ABSTRACT
Diabetes mellitus is a chronic metabolic disorder which alters several cellular, tissue, organ and system functions, often leading to death. This research, however, was designed to determine the effects of the methanol and aqueous extracts of V. doniana leaves on diabetes mellitus. The antioxidant parameters, membrane stabilisation, Na+/K+-ATPase activity, glycated haemoglobin and electrolytes concentrations were measured using spectrophotometric methods. Other parameters such as the fasting blood glucose concentration, body weights and histopathology of the pancreas were also determined. Thirty-five albino male rats of 4-7 weeks old, weighing 70-140 g were used for this study. The rats were housed in steel cages and acclimatised for one week prior to the experiment. The rats were induced with diabetes using streptozotocin at 65 mg/kg body weight (bwt). The rats were grouped into seven. Group 1 was the normal control, given animal feed and distilled water; group 2, the drug control, received 5 mg/kgbwt of glibenclamide. Group 3 was the negative control, diabetic but not treated. Groups 4 and 5 were given 400 and 800 mg/kgbwt of the aqueous extract respectively whereas groups 6 and 7 received 400 and 800 mg/kgbwt of the methanol extract respectively. The administration lasted for 28 days. Phytochemical analysis of the methanol extract of the leaves indicated the presence of flavonoids, alkaloids, tannins, glycosides, phenols, reducing sugars, steroids, saponins, soluble sugars, terpenoids and hydrogen cyanide. Acute toxicity study showed the relative safety of the extracts at 5000 mg/kgbwt. Generally, there were significant (p<0.05) increases in the fasting blood glucose concentrations of all diabetic rats compared to that of the rats in the normal control. However, prior to sacrifice, all the treated rats showed normal concentrations of fasting blood glucose. There was no significant (p>0.05) difference in body weights of rats across all groups. There was no significant (p>0.05) difference in the glycated haemoglobin concentration of rats across all groups compared to that of the rats in the normal control. Also, there was a significant (p<0.05) increase in the activity of Na+/K+-ATPase of rats in group 6 while rats in group 3 recorded a significant (p<0.05) low activity. The activities of SOD and CAT showed a significant (p<0.05) increase in rats of all treated groups while there was no significant (p>0.05) difference in GPX activities of rats in all treated groups compared to that of the rats in the normal control. Rats in group 6 showed a significant (p<0.05) reduction in MDA concentration. The methanol extract showed a higher membrane stabilising potential than the drug glibenclamide at 5 mg/kgbwt. Concerning electrolytes, there was generally a significant (p<0.05) reduction of Cl- in rats across all treated groups. A significant (p<0.05) reduction in HCO3- was recorded in group 7. Rats in groups 2 and 7 also showed significant (p<0.05) reductions in Na+ and Ca2+ across all treated groups compared to that of the normal control. Histopathology results revealed considerable regeneration of the pancreatic beta cells of the rats in groups treated with the methanol and aqueous extracts of V. doniana leaves. The results obtained from this research clearly indicate the effectiveness of the methanol and aqueous extracts of V. doniana leaves as antidiabetics.
TABLE OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables xi
List
of Figures xii
List
of Plates xiii
Abstract xiv
CHAPTER 1: INTRODUCTION
1.1
Background of the Study 1
1.2 Statement of the Problem 3
1.3 Aim of the Study 4
1.4 Objectives of the Study 4
1.5 Justification of the Study 5
CHAPTER
2: REVIEW OF RELATED LITERATURE
2.1 Diabetes Mellitus 7
2.1.1 Epidemiology and pathogenesis of type I
diabetes mellitus (IDDM) 7
2.1.2 Pathophysiology of type I diabetes mellitus 8
2.1.3 Epidemiology and pathogenesis of type II
diabetes mellitus 10
2.1.4 Pathophysiology of type II diabetes mellitus
(NIDDM) 10
2.1.5 Clinical characteristics of patients with
type I and II diabetes mellitus 11
2.1.6 Diabetes in pregnancy 12
2.1.7 Management of diabetes mellitus 12
2.1.7.1 Current pharmacological therapies 13
2.1.7.2 Oral therapies 13
2.1.7.3 Lifestyle modification, literacy and
self-management 14
2.1.8 Oxidative stress and DNA damage during
diabetes mellitus 15
2.2 Insulin Signalling Pathway 15
2.2.1 Glucose storage and uptake 15
2.2.2 Regulati7n of insulin signalling 17
2.2.3 Relationship between obesity and diabetes 17
2.3 Vitex doniana 18
2.3.1 Scientific classification 20
2.3.2 Botanic description 20
2.3.3 Biology 20
2.4 Health and Medicinal Benefits of V. doniana 21
2.5 Nutritional Components of V. doniana 21
2.6 Phytochemical Components of V. doniana 22
2.7 Glycated Haemoglobin (HbA1c) 23
2.7.1 Historical background of glycated
haemoglobin 23
2.7.2 Non-enzymatic
glycation compared with enzymatic deglycation 24
2.7.3 Clinical use of HbA1c 25
2.8 Antioxidant Enzymes 26
2.8.1 Properties and biological implications of
antioxidant enzymes 30
2.8.1.1 Superoxide dismutase (SOD) 30
2.8.1.2 Catalase (CAT) 32
2.8.1.2.1 Cellular role of catalase 33
2.8.1.3 Glutathione peroxidase (GPX) 34
2.9 Malondialdehyde (MDA) 35
2.10 Na+/K+-ATPase 36
2.11 Lethal Dose (LD50) 37
2.12 Electrolytes 37
2.12.1 Potassium 38
2.12.2 Sodium 38
2.12.3 Calcium 39
2.12.4 Chloride 40
2.12.5 Bicarbonate 40
2.13 Membrane Stabilisation 41
CHAPTER 3: MATERIALS AND
METHODS
3.1 Equipment 42
3.2 Reagents and Chemicals 43
3.3 Collection and Identification of the
Plant 43
3.4 Methanol Extraction 43
3.5 Aqueous Extraction 44
3.6 Animal Experiment 44
3.7 Animal Grouping 44
3.8 Determination of the Lethal Dose (LD50)
of the Extracts 45
3.9 Induction of Diabetes Mellitus 45
3.10 Administration of the Extracts 45
3.11 Determination of the Fasting Blood Glucose
Concentration 46
3.12 Assessment of the Body Weights of the Rats 46
3.13 Collection of Blood 46
3.14 Determination
of Glycated Haemoglobin 47
3.15 Assay of Na+/K+-ATPase
Activities 48
3.16 Estimation of Malondialdehyde (MDA)
Concentrations 49
3.17 Measurement of Catalase (CAT) 49
3.18 Assay of Superoxide Dismutase (SOD)
Activities 49
3.19 Assay of Glutathione Peroxidase (GPX)
Activities 50
3.20 Determination of Membrane Stabilisation 51
3.21 Estimation
of Bicarbonate Ions (HCO3-) using Titration Method 51
3.22 Determination of Sodium Ions (Na+)
Concentrations 52
3.23 Determination of Potassium ions (K+)
Concentrations 52
3.24 Determination of Chloride Ions (Cl-)
Concentrations 53
3.25 Estimation of Calcium Ions (Ca2+)
Concentrations 54
3.26 Histological Preparations 54
3.27 Statistical Analyses 54
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Results 55
4.1.1 Qualitative phytochemical composition of V. doniana leaves 55
4.1.2 Acute toxicity study 56
4.1.3 Effects of the methanol and aqueous extracts
of V. doniana leaves on
fasting
blood glucose concentration 57
4.1.4
Effects of the methanol and aqueous extracts of V. Doniana leaves on
body weights 59
4.1.5 Effects of the methanol and aqueous extracts
of V. doniana leaves on
glycated
haemoglobin concentrations 61
4.1.6 Effects of the methanol and aqueous extracts
of V. doniana leaves on
Na+/K+-ATPase
activities 63
4.1.7 Effects of the methanol and aqueous extracts
on malondialdehyde (MDA)
concentration 65
4.1.8 Effects of the methanol and aqueous extracts
on antioxidant enzymes
activities 67
4.1.9 Effects of the methanol extract on membrane
stabilisation 69
4.1.10 Effects of the methanol and aqueous extracts on
electrolytes
concentrations 71
4.1.11 Effect of the methanol and aqueous extracts of
V. doniana leaves
extract
on the histology of the pancreas 75
4.2 Discussion 82
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 89
5.2 Recommendations 90
References 91
Appendices
LIST OF TABLES
2.1: Clinical
characteristics of patients with type I and II diabetes mellitus 11
4.1: Qualitative
phytochemical constituents V. doniana leaves 55
4.2:
LD50 (oral) of the methanol and aqueous extracts of V. doniana leaves 56
LIST OF FIGURES
2.1: Insulin
signalling pathway 17
2.2: V. doniana
Leaves 19
2.3: Generation
of reactive oxygen species and the defense mechanisms
against damage by active oxygen 29
4.1: Effects of the methanol and aqueous
extracts of V. doniana leaves
on
fasting blood glucose concentrations 58
4.2: Effects
of the methanol and aqueous extracts of V. doniana leaves
on body weights 60
4.3: Effects of the methanol and aqueous
extracts of V. doniana leaves
on
glycated haemoglobin concentrations 62
4.4: Effects of the methanol and aqueous
extracts of V. doniana leaves
on
Na+/K+ ATPase activities 64
4.5: Effects of the methanol and aqueous
extracts of V. doniana leaves
on
MDA concentrations 66
4.6: Effects of the
methanol and aqueous extracts on antioxidant enzymes
activities 68
4.7: In
vitro membrane stabilising effects of the methanol and aqueous
extracts
of V. doniana leaves 70
4.8 Effects of the methanol and aqueous
extracts of V. doniana leaves
on
concentrations of Cl-, K+ and HCO3- 72
4.9: Effects of the methanol and aqueous
extracts of V. doniana leaves on
Ca2+ and
Na+ concentrations 74
LIST
OF PLATES
1: Photomicrograph
showing the pancreas of a normal rat (H and E, X40)
75
2: Photomicrograph
showing the effects of STZ-induced diabetes on the
pancreas
of untreated rats (H and E, X40) 76
3: Photomicrograph
showing the effects of the drug, glibenclamide
(5
mg/kgbwt) on the Pancreas of Treated Diabetic Rats (H and E, X40) 77
4: Photomicrograph
showing the effects of the 400 mg/kgbwt of the
methanol
extract on the pancreas of treated diabetic rats (H and E, X40) 78
5: Photomicrograph
showing the effects of the 800 mg/kgbwt of the
methanol
extract on the pancreas of treated diabetic rats (H and E, X40) 79
6: Photomicrograph
showing the effects of the 400 mg/kgbwt of the
aqueous
extract on the pancreas of treated diabetic rats (H and E, X40) 80
7: Photomicrograph
showing the effect of the 800 mg/kgbwt of the
aqueous
extract on the pancreas of treated diabetic rats (H and E, X40) 81
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Diabetes
mellitus is a chronic metabolic disorder which partially or completely blocks
the utilisation of glucose into useful form of energy often resulting in high
blood sugar concentrations over a long period of time. It could be a result
from the inability of the pancreas to produce enough insulin or the
unresponsiveness of the body cells to produced insulin. Diabetes mellitus has
been grouped into three main types. Type1 diabetes, also known as Insulin-Dependent
Diabetes Mellitus (IDDM) results from the failure of the pancreas to produce
enough insulin (WHO, 2013). It is regarded as an autoimmune disorder. Type 2
diabetes mellitus, previously known as non insulin dependent diabetes mellitus
(NIDDM) begins with insulin resistance as cells fail to respond properly to the
insulin produced. Type 2 diabetes mellitus is commonly caused by excessive body
weight and low exercise (WHO, 2013). Gestational diabetes, the third main form,
is temporary. It manifests as high blood sugar concentrations in pregnant women
without a previous history of diabetes.
Diabtes
mellitus is characterised by longterm hyperglycaemia. It is diagnosed when a
patient demonstrates one of the following: fasting plasma glucose concentrations
≥ 7.0 mmol/l (126 mg/dl); plasma glucose concentrations ≥ 11.1 mmol/l (200 mg/dl) two hours after a
75 g oral glucose load as in a glucose tolerance test; symptoms of high blood
sugar and casual plasma glucose concentrations
≥ 11.1 mmol/l (200 mg/dl) and glycated haemoglobin concentrations ≥ 48
mmol/mol (≥ 6.5 % Diabetes Control and Complications Trial) (Diabetes Care,
2010). However, glycated haemoglobin has been used as a better diagnostic tool
for determining diabetic condition as well as its accompanying risks of
cardiovascular diseases and death (Selvin et
al, 2010).
Symptoms
of diabetes mellitus include: frequent urination, increased thirst, and
increased hunger. If untreated, diabetes can lead to severe complications which
may include: diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death
(Kitabchi et al, 2009). There may
also be serious complications such as cardiovascular diseases, stroke, chronic
kidney diseases, foot ulcers and eye damage (WHO, 2013). Diabetes mellitus
usually leads to processes that generate reactive oxygen species (ROS). This is
usually accompanied with a decrease in both enzymatic and non enzymatic
antioxidants, causing oxidative stress (Gumieniczek et al, 2002), compromise of membranes and leakage of important
biological molecules from cells.
Type
2 diabetes can be prevented or delayed by maintaining normal weight, engaging
in physical activity and eating a healthful diet. Dietary measures to help
prevent diabetes mellitus include:
maintaining a diet rich in whole grains and fiber, choosing good fat
such as polyunsaturated fats found in nuts, vegetable oils and fish. Also
helpful are limiting sugary beverages and eating less red meat. Smoking
cessation can also form an important preventive measure (Willi et al., 2007).
Furthermore,
diabetes mellitus could be managed with the target of keeping blood sugar
concentrations as close to normal, without causing hypoglycaemia. This is
usually accomplished with a healthy diet, exercise, weight loss and use of
appropriate medications such as metformins and sulfonylurease (Ripsin et al., 2009). In rare cases, a pancreas
transplant and weight loss surgeries are often recommended (Picot et al., 2009). Insulin administration
has also been useful in managing diabetes especially type 1.
Recently,
biological research has taken a turn into the exploitation of natural
substances in plants for an improved management of diabetes, with its
accompanying complications. Abdulrahman et
al. (2015) demonstrated the antidiabetic effect of Moringa oleifera Lam. seeds in male rats. Salim et al, (2016)
showed the hypoglycaemic effect of Vitex
simplicifolia leaf extracts on
rats. Numerous other plants and plant materials have been shown to exhibit
ameliorative effects through different mechanisms that reverse diabetic
complications (Zhou et al., 2013).
V. doniana
is a perennial shrub highly distributed in high rainfall areas. It is variously
called vitex or black plump (English), Dinya (Hausa), Oriri (Yoruba), Ejiji
(Kogi) and Ucha okoro (Igbo) (Burkill, 2000). In north central and eastern part
of Nigeria, the young leaves of V.
doniana are employed as vegetables as well as sauces for meals especially
for patients suffering from diabetes. In rural Abia State, for example, the hot
aqueous extracts of these leaves have been used by diabetics.
Due
to the ethical issues involving human experiments, rat models of the Wistar
strain were used for this experiment. Thus, the pathophysiology of diabetes
mellitus, as well as the effects of both aqueous and methanol extracts of V. doniana leaves administered to
diabetic rats were carefully studied under controlled conditions.
1.2 STATEMENT OF THE PROBLEM
Diabetes
is a macro degenerative disease in the world today, affecting over 150 million
people and having complications which include: hypertension, atherosclerosis
and microcirculatory disorders (Ogbonna et
al., 2008).
Globally,
the prevalence of diabetes mellitus is on the increase. In African communities,
drastic lifestyle changes accompanying urbanisation and westernisation have
been a major factor in higher rates (Sobngwi et al, 2010). Also, there has been an identification of genes that
raise susceptibility to diabetes in communities of Ghana and Nigeria (Rotimi et al, 2010). Hence, it poses a great
burden on health care coupled with difficult economic situations.
The
impairment of energy metabolism by diabetes mellitus usually causes numerous
complications often resulting in death (Kitabchi et al, 2009). Beta oxidation, an alternative means of generating
energy results in overproduction of reactive oxygen species. This further leads
to a compromise of natural antioxidants activities and reduced membrane
integrity by lipid peroxidation (Rauscher et
al, 2001). Other biochemical pathways for glucose metabolism are also
affected.
1.3 AIM OF THE STUDY
The
aim of the research work was to determine the effects of the methanol and
aqueous extracts of V. doniana leaves
on Na+/K+-ATPase and some markers of oxidative stress in
streptozotocin-induced diabetic albino male rats.
1.4 OBJECTIVES OF THE STUDY
To fully achieve the aim of this research,
the following objectives have been set out to form a clean guide:
- To determine the phytochemical
content of the methanol and aqueous leaf extracts of V. doniana.
- To determine the lethal dose of
the methanol and aqueous leaf extracts V. doniana.
- To determine if the methanol and
aqueous leaf extracts of V. doniana could have some positive
effects on some biochemical processes that are affected by diabetic
conditions such as membrane stability and glucose metabolism.
- To investig.ate what happens to
the activity of Na+/K+-ATPase under diabetic
condition and consequent treatment with oral administration of the
methanol and aqueous leaf extracts of V. doniana.
- To study the effects that the
methanol and aqueous leaf extracts of V. doniana could have on Superoxide
Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPX)
and Malonyldialdehyde (MDA).
- To ascertain the effects of the
methanol and aqueous leaf extracts of V. doniana on electrolyte such as sodium,
potassium, chloride, calcium and bicarbonates.
- To investigate the effects of
diabetic condition and treatment with the methanol and aqueous leaf
extracts V. doniana on body weights of rats.
- To investigate if the methanol
and aqueous leaf extracts of V. doniana could have any
effects on the histology of the pancreas under diabetic condition.
1.5 JUSTIFICATION
OF THE STUDY
A
recent survey has concluded that 90-95% of all diagnosed cases of diabetes
mellitus are of the type 2 (O’Brien, 2011). Type 2 diabetes mellitus is
characterised by insulin resistance (Holt, 2010). Biochemical research however,
has sought new therapeutic avenues in the treatment of diabetes and all its
accompanying disorders. So far a wide array of pharmacological drugs is
available for managing diabetes. Some of these drugs even have more
life-threatening after effects. Prevalent economic hardship coupled with the
high cost of prescription medicine, especially in the developing countries requires
the need for natural herbal remedies that are cheaper.
Granted,
V. doniana leaves as well as other parts of the plant have been used successfully in
managing other health risks. However, little is known on the effects of V. doniana leaves in the management of
disorders associated with diabetes mellitus, especially bordering on the
activity of Na+/K+-ATPase and antioxidant enzymes.
The
study will further underscore the application of V. doniana leaves extracts in particular, and plant extracts as a whole,
in managing or even treating diabetic complications.
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