Isolation and identification of components from ixeris sonchifolia hance as potential anti stroke agents

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 Introduction 1.1 Stroke 1.1.1 Pathophysiology 1.1.2 Epidemiology 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 Preparation and bioactivity screening of fractions from Ixeris sonchifolia Hance extract 30 2.1 Introduction 31 2.2 Materials and methods 32 2.2.1 Preparation of Ixeris sonchifolia Hance extract 2.2.1.1 Reagents and plant material 32 32 II 2.2.1.2 Extraction procedures 2.2.2 Screening assays to evaluate the bioactivities of Ixeris sonchifolia Hance extract 32 32 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 fractions to 36 2.2.2.3a Determination of free radical scavenging activity 37 2.2.2.3b Determination of inducibility of cellular antioxidant enzymes and molecules 37 ARE-dependent luciferase reporter assay 37 Western blot analysis of cellular Nrf2 levels 38 GSH measurement 40 2.2.3 Statistical analysis 2.3 Results 41 42 2.3.1 Possible anti-coagulation activities of fractions to isolated from Ixeris sonchifolia Hance extract 42 2.3.2 Effects of fractions to isolated from Ixeris sonchifolia Hance extract on the viability of H2O2-exposed PC12 cells 42 2.3.3 Evaluation of antioxidant activities of fractions to isolated from Ixeris sonchifolia Hance extract 44 2.4 Discussion and conclusions Chapter Isolation and characterization of components from ethyl acetate fraction of Ixeris sonchifolia Hance 48 55 3.1 Introduction 56 3.2 Materials and methods 56 3.2.1 Isolation of the fractions from Ixeris Sonchifolia Hance extract 56 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 to and in fractions to isolated from Ixeris sonchifolia Hance extract 59 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 Ixeris sonchifolia Hance 61 3.3.2 Quantitative HPLC analysis of compounds to and in fractions to isolated from Ixeris sonchifolia Hance 72 3.4 Discussion and conclusions 75 Chapter Cytoprotective effects of flavonoid compounds isolated from ethyl acetate fraction of Ixeris sonchifolia Hance extract 78 4.1 Introduction 79 4.2 Materials and methods 79 4.2.1Preparation compounds of stock solutions of flavonoid 79 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 to on the viability of H2O2-exposed SH-SY5Y cells 82 4.3.2 Antioxidant activities of flavonoid compounds to isolated from Ixeris sonchifolia Hance extract 88 4.4 Discussion and conclusions 93 Chapter Anti-inflammatory effects of flavonoid compounds isolated from the ethyl acetate fraction of Ixeris sonchifolia Hance extract 98 5.1 Introduction 99 5.2 Materials and methods 99 5.2.1Preparation compounds of stock solutions of flavonoid 99 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 chain reaction 102 5.2.6 Western blot analysis 104 5.2.7 Statistical analysis 105 5.3 Results 105 5.3.1 Effects of flavonoid compounds to on the viability of RAW 264.7 cells and BV-2 cells 105 5.3.2 Effects of flavonoid compounds to on nitric oxide production in LPS-activated RAW 264.7 cells. 105 5.3.3 Effects of flavonoid compounds to on LPSinduced mRNA expression of cytokines in BV-2 cells 107 5.3.4 Effects of flavonoid compounds to on the expression of Cox-2 and cPLA2 in LPS-stimulated BV-2 110 V cells 5.3.5 Effects of flavonoid compounds to on the expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 cells 5.4 Discussion and conclusions Chapter Possible protective effects of luteolin in rats suffering from cerebral ischemia induced by middle cerebral artery occlusion 111 113 122 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 6.3.1 Effect of luteolin on mortality and neurobehavioral recovery of MCA-occluded rats 127 127 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 6.4 Discussion and conclusions 129 132 Chapter 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. To accomplish this objective, the aerial part of Ixeris sonchifolia Hance was extracted and the crude extract was partitioned into fractions. The fractions were then subjected to screening using both cell-based in vitro bioassays and biochemical reactions. The results had revealed that the ethyl acetate fraction, although failed to exhibit anticoagulant activities, dosedependently protected cells from H2O2-induced cytotoxicity, scavenged DPPH free radicals, induced ARE-dependent transcriptional activity and caused upregulation of Nrf2 protein levels. The follow-up isolation work focused on the ethyl acetate fraction revealed the presence of two classes of compounds: flavonoids and VII sesquiterpene lactones. In vitro bioassays conducted on the isolated flavonoids, which were identified to be Apigenin (1), Luteolin (2), Apigenin-7-O-βglucopyranoside (3), Luteolin-7-O-β-glucopyranoside (4), Luteolin-7-O-βglucruonopyranoside (5) and Luteolin-7-O-β-galacturonide (6) respectively, revealed that these compounds could protect cells from H2O2-induced cytotoxicity by scavenging free radical directly and inducing phase Ⅱ enzymes, suggesting that the purified flavonoid compounds might exert neuroprotective effects during the early response, characterized by excitotoxic damage and oxidative stress, to stroke occurrence. Considering that the second wave of cell death after a stroke incident stems from the neuroinflammatory response, the anti-inflammatory effects of these isolated flavonoids on the LPS-stimulated cells were next determined. The results suggested that these flavonoid compounds might exert antiinflammatory activities by regulating cytokine secretion, inhibiting Cox-2 expression, as well as reducing NO release and iNOS expression. As validation, the potential anti-stroke effects of Luteolin, a representative of these isolated flavonoid compounds, were investigated using rats suffering from cerebral ischemia induced by MCA occlusion. The results revealed that while treatment with Luteolin failed to neither reduce MCAOinduced mortality nor improve neurobehavioral recovery, it significantly reduced the infarct area, decreased the number of cells positively stained with anti-cleaved caspase-3 and anti-Cox-2 antibodies, suggesting that Luteolin might exert neuroprotective effects in an in vivo stroke model. VIII In conclusion, extraction and isolation works focused on Ixeris sonchifolia Hance revealed the presence of two categories of compounds in the ethyl acetate fraction of its raw extract: flavonoids and sesquiterpene lactones. The isolated bioactive flavonoids were found to possess potential anti-stroke effects, which at least in part were attributed to their antioxidant and anti-inflammatory activities. IX Hossmann K. A. (1996). "Periinfarct depolarizations." Cerebrovasc Brain Metab Rev 8(3): 195-208. Hu D. and Sun Y. (2008). "Epidemiology, risk factors for stroke, and management of atrial fibrillation in China." J Am Coll Cardiol 52(10): 865-8. Huang P. L. (1999). "Neuronal and endothelial nitric oxide synthase gene knockout mice." Braz J Med Biol Res 32(11): 1353-9. Huang Z., Huang P. L., Ma J. et al. (1996). "Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine." J Cereb Blood Flow Metab 16(5): 981-7. Huang Z., Huang P. L., Panahian N. et al. (1994). "Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase." Science 265(5180): 1883-5. Iadecola C., Forster C., Nogawa S. et al. (1999). "Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia." Acta Neuropathol 98(1): 9-14. Iadecola C., Sugimoto K., Niwa K. et al. (2001). "Increased susceptibility to ischemic brain injury in cyclooxygenase-1-deficient mice." J Cereb Blood Flow Metab 21(12): 1436-41. Ichikawa H., Wang L. and Konishi T. (2006). "Prevention of cerebral oxidative injury by post-ischemic intravenous administration of Shengmai San." Am J Chin Med 34(4): 591-600. Imai K., Mori T., Izumoto H. et al. (2006). "Hyperbaric oxygen combined with intravenous edaravone for treatment of acute embolic stroke: a pilot clinical trial." Neurol Med Chir (Tokyo) 46(8): 373-8; discussion 378. Jan A. T., Kamli M. R., Murtaza I. et al. (2010). "Dietary Flavonoid Quercetin and Associated Health Benefits - An Overview." Food Reviews International 26(3): 302-307. Jung H. J., Park J. W., Lee J. S. et al. (2009). "Silibinin inhibits expression of HIF-1alpha through suppression of protein translation in prostate cancer cells." Biochem Biophys Res Commun 390(1): 71-6. Kalluri H. S. and Dempsey R. J. (2008). "Growth factors, stem cells, and stroke." Neurosurg Focus 24(3-4): E14. Kano T., Harada T. and Katayama Y. (2005). "Attenuation of extravasation of tissue plasminogen activator by the free radical scavenger, edaravone: evaluation in a rat thromboembolic stroke model." Neurol Res 27(5): 499-502. Kao E. S., Wang C. J., Lin W. L. et al. (2005). "Anti-inflammatory potential of flavonoid contents from dried fruit of Crataegus pinnatifida in vitro and in vivo." J Agric Food Chem 53(2): 430-6. 155 Kaspar J. W. and Jaiswal A. K. "An autoregulatory loop between Nrf2 and Cul3-Rbx1 controls their cellular abundance." J Biol Chem 285(28): 21349-58. Kaspar J. W., Niture S. K. and Jaiswal A. K. (2009). "Nrf2:INrf2 (Keap1) signaling in oxidative stress." Free Radic Biol Med 47(9): 1304-9. Kaste M. (2005). "Use of Animal Models Has Not Contributed to Development of Acute Stroke Therapies." Stroke 36: 2323-2324. Kawasaki-Yatsugi S., Ichiki C., Yatsugi S. et al. (2000). "Neuroprotective effects of an AMPA receptor antagonist YM872 in a rat transient middle cerebral artery occlusion model." Neuropharmacology 39(2): 211-7. Khatri I. A., Rana A. and Kulsoom A. (2008). "Intraarterial thrombolysis with reteplase in acute ischaemic stroke in a Pakistani center." J Pak Med Assoc 58(7): 408-11. Kiernan J. A. (2008). Histological and Histochemical Methods: Theory and Practice (4th ed). Bloxham, UK: Scion. Kim M. R., Hwang B. Y., Jeong E. S. et al. (2007). "Cytotoxic germacranolide sesquiterpene lactones from Carpesium triste var. manshuricum." Arch Pharm Res 30(5): 556-60. Kitagawa Y. (2006). "Edaravone in acute ischemic stroke." Intern Med 45(5): 225-6. Kleinig T. J. and Vink R. (2009). "Suppression of inflammation in ischemic and hemorrhagic stroke: therapeutic options." Curr Opin Neurol 22(3): 294301. Koharyova M. and Kolarova M. (2008). "Oxidative stress and thioredoxin system." Gen Physiol Biophys 27(2): 71-84. Koziol J. A. and Feng A. C. (2006). "On the analysis and interpretation of outcome measures in stroke clinical trials: lessons from the SAINT I study of NXY-059 for acute ischemic stroke." Stroke 37(10): 2644-7. Kroemer G. and Reed J. C. (2000). "Mitochondrial control of cell death." Nat Med 6(5): 513-9. Kudo I. and Murakami M. (2002). "Phospholipase A2 enzymes." Prostaglandins Other Lipid Mediat 68-69: 3-58. Kwan J. (2001). "Clinical epidemiology of stroke." CME Journal Geriatric Medicine 3(3): 94-98. Lai A. Y. and Todd K. G. (2006). "Microglia in cerebral ischemia: molecular actions and interactions." Can J Physiol Pharmacol 84(1): 49-59. 156 Landino L. M., Crews B. C., Timmons M. D. et al. (1996). "Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis." Proc Natl Acad Sci U S A 93(26): 15069-74. Lanfranconi S., Locatelli F., S B. C. et al. (2009). "Growth factors in ischemic stroke." J Cell Mol Med. Lau A. J., Toh D. F., Chua T. K. et al. (2009). "Antiplatelet and anticoagulant effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with Panax ginseng and Panax quinquefolium." J Ethnopharmacol 125(3): 380-6. Lee-Hilz Y. Y., Boerboom A. M., Westphal A. H. et al. (2006). "Pro-oxidant activity of flavonoids induces EpRE-mediated gene expression." Chem Res Toxicol 19(11): 1499-505. Lee D. H., Jo K. D., Kim H. G. et al. (2002). "Local intraarterial urokinase thrombolysis of acute ischemic stroke with or without intravenous abciximab: a pilot study." J Vasc Interv Radiol 13(8): 769-74. Lee S. and EH L. (2003). "Interactions between p38 mitogen-activated protein kinase and caspase-3 in cerebral endothelial cell death after hypoxiareoxygenation." Stroke 34: 2704-2709. Lee S. R., Tsuji K., Lee S. R. et al. (2004). "Role of matrix metalloproteinases in delayed neuronal damage after transient global cerebral ischemia." J Neurosci 24(3): 671-8. Lee S. T., Chu K., Jung K. H. et al. (2005). "Granulocyte colony-stimulating factor enhances angiogenesis after focal cerebral ischemia." Brain Res 1058(12): 120-8. Lee Y. Y., Westphal A. H., de Haan L. H. et al. (2005). "Human NAD(P)H:quinone oxidoreductase inhibition by flavonoids in living cells." Free Radic Biol Med 39(2): 257-65. Lees K. R., Asplund K., Carolei A. et al. (2000). "Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke: a randomised controlled trial. GAIN International Investigators." Lancet 355(9219): 1949-54. Lees K. R., Barer D., Ford G. A. et al. (2003). "Tolerability of NXY-059 at higher target concentrations in patients with acute stroke." Stroke 34(2): 482-7. Leonardo C. C., Eakin A. K., Ajmo J. M. et al. (2008). "Versican and brevican are expressed with distinct pathology in neonatal hypoxic-ischemic injury." J Neurosci Res 86(5): 1106-14. Levine R. L., Dixit S. N., Dulli D. A. et al. (2003). "Aspirin "failures," clopidogrel added to aspirin, and secondary stroke prevention in veterans presenting with TIA or mild-to-moderate ischemic stroke." J Stroke Cerebrovasc Dis 12(1): 37-43. 157 Ley J. J., Vigdorchik A., Belayev L. et al. (2005). "Stilbazulenyl nitrone, a second-generation azulenyl nitrone antioxidant, confers enduring neuroprotection in experimental focal cerebral ischemia in the rat: neurobehavior, histopathology, and pharmacokinetics." J Pharmacol Exp Ther 313(3): 1090-100. Li R., Guo M., Zhang G. et al. (2006). "Neuroprotection of nicotiflorin in permanent focal cerebral ischemia and in neuronal cultures." Biol Pharm Bull 29(9): 1868-72. Li W. and Kong A. N. (2009). "Molecular mechanisms of Nrf2-mediated antioxidant response." Mol Carcinog 48(2): 91-104. Liebeskind D. S. (2009). "Reversing stroke in the 2010s: lessons from Desmoteplase In Acute ischemic Stroke-2 (DIAS-2)." Stroke 40(9): 3156-8. Lim H., Son K. H., Chang H. W. et al. (2006). "Inhibition of chronic skin inflammation by topical anti-inflammatory flavonoid preparation, Ato Formula." Arch Pharm Res 29(6): 503-7. Lin Y., Hu S., Xu D. et al. (2007). "Vasodilation effect of water extract from Ixeris sonchifolia (Bge.) Hance in rat thoracic aortic rings in vitro." CHINESE JOURNAL OF PHARMACOLOGY AND TOXICOLOGY 21(3): 202-206. Liu B., Gao H. M., Wang J. Y. et al. (2002). "Role of nitric oxide in inflammation-mediated neurodegeneration." Ann N Y Acad Sci 962: 318-31. Liu C. S., Chen N. H. and Zhang J. T. (2007). "Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae." Phytomedicine 14(7-8): 492-7. Liu M., Wu B., Wang W. Z. et al. (2007). "Stroke in China: epidemiology, prevention, and management strategies." Lancet Neurol 6(5): 456-64. Liu PY, Li K, Zhang J et al. (2010). "Who is the king? The alpha-hydroxybeta-oxo-alpha,beta-enone moiety or the catechol B ring: relationship between the structure of quercetin derivatives and their pro-oxidative abilities." Chem Biodivers 7(1): 236-44. Lloyd-Jones D., Adams R., Carnethon M. et al. (2009). "Heart disease and stroke statistics--2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee." Circulation 119(3): e21-181. Lloyd-Jones D., Adams R., Carnethon M. et al. (2009). "Heart disease and stroke statistics -- 2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee." Circulation 119(3): 480-6. Lloyd-Jones D., Adams R., Carnethon M. et al. (2009). "Heart disease and stroke statistics -- 2009 update: a report from the American Heart Association 158 Statistics Committee and Stroke Statistics Subcommittee." Circulation 119(3): e21-181. Lo E. H., Broderick J. P. and Moskowitz M. A. (2004). "tPA and proteolysis in the neurovascular unit." Stroke 35(2): 354-6. Lo E. H., Dalkara T. and Moskowitz M. A. (2003). "Mechanisms, challenges and opportunities in stroke." Nat Rev Neurosci 4(5): 399-415. Lodder J., van Raak L., Hilton A. et al. (2006). "Diazepam to improve acute stroke outcome: results of the early GABA-Ergic activation study in stroke trial. a randomized double-blind placebo-controlled trial." Cerebrovasc Dis 21(1-2): 120-7. Longa E. Z., Weinstein P. R., Carlson S. et al. (1989). "Reversible middle cerebral artery occlusion without craniectomy in rats." Stroke 20(1): 84-91. Lopez-Saura P. A. (2003). "Thrombolysis with recombinant streptokinase in Cuba." Bmj 326(7386): 448. Lucas S. M., Rothwell N. J. and Gibson R. M. (2006). "The role of inflammation in CNS injury and disease." Br J Pharmacol 147 Suppl 1: S23240. Lutsep H. L. (2005). "Repinotan, A 5-HT1A agonist, in the treatment of acute ischemic stroke." Curr Drug Targets CNS Neurol Disord 4(2): 119-20. Lyden P., Shuaib A., Ng K. et al. (2002). "Clomethiazole Acute Stroke Study in ischemic stroke (CLASS-I): final results." Stroke 33(1): 122-8. Lyden P. D., Jackson-Friedman C., Shin C. et al. (2000). "Synergistic combinatorial stroke therapy: A quantal bioassay of a GABA agonist and a glutamate antagonist." Exp Neurol 163(2): 477-89. Lyden P. D., Shuaib A., Lees K. R. et al. (2007). "Safety and tolerability of NXY-059 for acute intracerebral hemorrhage: the CHANT Trial." Stroke 38(8): 2262-9. Lynch E. D., Gu R., Pierce C. et al. (2004). "Ebselen-mediated protection from single and repeated noise exposure in rat." Laryngoscope 114(2): 333-7. Ma J., Endres M. and Moskowitz M. A. (1998). "Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischaemia in mice." Br J Pharmacol 124(4): 756-62. Ma J., Qiu J., Hirt L. et al. (2001). "Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia." Br J Pharmacol 133(3): 345-50. Ma Z., Lv Z., Wang S. et al. (2009). "Extraction method of flavones from Chinese medicine Ixeris and its application as cardioprotective agent." China Patent: CN 101343299 A 20090114. 159 Macias F. A., Galindo J. C., Castellano D. et al. (2000). "Sesquiterpene lactones with potential use as natural herbicide models. 2. guaianolides." J Agric Food Chem 48(11): 5288-96. Madrigal-Carballo S., Rodriguez G., Sibaja M. et al. (2009). "Chitosomes loaded with cranberry proanthocyanidins attenuate the bacterial lipopolysaccharide-induced expression of iNOS and COX-2 in raw 264.7 macrophages." J Liposome Res 19(3): 189-96. Maeda M., Furuichi Y., Noto T. et al. (2009). "Tacrolimus (FK506) suppresses rt-PA-induced hemorrhagic transformation in a rat thrombotic ischemia stroke model." Brain Res 1254: 99-108. Mandava P., Anderson J. A. and Kent T. A. (2006). "MRI-guided, open trial of abciximab for ischemic stroke within a 3- to 24-hour window." Neurology 66(7): 1132. Mandava P., Lick S. D., Rahman M. A. et al. (2005). "Initial safety experience of abciximab and heparin for acute ischemic stroke." Cerebrovasc Dis 19(4): 276-8. Marshall J. W., Duffin K. J., Green A. R. et al. (2001). "NXY-059, a free radical--trapping agent, substantially lessens the functional disability resulting from cerebral ischemia in a primate species." Stroke 32(1): 190-8. Martin-Schild S., Hallevi H., Shaltoni H. et al. (2009). "Combined neuroprotective modalities coupled with thrombolysis in acute ischemic stroke: a pilot study of caffeinol and mild hypothermia." J Stroke Cerebrovasc Dis 18(2): 86-96. Mattson M. P., Duan W., Chan S. L. et al. (2002). "Neuroprotective and neurorestorative signal transduction mechanisms in brain aging: modification by genes, diet and behavior." Neurobiol Aging 23(5): 695-705. Mendelsohn M. E. (2002). "Protective effects of estrogen on the cardiovascular system." Am J Cardiol 89(12A): 12E-17E; discussion 17E-18E. Mendelsohn M. E. and Karas R. H. (1999). "The protective effects of estrogen on the cardiovascular system." N Engl J Med 340(23): 1801-11. Mergenthaler P., Dirnagl U. and Meisel A. (2004). "Pathophysiology of stroke: lessons from animal models." Metab Brain Dis 19(3-4): 151-67. Metodiewa D, Jaiswal AK, Cenas N et al. (1999). "Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product." Free Radic Biol Med. 26(1-2): 107-16. Michael J. O Neill, Astles P. C., Allan S. M. et al. (2004). "Anti-inflammatory modulators in stroke." Drug Discovery Today: Therapeutic Strategies 1(1): 59-67. Millikan C. (1992). "Animal stroke models." Stroke 23(6): 795-7. 160 Mohr J. P., Orgogozo J. M., Harrison M. J. G. et al. (1994). "Meta-Analysis of Oral Nimodipine Trials in Acute Ischemic Stroke." Cerebrovasc Dis 4(3): 197203. Moinova H. R. and Mulcahy R. T. (1998). "An electrophile responsive element (EpRE) regulates beta-naphthoflavone induction of the human gamma-glutamylcysteine synthetase regulatory subunit gene. Constitutive expression is mediated by an adjacent AP-1 site." J Biol Chem 273(24): 14683-9. Mousa S. A. (2008). "Cell adhesion molecules: potential therapeutic & diagnostic implications." Mol Biotechnol 38(1): 33-40. Muir K. W., Lees K. R., Ford I. et al. (2004). "Magnesium for acute stroke (Intravenous Magnesium Efficacy in Stroke trial): randomised controlled trial." Lancet 363(9407): 439-45. Mulder G. J. and Ouwerkerk-Mahadevan S. (1997). "Modulation of glutathione conjugation in vivo: how to decrease glutathione conjugation in vivo or in intact cellular systems in vitro." Chem Biol Interact 105(1): 17-34. Murakami M. and Kudo I. (2002). "Phospholipase A2." J Biochem 131(3): 285-92. Murphy A. N., Fiskum G. and Beal M. F. (1999). "Mitochondria in neurodegeneration: bioenergetic function in cell life and death." J Cereb Blood Flow Metab 19(3): 231-45. Murphy S. (2000). "Production of nitric oxide by glial cells: regulation and potential roles in the CNS." Glia 29(1): 1-13. Murray P. J. (2005). "The primary mechanism of the IL-10-regulated antiinflammatory response is to selectively inhibit transcription." Proc Natl Acad Sci U S A 102(24): 8686-91. Muthian G. and Bright J. J. (2004). "Quercetin, a flavonoid phytoestrogen, ameliorates experimental allergic encephalomyelitis by blocking IL-12 signaling through JAK-STAT pathway in T lymphocyte." J Clin Immunol 24(5): 542-52. Na Z., Cho J. Y., Lee H. J. et al. (2007). "Complete 1H and 13C NMR assignments of sesquiterpene glucosides from Ixeris sonchifolia." Magn Reson Chem 45(3): 275-8. Namura S., Zhu J., Fink K. et al. (1998). "Activation and cleavage of caspase3 in apoptosis induced by experimental cerebral ischemia." J Neurosci 18(10): 3659-68. Obied H. K., Karuso P., Prenzler P. D. et al. (2007). "Novel secoiridoids with antioxidant activity from Australian olive mill waste." J Agric Food Chem 55(8): 2848-53. 161 Ogura Y., Sutterwala F. S. and Flavell R. A. (2006). "The inflammasome: first line of the immune response to cell stress." Cell 126(4): 659-62. Ohman E. M., Kleiman N. S., Gacioch G. et al. (1997). "Combined accelerated tissue-plasminogen activator and platelet glycoprotein IIb/IIIa integrin receptor blockade with Integrilin in acute myocardial infarction. Results of a randomized, placebo-controlled, dose-ranging trial. IMPACTAMI Investigators." Circulation 95(4): 846-54. Ono M., Oda E., Tanaka T. et al. (2008). "DPPH radical-scavenging effect on some constituents from the aerial parts of Lippia triphylla." J Nat Med 62(1): 101-6. Paciaroni M., Medeiros E. and Bogousslavsky J. (2009). "Desmoteplase." Expert Opin Biol Ther 9(6): 773-8. Packer M. A., Porteous C. M. and Murphy M. P. (1996). "Superoxide production by mitochondria in the presence of nitric oxide forms peroxynitrite." Biochem Mol Biol Int 40(3): 527-34. Pantano P., Caramia F., Bozzao L. et al. (1999). "Delayed increase in infarct volume after cerebral ischemia: correlations with thrombolytic treatment and clinical outcome." Stroke 30(3): 502-7. Pardridge W. M. (2005). "The blood-brain barrier and neurotherapeutics." NeuroRx 2(1): 1-2. Park E. M., Cho S., Frys K. A. et al. (2006). "Inducible nitric oxide synthase contributes to gender differences in ischemic brain injury." J Cereb Blood Flow Metab 26(3): 392-401. Pedersen M. M., Chukwujekwu J. C., Lategan C. A. et al. (2009). "Antimalarial sesquiterpene lactones from Distephanus angulifolius." Phytochemistry 70(5): 601-7. Pierre J. Magistretti, Luc Pellerin and Martin J. L. (2000). "Brain Energy Metabolism: An Integrated Cellular Perspective." Psychopharmacology: the Fourth Generation of Progress. Piriyawat P., Labiche L. A., Burgin W. S. et al. (2003). "Pilot dose-escalation study of caffeine plus ethanol (caffeinol) in acute ischemic stroke." Stroke 34(5): 1242-5. Pistelli L., Bertoli A., Noccioli C. et al. (2009). "Antimicrobial activity of Inga fendleriana extracts and isolated flavonoids." Nat Prod Commun 4(12): 167983. Puetz V., Weise M., von Kummer R. et al. (2006). "Effective treatment with abciximab for consecutive bilateral middle cerebral artery occlusion." Cerebrovasc Dis 21(5-6): 419-21. 162 Qiu J., Nishimura M., Wang Y. et al. (2008). "Early release of HMGB-1 from neurons after the onset of brain ischemia." J Cereb Blood Flow Metab 28(5): 927-38. Qiu J., Whalen M. J., Lowenstein P. et al. (2002). "Upregulation of the Fas receptor death-inducing signaling complex after traumatic brain injury in mice and humans." J Neurosci 22(9): 3504-11. Rajkapoor B., Murugesh N. and Rama Krishna D. (2009). "Cytotoxic activity of a flavanone from the stem of Bauhinia variegata Linn." Nat Prod Res 23(15): 1384-9. Rashidian J., Iyirhiaro G. O. and Park D. S. (2007). "Cell cycle machinery and stroke." Biochim Biophys Acta 1772(4): 484-93. Richard Green A., Odergren T. and Ashwood T. (2003). "Animal models of stroke: they have value for discovering neuroprotective agents?" Trends Pharmacol Sci 24(8): 402-8. Rong J., Wen J., Wu G. et al. (2007). "Effects of anisodamine, Inula britannica and Ixeris sonchifolia Hance on expression of Bcl-2 and Bax proteins in myocardium of overtrained rats." Zhonghua Mazuixue Zazhi 27(20): 944-946. Rosamond W., Flegal K., Friday G. et al. (2007). "Heart disease and stroke statistics--2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee." Circulation 115(5): e69-171. Rosamond W., Flegal K., Furie K. et al. (2008). "Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee." Circulation 117(4): e25-146. Rosenberg G. A. (1999). "Ischemic brain edema." Prog Cardiovasc Dis 42(3): 209-16. Rothwell N. J. (1997). "Cytokines and acute neurodegeneration." Mol Psychiatry 2(2): 120-1. Sacco R. L., Boden-Albala B., Abel G. et al. (2001). "Race-ethnic disparities in the impact of stroke risk factors: the northern Manhattan stroke study." Stroke 32(8): 1725-31. Sacco R. L., Chong J. Y., Prabhakaran S. et al. (2007). "Experimental treatments for acute ischaemic stroke." Lancet 369(9558): 331-41. Sae-wong C., Tansakul P. and Tewtrakul S. (2009). "Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals." J Ethnopharmacol 124(3): 576-80. 163 Saeed S. A., Memon R. A., Gilani A. H. et al. (1997). "Effects of lipoproteins on cyclo-oxygenase and lipoxygenase pathways in human platelets." J Pak Med Assoc 47(3): 84-8. Sairanen T., Ristimaki A., Karjalainen-Lindsberg M. L. et al. (1998). "Cyclooxygenase-2 is induced globally in infarcted human brain." Ann Neurol 43(6): 738-47. Salom J. B., Perez-Asensio F. J., Burguete M. C. et al. (2004). "Single-dose ebselen does not afford sustained neuroprotection to rats subjected to severe focal cerebral ischemia." Eur J Pharmacol 495(1): 55-62. Salte R. and Norberg K. (1991). "Effects of warfarin on vitamin K-dependent coagulation factors in Atlantic salmon and rainbow trout with special reference to factor X." Thromb Res 63(1): 39-45. Salvesen G. S. (2001). "A lysosomal protease enters the death scene." J Clin Invest 107(1): 21-2. Sandercock P. A., Counsell C. and Tseng M. C. (2008). "Low-molecularweight heparins or heparinoids versus standard unfractionated heparin for acute ischaemic stroke." Cochrane Database Syst Rev(3): CD000119. Sarker S. D. and Nahar L. (2005). "Methods in Biotechnology." Natural Products Isolation, Second Edition: 233. Scheller D. K., De Ryck M., Kolb J. et al. (1997). "Lubeluzole blocks increases in extracellular glutamate and taurine in the peri-infarct zone in rats." Eur J Pharmacol 338(3): 243-51. Schmid-Elsaesser R., Hungerhuber E., Zausinger S. et al. (1999). "Neuroprotective efficacy of combination therapy with two different antioxidants in rats subjected to transient focal ischemia." Brain Res 816(2): 471-9. Schmidt H. H., Lohmann S. M. and Walter U. (1993). "The nitric oxide and cGMP signal transduction system: regulation and mechanism of action." Biochim Biophys Acta 1178(2): 153-75. Schottelius A. J., Mayo M. W., Sartor R. B. et al. (1999). "Interleukin-10 signaling blocks inhibitor of kappaB kinase activity and nuclear factor kappaB DNA binding." J Biol Chem 274(45): 31868-74. Schwab J. M., Beschorner R., Meyermann R. et al. (2002). "Persistent accumulation of cyclooxygenase-1-expressing microglial cells and macrophages and transient upregulation by endothelium in human brain injury." J Neurosurg 96(5): 892-9. Sena E., Wheble P., Sandercock P. et al. (2007). "Systematic review and metaanalysis of the efficacy of tirilazad in experimental stroke." Stroke 38(2): 38894. 164 Seshadri S., Beiser A., Kelly-Hayes M. et al. (2006). "The lifetime risk of stroke: estimates from the Framingham Study." Stroke 37(2): 345-50. Shi F. L., Hart R. G., Sherman D. G. et al. (1989). "Stroke in the People's Republic of China." Stroke 20(11): 1581-5. Shrivastava C. P. and Devgarha S. (2007). "Urokinase thrombolysis in acuteon-chronic vascular occlusion of lower limb." Asian Cardiovasc Thorac Ann 15(5): 405-7. Silver J. and Miller J. H. (2004). "Regeneration beyond the glial scar." Nat Rev Neurosci 5(2): 146-56. Simon R. P. (2006). "Acidotoxicity trumps excitotoxicity in ischemic brain." Arch Neurol 63(10): 1368-71. Singer D. E., Chang Y., Fang M. C. et al. (2009). "The net clinical benefit of warfarin anticoagulation in atrial fibrillation." Ann Intern Med 151(5): 297305. Soriano S. G., Coxon A., Wang Y. F. et al. (1999). "Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury." Stroke 30(1): 134-9. Stampf J. L., Schlewer G., Ducombs G. et al. (1978). "Allergic contact dermatitis due to sesquiterpene lactones. A comparative study of human and animal sensitivity to alpha-methylene-gamma-butyrolactone and derivatives." Br J Dermatol 99(2): 163-9. Strong K., Mathers C. and Bonita R. (2007). "Preventing stroke: saving lives around the world." Lancet Neurol 6(2): 182-7. Sucher N. J. (2006). "Insights from molecular investigations of traditional Chinese herbal stroke medicines: implications for neuroprotective epilepsy therapy." Epilepsy Behav 8(2): 350-62. Sudlow C. (2007). "Dipyridamole with aspirin is better than aspirin alone in preventing vascular events after ischaemic stroke or TIA." Bmj 334(7599): 901. Sudlow C. L. and Warlow C. P. (1996). "Comparing stroke incidence worldwide: what makes studies comparable?" Stroke 27(3): 550-8. Sugg R. M., Pary J. K., Uchino K. et al. (2006). "Argatroban tPA stroke study: study design and results in the first treated cohort." Arch Neurol 63(8): 105762. Sugiyama Y. and Hirota A. (2009). "New potent DPPH radical scavengers from a marine-derived actinomycete strain USF-TC31." Biosci Biotechnol Biochem 73(12): 2731-4. 165 Sun C., Shan C. Y., Gao X. D. et al. (2005). "Protection of PC12 cells from hydrogen peroxide-induced injury by EPS2, an exopolysaccharide from a marine filamentous fungus Keissleriella sp. YS4108." J Biotechnol 115(2): 137-44. Suzuki K., Okasaka M., Kashiwada Y. et al. (2007). "Sesquiterpene lactones from the roots of Ferula varia and their cytotoxic activity." J Nat Prod 70(12): 1915-8. Suzuki M., Sasamata M. and Miyata K. (2003). "Neuroprotective effects of YM872 coadministered with t-PA in a rat embolic stroke model." Brain Res 959(1): 169-72. Swanson R. A., Morton M. T., Tsao-Wu G. et al. (1990). "A semiautomated method for measuring brain infarct volume." J Cereb Blood Flow Metab 10(2): 290-3. Swanson R. A., Ying W. and Kauppinen T. M. (2004). "Astrocyte influences on ischemic neuronal death." Curr Mol Med 4(2): 193-205. Swarnkar A. S., Jungreis C. A., Wechsler L. R. et al. (1999). "Combined intravenous and intraarterial thrombolytic therapy for treatment of an acute ischemic stroke: a case report." J Stroke Cerebrovasc Dis 8(4): 264-7. Tabopda T. K., Liu J., Ngadjui B. T. et al. (2007). "Cytotoxic triterpene and sesquiterpene lactones from Elephantopus mollis and induction of apoptosis in neuroblastoma cells." Planta Med 73(4): 376-80. Tebbe U., Bramlage P., Graf A. et al. (2009). "Desmoteplase in acute massive pulmonary thromboembolism." Thromb Haemost 101(3): 557-62. Theodori R., Karioti A., Rancic A. et al. (2006). "Linear sesquiterpene lactones from Anthemis auriculata and their antibacterial activity." J Nat Prod 69(4): 662-4. Thom T., Haase N., Rosamond W. et al. (2006). "Heart disease and stroke statistics--2006 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee." Circulation 113(6): e85-151. Tietze F. (1969). "Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues." Anal Biochem 27(3): 502-22. Toti P., C D. E. F., Schurfeld K. et al. (2001). "Cyclooxygenase-2 immunoreactivity in the ischemic neonatal human brain. An autopsy study." J Submicrosc Cytol Pathol 33(3): 245-9. Toyoda K., Fujii K., Kamouchi M. et al. (2004). "Free radical scavenger, edaravone, in stroke with internal carotid artery occlusion." J Neurol Sci 221(1-2): 11-7. 166 Traystman R. J. (2003). "Animal models of focal and global cerebral ischemia." Ilar J 44(2): 85-95. Trendelenburg G. (2008). "Acute neurodegeneration and the inflammasome: central processor for danger signals and the inflammatory response?" J Cereb Blood Flow Metab 28(5): 867-81. Triantafilou M. and Triantafilou K. (2005). "The dynamics of LPS recognition: complex orchestration of multiple receptors." J Endotoxin Res 11(1): 5-11. van Gijn J., Kerr R. S. and Rinkel G. J. (2007). "Subarachnoid haemorrhage." Lancet 369(9558): 306-18. van Rossem K., Vermarien H., Decuyper K. et al. (1992). "Photothrombosis in rabbit brain cortex: follow up by continuous pO2 measurement." Adv Exp Med Biol 316: 103-12. Velat G. J., Burry M. V., Eskioglu E. et al. (2006). "The use of abciximab in the treatment of acute cerebral thromboembolic events during neuroendovascular procedures." Surg Neurol 65(4): 352-8, discussion 358-9. Venketasubramanian N, Tan LC, Sahadevan S et al. (2005). "Prevalence of stroke among Chinese, Malay, and Indian Singaporeans: a community-based tri-racial cross-sectional survey." Stroke 36(3): 551-6. Wahlgren N., Ahmed N., Davalos A. et al. (2008). "Thrombolysis with alteplase 3-4.5 h after acute ischaemic stroke (SITS-ISTR): an observational study." Lancet 372(9646): 1303-9. Wahlgren N. G., Bornhov S., Sharma A. et al. (1999). "The clomethiazole acute stroke study (CLASS): efficacy results in 545 patients classified as total anterior circulation syndrome (TACS)." J Stroke Cerebrovasc Dis 8(4): 231-9. Wahlgren N. G., MacMahon D. G., Keyser J. D. et al. (1994). "Intravenous Nimodipine West European Stroke Trial (INWEST) of Nimodipine in the Treatment of Acute Ischaemic Stroke." Cerebrovasc Dis 4(3): 204-210. Wahlgren N. G., Ranasinha K. W., Rosolacci T. et al. (1999). "Clomethiazole acute stroke study (CLASS): results of a randomized, controlled trial of clomethiazole versus placebo in 1360 acute stroke patients." Stroke 30(1): 218. Wang C., Li Y., Liu Y. et al. (2006). "Protective effects of Ixeris sonchifolia injections on rats with acute myocardial ischemia." JOURNAL OF HEBEI MEDICAL UNIVERSITY 27(1): 40-42. Wang L., Chen M., Dayuan S. et al. (2002). "Protective effects of Diemailing injection on experimental myocardial infarction in dogs." CHINESE JOURNAL OF HOSPITAL PHARMACY 22(2): 80-82. Wang Q., Tang X. N. and Yenari M. A. (2007). "The inflammatory response in stroke." J Neuroimmunol 184(1-2): 53-68. 167 Wang Z. T., Ng T. B. and Xu G. J. (1995). "Recent advances in pharmacognosy research in China." Gen Pharmacol 26(6): 1211-24. Warach S., Kaufman D., Chiu D. et al. (2006). "Effect of the Glycine Antagonist Gavestinel on cerebral infarcts in acute stroke patients, a randomized placebo-controlled trial: The GAIN MRI Substudy." Cerebrovasc Dis 21(1-2): 106-11. Warlow C., Sudlow C., Dennis M. et al. (2003). "Stroke." Lancet 362(9391): 1211-24. Wei M. C., Zong W. X., Cheng E. H. et al. (2001). "Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death." Science 292(5517): 727-30. Wei X., Liu H., Sun X. et al. (2005). "Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action." Neurosci Lett 386(1): 58-62. Wu B. Q. (1989). "[Cerebral infarction in 141 cases treated by traditional Chinese medicine and Western medicine]." Zhong Xi Yi Jie He Za Zhi 9(11): 656-7, 644. Xagorari A., Papapetropoulos A., Mauromatis A. et al. (2001). "Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages." J Pharmacol Exp Ther 296(1): 181-7. Xia Y., Dawson V. L., Dawson T. M. et al. (1996). "Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury." Proc Natl Acad Sci U S A 93(13): 6770-4. Yamagata K., Ichinose S., Miyashita A. et al. (2008). "Protective effects of ebselen, a seleno-organic antioxidant on neurodegeneration induced by hypoxia and reperfusion in stroke-prone spontaneously hypertensive rat." Neuroscience 153(2): 428-35. Yamaguchi T., Sano K., Takakura K. et al. (1998). "Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group." Stroke 29(1): 12-7. Yamashima T. (2000). "Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates." Prog Neurobiol 62(3): 273-95. Yang N. Y., Qian S. H., Duan J. A. et al. (2007). "Cytotoxic sesquiterpene lactones from Eupatorium lindleyanum." J Asian Nat Prod Res 9(3-5): 339-45. Yang Y. L., Chang S. M., Wu C. C. et al. (2007). "Cytotoxic sesquiterpene lactones from Pseudoelephantopus spicatus." J Nat Prod 70(11): 1761-5. 168 Yenari M. A., Kunis D., Sun G. H. et al. (1998). "Hu23F2G, an antibody recognizing the leukocyte CD11/CD18 integrin, reduces injury in a rabbit model of transient focal cerebral ischemia." Exp Neurol 153(2): 223-33. Yilmaz G. and Granger D. N. (2008). "Cell adhesion molecules and ischemic stroke." Neurol Res 30(8): 783-93. Yoo J. C., Cho H. S., Park E. et al. (2009). "Nargenicin attenuates lipopolysaccharide-induced inflammatory responses in BV-2 cells." Neuroreport 20(11): 1007-12. Zeidler P., Hubbs A., Battelli L. et al. (2004). "Role of inducible nitric oxide synthase-derived nitric oxide in silica-induced pulmonary inflammation and fibrosis." J Toxicol Environ Health A 67(13): 1001-26. Zhang D. D. (2006). "Mechanistic studies of the Nrf2-Keap1 signaling pathway." Drug Metab Rev 38(4): 769-89. Zhang L., Chopp M., Jia L. et al. (2009). "Atorvastatin extends the therapeutic window for tPA to h after the onset of embolic stroke in rats." J Cereb Blood Flow Metab 29(11): 1816-24. Zhang L., Zhang Z. G., Liu X. S. et al. (2007). "The PI3K/Akt pathway mediates the neuroprotective effect of atorvastatin in extending thrombolytic therapy after embolic stroke in the rat." Arterioscler Thromb Vasc Biol 27(11): 2470-5. Zhang Q. J. (2007). "One case of asthma caused by Diemailing." Zhongxiyi Linchuang Zazhi 19(5): 527. Zhang Z., Zhang R. L., Jiang Q. et al. (1997). "A new rat model of thrombotic focal cerebral ischemia." J Cereb Blood Flow Metab 17(2): 123-35. Zhao X., Haensel C., Araki E. et al. (2000). "Gene-dosing effect and persistence of reduction in ischemic brain injury in mice lacking inducible nitric oxide synthase." Brain Res 872(1-2): 215-8. Zhao Y., Hao Y., Ji H. et al. (2010). "Combination Effects of Salvianolic Acid B with Low-Dose Celecoxib on Inhibition of Head and Neck Squamous Cell Carcinoma Growth In vitro and In vivo." Cancer Prev Res (Phila Pa) 3(6): 787-96. Zhao Z., Cheng M., Maples K. R. et al. (2001). "NXY-059, a novel free radical trapping compound, reduces cortical infarction after permanent focal cerebral ischemia in the rat." Brain Res 909(1-2): 46-50. Zheng Z., Lee J. E. and Yenari M. A. (2003). "Stroke: molecular mechanisms and potential targets for treatment." Curr Mol Med 3(4): 361-72. Zheng Z. and Yenari M. A. (2004). "Post-ischemic inflammation: molecular mechanisms and therapeutic implications." Neurol Res 26(8): 884-92. 169 Zhou H., Lutterodt H., Cheng Z. et al. (2009). "Anti-Inflammatory and antiproliferative activities of trifolirhizin, a flavonoid from Sophora flavescens roots." J Agric Food Chem 57(11): 4580-5. Zhou X., Jin W., Cao C. et al. (2007). "The protective effect of Kudiezi injection on the experimental acute myocardial infarction." Shizhen Guoyi Guoyao 122(18): 1. 170 [...]... in stroke management will be discussed Recently, the raw extract of Ixeris sonchifolia Hance was developed for use in the prevention and treatment of cardio- and cerebral-vascular diseases As the main focus of this Ph.D study, the chemical constituents of Ixeris sonchifolia Hance and their biological activities will also be addressed 1.1 Stroke 1.1.1 Physiopathology The brain cells have a high demand... occurrence of stroke and 2 the associated medical and social burdens are on the rise The principles in stroke treatment involve the restoration of blood supply to the affected brain area in the shortest possible time from the onset of stroke, as well as to reduce ischemia-caused tissue damage Unfortunately, despite decades of research, alteplase (a tissue plasminogen activator that catalyzes the conversion of. .. scavenging activity of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract 45 2.3.3.2 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract on ARE-dependent transcriptional activity 45 2.3.3.3 Representative Western blot analysis of protein levels of transcription factor Nrf2 in SH-SY5Y cells treated with fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract 46... quantity measurement of compounds 1 to 5 and 7 in fractions 1 to 4 by HPLC analysis 73 5.3.3.1 Effects of flavonoid compounds 1 to 5 on the mRNA expression of cytokines in BV-2 cells 109     X List of figures Figure Title Page 1.2.1 The aerial part of Ixeris sonchifolia Hance 25 2.3.2.1 Effects of fractions 1 to 4 isolated from Ixeris sonchifolia Hance extract on the viability of H2O2-exposed PC12...List of tables Table Title Page 1.1.3.1 Summary of anti- stroke agents in preclinical studies and in clinical applications 19 2.3.1.1 Anticoagulation activity of fractions 1 to 4 extracted from Ixeris sonchifolia Hance 42 3.3.1.1 13 C-NMR spectral data for compounds 1 to 7 65 3.3.1.2 13 C-NMR spectral data for compounds 8 to 13 70 3.3.2.1 Linear regression, r2 values, detection limits and quantity measurement... cells release several pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, as well as other potential cytotoxic molecules including NO, ROS and prostanoids (Lucas et al., 2006) The astrocytes are also capable of secreting inflammatory factors such as cytokines, chemokines, and NO (Swanson et al., 2004) The released proinflammatory cytokines and chemokines will further enhance recruitment of microglial... which they suffer from stroke attacks is higher and thus the stroke is more severe, with a 1-month case fatality of 24.7% as compared to 19.7% for men (Appelros et al., 2009) Among all the risk factors for stroke, increased age is a noncontroversial predictor Although stroke can occur at any age, including in 8 fetus, the incidence increases exponentially from 30 years of age with 95% of strokes occurring... reduction of about 1% per year until the late 1960s, followed by a more steeper fall of as much as 5% per year (Bonita, 1992) Recently, the rate of this decline has decreased greatly (Feigin et al., 2003) This decline in incidence and mortality is likely related to the improved control of stroke risk factors such as high blood pressure and cigarette smoking in particular, as well as an improvement in standards... 9 and 10 68 3.3.1.4 Chemical structures of compounds 11 to 13 71 3.3.1.5 Chemical structures of compounds 14 to 18 71 3.3.2.1 Representative HPLC profiles for purified compounds 1 to 5 and 7 and fractions 1 to 4 74 3.4.1 Structures of sesquiterpene lactones 75 3.4.2 Skeletal structures of flavonoids, isoflavonoids and neoflavonoids 76 3.4.3 Skeletal structures of subgroups of flavonoids 76 HMBC and. .. of plasminogen to plasmin) is still the only drug approved by FDA for stroke treatment Therefore, finding new effective reagents efficient in stroke treatment and management has become crucial The following sections will give a short review on the physiopathology and epidemiology associated with stroke, followed by a summary of advancements in anti- stroke drug therapy In addition, the application of . ISOLATION AND IDENTIFICATION OF COMPONENTS FROM IXERIS SONCHIFOLIA HANCE AS POTENTIAL ANTI- STROKE AGENTS ZHANG YAOCHUN B.Sc. (Pharm.), Shandong University M.Sc Characterization of purified compounds isolated from Ixeris sonchifolia Hance 61 3.3.2 Quantitative HPLC analysis of compounds 1 to 5 and 7 in fractions 1 to 4 isolated from Ixeris sonchifolia Hance. ethyl acetate fraction of Ixeris sonchifolia Hance 55 3.1 Introduction 56 3.2 Materials and methods 56 3.2.1 Isolation of the fractions from Ixeris Sonchifolia Hance extract 56 IV