EVALUATION OF PHYTOMEDICINAL ATTRIBUTES AND METABOLITES’ IDENTIFICATION IN LEAVES OF HYOPHORBE INDICA AND HYOPHORBE LAGENICAULIS James William SESSION 2015-2018 Registration No. 2015-GCUL-PhD-CHEM-62 DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE EVALUATION OF PHYTOMEDICINAL ATTRIBUTES AND METABOLITES’ IDENTIFICATION IN LEAVES OF HYOPHORBE INDICA AND HYOPHORBE LAGENICAULIS Submitted to GC University Lahore in partial fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY IN CHEMISTRY BY James William SESSION 2015-2018 Registration No. 2015-GCUL-PhD-CHEM-62 DEPARTMENT OF CHEMISTRY GC UNIVERSITY LAHORE DECLARATION i PLAGIARISM UNDERTAKING ii RESEARCH COMPLETION CERTIFICATE iii CERTIFICATE OF APPROVAL iv DEDICATION My Parents (Late), beloved wife, my sons Shah Rukh James, Hammad James and my daughter Aman James for their prayers and sacrifices to support me and to boost my morale for the achievement of this endeavor. v ACKNOWLEDGEMENT I am very thankful to Almighty God for providing me this opportunity to complete my Doctorate in the field of chemistry at the beautiful, historical, richest and best endowed campus of Government College University, Lahore, Pakistan. This experience was very virtuous, and I enjoyed healthy learning during my research work. The working not only improved my laboratory skills but also enhanced and updated the knowledge in the field. It was not as simple as it seems today but there were untiring efforts behind that made the things smooth and positive. Inspite of my efforts it was God’s grace that made me able to complete this endeavor. I would like to offer the heartiest gratitude to my PhD supervisor Prof. Dr. Peter John, who supported and guided me beyond the imagination. His affection and supervision were full of comfort and ease to run the work at a smooth and steady pace. I would also like to pay special thanks to the worthy Prof. Dr. Ahmad Adnan, Chairperson and Professor/Dean, Faculty of Chemistry and Life Science, Government College University, Lahore, Pakistan. His continuous support and guidance always provided me an urge to learn and to accomplish the achievements heartedly and devotedly. I offer my special thanks to Dr. Muhammad Waseem Mumtaz, Assistant Professor of Chemistry at the University of Gujrat, Pakistan, for his affection, technical support and professional guidance. I am also very thankful to Prof. Dr. Hamid Mukhtar, Director, the Institute of Industrial Biotechnology University, Lahore, for his technical assistance and special motivation. vi vii TABLE OF CONTENTS DECLARATION .................................................................................................................................. i PLAGIARISM UNDERTAKING ........................................................................................................... ii RESEARCH COMPLETION CERTIFICATE .......................................................................................... iii CERTIFICATE OF APPROVAL ........................................................................................................... iv DEDICATION .................................................................................................................................... v ACKNOWLEDGEMENT .................................................................................................................... vi LIST OF FIGURES ............................................................................................................................. xi LIST OF TABLES ...............................................................................................................................xv LIST OF ABBREVIATIONS ............................................................................................................... xvi AFFIRMATION ............................................................................................................................... xix ABSTRACT ...................................................................................................................................... xx INTRODUCTION................................................................................................ 1 1.5.1 Synthetic Drugs for Diabetes Mellitus Type-2 ....................................................................... 8 1.5.2 The Dietary Enzyme Inhibitors .............................................................................................. 9 1.5.3 Plant-Based Treatment of Diabetes Mellitus Type-2 .......................................................... 10 LITERATURE REVIEW ................................................................................. 18 EXPERIMENTAL WORK ............................................................................... 32 viii 3.7.1 The DPPH Assay ................................................................................................................... 36 3.7.2 Total Antioxidant Power Assay ............................................................................................ 37 3.7.3 Iron Chelating Activity ......................................................................................................... 37 3.8.1 The α-Glucosidase Inhibition Activity .................................................................................. 38 3.8.2 The α-amylase Inhibitory Assay .......................................................................................... 38 3.8.3 Acetylcholine Esterase Inhibition Assay .............................................................................. 39 3.9.1 The 1HNMR Prediction of Metabolites ................................................................................ 39 3.9.2 UHPLC-QTOF-MS/MS Analysis ............................................................................................ 40 3.11.1 Oral Glucose Tolerance Test ........................................................................................... 41 3.11.2 Anti-Hyperglycemic Assessment .................................................................................... 41 3.11.3 Hypolipidemic Activity .................................................................................................... 43 RESULTS AND DISCUSSION ........................................................................ 45 4.4.1 DPPH Activity ....................................................................................................................... 51 4.4.2 Total Antioxidant Power Assay (TAP). ................................................................................. 54 4.4.3 The β-Carotene Bleaching Assay ......................................................................................... 56 4.4.4 Iron Chelating Activity ......................................................................................................... 58 4.5.1 The α-Glucosidase Inhibition Assay ..................................................................................... 60 4.5.2 The α-Amylase Inhibition .................................................................................................... 63 4.5.3 The Acetylcholine Esterase Inhibition ................................................................................. 65 4.6.1 The 1HNMR Based Identification of Metabolite Class ......................................................... 67 4.6.2 Metabolite Identification by UHPLC-QTOF-MS/MS ............................................................ 72 ix 4.9.1 The Blood Glucose Level ..................................................................................................... 98 4.9.2 Blood Haemoglobin Level .................................................................................................. 105 4.9.3 Hypolipidemic Assessment in Diabetic Mice ..................................................................... 107 CONCLUSION ................................................................................................ 115 x LIST OF FIGURES Fig. 1-1 The role of ROS and antioxidant defense in diabetes mellitus type 2. .......... 7 Fig. 1-2 Hyophorbe lagenicaulis ............................................................................... 16 Fig. 1-3 Hyophorbe indica ......................................................................................... 16 Fig. 3-1 Schematic diagram of methodology ............................................................. 44 Fig. 4-1 IC50 values of H. indica extracts for DPPH scavenging ............................... 52 Fig. 4-2 IC50 values of H. lagenicaulis extracts for DPPH scavenging ..................... 53 Fig. 4-3 Antioxidant power of H. indica leaf extracts ............................................... 54 Fig. 4-4 Antioxidant power of H. lagenicaulis leaf extracts...................................... 55 Fig. 4-5 The % inhibition of β-carotene by leaf extracts of H. indica ....................... 57 Fig. 4-6 The % inhibition of β-carotene by leaf extracts of H. lagenicaulis ............. 58 Fig. 4-7 Iron chelating activity of H. indica leaf extracts .......................................... 59 Fig. 4-8 Iron chelating activity of H. lagenicaulis leaf extracts ................................ 60 Fig. 4-9 The α-glucosidase of H. indica leaf extracts ................................................ 62 Fig. 4-10 The α-glucosidase of H. lagenicaulis leaf extracts .................................... 62 Fig. 4-11 The α-amylase inhibtion of H. indica leaf extracts .................................... 64 Fig. 4-12 The