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PROJECT TOPIC AND MATERIAL ON EVALUATION OF NUTRITIVE AND NON-NUTRITIVE COMPONENTS AND ANTI-DIABETIC PROPERTIES OF EDIBLE PORTIONS OF Chrysophyllumalbidum FRUIT IN STREPTOZOTOCIN-INDUCED DIABETIC MALE ALBINO RATS

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  • Name: EVALUATION OF NUTRITIVE AND NON-NUTRITIVE COMPONENTS AND ANTI-DIABETIC PROPERTIES OF EDIBLE PORTIONS OF Chrysophyllumalbidum FRUIT IN STREPTOZOTOCIN-INDUCED DIABETIC MALE ALBINO RATS
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ABSTRACT

Diabetes is becoming a pandemic disease despite the recent surge in new drugs to manage the condition. The limitations of currentlyavailable oral anti-diabetic agents either in terms of efficacy/safety coupled with the emergence of diabetes into a global epidemic have encouraged a concerted effort to search indigenous, inexpensive botanical sources to manage diabetes more efficiently. Chrysophyllumalbidum (Linn) belongs to Sapotaceae family and is commonly called African star apple. Its various usesas anti-oxidant, antimicrobial and anti-hyperlipidemic agents have been described in the literature.This study aimed at evaluating the nutritive and non-nutritive components and anti-diabetic properties of the edible portions (seed-shell pericarp, pulp and skin) of C. albidum fruit in streptozotocin-induced diabetic male albino rats.

 

The nutritive and non- nutritive components in seed-shell pericarp, pulp and skin of C. albidum fruit were analyzed using standard methods.In addition, the anti-diabetic capacity in different solvents of seed-shell pericarp, pulp and skin extracts of C. albidum fruit using alpha-amylase and alpha-glucosidase inhibitory assays were investigated. Furthermore, animal studies were conducted, consisting of sixty rats and divided into four groups. Diabetes was induced using 50mg/kg streptozotocin (i.p.), and a 70g/kg C. albidum fruit skin (CAFS) formulated diet was fed to streptozotocin-induced diabetic rats for 28 days to evaluate its anti-diabetic efficacy against glibenclamide (2.5mg/kg b.w.) as standard. Fasting blood glucose levels and body weights of rats were measured on weekly intervals starting with base line untill the end of treatment. Other biochemical parameters such as serum insulin, glycosylated haemoglobin, hepatic glycogen, plasma and liver lipid profile contents were evaluated at the end of treatment.Histopathological examinations of liver and pancreatic sections were also carried out.

 

Results of nutritive composition revealed that CAFS contained the highest content of copper (0.55mg/100g) and manganese (2.25mg/100g).  Also the non-nutritive contents of CAFSshowed the highest of saponin (0.41%), pectin (0.44%), cellulose (2.76%), arabinose (4854.79mg/100g) and starch (29.15%), compared with seed-shell pericarp and pulp. Hexane extract of CAFS exhibited the highest inhibitory activities on alpha-amylase (96.70%) and alpha-glucosidase (86.93%) at a concentration of 0.04mg/ml compared with other extracts studied. In vivo studies indicated that CAFS-treated rats had increased body weight from 216.94g to 226.88g and decreased fasting blood glucose from 289.11mg/dl to 122.66mg/dlbefore- and after- treatment respectively. Results of studies at the end of treatment revealed that CAFS-treatment significantly (p<0.05) increased levels of serum insulin (7.40µU/ml) and hepatic glycogen (9.63mg/g wet tissue) while it decreased glycosylated haemoglobin (5.45%), compared with diabetic (untreated) control group. CAFS-treatment reduced plasma and liver lipids except high density lipoprotein-cholesterol compared with diabetic-untreated group. These results were comparable with the standard drug-treated group. Histopathological analysis on liver and pancreas of CAFS-treatedgroup showed regenerative effects.

 

As a result of anti-hyperglycemicand anti-hyperlipidemic potentials of CAFS,it can be concluded that CAFS may have a considerable impact inpreventing the ill effects of diabetes and lipid disorders in experimental diabetes. Thus, CAFS could be used as therapeutic adjunct in the management of diabetes.

Keywords:     Chrysophyllumalbidum, Steptozotocin,Glibenclamide, Anti-diabetic-, Anti-                                   hyperlipidemic-properties.

 

Word Count: 491

TABLE OF CONTENTS

Title Page                                                                                                                                            i

Certification                                                                                                                                        ii

Dedication                                                                                                                                          iii

Acknowledgements                                                                                                                            iv

Abstract                                                                                                                                              vi

Table of Contents                                                                                                                               vii

List of Tables                                                                                                                                      xiv

List of Figures                                                                                                                                     xvi

Abbreviations                                                                                                                                      xx

Appendices                                                                                                                                         xxiii

CHAPTER ONE: INTRODUCTION

1.1 Background to the Study                                                                                                             1

  • Statement to the Problem                                                             5

1.3 Objective to the Study                                                                                                                  6

  • Research Questions             6

1.5 Hypotheses                                                                                                                                   7

1.6 Significance of the Study                                                                                                             7

CHAPTER TWO: REVIEW OF LITERATURE

2.1Chrysophyllum albidumPlant:  Description and Local Applications                                             8

2.1.1    Description and local Names                                                                                                  8

2.1.2    Scientific classification                                                                                                           9

2.1.3    Ethno-medicinal values                                                                                                           9

2.2       Chrysophyllum  albidumPlant: Phytochemical and Nutritional Potentials                             9

2.2.1    Phytochemical Constituents                                                                                                   9

2.2.2    Nutrient Compositions                                                                                                            10

2.3       Pharmacological Activities                                                                                                     11

2.3.1    Antioxidant activities                                                                                                             11

2.3.2    Hypoglycemic and hypolipidemic effects                                                                              11

 

Content                                                                                               Page

2.4       Diabetes Mellitus                                                                                                                    12

2.4.1    Diabetes and Insulin                                                                                                               12

2.4.2    Diagnostic Criteria for Diabetes                                                                                             12

2.4.3    Hypoglycemic agents                                                                                                              13

2.4.4    Free Radical Formation and Antioxidants                                                                             15

2.2.5    Mechanisms of Hyperglycemia-induced Oxidative Stress                                                     18

2.2.5.1 Glyceraldehyde Autoxidation                                                                                                            19

2.2.5.2 Protein kinase C (PKC) Activation                                                                                        19

2.2.5.3 Methylglyoxal, Glycation and Sorbitol                                                                                  20

2.2.5.4 Hexosamine Pathway                                                                                                             21

2.2.5.4 Oxidative Phosphorylation                                                                                                     21

2.2.6    Effects of Chronic Oxidative Stress on Insulin Gene Expression                                          21

2.2.7    Diabetes Complications                                                                                                          22

2.2.7.1 Microvascular Complications of Diabetes                                                                              22

2.2.7.1.1 Diabetic Retinopathy                                                                                                           22

2.2.7.1.2 Diabetic Nephropathy                                                                                                          23

2.2.7.1.3 Diabetic neuropathy                                                                                                             23

2.2.7.2 Macrovascular Complications of Diabetes                                                                             23

2.2.7.2.1 Cardiovascular Disease                                                                                                        23

2.2.7.2 .2 Cerebrovascular Disease                                                                                                     24

2.2.7.2.3 Peripheral Arterial Disease                                                                                                   24

2.2.8 Streptozotocin and its mode of Action                                                                                     25

 

CHAPTER THREE: METHODOLOGY

3.1       Materials and Methods                                                                                                           28

3.1.1   Collection and Identification of Plant materials                                                                      28

3.2       Drugs and Chemicals                                                                                                              28

3.3       Preparation of Plant Materials                                                                                                28

3.4.      Preparation of the plant extracts                                                                                             28

3.5       Feed Formulation                                                                                                                    29

Content                                                                                                         Page

3.6       Animal Care and Approval                                                                                                     30

  1. 7 Induction of experimental diabetes             31

3.8       Experimental protocol                                                                                                             32

3.9       Analyses of the Nutritive and non-Nutritive Components                                                     32

3.9.1    Proximate analysis                                                                                                                  32

3.9.2    Characterization of Carbohydrate Content by High Performance Liquid

Chromatography (HPLC)                                                                                                       33

3.9.3.   Starch and Fiber Fractions Determination                                                                              34

3.9.4    Mineral and Vitamin C Analyses                                                                                            34

3.10     Gas Chromatography-Mass spectrometry (GC-MS) and Phytochemical

Analyses                                                                                                                                  35

3.10.1 Gas Chromatography-Mass spectrometry (GC-MS) Analysis                                     35

3.10.2 Phytochemical Analysis                                                                                                           36

3.10.2.1 Preliminary Qualitative Phytochemical Analysis                                                                  36

3.10.2.1.1 Test for Saponins (Foam test)                                                                                            36

3.10.2.1.2 Test for Tannins                                                                                                                 36

3.10.2.1.3 Test for phlobatannins                                                                                                       36

3.10.2.1.4 Test for terpenoids                                                                                                             36

3.10.2.1.5  Test for alkaloids                                                                                                              37

3.10.2.1.6Test for Flavonoids (Sodium Hydroxide test)                                                                    37

3.10.2.1.7Test for cardiac glycosides (Keller-killiani test )                                                                37

3.10.2.2Quantitative Determination of Phytochemical Constituents                                                 37

3.10.2.2.1 Quantitative Determination of Alkaloid                                                                            37

3.10.2.2 .2 Quantitative Determination of Tannin                                                                              38

3.10.2.2.3Quantitative Determination of Saponin                                                                              38

3.10.2.2.4Quantitative Determination of Flavonoids                                                                         39

3.11  In vitro Anti-oxidant study of the plant extracts                                                                       39

3.11.1 Determination of total phenol content                                                                                     39

3.11.2  Determination of Total Flavonoid Content                                                                            40

3.11.3  Determination of Total Antioxidant Capacity                                                                        40

3.11.4  DPPH Radical Scavenging Assay                                                                                          41

Content                                                                                                        Page

3.11.5 Ferrous Ion-chelating Ability Assay                                                                                        41

3.11.6 Assay of Hydroxyl Radical Scavenging Activity                                                                   42

3.11.7 Assay of Anti-lipid Peroxidation Activity                                                                              42

3.11.8 Hydrogen Peroxide (H2O2) radical scavenging activity                                                          43

3.12 In vitro Antidiabetic Study                                                                                                         44

3.12.1  In vitroα- Glucosidaseinhibitory assay                                                                                    44

3.12.2  In vitroα- Amylase inhibitory assay                                                                                        44

3.13     In vivo Assay                                                                                                                           45

3.13.1 Blood Collection and Preparation of Sample                                                                          45

3.13.1.1 Blood Plasma Preparation                                                                                                     45

3.13.1.2 Blood Serum Preparation                                                                                                      46

3.13.2 Tissues Preparation                                                                                                                  46

3.13.3 In vivo Antioxidant Assay                                                                                                       46

3.13.3.1 Determination of MDA                                                                                                        46

3.12.3.2 Estimation of reduced glutathione (GSH)                                                                            47

3.12.3.3  Estimation of  Vitamin E (α-Tocopherol) Contents                                                             47

3.12.3.4  Estimation of Vitamin C (ascorbic acid) Contents                                                              48

3.12.3.5 Determination of Superoxide dismutase (SOD) activities                                                      48

3.12.3.6 Determination of Catalase (CAT) activities                                                                         49

3.12.3.7 Protein Determination                                                                                                           50

3.12.4 Anti-Diabetic Studies                                                                                                              51

3.12.4.1 Physiological Parameters                                                                                                      51

3.12.4.2. Oral glucose tolerance test (OGTT)                                                                                     51

3.12.4.3 Determination of Urine Sugar                                                                                              51

3.12.4.4 Determination of Fasting Blood Glucose Levels                                                                 52

3.12.4.5 Glycosylated Hemoglobin                                                                                                    52

3.12.4.6  Determination of serum insulin levels                                                                                  53

3.12.4.7  Estimation of Liver Glycogen Content                                                                               53

3.12.5 Haematological l Studies                                                                                                         54

3.12.6 Tissue Toxicities Studies                                                                                                          55

3.12.6.1 Liver Functions                                                                                                                     55

Content                                                                                               Page

3.12.6.1.1 Estimation of Asparate aminotransferase (AST)                                                               55

3.12.6.1.2  Estimation of Alanine aminoferase (ALT )                                                                      56

3.12.6.1.3  Estimation of Alkaline Phosphatase (ALP)                                                                      56

3.12.6.1.4  Estimation of Total protein (Biuret method)                                                                    57

3.12.6.1.5 Estimation of Total Bilirubin                                                                                             58

3.12.6.1 .6 Estimation of Direct Bilirubin                                                                                          58

3.12.6.1.7Estimation of  Plasma Albumin                                                                                          58

3.12.6.1.8Estimation of Globulin                                                                                                       59

3.12.6.2. Renal Function Analysis                                                                                                      59

3.12.6.2. 1 Plasma Urea Determination                                                                                              59

3.12.6.2. 2  Plasma Creatinine Determination                                                                                    59

3.12.6.3  Plasma Electrolyte Ions Analyses                                                                                        60

3.12.7     Antihyperlipidemic Studies                                                                                     60

3.12.7.1 Estimation of Plasma Total Cholesterol (TC) (CHOD-PAP-

Phosphotungstate method)                                                                                         60

3.12.7.2 Estimation of Triglycerides (CHOD-PAP Phosphotungstate method)                                61

3.12.7.3 Estimation of HDL-c (CHOD-PAP-Phosphotungstate method)                                         62

3.12.7.4Estimation ofLDL-c & VLDL-c                                                                                           62

3.12.7.5Atherogenic and Coronary risk Indices                                                                                 63

3.12.8  Histological Examination                                                                                                        63

3.12.9  Statistical Analysis                                                                                                      63

 

CHAPTER FOUR: DATA ANALYSIS, RESULTS AND DISCUSSION

OF FINDINGS

4.1 Data Analysis and Results                                                                                                            64

4.1.1 Evaluation of the nutritive and non-nutritive contents of the edible

Portions of Chrysophyllum albidum fruit                                                                               64

4.1.1.1 Proximate Composition and Starch content                                                                           64

4.1.1.2 Fiber and Sugar contents                                                                                                        64

4.1.1.3 Mineral contents                                                                                                                     66

Content                                                                                               Page

4.2: Phytochemical constituents of the edible Parts of Chrysophyllum albidum fruit                       68

4.3: GC-MS Studies of different solvent extracts of the edible parts of C. albidum fruit                                 69

4.3.1: C. albidum seed shell pericarp extract                                                                                      69

4.3.2: C. albidum fruit-pulp extract                                                                                                    71

4.3.3: C. albidum fruit- skin extract                                                                                                    73

4.4: In vitro Antioxidant activities of different extracts of C. albidum edible parts                         78

4.4. 1: Total Phenol Contents                                                                                                             78

4.4. 2: Total Flavonoid Contents                                                                                                        79

4.4.3: Total Antioxidant Activity                                                                                                         79

4.4.4: Antiradical Assays                                                                                                                    80

4.4.4. 1: DPPH Radical Scavenging Effects                                                                                      80

4.4.4. 2: Ferrous-Ion Chelating Effects                                                                                              81

4.4.4. 3: Anti-lipid Peroxidation Effects                                                                                            82

4.4.4. 4: Hydroxyl Radical Scavenging Effects                                                                                 83

4.4.4. 5: Hydrogen Peroxide Scavenging Effects                                                                              84

4.5:In vitro Antidiabetic activities of different extracts of C. albidum edible parts                          85

4.6Evaluation of physiological Characteristics of Diabetes                                                               88

4.6.1 Food Intake of Normal control and Streptozotocin Induced Diabetic Rats Fed

with or without CAFS Diet                                                                                                    88

4.6.2 Body weight of Normal control and Streptozotocin Induced Diabetic

Rats Fed with or without CAFS Diet                                                                                        89

4.6.3 Urine glucose of Normal Control and STZ-Induced Diabetic Rats Fed with or

without CAFS Diet                                                                                                                   89

4.6.4 Relative Organs weight of Normal Control and STZ-Induced Diabetic Rats Fed

with or without CAFS Diet                                                                                                    90

4.6.5 Oral glucose tolerance test (OGTT) of Normal Control and

STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                                     90

4.6.6 Fasting Blood Glucose (FBG) (mg/dl) of Normal Control

and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                                          91

4.6.7 Hepatic glycogen, Glycosylated heamoglobin and serum insulin levels of

Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                92

Content                                                                                               Page

4.7 Evaluation of Liver Function activities of Normal Control and STZ-induced

diabetic rats                                                                                                                            93

4.7.1 Liver enzymes  activities                                                                                                            83

4.7.2 Plasma Total protein, albumin and bilirubin of Normal Control

and STZ-Induced Diabetic RatsFed with or without CAFS Diet                                                           95

4.7.3 Electrolyte Ionsconcentrations and Renal function parameters

of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                            95

4.8 Histopathological evaluation of rats Liver and Pancreatic Tissues of Normal

Control  Group and STZ-Diabetic Treated and Untreated Groups                                        98

4.8. 1 Liver Tissues                                                                                                                             98

4.8. 2 Pancreatic Tissues                                                                                                                     99

4.9Haematological Evaluation of Normal Control and STZ-Induced Diabetic Rats             99

4.10 Serum, Hepatic and Pancreatic oxidative stress biomarkers in

Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                101

4.10.1 Serum, hepatic and pancreatic superoxide dismutase and

catalase Activities                                                                                                                   101

4.10.2 Serum, hepatic and pancreatic reduced glutathione,

vitamins C and E concentations in Normal Control and STZ-Induced Diabetic

Rats Fed with or without CAFS Diet                                                                                        102

4.10.3 Serum, hepatic and pancreatic malondialdehyde concentrations

in Normal Control and STZ-Induced Diabetic Rats                                                                  103

4.11  Plasma and Liver Lipid profilein Normal Control and STZ-Induced

Diabetic Rats Fed with or without CAFS Diet                                                                          104

4.12 Discussion of Findings                                                                                                               106

 

CHAPTER FIVE: SUMMARY, CONCLUSION AND

RECOMMENDATIONS

5.1 Summary                                                                                                             121

5.2 Conclusion                                                                                                                                    123

5.3 Recommendations                                                                                                                                    124

 

Content                                                                                               Page

5.4 Contributions to Knowledge                                                                                                        124

References                                                                                                                                         126

Appendices                                                                                                                                        144

 

 

LIST OF TABLES

Table                                                                                                               Page

1:         Chemical composition of formulated Normal control diet (NCD)                                                and CAFS-supplemented diet                                                                                                       29

  1. Analyzed Nutrient Composition of the Formulated Diets (g/100g)                         30
  2. Proximate Composition and Starch content of the freeze dried edible                                     parts of C. albidum fruit                                                                                                   64
  3. 4. Fiber fractions of the edible parts of albidum fruit 65
  4. Sugar contents of the edible parts of C. albidum fruit                                                          66
  5. Macro elements content of the edible parts of freeze dried C. albidum fruit 67
  6. Micro elements content of the edible parts of freeze dried C. albidum fruit             68
  7. Quantitative phytochemical constituents of the freeze dried edible parts of C.                    albidum fruit                                                                                                                             69
  8. GC – MS Identified Phytochemical compoounds of the n-hexaneextract of Chrysopyllum  albidum Seed-shell pericarp                                                                70
  9. GC – MS Identified Phytochemical compoounds of the n- Hexane extract of Chrysopyllum  albidum Fruit Pulp                                                                                          71

11.:      GC – MS Identified Phytochemical compoounds of the n-Hexane extract                                      of Chrysopyllum  albidum Fruit- skin                                                                                         74

12        GC – MS Identified Compounds in Chrysophyllum albidum edible portions

with Antioxidant and Hypocholesterolemic properties                                                          77

13a:     Comparative Mean values of Total Phenol, Total Flavonoid and Total Antioxidant                         Expressed as Standards Equivalent in the Edible Portions of C. albidum Fruit              Aqueous Extract                                                                                                                     78

13b.     Comparative Mean values of Total Phenol, Total Flavonoid and Total                                 Antioxidant Expressed as Standards Equivalent in the Edible Portions of                                                C. albidum Fruit Methanolic Extract                                                                                         79

14:       Mean Food intake (g) of Normal control and Streptozotocin Induced Diabetic                               Rats Fed with or without CAFS Diet                                                                                        88

15:       Mean Body Weight (g) of Normal control and Streptozotocin Induced Diabetic                             Rats Fed with or without CAFS Diet                                                                                        89

Table                                                                                                               Page

16:       Relative Organs weight of Normal Control and STZ-Induced Diabetic                                           Rats Fed with or without CAFS Diet                                                                                        90

17:       Mean Fasting blood Glucose (FBS) of Normal Control and STZ-Induced                           Diabetic Rats Fed with or without CAFS Diet                                                                92

18:       Mean Hepatic Glycogen, Glycosylated Haemoglobin and Serum Insulin of                         Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                93

19:       Mean Plasma Liver Function Parameters of Normal Control and                              Streptozotocin Induced Diabetic Rats                                                                                      94

20:       Mean Plasma Electrolyte ions concentration of Normal Control and                                    STZ-Induced             Diabetic Rats Fed with or without CAFS Diet                                              96

21:       Mean Plasma urea and creatinine levels of Normal Control and STZ-Induced                     Diabetic Rats Fed with or without CAFS Diet                                                                97

22:       Mean heamatological indices of Normal Control and STZ-Induced Diabetic Rats               100      Fed with or without CAFS Diet

23:       Comparative Mean values of Serum oxidative stress biomarkers in Normal Control                        and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                           101

24:       Comparative Mean values of Liver oxidative stress biomarkers in Normal Control              and STZ-Induced Diabetic Rats Fed with or without CAFS Diet                                              102

25:       Comparative Mean values of Pancreatic Tissue oxidative stress biomarkers                                     in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet   103

26:       Mean Plasma lipid profile of Normal Control and STZ-Induced Diabetic                                        Rats Fed with or without CAFS Diet                                                                                        104

27:       Mean Liver lipid profile of Normal Control and STZ-Induced Diabetic Rats Fed                           with or without CAFS Diet                                                                                                    105

 

 

 

 

 

 

LIST OF FIGURES

Figure                                                                                                                                      Page

  1. Overview of the Most Significant Symptoms of Diabetes             2
  2. Chrysophyllum albidum Tree                                                                                                     4
  3. Chrysophyllum albidum fruits                         8
  4. Factors Regulating Insulin Secretion.                         12
  5. Action Sites of Western Medicine in Diabetes Treatment             13
  6. Structures of Selected Anti-Diabetes Drugs                                     15
  7. Summary of ROS types and sources, and action point of antioxidants                         16
  8. Direct reactions of vitamin E                         17
  9. Six biochemical pathways along which glucose metabolismcan form ROS 19
  10. Diagrammatic representation of advanced glycation end-product (AGE)                         20
  11. The glucotoxic effect on insulin gene expression via loss of PDX-1and MafA 22
  12. STZ Induced toxicity in pancreatic beta cells of rat                         27
  13. Research Framework                         31
  14. Urinary Glucose Screening by color – matching of test strip with color index on

test strip container                                                                                                                       52

  1. DPPH radical scavenging properties of the edible parts albidum fruit extracts

and standard                                                                                                                                81

  1. Ferrous-ion chelating properties of the edible parts of albidum        fruit extracts

and standard                                                                                                                                82

  1. Inhibition of lipid peroxidation properties of the edible parts of albidum

fruit extracts and standard                                                                                                          83

  1. Hydroxyl radical scavenging properties of the edible parts of albidum

fruit extracts and standard                                                                                                          84

  1. Hydrogen peroxide scavenging properties of the edible parts of albidum

fruit extracts and standard                                                                                                          85

  1. Alpha- Amylase Inhibitory propertiesof the edible parts of albidum fruit

extracts and standard                                                                                                                  86

  1. Alpha- Glucosidase Inhibitory propertiesof the edible parts of albidum

fruit extracts and standard                                                                                                          87

Figure                                                                                                                                      Page

  1. Oral Glucose Tolerance test of Normal Control and STZ-Induced Diabetic    Rats

Fed with or without CAFS Diet                                                                                                 91

  1. Proposed model for the anti-diabetic mechanism of action of albidum fruit skin             106

 

 

 

 

LIST OF PLATES

Plate                                                                                                                                        Page

1:   Photomicrograph of Rats Hepatic tissues of normal control,diabetic

treated anduntreated rats                                                                                                                    98

2:   Photomicrograph of Rats Pancreatic tissuesof normal control,diabetic

treated anduntreated rats                                                                                                              99

 

 

ABBREVIATIONS

AAE: Ascorbic acid equivalent

ADP:   Adenosine diphosphate

AI: Atherogenic index

AICI3:             Aluminum chloride

ALB:   Albumin

ALP:   Alkaline phosphatase

ALT: Alanine aminotransferase

ANOVA:  Analysis of Variance

AST: Asparate aminotransferase

ATP: Adenosine-5-triphosphate

BSA:   Bovine serum albumin

Ca2+:    Calcium Ion

CAFS: Chrysophyllum albidumFruit-skin

CAT:   Catalase

CDNB:  1-chloro-2, 4-dintrobenzene

CHE: Cholesterol esterase

CHO: Cholesterol oxidase

COX: Cycloxygenase

CRI: Coronary risk Index

CRT: Creatinine

CVD: Cardiovascular Disease

DCCT: Diabetes Control and Complication trial

DMSO: Dimethyl sulfoxide

DNP:  Dinitrophenol

DNSA: Dinitrosalicylic acid

DPPH:  1,1 –Diphenyl 2-picryl hydrazyl (oxidzed)

DPPHH:  1,1 –Diphenyl  2-picryl hydrazine (reduced)

DTNB: 5,5-Dithiobis(2-nitro-benzoic acid)

EDTA:  Ethylenediaminetetraacetic acid

ELISA: Enzyme-linked Immunosorbent Assay

FBG: Fasting blood glucose

FFA: Free fatty acid

G-3-P: Glycerol-3-phosphate

GAE: Gallic acid equivalent

GC-MS:  Gas chromatography mass spectrometry

GK: Glycerol kinase

GOPOD: Glucose oxidase-peroxidase

GPO: Glycerol phosphate oxidase

GSH: Reduced glutathione

H & E: Haematoxylin and Eosin

H2 SO4: Sulphuric Acid

H2O2: Hydrogen peroxide

H3PO4: Phosphoric acid

Hb: Haemoglobin

HbA1c: Glycosylated hemoglobin

HCI:Hydrochloric acid

HCT: Haematocrit

HDL-c: High Density Lipoprotein cholesterol

HMG-CoA: 3-hydroxy-3-methyl-gluaryl-CoA

IC50:    Fifty percent inhibitory concentration

IFN-y:  Interferon gamma

IL:       Interleukin

K2Cr207: Potassium dichromate

KCI:  Potassium hydroxide

L:       Liter

LDL-c: Low density lipoprotein-cholesterol

LDL-c:            Low Density Lipoprotein cholesterol

LSD: Least Significant Difference

LYM: Lymphocytes

MCH: Mean corpuscular haemoglobin

MCHC: Mean corpuscular haemoglobin concentration

MCV:  Mean Corpuscular Volume

MDA: Malondialdehyde

MDH: Malate dehydrogenase

MM: Millimolar

MS: Mass selective detector

NEU: Neutrophils

NF-Nuclear Factor kappa beta

NOS: Nitric Oxide synthase

.OH:Hydroxyl Radical

PCV: Packed cell volume

PLT: Platelet Count

POD: Peroxidase

QE: Quercetin equivalent

RBC: Red blood cell

SOD: Superoxide dismutase

SPSS: Statistical Software Package of Social Science

TAC: Total antioxidant activity

TBA: Thiobarbituric acid

TC: Total cholesterol

TG: Triglyceride

µm:      Micrometer

µMol:  Micromolar

V/V:Volume/volume

VLDL-c: Very low density lipoprotein-cholesterol

W/V:   Weight/volume

WBC: White blood cell

WHO: World Health Organization

 

 

 

 

 

APPENDICES

  1. Fiber fractions of the edible parts of albidum fruit
  2. Phytochemical screening of the edible parts of freeze dried albidum fruit
  3. Chromatogram of n- Hexane extract of albidum seed shell pericarp
  4. Chromatogram of n- Hexane extract of albidum Fruit pulp
  5. Chromatogram of n- Hexane extract of albidum Fruit skin
  6. Structures of compounds with both antioxidant and hypocholesterolemic properties identified from NIST database library
  7. Structures of compounds with hypocholesterolemic properties identified from NIST database library
  8. Total Phenol Standard Assay Absorbance values
  9. Standard Curve of Total Phenol
  10. Total Phenol content of seed shell pericarp aqueous extract as gallic acidEquivalent (GAE)
  11. Total Phenol content of seed shell pericarp methanol extract as gallic acidEquivalent (GAE)
  12. Total Phenol content of fruit pulp aqueous extract as gallic acidequivalent (GAE)
  13. Total Phenol content of fruit pulp methanol extract as gallic acidEquivalent (GAE)
  14. Total Phenol content of fruit skin aqueous extract as gallic acidEquivalent (GAE)
  15. Total Phenol content of fruit skin methanol extract as gallic acidEquivalent (GAE)
  16. Total Flavonoid Standard Assay Absorbance valuesStandard curve of Total Flavonoid
  17. Total Flavonoid content of seed shell pericarp aqueous extract as quercetin Equivalent (QE)
  18. Total Flavonoid content of seed shell pericarp methanol extract as quercetin Equivalent (QE)
  19. Total Flavonoid content of fruit pulp aqueous extract as quercetin Equivalent (QE)
  20. Total Flavonoid content of fruit pulp methanol extract as quercetin Equivalent (QE)
  21. Total Flavonoid content of fruit skin aqueous extract as quercetin Equivalent (QE)
  22. Total Flavonoid content of fruit skin methanol extract as quercetin Equivalent (QE)
  23. Total Antioxidant Activity Standard Assay Absorbance values
  24. Standard curve of Total Antioxidant Activity
  25. Total Antioxidant content of seedshell pericarp aqueous extract as ascorbic acid Equivalent (AAE)
  26. Total Antioxidant content of seed shell pericarp methanol extract as ascorbic acid Equivalent (AAE)
  27. Total Antioxidant content of fruit pulp aqueous extract as ascorbic acid Equivalent (AAE)
  28. Total Antioxidant content of fruit pulp methanol extract as ascorbic acid equivalent (AAE)
  29. Total Antioxidant content of fruit skin aqueous extract as ascorbic acid Equivalent (AAE)
  30. Total Antioxidant content of fruit skin methanol extract as ascorbic acid equivalent (AAE)
  31. Comparative Mean values of DPPH Radical Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standards
  32. Comparative Mean values of Ferrous ion Chelating Effects of the Edible Portions of albidum Fruit Extracts and Standard
  33. Comparative Mean values of Hydroxyl Radical Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standard
  34. Comparative Hydrogen Peroxide Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standard
  35. Comparative Mean values of Inhibitory Effects of the Edible Portions of albidum Fruit Extracts and Standard on alpha –Amylase activities
  36. Comparative Mean values of Inhibitory Effects of the Edible Portions of albidum Fruit Extracts and Standard on alpha –Glucosidase activities
  37. Food Intake of Normal Control and STZ-Induced Diabetic Rats- treated and –untreated
  38. Urine sugar screening of Normal control and Streptozotocin Induced Diabetic Rats
  39. Mean Relative Organs weight of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  40. Oral glucose tolerance test of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  41. Hepatic glycogen level of Normal control and STZ-induced Diabetic rat fed with or without CAFS diet
  42. Serum insulin level of Normal control and STZ-induced Diabetic rat fed with or without CAFS diet
  43. Glycosylated Haemoglobin levels of Normal Control and STZ-induced diabetic rats Fed
  44. with or without CAFS diet
  45. Plasma AST/ALT Ratio of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  46. Plasma Albumin/ Globulin (A/G) Ratio of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  47. Plasma Urea concentration of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  48. Plasma Creatinine concentration of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  49. Superoxide dismutase (SOD; Unit/mg protein) activities in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  50. Catalase (CAT; Unit/mg protein) activities in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  51. Serum Vitamin E level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  52. Liver and Pancreatic tissues Vitamin E level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  53. Serum Vitamin C level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  54. Liver and Pancreatic tissues Vitamin C level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  55. Liver and Pancreatic tissues reduced glutathione (GSH) concentations in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  56. Serum reduced glutathione concentations in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  57. Serum, Liver and Pancreatic tissues Malondialdehyde (MDA) concentration in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  58. Total cholesterol (TC) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  59. Triglyceride (TG) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  60. High density lipoprotein (HDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  61. Low density lipoprotein (LDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  62. VeryLow density lipoprotein (VLDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  63. TC/HDL-c (CRI) and LDL-c/ HDL-c (AI) ratios of plasma Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
  64. TC/HDL-c (CRI) and LDL-c/ HDL-c (AI) ratios of liver Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet

CHAPTER ONE

                                                    INTRODUCTION

1.1 Background to the Study

Dietary control is vital in the management of diabetes. Reports from FAO (1998); WHO and FAO (2003) have shown that diets with low saturated fat, cholesterol and glycemic index as well as high contents of soluble fiber, vitamins and minerals are effective in the management of diabetes. Low glycemic foods contain sugars that digest and absorbed slowly into the blood and thus control blood sugar levels. The fiber-like substances such as gums and pectin reduced blood postprandial glucose levels (Jenkins et al., 1978; Ou et al., 2001) while diabetic subjects fed xanthan gum have lower fasting, postprandial serum glucose levels and total plasma cholesterol (Osilesi et al., 1985). Jenkins et al. (1978) reported that daily intake of 5–10 g of soluble fiber from different sources reduced serum cholesterol by 5–10%.  Fruits serve as one of the best sources of dietary fiber, minerals, Vitamins A, C and E and frequent intake of vegetables and fruits have demonstrated a lowered risk of diabetes, heart disease, hypertension, stroke and cancer (Southon, 2000; Wargovich, 2000). Fruits supply carbohydrates in the form of soluble sugars, cellulose and starch (Nahar et al., 1998) and serve as source of nutrient, appetizer and food supplement in a world faced with problem of food scarcity.

 

Diabetes mellitus (DM) is a worldwide endemic disease in terms of occurrence, cost of medical care, and general complications (King et al., 1998). The metabo­lism of protein, carbohydrate and fat are affected in diabetic conditions, resulting in hyperglycemia. DM complication is mainly associated with a high risk of coronary heart disease (Giugliano et al., 1996), atherosclerosis, stroke and peripheral vascular disease. The incidence of DM world wide, is projected to increase from 4% in 1995 to 5.4% by the year 2025 (Mohamed et al., 2006), with the utmost increases set to occur in the devel­oping countries of Africa, Asia and South America (WHO, 2008).

 

According to WHO (1994) and American Diabetes Association (2008), diabetes mellitus can be classified into insulin-dependent diabetes mellitus, IDDM (type 1 diabetes mellitus) and non- insulin- dependent diabetes mellitus, NIDDM (type 2 diabetes mellitus). Insulin-dependent diabetes mellitus is caused by cellular-mediated autoimmune damage to beta cells of the pancreas, accounts for about 5% to 15% of diabetic cases and occurs mostly in children or adolescents (Ranjan and Ramanujam, 2002). Genetics and environmental factors are implicated in the formation of IDDM. Administration of exogenous insulin is thus required to avert ketosis and preserve life (Lokesh and Amit, 2006). Non- insulin- dependent diabetes mellitus starts as insulin resistance, accounts for 85-95% of cases globally and occurs usually in adults of 40 years and above (WHO Regional Office for the South-East Asia, 2009). It is associated with hyperglycemia and glycosuria. The risk factors increases with age, lack of physical activity, obesity and impaired glucose tolerance.

 

Insulin resistance occurs when glucose is not properly utilized by the cells leading to high blood glucose in circulation. To maintain blood glucose level, the kidney excretes exess blood glucose through the urine and glucosuria occurs with increased excretion of water and sodium when blood glucose level exceeds the renal threshold (160 – 180 mg/L). The failure to use glucose by the body cells, results to increase appetite (polyphagia) (Robinson et al., 1986). The summary of the symptoms of diabetes is shown in Figure 1.

 

Figure 1: Overview of the most Significant Symptoms of  Diabetes

Source:  Cooke and Plotnick (2008)

 

Insulin resistance is associated with decreased glucose uptake and stimulation of muscle glycogen synthesis (Cline et al., 1999). In addition, alteration of  enzymatic activities like increased phosphatase activity and/or seryl phosphorylation of the insulin  receptor substrate by glycogen synthesis kinase 3 (GSK- 3), have also been reported in some cases of type 2 diabetes mellitus (Begum et al., 1991; Nadiv et al.,1994; Eldar-Finkelman and Krebs, 1997). Insulin resistance plays an important role in the etiology of many disorders including obesity, NIDDM, glucose intolerance, hypertension and other related disorders. It has been reported that autophosphorylation of insulin receptor kinase and subsequent phosphorylation of its principal substrate, IRS-1, are significantly lowered in insulin-responsive tissues of patient with severe obesity or NIDDM (Nadiv et al., 1992). Increased lipolysis and decreased lipogenesis occurred when there is a fall in circulated insulin leading to fatty acids release from adipose tissues and subsequently oxidized to ketone bodies in the liver. The rapid release of fatty acids into the blood leads to increase level of blood cholesterol and the formation of atherosclerosis (Khan and Ahmad, 1993). In diabetics, there is increase in excreted nitrogen through deamination, which is accompanied by cellular potassium excretion in urine when the muscle protein is broken down to support gluconeogenesis in the liver.

 

Of the several approaches applied, to lower and control the occurrence of diabetes, drug and diet therapies form the most popular approaches. The most common approach are the drug therapy with four distinct classes of oral hypoglycemic agents (biguanides, sulfonylureas, thiazolidinediones and alpha-glucosidase inhibitors) currently being recommended for use to treat NIDDM.  In dietary therapy, dietary modifications with adequate exercise are used to prevent excessive weight gain and obesity (Derek, 2001).  Intake of diets with low total and saturated fat, limited protein with replacement by complex carbohydrate and/or mono unsaturated fatty acids are the recommended diets for type 2 diabetes patients. Controlled diets will improve the metabolic control in diabetic subject and lower the risk of diabetes complications (Griver and Henry, 1994).

 

A large number of plants with hypoglycemic activity have been reported in different animal models. Aloe vera, Acacia arabica, Allium sativum L., Bombax ceiba L., Allium cepa, Brassicajuncea (L.) Cassia auriculata L., Caesalpinia bonducella (L.) and Musa sapientum L. are some of the scientifically validated antidiabetic plants (Modak et al., 2007).

 

Plant of Study

Chrysophyllum albidum (Linn), commonly called African star apple is a forest tree species of Sapotaceae family (Figure 2). It is widely distributed in Nigeria, Niger Republic and Uganda (Bada, 1997). C. albidum has various ethno-medicinal uses (Dalziel, 1937; Amusa et al., 2003) and across Nigeria, it is locally called ‘‘agbalumo’’ in South Western Nigeria and “udara” in South Eastern Nigeria.

Figure 2: Chrysophyllum albidum Tree 

Source: Orwa et al. (2009)

 

The fleshy pulp of C. albidum fruit is taken as snack, the seeds serve as a source of oil for various uses and the fruit is a good source of ascorbic acid (Adisa, 2000; Adepoju and Adeniji, 2012). C. albidum plants are rich in natural antioxidants and can thus support health by preventing oxidative stress related disease such as diabetics, cancer and coronary heart diseases (Burits and Bucar, 2002). The antioxidants content in vegetables and fruits has been associated with the diminished risk to chronic diseases by scavenging free radicals and prevent cells damage (Halliwell, 1994).

 

The antimicrobial and phytochemical screening of C. albidum seed cotyledon (Idowu et al., 2003; Okoli and Okere, 2010), leaves (Duyilemi and Lawal, 2009; Okoli and Okere, 2010; Kamba and Hassan 2011), root (Okoli and Okere, 2010), and stem bark (Adewoye et al., 2010; Kamba and Hassan 2011) have been investigated. In addition, the anti-hyperglycemic and hypolipidemic effects of C. albidum seed cotyledon ethanolic extract (Olorunnisola et al., 2008) and leaf ethanolic extract (Adebayo et al., 2010) have been reported. Adebayo et al., 2010, 2011a and 2011b, have reported the antiplatelet, antioxidant and hepatoprotective effects of C. albidum leaf while Onyeka et al. (2012) and Omotosho et al. (2013) reported the antifertility and antioxidant effects of C. albidum root bark and fruit juice.

 

Nwadinigwe (1982); Edem et al. (1984); Adisa (2000); Ige and Gbadamosi (2007); Ureigho (2010); Christopher and Dosunmu (2011); Oyebade et al. (2011); Adepoju and Adeniji (2012), have independently analyzed the nutritional contents of C. albidum pulp. Similarly, Ige and Gbadamosi (2007) analyzed the nutrient compositions of C. albidum fruit-peel (skin) and fruit juice. Ewansiha et al. (2011), analyzed C. albidum seed shell pericarp for its nutritional compositions while Ajewole and Adeyeye (1990), studied the physico-chemical characteristics and fatty acid composition of the seed. However, information on the nutrient contents of seed shell pericarp, fruit skin (peel) and fruit pulp of C. albidum are scanty in available literature. In addition, there is dearth of information on the efficacy of either of these edible portions of C. albidum fruit as remedy for the management of DM. Therefore, this study was design to investigate the nutrtitive and non-nutritive components and the antidiabetic potentials of the edible portions of C. albidum fruit.

 

1.2 Statement of the Problem

Diet has a vital role in the causes and control of several obesity-associated chronic diseases, such as diabetes and cardiovascular diseases. Current research has increased on studying individual foods to understand their specific role(s) and the mechanisms of action in the diminished risk to diseases in humans. Diabetes has emerged into a global epidemic, inspite of the recent search in new drugs to manage and prevent the condition; its prevalence continues to soar with increased risks and diagnosis in both adult and children (Ludwig and Ebbeling, 2001). In addition, many synthetic hypoglycemic agents such as biguanides, sulfonylureas, α-glucosidase inhibitors and insulin, commonly used for the treatment of diabetes are expensive and associated with serious side effects (Gupta et al., 2010). Sulfonylureas (e.g., glibenclamide) cause severe hypoglycemia, biguanides (e.g., metformins) are unsafe for patients with kidney problem, while α-glucosidase inhibitors cause dose-related malabsorption, flatulence and abdominal bloating (Codario, 2005). In addition, these hypoglycemic agents are not effective in the control of hyperlipidemia condition, which usually accompanies the incidence of diabetes (Derek, 2001). These associated problems with the synthetic oral anti-diabetic agents in terms of inefficacy, non-safety coupled with the emergence of the disease into a global epidemy have necessitate the search for more efficient alternatives with little or no side effect (Ranjan and Ramanujam, 2002). The plant kingdom, thus become a target for the search to develop indigenous, inexpensive botanical sources by multinational drug and biologically active lead compounds (Evans, 1996).

 

Since ancient times, medicinal plants with various active principles and properties have been used by laymen and physicians to cure a variety of human diseases such as coronary heart disease, diabetes and cancer (Havsteen, 1984; Middleton et al., 2000). Medicinal plants offer exciting opportunity to develop them into novel therapeutics due to their multiple beneficial effects as manipulating carbohydrate metabolism by various mechanisms, enhancing glucose uptake and utilization, restoring integrity and prevention of pancreatic β-cells damage as well as the antioxidant properties. In Nigeria, the populace is unaware of the high nutritional and nutraceutical values of C. albidum fruit; it is however considered as snack for the low-income earners. This has resulted into an information gap of its utilization as a functional food for all and sundry.

 

  • Obective of the Study

The main objective is to evaluate the nutritive and non-nutritive components and antidiabetic properties of the edible portions of C. albidum fruit (seed-shell pericarp, pulp and skin) in streptozotocin-induced diabetic male albino rats.

The specific objectives are to:

  • determine the nutritive and non-nutritivecomponents of the edible portions of albidum fruit;
  • assess the potential capability of the edible portions of albidum fruit in improving glucose tolerance, insulin sensitivity and oxidative stress factors often associated with diabetes mellitus;
  • compare the antidiabetic potency of the edible portions of albidum fruit with that of referenced drug (glibenclamide) and
  • determine the atherogenic and coronary risk indices from the lipid profile of the edible portions of albidum fruit.

 

  • Research Questions
  • What are the nutritive and non-nutritive constituents of the edible portions of albidum fruit?
  • Can the edible portions of C. albidum fruit improve glucose tolerance, insulin sensitivity and oxidative stress factors often associated with diabetes mellitus?
  • Can the edible portions of C. albidum fruit be as potent as the referenced drug (glibenclamide) in the control of DM?
  • Will the atherogenic and coronary risk indices of the edible portions of albidum fruit be low or high?

 

1.5 Hypotheses

The null hypothesis and alternative hypothesis at p<0.05 are as stated below:

Ho1:The nutritive and non-nutritive components of C. albidum edible portions (seed-shell                          pericarp, pulp and skin) do not possess antihyperglycemic and hypolipidemic                                          properties.

Ho2:The nutritive and non-nutritive components of C. albidum edible portions (seed-shell

pericarp, pulp and skin) possess antihyperglycemic and hypolipidemic properties.

 

1.6 Significance of the Study

The result of this study would be of great significance to the scientific community because it would provide evidence-based information on C. albidum fruit for Nutritionists and Dietitians for dietary management of DM in various communities. C. albidum fruit is readily available in the West Africa sub-region, cheap and with adequate information on its contributions to dietary management of DM. The results would serve as basis for useful information gathering to Food scientists, Biochemists and Pharmacists to research further on the medicinal potentials of C. albidum fruit. Results would also provide valuable information for use in compiling the food composition table of Nigerian food staples. Results would create public awareness of the use of C. albidum fruit as remedy for the management of DM and improve its consumption when in season. Equally, the results would  draw positive attention to attract Scientists to do further investigations on the efficacy of the fruit as remedy for the management of diabetic vascular related complications and other nutritionally related diseases.

 

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