PHARMACOGNOSTIC STUDIES ON TRILLIUM GOVANIANUM WALL. Ex. ROYLE Ph. D Thesis By SHAFIQ UR RAHMAN DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN 2015 PHARMACOGNOSTIC STUDIES ON TRILLIUM GOVANIANUM WALL. Ex. ROYLE SHAFIQ UR RAHMAN A THESIS SUBMITTED TO THE DEPARTMENT OF PHARMACY, UNIVERSITY OF PESHAWAR IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN PHARMACEUTICAL SCIENCES DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN 2015 CERTIFICATE OF APPROVAL This thesis, entitled, “Pharmacognostic studies on Trillium govanianum Wall. Ex. Royle” submitted by Mr . Shafiq ur Rahman to University of Peshawar is hereby approved and recommended as partial fulfillment for the award of Degree of “Doctor of Philosophy in Pharmaceutical Sciences” . Prof. Dr. Muhammad Ismail __________________________ Research Supervisor Department of Pharmacy University of Peshawar Prof. Dr. Muhammad Saeed __________________________ Chairman Department of Pharmacy University of Peshawar Prof. Dr. Taous Khan _________________________ External Examiner Department of Pharmacy COMSATS Institute of Information Technology, Abbottabad DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR, PESHAWAR, PAKISTAN 2015 Acknowledgements First of all I bow down my head to the Omnipotent, the most Merciful and the Compassionate Al-Mighty ALLAH , Who gave me the courage and provided me all the resources to complete this Ph.D. Project. I wish to pay homage to the most perfect personality of the world Hazrat Muhammad (PBUH) , who enlightened our minds to recognize our Creator. My research work would not have been possible without the help, support, and guidance of many people to whom I want to convey my cordial gratitude. I would like to thank my supervisor, Prof. Dr. Muhammad Ismail , for his guidance, support, understanding and patience during the entire period of my studies. I am very thankful for his admirable supervision, continuous encouragement during my Ph.D. studies. I am thankful to Prof. Dr. Muhammad Saeed , sitting Chairman, Department of Pharmacy, University of Peshawar, for his support and encouragement throughout my research studies. I am also grateful to Meritorious Professor. Dr. Zafar Iqbal (T.I ) and Prof. Dr. Fazal Subhan for their inspiring guidance and support during the course of this PhD project. I am thankful to Dr. Muhammad Raza Shah , Dr. Achyut Adhikari , Dr. Itrat Anis , Dr. Muhammad Ateeq , Dr. Burhan and Mr. Farid , International Centre for Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry, University of Karachi, Karachi for their help and facilitation during this long course of research studies. I am obliged to Prof. Dr. Jamshaid Ali Khan , Dr. Amir Zada Khan , Dr. Fazal Nasir , Dr. Inam Ullah , Dr. Muhammad Ismail , Dr. Fazal Khuda , Dr. Gohar Ali and Dr. ZakiUllah Department of Pharmacy, University of Peshawar for their support. I would like to thank Dr. Muhammad Khurram (Chairman) , Mr. Shujat Ahmad , Mr. Asaf Khan , Mr. Abidullah , Mr. Imad Afzal and all Teaching, Clerical and Laboratory Staff, Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal Dir (U) for their cooperation. I feel indebted to Dr. Farman Ali and Dr. Abdul Khaliq Jan , Department of Chemistry, Shaheed Benazir Bhutto University, Sheringal Dir (U) for their assistance. I want to extend special thanks to my dear friends Dr. Saeed Ahmad Khan, Mr. Arsalan, Mr. Farhad Ullah , Mr. Khalid , Mr. Tahir Ali , Mr. Sajid Khan Sadozai, Mr. Muhammad Shahid , Mr. Irfan Ullah and Mr. Muzaffar Abbas. Last but not the least; I am very thankful to my sweet Parents , wife, brother, sisters, my uncle retired Principal Mr. Fazal Halim, and all relatives for their prayers, support and kindness throughout my studies. Shafiq ur Rahman Table of Contents List of Tables................................................................................................................ i List of Figures............................................................................................................... iv List of Abbreviations.................................................................................................... vi List of Publications from Thesis................................................................................... vii Summary....................................................................................................................... 1 1. Introduction.......................................................................................................... 4 1.1 Medicinal plants.......................................................................................................... 4 1.2 Plants metabolites ........................................................................................................ 5 1.3 Traditional medicines and drug discovery................................................................... 7 1.4 Biodiversity of Indo-Pak Subcontinent........................................................................ 10 1.5 The Family Trilliaceae................................................................................................. 11 1.6 Genus Trillium ............................................................................................................. 12 1.6.1 Species of genus Trillium ........ ........................................................................................ 13 1.6.2 Phytochemical profiling of genus Trillium ...................................................................... 17 1.6.3 Medicinal importance and biological studies of genus Trillium ...................................... 28 1.7 Trillium govanianum ................................................................................................... 30 1.7.1 Plant Morphology .. .......................................................................................................... 31 1.7.2 Distribution...................................................................................................................... 31 1.7.3 Ethnobotanical Uses ......................................................................................................... 31 1.8 Aims and Objectives ........................................................................... ......................... 32 2. Materials and Methods......................................................................................... 33 2.1 Drugs and Chemicals.................................................................................................. 33 2.2 Research centers for experimental studies................................................................... 33 2.3 Physical constants........................................................................................................ 34 2.4 Spectroscopic techniques............................................................................................. 34 2.4.1 UV technique.................................................................................................................... 34 2.4.2 IR technique ...................................................................................................................... 34 2.4.3 Mass technique................................................................................................................. 34 2.4.4 Nuclear Magnetic Resonance (NMR) technique.............................................................. 35 2.4.5 Gas Chromatography and G as Chromatography -Mass Spectrometry .............................. 35 2.4.6 GC-MS identification of components............................................................................... 35 2.5 Chromatographic techniques for isolation and purification of compounds............... 36 2.5.1 Column Chromatography (CC) ........................................................................................ 36 2.5.2 Thin layer Chromatography (TLC) ..................... ............................................................. 36 2.5.3 Reagents for visualizing the spots.................................................................................. 36 2.5.3.1 Ceric sulphate solution as reagent ............................................................................ 37 2.5.3.2 Vanillin solution as reagent ...................................................................................... 37 2.6 Ethnomedicinal study.................................................................................................. 37 2.6.1 Site selection .................................................................................................................... 37 2.6 .2 Sampling informants and ethnomedicinal data collection ................................................. 37 2.7 Plant materials.............................................................................................................. 38 2.7.1 Collection ......................................................................................................................... 38 2.7.2 Extraction and fractionation ..................................................... ........................................ 38 2.8 Macroscopic and microscopic features of rhizome..................................................... 40 2.9 Physicochemical parameters........................................................................................ 40 2.9.1 Total ash ...........................................................................................................................