ABSTRACT
This study investigated the effects of crude extract and fractions from Moringa oleifera leaves on glucose transport proteins, AMPkinase,Na+/K+-ATPase and other parameters in streptozotocin induced diabetes male wistar rats using acceptable chemical and biochemical methods. Crude extract and active fractions of M. Oleifera were subjected to gas chromatography- mass spectrophotometer (GCMS) for identification of bioactive compounds in the plant. The animal experiment was carried out in two phases. In Phase 1, 40 male Wistar rats placed in 8 groups of five (5) rats each were used. Group 1 (normal control), group 2(drug control), group 3 (diabetic control), group 4 (Dimethylesulphuroxide control).Group 5and 6 were administered 1000mg/kgbwt and500mg/kgbwt of aqueous extract while group7 and 8 were administered 1000mg/kgbwt and 500mg/kgbwt of methanol extract respectively. Result obtained showed no significant difference (P>0.05) in the relative heart, lungs, liver and intestinal weight. The blood glucose level, glycated heamoglobin, cholesterol, triacylglycerol, LDL-Cholesterol, VLDL-Cholesterol levels and α-amylase activities increased significantly (P< 0.05) in group 3 and 4 when compared to group 1, 2, 5, 6, 7 and 8. Treatment with glibenclamide and different doses of crude extract in groups 2, 5, 6, 7 and 8 indicated a significant(P<0.05) increase in HDL-Cholesterol level, lipoprotein lipase, phosphate dehydrogenase and Na+/K+-ATPase activities when compared to group 3 and 4. The result revealed a significant increase (P<0.05) in Glucose Transport Protein 1 and 4 and AMPkinase activities in group 2, 5, 6, 7 and 8 respectively when compared to group 3 and 4. A total of 45 animals placed in 9 experimental groups of 5 animals each were used in phase 2.Group 1 (normal control), group 2 (drug control), group 3 (diabetic control), group 4(1000mg/kgbwt of aqueous extract) while group 5-9 received fractions 1- 5 respectively for 28 days. Result obtained showed no significant difference (P>0.05) in the relative heart, liver, kidney, lungs and intestinal weight. Fractions 3,4 and 5 significantly decreased (P<0.05) the glucose level, α-amylase activity, cholesterol, triacylglycerol, LDL-Cholesterol and VLDL-Cholesterol except HDL-Cholesterol when compared with fractions 1 and 2 respectively. The result revealed that fractions 3, 4 and 5 showed a significant increase (P<0.05) in lipoprotein lipase, phosphate dehydrogenase and Na+/K+-ATPase activities when compared with group 3, fractions 1 and 2 respectively. GLUT 1,GLUT 4 and AMPkinase activities were significantly increased(P<0.05) in groups administered glibenclamide, aqueous extract and fractions 3, 4 and 5 when compared with group 3, fraction 1 and 2 respectively.Fractions1 and 2did not show any significant difference (P >0.05) with the diabetic control group in all the parameters studied. Histopathological investigation of the pancreas showed that the crude extract and fractions 3, 4and 5 regenerated and restored the histoarcheteture of the pancreatic acini and reduced the vacuolations of Langerhans islets cell resulting in the absence of necrosis near to normal. GC- MS analysis of crude extract and fractions 3,4 and 5 showed the presence of many bioactive compound out of which hexadecanoic acid, Octadecanoic acid, Octadecenoic acid, Palmitic acid, and Octadecenoic acid ethyl ester were part. This indicated that Crude extract and fractions of M.Oleifera leaves ameliorated the studied indicators of diabetes and its complications in STZ- induced diabetic male wistar rats. The possible synergetic action of the bioactive agents present in the leaves might be responsible for the effects.
TABLE OF
CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables vii
List of Figures xv
List of Plates xvi
Abstract xvii
CHAPTER 1: INTRODUCTION
1.1 Background of
the Study 1
1.2 Aim of the Study 6
1.3 Objectives of the Study 6
1.4 Statement of Problem 7
1.5 Justification
of the Study 7
CHAPTER 2: LITERATURE
REVIEW
2.1 Diabetes
Mellitus 9
2.2 Classification
of Diabetes Mellitus 9
2.2.1 Type
1 diabetes mellitus (insulin dependent diabetes mellitus,
childhood
diabetes) (T1DM) 10
2.2.2 Immune-mediated
diabetes 10
2.2.3 Type
2 diabetes (adult-onset diabetes, maturity-onset diabetes) (T2DM) 11
2.2.4 Type
3 diabetes mellitus 13
2.2.5 Gestational
diabetes 13
2.3 Epidemiology
of Diabetes Mellitus 14
2.4 Normal
Glucose Homeostasis and Transport 15
2.4.1 β-cell
hormones 16
2.5 Glucose
Transport Across Membrane (GLUT 1 and GLUT 4) 19
2.5.1 Insulin
signaling vs AMP kinase signaling 19
2.5.2 Glucose
transport and transporters 23
2.6 Role
of Na+/K+-ATPase Enzyme in Glucose Transport 25
2.7 Biological
Role of AMP Activated Protein Kinase in Diabetes 27
2.8 Enzymes
involved in Lipid/Carbohydrate Metabolism in Diabetes Mellitus 28
2.8.1 Lipoprotein
lipase 28
2.8.2 Pancreatic
lipase 29
2.8.3 Pancreatic
amylase 29
2.9
Relationship Between Fasting
Hyperglycemia and Postprandial
Hyperglycemia 30
2.10 Pathogenesis
and Associated Complications of Diabetes 31
2.10.1 Diabetic ketoacidosis (DKA) 33
2.10.2 Glycated haemoglobin 33
2.10.3 Dyslipidemia 34
2.11 General
Management of Diabetes Mellitus 35
2.12 Conventional
Drugs Used in the Management of Diabetes Mellitus 36
2.12.1 Insulin 36
2.12.2 Oral anti-diabetic agents 37
2.13 A
Shift from Modern Medicine to Traditional Medicine 39
2.14 Description
of Moringa oleifera Lam 40
2.15 Classification
of Moringa oleifera 42
2.16 Nutritional
and Phytochemical Composition of Moringa oleifera 42
2.17 Therapeutic
Effects of Moringa oleifera 43
2.17.1 Anti-cancer activity of Moringa
oleifera 43
2.17.2 Antioxidant properties of Moringa
oleifera 44
2.17.3 Anti-inflammatory effect of Moringa oleifera 45
2.17.4 Anti-diabetic properties of Moringa
oleifera 45
2.17.5 Anti-fibrotic/ulcer properties of Moringa oleifera 46
2.17.6 Anti-microbial activities of Moringa
oleifera 47
2.18 Medicinal
Plants with Potential Hypoglycemic and Antidiabetic Activities 47
2.19 Phytochemicals in Plants Exhibiting
Hypoglycemic and Antidiabetic
Activity 49
2.20 Mechanism of Action of Plants with
Hypoglycemic and Antidiabetic
Activity 50
2.21 Conventional
Drugs used in the Experimental Induction of Diabetes 51
2.21.1 Streptozotocin 51
2.21.2 Alloxan monohydrate (2, 4, 5, 6-tetraoxypyrimidine) 52
CHAPTER 3:
MATERIALS AND METHOD
3.1 Materials 54
3.1.1 Collection
of plants materials 54
3.
1.2 Selection of animals 54
3.1.3 Reagents
and chemicals 55
3.1.4
Apparatus 55
3.2 Methods 56
3.2.1 Lorke’s method (1983) 56
3.2.2 Pilot study 57
3.2.3 Preparation
of plant extracts of Moringa oleifera
leaves 57
3.2.4 Aqueous
extraction of Moringa oleifera leaves 58
3.2.5 Methanol
extraction of Moringa leaves 58
3.2.6 Determination
of percentage aqueous and methanol yield of
Moringa oleifera 58
3.2.7 Administration of plant extracts 58
3.2.8 Induction of diabetes for the main study 59
3.2.9 Determination
of fasting blood glucose concentration 60
3.2.10 Experimental procedure 60
3.2.11 Calculation of body weight gain/loss 62
3.2.12 Preparation
of total cellular membrane from skeletal muscle and blood 62
3.2.13 Human glucose transport 1(GLUT1) and GLUT 4 63
3.2.14 Assay procedure for GLUT 1 and GLUT 4 63
3.2.15 Preparation of liver homogenate for AMP-kinase activity 64
3.2.16 Assay of human adenosine
monophosphate (AMP) kinase 64
3.2.17 Extraction
of liver tissue for analysis of glucose 6-phosphate
dehydrogenase 65
3.2.18 Assay of glucose -6- phosphate dehydrogenase 66
3.2.19 Estimation of protein in serum 66
3.2.20 Estimation of glycosylated haemoglobin (HbA1c) 66
3.2.21 Lipid profile assay 67
3.2.21.1Cholesterol determination 67
3.2.21.2Triacylglycerol estimation 68
3.2.21.3High density lipoprotein
(HDL) cholesterol determination 70
3.2.21.4Low density lipoprotein (LDL)
- cholesterol estimation 70
3.2.21.5Very low density lipoprotein
(VLDL) -cholesterol estimation 71
3.2.22 Intestinal amylase estimation 71
3.2.23 Lipoprotein lipase determination 72
3.2.24 Determination of Na+ /K+-ATPase Activity 73
3.2.25 Phytochemical analysis procedures 74
3.2.25.1Determination of phyticacid 74
3.2.25.2Determination of oxalate 74
3.2.25.3Determination of tannic acid 75
3.2.25.4 Determination of flavonoid 76
3.2.25.5 Determination of saponin contents 76
3.2.25.6 Determination of phenolic
acid 77
3.2.25.7 Determination of alkaloid 77
3.2.25.8Determination of cyanide 78
3.2.26 Proximate analysis procedures 78
3.2.26.1 Determination of ash content 78
3.2.26.2 Determination of fat content 79
3.2.26.3 Determination of protein
content 79
3.2.26.4 Determination of moisture content 80
3.2.26.5 Determination of crude fiber 81
3.2.26 Determination of carbohydrate content 81
3.2.27 Fractionation
of crude methanol extract of Moringa oleifera
leaves 82
[[[[[[[[[[[[[[[
3.2.27.1 Column chromatography 82
3.2.27.2 Solvent systems 84
3.2.28 Thin layer chromatography monitoring of fractions 85
3.2.29 GC-MS analysis of fractions 85
3.2.30 Instruments and chromatography conditions 86
3.2.31 Identification
of phytocomponents 86
3.2.32 Histopathological
studies 87
3.3 Statistical
Analysis 88
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Results 89
4.1.1 Result of proximate composition of Moringa oleifera leaf 89
4.1.2 Result
of quantitative phytochemical screening of Moringa
oleifera
leaf 90
4.1.3 Compound
identified from the GC-MS Analysis of crude extract of
Moringa oleifera leaf
extract 91
4.1.4 Result of lethal dose studies (LD50) 92
4.1.5 Results
of blood glucose concentrations of rats after 3 days of
induction of STZ
at doses of 55, 65 and 70 mg/kg body weight
(Pilot Study) 93
4.1.6 Result
of fasting blood glucose level of normal and STZ- induced
Diabetic
male albino rats 94
4.1.7 Result of weight changes of animals 95
4.1.8 Result of relative weight of organs 96
4.1.9 Result of serum protein and glycated heamoglobin
of experimental
animals 97
4.1.10 Result of serum lipid profile (mg/dl) in the sera of rats 98
4.1.11Effect of administration of methanol and aqueous
extracts of Moringa
Oleifera leaf
on
Lipoprotein lipase (LPL) and Intestinal amylase activity
in STZ- diabetic male wistar rats 99
4.1.12 Result
of liver phosphate dehydrogenase activity in STZ-induced
diabetic
animals 100
4.1.13 Result
of Na+/K+-ATPase
Specific activity in STZ-induced diabetic
animals 101
4.1.14 Result of GLUT
4, GLUT 1 and AMP-Kinase expressions in
experimental animals 102
4.1.15: Histology
result of STZ-induced diabetic treated with Moringa
oleifera leaf 103
4.2 Result of experiment 2: Investigation of
stimulatory effects of fractions
of M. oleifera leaf
extract in streptozotocin induced diabetes male
wistar rats 110
4.2.1 Result of fractions and their retention
factor (Rf) obtained after
column chromatography and thin layer chromatography (TLC)
monitoring of the fractions 110
4.2.2 Result of administration of fractions of M. oleifera on Fasting blood
glucose levels of animals 111
4.2.3 Result of administration of fractions of M. oleifera on weight of
animals 112
4.2.4 Result of
relative organ weight of STZ induced diabetic rats
administered fraction of Moringa oleifera leaf 113
4.2.5: Result of effect of fractions of Moringa oleifera leaf extract on
Protein and glycated heamoglobin levels in STZ –induced
diabetic male
wistar rats 114
4.2.6: Result of administration of fractions of Moringa oleifera leaf extract
on lipid profile (mg/dl) parameters 115
4.2.7: Result of effect of fractions of Moringa oleifera leaf extract on
lipoprotein lipase and intestinal
amylase in STZ –induced diabetic
male wistar rats 116
4.2.8: Result of effect of fractions of Moringa oleifera leaf extract on
phosphate dehydrogenase in STZ-
induced diabetic male wistar rats 117
4.2.9: Result of effect of fractions of Moringa oleifera leaf extract on
Na+/K+-ATPase
Specific activity in STZ- induced diabetic male wistar rats 118
4.2.10: Result of effect of
fractions of Moringa oleifera leaf
extract on GLUT 4,
GLUT 1 and AMP-Kinase expressions in STZ- induced diabetic
male
wistar rats 119
4.2.11: Pancreas
histology of rats treated with fractions of Moringa
oleifera leaf extract
4.2.12: Phytochemical
components identified from the GC-MS Analysis of
fraction 3 of Moringa oleifera leaf extract 128
4.2.13: Phytochemical
components identified from the GC-MS Analysis of
fraction 4 of Moringa oleifera leaf extract 129
4.2.14: Phytochemical
components identified from the GC-MS Analysis of
fraction 5 Of Moringa oleifera leaf extract 130
4.3 Discussion 131
CHAPTER 5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion 153
5.2 Recommendations 153
References 155
Appendix 190
LIST OF TABLES
|
|
|
PAGES
|
|
3.1
|
Method for Analysis of amylase
|
71
|
|
3.2
|
Pooling of fractions from
Moringa oleifera leaves
|
86
|
|
4.1
|
Quantitative percentage proximate
composition of Moringa oleifera leaf
|
89
|
|
4.2
|
Phytochemical composition of Moringa oleifera
|
90
|
|
4.3
|
GC-MS analysis of crude extract of Moringa oleifera leaf extract
|
91
|
|
4.4
|
Showing
the LD50 of the methanol extract of Moringa oleifera leaf in normal albino mice
|
92
|
|
4.5
|
Fasting blood
glucose of rats (mg/dL) (Pilot study)
|
93
|
|
4.6
|
Fasting blood glucose levels (mg/dl) of
normal and STZ- induced Diabetic male albino rats administered methanol and
aqueous leaf extract Moringa oleifera
|
94
|
|
4.7
|
The weight changes of STZ- induced diabetic Male Albino
Rats Administered Methanol and aqueous leaf extract of Moringa oleifera
|
95
|
|
4.8
|
The % Relative Organ Weights of STZ- induced diabetic Male
Albino Rats administered methanol and aqueous leaf extract of Moringa oleifera
|
96
|
|
4.9
|
Effect of
administration of methanol and aqueous extract of Moringa oleifera leaf on serum protein and glycated heamoglobin
level in ST2-induced diabetic rats
|
97
|
|
4.10
|
Lipid profile of STZ-induced
diabetic male wistar rats administered Methanol and Aqueous leaf extract
of Moringa
oleifera
|
98
|
|
4.11
|
Lipoprotein and
intestinal Amylase level of rats administered methanol and aqueous
extracts of Moringa oleifera leaf
|
99
|
|
4.12
|
Effect of methanol and aqueous crude extracts of Moringa oleifera leaf extract on
phosphate dehydrogenase level in ST2-induced diabetic wistar rats.
|
100
|
|
4.13
|
Effect of methanol and aqueous crude extracts of Moringa oleifera leaf extract on Na+/K+-ATPase
Specific activity in STZ-induced
diabetic wistar rats
|
101
|
|
4.14
|
Effect of administration of crude
extract of methanol and aqueous extract of M. oleifera on GLUT 4, GLUT 1
and AMP- Kinase expression in
STZ – induced diabetic male wistar rats
|
102
|
|
4.15
|
The result of the Pooled fractions and
their percentage yield of the leaf extract
|
110
|
|
4.16
|
Fasting blood
glucose levels (mg/dl) of STZ- induced Diabetic male albino rats administered
fractions of Moringa oleifera leaf extract
|
111
|
|
4.17
|
The weight changes of STZ- induced diabetic Male Albino
Rats Administered fractions of leaf extract of Moringa oleifera
|
112
|
|
4.18
|
Relative organ weight of STZ induced diabetic animals
administered fractions of Moringa oleifera
leaf extract
|
113
|
|
4.19
|
The
effect of administrations of fractions of Moringa
oleifera leaf extract on protein and glycated heamoglobin in STZ- induced
diabetic wistar rats
|
114
|
|
4.20
|
The
effect of administration of fractions of M.
oleifera on lipid profile parameters in STZ- induced diabetic male wistar
rats
|
115
|
|
4.21
|
Effect
of administrations of fractions of Moringa
oleifera leaf extract on lipoprotein lipase and intestinal amylase in
STZ- induced diabetic wistar rats
|
116
|
|
4.22
|
The effect of administrations of fractions of Moringa oleifera leaf extract on
phosphate dehydrogenase activity in STZ- induced diabetic wistar rats
|
117
|
|
4.23
|
The effect of administrations of fractions of Moringa oleifera leaf extract on Na+/K+-ATPase
Specific activity in STZ- induced
diabetic wistar rats
|
118
|
|
4.24
|
The effect of administrations of fractions of Moringa oleifera leaf extract on GLUT
4, GLUT 1 and AMP-Kinase expressions in STZ- induced diabetic wistar rats
|
119
|
|
4.25
|
Phytochemical components of fraction 3
of Moringa oleifera leaf extract
identified through GC-MS analysis of the extract
|
128
|
|
4.26
|
Phytochemical components of fraction 4
of Moringa oleifera leaf extract
identified through GC-MS analysis of the extract
|
129
|
|
4.27
|
Phytochemical components of fraction 5
of Moringa oleifera leaf extract
identified through GC-MS analysis
|
130
|
LIST OF FIGURES
PAGES
2.1 Role
of insulin, glucagon, amylin and GLP-1 in glucose homeostasis 18
2.2 Signaling
pathway of insulin and AMPKinase leading to GLUT 4
and
GLUT 1 translocation and activation 22
2.3 Glucose transport mediated by insulin
dependent and non-insulin
dependent transporters 24
2.4 Structural Features of the
Insulin-Regulated GLUT4 Glucose
Transporter
Protein 25
LIST OF PLATES
|
|
|
PAGES
|
|
4.1
|
Photomicrograph of pancreas section of non-diabetic animal
fed only water (normal control)
|
103
|
|
4.2
|
Photomicrograph of
pancreas section of animal induced with diabetes but was not
treated(diabetic control)
|
104
|
|
4.3
|
Photomicrograph of pancreas section from experimental animals administered
5mg/kgbwt of glibenclamide
|
105
|
|
4.4
|
Photomicrograph of pancreas section from experimental animals administered
500mg/kgbwt of aqueous extract of Moringa
oleifera leaf extract
|
106
|
|
4.5
|
Photomicrograph of pancreas section from experimental
animal administered 1000mg/kgbwt of aqueous extract of Moringa oleifera leaf extract.
|
107
|
|
4.6
|
Photomicrograph of pancreas section from experimental animal administered
500mg/kgbwt of methanol extract of Moringa oleifera leaf extract
|
108
|
|
4.7
|
Photomicrograph of pancreas section from experimental animal administered
1000mg/kgbwt of methanol extract of Moringa oleifera leaf extract
|
109
|
|
4.8
|
Photomicrograph of pancreas section of non-diabetic animal
fed only water (normal control)
|
120
|
|
4.9
|
Photomicrograph of pancreas section of animal induced with
diabetes but was not treated(diabetic control)
|
121
|
|
4.10
|
Photomicrograph of pancreas section from experimental
animals administered 5mg/kgbwt of glibenclamide
|
122
|
|
4.11
|
Photomicrograph of pancreas section from experimental
animal administered Fraction 1 (F1) of Moringa
oleifera leaf extract
|
123
|
|
4.12
|
Photomicrograph
of pancreas section from experimental animal administered Fraction 2 (F2) of Moringa oleifera leaf extract
|
124
|
|
4.13
|
Photomicrograph of pancreas section from experimental
animal administered fraction 3 (F3) of Moringa
oleifera leaf extract
|
125
|
|
4.14
|
Photomicrograph of pancreas section from experimental
animal administered fraction 4 (F4) of Moringa
oleifera leaf extract
|
126
|
|
4.15
|
Photomicrograph
of pancreas section from experimental animal administered fraction 5 (F5) of Moringa oleifera leaf extract
|
127
|
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Diabetes mellitus (DM) is a
disease of the endocrine which give rise to complex and multifarious disorders that alter the
metabolism of bio-molecules such as carbohydrate, fat and protein (Goldenber
and Punthakee, 2013; Gopinathan and Naveenraj, 2014). The number of individuals
having diabetes globally is expected to rise from 171 million to 366 million in 2030 (Wild et
al., 2004). A similar study reported
the global prevalence of diabetes for adults at 6.4% affecting 285 million adults in 2010 and
which is expected to rise to 7.7% affecting 439million by 2030.Thus,Shaw et al., (2010) reported that between
2010 and 2030, there will be a 69% increase in diabetic adults in developing
countries and 20% increase in developed countries.In 2016, an estimated
1.6million death were directly caused by diabetes. Thus, almost half of the
death that occur in 2016 were attributed to high blood glucose and diabetes was
the seventh leading cause of deathin
2016(WHO, 2018)
Diabetes
is characterized by high level of fasting and post prandial blood sugar with
altered fuel metabolism which manifest mainly as hyperglycemia because of
abnormal insulin secretion and insulin action or both. The acute hyperglycemia
in diabetes results in long term dysfunction and damage of organs such as
heart, eyes, blood vessels, kidneys, and nerves (Holman et al., 2008; Gopinathan and Naveenraj, 2014).
A lot of
factors such as increase consumption of energy rich meals, sedentary lifestyle
and obesity are responsible for the rise in diabetes patients. Thus, these
metabolic disorder arise because of insulin deficiency or insulin resistance or
both. (Vinik and Vinik, 2003; Goldenber and Punthakee, 2013). The resultant abnormalities could
advance to lesions such as neuropathy, retinopathy, nephropathy and angiopathy
(Holman et al., 2008).
An
individual becomes diabetic when the blood glucose level is above 126mg/dl
after an overnight fast and more than 200mg/dl after an oral glucose load of
75g (oral glucose tolerance test, OGTT) (American Diabetes Association,
2018). The two major types of DM are type-1-DM (T1DM) which results due to the autoimmune
destruction of Beta (β) cells of the pancreas and accounts for only 5% of all
cases while type-2 DM (T2DM) arises due to tissue insensitivity to insulin and compensatory
secretion of the hormone by islet beta cells of the pancreas.(WHO, 2018)
In
its early stages, the basic symptoms of diabetes are chronic hyperglycemia and
hyperinsulinemia because of insensitivity of tissues to insulin and the compensatoryinsulin
secretion by islet beta cells. Its progression invokes an interacting cellular
and physiological alterations resulting to β-cell failure. The mechanisms that
give rise to this failure are glucotoxicity and lipotoxicity (Robertson et al.,2004).The
excess uptake of glucose by the islet beta (β)-cells results to glucotoxicity.
These excess sugar drives glycation reactions and the mitochondrial electron
transport chain, leading to imbalance in the antioxidant capacity of the cell
because of increase production of reactive oxygen species (ROS). The developing
oxidative stress causes reduction in the production of insulin and its secretion,
and thus initiating series of cellular events that majorly leads to apoptosis
(Kaneto et al.,2007).
On
the other hand, lipotoxicity results due to insensitivity of adipocytes to
insulin, hindering the ability of this hormone to modulate uptake of
non-esterified fatty acids (NEFA) that emanates from triacylglycerides (TG)
lipolysis in circulation and to inhibit the breakdown of endogenous TG to NEFA.
Gluconeogenesis is stimulated because of impaired insulin secretioncaused by
excess NEFA and
this also inhibits glucose clearance by skeletal muscle, further
exacerbating hyperglycemia (Stumvoll et al.,
2005).
Impaired TG storage into adipocytes enhanced theproduction ofcholesterol
ester-poor, TG-rich low-density lipoprotein (LDL) particles. The half-life of
these particles isextended due to hyperglycemic promotion of glycation in
circulation. Thus these particles are prone to oxidation and are basic
initiators of atherogenesis and its vascular damages. Most metabolic disorders such as neuropathy,
retinopathy, and nephropathy when caused by diabetes are some of the
consequences of these damages (Dokken, 2008).
When there is insulin resistance and impaired insulin secretion, it leads to
hyperglycemia, hyperlipidemia and a corresponding increase in hepatic glucose.
The symptoms of chronic hyperglycemia and abnormalities in serum lipids
associated with diabetes are long-term damage and dysfunction resulting in
failure of important organs, including the eyes, kidneys, nerves, heart and
blood vessels (Paneni et al, 2013).
The
epidemic spread of diabetes and the discovery of new therapeutic routes in the
treatment of diabetespose a greater problem in the development of current
biomedical research. Although wide varieties of pharmacological drugs are being
used for diabetic treatment but because they produce serious side effects, treatments
with synthetic drugs are not effective most times in maintaining normal blood
glucose level and avoiding late stage diabetic consequences. This hasled to
increasing use of medicinal plants with anti-diabetic activity with minor side
effects (Modak et al, 2007).
One
source is Moringa oleifera Lam., (M. oleifera) which is commonly called drumstick
tree and belongs to the family of Moringacaea.
It originated from India, where different parts of it have served as food and
medicine (Kasolo et al., 2010).
Dietary consumption of parts of this plant could serve as a way of maintaining
personal health status and self-medication in various diseases. In sub-Saharan
Africa, the use of M.oleiferais
limited although some of its medicinal benefits have been reported by traditional herbal medicine
dealers. But in many regions of Africa, it is widely consumed as
self-medication by patients suffering from diabetes, hypertension and HIV/AIDS
(Dieye et al., 2008;
Kasolo et al., 2010). It’s also used as abortifacent (Nath et al., 1992; Shigh and Kuma, 1999) and
diabetes (Gupta and Mishra, 2002).
Interestingly,
the glucose lowering action of M.
oleifera aqueous leaf extract has been reported in normal and sub mildly
and severely diabetic rats (Jaiswal et al.,
2009).Thus, the nutritional and
pharmacological importance of this plant are being extolled by various
scientists. Hypercholesterolemia and hyperlipidaemia are recorded complications
of diabetes mellitus resulting from alterations in lipid metabolism
characterized by elevated level of cholesterol and triglycerides and they are
responsible for vascular complications (Sharma et al.,1996). The liver cell shows a pronounced high level of lipid
concentration during diabetes. There is increase in catabolic processes such as
glycogenolysis and lipolysis which is as a result of lack of insulin or insulin
insensitivity (Raju et al., 2001).
Many
studies have demonstrated that insulin modulates glucose transport in fat depot
by rapid movement of glucose transporters from an intracellular membrane pool
to the cell surface (Kahn, 1996). At
least two of these transporters, GLUT1 and GLUT4, coexist in muscle and adipose
tissues in which glucose transport is markedly stimulated by insulin. Although
GLUT 1 is also expressed in numerous tissues in which glucose transport is not
highly regulated by insulin, GLUT4 is confined primarily to these markedly
insulin responsive tissues (Hauner, 1998). Most studies suggest that GLUT4 is
the major glucose transporter in muscle and adipose cells (James et al., 1989). To understand the biochemical
mechanisms behind in-vivo insulin resistance in states such as diabetes and
fasting, the regulation of glucose transporter expression was recently studied
in adipose cells. Initially, adipose cells were thought to be a model for
muscle because in both tissues, the acute increase in transport of glucose in
response to insulin appears to result, at least in part, due to the movement of
glucose transporter proteins from an intracellular domain to the cell surface(Resh,
1982). The expression of GLUT4 in adipose cells was reduced at both the mRNA
and protein levels with diabetes and restored with insulin treatment. GLUT1
expression was much less affected. These observations led to the hypothesis
that the level of expression of GLUT4 is a major determinant of
insulin-stimulated glucose transport in insulin-responsive tissues (Hauner,
1998; Yvan et al., 1997).
It has also been observed with 5-amino-imidazole-
4-carboxamide ribonucleotide (AICAR), an adenosine monophosphate (AMP) analogue
and known AMP Kinase (AMPK) activator suggesting that AMPKinase is responsible for contraction mediated
glucose disposal (Blerina et al.,
2008). Activation of AMPK by AICAR in rat or muscle cells that largely express
constitutively active AMPK modulates glucose uptake and also result in movement of the glucose
transporter proteins(GLUT1 and GLUT4) from inner membrane to outer cell surfaceproviding
a relationship between AMPK activation, glucose transport and glucose transporters movement (Parimal, and Ranjan, 2006). AMPK could be referred as enzyme that
controlsglucose and lipid metabolism. The AMPK is an enzyme that regulates the
energy levels of the cell, being activated when the high energy phosphate
depletes. AMPK is also activatedby contraction of skeletal muscle and
myocardial ischaemia, and has also been implicated in the modulation of glucose
transport and fatty acid oxidation. When the liver AMPKinase activity is
enhanced, it results in improved fatty acid oxidation and decrease in
production of glucose, cholesterol, and triglycerides (Buhl et al., 2002).
The enthusiasm for the health benefits of M.
oleifera is in dire contrast with the scarcity of strong experimental and
clinical evidence supporting them and in all its effect on glucose transport
proteins (Majambu, 2012). The detailed study of the effects of M. oleifera leaf extract on transport
proteins- GLUT I, GLUT 4, Na+/K+-ATPase and AMP-kinase activities as a possible
mechanism of action is yet to be reported. This forms the basis for this study.
1.2 AIM OF
THE STUDY
The aim of the study isto investigate the effects of crude
extract and fractions M. oleifera leaf
extract on glucose transport protiens-GLUT 1, GLUT 4, Na+-K+-
ATPase, AMP-Kinase activities and other biochemical parameters
in streptozotocin(STZ)-induced diabetes male albino rats.
1.3 OBJECTIVES OF THE STUD
1 To identify the bioactive compounds
using Gas chromatography-mass spectrometry (GCMS) analysis in both the
fractions and the crude extract
2 To determine the effect of aqueous,
methanol and fractions of M. oleifera
leaf extract on GLUT 4, GLUT 1, AMP-Kinase and Na+-K+-
ATPase activities in STZ –
induced diabetic male wistar rats
3 To determine the effect of aqueous,
methanol and fractions of M. oleifera
leaf extract on serum proteins and glycated heamoglobin in STZ – induced
diabetic male wistar rats.
4 To
determine the effect of aqueous, methanol and fractions of M. oleifera leaf extract on selected enzymes (lipoprotein lipase,
intestinal amylase and phosphate dehydrogenase) in STZ – induced diabetic male
rats.
5 To determine the effect of aqueous,
methanol and fractions of M. oleifera
leaf extract on lipid profile in STZ –
induced male diabetic rats.
6 To study the effects of aqueous,
methanol and fractions on the histology of the liver, kidney and pancreas in
normal and STZ- induced male diabetic rats
1.4 STATEMENT OF PROBLEM
Alternative therapy such as herbal
medicine has been popular since the ancient time for the treatment of diabetes.
Their popularity is based on the assumption that they are of natural source and
therefore not harmful. More importantly is the fact that they are readily accessible,
cheap and can be acquired without medical prescription.
The mechanism of action of most
herbal drugs has also been elucidated in some cases. M. oleifera is one plant that have gotten wide acceptance in the
international community for its anti-diabetic action. However, limited
scientific data has been reported on the effect of leaf extract on glucose
transport proteins-GLUT 1, GLUT 4, AMPKinase and Na+/K+-ATPase
activities as a possible mechanism of action. Thus this study is aimed at
effects of crude extract and fractions of M.
oliefera leaf on glucose transport proteins,AMPKinase, Na+-K+-ATPase
activities possible mechanism of action
in STZ-induced diabetic male wistar rats.
1.5 JUSTIFICATION OF THE STUDY
The epidemic spread of diabetes and
identification of new therapeutic avenues in the treatment of all pathological
aspects of this disorder remain a major challenge for current biomedical
research. Although wide varieties of pharmacological drugs are being used for
the management of diabetes but due to the adverse side effects with prolonged
treatment, conventional drugs are not always satisfactory in maintaining normal
level of blood glucose and avoiding late stage diabetic consequences. This has
led to an increase in the demand for natural products with anti-diabetic
activity with fewer side effects. The
benefits for the treatment or prevention of disease or infection that may
accrue from either dietary or topical administration of Moringa preparations (e.g. extracts, decoctions, poultices, creams,
oils, emollients, salves, powders, porridges) are not quite so well known.
Although the oral history here is also voluminous, it has been subject to much
less intense scientific scrutiny, and it is useful to review the claims that
have been made and to assess the quality of evidence available for a more
well-documented claims. There is need to balance the evidence from
complementary and alternative medicine (traditional medicine, tribal lore, oral
histories and anecdotes) with the burden of proof required in order to make
sound scientific judgments on the efficacy and mechanism of actions of these
traditional cures. There have been reported studies on the anti-diabetic
effects of this plant but there are no reported scientific study done on the
effects of crude leaf extract on insulin responsive GLUT 4 , non-insulin
responsive GLUT 1, Na+/K+-ATPaseand AMP kinase levels in
STZ-induced diabetic rats. This study will enable us understand the biological
effects and the mechanism of action of this plant in the management of diabetes
and will also serve as a documented evidence for future research and
referencing.
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