Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum By Kashif Maqbool Khan CIIT/FA12-R60-004/ATD PhD Thesis In Pharmacy COMSATS University Islamabad, Abbottabad Campus - Pakistan Fall, 2018 COMSATS University Islamabad Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum A Thesis Presented to COMSATS University Islamabad, Abbottabad Campus In partial fulfillment of the requirement for the degree of PhD (Pharmacy) By Kashif Maqbool Khan CIIT/FA12-R60-004/ATD Fall, 2018 ii Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum A Post Graduate Thesis submitted to the Department of Pharmacy as partial fulfillment of the requirement for the award of Degree of Ph.D in Pharmacy. Name Registration Number Kashif Maqbool Khan CIIT/FA12-R60-004/ATD Supervisor Dr. Abdul Manann Associate Professor Department of Pharmacy COMSATS University Islamabad, Abbottabad Campus Co-Supervisor Dr. Muhammad Arfan Associate Professor Department of Chemistry, School of Natural Sciences (SNS) National University of Sciences & Technology (NUST), Islamabad iii iv v vi vii DEDICATION Dedicated to my family and friends who were the pillars of support during my PhD study viii ACKNOWLEDGEMENTS I bow my head before Almighty Allah, The omnipotent, The omnipresent, The merciful, The most gracious, The compassionate, The beneficent, who is the entire and only source of every knowledge and wisdom endowed to mankind and who blessed me with the ability to do this work. It is the blessing of Almighty Allah and His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to achieve this goal. I would like to take this opportunity to convey my cordial gratitude and appreciation to my worthy, reverently and zealot supervisor Dr. Abdul Mannan, Associate Professor, Department of Pharmacy, COMSATS University Islamabad, Abbottabad campus, Pakistan. Without whose constant help, deep interest and vigilant guidance, the completion of this thesis was not possible. I am really indebted to him for his accommodative attitude, thought provoking guidance, immense intellectual input, patience and sympathetic behavior. I would like to pay my deepest gratitude and appreciation to one of the member of my supervisory committee and co-supervisor Dr. Muhammad Arfan, Associate Professor, School of Natural Sciences, National University of Sciences and Technology, Islamabad, Pakistan, for his generous cooperation and kind assistance and providing valuable suggestions during accomplishment of my Ph.D. I am thankful to foreign supervisors Professor Dr. Satyajit D. Sarker and Dr. Lutfun Nahar, Medicinal Chemistry and Natural Products Research Group, School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, England, UK for their kind guidance and research assistance towards completing a part of my Ph. D. research work in UK. I am also extremely grateful to Dr. Ihsan ul Haq, Assistant Professor, Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan, for his valuable assistance, technical suggestions and kind help during the course of my Ph.D studies. I am also very much grateful to Higher Education Commission (HEC), Pakistan, for awarding me six months foreign scholarship under International Research Support Initiative Program (IRSIP) to carry out a part of my research work in Liverpool John Moores University, England, United Kingdom. I am also highly indebted to my best ix friends and fellows especially, Kashif Bashir, Misbah-ud-Din Qamar, Muhammad Ubaid , Jawad Akbar Khan, and rest of my fellows for their assistance, good company, marvelous behavior and friendly attitude. I am also thankful to all the administrative and laboratory staff of the Department of Pharmacy, COMSTAS University Islamabad- Abbottabad campus for their kind support. Last but not least, I really acknowledge and offer my heartiest gratitude to all members of my family especially, father, mother, wife, elder and younger brothers, sister and sweet daughter, Bareera Khan and son Saad Khan, for their huge sacrifice, moral support, cooperation, encouragement, patience, tolerance and prayers for my health and success which enabled me to achieve this excellent goal. Kashif Maqbool Khan CIIT/FA12-R60-004/ATD x ABSTRACT Phytochemical and Biological Investigations of Asparagus adscendens and Trillium govanianum This PhD thesis envisages the phytochemical and biological investigation of two important indigenous species of Pakistan. The main objective behind this investigation was to authenticate the folkloric history of these species. Asparagus adscendens Roxb. (A. adscendens), is native to the Himalayas. This plant has been used in the prevention and effective treatment of various forms of cancers. Trillium govanianum Wall. (T. govanianum), is a native species of the Himalayas. In folk medicine the plant has been reported for the treatment of wound healing, sepsis and in various sexual disorder Finely ground roots of A. adscendens and T. govanianum were macerated in methanol and extracted through solid-phase extraction by using gradient solvent system (water: methanol) It was further proceeded for analysis of fingerprint high performance liquid chromatography - photodiode array and highly sensitive liquid chromatography- electrospray ionization-quadrupole time of flight- mass spectrometry to obtain insights into the possible chemical composition of the fractions, particularly, to have an indication whether they contain phenolic, flavonoids, saponins or spogenin as possible contributors to the significant antioxidant, antimicrobial, antileishmanial and cytotoxic activities of the extracts and its fractions. Reverse phase HPLC-PDA based quantification revealed the presence of significant amount of quercetin, myricetin and kaempferol ranging from 23.31 to 234.23 & 0.221to 0.528 μg/mg DW for A. adscendens and T. govanianum, respectively. Moreover, in this study about 154 compounds have been identified by using both positive and negative ion mode liquid chromatography - mass spectrometry and gas chromatography - mass spectrometry analysis. LCMS analysis of A. adscendens, revealed compounds (4-29) and (30-37) and most of them are biological active e.g. Levoglucosan (C-10), Brugine (C-14) and (C-20) Bergenin. LCMS analysis of T. govanianum identified various biological active saponins and sapogenins e.g. Digoxigenin (C-52), Alliospiroside D (C-53), Hovenoside D(C-55), Pisumsaponin I (C-57), Fistuloside A (C-58), Pitheduloside F (C-60), Durupcoside B (C-61), xi Cyclopassifloside I (C-68), Ophiopogonin D (C-72), Crosatoside B (C-99), Yayoisaponin B (C-107), Protodioscin (C-109), Isoeruboside B (C-111), Phytolaccasaponin B (C-114), Calendasaponin C (C-115), Calendasaponin D (C-116), Azukisaponin IV (C-119), Pseudoprotodioscin (C-121), Polypodoside A (C-122), Agavasaponin C (C-123), Schidigerasaponin B1 (C-124), Dioscin (C-125), Pitheduloside K (C-127), Fistuloside B (C-129) and Ophiopogonin B (C-130). The GC/MS analysis of n-hexane fraction of MeOH extract of the roots of A. adscendens and T. govanianum was performed to get the fatty profile of both extracts. GC/MS analysis revealed the presence of twelve components (C-141 to 155). In current study, all the phytochemical and biological assays were performed on methanolic extract and SPE of A. adscendens and T. govanianum. The total phenolic and flavonoid contents of A. adscendens and T. govanianum in terms of gallic acid and quercetin equivalent per gram dry weight exhibited different levels of significant phenolic and flavonoid contents. Antioxidant assays, including DPPH scavenging activity, total antioxidant capacity and ferric reducing antioxidant power of A. adscendens and T. govanianum exhibited different levels activity, which might be attributed to the presence of phenolic compounds and possible saponins. The MeOH extract and SPE of A. adscendens and T. govanianum exhibited mild antibacterial activity determined by the zone of inhibition (mm diameter) ranges from 7 to 13 mm, against Staphylococcus aureus (NCTC 7508); Bacillus subtilis (NCTC 1604); M. luteus (NCTC 75080); Escherichia coli (ATCC 25922). The antimicrobial potential of both A. adscendens and T. govanianum was further accessed to determine the minimum inhibitory concentration (MIC) values by using resazurin microtiter assay (REMA), which exhibited considerable level of antibacterial potential against gram-positive bacteria (MIC: 2.5-0.009 mg/mL) than against gram-negative bacteria (MIC: 1.25-2.5 mg/mL). The antifungal potential of A. adscendens and T. govanianum were established against four strains of filamentous fungi. i.e Aspergillus fumigatus FCBP- 66; Mucor species (FCBP-0300); Aspergillus niger (FCBP-0198) and Aspergillus flavus (FCBP- 0064) showed mild to moderate or weak antifungal activity. Antileishmanial capability of A. adscendens and T. govanianum against Leishmania tropica KWH23 strain were manifested mild to moderate results. xii Cytotoxicity potential of A. adscendens and T. govanianum were accessed by using brine shrimp lethality assay, protein kinase inhibition assay and in vitro 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. The distinguishable protein kinase inhibitory activity against Streptomyces 85E strain with 19± 1.06 mm bald, 9± 0.45mm clear phenotype was observed around the MeOH extract. The MeOH extract of the roots and SPE fractions of A. adscendens and T. govanianum displayed considerable