ISOLATION AND IDENTIFICATION OF COMPONENTS FROM IXERIS SONCHIFOLIA HANCE AS POTENTIAL ANTI-STROKE AGENTS ZHANG YAOCHUN B.Sc. (Pharm.), Shandong University M.Sc. (Pharm.), Peking Union Medical College A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgement I would like to express my sincere gratitude to my dear supervisor Dr Chew Eng-Hui for her continuous encouragement, wise advice and kind support. Her support, understanding, advice and mentoring have helped guide me through my doctoral program and my dissertation. Without her, none of this would have been possible. I know that I will continue to benefit from her advice and the skills that she has helped me achieve for the rest of my career. Deep appreciation also goes to my co-supervisor Dr Ng Ka-yun for his great help throughout the Ph.D project. Special thanks to Dr Chui Wai Keung for his kind agreement for me to use his HPLC equipment, and Dr Koh Hwee Ling for her kind agreement for me to use her Blood Coagulation Analyzer. Their expertise advice on equipment techniques and analyzing methods accelerated my research progress. A big thank-you owned to Dr Keith Rogers from Institute of Molecular and Cell Biology, who helped me process the tissue immunohistological staining. I would like to thank Dr Chan Lai Wah and Dr Go Mei Lin for their valuable guidelines on my Ph.D candidature and graduate study. Thanks to the department lab technicians namely, Ms Ng Sek Eng, Ms Ng Swee Eng, Mdm Oh Tang Booy, Ms Wong Meiyin, Ms Lye Peypey and Mr Goh Minwei. Their hard work provided great technical support for my research project. In addition, I wish to express my deep gratitude to National University of Singapore for offering the research scholarship that supported my candidature. A sincere thank to my dear friends in NUS, who never failed to give me great suggestions in numerous ways. They are Dr Zhou Liang, Dr Lin Haishu, Dr Ling Hui, Dr Gapter Leslie A, Dr Surajit Das, Dr Yang Hong, Dr Hu Zeping, Dr Tian Quan, Dr Huang Meng, Dr He Chunnian, Dr Wu Zhenlong, Miss Gan Feifei, Miss Amrita Abhay Nagle, Miss Shridhivya Reddy Amarr, Miss Jin Jing, Miss Zhang Caiyu, Miss Yang Shili, Mr Shelar Sandeep Balu, Mr Sun Feng, Mr Yue Bingde, Mr Wang Zhe, Mr Wang Likun, Mr Tan Jing, Mr Si Chengtao, Mr Sun Lingyi and Mr Li Jian. The precious time spent with them will be preserved in my memory forever. Last but not least, I would like to extend my appreciation to my families. My respected grandmother, industrious parents and kind sisters, though separated by an ocean, have never stopped their care. My dear wife, Li Guijun, has fulfilled my life outside the lab. My lovely daughter, Zhang Xiao, has rendered me a new angle to observe, feel and understand the world. I’m writing this thesis to share with all of them. I Table of contents Acknowledgements I Table of contents II Abstract VII List of tables X List of figures XI Abbreviations XIV Chapter 1 Introduction 1 1.1 Stroke 3 1.1.1 Pathophysiology 3 1.1.2 Epidemiology 7 1.1.3 Progress in stroke treatment 10 1.1.3.1 Thrombolysis 10 1.1.3.2 Anticoagulants and antiplatelet drugs 11 1.1.3.3 Neuroprotectants 13 1.1.3.4 Combination therapy 21 1.1.3.5 Traditional Chinese Medicine 23 1.2 Studies on Ixeris sonchifolia Hance 24 1.3 Rationale and aims 28 Chapter 2 Preparation and bioactivity screening of fractions from Ixeris 30 sonchifolia Hance extract 2.1 Introduction 31 2.2 Materials and methods 32 2.2.1 Preparation of Ixeris sonchifolia Hance extract 32 2.2.1.1 Reagents and plant material 32 II 2.2.1.2 Extraction procedures 32 2.2.2 Screening assays to evaluate the bioactivities of Ixeris 32 sonchifolia Hance extract 2.2.2.1 Anticoagulation activity assay 33 2.2.2.2 In vitro MTT cell viability assay 34 2.2.2.3 Evaluation of antioxidant activities of 36 fractions 1 to 4 2.2.2.3a Determination of free radical scavenging 37 activity 2.2.2.3b Determination of inducibility of cellular 37 antioxidant enzymes and molecules ARE-dependent luciferase reporter assay 37 Western blot analysis of cellular Nrf2 levels 38 GSH measurement 40 2.2.3 Statistical analysis 41 2.3 Results 42 2.3.1 Possible anti-coagulation activities of fractions 1 to 4 42 isolated from Ixeris sonchifolia Hance extract 2.3.2 Effects of fractions 1 to 4 isolated from Ixeris 42 sonchifolia Hance extract on the viability of H2O2-exposed PC12 cells 2.3.3 Evaluation of antioxidant activities of fractions 1 to 4 44 isolated from Ixeris sonchifolia Hance extract 2.4 Discussion and conclusions 48 Chapter 3 Isolation and characterization of components from ethyl 55 acetate fraction of Ixeris sonchifolia Hance 3.1 Introduction 56 3.2 Materials and methods 56 3.2.1 Isolation of the fractions from Ixeris Sonchifolia 56 Hance extract III 3.2.1.1 Materials and general analytical procedures 56 3.2.1.2 Isolation procedures 57 3.2.1.3 Reactions 58 3.2.2 Quantitative HPLC analysis of compounds 1 to 5 and 59 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract 3.1.2.1 HPLC system and mobile phase 59 3.1.2.2 Sample preparations 60 3.1.2.3 Calibration graphs 60 3.1.2.4 Quality control 60 3.3 Results 61 3.3.1 Characterization of purified compounds isolated from 61 Ixeris sonchifolia Hance 3.3.2 Quantitative HPLC analysis of compounds 1 to 5 and 72 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance 3.4 Discussion and conclusions 75 Chapter 4 Cytoprotective effects of flavonoid compounds isolated from 78 ethyl acetate fraction of Ixeris sonchifolia Hance extract 4.1 Introduction 79 4.2 Materials and methods 79 4.2.1Preparation of stock solutions of flavonoid 79 compounds 4.2.2 In vitro MTT cell viability assay 80 4.2.3 Determination of LDH leakage 80 4.2.4 Flow cytometric analysis of cell death 80 4.2.5 Determination of free radical scavenging activity 81 4.2.6 ARE-dependent luciferase reporter assay 81 Western blot analysis 82 IV GSH measurement 82 4.2.7 Statistical analysis 82 4.3 Results 82 4.3.1 Effects of flavonoid compounds 1 to 5 on the 82 viability of H2O2-exposed SH-SY5Y cells 4.3.2 Antioxidant activities of flavonoid compounds 1 to 5 88 isolated from Ixeris sonchifolia Hance extract 4.4 Discussion and conclusions 93 Chapter 5 Anti-inflammatory effects of flavonoid compounds isolated 98 from the ethyl acetate fraction of Ixeris sonchifolia Hance extract 5.1 Introduction 99 5.2 Materials and methods 99 5.2.1Preparation of stock solutions of flavonoid 99 compounds 5.2.2 Cell culture and treatments 100 5.2.3 In vitro MTT cell viability assay 101 5.2.4 Measurement of nitric oxide release 101 5.2.5 RNA extraction and reverse transcription-polymerase 102 chain reaction 5.2.6 Western blot analysis 104 5.2.7 Statistical analysis 105 5.3 Results 105 5.3.1 Effects of flavonoid compounds 1 to 5 on the 105 viability of RAW 264.7 cells and BV-2 cells 5.3.2 Effects of flavonoid compounds 1 to 5 on nitric oxide 105 production in LPS-activated RAW 264.7 cells. 5.3.3 Effects of flavonoid compounds 1 to 5 on LPS- 107 induced mRNA expression of cytokines in BV-2 cells 5.3.4 Effects of flavonoid compounds 1 to 5 on the 110 expression of Cox-2 and cPLA2 in LPS-stimulated BV-2 V cells 5.3.5 Effects of flavonoid compounds 1 to 5 on the 111 expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 cells 5.4 Discussion and conclusions 113 Chapter 6 Possible protective effects of luteolin in rats suffering from 122 cerebral ischemia induced by middle cerebral artery occlusion 6.1 Introduction 123 6.2 Materials and methods 123 6.2.1 Experimental animals 123 6.2.2 Surgical procedures 124 6.2.3 Evaluation of neurobehavior 125 6.2.4 Measurement of infarct area 126 6.2.5 Immunohistological analysis 126 6.2.6 Statistical analysis 127 6.3 Results 127 6.3.1 Effect of luteolin on mortality and neurobehavioral 127 recovery of MCA-occluded rats 6.3.2 Effect of luteolin on infarct area in the brains of rats 128 suffering from cerebral ischemia induced by MCA occlusion 6.3.3 Immunohistochemistry staining of brain slices 129 6.4 Discussion and conclusions 132 Chapter 7 General discussion and conclusions 137 References 147 VI Abstract Ixeris sonchifolia Hance is a small and bitter perennial herb widely distributed in the northeastern part of China. Its raw extract had recently been developed into an injectable formulation showing considerable therapeutic efficacy in stroke management. But the biological targets and the underlying chemical components remained mostly unknown. Accordingly, (1) the effectiveness of the herbal preparation may suffer from batch-to-batch variations since it is difficult to ensure the presence of similar amounts of active ingredients, and (2) some of the components present in the herbal formation may pose certain side effects that potentially limit the therapeutic benefits. This thesis aimed to isolate and identify constituents from Ixeris sonchifolia Hance that display anti-stroke activities and to elucidate the possible mechanism(s) through which the identified compound(s) exerted the neuroprotective effects.