ABSTRACT
Type 2 diabetes (T2D) occurs when there is an advanced determent in insulin action (insulin resistance, IR), which proceeds toβ-cell dysfunction. This present study assessed the modulatory effects of Buchholziacoriacea(B. coriacea) seeds extract in high fructose-fed, streptozotocin-induced T2D in male Wistar rats.
Methanolic extract (MEBC), hexane fraction (HFBC), ethyl acetate fraction (EFBC) and n-butanol fraction of BC (BFBC) were prepared using 70% methanol and successive solvent partitioning method respectively. Antioxidant activities of 1-1-diphenyl 2-picryl hydrazyl (DPPH), nitric oxide radical scavenging assay (NOSA) and hydroxyl radical scavenging activities (HRSA) were assessed as well as α-amylase inhibition in vitro. High fructose (20%, p.o) (2 weeks) followed by streptozotocin (STZ) (40 mg/kg, i.p.) (FRU + STZ) (day 14) administered to achieve T2D in vivo. Control normal and diabetic untreated (FRU + STZ) rats were administered carboxymethyl cellulose (CMC) (1 ml/kg, p.o). Diabetic treated rats received BFBC (20, 200, 400 mg/kg, p.o), metformin (7.14 mg/kg, p.o) and glibenclamide (0.07 mg/kg, p.o) respectively.
BFBC had the highest percentage yield, DPPH and α-amylase inhibition activities, although, EFBC had a better inhibitory activities on HRSA and NOSA respectively. Also, untreated diabetic rats showed increase (p< 0.05 – 0.001) in blood glucose levels (BGLs), insulin (6 folds) and lipid peroxidation (LPO) levels in pancreas when compared with normal group. BFBC (20, 200, 400 mg/kg) showed decrease (p< 0.05) in BGLs in a time dependent manner in the BFBC treated animals.Similarly, BFBC produced a dose dependent decrease in serum insulin levels by 51% (20 mg/kg), 54% (200 mg/kg) and 70% (400 mg/kg) respectively.These effects were also comparable to metformin and glibenclamide. BFBC treatments elevated (p> 0.05) high density lipoprotein, but decreased (p> 0.05) triglycerides, total cholesterol and low density lipoprotein levels compared with control group while it lowers plasma alkaline phosphatase activities and urea (p< 0.05) compared with untreated group. BFBC (400 mg/kg) elevated total protein levels in the pancreas and heart by 103% and 7% compared with the untreated rats. Treatment of diabetic rats with BFBC elevated the body weights by 21% (20 and 200 mg/kg) and 36% (400 mg/kg) respectively. BFBC when administered did not significantly alter hematological, electrolytes and antioxidant enzyme activities in all rats. Histological assessments showed that sections of the pancreas, liver, kidney and heart from BFBC treated animals had reduced tissue damage compared with the untreated groups. Fourteen (14) bioactive compounds highest in oleic, stearic, 2-methyl-pyrrolidine-2-carboxylic, n-hexadecanoic, and 13-docosenoic acids were present in BFBC given Gas-Chromatography/Mass-Spectrometry analysis.
Thediabetic animal model was able to present the natural history of the disease in human T2D. Also, BFBC doses used in this study demonstrate potentials against in vitro and in vivo oxidative stress, hyperinsulinaemia, dyslipidemia as well as declension in β-cell function in T2D rat experiment. Further, application of some derivatives of BFBC in the treatment of problems associated with T2Dmay be useful.
Keywords: Buchholziacoriacea, Streptozotocin, Fructose, Type 2 Diabetes, Metformin, Glibenclamide.
Word Count: 487
TABLE OF CONTENTS
Content Page
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Abstract v
Table of Contents vi
List of Tables x
List of Figures xi
List of Plates xii
CHAPTER ONE: INTRODUCTION
1.1: Background to the Study 1
1.2: Statement of the Problem 3
1.3: Objective of the Study 4
1.4: Significance of the Study 5
CHAPTER TWO: REVIEW OF LITERATURE
2.1: History of Traditional Medicine 6
2.2: Geographical Distribution 6
2.3: Taxonomy 7
2.4: Common Names 7
2.5: General Description 8
2.6: Uses 8
2.7: Ethno-Pharmacological Properties of BuchholziaCoriacea 9
2.7.1: Antimicrobial and Anthelmintic Properties 9
2.7.2:Antihypercholesterolemic Activity 9
2.7.3: Anti-Ulcer and Gastric Anti-Secretory Activities 9
2.7.4: Effects on Male Reproductive Parameters 9
2.7.5:Immunomodulatory Effect 10
2.7.6: Hypoglycemic Properties 10
2.7.7: Phytochemicals, Mineral and Proximate Analysis 10
2.8: Antioxidants 11
2.9: Antioxidant Enzymes 11
2.10: Diabetes Mellitus 12
2.10.1: Epidemiology and Etiology of Type 2 Diabetes 13
2.10.2: Pathogenesis of Type 2 Diabetes 13
2.10.3: Environmental Factors in the Pathogenesis of Type 2 Diabetes 14
2.10.4: Pathophysiology of Type 2 Diabetes 14
2.10.5: Complications of Diabetes Mellitus 15
2.10.6: Oxidative Stress in Diabetes Mellitus 15
2.11:Streptozotocin Mechanism of Action 16
2.12: Dietary Fructose 17
Content Page
2.12.1: Fructose Metabolism 17
CHAPTER THREE: METHODOLOGY
3.1: Materials 19
3.1.1: Collection and Identification of the Plant 19
3.1.2: Drugs and Chemicals 19
3.1.3: Equipment 20
3.1.4: Experimental Animals 20
3.2: Methods 21
3.2.1: Preparation of Extracts 21
3.2.1.1: Extracts Fractionation 21
3.2.2: Quantitative Phytochemical Analysis 21
3.2.2.1: Determination of Total Phenolic Content 21
3.2.2.2: Total Flavonoid Content 22
3.2.2.3:Determination of Tannin Concentration 22
3.2.3: Gas Chromatography/Mass Spectrometry (GC/MS) Analysis 22
3.2.4: In-Vitro Assays for Antioxidant Property of Buchholziacoriacea 23
3.2.4.1: Determination of DPPH Radical Scavenging Activity 23
3.2.4.2: Inhibition of Nitric Oxide Radical 23
3.2.4.3: Assay of Hydroxyl Radical Scavenging Activity 24
3.2.5: In-Vitro Antidiabetic Study 25
3.2.5.1: Alpha Amylase Inhibition Assay 25
3.2.6: Acute Toxicity Test 26
3.2.7: Experimental Design 26
3.2.8: Induction of Type 2 Diabetes 27
3.2.8.1: Preparation of Fructose Solution 27
3.2.8.2: Injection of Streptozotocin 27
3.2.8.3: Confirmation of Diabetes and Measurement of Weekly Blood Glucose 27
3.2.9: Physiological Measurements 27
3.2.10: Necropsy 27
3.2.11: Ethical Concerns in Animal Study 28
3.2.12: Blood Glucose Estimation 28
3.2.13: Lipid Profile Assessment 29
3.2.13.1: Determination of Serum Total Cholesterol 29
3.2.13.2: Determination of Serum Triglycerides 29
3.2.13.3: Determination of Serum HDL Cholesterol 30
3.2.13.4: Determination of Serum LDL Cholesterol 30
3.2.14: Liver Enzyme Markers 31
3.2.14.1: Determination of Plasma Alanine Amino TransferaseActivity 31
3.2.14.2: Determination of Plasma Aspartate Amino Transferase Activity 32
3.2.14.3:Alkaline Phosphatase Activity 33
3.2.15: Kidney Function Markers 33
3.2.15.1: Serum Creatinine 33
3.2.15.2: Serum Urea 34
3.2.16:Determination of Tissue Protein Concentration 35
Content Page
3.2.17: Oxidative Stress Markers 37
3.2.17.1: Assessment of Lipid Peroxidation 37
3.2.17.2: Determination of Catalase Activity 38
3.2.17.3: Determination of Superoxide Dismutase Activity 39
3.2.17.4: Determination of Reduced Glutathione Level 40
3.2.17.5: Assay of Glutathione Peroxidase Activity 42
3.2.18: Insulin Assay 43
3.2.19: Full Blood Count 44
3.2.20:Histopathological Study 45
3.2.21: Statistical Analysis 45
CHAPTER FOUR: DATA ANALYSIS, RESULTS AND
DISCUSSION OF FINDINGS
4.1: Percentage Yield of Solvent Fractions 46
4.2: Quantitative Phytochemical Analysis 47
4.3: In Vitro Antioxidant Studies 48
4.3.1: DPPH Inhibition Assay 48
4.3.2: Nitric Oxide Radical Scavenging Assay 49
4.3.3: Hydroxyl Radical Scavenging Assay 50
4.4: Alpha Amylase Inhibition Assay 51
4.5: Acute Toxicity Test 52
4.6: Blood Glucose Level 53
4.7: Lipid Profile 54
4.8: Serum Insulin 55
4.9: Liver Function Tests 56
4.10: Kidney Function Tests 57
4.11: Tissue Total Protein Concentration 58
4.12: In Vivio Antioxidant Assays 59
4.12.1 Lipid Peroxidation 59
4.12.2: Reduced Glutathione 60
4.12.2: Catalase 61
4.12.3: Glutathione Peroxidase 62
4.12.4: Superoxide Dismutase 63
4.13: Serum Electrolytes 64
4.14: Full Blood Count 64
4.15: Body Weight Measurement 64
4.16: Diet and Water Intake 65
4.17: Visceral Organ weight 65
CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS
5.1: Summary 73
5.2: Conclusion 77
5.3: Recommendations 77
REFERENCES 78
APPENDIX 93
LIST OF TABLES
Table Page
- Percentage weight yield 46
- Quantitative Phytochemical Analysis 47
- Acute Oral Toxicity Test 52
- Bioactivity of Compounds Detected through GC/MS Analysis 71
LIST OF FIGURES
Figure Page
- The mechanism of streptozotocin (STZ)-induced toxic events 16
- Fructose Metabolism 18
- Buchholziacoriaceaseed and identification details 19
- DPPH Radical Scavenging 48
- In vitro nitric oxide radical scavenging activities 49
- In vitro hydroxyl radical scavenging activities 50
- α-Amylase inhibitory activities 51
- Blood Glucose Level 53
- Lipid Profile 54
- Serum Insulin 55
- Liver Biomarker Enzymes 56
- Kidney Function markers 57
- Tissue Protein Concentration 58
- Lipid peroxidation 59
- Reduced Glutathione 60
- Catalase 61
- Glutathione Peroxidase 62
- Superoxide Dismutase 63
- GC/MS Chromatogram of butanol fraction of coriacea seed extract 70
LIST OF PLATES
Plate Page
- Histological Examination of the Pancreas 66
- Histological Examination of the Liver 67
- Histological Examination of the Heart 68
- Histological Examination of the Kidney 69
CHAPTER ONE
INTRODUCTION
1.1 Background to the Study
Diabetes mellitus is referred to as a metabolic disorder in which there is high glucose level in the blood as a result of insulin deficiency, resistance or both (American Diabetes Association, 2009). It has been deduced globally that the adult population with diabetes will rise by 69% for the year 2030 (Shaw et al., 2010). Type 2 diabetes (T2D) occurs when there is an advanced determent in insulin action (insulin resistance, IR), which proceeds to β-cell dysfunction due to the failed ability of pancreatic β-cells to compensate for IR (Srinivasan et al., 2005). The number of people suffering from diabetes worldwide is estimated to be 215 million and 80–90% of them from T2D (Procopiou and Philippe, 2005). Sedentary lifestyle such as taking high-calorie containing food, lack of exercise, ageing are all risk factors for T2D and hence conduce to the recent rising prevalence of obesity and T2D (Aude et al., 2004). Streptozotocin (STZ) has been utilized broadly for induction of diabetes both type 1 diabetes and T2D in experimental animals (Szkudelski, 2001). Unfortunately, it lacks the ability to induce IR directly which is one of the pathogenesis of T2D, rather, it induces diabetes from direct pancreatic β-cells damage which resembles a typical T1D (Srinivasan et al., 2005).
Reports gotten from various studies showed that high fat or fructose diet induced IR in experimental animals but failed to induce hyperglycemia (Srinivasan et al., 2005; Wilson and Islam, 2012). In as much as giving animals a high fructose load alone can induce IR, several weeks may be required to achieve this. Hence, the cost and duration of the study will be high. More so, the animals can naturally develop nutritional tolerance when exposed to longtime feeding with fructose without developing signs and symptoms of IR and impaired glucose tolerance (Stark et al., 2000). Therefore, the search and development of a suitable T2D rat model that will boycott the setbacks experienced in using fructose or STZ as single agents to induce IR and T2D came into place. Interestingly, the model has been achieved through a combined effect of fructose and STZ. High fructose load induced IR while a low dose of STZ caused the initial β cell dysfunction and subsequently hyperglycemia (Wilson and Islam, 2012; Stalin et al., 2016). This model is a close replica of the natural history of T2D and its metabolic features in humans. More so, it is cheaper, readily available and useful for investigation of various compounds. Plants have been used extensively for treatment of disease due to the fact that they can produce multifarious basic biochemical and organic substances such as carbohydrates, proteins, terpenes, steroids, alkaloids and glycosides (Andrews, 1982).
Buchholzia Coriacea (B. Coriacea) a perennial plant belonging to the family capparidaceae and genus Buchholzia is popularly known as wonderful kola (Quattrochi-Umbetto, 2007). Earlier studies carried out on different parts of this plant shows that it has great medicinal potentials (Oluseyi and Francisca, 2009; Fred-Jaiyesimi et al. 2011; Adisa et al., 2011; Obembe et al., 2012; Olaiya and Omolekan 2013; Ibrahim and Fagbohum, 2013; Enenchi and Nwodo 2014; Eze et al., 2015). However, there is currently no study carried out to assess the efficacy of B. Coriacea in T2D.
Therefore, this present study assessed the possible modulatory effects of BC on high fructose-fed, STZ-induced T2D in male Wistar rats in order to ascertain its involvement and as well characterize the active compounds that may be present.
1.2 Statement of the Problem
Insulin resistance, hyperlipidaemia, β-cell dysfunction and their associations are major risk factors for the development of T2D and cardiovascular complications (Lender and Sysko, 2006). To mitigate these serious complications and negative outcome of T2D, the control not only of blood glucose but also of lipids is essential (Moller, 2001). More so, the available therapeutic options such as a combination of synthetic hypolipidaemic and antidiabetic drugs have their own setbacks (Lender and Sysko, 2006). Therefore, there is a need for new agents which will be more effective, readily available and with minimal side effects.
1.3 Objective of the Study
The general objective of this study was to evaluate the possible modulatory effects of the B. coriacea seed extracts in high fructose-fed, streptozotocin-induced T2D in male Wistar rats. The specific objectives are to:
- Carryout preliminary invitro analysis on the B. coriacea seed extracts including phytochemical components, the antioxidant properties and characterization of the bioactive compounds using GC/MS;
- Carryout biochemical and oxidative stress assays including blood glucose levels, serum insulin, hematology, body electrolytes, liver function tests, renal function tests, lipid peroxidation, reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase.
- Carryout body and organ weights as well as food and water intake assessments.
- Carryout the histopathology of the pancreas, liver, heart and kidney.
1.4 Significance of the Study
This study will increase our understanding of the pathophysiological role of fructose-fed, streptozotocin-induced T2D in vivo as well as evaluate the possible modulatory role of B. coriacea. In addition, it is hope that constituent compounds present in B. coriacea would aid further scientific investigations while contributing to the field of diabetology and or harnessing drug discovery and development.
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