SONOGRAPHIC INVESTIGATION OF THE EFFECT OF GONGRONEMA LATIFOLIUM ON GASTRIC EMPTYING OF SEMI - SOLID MEALS IN HEALTHY, DIABETIC AND GASTROPARETIC DOGS

ABSTRACT

Gongronema latifolium is an edible tropical rainforest plant traditionally used as a spice, staple vegetable and in the treatment and management of diabetes. Extensive studies have established its hypoglycaemic effect. However, scanty information is available on the mechanism by which it exerts its hypoglycaemic effect. Studies have noted the presence of saponin in G. latifolium, a component noted to affect gastric emptying (GE). Presently, the possible effect of G. latifolium on GE has not been investigated. Chronic diabetes may precipitate gastroparesis. How such condition may respond to treatment with G. latifolium is yet to be investigated. Therefore, this study investigated sonographically the possible effect of G. latifolium on GE in healthy and diabetic dogs. It also investigated if the effect of G. latifolium on GE in healthy and/or diabetic dogs is dose-dependent. The study further ascertained the relationship between its hypoglycaemic effect and GE. Finally, it investigated the possible effect of G. latifolium on GE in gastroparetic dogs. In a randomized placebo-controlled experimental design, sixty mongrel dogs whose mean age of 6.07 ± 0.80 months and mean weight of 5.20 ± 1.23kg did not vary significantly (p > 0.05) were randomly allotted to healthy, diabetic and gastroparetic groups. Twenty dogs in each group were randomly allotted into five subgroups. The subgroups were the placebo that served as control, low dose (100mg/kg), moderate dose (250mg/kg) and high dose (500mg/kg) of G. latifolium. A prokinetic dose of 0.5mg/kg of metoclopramide also served as a positive control. Intravenous injection of 100mg/kg of alloxan monohydrate was used to induce diabetes and 0.03mg/kg of clonidine injected subcutaneously in the back of the neck immediately after ingestion of a test meal, was used to induce gastroparesis. G. latifolium was extracted with 80% methanol. The coarse powdered extracts were pulverised, sieved, weighed out in doses of 100mg, 250mg and 500mg and subsequently encapsulated. After a 12 - hour fast, each dog was dosed once orally with the appropriate extract or drug depending on the group, 30 minutes before the administration of the test meal. The test meal used consisted of 100g Nestle cerelac and 150ml of water. GE was estimated by ultrasound measurements of gastric antral area. Antral images were acquired 30 minutes before ingestion of test meal and at regular intervals for the next six hours. Initial baseline measurements of the antral area were taken, from which subsequent measurements after the ingestion of the test meal were subtracted. All measurements were expressed as percentage of the maximal antral area measured during each test and plotted against time. Gastric antral area was measured concurrently with blood glucose concentrations using glucometer. The percentage maximal antral area and incremental blood glucose concentrations were computed and plotted against time respectively. The time in minutes at which the antral area decreased to 50 percent of its maximal area (T50) was used to describe the rate of GE. The blood glucose area under the curve (AUC) in mmol/L x minutes was calculated from the incremental postprandial blood glucose curve. All values were expressed as mean ± standard deviation. ANOVA with Dunnett’s post test were used to compare the means of the groups with control. Paired t test was used to compare pre and post intervention values while Pearson correlation was used to assess the linear association between the values of two variables. These analyses were performed using SPSS version 15.0 and GraphPad prism version 5.03. Probability (p) values less than 0.05 were considered significant. The GE in minutes in healthy dogs were 135.5 ± 3.5, 155.5 ± 3.9 ,198 ± 5.3 and 59 ± 2.5 for low, moderate, high doses of G. latifolium and prokinetic dose respectively whereas for the diabetic dogs the values were 169.8 ± 3.8, 227.8

 

± 9.9, 261.3 ± 19.3 and 107.3 ± 13.2 respectively. The values for the control that received the placebo were 126.6 ± 8.2 for the healthy and 143 ± 17.8 for the diabetics. In both the healthy and diabetic dogs treated with G. latifolium the GE values were significantly longer (p < 0.0001) compared with the placebo groups for only moderate and high doses. There was no significant (p > 0.05) difference between the low dose and the control in health and diabetes. The prokinetic groups GE were significantly shorter in healthy (p < 0.0001) and diabetics (p < 0.001) compared with their placebo controls. Significant positive correlations between dose and GE were noted in healthy (r = 0.94; p < 0.0001) and 21 diabetics (r = 0.98; p < 0.0001) treated with G. latifolium. The dose trends of G. latifolium on GE were also significant (p < 0.0001) in healthy and diabetics. The blood glucose AUC in healthy dogs were 938.1 ± 40, 559.1 ± 101.8 , 223.5 ± 52.2 and 1426 ± 108.2 for low, moderate, high doses of G. latifolium and the prokinetic dose respectively whereas for the diabetic dogs the values were 3357 ± 539.1, 1905 ± 434 , 385.3 ± 298.1 and 6485 ± 322.4 respectively. The values for the controls were 1028.6 ± 204.2 for the healthy and 3453 ± 217.6 for the diabetics. A strong inverse relationship between the hypoglycaemic effect of G. latifolium and GE was noted in healthy (r = -0.95; p < 0.0001) and diabetics (r = -0.90; p < 0.0001). The GE for the control, low, moderate, high doses and prokinetic dose were 266.5 ± 16.9; 253 ± 20.2; 252 ± 14.1; 262.3 ± 17.6; 226 ± 8.7 respectively in gastroparetics. There was no significant (p > 0.05) difference between the GE of low, moderate and high doses of G. latifolium and the control whereas the prokinetic group GE values were significantly shorter (p< 0.001) compared with gastroparetic control.

 

TABLE OF CONTENTS

1.0 CHAPTER ONE: INTRODUCTION

1.1 Background of the study……………………………………………………………….1

1.2 Objective of study……………………………………………………………………...5

1.3 Hypotheses……………………………………………………………………………..6

1.4 Significance of the study……………………………………………………………….6

1.5 Scope of Study…………………………………………………………………………7

1.6  Limitations of the study…………………………………………………………………..7

2.0 CHAPTER TWO: LITERATURE REVIEW

2.1 Gongronema latifolium………………………………………………………………..8

2.1.1 Phytochemical analysis………………………………………………………………..8

2.1.2  Traditional Uses………………………………………………………………………..8

2.1.3  Mechanism of action…………………………………………………………………...9

2.2 Major determinants of Glycemia……………………………………………………..11

2.3 Gastric Emptying……………………………………………………………………..11

2.3.1 Mechanism…………………………………………………………………………...12

2.3.2 Assessment of Gastric emptying……………………………………………………..13

2.3.3 Factors that affect gastric emptying………………………………………………….15

2.3.4 Gastric emptying studies in dogs…………………………………………………….19

2.4 Diabetes Mellitus……………………………………………………………………..21

2.4.1 Pathogenesis………………………………………………………………………….22

2.4.2 Prevalence……………………………………………………………………………22

2.4.3 Symptoms…………………………………………………………………………….23

2.4.4 Types…………………………………………………………………………………23

2.4.5 Complications………………………………………………………………………...24

2.5 Delayed Gastric emptying (Gastroparesis)…………………………………………...25

2.5.1 Prevalence……………………………………………………………………………25

2.5.2 Causes………………………………………………………………………………..26

2.5.3 Symptoms and Complications of gastroparesis……………………………………...27

2.6 Prokinetic agents……………………………………………………………………..27

2.7 Justification for this study…………………………………………………………....28

3.0 CHAPTER THREE: RESEARCH METHODS

3.1 Design…………………………………………………………………………………29

3.2 Plant Preparation……………………………………………………………………...30

3.2.1  Plant Selection………………………………………………………………………...30

3.2.2  Instruments/Chemicals………………………………………………………………..32

3.2.3  Plant Extraction……………………………………………………………………….32

3.2.4  Encapsulation of Plant extracts……………………………………………………….33

3.2.5  Acute toxicity tests……………………………………………………………………35

3.3 Subjects……………………………………………………………………………….35

3.3.1  Subject Selection……………………………………………………………………...35

3.3.2  Ethical Clearance……………………………………………………………………...36

3.3.3  Instruments/Equipment……………………………………………………………….36

3.3.4  Drugs/foods/chemicals………………………………………………………………..37

3.3.5  Subject preparation……………………………………………………………………37

3.4 Categorization of subjects…………………………………………………………….39

3.5 Test meal……………………………………………………………………………...40

3.6 Procedures…………………………………………………………………………….40

3.6.1  Test meal preparation…………………………………………………………………40

3.6.2  Induction of Diabetes…………………………………………………………………42

3.6.3  Induction of gastroparesis…………………………………………………………….42

3.6.4  Interventions…………………………………………………………………………..44

3.7 Data Collection………………………………………………………………………..48

3.7.1  Measurement of gastric emptying…………………………………………………….48

3.7.2  Measurement of Blood glucose……………………………………………………….52

3.8 Data Analysis…………………………………………………………………………54

4.0 CHAPTER FOUR: PRESENTATION OF RESULTS

4.1 Gastric emptying study in healthy dogs……………………………………………….56

4.1.1  Sex of the healthy dogs……………………………………………………………….56

4.1.2  Age of the healthy dogs……………………………………………………………….56

4.1.3  Weight of the healthy dogs……………………………………………………………57

4.1.4 Fasting blood glucose concentrations of the healthy dogs…………………………...58

4.1.5 Gastric emptying curve of the healthy dogs………………………………………….60

4.1.6 Effect of treatment interventions on T50 (minutes) in healthy dogs…………………61

4.1.7 Test of hypothesis 1…………………………………………………………………..63

4.2 Gastric emptying study in diabetic dogs………………………………………………63

4.2.1  Sex of the diabetic dogs……………………………………………………………...63

4.2.2  Age of the diabetic dogs……………………………………………………………..64

4.2.3  Weight of the diabetic dogs………………………………………………………….65

4.2.4  Fasting blood glucose concentrations of the diabetic dogs………………………….69

4.2.5  Gastric emptying curve of the diabetic dogs………………………………………...72

4.2.6  Gastric emptying T50 (minutes) of the diabetic dogs……………………………….73

4.2.7  Test of hypothesis 2………………………………………………………………….75

4.3 Blood glucose response during the gastric emptying study in healthy dogs………......76

4.3.1  Incremental blood glucose curve of the healthy dogs………………………………...76

4.3.2  Incremental postprandial blood glucose concentrations of the healthy dogs…………77

4.3.3 Test of hypothesis 3…………………………………………………………………..80

4.3.4  Peak incremental postprandial blood glucose concentrations of the healthy dogs…...82

4.4 Blood glucose response during the gastric emptying study in diabetic dogs………….86

4.4.1  Incremental blood glucose curve of the diabetic dogs………………………………..86

4.4.2  Incremental postprandial blood glucose concentrations of the diabetic dogs………...87

4.4.3 Test of hypothesis 4…………………………………………………………………..89

4.4.4  Peak incremental postprandial blood glucose concentrations of the diabetic dogs…..92

4.5 Gastric emptying study in gastroparetic dogs…………………………………………95

4.5.1  Sex of the gastroparetic dogs…………………………………………………………95

4.5.2  Age of the gastroparetic dogs………………………………………………………...96

4.5.3  Weight of the gastroparetic dogs……………………………………………………..97

4.5.4  Fasting blood glucose concentrations of the gastroparetic dogs…………………….98

4.5.5  Gastric emptying curve of the gastroparetic dogs…………………………………....99

4.5.6  Effect of treatment interventions on T50 (minutes) of the gastroparetic dogs……...100

4.5.7  Test of hypothesis 5………………………………………………………………....101

4.5.8 Blood glucose response during the gastric emptying study in gastroparetic dogs…...103

4.5.9  Incremental postprandial blood glucose concentrations of the gastroparetic dogs…104

4.5.10 Peak incremental postprandial blood glucose concentrations of the gastroparetic dogs……………………………………………............................108

5.0 CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion……………………………………………………………………………112

5.1.1 Effect of G. latifolium on gastric emptying in healthy and diabetic dogs…………112

5.1.2 Hypoglycemic effects of G. latifolium and gastric emptying in healthy and diabetic dogs…….118

5.1.3 Effect of G. latifolium on gastric emptying and blood glucose response in gastroparetic dogs.121

5.2 Conclusion……………………………………………………………………….123

5.3  Recommendation………………………………………………………………………124

REFERENCES…………………………………………………………………………….125

APPENDICES……………………………………………………………………………..147