IDENTIFICATION, CHARACTERIZATION AND BIOEVALUATION OF POLYPHENOLS FROM MARTYNIA ANNUA LINN. A Dissertation Submitted to the Department of Chemistry, Quaid-i-Azam University, Islamabad In partial fulfillment of the requirements for the degree of Doctor of Philosophy In Organic Chemistry By Saba Muazzam Department of Chemistry Quaid-i-Azam University, Islamabad 2019 DEDICATION I dedicate my dissertation to my father Mr. Muazzam Ali for his love, endless support and encouragement ACKNOWLEDGEMENTS First and foremost, I am thankful to Almighty Allah, who gave me courage and strength to accomplish this task. This dissertation would be impossible without the contribution from many others. I will first express my sincere gratitude to my advisor Dr. Muhammad Farman, Associate Professor, Department of Chemistry, Quaid-i-Azam University Islamabad, for his guidance, influence and support. He has guided me through the ups and downs of my academic career. My sincere thanks are also due to Professor Dr. Shahid Hameed, Chairman, Department of Chemistry, Quaid-i-Azam University Islamabad and Professor Dr. Aamer Saeed, Head of Organic Section, Department of Chemistry, Quaid-i-Azam University Islamabad, for providing necessary facilities and their support during my research work. I would also like to thank Professor Dr. James McCullagh, Head of Mass Spectrometry Research Facility, Chemistry Research Laboratory, University of Oxford, United Kingdom for providing necessary facilities and for the invaluable discussions and advices regarding chromatography and mass spectrometry. It was honor to learn and work with him. I am also grateful to Dr. Naureen Rana, Assistant Professor, Department of Zoology and Fisheries, University of Agriculture Faisalabad, for her moral support. Completion of this dissertation would have been difficult without the financial support from Higher Education Commission, Islamabad, Pakistan. I would like to thanks my fellows, labmates, friends for their motivation, enthusiasm help and moral support throughout my research work. Last but not the least, I pay my thanks to my family, my father, brother, sister, my uncles and aunts, in-laws. I would like to give special thanks to my loving and caring husband who has tolerated the long distance separation for years but been standing by me throughout my academic career and has been a constant source of encouragement and support, and also thanks to my cute son who has been affected in every way possible by this quest. SABA MUAZZAM TABLE OF CONTENTS LIST OF ABBREVIATIONS...............................................................................................I LIST OF FIGURES .............................................................................................................III LIST OF TABLES ...............................................................................................................VI ABSTRACT........................................................................................................................VIII 1. INTRODUCTION ...........................................................................................................1 1.1. Natural Products.................................................................................................................1 1.2. Medicinal Plants.................................................................................................................1 1.3. Secondary Metabolites.......................................................................................................1 1.4. Polyphenols .......................................................................................................................2 1.5. Flavonoids .........................................................................................................................2 1.5.1. Biosynthesis ....................................................................................................................3 1.5.2. Chemical Structures ........................................................................................................3 1.5.3. Classification ..................................................................................................................4 1.5.4. Structural Diversity ........................................................................................................6 1.5.5. Biological Impact of Flavonoids ....................................................................................6 1.6. Analytical Methods in Research of Polyphenols ...............................................................7 1.6.1. High Performance Liquid Chromatography ....................................................................7 1.6.2. Electrospray Ionization in Mass Spectrometry.................................................................7 1.6.3. Mass Spectrometry ..........................................................................................................8 1.7. Objectives ...........................................................................................................................9 2. LITERATURE REVIEW…...........................................................................................10 2.1. Martynia .......................................................................................................................................................10 2.2. Martynia annua L…. ...............................................................................................................................10 2.2.1. Occurance ................................................................................................................................................10 2.2.2. Vernacular Names .................................................................................................................................10 2.2.3. Plant Taxonomy .....................................................................................................................................10 2.3. Morphology .................................................................................................................................................11 2.4. Ethnobotany ................................................................................................................................................11 2.5. Phytochemistry ...........................................................................................................................................13 2.6. Pharmacological Activities .....................................................................................................................15 2.6.1. Antifertility Activity .............................................................................................................................16 2.6.2. Analgesic and Antipyretic Activity ..................................................................................................16 2.6.3. Anthelmintic Activity ..........................................................................................................................16 2.6.4. Antinociceptive Activity and CNS Depressant Activity ...........................................................16 2.6.5. Antioxidant Activity .............................................................................................................................17 2.6.6. Anticonvulsant Activity ......................................................................................................................17 2.6.7. Antibacterial Activity ...........................................................................................................................18 2.6.8. Wound Healing ......................................................................................................................................18 2.6.9. Hair Growth Promoting Activity ......................................................................................................19 3. MATERIALS AND METHODS ......................................................................................20 3.1. Chemicals and Reagents ..........................................................................................................................20 3.2. Plant Material ...............................................................................................................................................20 3.3. Sample Preparation .....................................................................................................................................20 3.4. Untargeted/TargetedAnalysis..................................................................................................................20 3.4.1. Extraction Protocols................................................................................................................................20 3.4.2. Authentic Standard Sample Preparation….......................................................................................21 3.4.3. LC-ESI-MS Method/Instrumentation ......................................................................................................21 3.4.4. Tandem Mass Spectrometry.................................................................................................................22 3.4.5. Data Processing ......................................................................................................................................22 3.4.6. Quality Control (QC) Samples ...........................................................................................................22