ABSTRACT
Lemongrass (Cymbopogon citratus Stapf) tea is commonly consumed as food and for the traditional treatment of diabetes mellitus locally. This study was conducted to investigate the effect of lemongrass tea (LGT) in type 2 diabetes mellitus (T2DM) rats model. Forty-two (42) animals were randomly divided into 6 groups of seven animals each: Normal Control (NC), Diabetic Control (DBC), Diabetic Lemongrass Tea Low (DLTL, was administered 0.25g/100ml/kg BW of the lemongrass tea), Diabetic treated with metformin (DMF 150mg/kg BW), Diabetic Lemongrass Tea High (DLTH) and Normal Lemongrass Tea High (NLTH) were administered 0.5g/100ml/kg BW of the lemongrass tea. T2DM was induced in rats by feeding 10% fructose solution ad libitum for two weeks followed by intra-peritoneal (i.p) injection of streptozotocin (STZ, 40mg/kg BW) in all animals except NC and NLTH groups. The phytochemical analysis of lemongrass tea and cold water extract was compared using standard methods. From the results, the hot LGT showed higher phytochemical constituents compared to cold water extract, except for saponins. The LGT treatment at both dosages significantly (p < 0.05) reduced blood glucose level and dyslipidemia compared to DBC while NLTH was within the normal range. There were significant (p < 0.05) improvements in oral glucose tolerance ability, weight gain, decreased food and fluid intakes. Similarly, serum insulin concentration, pancreatic β-cell function and liver glycogen content were significantly (p < 0.05) increased in DLTL, DLTH groups when compared to DBC group. The results obtained in this study validate the traditional usage of LGT for management of diabetes treatment and suggest that both lower and higher (0.25/100ml/kg and 0.5/100ml/kg) doses of LGT are effective to reduce most of the diabetes associated complications in a T2D model of rats
Table of Contents
TITLE PAGE i
DECLARATION ii
CERTIFICATION iii
DEDICATION iv
ACKNOWLEDGEMENT v
ABSTRACT vii
LIST OF PLATES xi
LIST OF FIGURES xii
LIST OF TABLES xiii
LIST OF ABBREVIATIONS xiv
CHAPTER ONE
1.0 Introduction 1
1.1 Statement of Research Problem 2
1.2 Justification 3
1.3 Aim and Objectives 4
1.3.1 Aim 4
1.3.2 The Specific Objectives are: 4
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Diabetes Mellitus 5
2.1.1 Prevalence of Diabetes 6
2.1.2 Overview on Glucose Metabolism and Insulin Signalling 7
2.1.3 Classification of Diabetes Mellitus 7
2.1.4 Diagnostic Tests for Type 2 Diabetes Mellitus 15
2.1.6 Complications of Diabetes Mellitus 17
2.1.7 Therapeutic Agents for Treatment Options 18
2.2 Medicinal Plants 24
2.2.1 Lemongrass 26
2.2.2 Phytochemicals Compounds 27
2.2.3 Biological Activities of Lemongrass 28
CHAPTER THREE
3.0 MATERIALS AND METHODS 32
3.1 Materials 32
3.1.2 Plant material, Collection and Identification 32
3.1.3 Experimental Animals 32
3.2 METHODOLOGY 33
3.2.1 Induction of T2DM 33
3.2.2 Tea Preparation and Intervention (Islam, 2011) 34
3.2.3. Quantitative phytochemical analysis 34
3.2. 3.1 Alkaloids determination using Harborne (1998) method 34
3.2.3.2 Flavonoids determination by the method of Bohm and Koupai Abyazani (1994) 35
3.2.3.3 Saponins determination 35
3.2.3.4 Tannin determination by Van-Buren and Robinson (1981) method 36
3.2.3.5 Determination of total phenolics by spectrophotometric method (Chang et al., 2002) 36
3.2.6 Oral glucose tolerance test (OGTT) 38
3.2.7. Determination of Serum Insulin 38
3.2.8 Insulin Resistance and Pancreatic β-cell Function Indices 38
3.2.9. Biochemical Test 39
3.2.9.1. Serum lipid profile (Total Cholesterol, Triglycerides, High Density Lipoprotein and Low- Density Lipoprotein) 39
3.2.9.1.1 Triglyceride Level 39
3.2.9.1.2 Total Cholesterol 41
3.2.9.1.3 High Density Lipoprotein 42
3.2.9.1.4 Low Density Lipoprotein 43
3.2.9.2. Serum Creatinine 43
3.2.9.3 Serum Total Protein (Bradford, 1976) 44
3.2.9.4 Assay for Serum Albumin Concentration 45
3.2.9.5. Assay of Aspartate and Alanine Aminotransferases Activity 45
3.2.9.6. Assay for Alkaline Phosphatase (ALP, EC 3.1.3.1) Activity 46
3.2.9.7. Determination of Hepatic Glycogen Content 47
3.2.10 Histopathological study 48
3.2.11. Data Analysis 49
CHAPTER FOUR
4.0 RESULTS 50
4.1 Phytochemical Composition of Lemongrass Tea and Lemongrass Cold-Water Extract 50
4.2. Body Weight of Animal Groups During Four Weeks Experimental Period 52
4.3 Fluid Intake in Animal Groups During Four Weeks Experimental Period 54
4.4 Feed Intake in Animal Groups during Four Weeks Experimental Period 56
4.5 Weekly Fasting Blood Glucose levels in Animal Groups During Four Weeks Experimental Period 58
4.6. Oral Glucose Tolerance Test (OGTT) in Animal Groups during the Last Week of Four Weeks Experimental Period 60
4.7 The Influence of Lemon Grass Tea on Serum Insulin, HOMA IR and HOMA β Scores on Diabetic Rats. 62
4.8 Serum Lipid Profile in Animal Groups at the End of the Four Weeks Experimental Period 64
4.9 Serum Biochemical Parameters and Liver Glycogen Content in Different Animal Groups 66
CHAPTER FIVE
5.0 DISCUSSION 70
CHAPTER SIX 74
6.0 CONCLUSION AND RECOMMENDATION 74
6.1 CONCLUSION 74
6.2 RECOMMENDATION 74
REFERENCES 75
LIST OF PLATES
plate 1: lemongrass image 27
plate 2: histology of the pancreatic tissues of different animal groups at the end of the intervention period… 69
LIST OF FIGURES
Figure 3.1Experimental Design for the Study 33
Figure 4.1 Serum Lipid Profile in Animal Groups at the End of the Four Weeks Experimental Period… 65
LIST OF TABLES
Table 2.1The Standard Values of Diagnostic Test in T2DM 16
Table 4.1Phytochemical Content of Lemongrass Tea and Lemongrass Cold-Water Extract 51
Table 4.2Body Weights of Different Animal Groups during the Four Weeks Experimental Period 53
Table 4.3 Fluid Intake in Different Animal Groups during the Four Weeks of Experimental Period 55
Table 4.4 Feed Intake in Different Animal Groups during the Four Weeks of Experimental Period 57
Table 4.5 Weekly Blood Glucose Concentrations in Animal Groups during the Four Weeks Experimental Period 59
Table 4.6 Oral Glucose Tolerance Test (OGTT) in Animal Groups during the Last Week of Experimental Period 61
Table 4.7 Serum Insulin Level, HOMA-IR, HOMA-β in Animal Group 63
Table 4.8 Serum Biochemical Parameters and Liver Glycogen Content in Animal groups 67
LIST OF ABBREVIATIONS
ALP Alkaline Phosphatase
ALT Alanine Aminotransferase
AST Aspartate Amino Transferase
DBC Diabetic Control
DLTH… Diabetic Lemongrass Tea High
DLTL Diabetic Lemongrass Tea Low
DM… Diabetes Mellitus
DMF Diabetic with Metformin
GDM… Gestational Diabetes Mellitus
HDL High Density Lipoprotein
HOMA-β… Homeostatic Model Assessment of Beta Cell Function
HOMA-IR Homeostatic Model Assessment of Insulin Resistance
IDF International Diabetes Federation
LDL Low Density Lipoprotein
LGT… Lemongrass Tea
NC… Normal Control
NLTH Normal Lemongrass Tea High
OGTT Oral Glucose Tolerance Test
T1DM Type1 Diabetes Mellitus
T2DM Type 2 Diabetic Mellitus
TC Total Cholesterol
TG Triglycerides
CHAPTER ONE
1.0 Introduction
Diabetes mellitus (DM)is a condition that causes hyperglycaemia due to either decreased insulin secretion or insulin sensitivity of target tissue (Panini,2013).It is a progressive metabolic disorder that eventually leads to micro and macro vascular changes such as nephropathy, neuropathy, retinopathy and cardiovascular diseases (Osadebeet al., 2010). It is broadly classified into three major classes, type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus. T1DM results from inadequate production of insulin by β-cells of the pancreas, while T2DM occurs when the pancreas does not produce enough insulin or when the body does not effectively use the insulin that is produced (Goldenberg and Panthakee, 2013). Furthermore, it has been reported that T2DM is most prevalent, accounting for more than 90% of diabetes cases (International Diabetes Federation, 2015). As a multifactorial disorder, management of diabetes requires comprehensive and holistic approaches. The currently available synthetic oral hypoglycaemic drugs such as biguanides (example include metformin), insulin secretagogues (example include glipizide) and α-glucosidase inhibitors (acarbose, miglitol, voglibose) are widely used in the treatment of T2DM. However, in view of the expensive, non-availability coupled with adverse effects associated with the use of these drugs, an alternative source with less toxic effects is of enormous interest. The use of plant-derived products is receiving much attention attributed to their less adverse consequences and functional foods are receiving much attention (Gomathi et al., 2013).
Considerable amount of data has shown that functional foods play crucial role in the prevention and control of chronic diseases such as diabetes (Jacobson, 2004). The concept of functional foods entail utilizing food for the prevention and reduction of risk factors for several diseases or enhancing certain physiological function beyond adequate nutritional effects (Tahraniet al., 2010). Among the prominent functional foods is the Cymbopogon citratus Stapf, commonly known as lemongrass. It is an aromatic perennial plant belonging to the Poacae family, having a long slender green leaf. It is widely distributed and extensively used worldwide (Ekpenyonget al. 2015). It is frequently consumed for recreational as well as therapeutic purposes attributed to the ingredients, taste, distinctive lemony smell and colour. In some African countries, such as Nigeria and Egypt, lemongrass leaf tea is consumed in management of fever, jaundice, throat and chest infections, moderate-to-severe pain, hypertension, obesity and diabetes (Mansouret al. 2002). Interestingly, Boaduoet al. (2014) reported that various extracts from lemongrass inhibited α-glucosidase activity in vitro and improved insulin release. However, detailed anti-diabetic action of lemongrass tea in T2DM rats‟ model is not widely available.
1.1 Statement of Research Problem
The prevalence of diabetes is increasing annually estimated to be 463 million people globally with 19.4 million people in Africa and the global projection is well above 700 million by 2045 (International Diabetes Federation, 2019). However, the current estimation also suggests that in Nigeria there were more than 2.7 million cases of diabetes in 2019. With the current statistics, DM not only takes a heavy toll on lives around the world but imposes a serious financial burden on the sufferers and their family members (Nasli-Esfahani et al., 2017). These geometric increases in the number of diabetics in recent times cannot be dissociated from unhealthy life style, urbanization, aging and ravaging influence of free radicals (American Diabetes Association, 2015).
Diabetes burden and allied complications like amputation, stroke or end stage renal diseases are soaring.T2DM is managed by a combination of diet, exercise and conventional therapy. Some of these conventional or synthetic drugs can cause side-effects including haematological, gastrointestinal reactions, hypoglycaemic coma, and disturbances in the liver and kidney metabolism. In addition, these drugs are not ideal for use during pregnancy (Gomathiet al., 2013).
1.2 Justification
Like other parts of the world, diabetes has also become a serious issue in Africa. It is quite obvious that the prevalence of DM and its complications are increasing at an alarming rate, especially among the black race (Ahren, 2013). The International Diabetes Federation‟s (IDF) Diabetes Atlas estimates that, 60% of people with DM in Africa are undiagnosed (IDF, 2019). Among the types of DM, T2DM is known to have the highest prevalence, which is between 90% of all diabetes cases (IDF, 2019).
There has recently been a rapid increase in consumer interest in the health enhancing roles of specific foods or physiologically active food components, so called functional food (Hasler, 1998). The crucial role of functional food in the treatment and reduction of complications associated with T2DM cannot be denied. Extracts and essential oils from Lemongrass have been reported to reduce blood glucose and lipid profiles levels in normal (Adeneye and Agbaje, 2007; Ademuyiwa et al. 2015; Ekpenyonget al. 2015) and hyperlipidemic rats (Bhartiet al. 2013; Ekpenyonget al. 2015).Lemongrass as a functional food is consumed in form of tea for nourishment and in the traditional treatment of diabetes and related disorders (Lunyera et al. 2016).Also, several studies have already investigated various alkaloids, flavanoids and phytochemicals in Cymbopogoncitratus, to exhibit anti-diabetic effects (Ekpenyonget al. 2015; Boaduo et al., 2014). These effects include inhibition of α-glycosidase, amylase enzymes activity (Boaduo et al.2014), reducing carbohydrate metabolism and therefore lowering blood glucose levels (Boaduo et al. 2014).
1.3 Aim and Objectives
1.3.1 Aim
The aim of this study is to investigate the anti-diabetic potential of lemongrass tea in a T2DM model of rats.
1.3.2 The Specific Objectives are:
1. To compare the phytochemical composition of lemongrass tea and cold water extract.
2. The determine the effect of lemongrass tea on body weight, feed and fluid intakes, serum glucose levels, insulin, insulin resistance indices in T2DM induced rats.
3. To determine the effect of lemongrass tea on lipid profiles (total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol) and some serum biochemical parameters (creatinine, total proteins, albumin, ALT, ALP, AST, and liver glycogen) in T2DM induced rats.
4. To determine the pancreatic integrity of T2DM induced rats administered with the LGT.
1.4 Research Hypothesis:
Null hypothesis: Consumption of lemongrass tea has no anti-diabetic effects. Alternative hypothesis: Lemongrass tea consumption possess anti-diabetic potential in the management of diabetes.
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