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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
- 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
- Analyzed Nutrient Composition of the Formulated Diets (g/100g) 30
- Proximate Composition and Starch content of the freeze dried edible parts of C. albidum fruit 64
- 4. Fiber fractions of the edible parts of albidum fruit 65
- Sugar contents of the edible parts of C. albidum fruit 66
- Macro elements content of the edible parts of freeze dried C. albidum fruit 67
- Micro elements content of the edible parts of freeze dried C. albidum fruit 68
- Quantitative phytochemical constituents of the freeze dried edible parts of C. albidum fruit 69
- GC – MS Identified Phytochemical compoounds of the n-hexaneextract of Chrysopyllum albidum Seed-shell pericarp 70
- 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
- Overview of the Most Significant Symptoms of Diabetes 2
- Chrysophyllum albidum Tree 4
- Chrysophyllum albidum fruits 8
- Factors Regulating Insulin Secretion. 12
- Action Sites of Western Medicine in Diabetes Treatment 13
- Structures of Selected Anti-Diabetes Drugs 15
- Summary of ROS types and sources, and action point of antioxidants 16
- Direct reactions of vitamin E 17
- Six biochemical pathways along which glucose metabolismcan form ROS 19
- Diagrammatic representation of advanced glycation end-product (AGE) 20
- The glucotoxic effect on insulin gene expression via loss of PDX-1and MafA 22
- STZ Induced toxicity in pancreatic beta cells of rat 27
- Research Framework 31
- Urinary Glucose Screening by color – matching of test strip with color index on
test strip container 52
- DPPH radical scavenging properties of the edible parts albidum fruit extracts
and standard 81
- Ferrous-ion chelating properties of the edible parts of albidum fruit extracts
and standard 82
- Inhibition of lipid peroxidation properties of the edible parts of albidum
fruit extracts and standard 83
- Hydroxyl radical scavenging properties of the edible parts of albidum
fruit extracts and standard 84
- Hydrogen peroxide scavenging properties of the edible parts of albidum
fruit extracts and standard 85
- Alpha- Amylase Inhibitory propertiesof the edible parts of albidum fruit
extracts and standard 86
- Alpha- Glucosidase Inhibitory propertiesof the edible parts of albidum
fruit extracts and standard 87
Figure Page
- Oral Glucose Tolerance test of Normal Control and STZ-Induced Diabetic Rats
Fed with or without CAFS Diet 91
- 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
- Fiber fractions of the edible parts of albidum fruit
- Phytochemical screening of the edible parts of freeze dried albidum fruit
- Chromatogram of n- Hexane extract of albidum seed shell pericarp
- Chromatogram of n- Hexane extract of albidum Fruit pulp
- Chromatogram of n- Hexane extract of albidum Fruit skin
- Structures of compounds with both antioxidant and hypocholesterolemic properties identified from NIST database library
- Structures of compounds with hypocholesterolemic properties identified from NIST database library
- Total Phenol Standard Assay Absorbance values
- Standard Curve of Total Phenol
- Total Phenol content of seed shell pericarp aqueous extract as gallic acidEquivalent (GAE)
- Total Phenol content of seed shell pericarp methanol extract as gallic acidEquivalent (GAE)
- Total Phenol content of fruit pulp aqueous extract as gallic acidequivalent (GAE)
- Total Phenol content of fruit pulp methanol extract as gallic acidEquivalent (GAE)
- Total Phenol content of fruit skin aqueous extract as gallic acidEquivalent (GAE)
- Total Phenol content of fruit skin methanol extract as gallic acidEquivalent (GAE)
- Total Flavonoid Standard Assay Absorbance valuesStandard curve of Total Flavonoid
- Total Flavonoid content of seed shell pericarp aqueous extract as quercetin Equivalent (QE)
- Total Flavonoid content of seed shell pericarp methanol extract as quercetin Equivalent (QE)
- Total Flavonoid content of fruit pulp aqueous extract as quercetin Equivalent (QE)
- Total Flavonoid content of fruit pulp methanol extract as quercetin Equivalent (QE)
- Total Flavonoid content of fruit skin aqueous extract as quercetin Equivalent (QE)
- Total Flavonoid content of fruit skin methanol extract as quercetin Equivalent (QE)
- Total Antioxidant Activity Standard Assay Absorbance values
- Standard curve of Total Antioxidant Activity
- Total Antioxidant content of seedshell pericarp aqueous extract as ascorbic acid Equivalent (AAE)
- Total Antioxidant content of seed shell pericarp methanol extract as ascorbic acid Equivalent (AAE)
- Total Antioxidant content of fruit pulp aqueous extract as ascorbic acid Equivalent (AAE)
- Total Antioxidant content of fruit pulp methanol extract as ascorbic acid equivalent (AAE)
- Total Antioxidant content of fruit skin aqueous extract as ascorbic acid Equivalent (AAE)
- Total Antioxidant content of fruit skin methanol extract as ascorbic acid equivalent (AAE)
- Comparative Mean values of DPPH Radical Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standards
- Comparative Mean values of Ferrous ion Chelating Effects of the Edible Portions of albidum Fruit Extracts and Standard
- Comparative Mean values of Hydroxyl Radical Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standard
- Comparative Hydrogen Peroxide Scavenging Effects of the Edible Portions of albidum Fruit Extracts and Standard
- Comparative Mean values of Inhibitory Effects of the Edible Portions of albidum Fruit Extracts and Standard on alpha –Amylase activities
- Comparative Mean values of Inhibitory Effects of the Edible Portions of albidum Fruit Extracts and Standard on alpha –Glucosidase activities
- Food Intake of Normal Control and STZ-Induced Diabetic Rats- treated and –untreated
- Urine sugar screening of Normal control and Streptozotocin Induced Diabetic Rats
- Mean Relative Organs weight of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Oral glucose tolerance test of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Hepatic glycogen level of Normal control and STZ-induced Diabetic rat fed with or without CAFS diet
- Serum insulin level of Normal control and STZ-induced Diabetic rat fed with or without CAFS diet
- Glycosylated Haemoglobin levels of Normal Control and STZ-induced diabetic rats Fed
- with or without CAFS diet
- Plasma AST/ALT Ratio of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Plasma Albumin/ Globulin (A/G) Ratio of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Plasma Urea concentration of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Plasma Creatinine concentration of Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Superoxide dismutase (SOD; Unit/mg protein) activities in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Catalase (CAT; Unit/mg protein) activities in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Serum Vitamin E level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Liver and Pancreatic tissues Vitamin E level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Serum Vitamin C level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Liver and Pancreatic tissues Vitamin C level in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Liver and Pancreatic tissues reduced glutathione (GSH) concentations in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Serum reduced glutathione concentations in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Serum, Liver and Pancreatic tissues Malondialdehyde (MDA) concentration in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Total cholesterol (TC) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Triglyceride (TG) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- High density lipoprotein (HDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- Low density lipoprotein (LDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- VeryLow density lipoprotein (VLDL) levels of plasma and liver in Normal Control and STZ-Induced Diabetic Rats Fed with or without CAFS Diet
- 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
- 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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