ESTIMATION OF THE EFFECT OF CISAMPELOUS OWANRENSIS BENZENE FRACTION ON GLUCOSE ABSORPTION ACROSS THE INTESTINAL EPITHELIUM OF WISTER RATS ON ALOXAN INDUCED DIABETES
ABSTRACT
Diabetes is a global public health crisis that threatens the economies of all nations, particularly developing countries. It is generally characterized by hyperglycaemia and hyperlipidaemia culminating in severe morbidities. This study aimed to assess the effects of Cissampelos owanrensis on glucose absorption in alloxan-induced diabetic male Wistar rats. Twenty (20) adult Wistar rats were divided into four (4) groups of five rats each (n=5). Groups 1 and 2 served as the control groups and group 3 and 4 were induced with diabetes by an intraperitoneal injection of alloxan (40mg/kg). The normoglycaemic groups (1 and 2) and the alloxan-induced diabetic groups (3 and 4) were treated with 300mg/kg of Cissampelos owanrensis. The fasting glucose level of the animals was monitored at 1 hour, 3 hours and 4 hours. At the end of the experiment the rats were sacrificed by cervical dislocation, blood samples were centrifuged to obtain the serum for biochemical analysis. The duodenum, jejunum and ileum were excised for biochemical study. Data obtained were analyzed using SPSS statistical tool and expressed as mean±SEM. Results show that fasting blood glucose levels of diabetic rats was reduced significantly on administration of 300mg/kg of the plant extract(502.33±5.57 after 1 hour, 427.33±7.67 after 3 hours, 298.33±10.91 after 4 hours). In conclusion, benzene fractions of Cissampelos owanrensis demonstrated significant reduction in glucose absorption across the intestinal epithelium.
TABLE OF CONTENTS
TITLE PAGE. i
CERTIFICATION ii
DEDICATION iv
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vi
ABSTRACT ix
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.1 BACKGROUND OF STUDY 1
1.2 AIM OF THE STUDY 3
1.3 OBJECTIVES OF THE STUDY 3
1.4 SIGNIFICANCE OF THE STUDY 3
1.5 SCOPE OF THE STUDY 3
1.6 LIMITATION OF THE STUDY……..………………4
CHAPTER TWO 5
LITERAURE REVIEW 5
2.1 MEDICINAL PLANT 5
2.2 ORIGIN AND GEOGRAPHICAL LOCATION DISTRIBUTION 5
2.3 COMMON NAMES OF THE PLANT 6
2.4 DESCRIPTION OF THE PLANT 6
2.5 CHEMICAL COMPOSITION OF THE PLANT 7
2.6 USES OF THE PLANT 7
2.7 DIABETES MELLITUS…………………………...…8
2.8 CAUSES OF DIABETES……...…………………….10
2.9 PLANTS WITH HYPOGLYCEMIC PROPERTIES…………...10
2.10 HYPOGLYCEMIC EFFECT OF CISAMPELOUS OWANRIENSIS11
2.11 ALLOXAN……………………………………………………………12
CHAPTER THREE 13
MATERIALS AND METHODS 13
3.1 LIST OF MATERIALS 13
3.1.1 CHEMICALS USED………………………………………………………14
3.1.2 MACHINES USED………………………………………………………..14
3.2 COLLECTION OF PLANT 15
3.3 EXTRACTION OF PLANT 15
3.3.1 EXTRACTION PROCEDURE……………………………………………16
3.4 PREPARATION OF DIFFERENT REAGENTS 17
3.4.1 PREPARATION OF KREB'S BUFFER SOLUTION…………………….17
3.4.2 PREPARATION OF DINITRATE SILICATE ACID…………………….17
3.4.3 ALLOXAN PREPARATION……………………………………………..17
3.4.4 PREPARATION OF D GLUCOSE……………………………………….18
3.5 EXPERIMENTAL ANIMALS 18
3.6 ETHICAL CONSIDERATION 19
3.7 INDUCTION AND TREATMENT 19
3.7.1 GROUPING OF ANIMALS…………………………………………20
3.7.2 MEASUREMENT OF GLUCOSE LEVEL........………………........21
3.8 METHODOLOGY……………………………………………………..21
3.8.1 ADMINISTRATION OF EXTRACT………………………………..21
3.8.2 SACRIFICING AND SAMPLE COLLECTION……………………21
3.9 EVERTED GUT SACS……………………………………………...22
3.10 BIOCHEMICAL ANALYSIS OF PARAMETERS………………….25
3.10.1TEST OF D GLUCOSE LEVEL OF PLASMA AND TISSUE SAMPLE25
3.10.2 TEST OF SODIUM LEVEL OF PLASMA AND TISSUE SAMPLE.27
3.11 STATISTICAL ANALYSIS………………………………………….28
CHAPTER FOUR 29
4.0 RESULTS 29
CHAPTER FIVE 38
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS 38
5.1 DISCUSSION 38
5.2 CONCLUSION 39
5.3RECOMMENDATIONS 40
REFERENCES 41
APPENDIX I:RAW DATA FOR LEVEL OF BLOOD GLUCOSE AT I, III AND IV HOURS 45
APPENDIX II:RAW DAT FOR FLUID TRANSFER IN DUODENUM 45
APPENDIX III: RAW DATA FOR FLUID TRANSFER IN JEJUNUM………46
APPENDIX IV: RAW DATA FOR FLUID TRANSFER IN ILEUM………..46
LIST OF FIGURES
Figure 1:Values of Blood Glucose Level of experimental rats. 29
Figure 3:Fluid Transfer in Duodenum. 31
Figure 4: Fluid Transfer in Jejunum.................................…...32
Figure 5: Fluid Transfer in Ilium………………………….......33
LIST OF TABLES
Table 1: Effect of Plant Extract on Glucose Uptake…………….34
Table 2: Effect of Plant Extract on Sodium Uptake………..36
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background to the Study
The management of diabetes mellitus has since the past two decades received immense attention as the disease ranks second in the world’s burden of diseases (Tiwari and Madhusdana, 2002). Accounting for several disease related deaths, diabetes mellitus affects about 345million people (Ross, 2001) of the global population without prejudice to race, age, gender or social status.
Contemporary medical recommendations provide drugs such as metformin, glimepiride, glipizide, among others for the management of diabetes. Despite these medications; the disease seems to be on the increase beating the WHO prediction of 323million in 2030. More recently, more attention has been directed towards the use of herbal remediation for the management of the disease.
Herbal remedies such as Allium cepa preparations (Ojieh et al., 2015), Vernonia amygdalina (Ekam et al., 2013), Cissampelos owariensis (Ekeanyanwu et al., 2013), Gongronema latifolium (Iweala et al., 2013), coconut water and coconut milk (Nwangwa and Aloamaka, 2011) have been studied in animals to elucidate their antidiabetic property, some findings have demonstrated plasma glucose reduction (Ekeanyanwu et al., 2013), reduced hyperlipidemia and ameliorate diabetic complications in experimental animals (Ojieh et al. 2015).
However, only a few of such plants have been studied to determine the mechanism by which they exert their hypoglycemic effect. Studies suggests the inhibition of renal glucose reabsorption (Eddouks et al., 2002), inhibition of gluconeogenesis and promotion of glycolysis (Atangwho et al., 2013) and the restoration of pancreatic cyto-architecture and function (Nwangwa and Aloamaka, 2011) as some of the mechanism of action of some herbs.
Cisampelos owarensis a tropical herb of the family Menispermaceae has been shown in previous studies by Ekeanyanwu et al., (2012) to cause decreased blood glucose level of diabetic rats but the mechanism by which this is activated is not clear. This study was therefore carried out to investigate the effect of Cissanpelous owanriensis in glucose absorption across intestinal epithelium of wistar rats in diabetic and normoglycemic states.
1.2 AIM OF THE STUDY
This study aims at investigating the effect of cisanpelous owariensis benzene extract on glucose absorption across the intestinal epithelium in wistar rats in alloxan induced diabetes. 1.3 OBJECTIVES OF THE STUDY
The specific objectives of this study include;
I. To determine the effect of Cissanpelous owanriensis on intestinal glucose absorption in diabetic wistar rats.
II. To investigate the effect of Cissanpelous owanriensis benzene extract on glucose absorption across the intestinal epithelium inwistar rats in a normoglycemic state. 1.4 SIGNIFICANCE OF THE STUDY
The study seeks to provide information on the health benefits of Cissanpelous owanriensis in the management of diabetes mellitus. 1.5 SCOPE OF THE STUDY
This research work focused on the absorption of glucose across the intestinal epithelium using prepangial and postpangial blood glucose level as parameters. 1.6 LIMITATION OF THE STUDY
The study was limited by lack of availability of materials i.e availability of getting the plant, constraint of finances to get some equipment for the work.
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION.
5.1 DISCUSSION
The most challenging goal in the management of diabetes mellitus is to achieve blood glucose level close to normal as possible. Herbal therapy using Cissampelos owanrensis have been shown by Ekeanyanwu et al.,(2012) to reduce blood glucose level. In this study, plasma glucose was checked in diabetic rats treated with Cissampelos owanrensis extracts 1 hour, 3 hours and 4 hours after injestion of the extract and glucose orally. Results showed that fasting blood glucose level was significantly lower (502.33±5.57after 1 hour, 427.33±7.67 after 3 hours, 298.33±10.91 after 4 hours) when compared to the blood glucose level in the group without the plant extract (600±0.00). This shows that Cissampelos owanrensis extract possesses hypoglycemic property as suggested by Musabayane (2006) who observed that Cissampelos owariensisdecreased blood glucose level in diabetic rats after its administration.
The decreased blood glucose level in diabetic animal with administration of herbal therapy has been attributed to several mechanisms such as impaired gluconeogenesisand facilitated glycolysis (Eddokus, 2003).
Recent studies suggest that herbal plants may interfere in the intestinal absorption of glucose in diabetic rats. Results from this study showed reduced intestinal glucose transfer across the intestinal epithelium. This resonates with the assertion of Musabayane (2006),who suggested that the inhibition of glucose absorption might be due to decreased activity of glucose transporter transmembranal proteins in the small intestine of rat.
Results from this study however shows that Cissampelos owanrensis does not interfere with the Glucose-Sodium transport from the lumen across the mucosa epithelium of the intestinal tract ofalloxan- induced diabetic rat models but may interfere with glucose transport across the basolateral membrane by inhibiting GLUT-2 transporter. This may be attributed to the high affinity of Cissampelos owariensis for electrolytes Yadav (2008)
5.2 CONCLUSION
From the foregoing, Cissampelos owanrensis possess hypoglycemic property.Its possible mechanism of hypoglycemic action may be attributed to the inhibition of GLUT-2 transport preventing the free transport of glucose across the serosa layer into the blood. 5.3 RECOMMENDATION
More studies on the effect of the plant extract on the GLUT-2 transporter should be carried out to ascertain if benzene fraction of Cissampelos owariensis inhibits glucose absorbtion across the intestinal epithelium through this transporter.
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