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

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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

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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

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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

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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

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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.

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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

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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 reported procedure. Free radicals scavenging activities of extracts were determined by DPPH, ABTS and FRAP assays. Among extracts highest antiradical values against

DPPH were found to be 81.747 by CTSM while lowest value (36.124) revealed by

POLE. Identification and quantification of phenolic acids in methanolic extracts of CTS and POL were done by HPLC with UV/DAD that confirm the presence of

Sinapic acid, m-Coumaric acid, Gallic acid and Quercetin in CTSM along with Gallic acid and Quercetin in POLM.

In case of in vitro anti thrombotic potential, highest activity among extract fractions was shown by CTSM and minimum by POLE while the overall order of antithrombotic potential was CTSM > CTSE > POLM >POLE. The antibacterial activity of methnolic extracts of CTS and POL against Gram positive (Bacillus subtilis, Styphylococcus aureus and Styphylococcus epidermidis) and Gram negative (Pseudomonas aerugenosa, Escherichia coli and Salmonella typhi) strains of bacteria was determined by disc diffusion method. Percent inhibition with respect to positive control (Ciproflaxacin) indicated that both CTSM and POLM exhibited more than 60 % inhibition against Styphylococcus epidermidis.

Furthermore to identify the active ingredients, CTSE extract was also fractioned by column chromatography. Five compounds CT-EtOAc-01 i.e. Methyl(12-O-benzoyl- androstan-3-O-(14)-β-D-cymaropyranosyl-(14)-β-Dcymaropyranosyl-(14)-β-

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D-cymaropyranosyl) hexanoate, CT-EtOAc-02 i.e. Methyl (12-O-benzoyl-androstan- 3-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl--(14)-β-D- cymaropyranosyl) hexanoate, CT-EtOAc-03 i.e. Methyl (23-O-benzoyl-androst-6-en- 17-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl--(14)-β-D- cymaropyranosyl)hexanoate, CT-EtOAc-04 i.e. Phenyl (12-O-benzoyl-gonan-6-en- 5’’-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl)oxalate and CT- EtOAc-05 i.e. Trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside were isolated, purified and characterized by spectral data (UV-VIS, FTIR, EI-MS and NMR).

The results obtained in this study suggested that methanolic and ethyl acetate extracts of CTS and POL possess remarkable antiviral, antioxidant, antithrombotic, and antibacterial effects which might be due to the charisma of their bioactive compounds like glycosides, flavonoids, tannins, saponins and alkaloids etc. Further studies in vitro, in vivo and in silico assays are mandatory to decipher the thorough aspects of these phytocompounds. The novel antiviral activity of compounds from CTS presents an attractive lead for natural chemical entities meant for the development of prospective anti-HCV agents. Hence, POL and CTS extracts and its potential constituents alone or plus with interferon could offer a future option to treat chronic HCV.

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ABBREVIATIONS

ABI Applied Biosystem ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) Amp Ampicillin AMPK AMP-activated protein kinase APS Ammonium per sulfate ATP Adenosin triphosphate BHT Butylated hydroxytoluene BMI Body mass index bp Base pair CC50 Cytotoxic concentration CHC Chronic hepatitis C

CHCl3 Chloroform CHO Chinese hamster ovary CLDN1 Claudin1 Cox-2 Cyclooxygenase-2 CTS Caralluma tuberculata stem CTSM Caralluma tuberculata methanolic extract

CTSE Caralluma tuberculata ethyl acetate extract DAAs Direct acting antivirals dH2O Distilled water DMEM Dulbecco's Modification of Eagle's Medium DNA Deoxyribonucleic acid DMSO Dimethyl sulfoxide dNTPs Deoxynucleoside triphosphate DPPH 2,2-diphenyl-1-picrylhydrazyl dsRNA Double stranded RNA E1 Envelope glycoprotein 1 E2 Envelope glycoprotein 2 ECL chemiluminscent EDTA Ethylenediamine tetra acetic acid eIF Eukaryotic initiation factors ELISA Enzyme Linked Immuno Sorbent Assay ER Endoplasmic reticulum ESI-MS Electro spray ionization-mass spectrometry EtOAc ethyl acetate FAS Fatty acid synthase FBS Fetal Bovine Serum GAG Glycosaminoglycans GAPDH Glyceraldehyde-3-phosphate dehydrogenase GFP Green fluorescence protein GTP Guaninosine triphospahte

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HBV Hepatitis B virus HCC Hepatocellular carcinoma HCV Hepatitis C virus HPLC High performance liquid chromatography HSC Hepatic stellate cells Huh-7 Human hepatoma cell line-passage 7 HVR 1 Hypervariable region 1 IC Internal control IDUs Injection drug users IFN Interferon IR Infra-red IRES Internal ribosomal entry site ISDR IFN-sensitivity determining region ISGs Interferon Stimulated Genes JNK c-Jun N-terminal kinase Kb Kilo base pair kbp Kilo base pair kDa Kilo daltons MAPK Mitogen activated protein kinase MAVS Mitochondrial Inhibitor of Viral Signaling MeOH methanol mg Microgram mL Microlitre M-MLV Moloney murine leukemia virus mRNA Messenger RNA NAFLD Non-alcoholic fatty liver disease NANBH Non-A, non-B hepatitis ng Nano gram NMR Nuclear magnetic resonance NS Nonstructural nt Nucleotide NTR Non-translated region ORF Open reading frame P53 tumor supressor protein PAGE polyacrylamide gel electrophoresis PAPRα Peroxisomal proliferator activator receptor-alpha PBS Phosphate Buffered Saline PEG Polyethylene glycol PEG-INFα Pegylated interferon α PKR Protein Kinase R pM Pico-Mole PO Portulaca oleracea POL Portulaca oleracea leaves

POLE Portulaca oleracea L ethylacetate

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POLM Portulaca oleracea methanol RdRp RNA-dependent RNA polymerase RISC RNA induced silencing complex RNA Ribonucleic acid RNAi RNA interference RNase Ribonuclease (enzyme) ROS Reactive oxygen species rpm Revolution Per Minute RT Room Temperature RT-PCR Reverse transcriptase polymerase chain reaction SDS–PAGE Sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis ssRNA Single stranded RNA SVR Sustained virological response TAE Tris Acetate EDTA TBE Tris-boric acid EDTA (buffer) TGFB Transforming growth factor-B TMS Trimethylsilane TNF-α Tumor necrosis factor alpha TLC Thin layer chromatography UBC Ubiquitin WHO World health organization

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Table of Contents 1.0 Introduction ...... Error! Bookmark not defined. 1.1 Natural Products from Times of Yore to 21st Millennium Error! Bookmark not defined. 1.2 Status of Medicinal Plants and their Research in Pakistan Error! Bookmark not defined. 1.3 Preface to Genus Caralluma and Caralluma tuberculata . Error! Bookmark not defined. 1.3.1 Active Constituents ...... Error! Bookmark not defined. 1.3.2 Pharmacological worth of Caralluma tuberculata and other species of Caralluma ...... Error! Bookmark not defined. 1.4 Introduction to Portulaca oleracea ...... Error! Bookmark not defined. 1.4.1 Phytochemistry of Portulaca oleracea ...... Error! Bookmark not defined. 1.4.2 Pharmacological attributes of Portulaca oleracea .... Error! Bookmark not defined. 1.5 Hepatitis...... Error! Bookmark not defined. 1.6 Hepatitis C and its Virus ...... Error! Bookmark not defined. 1.6.1 Transmission of Pathogen and Genotype Prevalence Error! Bookmark not defined. 1.6.2 HCV Structure and Genome ...... Error! Bookmark not defined. 1.6.3 HCV Non-Structural and Structural Proteins ...... Error! Bookmark not defined. 1.6.4 HCV RNA Replication and Potential Drug Target...... Error! Bookmark not defined. 1.6.5 In vitro Infection Systems for HCV Replication ...... Error! Bookmark not defined. 1.6.6 Available Treatment Options against HCV ...... Error! Bookmark not defined. 1.6.7 NS3 Gene as Candidate Drug Target...... Error! Bookmark not defined. 1.6.8 Ayruvadic Extract and Plant Derived Compounds as anti HCV Drug ...... Error! Bookmark not defined. 1.7 Goals/Objectives ...... Error! Bookmark not defined. 2.0 Chemicals, Equipment and Techniques ...... Error! Bookmark not defined. 2.1 Chemicals ...... Error! Bookmark not defined. 2.1.1 Materials for Chromatography ...... Error! Bookmark not defined. 2.1.2 Chemicals and Reagents used for Determination of Antioxidant....Error! Bookmark not defined. 2.2 Instrument and Apparatus ...... Error! Bookmark not defined. 2.3 Materials used for Biological Activities ...... Error! Bookmark not defined. 2.3.1 Cell Lines ...... Error! Bookmark not defined. 2.3.2 Plasmids ...... Error! Bookmark not defined. 2.3.3 Reagents & Chemicals ...... Error! Bookmark not defined. 2.3.4 Designing of Primers ...... Error! Bookmark not defined. 2.4 Instrumentation & Techniques ...... Error! Bookmark not defined.

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2.4.1 Physical Constant ...... Error! Bookmark not defined. 2.4.2 Spectroscopy ...... Error! Bookmark not defined. 2.4.3 Thin Layer Chromatography ...... Error! Bookmark not defined. 2.4.4 Column Chromatography (CC) ...... Error! Bookmark not defined. 2.5 Methodology ...... Error! Bookmark not defined. 2.5.1 Biochemical Analysis of Plants ...... Error! Bookmark not defined. 2.5.2 Collection, Identification and Preservation of Medicinal Plant Materials ...... Error! Bookmark not defined. 2.5.3. Plant Extract Preparation...... Error! Bookmark not defined. 2.5.4 Screening of Plant Extracts for Cellular Toxicity Analysis via Trypan Blue Dye Exclusion Method ...... Error! Bookmark not defined. 2.6 Anti-HCV Analysis of Compounds in Liver Cells...... Error! Bookmark not defined. 2.6.1 Antiviral Analysis of Plant Extract against HVC Ns3 Protease via Transfection Assay ...... Error! Bookmark not defined. 2.7 Gene Expression studies via Reverse Transcriptase PCR (RT-PCR) ....Error! Bookmark not defined. 2.7.1 Extraction of Total RNA ...... Error! Bookmark not defined. 2.7.2 Synthesis of cDNA ...... Error! Bookmark not defined. 2.7.3 Reverse Transcription PCR (RT-PCR) for Gene Expression Analysis Error! Bookmark not defined. 2.7.4 Expression studies via Real-time PCR...... Error! Bookmark not defined. 2.8 Western Blotting of PO extracts ...... Error! Bookmark not defined. 2.8.1 Protein Isolation ...... Error! Bookmark not defined. 2.8.2 Protein Quantification ...... Error! Bookmark not defined. 2.8.3 Protein Samples Preparation ...... Error! Bookmark not defined. 2.8.4 SDS Gel Electrophoresis ...... Error! Bookmark not defined. 2.8.5 Transfer of Protein and Immunoblotting ...... Error! Bookmark not defined. 2.8.6 Enhanced Chemiluminescence (ECL) Detection ...... Error! Bookmark not defined. 2.9 Analysis of the Two Plants Extract That Exhibit Positive Potential against HCV .. Error! Bookmark not defined. 2.10 Phytochemical Screening ...... Error! Bookmark not defined. 2.10.1 Qualitative Analysis of the Extracts ...... Error! Bookmark not defined. 2.10.2 Quantitative Determination of the Chemical Constituency Specifically Antioxidants ...... Error! Bookmark not defined. 2.11 Anti-clotting Properties of CTS and POL Extracts ...... Error! Bookmark not defined. 2.11.1 Sample Preparation ...... Error! Bookmark not defined.

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2.11.2 Preparation of Anti-Clot Test Solution ...... Error! Bookmark not defined. 2.11.3 Clot Lysis ...... Error! Bookmark not defined. 2.12 Antibacterial assay of extracts ...... Error! Bookmark not defined. 2.13 Bioassay guided extraction of CTS and POL ...... Error! Bookmark not defined. 2.14 Isolation of Compounds via Column Chromatography .. Error! Bookmark not defined. 2.14.1 Isolation of Compounds ...... Error! Bookmark not defined. 2.14.2 Physico-chemical Characteristics of Isolated Compounds..... Error! Bookmark not defined. 2.14.3 Elucidation of Compounds Structure by spectroscopy ...... Error! Bookmark not defined. 3.0 Results and Discussion ...... Error! Bookmark not defined. 3.1 Collection of Botanical Samples and Preparation of Extract ...... Error! Bookmark not defined. 3.2 Percentage yield of plants extract...... Error! Bookmark not defined. 3.3. Toxicological Studies of Plant Extracts in CHO and Liver Cell Lines .Error! Bookmark not defined. 3.3.1 Trypan Blue Dye Exclusion Assay ...... Error! Bookmark not defined. 3.3.2 Cytotoxic Screening of Phyto Extracts via MTT Assay Error! Bookmark not defined. 3.4 Anti-HCV Assay ...... Error! Bookmark not defined. 3.5 Inhibition of HCV NS3 Gene Expression by Selected Organic ExtractsError! Bookmark not defined. 3.6 Western Blotting ...... Error! Bookmark not defined. 3.7 Phytochemical analysis...... Error! Bookmark not defined. 3.7.1 Qualitative Analysis of CTS and POL ...... Error! Bookmark not defined. 3.7.2 Quantitative Methods for Screening of Phytoconstituents Error! Bookmark not defined. 3.8 Antioxidant Potential of Biologically Active Plant Extract Fractions Error! Bookmark not defined. 3.8.1 Total Phenolic, Total Flavonoids and Total Carotenoid Contents ...Error! Bookmark not defined. 3.8.2 Free Radical Scavenging Activity of Extracts by DPPH, ABTS and FRAP Assays ...... Error! Bookmark not defined. 3.8.3 Quantitative Estimation of Phenolic Acid by HPLC . Error! Bookmark not defined. 3.10 Antithrombotic Potential of Extracts ...... Error! Bookmark not defined. 3.11 Screening of Antibacterial Activity of Samples ...... Error! Bookmark not defined.

3.12 Extraction, Purifcation and identification of Compounds Obtained from CTSE Extract ...... Error! Bookmark not defined. 16

3.12.1 Compound CT-EtOAc-01 ...... Error! Bookmark not defined. 3.12.2 Compound CT-EtOAc-02 ...... Error! Bookmark not defined. 3.12. 3 Compound CT-EtOAc-03 ...... Error! Bookmark not defined. 3.12.4 Compound CT-EtOAc-04 ...... Error! Bookmark not defined. 3.12.5 Compound CTEtOAc-05 ...... Error! Bookmark not defined. 3.13 Discussion ...... Error! Bookmark not defined. 4.0 Future Prospects ...... Error! Bookmark not defined. 5.0 References ...... Error! Bookmark not defined. 6.0 Appendix ...... Error! Bookmark not defined.

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LIST OF FIGURES

Fig. 1.1: Taxonomy of Caralluma tuberculata N.E.Br. and whole plant along with flower ………………………………………………………………………………….6 Fig 1.2: Chemical structures of some notable basic compounds of Caralluma genus..9 Fig. 1.3: Pharmacological properties of some significant Caralluma species ………14 Fig. 1.4: Taxonomy of Portulaca oleracea L and whole plant along with flower ….15 Fig. 1.5: Detailed life cycle of hepatitis C virus …………………………………….29 Fig 1.6: The HCV subgenomic replicon system …………………………………….30 Fig. 1.7: Scheme of HCV Pseudo-particle production and infection..………………31 Fig. 2.1: Cycling parameters for expression analysis of HCV NS3 gene in reverse transcription PCR ……………………………………………………………………52 Fig. 2.2: Thermal cycling conditions for Real-time PCR …………………………...53 Fig. 2.3: Scheme for bioassay-guided fractionation of Caralluma tuberculata and Portulaca oleracea L methanolic extracts……………………………………………66 Fig. 3.1: Percentage yield of methanolic extracts of plants collected from Soon- sakaser valley of Pakistan…………………………………………………………….71 Fig. 3.2 (A & B): Cellular toxicity analysis CTS and POL extracts through Trypan blue exclusion method …………………………………………………………...... 73 Fig. 3.3 (A & B): Toxic effect of plant (CTS and POL) crude extracts on Huh-7 and CHO cell lines by MTT assay …………………………………………………..…...74 Fig 3.4: Anti-HCV activity of methanolic extracts of selected fifteen different plants………………………………………………………………...... 76 Fig. 3.5 (A&B): Anti-HCV activity of organic extracts of POL and CTS…………..77 Fig. 3.6 (A and B): Antiviral Activity of methanolic and ethyl acetate extracts of POL against HCV NS3 protease. ………………………………………………...... 79 Fig. 3.7 (A and B): Qualitative & quantitative inhibition of HCV NS3 protease by IFN, methanolic and ethyl acetate extract of CTS. ………………………………….79 Fig. 3.8: Antiviral Activity of methanolic and ethyl acetate extracts of POL against HCV NS3 protease at protein level…………………………………………………..80 Fig. 3.9: Total Phenolic Contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments…………………………………………………………………………..84 Fig. 3.10: Total flavonoid Contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments…………………………………………………………………………..84 Fig. 3.11: Total carotenoid Contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments…………………………………………………………………………..85 Fig. 3.12: DPPH free radical scavenging potential of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments………………………………………………………………………86 Fig. 3.13: ABTS free radical scavenging potential of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.……………………………………………………………………...86 Fig. 3.14: Free radical scavenging potential of selected sample extracts by FRAP assay All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments. …………………………………………………………..87 Fig. 3.15: Identification and quantification of Phenolic acids in CTSM ………………………………………………………………………………………..88

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Fig. 3.16: Identification and quantification of Phenolic acids in POLM…………………………………………………………………………………88 Fig. 3.17: In vitro thrombolytic activity of selected fractions of plant extracts with respect to positive control (Streptokinase) and negative control (water). All results are presented as mean and standard deviation for the three individual experiments. ***P value < 0.001 vs Streptokinase (control). ……………………………………………89 Fig. 3.18: Percent inhibition of bacteria by selected extracts with respect to ciproflaxacin (positive control)………………………………………………………91

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LIST OF TABLES

Table 1.1: Historical perspectives of natural products...... 2 Table 1.2: Apocynaceae Meve classifications into subfamilies with their genera and species numbers …………………...... 5 Table 1.3: Global distribution and pharmacological activities of different species of Caralluma genus……………………………………………………………………..13 Table 2.1: Primers of GAPDH and HCV non-structural NS3 gene of 1a & 3a genotype was used in this study …………………………………………………….44 Table 2.2: Botanicals details chosen for screening against total RNA HCV and its gene NS-3 serine protease…………………………………………………………....47 Table 2.3: Composition of the gels used in SDS-PAGE ……………………………55 Table 3.1: Qualitative phytochemical screening of CTS and POL methanolic extracts. ………………………………………………………………………………………..81 Table 3.2: Percent phytochemical components in CTS and POL methanolic extracts………………………………………………………………………………..82 Table 3.3: Identification and quantification of Phenolic acids by HPLC……..…….88 Table 3.4: Zones of inhibition (mm) in Disc Diffusion Plate for the antibacterial activity of successively extracted alcoholic extracts (SAE) of plants that have also antiviral potential.…………………………………………………………………….91

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1.0 Introduction

A ‘natural product’ term widely covers the product of natural bio source like plant, animal and microbes. It may be in the form of extract or single pure compound

(secondary metabolites) like alkaloids, limonoids, steroid, flavonoid, lignana, terpenoids, organosulfur, tannin, furyl, polylines, chlorophyllins, thiophenes, saponins, sulphides, coumarins, etc. (Naithani et al., 2008). The worth of medicinal plants lies in these metabolites that are non-nutritive for plants, but produce certain physiological action in plants and human against different types of infectious disease and metabolic disorders (Edeoga et al., 2005). In the field of antiviral compound search these Phyto-compound are striking targets owing to their ability to hamper viral entrance, blocking or restricting the replication of the RNA / DNA genome of the virus (Naithani et al. 2008). Bioactive compounds are also generated by plants as a result of their own “defence mechanisms” against pathogens (Wedge et al., 2000). Human interest in natural products constantly spans the history because of their striking potential as medicines, food, agriculture, cosmetics, etc. (Edeoga et al., 2005).

1.1 Natural Products from Times of Yore to 21st Millennium

Phytotherapy history is as old as the history of humankind itself (Ahmad et al., 2006). System of alternative medicine remained enriched by traditional Ayurveda, Kampo, Unani and Chinese medicine throughout the history. All these systems are based on myths and reality based knowledge that decipher frequently modified theory and practices. All these approaches of natural component formulation are still widely accepted in this century, owing to their therapeutic strength (Harvey et al., 2000). Summarized authenticated information about natural product usage as medicine in historical perspective is appendices in table 1.1.

Mostly the formulation scheme was kept in secret by traditional healers, herbalist and shaman, so this thing is a major obstacle to check the authenticity of these formulations on a scientific basis. Hence, flair to access bioactive compounds, realizing their worth and potential has always remained a main driving force. The

21 incredible resurgence of this research field was noticed over the preceding decade or so due to their small size (<200 Da), incomparable structure, diversity and drug like characteristics (absorbed and metabolized easily) (Sarker et al., 2005).

Table 1.1: Historical perspectives of natural products Time period Category Elucidation Before 320 BC Ayurveda (Chinese old Information regarding the medicinal properties of system of medicine) plants and their products. 1550 BC Ebers papyrus Details about large number of crude drugs from plant source like gum Arabica, castor seeds etc. 460-377 BC Hippocrates Enlist medicinal properties of 400 plants 370-287 BC Theophrastus Describe usage of plants and animals as medicine 23-79 AD Pliny the Elder Plant and animals usage as medicine 60-80 AD Discorides Author of “De material medica” ( Provide information about medicinal potential of more than 600 plants) 131-200 AD Galen Author of “Galenicals” who practiced botanical medicine and familiarized the West to its usage Before 632AD Tib-E-Nabvi In this book Hazrat Muhammad (PBUH) suggested the treatment of different disease by natural sources 15th Century Krauterbuch Depicted elaborated information and picture of medicinal plants.

No doubt, the contribution of natural products is remarkable for the development of drug is either in its intact form like vincristine (Catharanthus roseus), or act as ‘‘building blocks’’ substance that further modified into more complex molecule like diosgenin (Dioscorea floribunda) used in oral contraceptives, or the novel analogs synthesis from them like synthetic analogs of penicillin (Penicillium notatum) (Sarker et al., 2005). Plant based medicine satisfying the prime health care requirement of 80 % of the populace of developing countries (WHO, 2002).

In the last couple of decades, traditional medicine has been remained appealing target by academia and the pharmaceutical industry in search of "potential leads" that can be further utilized in the synthesis of modern-day drugs (De Silva et al., 1997). More than 40 % of modem drugs have been derived from plants specifically anticancer and antibacterial drugs. In the beginning of this millennium, eight out of thirty top selling drugs (amoxicillin, azithromycin, clavulanic acid, cyclosporine, plastitaxel, ceftriaxone, pravastatin and simvastatin) were either natural product or their respective derivative (Cragg et al., 1997).

Owing to the ever increasing cost, adverse effects, multiple drug and microbial resistance of the synthetic drugs; people are now turning to the world of

22 ethnopharmacognosy that is literally more efficacious, safe, and inexpensive with a alleviated side effect (Sànchez-Lamar et al., 1999). Moreover, these natural products can also disclose new springs of economic resources such as gums, oils, tannins etc. and these can be further used as precursors of other synthetic complexes (Farnsworth et al., 1966). Not only in Asian and African countries but also in Europe and North America plant based therapies are extensively accepted in the form of food supplements, nutraceuticals, complementary and alternative medicines.

Along with the expansion of newest molecular targets the stipulation for novel biologically active molecules has been increased. The previous practice of natural product research mainly spans around straightforward extraction and recognition of chemistry instead of its in vitro bioactivity. Sometime, in vivo models were used to determine their biological activity on the basis of ethno-pharmacological information. But the modern strategies opted in vitro bioassay-guided separation along with detection of active ‘‘lead’’ from natural reservoir. More attention is given to bioactivity and libraries of natural products.

New horizons of interdisciplinary science strengthen the research on natural product by introducing the concept of genetic manipulation and natural combinatorial chemistry. Because of the latest updates in the hyphenated techniques of separation and purification like mass spectrometry, advanced spectroscopic techniques, and ultrasensitive in vitro micro plate-based assays have revolutionized the research of pre-isolation of crude extract, online detection, chemical finger printing, and metabolomic analyses (Sarker et al., 2005).

1.2 Status of Medicinal Plants and their Research in Pakistan

Pakistan is bestowed with exclusive floral array in nine different ecological zones owing to its ideal climate conditions. In Pakistan, approximately 600 species out of 6000 wild plants are considered highly significant from the medicinal potential point of view (Hamayun et al., 2003). Mostly rural and suburban population of Pakistan primarily depends on plants or their active ingredients for their healthcare (Shinwari

23 et al., 2003). Unani system of medicine is used widely by traditional healers or Hakims while they suggest plant based remedies (Goodman et al., 1992).

Soon-Valley of Pakistan is blessed by the unique biodiversity of plant species because of a wide range of unusual climatic precincts. Its climate is subtropical with unique geological formation due to high concentration of salt as well as other minerals in the subsoil. In the local vegetation, a least work has been conducted on natural product with respect to the medicinal point of view (Ahmad et al., 2008). The plant communities are losing their species richness at high rate due intensive deforestation and unlimited expansion of urban areas. Despite of ever increasing human knowledge and folklore regarding usage of natural product, appropriate modern scientific approaches have only been applied to a very little part of the world's flora. It is a matter of grave concern to exploit the potential of those plant species that are in waiting line before they become endangered (Sasidharan et al., 2011).

1.3 Preface to Genus Caralluma and Caralluma tuberculata

Caralluma is considered in the family Apocynaceae that comprises three subfamilies Rauvolfioideae, Apocynoideae and Asclepiadaceae holding 424 genera (Liede- Schumann et al., 2005). Ornamental plants like Allamanda, Frangipani, Oleander, Vinca of tropical area, large trees with special buttress roots of rainforests and deciduous or evergreen trees, climbers or shrubs of the world warm and temperate regions are included in these genera. Milky latex of most of the plants is considered important for medicinal purpose and rubber production. Later, in 1890 Robert Brown separated Asclepiadaceae (milkweed family) from Apocynaceae. Recent advances in genomic, molecular and morphological analysis merge the Asclepiadaceae and Periploceae into Apocynaceae family (Endress et al., 2005). Meve also categorized Asclepiadaceae into three tribes as fallow Table no. 1.2.

Caralluma tuberculata is a member of the milkweed family Asclepiadaceae which include about 2500 species from 200 genera and widely distributed in some tropical regions of Punjab, Khyber Pakhtoonkhawa and Baluchistan provinces of Pakistan. In Pakistan, Asclepiadaceae is represented by approximately 23 genera along with 40

24 species. In semi-arid and desert vicinity of the country this Caralluma species have been used as emergency victuals for centuries.

Table 1.2: Apocynaceae Meve classifications into subfamilies with their genera and species numbers Total Total Subfamily Distribution Genera Species Cosmopolitan particularly in Africa, Asia Asclepiadoideae 177 3000 and Europe Periplocoideae 45 190 Restricted to Africa and Asia only Secamonoideae 9 180 Restricted to Africa and Asia only (Meve et al., 2005; Verhoeven et al., 2001 & Muller et al., 2002)

The Caralluma turbeculata vernacular name is chunga or chung in Punjab. Plants belonging to the genus Caralluma are normally leafless (Dawidar et al., 2012). This perennial herb has 15-45 cm height with a succulent, fleshy, leafless, erect and 4 angled stems having grooves on it. Quadrangular stem bears small flowers in several varieties of dark colour (Madhuri et al., 2011). Sessile single or many flowers in lateral cymes having ovate-lanceolate five sepals and deeply divided, glabrous, lobe lanceolate, dark purple colour corolla of about 9 mm in diameter. Anthers are without appendages and each having pollen mass one. Glabrous Follicles with size about 9-11 cm are progressively tapering to the tip. The flowering season is June. It mostly grows widely on rocks after rain, but it is also cultivated in some areas. Its stem and roots are not only eaten raw as famine food, but also cooked due to its pharmacological potential. The complete classification of Caralluma tuberculata is deciphered in Fig.1.1.

1.3.1 Active Constituents

Caralluma, in general, is famous for the presence of its key component glycosides specifically pregnane and aglycone, megastigmane glycosides, flavonoids, saponins and triterpenes. Pregnanes (I) are the C-21 steroids having perhydro-1,2- cyclopentanophenanthrene ring as backbone that have β-oriented methyl group at two positions (C-10 and C-13) and two outside chains of carbon atoms at C-17. In derivative form of pregnane on C-14 atom β confirmation is present along with a hydroxyl group (Deepak et al., 1989). Genus Caralluma was 1st time explored for its constituents in 1967 and two compounds (dihydrosarcostin (3, 8, 12, 14, 17,

25

(20S) -hexahydroxypregnane and tomentogenin (3, 12, 14, 17, (20S)- pentahydroxypregnane) (II &III) were extracted from C. dalzielii (Deepak et al., 1997).

Domain Eukaryota Kingdom Plantae Subkingdo Viridaeplantae m Phylum Tracheophyta Subphylum Euphyllophytina Infraphylu Radiatopses m Class Magnoliopsida Subclass Asteridae Caralluma tuberculata: Whole plant & flower Superorder Gentiananae Order Gentianales Family Apocynaceae Subfamily: Asclepiadoideae Tribe Ceropegieae Genus Caralluma Specific tuberculata - N.E.Br. epithet Caralluma tuberculata Botanical name N.E.Br. Fig. 1.1: Taxonomy of Caralluma tuberculata N.E.Br. and whole plant along with flower

The literature survey indicates that the pregnane glycosides were isolated from various parts of the plant. With polar solvents like ethylacetate, n-butanol, methanol, ethanol and water, the compounds like C21 class of steroidal glycosides with high molecular weight compounds were obtained but on other hand compounds having low weight like cumarin, sterol, steroid, terpenoid were obtained when the same parts were extracted with non-polar solvents like n-hexane, benzene etc. In a couple of studies, two new pregnane glycosides carumbellosides I and II and steroidal glycosides i.e. carumbellosides III-V were isolated from the extract of Caralluma umbellate whole plant first reported the isolation and identification of (Kishore et al., 2010; Sheng-Xiang et al., 1997). New steroidal glycosides, stalagmosides I-V and indicosides I and II together with the known compounds Carumbelloside III, lasianthoside A and lasianthoside B were isolated from the whole plant of Caralluma

26 stalagmifera. The genus is also characterized by the presence of flavones glycosides (Sreelatha et al., 2010).

Two novel steroid glycosides were isolated from Caralluma tuberculata that exhibited fairly in vitro cytotoxic activity on breast cancer cells even in micromolar concentration. Pregnanes are C21 steroids and often found in nature conjugated as glycosides. A flurry of pregnane glycosides and esterified polyhydroxypregnane were extracted from Caralluma and other family member of Asclepiadaceae. Some of these glycosides appear as novel potential template for drug development against cancer and tumor (Waheed et al., 2011).

Chemical investigation of Caralluma tuberculata indicates that it was endowed with the flavone glycosides and several pregnane glycosides (Abdel-Sattar et al., 2011; Oyama et al., 2007). Five pregnane glycosides were isolated from Caralluma tuberculata, in addition to a known one (russelioside E, 6). All these six compounds were checked for their action against malaria and trypanosome. Moreover, their cytotoxic effect was also tested on the growing human MRC5 embryonic cell line (Abdel-Sattar et al., 2008). Caratuberoside C (I), and D (II), two new pregnane glycosides were isolated from Caralluma tuberculata (Rizwani et al., 1993). A medicinal herb, Caralluma tuberculata, furnished a pregnane type compound, caratuberside A2. The sugar linkage was at C-14 which is a rare site of substitution (Rizwani et al., 1993). Key phytochemical ingredients of Caralluma umbellate include pregnane glycosides, flavone glycosides, bitter principles, saponins and various flavonoids (Al-Massarani et al., 2012; Ray et al., 2012). Some acylated pregnane glycosides like russeliosides E–H were obtained from chloroform extract of Caralluma russeliana (Abdel-Sattar et al., 2008).

The aglycone pregnane (Pregnane-14β -formyl-5, 7-dien-20-one) (VII), with unusual formyl group at C-14, were extracted from of the non polar (petroleum ether) extract Caralluma umbellate stem (Babu et al., 2008). Similarly two more aglycone steroids Cur I (3-hydroxy-pregn-5-ene) and Cur II (3, 14-dihydroxy pregn-5-ene) were extracted from the non polar extract of the roots of same plant (Kishore et al., 2010). Furthermore, in other studies, one more compound, i.e. 20S-epimer of boucerin was

27 extracted 1st time from the toluene extract of the above said specie of Caralluma (Kunert et al., 2009).

The Phytochemistry of genus Caralluma is characterized by many pregnane glycosides, but recently megastigmane glycosides (IV & V) have also been isolated from Caralluma negevensis with few flavones. Saponins and flavonoids predominantly found in the Caralluma are of great interest because of the wide range of immunostimulating activities. As Caralluma attenuata, the fresh whole plant contains luteolin-4-O-neohesperidoside, a flavonoid (flavones glycoside) identified as the major chemical constituents of the plant. Caralluma adscendens is declared to contain saponin glycosides, bitters, pregnane glycosides (caratubersides A and B and various boucerosides) (Tatiya et al., 2010).

Several members of the genus Caralluma are rich in triterpenes, pregnane glycosides or their esters which may have found medicinal uses (Dawidar et al., 2012). Triterpenes squalene, lupeol, lupenone, lupeol acetate, β -sitosterol acetate and guimarenol were extracted from Caralluma buchardii. Glycone moieties include different sugars like glucose, digitalose, thevotose, quinvose etc.

21 CH3 18 20 OH OH 12 CH3 17 11 OH 19 C 13 H D 16 1 9 14 CH3 R

15 2 10 8 OH A H H OH 3 5 B 7

HO 4 6 HO H H

Pregnane (I) R= H, Tomentogin (II) R=OH, Dihydrosarcostin (III)

28

H H

H H

OH H H H

HO HO Crur I Crur II OH OH 11

O O 3 2 2

4 6 7 7 8 9 9 5 H H O Glc(1 6)rha O Glc(1 6)rha Megastigmane glycosides (IV & V)

O 21 20

H 17 O 14

OC3H O H H 22

O Triterpene (VI) Aglycone pregnane (VII)

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HOH2C OH O OH OH OH

Glucose H3C OH O OH OH OH

Quinvose OH H3C H3C O OH O OH OH H3CO H3CO OH OH

Digitalose Thevotose Common sugars present in Caralluma pregnane glycosides Fig 1.2: Chemical structures of some notable basic compounds of Caralluma genus

1.3.2 Pharmacological worth of Caralluma tuberculata and other species of Caralluma

Traditionally it is believed that Caralluma tuberculata have anti inflammatory and strong hypoglycaemic effect (Ahmad et al., 1988; Ahmed et al., 1993; Mahmood et al., 2010). In folklore, this is also reputed for the treatment of rheumatoid arthritis, paralysis and fever (Khan et al., 2008). Caralluma family members are rich with Pregnane that naturally conjugated with glycoside and its structure holds basic C21steroids backbone along with sugar moiety. These molecules appear as potential lead for drug development against cancer and diabetes (Deepak et al., 1989; Deepak et al., 1997). Pregnane glycosides series has been extracted from its organic extracts that explicit cytotoxic effect against cell line of Human diploid embryonic cell like MRC5 (Abdel-Sattar et al., 2009; Abdel-Sattar et al., 2008).

The antiproliferative potential of Caralluma tuberculata was observed in the development of two cancer cell lines of breast MCF-7 and MDA-MB-468 (oestrogen-

30 dependent and estrogen-independent), and colonic cells (U937 and Caco-2) (Waheed et al., 2011). Androstan and pregnane glycosides stimulate caspase mediated apoptosis. Caspase enzyme, dependent on Calcium, is activated via two possible pathways, intrinsic (e.g. mitochondria damage) and extrinsic (e.g. Cell surface death receptor ligation). This activated caspase along with caspase 3 triggers the DNA fragmentation by the cleavage of PARP (poly-ADP Ribose polymers). After PARP cleavage, cell irrevocably prone to apoptosis. Due to structural homology of Pregnane glycosides(12-O-benzoyl-20-O-acetyl-3,12,14,20-tetrahydroxy-pregnan3-ylO-D- glucopyranosyl-(1→4)-d-glucopyranosyl-(1→4)-3-methoxy-d-ribopyranoside) with estrogen agonist inhibits Calcium exchangers, as a result calcium concentration increased that stimulate caspases and apoptosis (Deepak et al., 1997). Another possibility is activation of the xenobiotic and steroid receptors that also induce the apoptosis process in cancerous cells of the breast (Verma et al., 2009). This process is not fully cleared hence further research is mandatory to decipher the activation of xenobiotic receptors.

Treatment of mice with Caralluma tuberculata extract stimulates composite biochemical and cytological variations in it (Al-Bekairi et al., 1992). The ethanolic extract of Caralluma tuberculata afforded potential protection against gastric mucosa injuries caused by ethanol (80 %), sodium hydro oxide (0.2 M), hypertonic salt solution and indomethacin in a dose-dependent manner (Abdel-Sattar et al., 2011; Alharbi et al., 1994).

Many flavonoids such as Quercetin, rutin, Kaempferol, flavone glycoside and hypolaetin-8-glucoside have been reported to have gastric ulcer protective effects. Similarly saponins, such as the derivatives of glycyrrhetinic acid and triterpenoid saponins were also reported to have antiulcer effects in rats. It therefore appears responsible to suggest that flavonoids and saponins in Caralluma tuberculata may be totally or partially responsible for its antigastric ulcer activity (Alharbi et al., 1994).

Pregnane glycosides, Penicilloside E displayed the maximum selectivity index (SI 12.04) which was demonstrated by its highest antitrypanosomal potential trailed by caratuberside C. it was also proved that acylation is required for the antitrypanosomal activity instead of the of glycosylation process at C-20 (Abdel-Sattar et al., 2009).

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The methanolic extracts of Caralluma tuberculata N. E. Br. as well as its n-butanol, chloroform and petroleum ether soluble portion were examined for cytotoxicity against human diploid embryonic cell line (MRC5) and different other pharmacological activities. Overall six compounds were extracted from the chloroform fraction and were further tested for their antimalarial, antitrypanosomal and antiprotozoal activity. As for the anti-malarial activity, only petroleum ether soluble fraction demonstrated moderate inhibitory effect (IC507.94l g/mL), this fraction showed high cytotoxicity on MRC5 (IC50 0.8l g/mL) (Waheed, 2001). Methanolic extract did not exhibited any potential against the trypanosoma as compare to the petroleum ether extract that demonstrated reasonable activity (IC50 0.5l g/mL) along with 1.6 selectivity index. In contrast, modest activity i.e. (IC50 3.5l g/mL) along with selectivity index (17.9) was shown by the extract of chloroform. Chloroform extract was chosen further for extraction of biologically active six compounds that exhibit strong potential against malarial and trypanosome (Abdel- Sattar et al., 2008).

Other members of this genus like Caralluma fimbriata and Caralluma siniaca were considered to reduce body weight and blood glucose level respectively (Habibuddin et al., 2008; Lawrence et al., 2004). In “The Wealth of India” (1992) Caralluma fimbriata was recorded as very useful medicinal plant because as indean tribes used it act as hypoglycemic agent, weight loss stimulator and suppressant of appetite, pain, fever and inflammation (Abdel-Sattar et al., 2007; Sreelatha et al., 2010).

Caralluma edulis is also famous for its anti-diabetic potential (Wadood et al., 1989). Similarly hypoglycemic synergistic effect was noticed when C. edulis and C. attenuate were used in combination along with extract of phlorizin for reducing blood and urine glucose level in conjunction with weight loss (Venkatesh et al., 2003). In Nepal and Sri Lanka, stem of wild Caralluma umbellata are used in stomach disorders and abdominal pains (Kishore et al., 2010).

Caralluma adscendens is a thick, succulent perennial herb found wild in Africa, Afghanistan, Ceylon, India, Southern Europe and Saudi Arabia. In India, it grows naturally in the dry hills of Andhra Pradesh, Warangal and various other districts. It may also consumed in form of pickle and eaten as vegetable during famine (Tatiya et al., 2010). It is locally known as “Makadshenguli/Shengulmakad”. The local people 32 use it in raw form for treatment of diabetes and it is also utilized as a vegetable (Mali et al., 2009). Caralluma attenuata is eaten raw because it acts remedy for diabetes while its juice along with black pepper is suggested for the cure of migraine (Kumar et al., 2011). It usually exhibits antinociceptive activity owing to its luteolin-49-O- neohesperidoside components (Kumar et al., 2011).

Caralluma dalzielii N. E. Br. is a succulent herb occurring wildly on the Sahelian region of West Africa from Senegal to Nigeria where is used in folk medicine as antispasmodic and analgesic remedy. Plant latex in Bandiagara area of Mali is used for wound healing, while the stems are grounded and eaten row as tonic remedy and for bodily exhaustion and cardiac problems. A previous phytochemical study on this plant reported the isolation of two tomentogenin esters (De Leo et al., 2005). In short, worldwide distribution and pharmacological properties of some significant species of Caralluma genus are represented in summarized form in Fig.1.3 and table 1.3.

Table 1.3: Global distribution and pharmacological activities of different species of Caralluma genus Species of Distribution Pharmacological Activities Part Used Citations Caralluma Genus in traditional medicine System C. adscendens India Rheumatic Pain, Paralysis Plant Stem (Gowri et al., 2011; Naik et al., 2012) C. attenuate Spain, India Anti-tumor, Pain, Whole Plant (Kumar et al., 2011) Hyperglycemia (Abdel-Sattar et al., 2008) C. edulis Pakistan, Iran Rheumatic Pain, Parasitic Whole Plant (Ali et al., 2011; diseases, Gastric problems, Mahmood et al., 2011) Leprosy, Hyperglycemia, (Ahmad et al., 2009) Hypertension, Hypercholesterolemia C. umbellate India Gastric Ulcers & Pain Roots (Karuppusamy et al., 2007; Kishore et al., 2010) C. laciantha India Wound Healing, Veterinary Plant Stem (Naik et al., 2012) Use C. stalagmifera India Wound Healing, Plant Stem (Naik et al., 2012) Malnutrition C. sinaica Saudi Arabia Hyperglycemia, Leaves (Habibuddin et al., 2008; Hypercholesterolemia Nawal et al., 2012) C. diffusa India Organogenesis, Whole Plant (Karthik et al., 2013; Hyperglycemia, Ramadevi et al., 2012) C. penicillata Saudi Arabia Trypanosomacidal, Whole Plant (Abdel-Sattar et al., 2009) Antivenom C. russeliana Saudi Arabia Medicinal Whole Plant (Abdel-Sattar et al., 2009)

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C. bhupenderiana India Ethnoveternary uses Whole Plant (Ugraiah et al., 2013) C. fimbriata India, Iran Obesity, Hyperglycemia, Plant Stem (Kamalakkannan et al., Hypercholesterolemia 2010; Lawrence et al., 2004; Naik et al., 2012) C. europaea Italy Pharmaceutical Fruits & (Zito et al., 2010) Stem C. negevensis Africa, Spain Anti Cancer, Lung Diseases (Braca et al., 2002) C. diazielli Nigeria Obesity, Hyperglycemia, (Tanko et al., 2013) Hypercholesterolemia C. nilagiriana India Infectious Diseases (Prabakaran et al., 2013) C. wissmannii Egypt Medicinal Above (Dawidar et al., 2012) Ground Parts C. Arabica UAE Vegetable, Liver Tonic, Whole Plant (Zakaria et al., 2001) Obesity, Hyperglycemia, Hypercholesterolemia, Wound Healing C. pauciflora India Vegetable Plant Stem (Ugraiah et al., 2013) C. quadrangular Oman, Vegetable, Wound Healing, Whole Plant (Shah et al., 2013) Muscat Tonic C. tuberculata Pakistan, Vegetable, Hepatitis B & C, Whole Plant (Ahmad et al., 2009; India, Iran, Blood Purification, Gastric Mahmood et al., 2010; Nigeria, Saudi Problems, Liver Tonic, Marwat et al., 2014; Rauf Arabia Obesity, Hyperglycemia, et al., 2013; Safa et al., Hypercholesterolemia, 2012; Shah et al., 2012; Shah et al., 2013; Tareen et al., 2010; Zabihullah et al., 2006)

Fig. 1.3: Pharmacological properties of some significant Caralluma species

1.4 Introduction to Portulaca oleracea

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Portulaca oleracea, frequently well-known as Purslane in English and Kurfa in Arabic and Persian, is found all the temperate zones of the globe (Burkill et al., 1997). The Portulaca name is considered to narrate from the Latin language word “porto” denotes to hold or carry and “lac” denotes milk overall milky juice holding plant and it is mentioned in official pharmacopoeias of Mexico, France and Spain (Eduardo et al., 1995). In traditional Chinese medicine (TCM), Purslane is a natural herb that may also be considered food during the famine years. Chinese usually dig this feral vegetable and consume it, even presently still procure it from herbal shops. Numerous authors elaborated the botanical morphology of Portulaca oleracea from time to time (Matthews et al., 1993; Mitich et al., 1997; Rydberg et al., 1932). Generally, purslane is depicted as an annual succulent with prevailing prostrate growth. This annual herbaceous weed is succulent having 15-20 cm long stem and 6 mm long green flashy leaf (Chan et al., 2000). Stems are reddish in color, glabrous, and branch radially from the central axis while Leaves position is alternate or sub-alternate and are succulent glabrous. Roots consist of many fibrous lateral roots and a long thick taproot. Flowers are few along with sessile terminal head; Flowers are yellow in colour and noticed in sunny morning. It is a self pollinated plant whose reproduction is either via fragments of stem or seeds (Zimmerman et al., 1976).

Domain Eukaryota Kingdom Plantae Subkingdo Tracheobionta m Phylum Spermatophyta Subphylum Magnoliophyta Class Magnoliopsida Subclass Caryophyllidae

Order Caryophyllales Portulac oleracea: Whole plant and its flowers Family Portulacaceae Genus Portulaca L. Specific oleracea L. epithet Botanical Portulaca name oleracea L.

Fig. 1.4: Taxonomy of Portulaca oleracea L and whole plant along with flower

35

1.4.1 Phytochemistry of Portulaca oleracea

Pursulane is also considered as a good source of alkaloids and five oleraceins alkaloids (A- E) are reported in this regard (Xiang et al., 2014). Similarly sixteen phenolic including N-cinnamoyl phenylethylamides, pyrrole alkaloid (portulacaldehyde), amides and five phenylpropanoid acids were secluded from its polar extract (Kokubun et al., 2012). In one study five compounds (caffeic acid, hesperidin, oleracein A, oleracein B, and oleracein E) were extracted from the ethanolic extract with the help of a range of column chromatography (Yang et al., 2007). Four monoterpene glycosidic compounds including two novel ((3S)-3-O-(- D-glucopyranosyl)-3,7-dimethyl-7-hydroperoxyocta-1,5-dien-3-ol and portuloside A) and two already reported compounds ((3S)-3-O-(-D-glucopyranosyl)-3,7- dimethylocta-1,5-dien-3,7-diol and (3S)-3-O-(-Dglucopyranosyl)-3,7-dimethylocta- 1,6-dien-3-ol ) were isolated from methanolic extract of Portulac oleracea (Sakai et al., 1996; Seo et al., 2003). In addition to these perence of allantoin, N,N`- dicyclohexylurea, β-sitosterol and β-sitosterol-glucoside in the extract of plant aerial part was also confirmed with the help of spectral and crystallographic data of these constituents (Rasheed et al., 2004). A notable discovery in phytochemistry was seen in the form of three types of polysaccharides that exhibited promising antiviral activities against influenza and herpes simplex viruses. These are an acidic polysaccharide (1,3-,1,6- and 1,3,6-linked galactopyranosyl & 1,5-linked arabinofuranosyl), a neutral polysaccharides (arabinoglucomannan) and a pectin polysaccharide (having galacturonic acid, Galactose, rhamnose and Arabinose) (Dong et al., 2010). High performance liquid chromatography (HPLC) was also used to determine the presence of four ccompounds like caffeic acid, p-coumaric acid, ferulic acid and hesperidin in this plant (Wang et al., 2011).

1.4.2 Pharmacological attributes of Portulaca oleracea

Ancients from most civilizations considered it as a holy herb possessing spiritual, anti-magical and marvelous medicinal attributes (Grieve et al., 1997). Chinese assume that it carries “vegetable mercury” (Cantwell et al., 1993). It is consumed as a vegetable in cooked form and in salad dressing in raw form nearly in all the countries. Purslane is famous for its unusual therapeutic attributes and has been used as a

36 remedy for oral ulcer, diabetes and urinary disorders. It has powerful analgesic, antibacterial and antifungal activities (Chan et al., 2000; Oh et al., 2000).

It has been reported that because of high concentration of omega 3 fatty acid it can be used relaxant for skeletal muscle (Parry et al., 1993). Mechanism of action of Portulaca oleracea aqueous extract was studied and proved to possess the relaxant potential of skeletal muscle that may be considered by the interference of Ca2+ recruitment in skeletal muscle. Oral administration of this extract produces relaxation in rat’s skeletal muscle (Okwuasaba et al., 1987). Of course, Purslane holds high concentration of poly-phenols and unsaturated fatty acids that are excellent scavengers and denote a promising antitumor potential (Ebrahimzadeh et al., 2009) (Ebrahimzadeh et al., 2009; Liu et al., 2000).

Purslane seed oil (PSO) exhibited convincing antitumor characteristic in albino mice bearing Ehrlich ascites carcinoma (EAC) due to the presence of flavonoids content. Hussein et al. administered PSO and 5-Flourourasil, both separately and in mixture to the EAC induced mice for 21days and observed reduction in tumor volume along with notable improvement in life span and different biochemical parameters (Hussein et al., 2014).

Omega-3 fatty acid occupy a significant position for anti-carcinogenic prospective of Portulaca oleracea due to its different role. It can rationalize the accessibility of Linoleic acid. Moreover it also acts as a competitive inhibitor of Ω-6. Both LA and Ω- 6 are essential materials required directly or indirectly in cancer metabolism. Ω-3 fatty acid causes the un-saturation in membrane of cancerous cell that cause disintegration and also slow down the growth of cancerous cell by the activation of gene that start the process of apoptosis. Ω-3 (fatty acid) has inhibiting upshot on spur of ras P21cancer genes. This put a stop to the proliferation by preventing the attachment of cancerous cells onto the basement membrane and production of collagenases. Clinical findings decipher that Ω-3 fatty acid can also prop up the effect of chemo and radiotherapy and enhance recovery process in cancer patients after operation.

According to Simopoulos and Robinson, who mentioned in their book “The Omega Diet”, that people having modern unhealthy lifestyle consuming diet especially modern western diet having Ω-6 fatty acid contents 20 times more than that of Ω-3

37 fatty acids in contrast to the conventional asian diet. Ratio of cancer mortality is higher in western countries than eastern countries (Simopoulos & Robinson, 2002).

The functioning of peripheral and central nervous system is directly affected by the concentration of Portulaca oleracea. When the Portulaca oleracea ethanolic extract was intra peritonealy administrated in mice, it reduced the locomotive action and increases the muscle relaxant potential and antinociceptive activity (Radhakrishnan et al., 2001).

In an automatic single-cell bioassay system the alcoholic and aqueous extracts of this plant exhibited antimicrobial potential especially against different species of gram negative bacteria and antifungal action against the growth of selected fungal hyphae like fungi Aspergillus and Trichophyton and the yeast Candida (Banerjee et al., 2002; Oh et al., 2000). Fungitoxicity of aqueous and organic solvent (e.g. hexane, ethanol and chloroform) extracts were tested against different species of fungi (Banerjee et al., 2002). Ethanolic extracts of aerial parts of Portulaca oleracea also exhibited noteworthy analgesic and anti-inflammatory attributes (Chan et al., 2000; Islam et al., 1998; Zakaria et al., 1998). It also has the potential to stimulate the healing process of the wound by minimizing the wound surface area and enhancing its tensile strength (Rashed et al., 2003). The aqueous extracts of the aerial part of Portulaca oleracea resulted relaxation of rabbit jejunum and guinea pig fundus in a dose dependent manner. Moreover extract also created a dosage dependent pressure reaction on rat blood pressure (Parry et al., 1993). In contrast daily uptake of Portulaca oleracea seeds extract showed anti-fertility action on male albino rat’s reproductive organs. It induced an effective impairment of spermatogenesis (Verma et al., 1982).

In the present decade, Purslane polysaccharides were also utilized to treat burns, headaches, liver, stomach and intestinal ailments, cough, shortness of breath and arthritis (Chen et al., 2010; Dong et al., 2010; Li et al., 2009). Polysaccharide from pursulane can also assuage fatigue persuaded by mice forced swimming. The plant positive potential was noticed to enhance the activity, timing of mouse by increasing concentration of hepatic glycogen along with decreasing concentration of blood lactic acid and serum urea nitrogen (Jingrong et al., 2009).

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Portulaca oleracea also has the ability to alleviate the oxidative stress provoking by the deficiency of vitamin A. Phenolic alkaloids are the latest class of antioxidants discovered in this plant (Yang et al., 2009). Alcoholic and aqueous extracts of the Portulaca oleracea has also the potential to hamper the gastric abrasions persuaded by ethanol or hydrochloric acid. By enhancing the dose of both extracts in mice, the severity of ulcers and reduces their effects on the secretion of gastric acid were also calculated. Therefore, Portulaca oleracea is used in folk medicines due to its gastroprotective action (Karimi et al., 2004).

Portulaca oleracea boiled aqueous extract causes a momentous increase in pulmonary action. The results of the study showed that it is relatively potent, but transitory bronchodilatory outcome on the asthmatic airways (Malek, Boskabady, Borushaki, & Tohidi, 2004). The plant is commonly utilized to remove intestinal worms due to the bioactive compounds present in it. This plant provides efficacious treatment for controlling intestinal parasite loads (Quinlan et al., 2002). Traditionally Portulaca oleraceae is seen as one of the most familiar herb for solving urinary problems. It is safe and effectual to facilitate stable clinical trials (Lans et al., 2006).

The portulaca oleracea extract has hypoxia neuroprotective effects (Wang et al., 2007). It enhances the expression of protein in the mouse cortices by raising levels of ATPs in cortices. In mouse it also decreases the brain inflammation (Dong et al., 2005). An aqueous extract of this plant improves the learning behaviour and memory ability in mice (Hongxing et al., 2007). Portulaca oleracea is also helpful to improve the disorder of lipid (Xiao et al.,2005). Its highly polar extract shows that, in high doses, it reduces the activity of blood urea nitrogen (nephrotoxicity indicators) (Karimi et al., 2010). Hydroalcoholic extract of Portulaca oleracea can also be considered for the cure of hypercholesterolemia (Movahedian et al., 2007). An Ethanolic extract of this plant also shows hepatoprotective activity in rats (Elkhayat et al., 2008). The oral administration of the homogenates of Portulaca oleracea reduces the blood-sugar level of alloxan-diabetic rabbits to normal (Akhtar et al.,).

Another exiting application of Portulaca oleracea is its land remedial action as it is used for landfill leachates and removal of nerotoxic compounds like bisphenol A (industrial effluent contaminant) from the waste water (Imai et al., 2007).

39

1.5 Hepatitis

The term hepatitis generally refers to the condition of liver inflammation that may self- limiting to severe chronic form along with liver cirrhosis and carcinoma (Ryder et al., 2001). Different types of viruses responsible for different types of Hepatitis along with some other contributing factors like autoimmune diseases, alcohol, nonalcoholic fatty liver disease (diabetes, obesity, hyperlipidemia and metabolic disorders, etc.), certain drugs (acetaminophen, antibiotics and central nervous system drugs), organic solvents and some herbal medicines etc. (Ghabril et al., 2010). There are six categories of hepatitis depending upon the type of hepatic virus like hepatitis viruses A to E and G.

In addition to this, hepatitis type may be Ischemic and Giant cell hepatitis. Ischemic hepatitis appears due to any sort of liver injuries caused by insufficient supply of oxygen or blood. This condition is frequently associated with heart failure, sepsis or shock. As a result the level of Alanine transaminase (ALT) and aspartate transaminase (ASP) enzymes elevated (Masuoka et al., 2013). A rare type of Giant cell hepatitis is usually observed among newly born babies in which multinucleate giant cells present in liver. The actual cause of this hepatitis is not known, but it is said to be associated with autoimmune disorders, drug toxicity and viral infections (Raj et al., 2011).

1.6 Hepatitis C and its Virus

Hepatitis C virus (HCV), as a causative driving force of non-A, non-B hepatitis, was first time discovered in 1989 which widely spread throughout the world population. HCV contagion is a global health dilemma in both developed and developing countries. More than 190 million individuals are infected worldwide and more than 70 % of the personnel’s’ results in the development of the liver diseases which are chronic, more than 15 % develops hepatocellular carcinoma and about 20 % develop liver cirrhosis. The rate of chronicity of HCV in humans is rationally increasing from 50 % to 80 % and it depends upon the age when infection appears. It was observed in both the strains (heterologous and homologous) of animal model (chimpanzee) that in certain cases an immune response was seen in animals during HCV infectivity that cannot resist against the development of chronic infection (Farci et al., 1994).

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The prevalence frequency of HCV diversifies significantly throughout the globe; the rate is low at Western hemisphere while higher at Eastern hemisphere. Overall, highly HCV prevalent areas are Cameroon, Central African region, South Asia and Western African (Hamid et al., 2004). Egypt is the country where the occurrence of this infection was noticed up to 20 % of its total population. Even the highly developed areas like the United States of America and Europe have 1-2 % of the population that carries HCV genes. It becomes very hard to find the HCV new infections because of lack of diagnosis of this infection at acute level.

1.6.1 Transmission of Pathogen and Genotype Prevalence

HCV has a narrow range of tissue tropism and host specificity. In humans, HCV transmition occurs by direct contact to blood. There are various courses of HCV transmission for infection like injectable drug users, sexual transmission especially Gay, people with multiple partners, sex workers, and partners of HIV/HCV co- infected individuals in developed countries (Sulkowski et al., 2002). On other side, international standards are not applied in developing countries about blood transfusion, shaving from barbers, injecting drug users, reuse of syringes, reuse of needles for ear and nose piercing, tattooing, unsterilized dental and surgical instruments are the focal cause to transmit HCV. Sharing eating or drinking utensil, coughing, hugging, sneezing, usual social contact, water or food are not the causative ways of transmit HCV (Mast et al., 1998). On the other way transmission through mother to her baby is rare but currently documented (Alter et al., 2007).

On the bases of heterogeneity of genomic sequences, classification of HCV is categorized into eleven main genotypes (designated 1-11), about 100 different strains (numbered 1, 2, 3, etc.) and many subtypes (designated a, b, c, etc.) (Simmonds et al.,1999). The variability is distributed throughout the genome especially non-coding regions at either end of the genome (5'-UTR and 3'-UTR; UTR-untranslated region) (Simmonds et al., 1999). Genotype 1-3 is globally distributed. Most common of all are 1b and 1a which is approximately 60 % of all world infectivity and prevail abundantly in North America and Northern Europe, then in Eastern and Southern Europe and subsequently in Japan. Type 1 is more commonly represented than type 2. Genotype 3 is distributed diversely in various countries and widespread in South East Asia. Type 4 primarily found in central Africa, Egypt and Middle East. Genotype 5 is

41 discovered in vicinity of South Africa, and type 6-11are spread in Asia (Houghton et al., 1991; Mondelli et al., 1999; WHO, 1999). It is debate able point that weather the genotype of virus has any effect on pathogenicity of disease or not. There are other factors observed in patients and are characteristic of disease, such as immunological, genetic, and environmental that cause variation in development of disease (Mondelli et al., 1999). For the treatment of viral disease, it is significant to know about the genotype and its specific response against medication. Type 2 and 3 give more efficient responses, type 1 connected with less effective output solely for interferon. In advanced gold parameters of therapy - the combined form of ribavirin and pegylated interferon-α, all types give actively enhanced response.

1.6.2 HCV Structure and Genome

The structural details of HCV infectious viruses remained insufficient for a longer period of time because it was very hard to maintain the cultivation of HCV viruses in cell culture systems which are important for producing adequate numbers of virions for thorough exploration under electron microscope. Furthermore, virus particles derived from serum or plasma are connected with low density lipoprotein (LDL) of serum, due to this reason the isolation of viruses from centrifugation becomes so difficult (Thomssen et al., 1992). It has been observed that those HCV viruses that were extracted from the cell cultures are having spherical envelope comprising dimer or tetramer of two HCV envelope glycoproteins (E1 and E2) (Heller et al., 2005; Wakita et al., 2005; Yu et al., 2007). A spherical shaped structure has been observed on inner side of virions which represents core (nucleocapsid) that harbors core proteins and viral genome (Wakita et al., 2005).

The positive RNA genome contains 3’ non-translated region (NTR), a single open reading frame (ORF), internal ribosome entry site (IRES) and 5’NTR are significant for efficient RNA replication. The 5' untranslated region (UTR) of HCV having 341 nt is highly conserved part of genome because of stem loop and pseudo-knot containing four structural domains and is placed at upstream of open reading frame (ORF) translation starting codon (Brown et al., 1992). A pseudo knot and number of step loops are present in four advanced structural domains, named I –IV domains. Very first core coding part of 12-30 nt form the IRES along with II to IV domains (Honda et al., 1996).

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The HCV IRES form the smooth pre-initiation complex by the direct attachment of 40S subunit of the ribosome, without any involvement of initiation factors of canonical translation. Binding of the subunit is the crucial stage, which contributes to formation of HCV polyprotein translation (Forton et al., 2002; Lerat et al., 2002).

About 225 nt are present in 3'UTR and these are arranged in three regions as following, a highly conserved 3'-terminal stretch of 98 nt (3'X region) containing three stem-loop structures SL1 to SL3 and an elongated poly(U)-poly(U/UC) tract and a variable part of about 30-40 nt. (Kolykhalov et al., 1996). The 3'UTR in addition to the four stable stem-loop structures is located at the 3' terminal coding sequence of the NS5B and with the NS5B RNA dependant RNA polymerase (RdRp). While the 52 upstream nt of the poly (U/C) tract and the 3'X region were seen significant for RNA replication, although viral replication increased by remaining sequence of the 3'UTR (Friebe et al., 2002; Ito et al., 1996).

Proteolytically progression of polyprotein is done by enzymes; viral and cellular proteases, into ten particular proteins, comprising of four structural proteins such as NS5B, NS5A, NS4B, NS4A, NS3 and NS2 and remaining non structural (Lindenbach et al., 2005). Four viral enzymes encoded by these NS genes are as follows: NS3.4A serine protease, NS2.3 cysteine protease, NS5B RNA-dependent RNA polymerase and NS3 RNA helicase. These all four enzymes are vital for infectivity (HCV replication) in animal model (Kolykhalov et al., 2000). In general two enzymes considered as striking goals for developing an oral drug for HCV, these enzymes are NS5B RNA-dependent RNA polymerase and NS3.4A serine protease.

1.6.3 HCV Non-Structural and Structural Proteins 1.6.3.1 Structural proteins

1.6.3.1.1 Core protein

Core protein is a membrane bounded present in the cytosol and found to be coupled with mitochondria, lipid droplets endoplasmic reticulum and nucleus. As a result of these interactions of core protein, it is considered to be involved in various pathways like apoptosis, cell signalling, and transcription of genes and metabolism of lipid (Tellinghuisen et al., 2002). Core proteins have direct or indirect relation with hepatocellular carcinoma and hepatitis (Hope et al., 2002; Lerat et al., 2002).

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Nucleocapsid of HCV comprises conserved core protein, which is folded into three domains. All domains differ on the basis of hydrophobic interactions like domain one has two small hydrophobic regions of 117 amino acids. Domain two is comparatively less basic while 3rd domain is highly hydrophobic among all and also carrying signal sequences of E1 (envelope) protein (Bukh et al., 1994).

1.6.3.1.2 Envelope protein

Next to the core protein, highly glycosylated two envelope proteins E1 and E2 are present that have significant roles during cell entry. The conserved glycosylated site of envelope proteins is rich in mannose concentration. The weight of E1 is 35 kDa and has five N-linked glycpsylation sites. Its role is as fusogenic subunit (Bartosch et al., 2003). The molecular weight of E2 is 72 kDa and having eleven N-linked glycosylation points that directly interlink with cell surface receptors which support entrance the virus in the host cell (Nielsen et al., 2004). Moreover owing to its hyper variable regions of amino acids (HVR1 and HVR2) it is highly susceptible to mutations and thus acts as one of the contributing factors of HCV genotype variability. Conserved epitopes of B-cell and T-cell in E2 proteins of HCV will a potential target for developing anti-HCV vaccine in the future (Idrees et al., 2013).

1.6.3.1.3 P7 Protein

P7 is a transmembrane protein, having 63 amino acids, whose N-terminal domain is toward cytoplasmic region and C-terminal domin is suspended in Endoplasmic reticulum. It forms a bridge between the genes of envelope protein (E2) and nonstructural protein (NS2). Its role considered as signal sequence which stimulate NS2 translocation into the lumen of endoplasmic reticulumn. Furthermore, ion channels formed by this protein facilitate the virus infection (Griffin et al., 2003). Some characteristics of P7 are similar to viroporin. P7 role also considers significant during assembly and the liberation of virons according to specific genotype (Steinmann et al., 2007).

1.6.3.2 HCV non-structural proteins

1.6.3.2.1 NS2

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HCV non structural protein NS2 is a trasmembrane protein of 23kDa which is considered very important for the in vitro and in vivo completion of the HCV replication cycle (Pietschmann et al., 2006). N-terminus hydrophobic residues of NS3 forming 3-4 transmembrane domains enter into the membrane of the endoplasmic reticulum. The N- and C-terminus of NS2 (having two active sites and reside in the cytoplasm) plays an important role autoproteolytic action of NS2/NS3 (Grakoui et al., 1993; Lorenz et al., 2006). NS2-3 also called metalloprotease because Zinc metal stabilizes the structure of NS3 at its active site. Moreover Zinc stimulates the protease activity (Reed et al., 1995).

1.6.3.2.2 NS3

The NS3 is chymotrypsin-like serine protease that has the catalytic triad of amino acids (Ser-1165, Asp-1107 and His- 1083). It belongs to the trypsin/chymotrypsin protease superfamily. Replacement of Ser-1165 and His-1083 with another amino acid such as alanine stops the cleavage of NS3 of HCV polyprotein without interfering with NS3 structure (Bartenschlager et al., 1994; Grakoui et al., 1993b).

NS3 can cleave consensus sequence of decapeptide (DXXXXC-SXXX) due to its wide range of specificity for the substrate (Grakoui et al., 1993b). The substrate- binding pocket can keep six amino acid residues. Sometimes, when the substrate adds 10 amino acid residues, its cleavage proficiency becomes best which suggest that at the surface of NS3 the substrate interactions occur. At the N-terminus, the last 185 amino acids of HCV NS3 protease engaged in the cleavage involving NS3, 4A, 4B, 5A and 5B (Bartenschlager et al., 1994). The short consensus sequence of NS3 protease connected with the catalytic subunit of protein kinase A (PKA). This leads to PKA catalytic subunit retention in cytoplasm that prevents its entrance in nucleus. PKA alters the target protein function by adding phosphate groups, thus regulating intracellular signaling (Borowski et al., 1997).

NS4A is a 54 amino acid viral protein which binds to the protease domain as a cofactor (Satoh et al., 1995). NS4A has two roles. It supports NS3 to membranes and it stabilizes the structure of NS3, as a result protease is activated (Urbani et al., 1999). NS3 has a structural zinc ion that plays a significant role for domain of protease NS3 as well as auto-protease (NS2-3) (Love et al., 1996). This zinc ion is coordinated with

45

NS3 by three cysteine residues and one water molecule (Satoh et al., 1995). It is demonstrated through studies that only one fragment is needed for full protease activation. In vitro which is small, predominantly hydrophobic 13 amino acid long fragment of NS4A that binds directly to NS3 (Tomei et al., 1993). All of the evaluated studies have shown that the substrate specificity of NS3 protease has used steady-state kinetics and parameters such as kcat, Km, and kcat/Km to define this specificity.

It is important to note through studies by De Francesco and co-workers that some Pn- P1 product peptides show substantial inhibitory activity which can be enhanced by amino acid substitution. Thus, it is feasible that for the N-terminal product peptide, the NS3 protease affinity might amend the rate-limiting step in fixed circumstances. The base of catalytic/chemical mechanism of NS3 protease-catalyzed cleavage of peptide bonds is to maintain the catalytic triad of NS3, similar to another enzyme which is serine proteases. It was studied that the NS4A is present usually in complex form with 3-dimensional structure of NS3 serine protease (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). The presence Oxyanio hole and catalytic triads are the main requirements of protease activity. Oxyanion hole basically constitutes the backbone amides of Ser-139 along with Gly-137 while catalytic triad is a combination of Asp-81,Ser-139 and His- 57. In the proper location of this catalytic triad and the substrate, there exists major involvement of NS4A (Bartenschlager et al., 1993; Grakoui et al., 1993a; Tomei et al., 1993). His-57 is known to act as a general base to activate the Ser-139 nucleophile. When the carbonyl carbon of the scissile bond of Ser-139 exemplify nucleophilic attack, it escorts the development of tetrahedral transitional state having an oxyanion which becomes stable by this oxyanion hole of the enzyme at once when a substrate binds to it.

The NS3-NS4A protease is regarded as the most peculiar target for anti-HCV drugs. The HCV antiviral drugs posses two enzymatic components of the replicase that are the most popular targets for their development; the NS5B RdRp and NS3- NS4A serine protease.

These are attractive in part because both these enzymes are indispensable for HCV replication and used for the development of biochemical assays for inhibitor screening, which is an important consideration in the era particularly before cell-based 46 systems (Pawlotsky et al., 2006; Pawlotsky et al., 2004). In recent times, to provoke the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathways, the HCV NS- 3NS4A were shown in vitro that is essential arbitrator of interferon associated response during viral infection (Foy et al., 2003).

1.6.3.2.3 NS4A and NS4B

NS4A protein of HCV RNA comprises 54 amino acids and work as cofactor for the activity of NS3 due to its N-terminal hydrophobic residues (Wolk et al., 2000). It is assumed that last twenty amino acid residues of NS4A make a helix that provide an anchoring surface for binding of the complex of NS3/NS4A onto the membrane of endoplasmic reticulumn (Kim et al., 1996). The posphorylation activity of NS5A is also modulated by NS4A which is confirmed by deletion analysis of NS5A central amino acids (2135 to 2139) (Asabe et al., 1997).

NS4B is considered very important because recruitment of different proteins of HCV dependent upon it. NS4B interlinked with NS4A thus indirectly regulate its function (Lin et al., 1997). Molecular weight of NS4B is 27 kDa and it comprises four transmembrane helixes of hydrophobic amino acids (Lundin et al., 2006). Being an essential membrane protein it is found to be restricted along with remaining non structural proteins inside the endoplasmic reticulum (Hugle et al., 2001). Inside endoplasmic reticulum a membranous plexus is formed because of the action of NS4B. All the viral proteins were gathered here and form the replication complex (Egger et al., 2002).

1.6.3.2.4 NS5A and NS5B

NS5A always remain a potential target for anti-HCV therapies due to its significant role in replication, apoptosis, modulation of different pathways of cell signaling and enhancement of interferon response. Cell signaling pathways like ROS (Reactive oxygen species), MAPK (mitogen-activated protein kinase) and PIK(phosphatidylinositol 3-kinase signaling) pathways lead to hepatocellular carcinoma and modulation of hepatocytes are regulated directly and indirectly by NS5A (Macdonald et al., 2004). NS5A have interferon-α sensitivity-determining region (ISDR) which interacts with protein kinase receptor i.e. gene product stimulated by interferon- α (Enomoto et al., 1995). This region causes resistance

47 during treatment with interferon (Gale et al., 1997). Being a hydrophilic phospho- protein it does not span across the membrane like other non structural proteins of RNA. A distinctive amphipathic α-helix present at the N-teminal of NS5A helps its association with membrane and presence in replication complex (Penin et al., 2004). Any sort of mutation in α-helix region totally disrupts the replication of HCV. Such sort of mutations is essential for creating replicon cell line (Elazar et al., 2003; Lohmann et al., 1999).

An important RNA dependent RNA polymerse (RdRp) i.e. NS5B involves the production of new strand of RNA. It molecular weight is 65 KDa. It is prone to error due to absence of proof reading ability and thus contributes in genetic diversity of HCV. NS5B hydrophobic profile indicates that at its c-terminus sequence of 21 amino acids present that involve in membrane penetration (Yamashita et al., 1998). Structural confirmation decipher that this polymerase having the shape like ‘right hand’ in which active cleft of palm completely encircled by two sub domains appear with shape like thumb and finger (Lesburg et al., 1999). Its aspartic acid residues combine with metallic divalent ion in order to facilitate the attachment of primer and polymerization of priming nucleotide along with the release of pyrophosphate product. It is also proficient for RNA ‘de novo’ synthesis in the absence of primer. Due to the central role of NS5B it appears as notable target along with NS3 Serine protease for antiviral therapies of this millennium (De Francesco et al., 2005; Moradpour et al., 2005).

1.6.4 HCV RNA Replication and Potential Drug Target

The high level of diversity is a result of both the error prone RNA dependent RNA polymerase (RdRp), which is lacking a proofreading functional, and higher replication rate of HCV, together leading to a very high mutation rate (Lindenbach et al., 2005). This remarkable degree of variability manifests itself in that within an infected host HCV exists not as a single genetic homogenous virus, but rather a population of closely related but yet distinct virions, referred to as the “quasispecies swarm” (Pawlotsky et al., 2006). Within the swarm exist many preformed variants that often resistant to DAAs and these emerge when the drugs are present. Resistance to DAAs will likely be the major challenge of HCV therapy in years to come.

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Besides the unspecific peginterferon and RBV that make up the current standard of care (SOC) and the DAAs that target viral enzymes and are thus prone to resistance development, there is a third group of therapeutics in various stages of development: these compounds target host factors that the virus needs to replicate and have hence been designated host-targeting agents (HTAs). The development of these compounds is based on our increasing understanding of the molecular biology of HCV and its interaction with the host cell. Theoretically, targeting host cell factors instead of viral gene products should combine a specific anti-viral action with a higher barrier to resistance and broader genotype specificity. This is because, different from viral targets, host cell factors are encoded in the host genome and hence not subject to the high genetic variability of the viral genome. While this may be a major advantage of HTAs compared to DAAs, it may come at the price of an increased potential for toxic side effects.

Fig. 1.5: Detailed life cycle of hepatitis C virus

1.6.5 In vitro Infection Systems for HCV Replication

In previous decades the development of targeted drug against HCV always remains a tedious task due to crave of highly efficient in vitro cell culture system for viral infection. Moreover, complex organization of liver shortage of HCV in vivo models and limited tropism of HCV may also facilitate the discovery in vitro infection system. In vitro culture system is a fundamental necessity for particulars of HCV lifecycle like viron robustic entry, replication, and then budding viron assembly and

49 their release and afterward development of therapeutic accordingly (Wilson & Stamataki, 2012). However in present decade notable efforts have been made in this regard e.g. HCV Replicons, HCV Pseudoparticles, Cell-Culture-Derived HCV (HCVcc), Primary Cell Culture and Immortalized Primary Hepatocytes, Hepatoma Cell Lines (Hep3B cells, PLC/PRF/5 cell, Huh-6 cells, Huh-7 clones : Huh-7, Huh- 7.5, & Huh-7.5.1) and liver slices.

1.6.5.1 Sub-genomic replicons system of HCV

Sub-genomic replicons (total RNA or genome elements of structural & non structural proteins) are high level replicas of HCV genomes that were studied in a Huh-7 cell system (Blight et al., 2000). Replicon constructs of HCV having the ability to replicate independently in various hepatoma cell lines and used for identification of adaptive mutation and resistant cell types. All types having a gene for neomycin phosphotransferase used for selection.

Bi-cistronic replicon having Encephalo myo crditis virus (EMCV) internal ribosome entry site (IRES) prior to the onset of genes of non-structural protein. T7 promoter position is next to all these above mentioned genes in RNA transfected in Huh-7 cell lines. In the presence of G418 antibiotic, RNA replicates rapidly to form colonies (Fig. 1.6). Interferon resistance originated by adaptive mutations that boost up the rate of HCV RNA replication (Bartenschlager, 2002). Transit assay replicon system is used for identification of these mutations in which gene of luciferase or neomycin phosphotransferase replaced the gene of phosphoyransferase. Such two mutations are noticed in NS3 and one mutation in NS5A (Blight et al., 2000; Lohmann et al., 2003). In short, viral and host signaling require for replication can be studied by this in vitro system but this system fail to answer that how virus enter into host cell and its assembly process.

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HCV IRES EMCV IRES

T7 NEOR 3 4A 4B 5A 5B

In vitro Transcription Removal of DNA

NEOR 3 4A 4B 5A 5B

Transfection Huh-7 Hepatoma

No RNA No Replication Replication G418 Selection

Cell Death Colony Cell Death Fig. 1.6: The HCV Thesubgenomic HCV repliconSubgenomic system Replicon System

1.6.5.2 Hepatitis C Virus pseudoparticals (HCVpp)

This model system helps to study initial phases of viral life cycle like attachment to host cell membrane and then internalization. HCVpp are produced as a result of transfection of 293T cells (embryonic cells of human kidney) by three vectors. The 1st vector encode genes of Pol and Gag proteins and liable for budding of particles at plasma membrane and encapsulation of RNA while 2 nd vector encode genes of luciferase reporter protein. The 3rd vector encodes E1 and E2 envelope glycoproteins that are essential for HCV entry into host cell membrane. These tranfected kidney cells secrete pseudoparticles that can be utilized to infect Huh-7 cells. Level of infectivity can be checked by quantification of luciferase or expression of GFP in hepatoma cells. Furthermore HCVpp also use to recognize Claudin-1 and Occludin (HCV coreceptors) and neutralize the monoclonal antibody action against heteroogus HCV glycoprotein (E1, E2) and sera of HCV infected person (Cai et al., 2005; Hsu et al., 2003). HCV pseudoparticles are also essential to find out fusion mechanism of virus and also utilized to identify the inhibitors that can block/hamper entry of HCV (Fig. 1.7).

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Fig. 1.7: Scheme of HCV Pseudo-particle production and infection.

HEK 293T cells are transfected with CMV-Gag-Pol, Luciferase vector and HCV GP expression constructs. Successfully transfected 293T cells assemble HCVpp intracellularly and secrete them into the supernatant. The HCVpp-containing supernatant can be harvested. The supernatant can then either be used unmodified or conditioned (centrifugation, drug treatement etc.) to infect target cells. HCVpp attach to the target cells, become endocytosed and fuse with the endocytic membranes to release the retroviral core containing the luciferase vector into the cytoplasm. The Luciferase vector is then reverse transcribed and integrated into the host-cell genome. 24 to 96 h after infection, transgene expression can be analyzed.

1.6.5.3 Cell culture derived HCV (HCVcc)

Cell culture based HCV system appears as the insurgency in the HCV research field that not only verifies the findings of HCVpp like HCV co-receptors identification but also enhance the HCV full lifecycle niceties. Strain of HCV replicates and liberates infectious particles in cell culture called (HCVcc). This strain was cloned with HCV (genotype 2a) obtained from Japanese patient having an acute phase infection. It was

52 also appear infectious within in vivo animal models (chimpanzee and mice) that were transplanted with human hepatocyte.

1.6.5.4 Immortalized Primary Hepatocytes and Primary Cell Culture

Prime hepatocyte cell cultures obtained from chronically infected chimpanzees or humans by HCV were exploited for HCV infectivity studies. But such primary culture are considered best option because of its diverse viral population, reduced level of HCV RNA replication, presence of HCV specific Abs in patient serum and poor reproducibility of data.

1.6.5.5 Hepatoma Cell Lines

These hepatic cell clone (Huh-7 clones) are considered as best ideal in vitro system to date that having high susceptibility for viral infection. The discovery of Huh-7 clones (Huh-7, Huh-7.5, and Huh-7.5.1) opened a new horizon in research on HCV. Proficient replication viral genome in Huh-7.5 cells may fairly credited to a major defected retinoic-acid-inducible gene-I (RIG-I) pathway of antiviral immune response. Human hepatocellular carcinoma derived Huh-7 clones are poorly differentiated; exhibit atypical proliferation, deviant gene regulation, and tainted signaling pathways initially raised the questions about their physiological implication to the in vivo setting.

1.6.6 Available Treatment Options against HCV

Interferon boosts up the action innate immune response because it acts like cytokine that after binding of receptor against HCV regulate tyrosine kinase mediated phosphorylation cascade. As a result transcriptional factor in nucleus persuade the synthesis of various antiviral proteins. There are chances that autoimmune diseases (autoimmune hepatitis or diabetes, etc.) may also be provoked due to immune modulator action of interferon.

First successful chemotherapeutic against HCV is a guanosine analogue called ribavirn. This broad spectrum drug reduced the infectivity of HCV not only by acting against RNA dependent RNA polymerase but also ensuing mutations in HCV genome. It also acts in synergism by enhancing the immune modulation process of Interferon by affecting the signalling pathways.

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During HCV infection oxidative stress, increased that minimizes the antiviral effect of interferon and ribavirin by jamming the JNK/STAT signalling pathways. But the limitations that are associated with the protracted use of ribavirin are its side effects like haemolytic anaemia, insomnia and low grade fever, etc. Other additional contributing factors are immense viral titer baseline and fibrosis stage, age, alcohol intake, gender, African and American race, baby boomers, and changed immune response of the host due to raised levels of interleukin-8 (IL-8) and interleukin-10 (IL- 10) in serum. Moreover vaccine development against still facing hindrance owing to the capacity to HCV genome for undergoes sequence variation during evasion strategies.

Further progress is looming on the horizon. Knowledge of the molecular structure of the hepatitis C proteins has allowed the design of new drugs targeting the sites of HCV-encoded enzymes that are important for the replication of the virus.

1.6.7 NS3 Gene as Candidate Drug Target

The NS3 is chymotrypsin-like serine protease (Hahm et al., 1995) of 69 kDa that has the catalytic triad of His- 1083, Asp-1107 and Ser-1165. It belongs to the trypsin/chymotrypsin protease superfamily. When His-1083 and Ser-1165 are replaced with alanine, it stopped NS3 cleavage of the HCV polyprotein without affecting the NS3 protein structure (Bartenschlager et al., 1994; Grakoui et al., 1993). NS3 can cleave consensus sequence of decapeptide (DXXXXC-SXXX) due to its wide range of specificity for the substrate (Grakoui et al., 1993a). The substrate- binding pocket can keep six amino acid residues. Sometimes, when the substrate adds 10 amino acid residues, its cleavage proficiency becomes best which suggest that at the surface of NS3 the substrate interactions occur. At the N-terminus, the last 185 amino acids of HCV NS3 protease engaged in the cleavage involving NS3, 4A, 4B, 5A and 5B (Bartenschlager et al., 1993). The short consensus sequence of NS3 protease connected with the catalytic subunit of protein kinase A (PKA). This results in the retention of the catalytic subunit of PKA in the cytoplasm that prevents it entering the nucleus. PKA alters the target protein function by adding phosphate groups, thus regulating intracellular signaling (Borowski et al., 1997).

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NS4A is a 54 amino acid viral protein which binds to the protease domain as a cofactor (Satoh et al., 1995). NS4A has two roles. It supports NS3 to membranes and it stabilizes the structure of NS3, as a result protease is activated (Tanji et al., 1995; Urbani et al., 1999). NS3 has a structural zinc ion that is of significant for the NS3 protease domain and NS2-3 auto-protease (Love et al., 1996). This zinc ion is coordinated with NS3 by three cysteine residues and one water molecule (Satoh et al., 1995). It is demonstrated through studies that only one fragment is required for full activation of the protease in vitro which is small, predominantly hydrophobic 13 amino acid long fragment of NS4A that binds directly to NS3. It is important to note that some Pn-P1 product peptides show substantial inhibitory activity which can be enhanced by amino acid substitution. Thus, it is feasible that for the N-terminal product peptide, the NS3 protease affinity might amend the rate-limiting step in fixed circumstances. The catalytic/chemical mechanism of NS3 protease-catalyzed cleavage of peptide bonds is based on the conservation of the catalytic triad which likely similar to that observed for other serine proteases (De Francesco et al., 2003).

The 3-dimensional structure of the NS3 serine protease domain complexed with NS4A has been determined (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). Protease activity requires an oxyanion hole (backbone amides of Gly-137 and Ser- 139) and a catalytic triad (Ser-139, His-57 and Asp-81). In the proper location of this catalytic triad and the substrate, there exists major involvement of NS4A (Bartenschlager et al., 1993; Grakoui et al., 1993b; Tomei et al., 1993). His-57 is known to act as a general base to activate the Ser-139 nucleophile. When the carbonyl carbon of the scissile bond of Ser-139 exemplify nucleophilic attack, it escorts the development of tetrahedral transitional state having an oxyanion which becomes stable by this oxyanion hole of the enzyme at once when a substrate binds to it (Pause et al., 2003).

The NS3-NS4A protease is one of the most popular viral targets for anti-HCV therapeutics. The HCV antiviral drugs have been two enzymatic components of the replicase that are the most popular targets for their development, the NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase. These are attractive in part because both these enzymes are required for HCV replication and for the development of biochemical assays for inhibitor screening which is an important consideration in

55 the era particularly before cell-based systems (Pawlotsky, 2006; Pawlotsky & McHutchison, 2004). In recent times, to provoke the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathways, the HCV NS-3NS4A were shown in vitro that is essential mediator of interferon induction in response to a viral infection (Foy et al., 2003). Furthermore, NS3-NS4A comes into view to avert dsRNA signaling through the toll-like receptor 3 upstream of IRF-3 (Li et al., 2005).

1.6.7.1 NS3 Serine protease as potential HCV inhibitors

Finally, two 1st generation direct acting antiviral (DAA) protease inhibitors (boceprevir and telaprevir) with their marvelous effect in chronically HCV affected patent comes in market twenty year following the discovery of virus responsible for Hepatitis C.

In 2011 FDA approved two peptidomimetic ketoamide protease inhibitors, Telaprevir (VX-950) by Vertex Pharmaceuticals and Tibotec and Boceprevir, (SCH 503034) by Schering-Plough, as orally administered DAA drugs that selectively inhibit NS3/4A protease of HCV (Venkatraman et al., 2006). This peptidomimetic linear inhibitor along with gold standard care of therapy exhibits marvelous viral cure results in African-American Hepatitis C patients (62 %) in term of ideal effectiveness and tolerability. These regime results of these small molecule inhibitors along with interferon are >70 % in patients affected with genotype II and III and 40-70 % in remaining other genotypes of HCV. But certain confines like daily three time dose along with food, side effects like anemia, rashes on skin etc. interaction with other drugs, genetic barrier aligned with resistance etc. These precincts signify the need of 2nd generation of inhibitors that improvement chances in pharmacokinetics along with minor side effect.

Second wave of protease inhibitors include Simeprevir (TMC435); BI201335 of Boehringer Ingelheim Pharmaceuticals, Ingelheim, Germany; Danoprevir/r (RG7277) of Intermune Pharmaceuticals, Brisband, CA; Asunaprevir (BMS-650032; Bristol- Myers Squibb, New York, NY; ABT-450/r of Abbott, Abbott Park, IL; Enanta Pharmaceuticals, Watertown, MA; Sovaprevir (ACH-1625; Achillion Pharmaceuticals, New Haven, CT; MK-5172 (Merck & Co., Inc, Whitehouse Station, NJ) and ACH-2684 of Achillion Pharmaceuticals etc. These inhibitors are at the 2 nd or

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3rd phase of clinical trial and offering potent effect with better dosage options along with bearable side effects.

1.6.8 Ayruvadic Extract and Plant Derived Compounds as anti HCV Drug

Ethnopharmacological data shows that large number of natural products of plant origin has been used throughout the history for infections caused by viruses like Hepatitis, Herpes simplex virus, Human immune deficiency virus and influenza virus. High Throughput screening of the products show the way to the discovery of potential lead compounds as inhibitors of viral growth during in vitro assays.

In the current decade, numerous traditional plants were analysed scientifically in order to confirm their utilization in anti HCV therapy. For example Herbal extract from Acacia nilotica and Phyllanthus amarus abrogate the replication of HCV RNA (Ravikumar et al., 2011; Rehman et al., 2011). Similarly, some other plant extracts like Acacia confusa and San-Huang-Xie-Xin-Tang exhibited anti-HCV potential via suppressing the expression of cyclooxygenase 2 (Lee et al., 2000). Glycyrrhizin obtained from roots extract of licorice showed potent effect in chronically suffered hepatic patients by plummeting hepatic cancer (Ashfaq et al., 2011).

Different plant secondary metabolites like flavonoids, lignin and polyphenols were found to have potential to abrogate the growth of HCV via in vitro and in vivo analysis. Different flavonoids like Apigenin, Luteolin, Ladanein-BJ486K, Silibinin, Naringenin, Quercetin, (−)-Epigallocatechin-3-gallate (EGCG) Honokiol etc. and lignin compounds like Honokiol and 3-Hydroxy Caruilignan C were reported to have the potential against HCV.

Apigenin and Luteolin appear as inhibitors of NS5B polymerase in cell culture assay as well as in notional depiction of its molecular attributes used for biological molecule and ligand interaction. Another flavnoid molecule, Ladanein, that was extracted from the plant Marrubium peregrinum L. was reported to have anti HCV activity when used in combination therapy with cyclosporin A. But the mechanism how it prevent the iron from entry and how it inhibit its replication is not still clear (Haid et al., 2012).

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Silibinin of Silybum marianum seed appear as inhibitor of HCV at different stages of virus life cycle in cell culture like NS3 serine protease and RNA-dependent RNA polymerase (NS5B) (Ashfaq et al., 2011). Another significant inhibition response was noticed at place of entry with HCVpp and fusion assay of liposome (Wagoner et al., 2010). Silibinin Low bioavailability problem was overcome by the injectable formulation of succinate-conjugated silibinin that exhibited considerable inhibition along with reduced viral titer (Rutter et al., 2011). Thus the intravenous formulation of silymarin instead of oral is thought to be very effective (Payer et al., 2010).

The use of Naringenin in nutraceutical is already famous due to its both in vitro and in vivo attributes like anti-carcinogenic, anti-inflammatory and anti-oxidant. This flavonoid predominantly present in Grapefruit. As assembly and secretion of HCVs have some association with metabolism of lipoprotein. An obvious Naringenin dose dependent inhibition of core protein, HCV + RNA strand and ApoB was noticed in Huh-7 cells (Nahmias et al., 2008).

Quercetin is derived from fruits, grains, and vegetable and having direct link with HCV virulence instead of sub-genomic direct inhibition. It was confirmed by co- immuno precipitation assay and colacalization assay that interact directly with heat shock proteins-40 (HSP-40) and heat shock proteins-70 (HSP-70). Another significant effect of Quercetin (50 μM) was noticed in cell culture-based bicistronic reporter method is the reduced action of Internal ribosomes entry sites (IRES) of HCV both in presence and absence of NS5A (Bachmetov et al., 2012; Gonzalez et al., 2009).

EGCG, a catechin molecule, of green tea come as another inhibitor of HCV entry into host cell. Pseudo particles of HCV were utilized to confirm the EGCG effect on HCV entry. It inhibits the entry of HCV polyprotein regardless to its genotype in human primary hepatocytes and cell line derived from liver. Another potential of EGCG is its ability to inhibit cell-to-cell spread, a most important route of transmission of HCV in the infected liver. For efficient results of complete viral eradication EGCG was used along with direct acting drugs like cyclosporin A or boceprevir.

Liginin compounds like honokiol extracted from Magnolia officinalis inhibit the viral entry and NS5A, NS5B and NS3 serine protease during sub genomic replication in Huh-7 cells culture system along with HCVpp, HCVcc, and replicons of subgenom of

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HCV 1a and 2a genotype. Similarly 3-hydroxy caruilignan C compound of Swietenia macrophylla was used for anti-HCV therapy owing to its ability to decrease the level of HCV RNA. It was also having synergistic effect and utilized in combination therapy along with IFN α and Telapvir (Calland et al., 2012).

Polyphenol inhibitors of HCV like Excoecariphenol D, Corilagin separated Excoecaria agallocha L extract and 3-hydroxy caruilignan C extracted from Swietenia macrophylla inhibited NS3 serine protease with IC50 (in vitro) value 13.5 μM , 37.5 μM and 12.6 μM respectively (Wu et al., 2012).

Natural medicinal flora have been explored for their antiviral potential against numerous viruses specifically hepatitis B and C viruses, human immunodeficiency virus, herpes simplex virus and influenza virus because it appear potent in vitro inhibitor of viral growth in the cell culture. But there are still flurry plants that are in queue to assess and exploited for therapeutic role against functionally and genetically diverse and worse families of virus. Nevertheless, these plants can be used to reduce Hepatitis C virus titer in mammalian cells.

Keeping in view the worth of medicinal flora the present study was designed to screen the widely growing selected varieties plants against HCV. There are different parts of this study. Firstly, for this purpose plants was collected from the Soon sexier valley of Punjab, Pakistan on the basis of available ethnopharmacological data. Alcoholic extracts of selected plants were first utilized to check their in vitro toxicological effects in hepatoma cell line by cell counting via hamocytometer and trypan blue exclusion methods. Then HCV (3a genotype) infected liver cells were employed for in vitro antiviral screening of these phytoextracts at their non toxic dose and viral titer was counted with quantitative RT-PCR. After this dose response of promising extracts were studied in order to find out 50% inhibitory concentration (IC50) and their synergistic effect if any with interferon alpha 2α. After this results were also verified against subgenomic non structural protein (NS3). In second half potential extracts were examined thoroughly for their chemical constituents by qualitative and quantitative analysis. Then different fractions of these potential extracts on the basis of polarity were obtained through bioassay guided extraction and theses fractions were again screen against HCV whole genome and specifically its gene NS3.

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Thirdly active fractions of extracts were subjected to column chromatography in order to extract the compounds present in them. Presence of sigle and pure compound in eluted fractions was confirmed by thin layer chromatography (TLC). Copounds were identified by the spectral data obtained from UV-Vis spectroscopy, FTIR, Electrospray ionization mass spectrometry (ESI-MS), one and two dimensional Nuclear magnetic resonance (NMR).

Findings of present study depicts that two out of one thirteen plants designated as CTF and POL exhibit anti-HCV activity in a dose-dependent fashion at non toxic concentration and had synergistic effect when combined with interferon alpha 2a. Moreover the domino effect corroborate with data, using sera from HCV patients and HCV sub-genomic replicon. Furthermore, after bioassay guided extraction and identification of phytochemicals the compounds were isolated from active extract fractions of both plants by column chromatography. Purified fractions were tested again for activity against HCV in our in-vitro assay.

1.7 Goals/Objectives

The purpose of this study was to explore new therapeutic agents against target Hepatitis C virus RNA from natural flora in order to elucidate the control of pathogenesis of HCV infection. Overall objectives of this project are following:

 Main goal of project is in vitro antiviral screening of undocumented Soon- Valley medicinal plants against HCV whole genome and its NS-3 serine protease.

 Toxicological analysis of all the selected plant extracts in hepatoma cell line.

 Investigation of dose response and synergistic studies along with IFNα against HCV RNA.

 Bioassay guided extraction followed by the purification and characterization of biologically active constituents from selected flora that inhibits HCV RNA in vitro.

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 Preliminary analysis (qualitative and quantitative) of phytoconstituents in the selected extracts.

 Antioxidant scavenging potential of extracts through DPPH free radical scavenging assay, ABTS radical cation scavenging assay, Ferric reducing antioxidant power assay and quantification of phenolic acids by HPLC.

 Other pharmacological analysis like antithrombitic and antibacterial potential of the extracts.

 Extraction of compounds from active fractions of plant extract through column chromatography.

 Identification of compounds on the basis of spectral analysis obtained by UV- Vis, FTIR, and NMR analysis.

2.0 Chemicals, Equipment and Techniques

2.1 Chemicals

The majority of chemicals used were obtained from commercial sources and used as received without more purification. Analytical grade solvents petroleum ether (60-80

°C), n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc), methanol (MeOH), acetone and dimethylsulfoxide (DMSO) were used for extraction and chromatography procedures. For NMR experiments, deuterated solvents were used such as

Chloroform-d1 (CHCl3-d1) and methanol-d4 (CH3OH-d4) were purchased from Armar Chemicals (Buchs, Switzerland).

2.1.1 Materials for Chromatography

a. Thin layer chromatography (TLC): TLC analyses were carried out on pre- coated Aluminium-backed TLC plates with 0.2 mm thickness (Merck,

Germany). Different solvent systems were selected for this purpose like C6H6:

EtOAc (90:10, 80:20 and 70:30), C6H6: EtOAc: MeOH (70:20:10), Chloroform : EtOAc (50:50), EtOAc : MeOH (50:50), EtOAc : MeOH

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(30:70), CHCl3 : MeOH (95: 5, 90: 10 and 85: 15), EtOAc: MeOH: H2O (80: 10:10) etc.

b. Column chromatography: Silica gel 60 F245 particle size 0.04-0.63 mm 230 - 400 mesh, (Fluka, Buchs, Switzerland) and activated by heating at 105 °C for one hour prior to utilize for column chromatography. c. Reagents for identification Anisaldehyde-sulfuric acid spray reagent: p-Anisaldehyde (0.5 mL) was mixed with 10 mL of glacial acetic acid, followed by the addition of methanol (85.0 mL) and of concentrated sulfuric acid (5.0 mL). The reagent was sprayed on TLC plates, which were then heated in an oven at 105 °C until color development was completed. Phosphomolybedic acid reagent: Phosphomolybedic acid (10 g) was dissolved in ethanol (100.0 mL). This solution was sprayed on TLC plates, which were then dried and heated till the color appeared. Vanillin reagent: Vanillin (1.00 g) was dissolved in concentrated sulfuric acid (100 mL). Reagent was sprayed on TLC plates, dried and then heated in an oven at 105 °C until color development was completed.

Ceric sulfate spray reagent Ce(SO4)2: Ceric sulfate (1.0 g) was dissolved in concentrated sulfuric acid (10 mL ). The mixture was added to distilled water to a final volume of 250 mL. The reagent was sprayed on TLC plates, which were then heated in an oven at 105 °C until colour development was completed. Ammonia reagent: Ammonium chloride (1g) was dissolved in distilled water (100 mL). TLC plates sustaining tests spot were exposed to the vapors of ammonia appearance of yellow colour spot indicates the presence of the flavonoid.

2.1.2 Chemicals and Reagents used for Determination of Antioxidant

All sort of the chemicals and reagents utilized in this project were of analytical grade and procured from Sigma-Aldrich or E. Merck or else specified. Acetone, Chloroform, Ethanol, Glacial Acetic acid, Hexane, Hydrochloric acid, Iso-octane, Methanol, Sodium carbonate, Folin-Ciocalteu reagent, 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid)(ABTS), Aluminium chloride, Ascorbic acid, Butylated hydroxytoluene (BHT), Catechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 62

Disodium hydrogen phosphate, Ferric chloride, Gallic acid, Manganese dioxide, Phenolphthalein, Potassium iodide, Potassium ferricyanide, Sodium dihydrogen phosphate, Sodium hydroxide, Sodium thiosulphate, Starch, Sodium nitrite, Trichloroacetic acid.

2.2 Instrument and Apparatus

Analytical balance (Sheimadzu Ay 220), Rotary evaporator, Oven (LabTech. Korea), Heating mantle, UV-visible spectrophotometer (Perkin-Elmer Lambda-2 Spectrophotometer), Orbital shaker. Soxhlet assembly, conical flasks, beakers, and all other glassware used in this research work were of A-grade Pyrex.

2.3 Materials used for Biological Activities

2.3.1 Cell Lines

Human hepatocyte derived cellular carcinoma (Huh-7) cell line cells and Chinese hamster ovary (CHO) cell lines were received as gift from Dr. Zafar Nawaz (Department of Biochemistry and Molecular Biology, University of Miami, USA) and Dr. Ahmed Usman Zafar (Biopharmaceuticals lab, CEMB, Pakistan) respectively. Huh-7 cells were cultured and reserved in Dulbecco's modified Eagle medium (DMEM) (Sigma, USA) having high concentration of glucose (4500 mg/mL). DMEM high glucose supplemented with fetal bovine serum (12 %) (Sigma, USA). Media was made highly selective by the use of antibiotics like streptomycin (100 μg/mL) along with penicillin (100 IU/mL) (Sigma, USA) prior to use for culturing of Huh-7 cell. Complete culture medium having inoculums’ of cells was incubated at 37 oC in an atmosphere of 5 % CO2 in humidified incubator. Similarly CHO cells were cultured in DMEM Hams F-12 supplemented with fetal bovine serum (10 %), penicillin (100 IU/mL) and streptomycin (100 μg/mL).

2.3.2 Plasmids

Nonstructural genes (NS2, NS3/4A) of HCV genotype 3a along with FLAGtag were cloned in mammalian expression vector pCR3.1. HCV NS3 gene of genotype 1a

63 along with FLAGtag was also cloned in pCR3.1 expression vector. These vectors were transformed into competent Top10 cells of E.coli and plasmids were purified by mini-prep and maxi-prep by using Qiagen DNA purification kit. These vectors (pCR3.1) were compassionately offered by Functional Genomics laboratory, CEMB.

2.3.3 Reagents & Chemicals

Solvent dimethyl sulfoxide (DMSO) of Sigma Aldrich (Cat # D2650, Sigma) and MTT reagent (Cat # M565) of PhytoTechnology Laboratories (USA) were used. Maxima SYBR Green/ROX qPCR Master Mix (2X) (Cat # K0221) and HCV Real- time PCR Quantitative kit were procured from Thermo Scientific Inc. and Sacace biotechnologies (Italy) respectively. Housekeeping gene of human tissues i.e. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (Cat # sc25778) was purchased from Santa Cruz Biotechnology.

2.3.4 Designing of Primers

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and HCV specific non- structural (NS3) gene sequences of genotype 1a and 3a were retrieved from NCBI. The NCBI sequence was transformed into the FASTA format and then primer 3 software, (http://gmdd.shgmo.org/primer3/?seqid=47) was used for designing of primers. The oligonucleotide primer sequences for selected genes are presented in table 2.1.

Table 2.1: Primers of GAPDH and HCV non-structural NS3 gene of 1a & 3a genotype was used in this study Sr. No. Primers Sequences 5’-3’ Length

1 GAPDH F ACCACAGTCCATGCCATCAC 20

2 GAPDH R TCCACCACCCTGTTGCTGTA 20

3 NS3-1a F GGACGACGATGACAAGGACT 20

4 NS3-1a R CCTCGTGACCAGGTAAAGGT 20

5 NS3-3a F GACCATTGTGACCAGCTTGA 20

6 NS3-3a R GCGGGTGACCAAGTACAAGT 20

2.4 Instrumentation & Techniques

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2.4.1 Physical Constant

Melting points were measured on Stuart Scientific SMP10 apparatus and are uncorrected. Optical rotation was calculated via JASCO-DIP360 digital polarimeter

2.4.2 Spectroscopy

Infrared spectra were obtained by using ALPHA-P FTIR spectrometer and Mass spectra (MS) were recorded using a Micromass Platform II spectrometer using an electrospray ionization source. Nuclear magnetic resonance (1H NMR, 13C, 13C- DEPT, COSY, HSQC, and HMBC NMR) spectra spectra were recorded at 300 MHz and 400 MHz NMR on a Bruker DPX 400 spectrometer, expressed in ppm (δ) relative to trimethylsilane (TMS) as an internal standard. Spectra were recorded after dissolving sample in CHCl3-d1 and CH3OH-d4 on the basis of their solubility. These facilities were availed from the Manchester Institute of Biotechnology and Department of Chemistry, University of Manchester, United Kingdom.

2.4.3 Thin Layer Chromatography

Thin layer chromatography (TLC) is an economical technique utilized for the separation, preliminary qualitative detection or partly a quantitative visual analysis of samples. The TLC chromatography technique gives a clue as to how many components are in a mixture or an extract. It is also used to determine the purity of the isolated compound. Commercially prepared TLC aluminum sheets of 20 x 20cm

Silica gel 60 F254 was used. The plate was cut to size of 5 x 5cm and the extract was spotted at the bottom the TLC plate (about 0.5cm from the base). The plate was then placed in a developing tank containing chosen solvent system. The spots were developed using DV light, anasaldehyde and iodine vapor.

2.4.4 Column Chromatography (CC)

The preparatory role of column chromatography helps to separate desired pure compound from crude mixture. The concentration of extracting the compound may vary from very low high concentration depending upon the column size and type of mobile phase. Cotton wool plug and small amount of sand was put at the column

65 bottom in order to provide a uniform base to stationary phase otherwise stationary phase gel will block the column.

The adsorbent which is the silica gel (7~230 mesh size) was weighed using a ratio of 30 gm of the adsorbent to 1.00 gm of the crude. The column was filled with a little quantity of hexane. Slurry of weighted adsorbent was prepared using 100% hexane and then carefully poured into the column. Air bubbles were prevented from forming. A solution of the extract was then pipetted on top of the stationary phase. This layer was topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the flow of the eluting solvents. The eluting solvent initially was 100 % hexane and the polarity was gradually increase at a 95:5 hexane:ethyl acetate ratio until a 50:50 ratio was used. About 25 mL of eluting mixture was collect until there appears to be no more solute in the column. The fractions collected were monitored by TLC and similar fractions were combined together. Solvent was then removed from the bulked fraction, allowed to dry and weighed. TLC analysis was carried out on the semi dry bulked fraction using various polar solvent to ensure the purity of the fraction.

2.5 Methodology

2.5.1 Biochemical Analysis of Plants

Pharmacological attributes of flora are owing to the potential of its bioactive constituents. In Pakistan, more than fifty percent of the population relies on plant directly and indirectly for the treatment of different disorders. By considering the ethno pharmacological usage, plant assessment for their biochemical constituency is highly crucial.

2.5.2 Collection, Identification and Preservation of Medicinal Plant Materials

Fifteen plants having medicinal value in folkary was collected from the vicinity of the Soon-Sakaser valley of Punjab, Pakistan in September to November 2010. Taxonomic identification was authenticated by Mr. Amin Ullah Shah, Department of biological science, University of the Sargodha, Sargodha. Voucher specimens were deposited also at Herbarium, University of the Sargodha, Sargodha. All sorts of information

66 concerning plant scientific names, family names, vernacular uses and parts used from fifteen medicinal plants are shown in Table 1. Freshly collected plant samples were rinsed with tap water and then with de-ionized water in order to make it free from dust and any metal contamination. Then roots and leaves were separated manually from the stem. All sample materials were chopped into smaller pieces with a sharp steel knife and dried under shade at room temperature till dryness. Air dried plant samples were pulverized with an electric grinder into fine, coarse powder, and then the dried, powdered samples were stored in polyethylene air sealed bags at 4°C for further in vitro and in vivo assessment.

Table 2.2: Botanicals details chosen for screening against total RNA HCV and its gene NS-3 serine protease. Sr. Botanical Common Family Local use Part(s) Plant’s No. Name Name used Code 1 Achyranthus Prickly Amaranthaceae Diuretic, purgative, Leaves AAL aspera Chaff hepato-protective, Flower laxative, anti-allergic 2 Arjuna Arjun Combretaceae cardiovascular Bark ATB Terminalia ailments, wounds healing, hemorrhages and ulcers 3 Bacopa Brahmi boti Plantagenaceae Ulcers, tumors, Leaves BMB monnieri ascities, leprosy, enlarged spleen, inflammations, anemia, biliousness 4 Caralluma Chunga Asclepadaceae Diabetes, Cancer, Ariel part CTS Tuberculta Rheumatoid arthritis i.e. stem 5 Caralluma Asclepadaceae Diabetes, Cancer, Roots CTR Tuberculta Rheumatoid arthritis 6 Chichorium Kasni Compositae Gallstone, jaundice, Leaves CIL intybus hypertension 7 Chlorophytum Safed Liliaceae Diabetes, sex tonic, Roots CBR borivilianum moosli pre and post natal problems, arthritis 8 Corchorus Bauphali, Tiliaceae. treachery troubles, Leaves CDL depressus Munderi antipyretic, diabetes (Linn.) laxative, gonorrhea,

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chronic cystitis 9 Myristica Nutmeg, Myristiceae Carminative, liver Leaves MFHL fragrans Jaiphal, diseases, respiratory Houtt. Javitri ailments, tuberculosis 10 Portulacea Purslane, Portulacaceae Anticancer, boils, Leaves POL oleracea L Hurfa haemorrhoids, dysentery 11 Santalum Sandal Santalaceae colds, Bark SAB album bronchitis, ailments of gallbladder and liver 12 Sesamum Sesame, Til Pedaliaceae Anticancer, laxative, Seed SIS indicum emollient 13 Sonchus Field milk Asteraceae Gallstone, Gout Leaves SAL Arvensis thistle, haemorrhoid, Dodhak hypertension, wound healings 14 Rheum emodi Raiwand Polygonaceae liver diseases, diuretic, Roots RER cheeni Purgative 15 Zizyphus Chinese Rhamnaceae Antiulcer, sedative, Seeds ZJS Jujuba date, Unab immunostimulant, anti-inflammatory

2.5.3. Plant Extract Preparation

All samples were weighed and immersed in methanol (70 %) separately for overnight at 38 °C which is the most suitable temperature for enzymatic activity. After 24 hours, they were filtered through Whatmann filter paper No.42 (125 mm) and cotton wool; the residue was soaked over again in fresh methanol (70 %). Process of successive extraction and filtrations was carried out three times. After final filtration, samples were concentrated in vacuum under reduced pressure using a rotary flask evaporator. Greenish brown gummy solid of crude methanolic extracts were obtained which is then dried in desiccator. The final percentage yield of each extract was calculated. All extracts were stored in pre weighed air tight glass vials for phytochemical analysis at 4 oC in refrigerator.

2.5.4 Screening of Plant Extracts for Cellular Toxicity Analysis via Trypan Blue Dye Exclusion Method

Hepatoma liver cell line (Huh-7) and standard CHO fibroblast cells were employed as in vitro models for numerical elucidation of cell viability under the effect of plant crude extracts. CHO cells were utilized as positive control for cytotoxic analysis of Phyto extracts of the body cells additional to liver cells. Cell counting was carried out to determine the 50 % cytotoxic concentration (CC50) of phyto extracts as illustrated earlier (Ashfaq et al., 2011).

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2.5.4. 1 Preparation of plant extract stock solution

About 50 mg of every plant organic extract was mixed meticulously in 1.00 mL of Dimethyl sulfoxide (DMSO) that confers 50 µg/µL stock concentrations. All stock solutions were sieved by means of 0.22 µm filter and stocked up at -20 ºC.

2.5.4.2 Culturing of Huh-7 and CHO Cell

The cell lines (Huh-7 and CHO) were cultured in Dulbecco's modified Eagle medium (DMEM) along with Fetal bovine serum (10 %), streptomycin (100 µg/mL) and penicillin (100 IU/mL), afterward nurtured at 37 ºC along with continuous supply of

CO2 (5 %).

2.5.4.3 Cell viability assay

The crude extracts of all selected plants were scrutinized for their toxicological assessment against Huh-7 and CHO cell lines by using Trypan blue dye. Liver and fibroblast cells were seeded in 24 well plates with density of 8×104/well. By considering the first three wells of culture plate as control, in the remaining wells different concentrations of the test samples (1.5 µg/mL to 200 µg/mL) were added in triplica. After 24 h incubation, cells were washed with phosphate buffer saline (1 % PBS) and then treated with the solution of trypsin EDTA. After trypsanization, fresh culturing media was added to each week in order to mimick the trypsin activity. Then suspension of these cells along with trypan blue dye was prepared with 1:1 ratio. 10 µL of the cell suspension was dispensed onto a glass slide and viable cells were calculated using haemocytometer.

2.5.4.4 Cell proliferation & toxicity assay

Cellular toxicity of plant extracts was also assessed by utilizing enzyme based methods, one of them known as MTT assay as described previously (Ashfaq et al., 2011). ‘MTT [3-{4, 5-dimethylthiazol-2-yl}-2, 5-diphenyltetrazolium bromide] assay is a well-known in vitro method to analyze mitochondrial dehydrogenase activities in live cells. In metabolically active cells, yellow tetrazoluim dye, MTT, is reduced to water insoluble purple formazon crystals by NADH. These insoluble farmazon crystals were dissolved in organic solvents such as DMSO or isopropanol before measuring absorbance. Absorbance in the visible region is proportional to the number

69 of metabolically active cells. Briefly, in order to valuate cytotoxicity, Huh-7 cells were plated in 96 wells plate at a density of 2×104 cells/well. After 24 h, cells were serially diluted in triplicate with different concentrations of plant extracts ranges from 0.8 µg/mL to 200 µg/mL. Control was considered in triplicate by adding DMSO instead of plant extracts. Plate was incubated for 20 h at 37 oC in an atmosphere of 5 % CO2. After 20 h of treatment, medium was replaced by fresh medium (100 μL) and 30 µL MTT solution (5.00 mg/mL in 1X PBS) was added in each well. Plate was wrapped with aluminum foil and kept for another 3-4 h of incubation at 37 oC in humidified atmosphere. After carefully removing medium, 100 µL of DMSO was added to solubilize formazon crystals in each well. Absorbance measurements of reduced MTT were taken by microtitre plate reader at wavelength of 570 nm and a reference wavelength of 620 nm. Proliferation was analyzed as the fraction of survived treated cells relative to untreated.

Cell viability was calculated by using following formula;

% Cell viability = Treated 570 nm – 620 nm X 100 Control 570 nm – 620 nm

2.6 Anti-HCV Analysis of Compounds in Liver Cells

Huh-7 cells (3×105 cells per well) were cultured in six well culture plate. Cells were swabbed two times with 1XPBS after 24 hrs. Each well was inoculated with 1×105 IU/ml of HCV-3a genotype infected serum. Specific dose of test compound (least amount of cell killing) was added in the wells. Total RNA extraction was carried out by using Gentra RNA extraction kit (Gentra System Pennsylvania, USA) according to the manufacturer’s directions of 24 h post infection. Afterward, cells were lysed with cell lysis solution having 5 µL internal control (Sacace Biotechnologies Caserta, Italy). RNA pallet was dissolved thoroughly in 1 % DEPC (Diethyl pyrocarbonate) treated water. Quantifications of HCV RNA were carried out by Real Time PCR Smart Cycler II system (Cepheid Sunnyvale, USA) using Sacace HCV quantitative analysis kit (Sacace Biotechnologies Caserta, Italy).

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2.6.1 Antiviral Analysis of Plant Extract against HVC Ns3 Protease via Transfection Assay

Huh-7 cells were cultured at the density of 3× 105 in 6-well plates for 24 hrs designed for transfection studies. . After the removal of media, cells were washed thoroughly with 1×PBS and transfected immediately with the expression vector pCR3.1/Flag/NS3-NS4A containing NS3 full length gene (Accession # HCV-PK- HQ202536) and NS4A (Accession # HCV-PK-HM135518) gene kindly provided by Applied and Functional Genomics lab, CEMB. The cells were co transfected with pCR3.1/Flag/NS3-NS4A in the presence and absence of 100 μg of plant extract and interferon along with Lipofectamine™ 2000 (Invitrogen life technologies, Carlsbad, CA) as per the company's standard procedure. In brief, in 300 μL of DMEM culturing media LipofectamineTM (9.00 μL for 6-wells) was added and then this media was incubated at 37 oC for 5.00 mins. Mammalian expression plasmid (pCR3.1/FLAGtag/HCV NS3) construct (800 ng/well) was added into DMEM (300 μL) separately and incubated at 37 oC for 5 mins. After incubation, both solutions were mixed thoroughly and again incubated for 25 mins at 37 oC. When culturing medium in six well plate attain 80 % confluence of cells, it was removed from each well of the plate and wells were rinsed with 1XPBS. Afterward, 900 uL of fresh media was poured into each well. After 25 mins, DMEM holding transfection complex (100 μL) was added carefully to all wells and plate was again incubated in humidified incubator at 37 oC for 24 h. For ideal transfection outcome, cells were collected usually after 24 h to 48 h for future gene transcription and expression studies.

2.7 Gene Expression studies via Reverse Transcriptase PCR (RT- PCR)

2.7.1 Extraction of Total RNA

After 24 h total RNA was extracted using Trizol reagent (Invitrogen life technologies, Carlsbad, CA) in accordance with the manufacturer's protocol. In brief, Cells in each well were washed thoroughly with 1X PBS, lysed by TRIzol® (1.00 mL) and then transferred to eppendorfs (1.5 mL). About 200 μL chloroform was added into each well having cell lysate. Samples were overturned carefully with hand for 5.00 mins 71 and centrifuged for 15 mins at 12,000 rpm. After the centrifugation, uppermost aqueous layer holding RNA was pipette out cautiously into an uncontaminated eppendorfs that already having absolute isopropanol (500 μL). Material was again inverted for 3 mins with hand and centrifuged for 10 mins at 12,000 rpm. A little noticeable lucent RNA pellet become visible and that was washed with ethanol (70 %) and dried up at room temperature. RNA pellet was immersed in 40 μL of Diethyl pyrocarbonate (DEPC) treated water. Purified RNA was stored for 30 mins at -20 oC. RNA quantification was carried out by Nanodrop spectrophotometer (Nanodrop ND- 1000, Optiplex, USA).

2.7.2 Synthesis of cDNA

The RNA template (1.00 μg) was utilized to synthesize the cDNA strand by 200 U/μL M-MLV Reverse Transcriptase (Invirtogen, Life technologies, Carlsbad, CA) according to company’s protocol via Reverse Transcription System. Shortly, 1.00 μL of oligodT primer, 1.00 μL of dNTPs and 1.00 μg of RNA were added into DEPC treated H2O in order to make 12 μL final volume. Reaction mixture was incubated for 5.00 mins at 65 oC and then for 1.00 min at -20 oC. About 4.00 μL of 5X first strand buffer, 2.00 μL of 0.1M DTT and 1.00 μL RNase free water were added into every ice-cold reaction mixture tube and then incubated this mixture at 37 oC for 2 mins. Afterward, 1.00 μL of M-MLV was added, furthermore incubated for 1.00 h at 37 oC and 70 oC for 15 mins subsequently. Finally sample solution was stored at -20 oC.

2.7.3 Reverse Transcription PCR (RT-PCR) for Gene Expression Analysis

Specific sequence forward and reverse primers for GAPDH (internal control) and HCV NS3 genes were designed to investigate the effect of plant extract on the expression of genes through RT-PCR (Applied Biosystems Inc. USA). Primers were used for the amplification of HCV NS3 and GAPDH genes and their sequence is already depicted in Table 2.1. Semi-quantitative and quantitative gene expression data is mostly normalized to expression level of internal control/housekeeping genes. Housekeeping genes (GAPDH) expression remains unaffected in cells during this study. GAPDH and β-actin are the most commonly used internal control/housekeeping genes in comparing gene expression data (Barber et al., 2005). PCR reaction was carried out by using 5.00 μL of 2X PCR master mix (Fermentas), 72

1.00 μL of cDNA template, 10 pmol of reverse and forward primers and water (DEPC treated) to make final 10 μL volume. Thermal cycling parameters were expressed in the fig no. 3.1. The reaction conditions of thermocycler for expression study of both GAPDH and HCV NS3 gene were same. Amplified products of PCR were analyzed by Agarose gel electrophoresis using agarose gel (2.00 %). The image of DNA products were captured in gel documentation apparatus under the luminescence of short wavelength ultra violet light.

Fig. 2.1: Cycling parameters for expression analysis of HCV NS3 gene in reverse transcription PCR

2.7.4 Expression studies via Real-time PCR

Reverse transcription tagged along by real-time PCR (RT-qPCR) is considered as the gold standard for the quantification of gene expression because of its remarkable sensitivity and sequence specificity characteristics. Relative Quantification was also conceded out by means of quantitative Real-time PCR on ABI 7500 Real time PCR machine (Applied Biosystems Inc. USA) using SYBR Green Chemistry (Fermentas, USA). Reported protocol of expression analysis was followed with little modifications (Jahan et al., 2011). Briefly in 7.5 µL of SYBR Green/ROX qPCR Master Mix (2X) (Fermantas), relevant forward and reverse primers (1.00 µL each) and RNase free water (5.00 µL) were mixed and reserved in ice. After mixing all regeants cDNA template (0.5 µL) was added in the end and final reaction mixture was added in the reaction wells of PCR plate. Final data was acquired in extension step. The assay was done in triplicate and comparative CT method (ΔΔ Ct value) was utilized for targeted gene relative quantification against GAPDH (internal control).

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Fig. 2.2: Thermal cycling conditions for Real-time PCR

2.8 Western Blotting of PO extracts

A specific protein in any given sample could be detected by using specific antibody for that protein & this analytical technique is known as western blotting. It involves the separation of denatured proteins by using SDS gel electrophoresis, transfer of those proteins on a nitrocellulose membrane & finally treating with specific monoclonal antibodies.

2.8.1 Protein Isolation

Cell culture was prepared in 6 well plate/ 60 mm culture dish to conduct transfection assay. A transfection test was executed by either including or excluding the plant extracts after 24 h of cell culturing. Then, after 48 h, cells were rinsed with 1XPBS (phosphate buffer saline). A cocktail of cell lytic buffer comprising of 10 µL (PMSF) along with 1.00 µL protease inhibitor (Sigma Aldrich, USA) mixed with 1.00 mL of cell lyses buffer was poured in each well. This cell lystae was kept at -20 0C for 30 min after transferring to the eppendorf, then centrifuged for 25 min at the rate of 13,000 rpm for 4 0C. The supernatant containing protein was collected & stored in new eppendorfs at -20 0C.

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2.8.2 Protein Quantification

Quantification of the isolated protein was done by spectrophotometer using Bradford reagent (Fermentas, ready-to-use, #R1271). This was done by mixing of 10 µL protein with 990 µL Bradford reagent & subjected to quantification at 595 nm.

2.8.3 Protein Samples Preparation

For protein sample preparation, the pre-calculated sample volume (containing 60 µg of total protein as per yield of protein extraction quantified by Bradford assay) was mixed with 5X protein loading buffer (Genei) plus 20X of reducing dye (Fermantas). After 3-4 min heating of reaction tubes in hot water, those tubes were kept on ice for subsequent 2-3 mins. Then, after a short spin, samples along with 10 µL of the protein marker (Fermentas) were loaded into protein gel.

2.8.4 SDS Gel Electrophoresis

After heat shock of samples, 60 µg protein along with the sample buffer (5X) & reducing agent (20X to remove unwanted structures) were loaded onto SDS-PAGE for analysis of HCV NS3 protein in presence or absence of plant extracts. The acrylamide gel apparatus, used for electrophoresis, was positioned vertical in holder assembly. Except 10 % APS & TEMED (used as polymerization catalyst), all other reagents were thoroughly mixed. Resolving gel was poured onto the 2/3 portion of apparatus followed by 70 % ethanol to avoid any bubble formation.

Table 2.3: Composition of the gels used in SDS-PAGE Solutions Resolving gel 10 % Stacking gel 5.00 % Acrylamide/bis-acrylamide (30 %) 3.00 mL 0.65 mL Tris buffer 0.1 M (pH 8.8) 0.5 M (pH 6.8) 10% SDS 0.15 mL 0.08mL 10% APS 0.15mL 0.08 mL dH2O 2.5 mL 2.75 mL TEMED 0.006 mL 0.008 mL

After the gel polymerization, ethanol was removed followed by washing with water. Water was wiped with a filter or tissue paper before inserting combs. After loading stacking gel, 1XSDS-PAGE tank buffer was poured into Western blot tank (Bio-Rad,

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Germany). Initially, gel was run at 60 V till samples passed from the stacking gel, then voltage was switched up to 80 V to allow complete separation of protein samples.

2.8.5 Transfer of Protein and Immunoblotting

To detect by antibody, proteins were made available at nitrocellulose membrane (Hybond C extra, Amersham). To acquire this goal, six sheets of blotting paper & one sheet of nitrocellulose was cut as per the gel dimension. After soaking these papers in transfer buffer, the gel was carefully placed over nitrocellulose sheet & was covered by the blotting papers. Then the whole set was placed in electro blot and run at 16 V for 1.5 h. By the use of 5 % skimmed milk (prepared in TBST at 4 0C overnight), the membrane was blocked. 1X TBST was used to wash the membrane & then membrane was incubated for 1h at room temperature with the monoclonal antibody(Primary) specific to HCV NS3 gene along with GAPDH (Santa cruz, Biotechnology). Again, 1X TBST was used for three times washing of membrane and each washing continued for 5.0 minutes. The membrane was incubated with anti-mouse antibody (Secondary) for 45 minutes. Finally, the membrane was washed for 3 more times by using 1X TBST.

2.8.6 Enhanced Chemiluminescence (ECL) Detection

After removal of excess buffer, the membrane was kept over cling film/flat surface. Both ECL solutions (chemiluminescence peroxidase substrate; Sigma, Cat # CPS- 3500) were mixed as 1:2 ratios & poured over the membrane for about 4-5 minutes. The membrane was wrapped with cling film after draining of excessive substrate. The membrane was exposed to X-ray film (Kodak) for 5.00 minutes on a cassette. Kodak developer & fixer solutions were used for the film development. Initially, the X-ray film was dipped in developer solution for about 30-40 seconds, then washing with distilled water, followed by dipping & hand shaking of the film in fixer solution for about 30 Sec.

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The exposed & developed X-ray film showed the protein bands. A comparative study of protein bands between sample (with plant extracts) and control (without plant extracts) indicated the inhibition. The protein expression levels were checked using chemiluminescence’s detection kit (Sigma Aldrich, USA). The protein was quantified from X-ray images using freely available software Gel-quant-express (http://gel- quant-express.software.informer.com/4.1/).

2.9 Analysis of the Two Plants Extract That Exhibit Positive Potential against HCV

All fifteen plant methanolic extract were screened for their activity against the expression vector (pCR3.1/FLAGtag/HCV NS2-NS3/4A) of genotype 3a under the uniform conditions explained above. Only two plants (plant codes CTS and POL) out of exhibiting positive potential during antiviral screening against HCV. So these plants were further selected for bioassay directed extraction in different solvents and then qualitative and quantitative analysis of the phytoconstituents of all extract and their screening against HCV RNA and its protein NS-3 serine protease. In addition to this antioxidant, antithrombic and antibacterial potential of these promising extracts methanolic extracts was also carried out. Finally the active fractions were also subject to the column chromatography for extraction and purification of some compounds present in them. These compounds were then identified with the help of different techniques like FTIR, Mass spectrometry and NMR etc.

2.10 Phytochemical Screening

Preliminary qualitative and quantitative phytochemical screening was carried out to determine the presence of various biochemically active compounds in methanolic extract.

2.10.1 Qualitative Analysis of the Extracts

Various known chemical tests were used to detect the presence of bioactive constituents like alkaloids, fatty acids, steroids, glycosides, terpenids, saponins, flavonoids, resins, phenols, tannin, reducing sugars, carbohydrates, and proteins etc. in each extract of both plants by using standard procedures.

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2.10.1.1 Glycosides

Presence of glycoside in the extract was detected by Keller-Killani test. For this purpose extracts solutions (5.00 mL each) were treated with glacial acetic acid (2.00 mL) having one drop of ferric chloride solution. This mixture was underlaid with of concentrated sulphuric acid (1.00 mL). A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer that signify the presence of glycosides.

2.10.1.2 Steroid

Methanolic extract (0.5 g) of each sample was added into the solution of acetic anhydride (2.00 mL) and sulphuric acid (2.00 mL). The colour changed from violet to blue or green in some samples indicating the presence of steroids.

2.10.1.3 Alkaloids

Alkaloids in various fractions were detected by adding the extract (0.4 gm) into of 1.00 % HCl (8.00 mL), then warmed and filtered. Each filtrate (2.00 mL) was titrated separately by means of (a) potassium mercuric iodide (Mayer’s reagent) and (b) potassium bismuth (Dragendroff’s reagent). Turbidity indicated the presence of alkaloids (Harborne et al., 1984).

2.10.1.4 Terpenoids

Extract solution (1.00 mL) was added in of chloroform (10 mL). Concentrated H2S04 (3.00 mL) was carefully added along the sides of the test tube in order to form a layer. A reddish brown colouration of the interface indicated the presence of terpenoids (Gibbs et al., 1974).

2.10.1.5 Fatty Acids

Extract solution (0.5 mL) was mixed with ether (5.00 mL). These extract allowed to evaporate on filter paper and then dried the filter paper. The appearance of transparence on filter paper indicates the presence of fatty acids (Ayoola et al., 2008).

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2.10.1.6 Saponins

Detection of saponin was conceded by dissolving the extract solution (5.00 mL) into distilled water (20 mL) and then agitated in a graduated cylinder for 15 minutes till the formation of steady persistent foam. Few drops of olive oil were added into the frothing. Formation of emulsion points toward the existence of saponins (Kumar et al., 2012).

2.10.1.7 Flavonoids

Three methods were used to test for flavonoids. First, dilute ammonia (5.00 mL) was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1.00 mL) was added. A yellow colouration that disappears on standing indicates the presence of flavonoids. Second, a few drops of 1.00 % aluminium solution were added to a portion of the filtrate. A yellow colouration indicates the presence of flavonoids. Third, a portion of the extract was heated with ethyl acetate (10 mL) over a steam bath for 3 min. The mixture was filtered and the filtrate (4.00 mL) was shaken with dilute ammonia solution (1.00 mL). Appearnce of yellow colouration indicates the presence of flavonoids (Ayoola et al., 2008; Harborne et al., 1984).

2.10.1.8 Tannins

Sample extract (0.5 mL) was taken along with one ml of water and 1-2 drops of ferric chloride solution. The result was in the form of blue colour for gallic tannins and for catecholic green black for catecholic tannins (Iyengar et al., 1995).

2.10.1.9 Coumarins

In extract solution (2.00 mL) of selectected plants 10 % NaOH (3.00 mL) was added and observed the formation of yellow colour that indicated the presence of coumarins (Rizk et al., 1982).

2.10.1.10 Anthraquinone (Bornatrager’s test)

In a dry test tube extract (0.5 mg) was taken and chloroform (5.00 mL) was added. After shaking of 5 min the extract was filtered and equal volume of 10 % ammonium

79 solution was added. Appearance of pink, violet or red colour confirmed the presence of anthraquinone.

2.10.1.11 Anthocyanins

A mixture of 2 N HCl and ammonia (2.00 mL) was added in the extract solution (2.00 mL) of the plant. The emergence of pinkish red colour and then its conversion into blue-violet colour indicated the occurrence of anthocyanins (Paris et al., 1969).

2.10.1.12 Emodins

Solutions of NH4OH (2.00 mL) and Benzene (3.00 mL) were added into the extract solution and observed the emergence of red colour that indicates the presence of emodins (Rizk et al., 1982).

2.10.1.13 Resin Identification

For the confirm presence of resin in selected plant, distilled water (5.00 mL) was added into the extract solution and observed the appearance of turbidity.

2.10.2 Quantitative Determination of the Chemical Constituency Specifically Antioxidants

2.9.2.1 Determination of alkaloids

For alkaloids determination, 5.00 gm of each sample was weighed into a 250 mL beaker, and 200 mL of 20 % acetic acid in ethanol was added and was covered to stand for 4 hrs. This was filtered and the extract was concentrated using a water bath to evaporate one-quarter of the original volume. The concentrated ammonium hydroxide solution was added drop-wise to the extract until the precipitation was completed. The entire solution was allowed to settle and the precipitate was collected by filtration, after which it was weighed (Harborne et al., 1984).

2.10.2.2 Determination of Pectin

To determine pectin, 5.00 gm of the plant sample was dispersed in 40 mL water in a beaker and boiled gently for one hour. The content of the beaker was transferred to a

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50 mL volumetric flask and diluted up to the mark with distilled water. The contents of the flask were thoroughly mixed and filtered into a 50 mL volumetric flask, after which 10 mL of the filtrate was taken into 80 mL beaker, where 30 mL de-ionized water and 1.00 mL sodium hydroxide were added to it along with a constant string. It was then allowed to stand overnight. To this solution, 5.00 mL of acetic acid (1.00 N) was added with continuous stirring and was allowed to stand for 5 mins. Then 2.5 mL of the calcium chloride solution were allowed to stand for one hour and the solution was boiled for one minute. The solution was filtered through Whatman No. 41 filter paper and washed with hot de-ionized water until the solution become free from chloride. The residue was transferred to the previously weighed watch glass. The watch glass, along with the residue, was placed on a water bath and dried in the oven at 100 °C until a constant weight was obtained (Horwitz et al., 2000).

2.10.2.3 Determination of saponins

For the saponins determination, 5.00 gm of each plant samples was weighed and was dispersed in 100 mL of 20 % ethanol. The suspension was heated over a hot water bath for 4.00 h with continuous stirring at about 55 °C. The filtrate and the residue were re-extracted with another 100 mL of 20 % ethanol. The combined extracts were reduced to 40 mL over water bath at about 90 °C. The concentrate was transferred into a 250 mL separating funnel and 20 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated and about 30 mL of n-butanol was added. The combined n-butanol extracts were washed twice with 10 mL of 5.00 % aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation, the samples were dried in the oven to a constant weight. The saponin content was calculated in percentage (Obadomi et al., 2001).

2.10.2.4 Total phenolic contents

Evaluation of total phenolic contents was carried out by modifying the reported method (Kalpna et al., 2011). Shortly, freshly prepared 0.5 N Folin-Ciocalteu reagent (0.1 mL) was added into 0.5 mL of diluted extract sample (10 mg/5mL distilled water) and incubated for 15 minutes. Then the solution was again incubated in dark for half an hour after the addition of 2.5 mL of 7 % Na2CO3 solution. Finally noticed

81 the absorbance of at 700 nm and expressed the results as Gallic Acid Equivalent mg/g dry weight of the plant extract.

2.10.2.5 Total flavonoid contents

Total flavonoid contents were determined the reported method (Zhishen et al., 1999).

In brief, 0.3 mL of fresh prepared NaNO2 solution (1:20) was added into the 1.00 mL of plant extract aqueous solution (10 mg/mL). Then, after 5 minutes of room temperature incubation, 0.3 mL of freshly prepared AlCl3 (1:10) was poured into that solution. Then wait for six minutes for the accomplishment of reaction and then added 2.00 mL of 1.00 M NaOH. Finally, make the total volume upto 10 mL by the addition of distilled water and immediately measured the absorbance at 510 nm. Final results were articulated as Catechin Equivalent mg/g of the plant dry weight.

2.10.2.6 Total carotenoid contents

Total carotenoids were determined following a method reported by (Lichtenthaler et al., 2001). In brief, plant powder samples (5.0 gm) were immersed in 80 % acetone separately and incubated at 4 0C in dark for three days. After incubation samples were filtered and centrifuged at 2500 rpm for 15 minutes and separate supernatant. Finally measure the supernatant absorbance at three different wavelength i.e. 663.2 nm, 646.8 nm and 470 nm. Concentrations of total carotenoid and Chlorophyll a, Chlorophyll b were calculated via following equations and results were articulated in µg/g of leaf.

Ca= 12.25A663.2- 2.79A646.8 Cb = 21.50A646.8 – 5.10A663.2 ퟏퟎퟎퟎ퐀ퟒퟕퟎ – ퟏ.ퟖퟐ퐂퐚 – ퟖퟓ.ퟎퟐ퐂퐛 Cx+c = ퟏퟗퟖ 2.10.2.7 Total tannin contents

Total tannin contents were determined according to the reported protocol (Hosu et al., 2014) with a few modifications. Plant extract (10 mg) was diluted with 4.00 mL distilled water, which was divided into two equal fractions. In both factions concentrated HCl (3.00 mL) and distilled water (1.00 mL) were added. In one fraction ethanol (0.5 mL) was added while the second one in which ethanol was not added was incubated at 100 0C for 30 min. Then measured the absorbance difference of these

82 two fractions at three different wavelength 470, 520 and 570 nm and calculated the total tannin contents with the help of following formula.

∆A520 = 1.1 x ∆A470 ∆A520 = 1.57 x ∆A570 TTC (g/l) = 15.7 x minimum (∆A520)

2.10.2.8 DPPH radical scavenging activity

Reported DPPH assay along with certain modification was used to determine the DPPH radical scavenging capacity of plant extracts (Proestos et al., 2013). DPPH radical exhibits utmost absorbance at 517 nm while drop in absorbance values is calculated as radical scavenging potential of phyto extract. For this assay, 0.2 mM DPPH (2.00 mL) was added into 2.00 ml of methanolic solution of plant extracts (5:1). Similarly in case of control, DPPH solution (2.00 mL) was added into methanol (2.00 mL) and incubated the reaction mixtures for one hour in dark. Then measure the absorbance at 517 nm against blank (methanol). All measurements were made in triplicate and DPPH radical percent inhibition was calculated with the aid of subsequent formula:

푨풄풐풏풕풓풐풍−푨풔풂풎풑풍풆 % Inhibition = ×ퟏퟎퟎ 푨풄풐풏풕풓풐풍

2.10.2.9 ABTS radical cation scavenging activity

Reported ABTS assay along with slight modification was used to determine the ABTS radical cation scavenging capacity of plant extracts (Proestos, et al., 2013). In brief, 7.00 mM solution of ABTS and 2.45 mM solution of MnO2 were prepared separately and mixed with ratio of 1:1 and incubated in dark for dark for 48 hours. Then diluted the solution of ABTS with aqueous methanol (1:25) and finally of this solution (2.00 mL) was added in 200 μL of plant extract prepared in aqueous methanolic (1:10). Similarly control was prepared and absorbance was measured at 734nm. Percent scavenging of ABTS radical cation was calculated using the relation:

푨풄풐풏풕풓풐풍−푨풔풂풎풑풍풆 % Inhibition = ×ퟏퟎퟎ 푨풄풐풏풕풓풐풍

All experiments and recordings were performed thrice and results were averaged.

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2.10.2.10 Ferric reducing antioxidant power

Plant extracts reducing power was determined according to the method reported by Zhuang (Zhuang et al., 2012). Briefly, plant extract (10.00 mg) was added into 3.5 mL phosphate buffer (0.2 M, pH 6.6), followed by subsequent addition of 1.00 % potassium ferricyanide (2 mL) and incubation at room temperature for 20 minutes. Afterward, 10 % trichloroacetic acid (2.5 mL) was added into the mixture and the mixture was centrifuged at 3000 rpm for 10 minutes. Then distilled water (2.5 mL) and 0.1 % ferric chloride (0.5 mL) was added into the 2.5 mL solution of supernatant and finally noted the absorbance of mixtures at 700 nm. Results were expressed as Ascorbic Acid Equivalent mg/g dry weight.

2.10.2.11 Identification and Quantification of Phenolic Acids by HPLC

High performance liquid chromatographic method was utilizes for identification and quantification of phenolic acids in selected plant extracts. In brief, sample was prepared by dissolving the plant extract (50 mg) into of distilled water (15 mL) followed by subsequent addition of 24 mL methanol (Merck, HPLC grade) and shaken the mixture for 5 minutes in shaker. Then 10 mL of 6.00 M HCl (Merck, Analytical grade) was added and mixture was incubated in oven at 950 C for two hours. Samples were filtered through 0.2 μ syringe filter paper and 5.00 mL of each extract was injected on a Shim-Pack CLC-ODS (C-18), (25 cm x 4.6 mm, 5 μm) for gradient elution. Working solvent were A(H2O:Acetic Acid 94:6, pH=2.27), B(Acetonitirle 100 %). The gradient profile was 0-15 min= 45 % B, 30-45=100 %B with flow rate of 1ml/min. UV/Visible detector was set at 280nm for quantitative determination. The temperature of the column was 25 ◦C. Concentrations of phenolic acids in each sample were determined with the help of calibration curve.

2.11 Anti-clotting Properties of CTS and POL Extracts

In vitro thrombolytic potential of methanolic and ethylacetate fractions of CTS and POL were determined by following the already reported method (Prasad et al., 2007))

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2.11.1 Sample Preparation

Positive control: Stock of positive control was prepared from commercially available Streptokinase (SK) vial (15, 00,000 I.U) (Polamin Werk GmbH, Herdecke, Germany) that was dissolved in 5.00 mL of sterile water and from which 100 μL (30,000 I.U) was used for in vitro thrombolysis.

Specimens: Whole blood (5.00 mL) was drawn from healthy human volunteers (n = 10) without a history of oral contraceptive or anticoagulant therapy. Blood sample (500 μL) was transmitted to pre weighed alpine tube for the formation of clot.

2.11.2 Preparation of Anti-Clot Test Solution

Selected plant extract (100 mg) was suspended in 10 mL of distilled water, mixed vigorously on vortex mixer and kept overnight at room temperature. Then mixture was filtered through 0.22 micron syringe filter and used to check in vitro thrombolytic activity.

2.11.3 Clot Lysis

After the formation of clot, serum was completely aspirated without disturbing the colt and each tube having clot is again weighed for determination of clot weight.

Weight of clot = weight of clot containing tube –weight of empty tube

Each tube having clot was labeled properly and to each tube containing pre weighed clot, 100 µL of aqueous plant extract added separately. As a positive control, 100 µL of Streptokinase and as a negative control, 100 µL of distilled water were separately added to the control tubes. All the tubes were then incubated at 37 °C for 90 minutes and observed for clot lysis. After incubation, fluid obtained was removed and tubes were again weighed to examine the difference in weight before and after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The experiment was repeated numerous times with the blood samples of different volunteers (Prasad et al., 2006).

% clot lysis = (Weight of the lysis clot / Weight of clot before lysis) × 100

2.12 Antibacterial assay of extracts

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The antibacterial action of selected phyto extract was performed on active cultures of different bacterial strain by following the Kirby Bauer disc diffusion method (Bauer et al., 1966). For this purpose six bacterial strains (three gram positive and three gram negative strains) were procured from the microbiology laboratory of department of pharmacy, University of Sargodha and were as follows: Bacillus subtilis 6633, Staphylococcus aureus ATCC 25923, Styphylococcus epidermidis ATCC 12228, Pseudomonas aerugenosa ATCC 27853, Escherichia coli ATCC3411 and Salmonella typhi ATCC 26823. The bacterial stock cultures were preserved on nutrient agar plates. A loop full of bacterial cells from the nutrient agar plates was inoculated into 10 mL nutrient broth and incubated at 37 oC for 24 h. After incubation, the culture suspension was compared with the McFarland standard. In the presence of visible light visually compared the turbidity of bacterial suspension to the 0.5 McFarland standards and adjusted the turbidity of bacterial suspension. Nutrient agar media of (Oxoid, UK) was prepared and sterilized by autoclaving at 121 oC for 15 minutes at 15 psi pressure and petri plates were prepared. Then Wicks paper disk (6.00 mm diameter) that was carrying sample solution was placed smoothly on the respective growth media. Afterward 100 µL of innoculum was added, mixed thoroughly and poured into the petri plates using a sterile glass spreader. The plates were incubated for 24 h at 37 °C and the diameter of the inhibition zones was recorded in millimeters by using zone reader. Each antimicrobial assay was performed in triplicate & mean values were taken (NCCLS. 2003).

2.13 Bioassay guided extraction of CTS and POL

For bioassay-directed extraction, crude methanolic extracts of each plant was dissolved in 70 % n-hexane and then separate the aqueous layer from non polar hexane layer with the help of separating funnel. In aqueous faction add the ethyl acetate repeated the similar procedure for separation of ethyl acetate soluble compounds. Then chloroform and n-butanol was added respectively in order to obtain the chloroform and butanol extract. Final remaining aqueous extract was not used in any of the further analysis. The bioassay guided extraction scheme used for the separation of polar, slightly polar and non polar compounds is shown in fig. 3. The polar and non polar organic layers were concentrated in vacuum to give four fractions (H, C, E and B) which were screened for HCV NS3-SP inhibition activities.

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Fig. 2.3: Scheme for bioassay-guided fractionation of Caralluma tuberculata and Portulaca oleracea L methanolic extracts.

2.14 Isolation of Compounds via Column Chromatography

2.14.1 Isolation of Compounds

Flash chromatography was executed to isolate the compounds from Ethyl acetate fraction on a glass column (60 x 3.5cm) manually packed with silica gel 60. After the column packing it was equilibrated with CHCl3 that remove all the trapped air bubbles from silica gel.10 gm of EtOAc extract was adsorbed on silica and this slurry was loaded on the top of the packed column. Sample elution was carried out stepwise using a gradient CHCl3-MeOH solvent system. 110 fractions of 250 mL were collected successively with increasing concentrations of methanol in chloroform and concentrated. Presence of compounds in fractions was monitored by thin layer chromatography (TLC) by using commercially available TLC plate coated with polyamide film (China). Spots were observed directly under ultraviolet light (254 nm) and then beneath visible light after using different reagents. Fractions possessing component with same Rf values were combined together. Fractions that have show two spots on TLC again chromatographed on silica column using Ethyle actate: methanol solvent system.

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2.14.2 Physico-chemical Characteristics of Isolated Compounds

After examining the physical appearance and state of extracted compounds and their melting points were determined on Stuart Scientific SMP10 apparatus. Optical rotation was calculated via JASCO-DIP360 digital polarimeter.

To verify the steroidal character of the compounds, the EtOAc fractions and the isolated Compounds were examined by Liebermann-Burchard test (Burke et al., 1974; Halim et al., 1996). Among the many colour reactions for steroids, the Liebermann- Burchard procedure is perhaps the most widely used test. Liebermann- Burchard colour reactions for steroids have oxidative mechanisms, yielding oxidation products. This reaction was illustrated firstly by in 1885 by Liebermann and then applied to cholesterol analysis soon after by Burchard. In the early studies Chloroform was used as a solvent, but the Liebermann-Burchard (L-B) reaction, as carried out today, is done in an acetic acid, acetic anhydride medium and sulphuric acid (Halim et al., 1996). Ten drops of 1.00 % solution of the EtOAc fraction in chloroform were added to a dry test tube. Then 5 drops acetic anhydride was added to the test tube and the solution was mixed well. Then three drops of concentrated sulphuric acid was added and the solution was mixed thoroughly. Immediate change in colour was recorded and even after passage of 10 minutes change is noticed and recorded.

2.14.3 Elucidation of Compounds Structure by spectroscopy

Spectroscopy is the study of the absorption and emission of radiation by matter. In physical and analytical chemistry, spectrometry is often used in the identification of substances through the spectrum emitted from or absorbed by them. Fractions having confirmed single compound were dried thoroughly in vacuum line for 4-6 hrs in order to make sure that complete evaporation of solvent. Then the structures of these purified compounds were characterized by FTIR, ESI-MS and NMR.

2.14.3.1. Electro Spray Ionisation (ESI)

Electro Spray Ionisation (ESI) is perhaps the most common method of choice for analysis of molecular weight of broad range of chemical and biological analyte. It is considered more sophisticated ionization that does not cause extensive fragmentation

88 of molecular ions. Here ions are generated in form of charge droplet from the liquid and then subsequently evaporated. The analyte was prepared in methanol (80 %) along with formic acid (0.1 %) in order to enhance protonation and increase sensitivity. Mass spectra (MS) were recorded using a Micromass Platform II spectrometer using an electrospray ionization source.

2.14.3.2 Infra-Red spectroscopy

Infra-red (IR) spectroscopy offers an approach of unearthing the functional moieties in any molecule, as it identifies the bending and stretching vibrations of bonds specifically the asymmetrical bonds stretching of the functional groups such as C=O,

NO2, NH2, and OH. Infrared spectra were obtained by using ALPHA-P FTIR spectrometer

2.14.3.3 Nuclear Magnetic Resonance (NMR) spectroscopy

NMR spectroscopy work on the basis of nuclear magnetic resonance that provides full insight not only about the structure of unknown compounds but also for reaction dynamics and environment of molecule. Resonance frequency due to intramolecular magnetic field created around atoms of molecule. Compound absorbs electromagnetic rays under suitable conditions of applied magnetic field. Absorption is the basic property of the atom’s nuclei. NMR spectrum is obtained from peak intensities plot derived from absorption peaks and frequencies.

Extracted purified compounds were dissolved in CDCl3 or MeOH-D3 (CD3OD) depending upon the nature of compounds and then their structure were interpreted from the spectra obtained from one dimensional (1H, 13C, and 13C DEPT) and two dimensional (COSY, HMBC and HSQC) NMR spectras determined on 300 MHz and 400 MHz NMR on a Bruker DPX 400 spectrometer, expressed in ppm (δ) relative to trimethylsilane (TMS) as an internal standard.

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3.0 Results and Discussion

In recent era, research on medicinal plants focuses not only the documentation of their ethnopharmacological detail but also deciphering the characteristics of their potentially active principals (Fennell et al., 2004). As we know that plants are complex mixture of the active and inactive components and the separation of active from inactive is always remain arduous and tedious task (Sticher, 2008). Five different approaches usually opted for phyto research like random, ecological, phylogenetic or chemotaxonomic, ethno-guided and bioassay directed (Albuquerque

90 et al., 2006; Elisabetsky et al., 1994). Different aspects like evidence based information about ethno pharmacological use by natives; plant part along with disease name and its availability in natural habitat etc were kept under consideration when medicinal plants were chosen for scientific assessment of their biological potential and rationalizing their ethno pharmacological usage (Matos, 2000). This information should be complete enough to fill the gap between traditional knowledge of plant and present modern scientific utilization.

3.1 Collection of Botanical Samples and Preparation of Extract

Different samples from fifteen plants were collected from Soon-sakaser valley of Khushab, Pakistan on the basis of conventional knowledge of local community and herbal practioners. All indigenous botanical samples were used as primary source of health care against viral and liver disease specifically for hepatitis viruses. Clean air dried botanicals were pulverized and their methanolic extracts were obtained by maceration method.

Keeping under consideration the limited resources and huge amount medicinal plant sample a proficient system of extraction is needed for the separation and the biological screening of compounds from the cellular matrix of plant (Sticher, 2008). Preferred method (solid-liquid extraction) should be convenient, meticulous, swift, efficient, pollution free and inexpensive etc. Drawbacks associated with such method of extraction yield are laborious, prolonged extraction time, high consumption of solvent and poor yield. A wide variety of the solvents can be used ranging from non polar to slightly polar and highly polar solvents depending upon the nature of compound we want to extract. As usually we are working with unknown compounds and we should know that extractant nature effect the composition of crude extract. During traditional practice usually water and ethanol are solvent of choice. But in modern scientific research petroleum ether, hexane, ethyl acetate, chloroform, acetone, methanol, propanol, butanol etc. some factors like temperature, light and storage conditions also affect the process of extraction and composition of constituents.

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3.2 Percentage yield of plants extract

Method of extraction is an important step for any type of natural product studies because it has direct effect on the extractant capacity, amount of yield and quality of extract (Berlin et al., 2005). Non polar and slightly polar solvent like petroleum ether, hexane, dichloromethane, ethyl acetate extract sterol, terpenoids and some phenolics while highly polar solvent like methanol, acetone extract wide range of polar compound for example phenolics. After the calculation of percentage yield of all samples separately all extracts were stored in contamination free vials and stored in refrigerator at 4 ᵒC. Percentage yield of methanolic extracts of plants is shown in Fig. 3.1.

30

20

% % Yield 10

0

CIL CTS BML CBR CDL SAB SIS CTR AAL SAL RER ZJS POL ATB MFHL Methanolic extract of plants

Fig. 3.1: Percentage yield of methanolic extracts of plants collected from Soon- sakaser valley of Pakistan

3.3. Toxicological Studies of Plant Extracts in CHO and Liver Cell Lines

Due to the ever increasing interest of man natural thing utilization and emphases of research on medicinal plant has focus the dire need of gathering information regarding quality, therapeutic efficacy, safety and toxicity potential of botanical extracts before the treatment of any disease. Therefore cytotoxic trials become the gist of any medicinal plant research that focus on its biological activity either the crude extract of plant or any of its bioactive constituent.

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For this purpose stock solutions (40 mg/mL) of each extract were prepared in DMSO (Cell culture grade) separately for in vitro toxicological and antiviral studies in CHO and hepatoma cell line (Huh-7).

3.3.1 Trypan Blue Dye Exclusion Assay

Prior to antiviral screening against Hepatitis C virus, cytotoxic effects of all the samples were determined on liver Huh-7 cells through Trypan blue exclusion method. After 24 h incubation of Huh-7 with different concentrations (ranging from 1.5 µg/mL to 200 µg/mL) of all extract separately, cell viability was evaluated using trypan blue dye and cells were counted using haemocytometer. Concentration of CTS and POL did not exert any effect on viability of CHO and Huh-7 cells (Fig. 3.2 A & B). Similar results were observed for other 13 extracts most of them appeared as non toxic (data not shown).

3.3.2 Cytotoxic Screening of Phyto Extracts via MTT Assay

Another In vitro assay performed to check the viability of cell under the effect of plant extract was MTT assay. In this assay, a cellular enzyme (oxidoreductase) in the presence of NADPH reduce the substrate tetrazolium dye MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide into purple colour insoluble formazan crystals in the alive cells. Crystals of formazon was dissolved adding the solution of DMSO. Absorption of solution in visible range of light directly reflected the number of alive cells. MTT assay results confirmed that both cell lines remain viable to the concentration (200 μg/mL) of plant extract. Toxic effects of plant (CTS and POL) crude extracts are shown in Fig. 3.3 (A & B).

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Fig. 3.2 (A & B): Cellular toxicity analysis CTS and POL extracts through Trypan blue exclusion method For toxicological analysis of CTS (A) and POL (B), Huh-7 liver cells were seeded at a density of 3 × 105 in six well plate. After considering first well as control, varying concentrations of the CTS/POL were added in wells. Subsequent to 24 h incubation, viable cells were counted with haemocytometer via Trypan blue dye exclusion method. For the treatments, mean (n=3) + SD (n=3) with P value < 0.05 were found significant with control.

CHO cell lines, derived from Chinese hamster ovary, were chosen usually for toxicity screening of different products, gene expression, genetics and nutrition studies because of their ability of fast growth, long-lasting stable expression of gene and

94 elevated protein production. Huh-7 cells (hepatoma cell line) are also considered a reliable source for study of anti-cancer, antiviral agent and hepatic coupled malady.

150 Huh-7 CHO

100

50

%age Cell Survived

0

0.8 1.5 25 50 Ctrl. 3.25 6.25 100 200 Concentration (µg/mL) A

Fig. 3.3 (A & B): Toxic effect of plant (CTS and POL) crude extracts on Huh-7 and CHO cell lines by MTT assay. All values were expressed as mean ± SEM for experiments done in triplicate.

Medicinal plant because of their natural origin are usually considered as safe and this is a logic that is behind the prolonged and continuous usage of medicinal plant for curative and nutrition purpose since from all historic era to present (Fennell et al., 2004). Administration of plant based preparation along with the hope of their therapeutic ability against a number of different diseases like microbial infections, cancer, cold, metabolic disorders, immunological disorders, insomnia and inflammation etc. (Chen et al., 2002). But in reality according to some authentic studies on the cytotoxicity, safety and efficacy of some plants extract it was proved that prolonged usage of many plant extract cause cancer, cytotoxicity and

95 genotoxicity (Ernst et al., 2011). Medicinal plant adverse reactions are obvious in form of adulteration, heavy metal contamination, herb drug interaction and toxicity of organs like liver, heart, gastrointestinal tract, central nervous system and kidney etc. (Jordan et al., 2010). Most of the herbal products or formulation of so called herbal industries do not fulfill the criteria of quality assurance, cytotoxic trials and good quality manufacturing practice (Palombo, 2006). Furthermore, solvent choice used for extraction can also cause problem of cytotoxicity. Sometime cytotoxic effect becomes severe when botanicals are mixed with synthetic drug, microbial byproducts like toxins etc without any standard clinical trial (Palombo, 2006). Especially when botanical extract is administered along with narrow range spectra drug it potentially hampers the response of drug and result cytotoxicity (Chen et al., 2011). In fact during herbal-drug interaction the active constituent of herb exhibit their action because these constituent having potential to undergo different enzymatic transformations and produce toxic and non toxic metabolites (Chen et al., 2011). Keeping in mind the significance of extract toxicity effect these studies were carried out and all selected plant extracts appear non toxic and suitable for further biological studies.

3.4 Anti-HCV Assay

Medicinal plants are utilized for premier health care need against various dreadful infections throughout the globe. Therapeutic potential of medicinal plants lie behind the separate or synergistic action of complex mixture of secondary metabolites (Voravuthikunchai et al., 2006).

It has been reported that HCV can replicate in Huh-7 cells by the detection of viral genes as well as viral copy number by Real Time PCR in both cells and supernatant, whereas, HCV replication in cell culture is strictly limited to human hepatocytes and their derivatives. In the present study, methanolic extracts from 15 different plants were tested to determine the antiviral activity against HCV. Real time RT-PCR results showed that only methanolic extracts of CTS and POL out of 15 medicinal plants exhibited convincing potential (57 % and 70 % respectively) against HCV-RNA at non-toxic concentration (Figure 3.4). This Methanolic extract further on the basis of biassay-guided directions subdivided into four different solvent fractions. Both plant

96 methanolic extracts (POLM and CTSM) suspended in water further fractioned with n- hexane (POLH and CTSH), ethyl acetate (POLE and CTSE), chloroform (POLC and

CTSC) and n-butanol (POLB and CTSB). Among these only ethyl acetate fraction (CTSE & POLE) in both cases significantly reduced (68 % and 74 %) of the viral titer (Figure 3.5 (A&B).

Fig. 3.4: Anti-HCV activity of methanolic extracts of selected fifteen different plants Huh-7 cells infected with HCV serum was cultured and incubated with 1.5 µg/well concentration of different plants extracts for 24 h. After incubation total RNA was extracted and the levels of HCV RNA were determined by Quantitative RT-PCR assay and are shown as percentage to the levels of HCV RNA in cells in comparison of control (without treatment of plant extract). Control (Ctrl.) Achyranthus aspera (AAL), Bacop monnieri (BMB), Chichorium intybus (CIL), Chlorophytum borivilianum (CBR), Corchorus depressus (Linn.) (CDL), Myristica fragrans Houtt (MFHL), Portulaca oleracea L (POL), Sesamum indicum (SIS), Sonchus Arvensis (SAL), Caralluma Tuberculta stem (CTS), Caralluma Tuberculta root (CTR), Santalum album (SAB), Rheum emodi (RER), Zizyphus Jujuba (ZJB). All the treatments were made in triplicate (n=3) and results are exemplified as mean ± standard deviation. ***P value < 0.001 vs. control.

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A.

B.

Fig. 3.5 (A&B): Anti-HCV activity of organic extracts of POL and CTS. Huh-7 cells were incubated with HCV serum (1×105/ml) and 1.5 µg/well concentration of POLM, POLE, CTSM and CTSE extracts for 24 h. After incubation, total RNA was extracted and the levels of HCV RNA were determined by Quantitative RT-PCR assay and are shown as percentage to the levels of HCV RNA in cells incubated without extract (control). All the treatments were made in triplicate (n=3) and findings were found statistically significant at P< 0.001 with respect to control (Ctrl.).

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3.5 Inhibition of HCV NS3 Gene Expression by Selected Organic Extracts

In order to determine the effect of CTS and POL extracts against HCV NS3 protease, Huh-7 cells were transfected with NS3 protease plasmid in the presence and absence of herbal extracts. After 48 h incubation, cells were harvested, RNA was extracted and cDNA were generated by oligo dT primers. cDNA was amplified by PCR using primers specific to the HCV NS3 protease. Fig. 3.6 (A & B) and Fig. 3.7 (A & B) represent the qualitative and quantitative inhibition of HCV NS3 gene by POL and CTS extracts.

It is obvious from the figures that ethyl aceatate and methanolic extract respectively inhibit the 69 % and 53 % expression of HCV NS3 protease. Similarly the POLM reduced the NS3 gene expression to 75 %, whereas the ethyl acetate extract reduced the level up to 85 %. The effect of POL on NS3 gene was also compared to INF (101 U) and it is shown that POL is more active in the reduction of NS3 gene expression Fig. 3.6 (A & B). GAPDH mRNA expression was used as internal control, which remained unaffected after the addition of extract. Present response is comparable to the in vitro inhibition of HCV NS3-SP with Rhodiola kirilowii ethanolic extract of and Camellia sinensis Epigallocatechin-3-gallate compound.

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A B. Fig. 3.6 (A and B): Antiviral Activity of methanolic and ethyl acetate extracts of POL against HCV NS3 protease. A) Huh-7 cells were transfected with 0.5 µg/well of constructed HCV NS3 protease vector in the presence and absence of PO and interferon (Roche) for 24 h. Cells were harvested and relative RNA determinations were made using semi-quantitative RT-PCR. The results reflected that PO and interferon inhibited HCV NS3 expression, while expression of GAPDH remained constant. Lane 1; Control, Lane 2; Huh-7 cells, Lane 3; POLM, Lane 4; POLE, Lane 5; IFN. B) Quantitative inhibition of HCV NS3 protease by methanolic and ethyl acetate extracts of Portulac oleracea studied through Real Time RT-PCR assay by using SYBR Green master mix. Statistically significant inhibitions (P< 0.001) in mRNA expression levels of HCV NS3-SP expression in POLM, POLE, and IFN treated cells relative to control were determined experimentally (n=3).

A. B. Fig. 3.7 (A and B): Qualitative & quantitative inhibition of HCV NS3 protease by IFN, methanolic and ethyl acetate extract of CTS. A) Huh-7 cells were transfected with 0.5 µg of constructed HCV NS3 protease vector in the presence and absence of CTS and interferon (Roche) for 24 h. Cells were harvested and relative RNA determinations were made using semi- quantitative RT-PCR. The results reflected that CTS and interferon inhibited HCV NS3 expression, while expression of GAPDH remained constant. Lane 1; Control, Lane 2; Huh-7 cells, Lane 3; CTSM, Lane 4; CTSE, Lane 5; IFN. B) Quantitative inhibition of HCV NS3 protease by methanolic and ethyl acetate extracts of CTS studied through Real Time RT-PCR assay by using SYBR Green master mix. Statistically significant inhibitions (P< 0.05) in mRNA

100

expression levels of HCV NS3-SP expression in CTSM, CTSE, and IFN treated cells relative to control were determined experimentally (n=3).

3.6 Western Blotting

The effect of inhibition of Portulaca oleracea extract against HCV NS3 protease, Huh-7 cells were transfected with NS3 protease plasmid in the presence and absence of herbal extracts was also checked at protein level. Ethyl acetate and methanol extracts of POL having antiviral potential for the inhibition of HCV NS3 protein were also studied. Huh-7 cells were transiently transfected with pCR3.1/FLAGtag/HCV NS3 of genotype 3a. Subsequent to 48h of incubation, the transfected cells were lysed & western blotting technique along with monoclonal antibodies of HCV NS3 and GAPDH was used to study the extracted proteins. For setting internal control, GAPDH was used (Fig. 3.8). There was a dramatic reduction in the protein expression of the NS3 gene after treatment of Portulaca oleracea extracts (Fig. 3.8). There were

60 % and 70 % inhibition in Huh-7 cells treated POM and POE respectively.

Fig. 3.8: Antiviral Activity of methanolic and ethyl acetate extracts of POL against HCV NS3 protease at protein level Huh-7 cells were transiently transfected with NS3 gene of HCV and subsequently treated with POL methanolic (POLM) and ethyl acetate (POLE) extracts DMSO and interferon. Total protein was isolated 24h post transfection and subjected to SDS-PAGE, and western blot. Results shows that POLM and POLE significantly reduced the expression of NS3 gene as compared to Control, DMSO and interferon treated cells. Lane 1; Control, Lane 2; DMSO, Lane 3 IFN; Lane 4; POLM, Lane 5; POLE, Lane 6; Mock. Blots were also normalized by measuring the expression of GAPDH used as internal control. The quantitative expression of protein on blots was measured using freely available software Gel- 101 quant-express. All experiments were performed in triplicate. Error bars indicate mean S.D, ***P≤0.001.

3.7 Phytochemical analysis

Plants exhibit their therapeutic potential by the charisma of secondary metabolites. Secondary metabolite concentration of plant is greatly affected by climate conditions, geographic and genetic variations, soil composition, pollution, season of plant collection, treatment of plant before extraction, etc. From socioeconomic point of view phytotherapy is also preferable over costly synthetic drug (Nunes et al., 2003). Biochemical investigation of the extracts attained with various solvents is the utmost significant relating to a variety of diseases. Results acquired in this perspective are given as under.

3.7.1 Qualitative Analysis of CTS and POL

Standard procedures were used for the phytochemical screening of plants. Phytochemical screening of the extract shows that many natural products were present in these extracts. Qualitative screening of methanolic extracts of CTS and POL pointed toward the occurrence of various constituents as revealed in Table 3.1. CTS extract was found to be rich with glycosides, steroids, saponin, coumarine and resins, but on other hand POL extract was rich in anthraquinones, fatty acids, flavonoids, tannin and resins.

Table 3.1: Qualitative phytochemical screening of CTS and POL methanolic extracts

Sr. No. Constituents CTS POL 01 Glycosides +++ + 02 Steroids +++ ++ 03 Alkaloids ++ + 04 Terpenoids + ++ 05 Fatty acids + +++ 06 Saponin +++ + 07 Flavonoids + +++ 08 Tannins + ++ 09 Coumarine +++ ++ 10 Anthraquinones - +++ 11 Anthocyanin - - 12 Endomine - + 13 Resins +++ ++ +++, high concentration; ++, medium concentration; +, low concentration; -, not detected

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3.7.2 Quantitative Methods for Screening of Phytoconstituents

Screening assay disclose presence of plant primary or secondary metabolites (phytochemicals). Phyto-constituent screening assays, either the qualitative or quantitative, are easy and inexpensive protocols used for rapid clue about the presence of particular phyto compound in plant matrix. Table 3.2 demonstrates the percentage of alkaloids, pectin, soluble pigments, saponin, flavonoids and tannins in methanolic crude extracts of CTS and POL. The concentration of alkaloids was found to be very high i.e. 15.82+0.06 in POL extract and while the CTS methanolic extract also has the highest percentage of alkaloids. Pectin and saponins was also found in significant quantity.

Table 3.2: Percent phytochemical components in CTS and POL methanolic extracts. Sr. No. Constituents % CTS POL

1 Alkaloids 7.84± 0.001 15.82+0.06

2 Pectin 5.4± 0.431 6.61± 0.031 3 Soluble pigments 2.9740± 0.004 1.035+0.013

4 Saponin 3.08± 0.011 1.76± 0.008

5 Tannins 0.169± 0.004 0.898+0.001

3.8 Antioxidant Potential of Biologically Active Plant

Extract Fractions

The antioxidant potential of metanolic and ethyl acetate extracts of CTS and POL was determined by following the different reported procedure for total phenol, flavonoids, tannin, carotenoid and free radical scavenging potential etc.

3.8.1 Total Phenolic, Total Flavonoids and Total Carotenoid Contents

Phenolic compounds are produced in plants in response various factors like UV radiation, stress like drought, nutrition deficit, harsh temperature and pressure condition and defensive action against pathogens. These compounds are considered as

103 significant therapeutically active constituent that deciphered a wide range of activities like anticancer, antiviral, antibacterial, antifungal, anti-allergic etc. The total phenolic content of the different fractions of CTS and POL were determined by a modified Folin-Ciocalteu method and results were expressed in graphical forms (Fig. 3.9). TPC were resolute in this study weighed against with standard (gallic acid) and final results are articulated in term of milligrams of gallic acid equivalent (mg GAE/g dry sample). In present study phenolic contents were found to be highest in concentration methanolic extract of POL and CTS while their concentration is reduced in case of ethyl acetate extracts of both plants. So the methanol was found to be the best extraction solvent for phenols in this study.

Flavonoids being phenolic glycosides donate elevated antioxidant bustle of plant owing to their metal chelating and scavenging attribute thus act to control pathophysiology related to oxidative stress. Structure of flavonoid contain a central backbone of three rings and sometime occur naturally in conjugated form along with a glycone moiety. Oligomeric form of flavonoid is Proanthocyanidin. Due to the presence of phenolic group both they operate like antioxidant, phytoestrogen, reduce the risk of inflammation and cancer (Middleton et al., 2000; Noroozi et al., 1998).

Flavonoids concentration in sample STSM, STSE, POLM, and POLE is 42.15, 18.35, 47 and 35 respectively present as mg/g Catechin equivalents. The projected flavonoids content of CTS and POL extracts are expressed as mg/g Catechin equivalents in fig. 3.10.

In plants the carotenoid play an important role in photosynthesis process. The concentration of carotenoid is also increased during ripening of fruits. These are main precursor required for the formation of vitamin A and β-carotene in mammals. Owing to their free radical scavenger and antioxidant potential they have the capacity to reduce cancer by modulating immune response. In the present study carotenoid contents were found in agreement with the previous findings. Results of total carotenoids contents are expressed as μg/g of the samples also revealed a correlation among antioxidant potential and total carotenoid contents of plant samples which is also evident from the graphs (Fig. 3.11). All measurements were done in triplicate and results were averaged.

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Fig. 3.9: Total Phenolic Contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

Fig. 3.10: Total flavonoid Contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

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Fig. 3.11: Total carotenoid contents of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

3.8.2 Free Radical Scavenging Activity of Extracts by DPPH, ABTS and FRAP Assays

H-ion-donating power of polyphenols is considered an imperative biologically property that convert potentially damaging species of reactive oxygen into the nontoxic one. Particularly, DPPH• appear quick in action in determining antioxidant potential for rapid conversion of labile hydrogen atom into radicals. CTSM, CTSE,

POLM and POLE extracts antiradical values against DPPH were found to be 81.747, 58.407, 45.511 and 36.124 respectively.

In vitro antioxidant potential like free radical scavenging action of CTS and POL methanolic and ethyl acetate extracts was also resolute by the ABTS•+ assay. The ABTS•+ cation radical is preferred over DPPH free radical based assay when sample interference or solubility come up. The ABTS•+ scavenging data (Fig 3.13) propose that extracts components have the potential to scavenge free radicals of ABTS and thus protect susceptible subject from free radical mediated oxidative damage.

Among all the assays use for determination of antioxidant potential of compounds and phytoextract, the FRAP assay is the most simple, fast, inexpensive, reproducible and highly reliable method (Benzie & Strain, 1999). Antioxidant compounds can

106 effectively reduce Fe (III) to Fe (II) exhibiting high reducing power values. Ferric reducing antioxidant power of selected plant extracts is shown in Fig.3.14.

Fig. 3.12: DPPH free radical scavenging potential of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

Fig. 3.13: ABTS free radical scavenging potential of selected sample extracts. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

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Fig. 3.14: Free radical scavenging potential of selected sample extracts by FRAP assay. All experiments were performed in triplicate. Data are presented as the mean ± SEM of n = 3 experiments.

3.8.3 Quantitative Estimation of Phenolic Acid by HPLC

The extraction of phenolics from the plant cell matrix is greatly influenced by extraction protocol, nature of chemical and solvent, particle size of sample and interfering moieties etc. Identification and quantification of phenolic acids in methanolic extracts of CTS and POL was done by HPLC with UV/DAD. Identification was done on the basis of retention time in comparison with the standard curves at 280 nm. Fig.3.15, Fig.3.16 and Table 3.3 representing the phenolic acids identified in CTSM and POLM. Thus in case of CTSM Sinapic acid, m-Coumaric acid,

Gallic acid and Quercetin were detected while in POLM were Gallic acid and Quercetin were seen.

Secondary metabolites are produced via methabolic pathway as side tract or side product which has great pharmacological and cytotoxic effect on animal and human. These are beneficial to some extent for plants itself because different processes like plant cell signaling, pollination, free radical scavenging activity monitored by alkaloid, terpenoids and flavonids, etc.

Table 3.3: Identification and Quantification of Phenolic acids by HPLC

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trans-4- hydroxy- 3- m- Galli methoxy SAMPLE Sinapi Coumari c Querceti Syringi Cinnami Feruli S c acid c acid acid n c acid c acid c acid 6.16.39 4.62 4.39 CTSM mg/g mg/g mg/g 0.39mg/g - - POLM 2.44 0.29mg/g - - - - - mg/g

Fig. 3.15: Identification and quantification of Phenolic acids in CTSM

Fig. 3.16: Identification and quantification of Phenolic acids in POLM

3.10 Antithrombotic Potential of Extracts

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An in vitro thrombolytic method was used to scrutinize the thrombolytic potential of selected fractions of plant extracts in blood sample drawn from healthy human volunteers, along with streptokinase as a positive control and water as a negative control (Prasad et al., 2006). Streptokinase, a known thrombolytic drug is used as a positive control (Tillett et al., 1933). The comparison of positive control with negative clearly demonstrated that clot dissolution does not occur when water was added to the clot. On the basis of the result obtained in this present study we can say that the CTS and POL extracts have moderate thrombolytic activity compared to control. Moreover, highest activity among extract fractions was shown by CTSM and minimum by POLE. The order antithrombotic potential was CTSM > CTSE > POLM

>POLE as shown in fig. 3.17

Fig. 3.17: In vitro thrombolytic activity of selected fractions of plant extracts with respect to positive control (Streptokinase) and negative control (water). All results are presented as mean and standard deviation for the three individual experiments. ***P value < 0.001 vs Streptokinase (control).

3.11 Screening of Antibacterial Activity of Samples

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Infectious diseases are ailment caused by the attack of particular pathogens like viruses or bacteria or fungus or protozoan parasite and their byproducts like toxin, capsule etc. These infections may also appear as a result of pathogen transmission as causative agent from infected person to the other host including human, animal etc. Host susceptibility for infection, severity and progression of infection, depend several factors like age, gene, gender, status of immunity and nutrition etc. Infectious disease considered leading cause of mortality in developing and underdeveloped countries accounting above 26 % of total reported deaths (Becker et al., 2006). No doubt after the discovery of antibiotic in 1926 and later development of its various kinds totally revolutionize the therapeutic of infections. Although marvelous effect of antibiotic is to cure the infection but on other hand it create resistance problem in microbe. Horrible examples of resistant mutated microbe emerge that become serious problem of today because they become difficult to treat (Barbour et al., 2004). Resistance problem in microbe come into sight as a result of microbe gene transformation between different species of host, gene mutagenesis or by the involvement of transposons, cryptic genes activation and chromosomal aberration as result of interaction between antibiotic induce oxidative stress and chromosome. Moreover, another main issue linked with the use of antibiotic is side effects that appear after its use. The domino effect of the arbitrary misuse of antibiotic is the development of resistant strains in animals and human (Barton, 2000). The cost issue is also another hindrance that cannot be ignored, especially in low income community.

The antimicrobial activity of methnolic extracts of CTS and POL against Gram positive and Gram negative bacteria was determined by disc diffusion method. Zones of inhibition were measured and tabulated in Table 3.5. Gram negative strains of bacteria such as Pseudomonas aerugenosa was inhibited by both extracts while Escherichia coli was not inhibited by extract of POL. Similarly CTS does not exhibit potential against Salmonella typhi. While on other hand, both CTS and POL exhibit significant potential against all selected strains of gram positive bacteria (Bacillus subtilis, Styphylococcus aureus, and Styphylococcus epidermidis). Percent inhition with respect to the positive control (Ciproflaxacin) is also expressed in Fig. 3.18.

Table 3.4: Zones of Inhibition (mm) in Disc Diffusion Plate for the antibacterial activity of successively extracted alcoholic extracts (SAE) of plants that have also antiviral potential.

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Bacterial Test organism Zone of inhibition (M+SD) mm Strains CTS POL Ciproflaxacin (Positive control) Gram positive Bacillus subtilis 8.5 ± 0.1 12± 0.4 23

Styphylococcus aureus 7.33 ± 11± 0.02 19 0.05 Styphylococcus 12.66 ± 12± 0.16 18 epidermidis 0.02 Gram Pseudomonas 8.30 ± 11.86 ± 20 negative aerugenosa 0.06 0.05 Escherichia coli 6.23± - 20 0.04 Salmonella typhi - 8.35± 25 0.03 The results are mean ± S.D. diameter of average of growth inhibition zone of three replicate. SD= Standard deviation

Fig. 3.18: Percent inhibition of bacteria by selected extracts with respect to ciproflaxacin (positive control)

3.12 Extraction, Purifcation and identification of

Compounds Obtained from CTSE Extract

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3.12.1 Compound CT-EtOAc-01

5' O 3' O 2'' 6'' 1' O 1' O 11 12 17 4'' 13 O 15 5''' 2''' 1 8 O O 1''' 4''' O 10 1''' 4''' 1''' 3 5 7 2''' O 2''' O O O O

CymIII Cym II Cym I

1H-NMR 1 ’’ H-NMR (400 MHz, CDCl3) δ (ppm): 8.10-8.12 (m, 1H, H-5 ), 7.85-7.86 (dd, J = 1.6, 9.6 Hz (2H, H-3’’ & H-7’’), 7.83-7.84 (m, 2H, H-4’’ & H-6’’), 4.59-4.61 (m, 1H, ’ H-3), 3.86-3.90 (m, 1H, H-12), 3.67 (s, 3H, CO2CH3), 2.17-2.72 (m, 1H, H-1 ), 2.12- 2.16 (m, 1H, H-17), 2.01-2.03 (m, 4H, H-6 & H-7), 1.85-1.89 (m, 1H, H-8/H-14), 1.387 (m, 1H, H-8/H-14), 1.78-1.87 (m, 2H, H-16), 1.71-1.76 (m, 2H, H-15), 1.63- 1.66 (m, 2H, H-2’), 1.68 (br. d, J = -10 Hz, 2H, H-11), 1.55-1.61 (m, 1H, H-14), 1.50 (dddd, J = -12.0, -12.0, 4.5, 4.5 Hz, 1H, H-5), 1.45 (m, 3H, H-1 & H-9), 1.24-1.28 (m, 2H, H-2), 1.23 (s, 3H, H-18), 1.12-1.15 (m, 2H, H-3’), 1.098 (s, 3H, H-19), 1.03-1.00 (3, 3H, H-5’) ’’' ’’’ ’’’ Cym-I: 4.48-4.51 (dd, J =1.6, 9.6 Hz, 8H, H-1 , H-2 , H-4 ), 3.40-3.55 (m, 4H, H- ’’’ ’’’ ’’’ 3 ), 3.39 (s, 3H, O-CH3), 1.30-1.33 (m, 1H, H-5 ), 1.45-1.60 (m, 9H, CH3 at C-5 ) Cym-II: 4.48-4.51 (dd, J=1.6, 9.6 Hz, 8H, H-1’’’, H-4’’’, H-5’’’), 3.40-3.55 (m, 4H, H- ’’’ ’’’ 6 ), 3.43 (s, 3H, O-CH3), 1.45-1.60 (m, 9H, CH3 at C-2 ) ’’' ’’’ Cym-III: 4.48-4.51 (dd, J=1.6, 9.6 Hz, 8H, H-1 , H-2 ), 3.45 (s, 3H, O-CH3), 2.28- ’’’ ’’’ ’’’ 2.32 (m, 1H, H-5),1.82-1.89 (m, 4H, H-3 , H-4 ), 1.45-1.60 (m, 9H, CH3 at C-5 ).

13C-NMR 13 ’’ ’’ C-NMR (300MHz, CDCl3) δ (ppm): 170.2 (CO2Me), 160.0(C-1 ), 133.0(C-5 ), 132.0(C-1’’), 129.6(C-3’’ & C-7’’), 129.7 (C-4’’ & C-6’’), 82.8 (C-3), 68.3 (C-12), 40.7 (C-5), 49.9 (C-1’), 55.0 (C-13), 46.1(C-14), 46.1(C-8), 46.1 (C-9), 43.7 (C-17), 40.7 (C-5), 39.0 (C-10), 37.1(C-7), 35.7 (C-4, C-11), 31.8 (C-1), 29.1(C-6), 28.5 (C-2, C- 16), 27.5 (C-2’, C-15), 26.2(C-3’), 25.6 (C-4’), 17.9 (C-5’), 12.30 (C-19), 10.0 (C-18) Cym-I ’’’ ’’’ ’’’ ’’’ ’’’ 101.2 (C-1 ), 76.9 (C-2 ), 72.8 (C-4 ), 71.3 (C-5 ), 55.7 (O-CH3), 34.3 (C-3 ), ’’’ 18.6 (CH3 at C-5 )

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Cym-II ’’’ ’’’ ’’’ ’’’ ’’’ 99.1(C-1 ), 77.0 (C-4 ), 73.9 (C-5 ), 71.4 (C-2 ), 58.1 (O-CH3), 34.3 (C-5 ), 19.2 ’’’ (CH3 at C-2 ) Cym-III ’’’ ’’’ ’’’ ’’’ ’’’ 95.6(C-1 ), 81.0(C-2 ), 71.7(C-5 ), 62.0(O-CH3), 35.6(C-3 ), 35.6 (C-4 ), 21.5 ’’’ (CH3 at C-5 )

The compound CT-EtOAc-01 was isolated as colorless amorphous powder (24mg)

25 with melting point 217-220°C, Rf value of 0.42 and [α]D of 18.61° (MeOH, C = 0.05). The appearance of absorption signals at 1710, 1608, 1504, 1235 cm-1 in IR indicates the presence of C=O (ester), C=C (aromatic) and C-O functionalities respectively.

The positive Lieberman-Burchard’s Molisch and Keller Kiliani tests confirmed the compound to be steroidal glycoside with 2-deoxysugars. The HR ESI MS exhibited

[M+Na] at 964 amu which was assigned to molecular formula C54H84O13 with 13 double bond equivalent (DBE). The 13C NMR revealed the presence of 54 carbon resonances that appeared as: 10 methyl, 24 methine, 15 methylene and 5 quaternary carbons. The Broadband (BB), DEPT (45°, 90° and 135°) and COSY analyses confirmed aglycon to be comprised of 33 carbons. The detailed NMR analyses also revealed the presence of two ester carbonyl carbons which appeared at 170.2 and 160 ppm in 13C NMR and were rationalized to be methyl ester moiety and benzoylic carbonyl respectively. The ester linkages were located to be at C-12 and C-1’ due to a significant downfield shift of the methine protons to which these ester functionalities were bound. C-17 appeared at 2.12-2.16 ppm while C-12 appeared at 3.86-3.90 ppm which is quite low field as compared to other methine protons.

The structure of the aglycone was solved from NMR analyses. The three methyls of the aglycone appeared at: 1.23 (s, H-18), 1.098 (s, H-19) and 1.03-1.00 (m, H-5’). The side chain at C-17 was identified by HSQC analysis which indicated presence of one methyl, one oxygenated methyl, 3 methylene, 1 methine and one quaternary carbon (carbonyl carbon). The HSQC confirmed the assignment of these protons which were found correlate with methyl carbons at 12.0, 9.5 and 17.9 ppm respectively. The HSQC spectrum confirmed C-10 and C-13 to be quaternary carbon while the C1’ was inferred to be attached with a triplet carbon (i.e. CH2) and a methine (C-17). The oxygenated methine carbons of the aglycon moiety (i.e., C-3 & C-12) appeared at

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3.86-3.90 ppm and 4.59-4.61 ppm which were correlated with oxygenated methine carbons at 82.8 and 68.3 ppm respectively by HSQC experiment.

The UV spectrum showed λmax 227, 268 and 283 nm which indicated presence of benzoyl system. The appearance of 5 protons (as three sets of multiplets with 2, 2 and 1 H integration), at 7.85-7.86, 7.83-7.84 and 8.09-8.12 ppm indicated the presence of a deshielded aromatic system which correlated with carbon resonances at 129.6, 129.7 and 133.0 ppm respectively. The singlet aromatic carbon appeared at 132.0 ppm. The 3 HMBC assignment showed a clear J correlation between the benzoyl carbonyl (δC:

160.0) and C-12 (δC: 82.8; δH: 3.86-3.90 ppm) which confirmed the benzoyl moiety to be attached at C-12. Similarly the location of methyl ester was confirmed by the correlation with C-1’. The absolute configuration of the isolated compound was left unresolved.

In addition to the aglycone signals, the 13C NMR also exhibited 21 signals which were assigned to the sugar part which was found to comprise of three hexoses. The 13C NMR showed the presence of three anomeric carbons which appeared at 95.6, 99.1 and 101.2 ppm. These values were correlated in HSQC experiment with the multiplet signal of 8H integration at 4.48-4.51 ppm. Since the peaks were not well resolved, exact and detailed assignment could not be performed.

The attachment of sugar moiety was confirmed to be at C3 by the significant 3 1 downfield shift of H in H NMR (δH: 4.59-4.61 ppm) and its corresponding carbon 3 resonance (δC: 68.3 ppm). The long range correlation between C and Cym-I also confirmed the attachment of sugar to be at C17. Therefore the compound was determined to be methyl(12-O-benzoyl-androstan-3-O-(14)-β-D-cymaropyranosyl- (14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl)hexanoate.

3.12.2 Compound CT-EtOAc-02

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O

2'' 6'' 1' O 1' O 18 O 11 12 17 4'' 19 13 O 15 5''' 2''' 1 8 O O 1''' 4''' O 10 1''' 4''' 1''' 3 5 7 2''' O 2''' O O O O

CymIII Cym II Cym I

1H-NMR(400 MHz, CDCl3): δ (ppm): 8.09-8.12 (m, 1H, H-5’’), 7.85-7.86 (dd, J = 1.6, 9.6 Hz (2H, H-3’’ & H-7’’), 7.83-7.84 (m, 2H, H-4’’ & H-6’’), 5.18 (br d, J = 5.4 Hz, H-6), 4.88-4.90 (m, H-1’), 4.59-4.61 (m, 1H, H-3), 3.86-3.90 (m, 1H, H-12), 2.73 ’ (s, 3H, COCH3), 2.06 (s, 3H, CH3 at H-1 ), 2.01-2.04 (m, 4H, H-7), 1.85-1.89 (m, 1H, H-8/ H-14), 1.78-1.87 (m, 2H, H-16), 1.71-1.76 (m, 2H, H-15), 1.65 (br d, J = 10 Hz, 2H, H-11), 1.42-1.45 (m, 1H, H-17), 1.38 (m, 1H, H-8/H-14), 1.28 (s, 3H, H-18), 1.03-1.01 (m, 5H, H-1, H-2, H-9), 1.00 (s, 3H, H-19).

’’' ’’’ ’’’ Cym-I: 4.48-4.51 (dd, J =1.6, 9.6 Hz, 8H, H-1 , H-2 , H-4 ), 3.40-3.55 (m, 4H, H- ’’’ ’’’ ’’’ 3 ), 3.39 (s, 3H, O-CH3), 1.30-1.33 (m, 1H, H-5 ), 1.45-1.60 (m, 9H, CH3 at C-5 ) Cym-II: 4.48-4.51 (dd, J=1.6, 9.6 Hz, 8H, H-1’’’, H-4’’’, H-5’’’), 3.40-3.55 (m, 4H, H- ’’’ ’’’ 6 ), 3.43 (s, 3H, O-CH3), 1.45-1.60 (m, 9H, CH3 at C-2 ) ’’' ’’’ Cym-III: 4.48-4.51 (dd, J=1.6, 9.6 Hz, 8H, H-1 , H-2 ), 3.45 (s, 3H, O-CH3), 2.28- ’’’ ’’’ ’’’ 2.32 (m, 1H, H-5),1.82-1.89 (m, 4H, H-3 , H-4 ), 1.45-1.60 (m, 9H, CH3 at C-5 ).

13C-NMR 13 ’’ C-NMR (300MHz, CDCl3) δ (ppm): 170.2 (CO2Me), 160.0(C-1 ), 140.7 (C-5), 133.0(C-5’’), 132.0(C-1’’), 129.6(C-3’’ & C-7’’), 129.5 (C-4’’ & C-6’’), 129.1 (C-6), 82.8 (C-3), 75.3 (C-1’), 68.3 (C-12), 55 (C-12), 49.9 (COCH3), 49.9 (C-1’), 55.0 (C- 13), 46.1 (C-14), 46.1(C-8), 46.1 (C-9), 43.7 (C-17), 39.0 (C-10), 37.1 (C-7), 35.7 (C- 4, C-11), 31.8 (C-1), 28.5 (C-2, C-16), 27.5 (C-15), 25.6 (C-4’), 17.9 (C-5’), 12.30 (C-19), 10.0 (C-18) Cym-I ’’’ ’’’ ’’’ ’’’ ’’’ 101.2 (C-1 ), 76.9 (C-2 ), 72.8 (C-4 ), 71.3 (C-5 ), 55.7 (O-CH3), 34.3 (C-3 ), ’’’ 18.6 (CH3 at C-5 ) Cym-II ’’’ ’’’ ’’’ ’’’ ’’’ 99.1(C-1 ), 77.0 (C-4 ), 73.9 (C-5 ), 71.4 (C-2 ), 58.1 (O-CH3), 34.3 (C-5 ), 19.2 ’’’ (CH3 at C-2 ) Cym-III ’’’ ’’’ ’’’ ’’’ ’’’ 95.6(C-1 ), 81.0(C-2 ), 71.7(C-5 ), 62.0(O-CH3), 35.6(C-3 ), 35.6 (C-4 ), 21.5 ’’’ (CH3 at C-5 )

The compound CT-EtOAc-02 was isolated as colorless amorphous powder (28.32

25 mg) with melting point 205°C, Rf value of 0.39 and [α]D of [+18.2°] (MeOH,

116

C=0.05). The IR absorption signals at 1734, 1724, 1456, 1377, 1274 cm-1 in indicates the presence of two C=O (ester), C=C (aromatic) and C-O functionalities.

Positive Molisch, Lieberman-Burchard, and Keller Kiliani tests confirmed the compound to be steroidal glycoside comprising 2-deoxysugars. The HR ESI MS exhibited [M+Na] at 919 amu which was assigned to molecular formula C51H76O13 with 14 double bond equivalent (DBE). The 13C NMR and the DEPT spectras of compound revealed the presence of 51 carbon resonances that appeared as: ten methyl, twenty two methine, eleven methylene and six quaternary carbons. The Broadband (BB), DEPT (45°, 90° and 135°) and COSY analyses confirmed aglycon to be comprised of 33 carbons. The detailed NMR analyses also revealed the presence of two ester carbonyl carbons which appeared at 171.2 and 161 ppm in 13C NMR and were rationalized to be acetoxy group at C-1’ and benzylic carbonyl respectively. The ester linkages were located to be at C-12 and C-1’ due to a significant downfield shift on the methine protons to which these ester functionalities were bound. C-1’ appeared as multiplet at 4.88-4.90 ppm or δ while C-12 appeared at 3.86-3.90 ppm which is quite low field as compared to other methine protons. Moreover, appearance of singlet for three protons of at 2.73 ppm also specifies the presence of methyl of acetoxy group. Appearance of signal of olefinic proton at 5.18 δ (br d) in 1H NMR spectrum point toward the confirm presence of Δ5 pregnane glycoside. Presence of aromatic rings in aglycone backbone was also confirmed by UV which exhibited max at 270 nm.

The structure of aglycone part was solved from NMR analyses. The three methyls of 19 the aglycone appeared at 1.00 (methyl of C ), 2.73 ppm (COCH3) and 1.24-1.28 ppm (methyl of C18). The side chain at C-17 was identified by HSQC analyses which indicated presence of one methine to which one methyl is bound. The HSQC spectrum confirmed C-10 and C-13 to be attached with quaternary carbon while the C-1’ was inferred to be attached with acetoxy moiety. The oxygenated methine carbons of the aglycon moiety appeared at 3.86-3.90 ppm (H-12) and 4.59-4.61 ppm (H-3) which was correlated with oxygenated methine carbons at 68.3 and 82.8 ppm respectively by HSQC experiment.

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In addition to the aglycone signals, the 13C NMR also exhibited 21 signals which were assigned to the sugar part which was found to comprise of three hexoses. The 13C NMR showed the presence of three anomeric carbons which appeared at 95.6, 99.1 and 101.2 ppm. These values were correlated in HSQC experiment with the multiplet signal of 8H integration at 4.48-4.51 ppm. Since the peaks were not well resolved, exact and detailed assignment could not be performed.

The attachment of sugar moiety was confirmed to be at C-3 by the significant 3 1 downfield shift of H in H NMR (δH: 4.59-4.61 ppm) and its corresponding carbon 3 resonance (δC: 68.3 ppm). The long range correlation between C and Cym-I also confirmed the attachment of sugar to be at C17. Therefore the compound was determined to be methyl (12-O-benzoyl-androstan-3-O-(14)-β-D-cymaropyranosyl- (14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl) hexanoate.

3.12. 3 Compound CT-EtOAc-03

5'

O 3'

2'' 6'' 1' 1' 2'''' O 18

11 12 17 4'' 19 13 4'''' O 15 5''' 2''' 1 8 O O 1''' 4''' O 10 1''' 4''' 1''' 3 5 7 2''' O 2''' O O O O

CymIII Cym II Cym I

Compound CT-EtOAc-03 (C55H86O11) was obtained from the ethyl acetate extract of 25 Caralluma tuberculata as colourless, amorphous powder in 33 mg along with [α]D

[+17.2°] (MeOH, C=2). Rf value calculated from TLC was 0.41.

Significant peaks obtained from IR spectra are at 1720, 1507, 1200 indicate the presence of ester, and aromatic functional groups. The molecular weight of the compound was determined by obtained by high resolution ESI-MS which exhibited [M+Na] at 946 amu which suggested molecular formula of compound to be

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C54H84O13 with 13 double bond equivalent (DBE). In this structure total number of hydrogen atoms are eighty six and carbon atoms are fifty five among which quaternary carbons are four, tertiary carbon (methine group) are nineteen, secondary carbon (methylene group) are fifteen and primary carbon (methyl group) are seven. Moreover three methoxy groups are also present.

Most of spectral signal appeared on the same position as in the previous one with a little bit variation only the difference is of the disappearance of singlet peak of methyl ester protons at 3.37 δ. And this position was occupied by appearance of additional protons due to isopropylic group indicate the given compound. A multiplet peak ranging from 1.01 to 0.98 indicates H-1’. Presence of double bond between C-6 & C- 7 is indicated by doublets at position 5.73 & 6.12 δ for H-6 & H-7.

In addition to the aglycone signals, the 13C NMR also exhibited 21 signals which were assigned to the sugar part which was found to comprise of three hexoses. The 13C NMR showed the presence of three anomeric carbons which appeared at 95.6, 99.1 and 101.2 ppm these values were correlated in HSQC experiment with the multiplet signal of 8H integration at 4.48-4.51s ppm. Since the peaks were not well resolved, exact and detailed assignment could not be performed. Therefore the compound was determined to be Methyl (23-O-benzoyl-androst-6-en-17-O-(14)-β-D- cymaropyranosyl-(14)-β-D-cymaropyranosyl--(14)-β-D-cymaropyranosyl) hexanoate.

3.12.4 Compound CT-EtOAc-04

O

2''' O 2'' O O 1''' 4''' O 6'' 1'' O O 4''' 1''' 1' 2' 2''' 4' 11 12 17 O 4'' 13 O O 15 1 8 6' 10 Cym II Cym I 3 5 7

119

The compound CT-EtOAc-04 (C45H58O10 ) was isolated as colorless amorphous 25 powder (28.3 mg) with melting point 196°C, Rf value of 0.54 and [α]D of [+24.6°] (MeOH, C=0.1). The IR absorption signals at 1716, 1698, 1444, 1298 cm-1 in indicates the presence of two C=O (ester), C=C (aromatic) and C-O functionalities.

Positive Molisch, Lieberman-Burchard, and Keller Kiliani tests confirmed the compound to be steroidal glycoside comprising 2-deoxysugars. The HR ESI MS exhibited [M+Na] at 782 amu which was assigned to molecular formula C45H58O10 with 17 double bond equivalent (DBE). The 13C NMR and the DEPT spectras of compound revealed the presence of 45 carbon resonances that appeared as: four methyl, twenty five methine, nine methylene and five quaternary carbons. The Broadband (BB), DEPT (45°, 90° and 135°) and COSY analyses confirmed aglycon to be comprised of 31 carbons. The detailed NMR analyses also revealed the presence of two ester carbonyl carbons which appeared at 168.5 and 161 ppm in 13C NMR and were rationalized to be benzoyl group at C-12 and benzyloxy carbonyl at C-1’ respectively. The ester linkages were located to be at C-12 and C-1’ due to a significant downfield shift on the methine protons to which these ester functionalities were bound. Therefore the compound was determined to be Phenyl (12-O-benzoyl- gonan-6-en-5’’-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl) oxalate.

3.12.5 Compound CTEtOAc-05

O 3''

1'' HO O 5 7 O O 9 HO 1' 8 3 HO OH H3CO OH

120

1 H-NMR (400 MHz, CDCl3): δ (ppm): 8.09 (d, j=1.45 HZ, 2H), 7.84 (d, j=7.2Hz, 1H), 7.63-7.45 (m, 2H), 7.50-7.40 (m, 3H), 7.24-7.105 (m, 1H), 4.89-4.72 (m, 5H), 4.47 (d, j=10.43, 1H), 3.79 (t), 3.66 (s, 3H, OMe),2.33 (t, 5H, J=7.63H), 1.99-2.11 (m, 1H),1.55-1.68 (m, 2H). 13C-NMR Aglycone 13 C-NMR (300MHz,CDCl3): δ (ppm): 144 (C-4), 138 (C-1), 137 (C-3), 132 (C-5), 130 (C-7), 127 (C-8), 126 (C-6), 122 (C-2), 60 (C-9), 58 (OME) Glycone 13 C-NMR (300MHz,CDCl3): 103 (C-1’), 80 (C-3’), 72 (C-5’), 71.8 (C-2’), 68 (C-4’), 65.2 (C-6’) Glycone substituent: 13 C-NMR (300MHz,CDCl3): 174 (C-1’’), 60 (C-3’’), 58 (C-2’’), 18 (C-4’’)

Compound 5 was obtained from the initial fractions of ethyl acetate extract of

Caralluma tuberculata during column chromatography using Hexane and ethyl acetate as mobile fraction. This compound was colourless, amorphous powder of 22.51 mg

25 along with [α]D [+17.2ο] (MeOH, C=2). Rf value calculated from TLC was 0.61.

Significant peaks obtained from IR spectra are at 3500, 2810, 1740, 1470, 1230 indicate the presence of alcoholic, methane, ester, carbonyl and aromatic functional groups. The molecular weight of the compound was determined by molecular ion peak obtained by high resolution ESI-MS and it was [M]= 414 amu. Empirical formula i.e C20H26O10 was calculated after the interpretation of spectral data of NMR and with the unsaturation 13 double bond equivalent (DBE). The presence of total twenty carbons in the glycone and aglycone moieties of the structure was confirmed by 13CNMR. Presence of substituent at position 6’ was confirmed due to downfield shift observed in 13CNMR at 65.2 ppm. Present compound was already reported but in different plant species Capsicum annuum L.(Materiska et al., 2003) and its name was determined to be 3-Hydroxy-2-methyl-propionic acid 3,4,5-trihydroxy-6-[4-(3- hydroxy-propenyl)-2-methoxy-phenoxy]-tetrahydro-pyran-2-ylmethyl ester or commonly called Trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside.

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3.13 Discussion

The momentous spread of life threatening disease like HCV, insufficient virus structure targeted treatment, dearth of vaccination, greater side effects and high cost of authenticated therapy prompted the researchers and health professional to explore some suitable alternative but better targeted approaches to control this dilemma. These alternatives must be efficacious, economical and free of such side effects. Natural products either plant extract or the isolated pure compounds or the standardized herbal extract offers the indefinite prospects for novel drug discovery owing to their matchless chemical miscellany (Cos et al., 2006). Botanical extracts in conjunction with their flurry of active constituents occupied a central position and were used successfully against dreadful infectious diseases, various cancers and metabolic disorders (Duraipandiyan et al., 2006). Turning toward ethnopharmacognosy is helping us to discover numerous plant based constituents having antioxidant, anticancer, antifungal, antibacterial and antiviral potential.

On the basis of folk knowledge, eastern system of medicine and exploration of plants from the valleys and unexplored areas is the focus of research now a day (Ahmad et al., 2008). The plant communities are losing their species richness at a high rate due to intensive deforestation and unlimited expansion of urban areas. Through bioassay- directed screening, structure–activity relationship, and biochemical investigation, identification of bioactive compounds as potential molecular targets of HCV-NS3 is a hot issue, which may offer impending expansion of potential anti-HCV regimens.

In the present study, fifteen indigenous medicinal plants (listed in Table 2.1) were scrutinized for their therapeutic potential like anti-HCV activities (HCV whole virus and its non-structural proteins NS3/NS4). Recently non-structural proteins of HCV (NS3 and NS5B) are centres of attraction as promising targets for drug development against HCV owing to their key role in HCV replication. The binding of anti HCV agents to the extremely conserved domain at the junction of NS3 protease and helicase domain results the discovery of distinctive class of DAAs (Saalau-Bethell et al., 2012).

In first part of the study, plants were collected from the Soon-sakaser valley of Punjab, Pakistan, after primer folkare information. The pre washed and dust free plant

122 samples were shadow dried at room temperature and ground in the form of fine, homogeneous, coarse powder and their methanolic extracts were obtained after successive extraction. Proper operation must be followed in order to obtain the maximum surface area of the sample for solvent action and finally improve the kinetics of analytical extraction. Extraction is the essence of medicinal plant research that helps to obtain maximum desirable constituents from plants to utilize further for mining and depiction of all details. Extraction should be cautious enough to assure that no active principal or compound was lost or damaged or distorted during this procedure (Farnsworth, 1966). Selection of solvent type depends upon the nature of targeted compound, for example polar solvents (ethanol, methanol and ethyl acetate) are usually preferred for isolation of hydrophilic compounds (Cos et al., 2006). Similarly, sorts of extraction schemes were used to obtain crude extracts depending upon the type of test material, i.e. fresh or dry plants and their powder.

In vitro cellular toxicity studies were done for all plant extracts using Huh-7 and CHO cells by Trypan blue exclusion method and MTT assay. Phyto extracts exhibited a broad range of cytotoxic amount varying from the 1.5 µg/mL to the 200 µg/mL. Cytotoxicity data of only two promising plants POL and CTS, which latter exhibit strong anti HCV potential in activity guided assay, is reported in Fig. 3.2 (A & B) while Cytotoxicity data through MTT cell proliferation method is reported in Fig. 3.3 (A & B). Data from both assays show that concentration of CTS and POL did not exerted any effect on viability of CHO and Huh-7 cells. Cellular toxicity data of other plants that did not show convincing potential against HCV is not presented. After the toxicological analysis of plant extracts, the in vitro screening bioassays of plants against HCV facilitate to explore the anti HCV agents from these phyto sources. In case of serum inhibition assay, hepatic cell line was infected with positive serum (high titre of HCV) of 3a genotype for screening of plants against whole virus. Crude methanolic extracts of selected plants were screened against HCV infected liver cell line and the results are displayed in Fig. 3.4. Only two methanolic extracts (CTS and POL) exhibit potent and convincing anti HCV potential as shown in Fig. 3.5 (A&B). The viral load was reduced to more than 60 % in case of POL while 57 % in case of CTS which confirm that both possess appreciable antiviral potential. Both plant’s methanolic extracts (POLM and CTSM) were suspended in water then further fractionated with n-hexane (POLH and CTSH), ethyl acetate (POLE and CTSE),

123 chloroform (POLC and CTSC) and n-butanol (POLB and CTSB). These different solvent fractions were screened for their anti HCV activity as well. Among these, only the ethyl acetate fractions (CTSE & POLE) from both plants significantly reduced the viral titer (68 % and 74 %) as presented in Fig. 3.5 (A&B).

For the purpose of in vitro protease inhibition, Huh-7 cells were transfected with mammalian expression vector (PCR3.1/FLAGtag/HCV NS3) in the absence and presence of selected plant extracts. Fig. 3.6 (A&B) and Fig. 3.7 (A&B) represents the qualitative & quantitative inhibition of HCV NS3 protease by IFN, methanolic and ethyl acetate extract of CTS and POL. Moreover in case POL, its inhibitory effect on the protein expression level of NS3 transfected into Huh-7 cells was also confirmed by Western blot. There was a dramatic reduction in the protein expression of the NS3 gene after treatment of POL extracts (Fig. 3.8). There were 60 % and 70 % inhibition in Huh-7 cells treated POM and POE respectively. This response is likely to be comparable to in vitro inhibition of HCV NS3-SP with ethanolic extract of Rhodiola kirilowii (Chinese herb) and Camellia sinensis Epigallocatechin-3-gallate compound (Chen et al., 2012; Zuo et al., 2007). NS3-mediated processing of protein junctions among the non-structural proteins is indispensible for HCV replication and hence recognized as striking target for antiviral agents’ development (Pause, et al., 2003). Moreover, NS3 may also interfere with the functions host cell like protein kinase A arbitrated signal transduction and inhibition of cell transformation (Borowski et al., 1996; Sakamuro et al., 1995). Besides POL and CTS, a number of studies have explored bioactive products from Galla Chinese, Rhodiola kirilowii, Lonicera hypoglauca Miq. Swietenia macrophylla, Solanum nigrumand Camellia sinensis with anti-NS3 protease activity (Duan et al., 2004; Wang et al., 2009; Zuo et al., 2007). Previous reports have verified that Epicatechin and Epigallocatechin compounds from rhizomes of the Chinese therapeutic herb have remarkable activity against NS3 serine protease of HCV (Chen et al., 2012).

The phytochemical analysis of methanolic extracts of CTS and POL confirms the presence of specific constituents in significant amounts as shown in table Table 3.1 and Table 3.2. Qualitative screening of methanolic extracts of CTS and POL pointed toward the occurrence of various constituents as revealed in Table 3.1. CTS extract was found to be rich with glycosides, steroids, saponin, coumarine and resins, but on

124 other hand, POL extract was rich in anthraquinones, fatty acids, flavonoids, tannin and resins. The Preliminary phytochemical screening demonstrated that hexane fractions were rich in non-polar compounds like steroids etc., while ethyl acetate fractions sustained high concentration of flavonoids. Presence of these compounds provided a foundation for identification of unknown but excellent therapeutic agents and to structure those through spectral data.

As we know that antioxidants exhibit strong anti cancer potential and hepatocellular carcinoma appear in 15 % cases of patients of HCV, which leads us for the verification of antioxidant potential of POL and CTS extracts. Fig 3.9 to 3.11 contain the data for antioxidant potential in term of total phenolic, flavonoids and total carotenoids contents of biologically active plant extract fractions while Fig. 3.12-3.14 represent the free radical scavenging activity of extracts by DPPH, ABTS and FRAP assays. In case of in vitro anti thrombotic potential, highest activity among extract fractions was shown by CTSM and minimum by POLE while the overall order of antithrombotic potential was CTSM > CTSE > POLM >POLE (Fig. 3.17). The antibacterial activity of methnolic extracts of CTS and POL against Gram positive (Bacillus subtilis, Styphylococcus aureus and Styphylococcus epidermidis) and Gram negative (Pseudomonas aerugenosa, Escherichia coli and Salmonella typhi) strains of bacteria was determined by disc diffusion method (Table 3.4). Percent inhibition with respect to the positive control (Ciproflaxacin) indicated that both CTSM and POLM exhibited more than 60 % inhibition against Styphylococcus epidermidis.

Carlluma tuberculata (CTS) is widely grown succulent perennial herb that is not only eaten as raw famine food, but also cooked as vegetable in some area. It is considered valuable due to its potential use against diabetes, cancer and inflammation. Prior to our work, CTS and POL antiviral specifically anti HCV potential was not reported. The most potent ethyl acetate fraction of CT was further utilized to isolate the compounds through flash chromatography using silica gel 60. The structure of steroidal glycosides was deduced by 1H,13C-NMR, DEPT, COSY and mass spectral data. Five different compounds CT-EtOAc-01 i.e. ethyl(12-O-benzoyl-androstan-3-O- (14)-β-D-cymaropyranosyl-(14)-β-Dcymaropyranosyl-(14)-β-D- cymaropyranosyl) hexanoate, CT-EtOAc-02 i.e. Methyl (12-O-benzoyl-androstan-3- O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl--(14)-β-D-

125 cymaropyranosyl) hexanoate, CT-EtOAc-03 i.e. Methyl (23-O-benzoyl-androst-6-en- 17-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl--(14)-β-D- cymaropyranosyl)hexanoate, CT-EtOAc-04 i.e. Phenyl (12-O-benzoyl-gonan-6-en- 5’’-O-(14)-β-D-cymaropyranosyl-(14)-β-D-cymaropyranosyl)oxalate and CT- EtOAc-05 i.e. Trans-p-ferulylalcohol-4-O-(6-(2-methyl-3-hydroxypropionyl) glucopyranoside were isolated, purified and characterized. which have potential to be further utilized for docking studies with the structure of NS3-SP to confer further mechanism.

The potency of bioactive compounds from CTS and POL is very important and well proven with an additional edge of being wildly grown natural source. The novel antiviral activity of compounds from these plants presents an attractive lead for natural chemical entities for the development of potential anti-HCV agents. Additionally, if natural molecules cannot be replaced in the present anti-HCV treatment, these could be used in combination or supplementation to interferon thus reducing overall treatment cost. The above data also proposes that the therapeutic importance of extracts may lead to an alternative, more potent oral treatment for chronic HCV.

4.0 Future Prospects

Findings of the present project “Bioassay directed characterization of selected indigenous medicinal plants for their anti-HCV potential” indicates that both plant

126 species having code CTS and POL are very useful and non toxic medicinal plants that have strong therapeutic potential against HCV specifically its protein NS3. In short, it can be speculated that therapeutic spur of antiviral plant extracts or purified fractions alone or in mixture with IFN could offer an effective therapy alternative against HCV whole virus and it NS3 protease. Structural analysis of correspondent antiviral active fraction might prove supportive for identification of new antiviral compounds.

As both species fall mostly in the category of uncultivated and uncommon vegetables, so these offer a very inexpensive source of medication, especially to poor rural peoples who live in such vicinity where these grow. There is dire need of hour to grow these plants commercially in order to attain the benefit from their medicinal potential and to fulfill the global demand.

Although very few efforts has been made to explore biologically active constituent of these flora, but these are thoroughly known for their therapeutic potential against infectious and metabolic disorders. Focus was only given to isolate compound and identify them but not explored the effect of these compounds at molecular level and their possible mechanism of action.

Docking studies are considered very important in rational drug designing as it decide the preferred orientation of drug or compound toward the target (NS3 serine protease). So such finding would strengthen our goal to develop target based drugs and then their examination on in vivo animal model. Docking of antiviral compounds will determine the preferred orientation of antiviral compounds towards NS3 protease. Docking is important in rational drug designing. Designing structures by docking marvel ligands to create potent active sites and then compare docked compounds to known active compounds is useful for designing novel candidate drugs.

The climate of the Soon-valley is subtropical with unique geological formation due to salt as well as other minerals in the area. In the local vegetation, at least work has been conducted with respect to medicinal point of view. Intensive efforts needed to devote toward the discovery of innovative HCV antiviral agents. Owing to the marvellous therapeutic potential of these extracts there is a possibility to develop such drugs from these. There are innumerable plants in this valley waiting to be explored

127 for their therapeutic applications against genetically and functionally diverse virus families.

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6.0 Appendix

Table of Contents 1.0 Introduction ...... 21 1.1 Natural Products from Times of Yore to 21st Millennium...... 21 1.2 Status of Medicinal Plants and their Research in Pakistan ...... 23 1.3 Preface to Genus Caralluma and Caralluma tuberculata ...... 24 1.3.1 Active Constituents ...... 25 1.3.2 Pharmacological worth of Caralluma tuberculata and other species of Caralluma ...... 30 1.4 Introduction to Portulaca oleracea ...... 34 1.4.1 Phytochemistry of Portulaca oleracea...... 36 1.4.2 Pharmacological attributes of Portulaca oleracea ...... 36 1.5 Hepatitis...... 40 1.6 Hepatitis C and its Virus ...... 40 1.6.1 Transmission of Pathogen and Genotype Prevalence ...... 41 1.6.2 HCV Structure and Genome ...... 42 1.6.3 HCV Non-Structural and Structural Proteins ...... 43 1.6.4 HCV RNA Replication and Potential Drug Target...... 48 1.6.5 In vitro Infection Systems for HCV Replication ...... 49 1.6.6 Available Treatment Options against HCV ...... 53 1.6.7 NS3 Gene as Candidate Drug Target...... 54 1.6.8 Ayruvadic Extract and Plant Derived Compounds as anti HCV Drug ...... 57 1.7 Goals/Objectives ...... 60 2.0 Chemicals, Equipment and Techniques ...... 61 2.1 Chemicals ...... 61 2.1.1 Materials for Chromatography ...... 61 2.1.2 Chemicals and Reagents used for Determination of Antioxidant...... 62 2.2 Instrument and Apparatus ...... 63 2.3 Materials used for Biological Activities ...... 63 2.3.1 Cell Lines ...... 63 2.3.2 Plasmids ...... 63 2.3.3 Reagents & Chemicals ...... 64 2.3.4 Designing of Primers ...... 64

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2.4 Instrumentation & Techniques ...... 64 2.4.1 Physical Constant ...... 65 2.4.2 Spectroscopy ...... 65 2.4.3 Thin Layer Chromatography ...... 65 2.4.4 Column Chromatography (CC) ...... 65 2.5 Methodology ...... 66 2.5.1 Biochemical Analysis of Plants ...... 66 2.5.2 Collection, Identification and Preservation of Medicinal Plant Materials ...... 66 2.5.3. Plant Extract Preparation...... 68 2.5.4 Screening of Plant Extracts for Cellular Toxicity Analysis via Trypan Blue Dye Exclusion Method ...... 68 2.6 Anti-HCV Analysis of Compounds in Liver Cells...... 70 2.6.1 Antiviral Analysis of Plant Extract against HVC Ns3 Protease via Transfection Assay ...... 71 2.7 Gene Expression studies via Reverse Transcriptase PCR (RT-PCR) ...... 71 2.7.1 Extraction of Total RNA ...... 71 2.7.2 Synthesis of cDNA ...... 72 2.7.3 Reverse Transcription PCR (RT-PCR) for Gene Expression Analysis ...... 72 2.7.4 Expression studies via Real-time PCR...... 73 2.8 Western Blotting of PO extracts ...... 74 2.8.1 Protein Isolation ...... 74 2.8.2 Protein Quantification ...... 75 2.8.3 Protein Samples Preparation ...... 75 2.8.4 SDS Gel Electrophoresis ...... 75 2.8.5 Transfer of Protein and Immunoblotting ...... 76 2.8.6 Enhanced Chemiluminescence (ECL) Detection ...... 76 2.9 Analysis of the Two Plants Extract That Exhibit Positive Potential against HCV ...... 77 2.10 Phytochemical Screening ...... 77 2.10.1 Qualitative Analysis of the Extracts ...... 77 2.10.2 Quantitative Determination of the Chemical Constituency Specifically Antioxidants ...... 80 2.11 Anti-clotting Properties of CTS and POL Extracts ...... 84 2.11.1 Sample Preparation ...... 85 2.11.2 Preparation of Anti-Clot Test Solution ...... 85 2.11.3 Clot Lysis ...... 85

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2.12 Antibacterial assay of extracts ...... 85 2.13 Bioassay guided extraction of CTS and POL ...... 86 2.14 Isolation of Compounds via Column Chromatography ...... 87 2.14.1 Isolation of Compounds ...... 87 2.14.2 Physico-chemical Characteristics of Isolated Compounds...... 88 2.14.3 Elucidation of Compounds Structure by spectroscopy ...... 88 3.0 Results and Discussion ...... 90 3.1 Collection of Botanical Samples and Preparation of Extract ...... 91 3.2 Percentage yield of plants extract...... 92 3.3. Toxicological Studies of Plant Extracts in CHO and Liver Cell Lines ...... 92 3.3.1 Trypan Blue Dye Exclusion Assay ...... 93 3.3.2 Cytotoxic Screening of Phyto Extracts via MTT Assay ...... 93 3.4 Anti-HCV Assay ...... 96 3.5 Inhibition of HCV NS3 Gene Expression by Selected Organic Extracts ...... 99 3.6 Western Blotting ...... 101 3.7 Phytochemical analysis...... 102 3.7.1 Qualitative Analysis of CTS and POL ...... 102 3.7.2 Quantitative Methods for Screening of Phytoconstituents ...... 103 3.8 Antioxidant Potential of Biologically Active Plant Extract Fractions ...... 103 3.8.1 Total Phenolic, Total Flavonoids and Total Carotenoid Contents ...... 103 3.8.2 Free Radical Scavenging Activity of Extracts by DPPH, ABTS and FRAP Assays ...... 106 3.8.3 Quantitative Estimation of Phenolic Acid by HPLC ...... 108 3.10 Antithrombotic Potential of Extracts ...... 109 3.11 Screening of Antibacterial Activity of Samples ...... 110

3.12 Extraction, Purifcation and identification of Compounds Obtained from CTSE Extract ...... 112 3.12.1 Compound CT-EtOAc-01 ...... 113 3.12.2 Compound CT-EtOAc-02 ...... 115 3.12. 3 Compound CT-EtOAc-03 ...... 118 3.12.4 Compound CT-EtOAc-04 ...... 119 3.12.5 Compound CTEtOAc-05 ...... 120 3.13 Discussion ...... 122 4.0 Future Prospects ...... 126 5.0 References ...... 128 148

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