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Evaluation of the Modulatory Effect Of EVALUATION OF THE MODULATORY EFFECT OF METHANOLIC EXTRACT OF CADABA FARINOSA Forssk ON CARBONIC ANHYDRASE IN STREPTOZOTOCIN INDUCED DIABETIC RATS BY Salihu Ismail IBRAHIM DEPARTMENT OF BIOCHEMISTRY FACULTY OF LIFE SCIENCES AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA. JULY, 2017 i EVALUATION OF THE MODULATORY EFFECT OF METHANOLIC EXTRACT OF CADABA FARINOSA Forssk ON CARBONIC ANHYDRASE IN STREPTOZOTOCIN INDUCED DIABETIC RATS BY Salihu Ismail IBRAHIM B.Sc (2005), M.Sc (2011) BUK PhD/SCIE/1254/2011-2012 THESISSUBMITTED TO THE SCHOOL POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA. IN PARTIAL FULFILLMENT FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE (Ph.D) IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY AHMADU BELLO UNIVERSITY, ZARIA-NIGERIA. JULY, 2017 ii DECLARATION I declare that the work in this Project Thesis entitled „EVALUATION OF THE MODULATORY EFFECT OF METHANOLIC EXTRACT OF CADABA FARINOSA ForsskON CARBONIC ANHYDRASE ACTIVITY IN STREPTOZOTOCININDUCED DIABETIC RATS‟has been carried out by me in the Department of Biochemistry, Ahmadu Bello University, Zaria.The information derived from the literature has been duly acknowledged in the text and a list of references provided. No part of this thesis was previously presented for another degree or diploma at this or any other Institution. Salihu Ismail Ibrahim………………………… ……………………… PhD/SCIE/1254/2011-2012Signature Date iii CERTIFICATION This thesis titled „EVALUATION OF THE MODULATORY EFFECT OF METHANOLIC EXTRACT OF CADABA FARINOSA ForsskON CARBONIC ANHYDRASE ACTIVITY IN STREPTOZOTOCININDUCED DIABETIC RATS‟ by Salihu Ismail IBRAHIM meets the regulations governing the award of the degree of Doctor of Philosophy (Ph.D) of the Ahmadu Bello University, and is approved for its contribution to knowledge and literary presentation. Prof. D. A. Ameh ……………………………….. ……………………. …………… Chairman, Supervisory Committee Signature Date Prof. S. E. Atawodi FAS …………………………………. ……………………… ……………… Member, Supervisory Committee Signature Date Prof. I. A. Umar …………………………………. ……………………… ……………… Member, Supervisory Committee Signature Date Prof. M.N. Shuaibu ……………………. ………………………… ………………. Head of Department SignatureDate Prof. S.Z. Abubakar ……………………… …..………………….. ……………….. Dean, School of Postgraduate Studies Signature Date iv DEDICATION I dedicate this work to my family for their abundant support, understanding and love v ACKNOWLEDGEMENTS All praise is due to Allah for bestowing me with the knowledge and wisdom to carry out this research. Firstly, I would like to acknowledge my supervisor(s) Prof. D.A Ameh, Prof. S.E. Atawodi and Prof. I.A. Umar for the opportunity they have provided me in putting this thesis together.I am appreciative of their encouragement, insight and their diligence towards the successful completion of this project. Without their guidance, this project would nothave gone forward or been as interesting. I would like to express deep gratitude to the Head Department of Biochemistryfor his intellectual advice. I would like to also thank Prof A.J. Nok, Dr Auwal Ibrahim and all the staffs of the Department of Biochemistry for their support and help. I would like to recognize Bashir Musa of Multiuser Research Laboratories who was there in the beginning to show me the fundamentals of carrying out laboratory analysis. I would like to recognize Dr. James Habila of the Department of Chemistry, Mal Muazu, Mal. Abdullahi and Aliyu for theirassistance with the laboratory analysis and animal work. I will not end without showing appreciation to my fellow PhD research students for providing an important support network. Finally the biggest thanks go to my family, May Allah rewards them abundantly. vi TABLE OF CONTENTS Contents Page Title Page - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - ii Declaration - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - iii Certification - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -iv Dedication - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - v Acknowledgments - -- - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - vi Table of Contents - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - vii List of Tables - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - xii List of Figures - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - -xiii List of Plate - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - -xv List of Abbreviation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - xvi Abstract - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - xvii CHAPTER ONE 1.0 INTRODUCTION - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 1.1 Statement of Research Problem - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 5 1.2 Justification for the Study - - - - - - - - - -- - - - - - - - - -- - - - - - - - - - - - - - - - - - 6 1.3 Aims of the study- - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - 7 1.4 Hypothesis - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - -- - - - - - - - - - - - - - - - - - 8 vii CHAPTER TWO 2.0 LITERATURE REVIEW - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -8 2.1 Carbonic Anhydrase - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - -- - - - - - - - -8 2.1.1 Catalytic mechanismof carbonic anhydrase - - - - - - - - - - - - - - -- - - - - - - - - 10 2.2 Carbonic anhydrase and lactate uptake - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - --11 2.3Carbonic anhydrase in Hepatic gluconeogenesis - - - - - - - - - - - - - - - - - - - - - - - -13 2.4Carbonic Anhydrase Inhibitors - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 15 2.4.1 Carbonic anhydrase inhibition - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -16 2.4.2 Carbonic anhydrase inhibition in the liver- - - - - - - - - - - - - - - - - - - - - - - - - - - - - -18 2.4.3 First Generation inhibitors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 20 2.4.4 Second Generation inhibitors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 21 2.4.5 Third Generation inhibitors - -- - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - 21 2.4.6 Acetazolamide - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - 22 2.5 Diabetes Mellitus - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - -24 2.6 Drugs used in the treatment of Diabetes - - - - - - - - - - - - - - - - - - - - - - - - - -25 2.6.1 Metformin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -26 2.6.2 Metformin and Carbonic anhydrase inhibition - - - - - - - - - - - - - - - - - - - - - - - - - -27 2.7 Natural Products as Medicine - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - -29 2.7.1 Natural Products that inhibit Carbonic anhydrase - - - - - - - - - - - - - - - - - - - - -30 2.8 Cadaba farinosa Forrsk - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -32 2.8.1Taxonomical classification - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 32 2.8.2Vernacular names/Local names - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 33 2.8.3Botanical description - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 33 2.8.4Phytochemical studies - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 33 viii 2.8.5 Mineral and Amino acid content - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - 34 2.8.6 Ethno-MedicinalUses ofCadaba farinosa - - - - - --- - - - - - - - - - - - - - - - - 34 2.8.7 Pharmacological activity - - - -- - - - - - - - - - -- - - - -- - - - - - - - - - -- - - - - 35 2.8.8 Toxicological and safety assessment- - - - - - -- - - - - - - - - - - - - - - - - -- - - - - 36 CHAPTER THREE 3.0 MATERIALS AND METHODS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 38 3.1 Materials - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - 38 3.1.1 Chemicals/Reagents- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 38 3.1.2Equipment - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 38 3.1.3 Plant Samples Collection and Identification - - - - - -- - - - - - - - - - - - - - - - - - 38 3.1.4 Animals - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 39 3.2. Methodology- - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - -- - - 39 3.2.1Extraction and fractionation- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - 39 3.2.2Column Chromatography - - - - - - -- - - - - - - - - - -- - - - - - - - - - - - - - - - - - 39 3.2.3Thin Layer Chromatography (TLC) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 40 3.2.4 PreparativeThin Layer Chromatography (PTLC) - - - - - - - - - - - - - - - - - - - 40 3.2.5GC-MS Analysis - - - - - - - - - - - - - - - - - - - - - -
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