In the Name of Allah, the Most Gracious, the Most Merciful EXTRACTION AND EVALUATION OF SECONDARY METABOLITES AS GREEN ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITOR By Farieha 2001-ag-622 A Thesis Submitted in Partial Fulfilment of the Requirement for the Degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY DEPARTMENT OF CHEMISTRY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE FAISALABAD 2017 DECLARATION I hereby declare that the contents of the thesis “Extraction and Evaluation of Secondary Metabolites as Green Angiotensin Converting Enzyme (ACE) Inhibitor” are product of my own research and no part has been copied from any published source (Except the reference, standard mathematical or genetic models/questions/ formulae/ protocols etc.). I further declare that this work has not been submitted for award of any other diploma/degree. The University may take action if the information provided is found inaccurate at any stage. Farieha 2001-ag-622 To The Controller of Examinations, University of Agriculture, Faisalabad. “We, the Supervisory Committee, certify that the contents and form of thesis submitted by Mrs. Farieha, Regd. 2001-ag-622 have been found satisfactory and recommended that it be processed for evaluation, by External Examiner (s) for the award of degree”. Supervisory Committee Supervisor _____________________________ Dr. Nazish Jahan Member _____________________________ Dr. Shaukat Ali Member _____________________________ Prof. Dr. Khalil-Ur-Rehman DEDICATED TO MY PARENTS, MY SIBLINGS, MY HUSBAND AND TEACHERS FOR ENABLING ME TO BE THE WAY I AM WHOSE LOVE, CARE AND GUIDANCE HAS ALWAYS MADE MY LIFE EASIER AND THIS GOAL POSSIBLE WHO ALWAYS INSPIRED AND ENCOURAGED ME FOR WHAT I WANT AND GRAFTED IN THE UNTIRING TO GET ON HIGHER IDEAS OF LIFE. A SPECIAL DEDICATION TO MY LOVING DAUGHTER WHO SACRIFICES A LOT DUE TO MY Ph.D. ACKNOWLEDGMENT In the name of ALLAH, the all corroborate possessor of Majesty and Splendor, the Omnipotent, the Omnipresent the whole benevolent caprice to make this humble contribution to already existing Ocean of knowledge. Next all praises are due to His last messenger HAZARAT MUHAMMAD (Peace Be Upon Him) Who is an internal torch of guidance and knowledge for humanity as a whole and is an ever inspiring for all the learned person. So this is the time for a last and personal word. During those 4 years of my Ph.D. journey I have met with lot of people outside and inside the work sphere that contributed to make this adventure possible and enjoyable. This dissertation would not have been possible without the guidance and the help of these people who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, my utmost gratitude to my Supervisor Dr. Nazish Jahan, Assistant Professor, University of Agriculture, Faisalabad, for her encouraging and their help in overcoming all obstacles in the completion of this research work. I appreciate all her contributions of time, ideas, and funding to make my Ph.D. experience productive and stimulating. My sincere thanks to Prof. Dr. Khalil- Ur- Rehman, Chairman Department of Biochemistry, University of Agriculture, Faisalabad, his personal guidance have provided a good basis for the present thesis. I warmly thank Dr. Shoukat Ali, Assistant professor, University of Agriculture Faisalabad, for his support and his kind concern and consideration regarding my academic requirements. My sincere thanks to Dr. Misbah Ijaz Lecturer, Dr. Asad Manzoor Lecturer and students of Department of Clinical Medicine and Surgery (CMS) of University of Agriculture, Faisalabad for their help and support to complete my research work. Special thanks are extended to Lab. fellows Zill-e-Huma and Fatika Zafar for their support. I am thankful to Lab. Assitants and attendants of Organic Chemistry Lab. of Department of Chemistry, University of Agriculture, Faisalabad, for their cooperation and assistance during research work. Words are lacking to express thanks to my Loving Parents Ch. Muhammad Hussain and Khursheed Hussain, as my entire personality to this stage is the result of their efforts, prayers, inspiring sentiments and encouragement throughout my life. I am also feeling thankful to my brothers Adnan Hussain and Farhan Murtaza and their families for all their love and encouragement. Special thanks to, my Brother in laws Atif Naseem, Adil Naseem and my sister in law Iqra Naseem, who always supported me during the whole duration of my studies. And most of all to my loving, supportive, encouraging, and patience husband Muhammad Shahid Naseem, my daughter Ayesha Fatima They have lost a lot due to my research. Without their encouragement and understanding it would have been impossible for me to succeed this work. Farieha CONTENTS List of Detailed Contents i List of Tables vi List of Figures viii List of Abbreviations xvi Abstract xxi Chapters NO. TITLE PAGE 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 3 MATERIALS AND METHODS 40 4 RESULTS AND DISCUSSION 52 5 SUMMARY 199 LITERATURE CITED 202 Table of Contents CHAPTER 1 INTRODUCTION ...................................................................................... 1 CHAPTER 2 REVIEW OF LITERATURE..................................................................... 5 2.1 Pathogenesis of Hypertension .................................................................................... 6 2.2 Management of Hypertension .................................................................................... 7 2.2.1 Non- Medicated Management of Hypertension .................................................. 7 2.2.2 Medicated Management of Arterial Hypertension.............................................. 7 2.3 Ranin Angiotensin Aldosterone System (RAAS) ...................................................... 8 2.4 RAAS as Therapeutic Target ..................................................................................... 8 2.5 Angiotensin Converting Enzyme (ACE) ................................................................... 9 2.6 Synthetic ACE Inhibitors ......................................................................................... 11 2.7 Natural ACE Inhibitors ............................................................................................ 12 2.8 Secondary Metabolites of Plants as Green ACE Inhibitors ..................................... 13 2.8.1. Flavonoids ........................................................................................................ 14 2.8.1.1 Extraction and Characterization of Flavonoids from Plants ....................... 15 2.8.1.2 Flavonoids as ACE Inhibitors ..................................................................... 16 2.8.2 Tannins .............................................................................................................. 18 2.8.2.1 Extraction and Characterization of Tannins from Plants ............................ 20 2.8.2.2 Tannins as ACE Inhibitors .......................................................................... 21 2.8.3 Alkaloids ........................................................................................................... 22 2.8.3.1 Extraction and Characterization of Alkaloids from Plants ......................... 25 2.8.3.2 Alkaloids as ACE Inhibitors ....................................................................... 26 2.8.4 Steroids ............................................................................................................. 26 2.8.4.1 Extraction and Characterization of Steroids from Plants ............................ 27 2.8.4.2 Steroids as ACE Inhibitors ......................................................................... 28 2.9 Synergistic Effects of Plants and Secondary Metabolites ....................................... 28 2.10 In vivo Antihypertensive Effect ............................................................................. 29 2.10.1 Renovascular Hypertension ............................................................................ 29 2.11 Therapeutic Effects of Selected Medicinal Plants ................................................. 31 2.11.1 Phoenix dactylifera ......................................................................................... 31 2.11.2 Curcuma longa L. ........................................................................................... 31 2.11.3 Piper nigrum L. ............................................................................................... 32 2.11.4 Cuminum cyminum .......................................................................................... 32 2.11.5 Coriandrum sativum L. ................................................................................... 32 2.11.6 Coffea Arabica ................................................................................................ 33 2.11.7 Ferula asafoetida ............................................................................................ 33 2.11.8 Rauvolfia serpentina L. ................................................................................... 33 2.11.9 Amomum subulatum Roxb ............................................................................... 34 2.11.10 Cassia fistula ................................................................................................. 34 2.11.11 Plantago ovata .............................................................................................. 35 2.11.12 Brassica juncea ............................................................................................