Bioassay Directed Characterization of Selected Indigenous Medicinal Plants for their Anti-HCV Potential A thesis submitted to University of Sargodha In partial fulfillment of the requirements for the degree of Doctor of Philosophy in CHEMISTRY by Sobia Noreen Department of Chemistry University of Sargodha Sargodha November 2014 1 CONTENTS Page DEDICATION I DECLARATION II CERTIFICATE III APPROVAL CERTIFICATE IV ACKNOWLEDGEMENT VI ABSTRACT VII-VIII ABBREVIATIONS IX-XI CONTENTS XII-XV LIST OF FIGURES XVI-XVII LIST OF TABLES XVIII 2 Dedication The fruit of my work is dedicated to My Holy Prophet Hazrat Muhammad (SAW) & My Uncle Ch. Javed Iqbal (Late) His abundant affections, patronizing encouragement and secret prayers have enabled me to accomplish this task. DECLARATION 3 I hereby declare that the work described in this thesis was carried out by me under the supervision of Prof. Dr. Ishtiaq Hussain, Professor, Department of Chemistry, University of Sargodha, Sargodha. 40100, Pakistan for the degree of Doctor of Philosophy in Chemistry. I also hereby declare that the substance of this thesis has neither been submitted elsewhere nor is being concurrently submitted for any other degree. I further declare that the work embodied in this thesis is the result of my own research and where work of any other investigator has been used that has been duly acknowledged. ________________ Sobia Noreen Registration No.: 09/UOS/ Ph.D/ CHM/01 Department of Chemistry University of Sargodha 4 CERTIFICATE OF ORIGINALITY OF RESEARCH WORK In accordance with Revised Rules & Regulations 2008 for MS/MPhil, MS/MPhil leading to PhD and PhD, I hereby certify that Ms. Sobia Noreen has conducted research work under my supervision, and is qualified to submit the thesis entitled, “Bioassay directed characterization of selected indigenous medicinal plants for their anti-HCV potential’’ for the degree of Doctor of Philosophy. It is further certified that the research work carried out by the scholar is original and nothing is plagiarized. Prof. Dr. Ishtiaq Hussain, Prof. Dr. Muhammad Ilyas Tariq Supervisor Supervisor-II Department of Chemistry, Department of Chemistry, University of Sargodha,Sargodha University of Sargodha, Sargodha Dr. Muhammad Sher Chairman Department of Chemistry, University of Sargodha, Sargodha 5 APPROVAL CERTIFICATE This thesis entitled “Bioassay directed characterization of selected indigenous medicinal plants for their anti-HCV potential” submitted by Sobia Noreen, Session 2009-2014, in the Partial fulfillment of the requirement for the degree of Ph.D. in Chemistry is hereby approved. Supervisors: Prof. Dr. Ishtiaq Hussain , Supervisor Department of Chemistry, University of Sargodha, Sargodha. Prof. Dr. Muhammad Ilyas Tariq Supervisor-II Department of Chemistry, University of Sargodha, Sargodha. External Evaluator Prof. Dr. Kausar Malik Director ORIC Lahore College for Women University Lahore. Chairman: Dr. Muhammad Sher Department of Chemistry University of Sargodha, Sargodha. 6 ACKNOWLEDGMENT Prior to acknowledgment, I glorify, almighty Allah, the most merciful, the most gracious who gave me the power, health, strength and means to complete my research. First of all, all my acknowledgements and praises are for the Prophet Muhammad, thousands salutations and benedictions to his sublime Holiness, the chosen, the benefactor – the blessing and peace of God be with Him and his AAL A.S through whose grace the sacred Quran descended. It is an honor for me to owe my deepest gratitude to my supervisor, Prof. Dr. Ishtiaq Hussain, his supervision and support, guidance, correction and valuable comments made me to accomplish present study. I am also highly obliged to my co-supervisor Prof. Dr. Ilyas Tariq for her excellent guidance and enthusiastic encouragement throughout this research work. I would also like to express my gratitude to the Vice Chancellor, Prof. Dr. Muhammad Akram Chaudhary, whose motivation as a role model geared through the challenges. It is my pleasure to express my thanks to Prof. Dr. Nazra Sultana, Dean, Faculty of Science and Technology; Dr. Muhammad Sher, Chairman, Department of Chemistry, University of Sargodha for his support and encouragement during the course of studies. Special thanks for my honorable teachers Prof. Dr. Tayyab Husain, Diretor, CEMB, University of the Punjab, Lahore and Dr. Shahid Iqbal, Dr. Ashraf Shaheen, University of Sargodha for their every possible cooperation and constructive criticism during this study. Special thanks reserve to Dr. Jhon M. Gardiner, MIB, University of Manchester, UK for his generous support of his lab analytical facilities for six months during my research visit to accomplish this task. I am also grateful to my all other colleagues from my department or other department of my university for their assistance and guidance for achievement of this task. I am thankful to all my teachers my mender who taught me even a single word. I am here only because of you. My sincere thanks to Mr. Asif, Mr. Afzal, Mr. Farhan Ahmad (Hightech.lab) and other staff, who was really helpful and provides me every possible facility needed to accomplish this task. My special thanks to my all my friends, colleagues and students (Dr. Tusneem, Bushra Ijaz, Fozia, Usman Ali ashfaq, Dr. Anjum Murtaza, Aqeel khan, Rehana, Sadia, Farhana, Ayesha and Iffat for their sincere support, guideline and their cheerful company which can never be forgotten by me. My heartiest acknowledgements are for the untiring efforts of my family members especially my Mother, my anti, my sisters and brothers who had been encouraging through entire period of my studies and because of their prayers I am at this destiny. I owe my special gratitude to my dear loving husband Qamar-ul-zaman, whose enthusiastic encouragement and sacrifices made me able to complete this project. At the end I cannot forget my sweet baby Basil Raffan, whose cheerful company always refresh me and provide me strength to achieve my goals. My all success is due to prayers of my dear ones. Sobia Noreen 7 ABSTRACT Hepatitis C is the most common chronic blood born infection affecting approximately 3 % population of the world and about 6 % population of the Pakistan. About one– fifth of the individuals with this infection results in the development of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The present treatment standard is pegylated INF-a along with ribavirin, direct-acting antivirals (DAAs) like telaprevir and boceprevir for definite period according to viral genotype. The main goal of therapy is to achieve a stable virological response along with eradication of HCV infection and cure of the fundamental HCV induced liver disease. Unfortunately, only less than 50 % of HCV patients get benefit to some extent from this therapy due to side effects, resistance and high cost. Hence, there is a need to develop anti-HCV agents, both from herbal sources and synthetic chemicals which are non toxic, more efficacious and cost-effective. Soon-Valley of Pakistan is bestowed with a unique biodiversity, comprising of different climatic zones and wide range of plant species especially that have hepatoprotective effect. No doubt phyto-constituents having the remarkable potential inhibit the replication cycle of various types of DNA or RNA viruses. The present work is intended to explore plants with anti-HCV potential, leading to natural chemical entities as lead compounds. In current study, methanolic extracts of shade air dried selected parts of fifteen medicinal plants of Soon Valley were screened against HCV NS3 protease (genotype 3a) and as well as whole virus. The cellular toxicity effects of organic extracts on the viability of Huh-7 were studied through Trypan blue exclusion method and MTT assay. In serum inhibition assay, liver cells were infected with high titre of HCV positive serum of genotype 3a for screening of antiviral plants against whole virus. In in vitro protease inhibition assay, Huh-7 cells were transfected with HCV NS3 protease by introducing mammalian expression construct PCR3.1/FLAGtag/HCV NS3 in the presence and absence of plants extracts. Only the methanolic extract of Caralluma tuberculata (CTS) and Portulaca oleracea L. (POL) exhibited 57 % and 70 % inhibition. Four fractions of each CTS and POL extract were obtained through bioassay-guided extraction. Subsequent inhibition of all organic extract fractions against NS3 serine protease were checked to track the 8 specific target inside the genome of the virus and in this case only ethyl aceatate and methanolic of CTS extracts inhibit the 69 % & 53 % and the POL extracts inhibit 80 % & 85 % expression of HCV NS3 protease respectively while keeping GAPDH expression constant. The results showed that the POL and CTS methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. The plant organic extract was screened for the presence of phytochemical through standard procedures. Phytochemical analysis of selected fractions confirmed the presence of alkaloids, coumarins, flavonoids, glycosides, saponins, terpenoids, and tannins etc. In addition, the active fractions of both plant extracts were also checked for their some other biological activities like antioxidant potential, in vitro anti thrombotic and antibacterial activities. Antioxidant potential of CTSM, CTSE, POLM and POLE was determined in term of total phenol, flavonoids, tannin, carotenoid and free radical scavenging potential by following the different