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EVALUATION of the ANTIDIABETOGENIC EFFECTS of the FREEZE-DRIED EXTRACTS of Rotheca Myricoides on TYPE 2 DIABETES in an ANIMAL MODEL

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EVALUATION of the ANTIDIABETOGENIC EFFECTS of the FREEZE-DRIED EXTRACTS of Rotheca Myricoides on TYPE 2 DIABETES in an ANIMAL MODEL EVALUATION OF THE ANTIDIABETOGENIC EFFECTS OF THE FREEZE-DRIED EXTRACTS OF Rotheca myricoides ON TYPE 2 DIABETES IN AN ANIMAL MODEL BY BONIFACE MWANGI CHEGE REG. NO. H56/89241/2016 A RESEARCH THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE AWARD OF A DEGREE IN MASTER OF SCIENCE IN MEDICAL PHYSIOLOGY OF THE UNIVERSITY OF NAIROBI. APRIL, 2019 DECLARATION I hereby declare that this thesis is my original work and to the best of my knowledge has not been presented elsewhere for approval and for the award of a degree, diploma or certificate. I further declare that all material cited in this thesis that is not my own work has been duly referenced. Signature: _______________________________ Date: _________________ Boniface Mwangi Chege (Investigator) Approval by Supervisors This thesis has been submitted for examination with our approval as university supervisors. Signature: _______________________________ Date: _________________ Dr Peter Waweru Mwangi. Department of Medical Physiology, University of Nairobi. Signature: _______________________________ Date: _________________ Dr Frederick Bukachi. Department of Medical Physiology, University of Nairobi ii ACKNOWLEDGEMENTS I raise my heart in gratitude to God almighty for all the blessings. He has been the guiding force behind my efforts. I wish to express my sincere thanks and appreciation especially to the following, My supervisors, Dr Peter Waweru Mwangi and Dr Frederick Bukachi for their scholarly guidance, availability at all times, constant encouragement and keen interest during the course of this study. Prof. Owino Okong’o, Prof. Nilesh Patel for the teaching in various areas of medical physiology and research. All members of the Department of Medical Physiology, for providing a very friendly working environment, especially to the department administrators i.e. Caroline Makandi and Elizabeth Juma. The technical staff, Mr. David Wafula, Mr Horo Mwaura, Mr. Jackson Mugweru and Mr. Thomas Arani, for their encouragement, availability at all time and all the support given during this study. Dr Zephaniah Birech and Mr. Moses Juma for their assistance with Raman Spectroscopy. My colleagues, Prabjot Sehmi and Nelly Nyaga for the cheerful atmosphere and inputs they provided throughout the study. My Parents Mr. Samson Chege & Mrs. Jane Chege for their warm care and support, my beautiful wife Christine Mwangi, my handsome boys Ethan Chege and Ellis Githae for always staying by my side. iii TABLE OF CONTENTS DECLARATION ......................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................ iii TABLE OF CONTENTS ............................................................................................ iv LIST OF FIGURES .................................................................................................. viii LIST OF TABLES ....................................................................................................... x LIST OF ABBREVIATIONS AND ACRONYMS .................................................... xi ABSTRACT ............................................................................................................... xii CHAPTER ONE .......................................................................................................... 1 1.1 Background ........................................................................................................ 1 1.2 Problem statement .............................................................................................. 2 1.3 Study justification ............................................................................................... 3 1.4 Hypothesis .......................................................................................................... 4 1.5 Objectives ........................................................................................................... 4 1.5.1 Specific objectives ........................................................................................... 4 1.6 Research question ............................................................................................... 5 CHAPTER TWO ......................................................................................................... 6 2.0 LITERATURE REVIEW ................................................................................... 6 2.1 Rotheca myricoides (Hochst.) Steane & Mabb plant ...................................... 6 2.2 Phytochemistry screening of Rotheca myricoides (Hochst.) Steane & Mabb. 8 2.3 Classification and diagnosis of diabetes mellitus ........................................... 8 2.4 Pathophysiology of type 2 diabetes mellitus .................................................. 9 2.5 Mechanism of action of hypoglycaemic herbs ............................................. 11 2.6 Induction models of type 2 diabetes mellitus ............................................... 12 2.7 Measurement of branch chain amino acids using Raman spectroscopy ....... 12 iv CHAPTER THREE .................................................................................................... 14 3.0 MATERIALS AND METHODS ..................................................................... 14 3.1 Extraction of Rotheca myricoides (Hochst.) Steane & Mabb and diet preparation .......................................................................................................... 14 3.2 Animal procedures and protocols ................................................................. 14 3.3 Determination of the efficacy of the Rotheca myricoides extract ................ 15 3.4 Measurement of fasting blood glucose and body weight of experimental rats ............................................................................................................................ 15 3.5 Determination of insulin sensitivity using oral glucose tolerance test (OGTT) ............................................................................................................................ 16 3.6 Measurement of fasting serum insulin levels ............................................... 16 3.7 Quantification of lipid profile and serum uric acid levels ............................ 16 3.8 Estimation of hepatic triglycerides levels in rats .......................................... 16 3.9 Determination of branched chain amino acids using Raman spectroscopy.. 18 3.10 Mechanism of action of the freeze-dried extract of Rotheca myricoides ... 18 3.11 Ethical approval .......................................................................................... 19 3.12 Data analysis ............................................................................................... 19 CHAPTER FOUR ...................................................................................................... 20 4.0 RESULTS ......................................................................................................... 20 4.1 Baseline characteristics of the rats and percentage yield of the extract ........ 20 4.2 Effects of the freeze-dried extract of Rotheca myricoides on body weight of the rats ................................................................................................................. 21 4.3 Outcome of freeze-dried extract of Rotheca myricoides on fasting blood glucose ................................................................................................................ 25 4.4 Results of Rotheca myricoides extract on glucose tolerance ........................ 31 4.5 Effect of the Rotheca myricoides extract on fasting insulin levels. .............. 35 4.6 Outcome of Rotheca myricoides extract on the homeostatic model assessment of insulin resistance (HOMA-IR) .................................................... 36 4.7 End result of Rotheca myricoides extract on hepatic triglycerides. .............. 38 4.8 Effect of Rotheca myricoides extract on plasma lipids................................. 39 v 4.9 Changes on liver weight after administration of Rotheca myricoides extract ............................................................................................................................ 44 4.10 Liver weight: body weight ratio changes after administration of Rotheca myricoides extract. .............................................................................................. 45 4.11 Effect of Rotheca myricoides extract on serum uric acid ........................... 47 4.12 Outcome of Rotheca myricoides on retroperitoneal adipose tissue ............ 48 4.13 Outcome of Rotheca myricoides extract on pericardial adipose tissue ...... 49 4.14 Effect of the Rotheca myricoides extract on mesenteric adipose tissue ..... 50 4.15 Mechanism of action of the Rotheca myricoides extract by using a Glut-4 blocker Indinavir sulphate .................................................................................. 51 4.16 Acute response of the Rotheca myricoides extract after oral glucose tolerance test. ...................................................................................................... 54 4.17 Effect on Rotheca myricoides extract on the branched chain amino Raman spectra. ................................................................................................................ 56 CHAPTER FIVE ........................................................................................................ 63 5.1 DISCUSSION, CONCLUSION AND
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