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OKOLIE, AMANDA CHIKAOSOLU

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Download the complete Biochemistry topic and material (chapter 1-5) titled MODULATION OF HIGH FRUCTOSE FED, STREPTOZOTOCIN-INDUCED TYPE 2 DIABETES BY WONDERFUL KOLA (BUCHHOLZIA CORIACEA) SEED EXTRACTS IN MALE WISTAR RATS here on PROJECTS.ng. See below for the abstract, table of contents, list of figures, list of tables, list of appendices, list of abbreviations and chapter one. Click the DOWNLOAD NOW button to get the complete project work instantly.

 

PROJECT TOPIC AND MATERIAL ON MODULATION OF HIGH FRUCTOSE FED, STREPTOZOTOCIN-INDUCED TYPE 2 DIABETES BY WONDERFUL KOLA (BUCHHOLZIA CORIACEA) SEED EXTRACTS IN MALE WISTAR RATS

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  • Name: MODULATION OF HIGH FRUCTOSE FED, STREPTOZOTOCIN-INDUCED TYPE 2 DIABETES BY WONDERFUL KOLA (BUCHHOLZIA CORIACEA) SEED EXTRACTS IN MALE WISTAR RATS
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  • Length: [71] Pages

 

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

 

 

 

 

Content                                                                                                                       Page

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

 

  1. Percentage weight yield 46
  2. Quantitative Phytochemical Analysis 47
  3. Acute Oral Toxicity Test 52
  4. Bioactivity of Compounds Detected through GC/MS Analysis 71

 

LIST OF FIGURES

Figure                                                                                                                    Page

  1. The mechanism of streptozotocin (STZ)-induced toxic events 16
  2. Fructose Metabolism                                                                                     18
  3. Buchholziacoriaceaseed and identification details 19
  4. DPPH Radical Scavenging                                                                         48
  5. In vitro nitric oxide radical scavenging activities 49
  6. In vitro hydroxyl radical scavenging activities                                     50
  7. α-Amylase inhibitory activities             51
  8. Blood Glucose Level                                                                         53
  9. Lipid Profile                                                                                     54
  10. Serum Insulin                                                                                                 55
  11. Liver Biomarker Enzymes                                                             56
  12. Kidney Function markers                                                                                     57
  13. Tissue Protein Concentration                                                 58
  14. Lipid peroxidation                                                                                     59
  15. Reduced Glutathione                                                                         60
  16. Catalase                                                                                     61
  17. Glutathione Peroxidase                                                 62
  18. Superoxide Dismutase                                                 63
  19. 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:

  1. 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;
  2. 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.
  3. Carryout body and organ weights as well as food and water intake assessments.
  4. 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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