Deanship of Graduate Studies Al-Quds University Phytochemical screening of Cuscuta palaestina, Gundelia tournefortii, Pimpinella anisum and Ephedra alata natural plants and their in-vitro cytotoxic effects Sadam Ahmad Mahmoud Makhamra M.Sc. Thesis Jerusalem-Palestine 1437-2016 Phytochemical screening of Cuscuta palaestina, Gundelia tournefortii, Pimpinella anisum and Ephedra alata natural plants and their in-vitro cytotoxic effects Prepared By: Sadam Ahmad Mahmoud Makhamra B.Sc., Applied chemistry, Palestine Polytechnic University, Palestine. Supervisor: Prof. Dr. Saleh Abu-Lafi A thesis submitted in partial fulfillment of requirements for the degree of the Master of Pharmaceutical Industries technology in the Department of Chemistry / Program of Postgraduate Studies in Applied and Industrial Technology Faculty of Science and Technology/ Al-Quds University. 1437/2016 Declaration I certify that this thesis submitted for the degree of master is the result of my own research, except where otherwise acknowledged, and this thesis has not been submitted for the higher degree to any other university or institute. Signed: ………………….. Sadam Ahmad Mahmoud Makhamra Date: 3-8-2016 I Abstract The aim of this research is to assess the in-vitro antiproliferative activities of the crude extract, preparative HPLC fractions and pure active compounds in selected Palestinian plants, namely Cuscuta palaestina, Gundelia tournefortii, Pimpinella anisum and Ephedra alata. Chromatographic and spectroscopic techniques such as analytical HPLC-PDA, preparative HPLC-PDA, GC-MS, 1H-NMR, and LC-MS and biological assays were used to achieve this goal. Cuscuta palaestina is listed as native plant of flora palaestina. The methanolic and hexane extracts were used to determine its activity against human colorectal carcinoma cell line. The in-vitro results using MTT and the LDH leakage assays, gave EC50 values of 71.55 ± 4.75 µg/ml for methanol extract and 175.21 ± 3.89 µg/ml for hexane extract. The methanolic extract exerted a significant anti-proliferative effect on the HCT-116 cancer cell line. Sesamin and two other Phytosterols namely Campesterol and Stigmasterol, have been identified in this plant and therefore could be the major source for their anticancer activity. The Sesamin percentage in the methanolic extract is found to be three times more than Sesame seeds. Gundelia tournefortii is an Artichoke-like edible wild thistle is commonly used in Palestinian Cuisine. Its anticancer effects on the Human colon carcinoma cell line was investigated. Methanol and hexane extracts were found to exert considerable antitumor activity against HCT-116 cancer cell line with EC50 values of 303.3 ± 12 µg/ml, 313.3 ± 18.6 µg/ml respectively, while the aqueous extract was inactive. Phytochemical identities in both methanol and hexane extracts using GC-MS revealed about seventy compounds. Six potential compounds are β-Sitosterol, Stigmasterol, Lupeol, Gitoxigenin, α-Amyrin and Artemisinin. Eight fractions were collected by preparative-HPLC and tested in-vitro. The cytotoxic effect of fraction VIII was found to be the highest against HCT-116 cancer cell line. Pimpinella anisum L. (Anise) was found to have potential anticancer activity on human prostate cancer cell line (PC-3). Six fractions of anise methanolic extract were collected using preparative-HPLC. The obtained EC50 value as measured for fractions II, III, IV, and V by the MTT assay was 0.5 ± 0.10 mg/ml, 0.3 ± 0.11 mg/ml, 0.49 ± 0.12 mg/ml, 0.23 ± 0.10 mg/ml, respectively. II Ephedra alata acquired recently a tremendous reputation after the use of its stems to heal a Palestinian farmer from Jenin. The in vitro antiprolliferative activity of its methanolic extract gave EC50 value of 25 ± 1.2 µg/ml by using MTT assay against HepG2 cell line (human liver cancer). Therefore, the four investigated Palestinian plants could be further investigated to reach new anticancer lead or drug and to study mechanism of action and evaluate efficiency and toxicity for in vivo models. Keywords: Cytotoxicity, Cuscuta palaestina, antiproliferation, anticancer, HCT-116 (human colon cancer cell line); MTT, LDH, EC50, GC-MS analysis. Gundelia tournefortii, phytochemicals, Pimpinella Anisum, Anise PC-3 (human Prostate cancer cell line); Ephedra alata HepG2 (human liver cancer cell line); III Acknowledgment First and foremost praise be to Almighty Allah for giving me courage, determination and guidance in my life and never less in my challenging academic journey. I would like to express my special appreciation to my supervisor, Professor Dr. Saleh Abu-Lafi, for his supervision and constant support. His immeasurable constructive comments and suggestions throughout the experimental design, analytical analysis and thesis works have contributed to the completion of this piece of research. I would like to express my appreciation to Al-Quds University for the role it plays to fulfill the needs of Palestinian society through the master program in Applied and Industrial Technology. My deepest respect and thanks to Prof. Dr. Anwar Rayan, head of drug discovery informatics laboratory, QRC-Qasemi Research Center for his collaboration in the test of anticancer activities of plants fractions. Finally, special thanks is extended for my parents for encouraging me to seek knowledge and for their help and continuous support. IV Dedication This thesis is dedicated to: The sake of Allah, my Creator and my Master, The teacher of teachers, Prophet of humanity Mohammad -peace be upon him, My beloved parents, who never stop giving of themselves in countless ways, and for endless support, and encouragements. V Table of Contents Subject Page Declaration I Abstract II-III Acknowledgment IV Dedication V Table of Contents VI-VIII List of Abbreviations and symbol IX List of Table X List of Figures XI-XV List of Pictures XVI Chapter One: Introduction 1-18 1. Introduction 1 1.1.Cancer disease and their therapy 2 1.2.Secondary metabolites from natural product 4 1.3.Medicinal plants and traditional medicine 7 1.4.Natural products used as anticancer agent, isolation and identification 8 1.5.Screening anticancer activity from four Palestinian Plants 9 1.5.1. Cuscuta palaestina plant 9 1.5.2. Gundelia tournefortii plant 11 1.5.3. Pimpinella anisum plant 13 1.5.4. Ephedra alata plant 14 1.6.Analytical methods for the analysis of plant constituents 16 1.6.1. High Performance Liquid Chromatography (HPLC) 16 1.6.2. Gas Chromatography - Mass Spectroscopy (GC-MS) 16 1.7.Research problem 17 1.8.Aim of the study 18 1.9.Objective of the study 18 1.10. Question of the study 18 1.11. Hypotheses 18 Chapter Two: Literature Review 19-22 2. Literature Review 19 Chapter Three: Methodology 23-37 VI 3. Methodology 23 3.1.Materials, and collection Plant 24 3.2.Reagents 26 3.3.Instrumentation system 26 3.4.Equipment's 27 3.5.Extraction Methods and analytical procedures 27 3.5.1. Cuscuta palaestina plant 27 3.5.2. Gundelia tournefortii plant 29 3.5.3. Pimpinella anisum plant 30 3.5.4. Ephedra alata plant 30 3.5.5. GC-MS analysis conditions for Cuscuta palaestina 31 3.5.6. GC-MS analysis conditions for Gundelia tournefortii 31 3.5.7. Chromatographic conditions of HPLC-PDA 32 3.5.7.1. Analytical HPLC-PDA 32 3.5.7.2. Prep-HPLC-PDA 33 3.5.8. Cell cultures and treatments 33 3.5.9. Micro-culture tetrazolium assay (MTT) 34 3.5.10. Silylation derivatization 36 3.5.11. Lactate dehydrogenase (LDH) leakage assay 36 Chapter Four: Results and Discussion 38-82 4. Results and Discussion 38 4.1.Cuscuta palaestina plant 39 4.1.1. Analysis by GC-MS 39 4.1.2. Quantitation of Sesamin in Cuscuta Palaestina samples by HPLC-PDA 46 4.1.3. Quantitation of extracted Sesamin using different solvents by HPLC-PDA 47 4.1.4. Anticancer activity of Cuscuta palaestina methanolic and hexane extracts 54 4.2.Gundelia tournefortii plant 56 4.2.1. GC-MS phytochemical analysis of Gundelia tournefortii 56 4.2.2. Gundelia tournefortii methanol extract fractionation by preparative HPLC-PDA 70 4.2.3. Anticancer activity of the Gundelia tournefortii methanolic and hexane extracts 72 4.3.Pimpinella anisum plant 74 4.3.1. Preparative HPLC separation of Pimpinella anisum extract 74 4.3.2. Anticancer activity of the Pimpinella anisum methanolic extract 75 4.4. Ephedra alata plant 80 4.4.1. Preparative HPLC fractionation of Ephedra alata extract 80 4.4.2. Anticancer activity of Ephedra alata methanolic extract 81 Chapter Five: Conclusion and Recommendation 83-85 5.1. Conclusion 84 5.2. Recommendation 85 References 86-93 Appendices 94 Appendix 1 94 VII Appendix 2 95 Appendix 3 95 Abstract in Arabic 96-97 VIII List of Abbreviations and symbol Abbreviations Theme WHO World Health Organization HPLC High-Performance Liquid Chromatography PDA Photodiode array GC-MS Gas Chromatography - Mass Spectrometry ATCC American Type Culture Collection LOD Limit of Detection LOQ Limit of Quantitation NIST National Institute of Standards and Technology m/z Mass-to-Charge ratio 1H-NMR Proton Nuclear Magnetic Resonance HR-ESi-MS High Resolution-Electrospray Ionization-Mass Spectrometry 13C-NMR Carbon-13 Nuclear Magnetic Resonance 2D-NMR Two Dimensional Nuclear Magnetic Resonance COSY Two-dimensional 1H-1H correlation spectrometry HMQC Heteronuclear Multiple-Quantum Correlation spectroscopy MTT Micro-culture tetrazolium LDH Lactate dehydrogenase ELSD Evaporative light scattering detector Fig. Figure µ Micro EC50 Half maximal effective concentration IC50 Half maximal inhibitory concentration DMSO Dimethyl sulfoxide DMEM Dulbecco's Modified Eagle Medium EDTA Ethylenediaminetetraacetic acid IU International unit U Unit ml Milliliter LC-QTOF Liquid Chromatography - Quadrupole Time-of-Flight Da Dalton (Molecular weight unit) PUBMED A bibliographic database, comprises more than 26 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full- text content from PubMed Central and publisher web sites.