Cytotoxicity of Extract of Malaysian Mitragyna Speciosa Korth and Its Dominant Alkaloid Mitragynine”

“ Cytotoxicity of extract of Malaysian Mitragyna speciosa Korth and its dominant alkaloid mitragynine” Ph.D thesis by Dr. Nor Aini Saidin, dedicated To, My hubby,my kids, my mum and in loving memory of my father Thank you for everything Cytotoxicity of extract of Malaysian Mitragyna speciosa Korth and its dominant alkaloid mitragynine. A thesis submitted by Nor Aini Saidin, D.A.H.P, D.V.M, MSc. Toxicology For the degree of DOCTOR OF PHILOSOPHY In the FACULTY OF MEDICINE IMPERIAL COLLEGE LONDON Department of Biomolecular Medicine Imperial College London London SW7 2AZ November, 2008 i ABSTRACT Mitragyna speciosa Korth (Kratom), a herb of the Rubiaceae family is indigenous in southeast Asia mainly in Malaysia and Thailand. It is used as an opium substitute and has been increasingly abused by drug addicts in Malaysia. Recently, the potent analgesic effect of plant extract and its dominant alkaloid mitragynine (MIT) were confirmed in vivo and in vitro. MIT acted primarily on µ- and δ-opioid receptors, suggesting that MIT or similar compounds could be promising alternatives for future pain management treatments. However the potential cytotoxicity of this plant is unknown. Therefore, the cytotoxicity of methanol-chloroform extract (MSE) and MIT on human cell lines (HepG2, HEK 293, MCL-5, cHol and SH-SY5Y cells) has been examined. MSE appeared to exhibit dose-dependant inhibition of cell proliferation in all cell lines examined, at concentration > 100 µg/ml with substantial cell death at 1000 µg/ml. SH-SY5Y was the most sensitive cell line examined. MIT showed a similar response. Clonogenicity assay was performed to assess the longer- term effects of MSE and MIT. The colony forming ability of HEK 293 and SH-SY5Y cells was inhibited in a dose-dependant manner. Involvement of metabolism in cytotoxicity was further assessed by clonogenicity assay using rat liver S9 (induced by Arochlor 1254); toxicity increased 10-fold in both cell lines. To determine if cytotoxicity was accompanied by DNA damage, the Mouse lymphoma tk gene mutation assay was used. The results were negative for both MSE and MIT. Studies on the involvement of metabolism in cytotoxicity of MSE and MIT were performed using MCL-5 and it appeared that CYP 2E1 is involved in activation of cytotoxicity. Studies with opioid antagonists were performed using SH-SY5Y cells treated with MSE and MIT. Naloxone (µ and δ receptor antagonists), naltrindole (δ receptor antagonist) and cyprodime hydrobromide (µ receptor antagonist) confirmed that MSE cytotoxicity was associated with µ and δ receptor while MIT mainly acted on µ receptor. Studies on mechanism of MSE and MIT cytotoxicity showed that cell death observed at high dose was preceded by cell cycle arrest, however MSE cell arrest was independent of p53 and p21 while MIT showed opposite result. Studies have been undertaken to examine the nature of this cell death. Morphological examinations showed that cell death induced by MSE was cell type dependant, in which SH-SY5Y cells appeared to die via apoptosis-like cell death while HEK 293 and MCL-5 cells predominantly via necrosis. Biochemical assessments confirmed that MSE induced cell death independent of p53 or caspases pathway while MIT cell death appeared to be associated with p53 and caspases pathway. The involvement of reactive oxygen species (ROS) generation in MSE and MIT mediating cell death was performed using SH-SY5Y cells. The results appeared negative for both MSE and MIT treated cells. Collectively, the findings of these studies suggest that MSE and its dominant alkaloid MIT produced cytotoxicity effects at high dose. Thus, the consumption of Mitragyna speciosa Korth leaves may pose harmful effects to users if taken at high dose and the evidence for involvement of CYP 2E1 in increasing the MSE cytotoxicity suggests that caution may be required if the leaves are to be taken with CYP 2E1 inducers. ii ACKNOWLEDGEMENTS This thesis is the account of my three years of devoted work in the field of toxicology at the Department of Biomolecular Medicine, Faculty of Medicine, Imperial College London which would not have been possible without the help of many. First and foremost, I wish to express my sincere gratitude to my direct supervisor, Prof. Dr. Nigel J. Gooderham for his constant encouragements, invaluable suggestions and who provide support in the most difficult times which have been essential to my success throughout the last three years. With his enthusiasm, his inspiration, his great effort to explain things clearly and simply, his sound advice and lots of good ideas has made my study and my thesis-writing period running smoothly and enjoyable. It has been a distinct privileged to work with him. I am also deeply honoured to my second supervisor, Prof. Elaine Holmes who gave me a chance to learn a NMR-based metabonomic work during my first year which is totally a new area for me to experience with. I am indebted to my NMR mentor, Prof. Yulan Wang who helped me in understanding and running the NMR analysis. To my colleagues in the Molecular Toxicology group, James, Lucy, Michalis, Costas and Nurul, many thanks for your help and support throughout my laboratory work. I wish to thank the member of Leucocyte Biology laboratory for allowing me to use your flow cytometry facilities. My thanks will also go to Sachinta Jayasinge and Norhaslinda for helping me in flow cytometry analysis, Siti Hamimah for the western blot analysis, Dr. Martin Spitaler for the microscopy examinations and histopathology group from Hammersmith campus of ICL especially Fatimah Jaafar for the interpretation of my microscopic slides and to GlaxoSmithKline staff, especially Dr. Sharon Robinson and Bibi for a wonderful training in MLA testing. To members of Biomolecular Medicine department who directly or indirectly help me these years and those names not listed here, rest assured that my gratitude is not less than for those listed here. I am very grateful to my sponsorships, Ministry of Higher Education Malaysia and International Islamic University Malaysia for providing the financial support for this study. I greatly appreciate to my deputy dean, Dato’ Dr. Syed Zahid Idid for introducing me to this plant, Mitragyna speciosa Korth for the subject of this study, to Assoc. Prof. Dr. Taufik Yap for helped in the extraction process of this plant and to police officers from Narcotic department of Kuala Kubu, Selangor, Malaysia for assistance in getting the leaves of the plant. I owe special gratitude to my family; hubby (Aziz), my lovely kids (Akbar, Ain, Alif and Arif) and my mum (Sopiah) for their patience, understanding, love, support and amazing sacrifice throughout these years. To them I dedicated this thesis. Finally, thanks to Almighty Allah S.W.T for showering His blessing, giving me strength and patience during hard times and for this amazing opportunity in my life. iii STATEMENT OF ORIGINALITY I certify that this thesis, and the research to which it refers, are the production of my own work, and that any ideas or quotations from the work of other people, published or otherwise, are fully acknowledged in accordance with the standard referencing practices of the discipline. iv PUBLICATIONS Published Abstracts Saidin, N. and Gooderham N.J. (2007). In vitro toxicology of extract of Mitragyna speciosa Korth, a Malaysian phytopharmaceutical of abuse. Toxicology, 240, 166-167. Saidin, N., Takayama, H., Holmes, E. and Gooderham, N.J. (2008). Cytotoxicity of extract of Malaysian Kratom and its dominant alkaloid mitragynine, on human cell lines. Planta Medica, 74: DOI: 10.1055/s-2008-1075279 Saidin, N., Randall, T., Takayama, H., Holmes, E. and Gooderham, N.J. (2008) Malaysian Kratom, a phyto-pharmaceutical of abuse: Studies on the mechanism of its cytotoxicity. Toxicology, 253, 19-20. v CONTENTS Dedication Title i Abstract ii Acknowledgements iii Statement of originality iv Publications v Contents vi List of figures x List of tables xii Abbreviations xiii Contents Chapter 1 General introduction 1 1.0 Overview 2 1.1 Pharmaceuticals from plants 2 1.1.1 Drug discovery from plants and the central nervous system 2 1.1.2 Safety concern on the use of pharmaceutical from plant 4 1.2 The plant Mitragyna speciosa Korth and Mitragynine 5 1.2.1 Description of the plant 5 1.2.2 Chemical constituents of the plant 7 1.2.3 Biological activity of this plant 8 1.3 Xenobiotic-induced cytotoxicity 10 1.4 The cell cycle 10 1.4.1 Review of the cell cycle 10 1.4.2 The cell cycle control system and checkpoints 11 1.4.3 Cell cycle arrest: Roles of p53 and its target gene, p21 protein 13 1.5 Genotoxicology 15 1.5.1 Overview of DNA damage and repair 16 1.5.2 Carcinogenesis 18 1.5.3 Genotoxicity testing 20 1.6 Cell death 23 1.6.1 Various ways of cell death: Apoptosis vs necrosis 23 vi 1.6.2 Mechanisms of apoptotic and necrotic cell death 26 1.6.2.1 Apoptosis pathways 26 1.6.2.2 Necrotic cell death 30 1.6.3 In vitro cell death assessment 32 1.7 Justification, Objectives and Hypothesis 34 1.7.1 Justification 34 1.7.2 Hypothesis 34 1.7.3 Aims and Objectives 34 Chapter 2 Effects of MSE and MIT on the growth and survival of human cell lines 35 2.1 Introduction 36 2.2 Materials and methods 37 2.2.1 Chemicals and reagents 37 2.2.2 Cell lines and culture conditions 37 2.2.3 Resuscitation of frozen cells 39 2.2.4 Cell quantification and viability 39 2.2.5 Preparation and analysis of methanol-chloroform extract of Mitragyna speciosa Korth (MSE) 40 2.2.5.1 Chemicals and reagents 40 2.2.5.2 Sample 40 2.2.5.3 Extraction using organic solvent

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