analytical Thin Layer Chromatography 32
3.2.10.3 Bioassay Screening of the Fractions of HAE for Bioactivity in Alloxan- Induced Hyperglycemic Rats 33
3.2.11 Phytochemical Spot Test: 34
3.2.12 In-vitro Anti-oxidant Assay of Crude and Fractions of HAE 36
3.2.12.1 DPPH Photometric Assay 36
3.2.12.2 Ferric Reducing Antioxidant Power. 37
3.2. 13 Data Analysis 37
CHAPTER FOUR: RESULTS 39
4.1 Plant Extraction 39
4.2 Acute Toxicity Test 39
4.3 Effect of HAE on Blood Glucose Level in Alloxan-induced Hyperglycemic Rats 39
4.4 Effect of HAE on OGTT in Normoglycemic Rats 39
4.5 Effect of HAE on OGTT in Alloxan-induced Hyperglycaemic Rats. 40
4.6 Yield of Fractions of HAE 40
4.7 Bioassay of Effects of the Fractions of HAE on Blood Glucose Level in
Alloxan-induced Hyperglycemic Rats. 40
4.8 Phytochemical Spot Test of Crude Extract and Fractions of HAE 41
4.9 DPPH Photometric assay for Anti-oxidant Activity of Crude and Fractions of HAE 41
4.10 Ferric Reducing Anti-oxidant Power of Crude and Fractions of HAE 41
CHAPTER FIVE: DISCUSSION AND CONCLUSION 51
5.1 Discussion 51
5.2 Conclusion 57
5.3 Recommendation 57
5.4 Challenges and Solutions 57
Reference 59
Appendix 69
INTRODUCTION
Herbal medicine has been used by many people in the developing countries for the treatment of various ailments (Akah et al., 1998). This is because herbal medicines are cheaper, less toxic and easily accessed than most orthodox medication.
Some orthodox medicines have reduced efficacy due to drug resistance, hypersensitivity reactions and other side effects (Sofowora, 2008). In recent times, plants with medicinal values have provided interesting area of research, as source of novel drug for the treatment of plethora of disease.
One of such disease, which from ancient times has posed threat to both humans and animals, is diabetes mellitus. Diabetes mellitus was used to describe the disease condition characterized by sweet urine since 1675 by Thomas Willis (Medvei, 1993).
In medieval Persia, Avicenna treated diabetes mellitus using a mixture of Lupine, Trigonella and Zedoary seed (Banting et al., 1991).
Their work led to the availability of effective treatment of diabetes. Insulin injection, though expensive, also has its own side effects, which include hypoglycaemic coma and hypersensitivity due to the development of antibodies against animal insulin in humans (Munson et al., 1995).
Many synthetic oral hypoglycaemic agents have also been developed for the management of diabetes.
These hypoglycaemic agents are expensive and also have unwanted side effects which include nausea, edema, haemodilution, hepatic failure, lactic acidosis, anorexia etc (Rang et al., 2003). They are also contraindicated in pregnancy.
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