FOLK MEDICINE- Cratoxylum cochinchinense ANTIOXIDANT BUT CYTOTOXIC TANG SOON YEW (BSc (Hons), UNSW, Australia) A thesis submitted for the degree of Doctor of Philosophy Department of Biochemistry NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS Firstly, special thanks to my Family for their support throughout this study. I also wish to thank my supervisors, Professor Barry Halliwell and A/P Matthew Whiteman, for their invaluable guidance and advice as well as patience throughout this study. Special thanks to Professor Yong Eu Leong and Dr. Yap Sook Peng from the Department of Obstetrics and Gynecology, Faculty of Medicine, National University of Singapore for supplying us the Chinese medicinal extracts. This project would not be possible without these extracts, especially Cratoxylum cochinchinense which was harvested from Sook Peng’s garden in Malaysia. My sincere thanks to Professor Sit Kim Ping, Department of Biochemistry, Faculty of Medicine, National University of Singapore for her generous gifts of a number of cell lines used in this study. My sincere thanks also extend to members of the Antioxidants and Oxidants Research Group, in one way or the other, they have been a part of making this thesis possible. Members of the group include Dr. Peng Zhao Feng, Siau Jia Ling, Dr. Andrew Jenner, Lim Kok Seong, Wang Huang Song, Sherry Huang, Long Lee Hua, Chua Siew Hwa, Dr. Wong Boon Seng and Dr. Jetty Lee. Last but not least, many thanks to my friends Chai Phing Chian, Ng Kian Hong, Dr. Mirjam Nordling, members of the Flow Cytometry and Confocal Units and …… ii List of journal publications Tang, S. Y., Whiteman, M., Jenner, A., Peng, Z. F., and Halliwell, B. (2004) Mechanism of cell death induced by an antioxidant extract of Cratoxylum cochinchinense (YCT) in Jurkat T cells: the role of reactive oxygen species and calcium. Free Radic. Biol. Med. 36:1588-1611. Tang, S. Y., Whiteman, M., Peng, Z. F., Jenner, A., Yong, E. L., and Halliwell, B. (2004) Characterization of antioxidant and antiglycation properties and isolation of active ingredients from traditional chinese medicines. Free Radic. Biol. Med. 36:1575-1587. International conference attended during 2001-2005 1st Asia Pacific Conference and Exhibition on Anti-Aging Medicine 2002: From Molecular Mechanisms to Therapies. June 23-26. The 2nd International Conference on Natural Products 2002. 2st Asia Pacific Conference and Exhibition on Anti-Aging Medicine 2003: Diet, Disease, and Lifestyle. Sep 08-11. The 3nd International Conference on Natural Products- A Must for Human Survival. Oct 23-25, 2004. ICCAM (International Congress on Complementary and Alternative Medicines) Herbal Medicines: Ancient Cures, Modern Science. Feb 26-28, 2005. International Networking for Young Scientists Symposium. Cancer Biology-Cell Apoptosis. Mar 1-2, 2005. iii TABLES OF CONTENTS Page Acknowledgements i List of journal publications iii International conference attended during 2001-2005 iii Table of contents iv Abstracts x List of tables xii List of figures xiii List of abbreviations and keywords xvi CHAPTER 1. INTRODUCTION 1.1. Introduction 1 1.2. Traditional Chinese medicines 2 1.3. Reactive oxygen species 4 1.3.1. Hypochlorous acid 7 1.3.2. Peroxynitrite 9 1.4. Oxidative stress 10 1.5. Antioxidant defence system 11 1.6. Antioxidant and polyphenolic compounds 13 1.7. Reactive oxygen species in cell signaling 15 1.7.1. Introduction 15 iv 1.7.2. Cellular redox state 17 1.8. Calcium and its role in cell signaling and cell death 19 1.8.1. Introduction 19 1.8.2. Cellular calcium homeostasis 19 1.8.3. Roles of endoplasmic reticulum in cytosolic calcium homeostasis 21 1.8.4. Role of mitochondria in cytosolic calcium homeostasis 22 1.8.5. Calcium and oxidative stress 22 1.8.6. Calcium and cytotoxicity 23 1.9. Mitochondria 25 1.9.1. Mitochondrial structure and function 25 1.9.2. Mitochondrial reactive oxygen species 26 1.10. Cell death 27 1.10.1. Introduction 27 1.10.2. Differences between apoptosis and necrosis 28 1.10.3. Mitochondrial permeability transition and cell death 34 1.10.4. Calcium overload and mitochondrial permeability transition 37 1.10.5. Bcl-2 proteins and cell death 38 1.11. Plasma membrane NADH reductase/ Plasma membrane redox system 39 1.12. Aims of this study 40 CHAPTER 2. EXPERIMENTAL PROCEDURES 2.1. Materials 41 v 2.2. Methods 43 2.2.1. Extract preparation 43 2.2.2. ABTS assay 43 2.2.3. Ascorbate-iron induced lipid peroxidation 44 2.2.4. Total phenolic content 45 2.2.5. Scavenging of DPPH• (2,2-Diphenyl-1-picrylhydrazyl) 45 •- 2.2.6. Superoxide anion (O2 ) scavenging effect 45 2.2.7. Xanthine oxidase (XO) activity 46 2.2.8. Iron-binding activity 46 2.2.9. Non-enzymatic protein glycation 47 2.2.10. Inhibition of hypochlorous acid-induced DNA damage 47 2.2.11. Isolation, purification, and identification of active ingredient from Cratoxylum cochinchinense (WN) 49 2.2.12. Bleomycin-iron dependent DNA damage 50 2.2.13. Peroxynitrite scavenging assays 50 2.2.13.1. Synthesis of Peroxynitrite 50 2.2.13.2. Assessment of pyrogallol red (PR) bleaching by peroxynitrite (ONOO-) 51 2.2.13.3. Measurement of tyrosine nitration 51 2.2.14. Cell culture 52 2.2.15. Cell counting 52 2.2.16. Isolation of human lymphocytes 53 2.2.17. Assessment of cell viability 53 vi 2.2.17.1. MTT assay 54 2.2.17.2. Trypan blue exclusion assay 54 2.2.18. Treatment of Jurkat T cells with YCT 55 2.2.19. O2 electrode assay 55 2.2.20. Cell cycle analysis using flow cytometry 56 2.2.21. Annexin V-FITC and propidium iodide staining of Jurkat T cells 56 2.2.22. Release of lactate dehydrogenase 57 2.2.23. Assessment of cellular and nuclear morphology 57 2.2.24. Caspase 3 and 9 activities 58 2.2.25. Western blot analysis 59 2.2.26. Internucleosomal DNA fragmentation assay 60 •- 2.2.27. Cytofluorimetric measurement of superoxide radicals (O2 ) and other reactive oxygen species (ROS), lipid peroxidation (LPO), 2+ 2+ 2+ 2+ intracellular Ca ([Ca ]i), mitochondrial Ca ([Ca ]m), mitochondrial + transmembrane potential (ΔΨm), intracellular potassium ([k ]i) and + sodium ([Na ]i) 60 2.2.28. Multiwell plate reader measurement of lipid peroxidation (LPO) and mitochondrial transmembrane potential (ΔΨm) 63 2.2.29. Intracellular ATP determination 63 2.2.30. Intracellular glutathione measurement 64 2.2.31. Cytochrome c levels 64 2.2.32. Kinetic study of rise in intracellular Ca2+ with Fluo3/ AM 66 vii 2.3. Data analysis 66 CHAPTER 3. RESULTS AND DISCUSSION RESULTS 3.1. Radical scavenging by TCM extracts 67 3.2. Inhibition of ascorbate-iron induced phospholipid peroxidation 70 3.3. Correlation between TEAC values, total phenolic content and DPPH reducing ability 71 3.4. Superoxide scavenging and xanthine oxidase inhibition 72 3.5. Iron-binding activity 73 3.6. Scavenging of hypochlorous acid (HOCl); protection against HOCl-mediated DNA damage 74 3.7. Inhibition of protein glycation 75 3.8. Purification and identification of ‘active’ ingredient from extract WN 76 3.9. Low pro-oxidant effect of YCT extract 79 3.10. Inhibition of PR bleaching and 3-nitrotyrosine formation by YCT 80 3.11. A comparison of our semi-purified extract (YCT) with pure mangiferin 83 DISCUSSION 86 CHAPTER 4. RESULTS AND DISCUSSION RESULTS viii 4.1. Cytotoxicity of YCT by MTT assay and propidium iodide staining 93 4.2. YCT-induced loss of viability in Jurkat T cells but not in normal lymphocytes 97 4.3. Changes in cellular morphology 99 4.4. YCT-induced phosphatidylserine exposure and LDH release 101 4.5. Caspase activities and internucleosomal DNA fragmentation 105 4.6. YCT induces rapid oxidative stress 108 4.7. YCT-induced lipid peroxidation in Jurkat T cells 112 2+ 4.8. Mitochondrial Ca overloading and ΔΨm dissipation 115 2+ 4.9. Effects of ruthenium red on mitochondrial Ca and ΔΨm 118 4.10. YCT-mediated changes in cytochrome c and ATP levels 120 4.11. Intracellular glutathione content 124 4.12. YCT-mediated mobilization of extracellular Ca2+ 127 4.13. Ca2+ influx through a non-selective cation channel? 131 4.14. The relationship between ROS generation and Ca2+ influx 133 4.15. Ferricyanide inhibit YCT-induced apoptotic signals 24 h after treatment 135 DISCUSSION 138 CHAPTER 5. CONCLUSION 143 CHAPTER 6. REFERENCES 146 APPENDIX A ix ABSTRACT There is considerable interest in the isolation of more potent antioxidant compounds to treat diseases involving oxidative stress. Thirty-three Traditional Chinese Medicine (TCM) extracts were examined for their antioxidant activity using the ABTS (2,2’-azinobis[3-ethylbenzothiazoline-6-sulphonate]) assay. Five extracts with high activity (Cratoxylum cochinchinense, Cortex magnoliae officinalis, Psoralea corylifolia L, Curculigo orchioides Gaertn, and Glycyrrhiza uralensis Fisch.) were selected for further characterization. C. cochinchinense out-performed other extracts in most of the assays tested except phospholipid peroxidation inhibition, where P. corylifolia L showed a higher activity. C. cochinchinense was particularly potent in inhibiting the formation of advanced glycation end products on proteins and strongly inhibited hypochlorous acid- induced DNA damage. We attempted to isolate the active ingredients from C. cochinchinense and obtained an extract (YCT) containing at least 90% mangiferin (MGF) as identified by HPLC and mass spectrometry. However, YCT showed significantly higher activity in assays of phospholipid peroxidation, inhibition of protein glycation, •− − superoxide (O2 ) and peroxynitrite (ONOO ) scavenging as compared to MGF, suggesting that the non-mangiferin constituents of YCT contribute to its additional antioxidant activities. In cell culture experiments, we show that YCT is selectively toxic to certain cell types and investigated the mechanisms of this toxicity in Jurkat T cells.