Pharmacological therapy of insulin response or release

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Introduction

In many developing countries, herbal medicines are of vital importance in primary health care (Kamboj, 2000). This is supported by literature in behavioural and pharmacological sciences with animals and people using a number of different plants for the control of disease symptoms and related illnesses in their environment (Hart, 2004; Cousins and Huffman, 2002). One of such disorder is diabetes mellitus, which is a chronic disease characterised by prolonged hyperglycaemia,especially post-prandial,in association with the consumption of diets that promote obesity.

Digestion

Digestion is the mechanical (chewing) and chemical (enzymatic action) breakdown of food into smaller components that are more easily absorbed into the blood stream (Reed and Wickham, 2009).

Absorption and transport of glucose

The newly formed glucose is rapidly absorbed across the wall of the small intestine with absorption being complete before reaching the terminal ileum. Due to the water soluble nature of glucose, its absorption is reliant on the presence of a specific co-transport protein that is driven by the facilitated absorption of sodium (symport dependent on sodium transport) i.e. in the absence of sodium no glucose absorptionresults (Nelson and Cox, 2005). The transporter has been named the Sodium – Glucose symporter (SGLUT). This driving force for the movement of sodium into the cell is created by the active sodium-potassium pump (Na+/K+ATPase) which depletes the cytoplasm of sodium by moving three sodium molecules out of the cytoplasm and two potassium molecules therein in order to maintain electrical neutrality (Figure 2-4). The glucose then leaves the cell via the basal cell membrane through the passive and specific glucose transporter GLUT2(Nelson and Cox, 2005) and enters into the blood capillaries.

Entry of Glucose into cells

Glucose has to be transported from the blood across the cell membrane into the cellular cytoplasm before it can be used by the body’s tissue cells (Guyton and Hall, 2000). However, with the membrane of cells being impermeable to glucose, the cell is reliant on transmembrane proteins transporters fordiffusion into the cell. The glucose transporters in non-epithelial mammalian tissues are part of the family of glucose transporters known as the GLUT receptors.

DECLARATION
DEDICATION
ACKNOWLEDGEMENT
ABSTRACT
CHAPTER 1
1.1 Introduction
1.2 Hypothesis
1.3 Aim
1.4 Objectives
CHAPTER 2
2 LITERATURE REVIEW
2.1 Introduction
2.2 Glucose Metabolism
2.2.1 Digestion
2.2.2 Absorption and transport of glucose
2.2.3 Entry of Glucose into cells
2.3 Insulin
2.3.1 Structure and biochemistry
2.3.2 Mechanism of insulin secretion
2.3.3 Mechanism of insulin action
2.3.4 Physiology of normal insulin secretion
2.4 Diseases of improper glucose metabolism
2.4.1 Type I diabetes mellitus
2.4.2 Type II diabetes mellitus
2.4.3 Other forms of diabetes
2.4.4 Complications of diabetes
2.5 Management and treatment of diabetes
2.5.1 Therapy for weight loss
2.5.2 Pharmacological therapy of insulin response or release
2.6 Medicinal plants and diabetes
2.6.1 Polyphenols
2.6.2 Polyphenols and diabetes
2.6.3 Polyphenols and obesity
2.6.4 Polyphenols and antioxidant defence
2.6.5 Potential risk associated with polyphenols
2.7 Selection of plant species
2.7.1 Ethnomedical uses of Ficus species
2.8 Conclusion
CHAPTER 3
3 MATERIALS AND METHODS
3.1 Materials
3.1.1 Reagents and chemicals
3.1.2 Cell lines and primary cell cultures
3.1.3 Plant material
3.1.4 The selected Ficus species
3.2 Methods
3.2.1 Preparation of leaf extracts
3.2.2 Phytochemical analysis of extracts
3.2.3 Total polyphenolic content
3.2.4 Trolox equivalent antioxidant capacity (TEAC)
3.2.5 αAmylase inhibition assay
3.2.6 α Glucosidase inhibition assay
3.2.7 Kinetics of inhibition against α amylase and α glucosidase activities
3.2.8 Calculation of EC50
3.2.9 Cytotoxicity assay
3.2.10 Glucose uptake in primary cell cultures
3.2.11 Glucose uptake in established cell lines
3.2.12 Insulin secretion assay
3.2.13 Solventsolvent fractionation and isolation of compounds
3.2.14 Isolation of compounds
3.2.15 General experimental procedures
3.2.16 In vivo assay
3.2.17 Statistical analyses
CHAPTER 4
4 RESULTS
4.1 Crude extracts
4.1.1 Extraction of plants
4.1.2 Phytochemical analysis
4.1.3 Antioxidant activity
4.1.4 Total polyphenol content and antioxidant activity
4.1.5 αAmylase inhibitory activity of extracts of Ficus species
4.1.6 αGlucosidase inhibitory activity of extracts of Ficus species
4.1.7 The enzyme kinetics of α amylase and α glucosidase inhibition by extract of F. lutea
4.1.8 Cytotoxicity of the acetone extracts of the ten Ficus species
4.1.9 Glucose uptake activity in primary rat abdominal muscle culture
4.1.10 Glucose uptake in primary rat epididymal fat cells
4.1.11 Glucose uptake activity in C2C12 muscle cells
4.1.12 Glucose uptake activity in H411E liver cells
4.1.13 Glucose uptake in 3T3L1 preadipocytes
4.1.14 Insulin secretion in RINm5F pancreatic cells
4.2 Solventsolvent fractionation of extract of F. lutea
4.2.1 Percentage yield of fractions
4.2.2 Antioxidant activity
4.2.3 Total polyphenol content
4.2.4 Inhibition of αamylase activity by the fractions from the acetone extract of F. lutea
4.2.5 Inhibition of αglucosidase activity by the fractions of acetone extract of F. lutea
4.2.6 Cytotoxicity of the fractions from acetone extract of F. lutea
4.2.7 Glucose uptake in C2C12 muscle cells
4.2.8 Glucose uptake activity in H411E liver cells
4.2.9 Insulin secretion in RINm5F pancreatic cells
4.3 Structure Elucidation of Compounds from the ethyl acetate fraction of F. lutea
4.3.1 Structure Elucidation of AFL1 or Lupeol (1)
4.3.2 Structure Elucidation of AFL2 or Stigmasterol (2)
4.3.3 Structure Elucidation of AFL3 or αAmyrin Acetate (3)
4.3.4 Structure elucidation of AFL4 or Epicatechin (4)
4.3.5 Structure elucidation of AFL5 or Epiafzelechin (5)
4.3.6 Inhibition of αglucosidase activity by the compounds
4.3.7 Glucose uptake in C2C12 muscle cells
4.3.8 Glucose uptake in H411E liver cells
4.3.9 Insulin secretion in RINm5F pancreatic cells
4.4 In vivo study
4.4.1 The effect of different diets on body weight
4.4.2 The effect of different diets on food intake
4.4.3 The effect of different diets on faecal weight
4.4.4 The effect of high calorie diet on blood glucose concentration
4.4.5 The effect of different diets on full blood count parameters
4.4.6 The effect of different diets on serum chemistry parameters
4.4.7 The effect of diets on gross pathological changes in CD1 mice.
CHAPTER 5
5 DISCUSSION
5.1 Selection of solvent for extraction of plants
5.2 Efficacy of crude extracts of the ten Ficus species
5.3 Isolation of the active components from the acetone extract of F. lutea
5.4 The isolated compounds from the ethyl acetate fraction of F. lutea
5.5 In vivo assay
CHAPTER 6
6 CONCLUSION
6.1 Future Work
REFERENCES
APPENDIX