Wu Wai Ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 163-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.vii / Abbreviations --- p.x / Chapter Chapter 1 --- General introduction --- p.11 / Chapter 1.1 --- Introduction --- p.11 / Chapter 1.1.1 --- "Origin, processing and delivery form of TCM" --- p.11 / Chapter 1.1.2 --- Problems about the uses of TCM --- p.13 / Chapter 1.1.2.1 --- Quality control --- p.13 / Chapter 1.1.2.2 --- Efficacy --- p.14 / Chapter 1.1.2.3 --- Herb-drug interactions --- p.14 / Chapter 1.1.2.4 --- Authentication --- p.15 / Chapter 1.1.3 --- Commonly used Traditional Chinese Medicine --- p.15 / Chapter 1.2 --- Interactions between TCM and warfarin --- p.17 / Chapter 1.2.1 --- Danshen-warfarin interactions --- p.19 / Chapter 1.3 --- Danshen --- p.20 / Chapter 1.3.1 --- Chemical constituents --- p.20 / Chapter 1.3.2 --- Pharmacological effects of danshen --- p.24 / Chapter 1.3.2.1 --- Anti-oxidant effects --- p.24 / Chapter 1.3.2.2 --- Effects on liver fibrosis --- p.25 / Chapter 1.3.2.3 --- Effects on tumours --- p.26 / Chapter 1.3.2.4 --- Effects on cardiovascular system --- p.26 / Chapter 1.3.2.5 --- Effect on platelet aggregation --- p.27 / Chapter 1.4 --- Warfarin --- p.27 / Chapter 1.4.1 --- Pharmacology --- p.28 / Chapter 1.4.2 --- Pharmacokinetics --- p.30 / Chapter 1.4.3 --- Metabolism --- p.30 / Chapter 1.4.4 --- Warfarin-drug interactions --- p.32 / Chapter 1.4.4.1 --- Pharmacokinetic Interactions --- p.32 / Chapter 1.4.4.2 --- Pharmacodynamic interactions --- p.34 / Chapter 1.5 --- Aim of study --- p.35 / Chapter Chapter 2 --- Effects of danshen extract and some of its active ingredients on warfarin metabolism in rat liver microsomes --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Materials and methods --- p.39 / Chapter 2.2.1 --- Chemicals and reagents --- p.39 / Chapter 2.2.2 --- Animals --- p.39 / Chapter 2.2.3 --- Preparation of rat hepatic microsomes --- p.40 / Chapter 2.2.4 --- Protein assay --- p.40 / Chapter 2.2.5 --- Preparation of aqueous fraction and ethanolic fractions of danshen from danshen roots --- p.41 / Chapter 2.2.5.1 --- Aqueous extract of danshen --- p.41 / Chapter 2.2.5.2 --- Ethanolic extract of danshen --- p.42 / Chapter 2.2.6 --- Incubation condition for warfarin metabolism --- p.42 / Chapter 2.2.7 --- Effects of danshen extract and some of its active ingredients on warfarin metabolism in vitro --- p.43 / Chapter 2.2.8 --- Effects of danshen extract and some of its sctive ingredients on enzyme kinetics of warfarin metabolism in vitro --- p.44 / Chapter 2.2.9 --- High Pressure Liquid Chromatography (HPLC) analysis --- p.45 / Chapter 2.2.10 --- Calibration curves and validation of the HPLC systems --- p.48 / Chapter 2.2.11 --- Data analysis --- p.52 / Chapter 2.3 --- Results --- p.53 / Chapter 2.3.1 --- Effects of danshen extract on warfarin metabolism --- p.53 / Chapter 2.3.2 --- Effects of aqueous extract of danshen on warfarin metabolism --- p.60 / Chapter 2.3.3 --- Effects of ethanolic extract of danshen on warfarin metabolism --- p.62 / Chapter 2.3.4 --- Effects of tanshinone I on warfarin metabolism --- p.64 / Chapter 2.3.5 --- Effects of tanshinone IIA on warfarin metabolism --- p.70 / Chapter 2.3.6 --- Effects of cryptotanshinone on warfarin metabolism --- p.76 / Chapter 2.3.7 --- IC20 of danshen extract and its components on warfarin metabolism --- p.82 / Chapter 2.4 --- Discussion --- p.84 / Chapter Chapter 3 --- Effects of danshen extract and some of its active ingredients on warfarin metabolism in human pooled liver microsomes and the human CYP2C9 isoform --- p.89 / Chapter 3.1 --- Introduction --- p.89 / Chapter 3.2 --- Materials and methods --- p.92 / Chapter 3.2.1 --- Chemicals and reagents --- p.92 / Chapter 3.2.2 --- Incubation conditions for warfarin metabolism --- p.92 / Chapter 3.2.3 --- Effects of danshen extract and its components on warfarin metabolism in vitro --- p.93 / Chapter 3.2.4 --- High Pressure Liquid Chromatography (HPLC) analysis --- p.94 / Chapter 3.2.5 --- Calibration curves --- p.95 / Chapter 3.2.6 --- Data analysis --- p.95 / Chapter 3.3 --- Results --- p.96 / Chapter 3.3.1 --- Effects of danshen extract and its components on warfarin metabolism by using human pooled liver microsomes --- p.96 / Chapter 3.3.2 --- Effects of danshen extract and its components on S-warfarin metabolism by using human lymphoblast CYP2C9 isoform --- p.103 / Chapter 3.4 --- Discussion --- p.111 / Chapter Chapter 4 --- Effects of acute and subchonic pretreatment of danshen extract on the pharmacokinetics of warfarin in the rats in vivo --- p.115 / Chapter 4.1 --- Introduction --- p.115 / Chapter 4.2 --- Materials and methods --- p.118 / Chapter 4.2.1 --- Chemicals and reagents --- p.118 / Chapter 4.2.2 --- Animals Table of Contents --- p.118 / Chapter 4.2.3 --- Effects of acute danshen extract pretreatment on the pharmacokinetics of warfarin --- p.119 / Chapter 4.2.4 --- Effects of subchronic danshen extract pretreatment on the pharmacokinetics of warfarin --- p.119 / Chapter 4.2.5 --- Steady state warfarin study --- p.120 / Chapter 4.2.6 --- Sample extraction --- p.120 / Chapter 4.2.7 --- High Pressure Liquid Chromatography (HPLC) analysis --- p.121 / Chapter 4.2.8 --- Calibration curve --- p.121 / Chapter 4.4 --- Results --- p.123 / Chapter 4.3.1 --- Effects of acute danshen extract pretreatment on the pharmacokinetics of warfarin --- p.123 / Chapter 4.3.2 --- Effects of subchronic danshen extract pretreatment on the pharmacokinetics of warfarin --- p.128 / Chapter 4.3.3 --- Steady state warfarin study --- p.136 / Chapter 4.5 --- Discussion --- p.138 / Chapter Chapter 5 --- Effects of danshen extract on the absorption of warfarin by using Caco-2 cells model --- p.142 / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Materials and methods --- p.144 / Chapter 5.2.1 --- Materials for Caco-2 cells culture experiment --- p.144 / Chapter 5.2.2 --- Preparation of Caco-2 monolayer --- p.144 / Chapter 5.2.3 --- High Pressure Liquid Chromatography (HPLC) analysis for warfarin --- p.145 / Chapter 5.2.4 --- Calibration curve --- p.145 / Chapter 5.2.5 --- Stability test for warfarin and danshen extract --- p.145 / Chapter 5.2.6 --- Toxicity test of danshen extract on Caco-2 cells --- p.146 / Chapter 5.2.7 --- Transport study --- p.146 / Chapter 5.2.8 --- Data Analysis --- p.147 / Chapter 5.3 --- Results --- p.149 / Chapter 5.3.1 --- Stability of warfarin and danshen extract --- p.149 / Chapter 5.3.2 --- Toxicity test of danshen extract on Caco-2 cells --- p.149 / Chapter 5.3.3 --- Integrity of Caco-2 cells monolayer --- p.152 / Chapter 5.3.4 --- Transport study --- p.152 / Chapter 5.4 --- Discussion --- p.154 / Chapter Chapter 6 --- General discussion --- p.156 / References --- p.163

Hung Shieh-Jung Fanny. / Thesis submitted in: August 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-109). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Content --- p.vi / List of Abbreviations --- p.xiii / List of Figure --- p.xv / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease (AD) --- p.1 / Chapter 1.1.1 --- Clinical features --- p.2 / Chapter 1.2 --- Histopathological studies of AD --- p.2 / Chapter 1.2.1 --- Neuritic plaques --- p.2 / Chapter 1.2.2 --- Neurofibrillary tangles (NFT) --- p.4 / Chapter 1.2.3 --- Tau --- p.5 / Chapter 1.3 --- Kinases and Alzheimer's Disease --- p.7 / Chapter 1.4 --- Free radical damage --- p.8 / Chapter 1.5 --- Available treatment for AD --- p.7 / Chapter 1.6 --- A Chinese medicinal material 一 Danshen （（Salviae miltiorrhizcie) --- p.11 / Chapter 1.6.1 --- Chemical constituents --- p.11 / Chapter 1.6.1.1 --- Lipophilic Compounds of Danshen --- p.12 / Chapter 1.6.1.2 --- Water-soluble Compounds of Danshen --- p.17 / Chapter 1.6.2 --- Pharmacological usage --- p.20 / Chapter 1.6.2.1 --- Action on Coronary system --- p.20 / Chapter 1.6.2.2 --- Bacteriostatic action --- p.21 / Chapter 1.6.2.3 --- Actions on the immune system --- p.21 / Chapter 1.6.3 --- Biological activity on brain --- p.22 / Chapter 1.7 --- Objectives and scope of the project --- p.23 / Chapter Chapter 2 --- General Materials and Method --- p.24 / Chapter 2.1 --- Recombinant DNA techniques --- p.24 / Chapter 2.1.1 --- Preparation of E. coli strain DH-5a competent cells --- p.24 / Chapter 2.1.2 --- Transformation of plasmid DNA into competent cells --- p.25 / Chapter 2.1.3 --- Preparation of plasmid DNA using QIAGEN Plasmid Maxipreps kit --- p.25 / Chapter 2.1.4 --- Phenol/ choroform extraction of DNA --- p.26 / Chapter 2.1.5 --- Spectrophotometric quantitation of the amount and purity of DNA --- p.27 / Chapter 2.2 --- Drugs preparation --- p.27 / Chapter 2.2.1 --- Preparation of aqueous extracts of Traditional Chinese Medicine (TCM) --- p.27 / Chapter 2.2.2 --- Preparation of ethanol extracts of Traditional Chinese Medicine (TCM) --- p.27 / Chapter 2.3 --- "3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay " --- p.28 / Chapter 2.4 --- Analysis of proteins from culture cells --- p.28 / Chapter 2.4.1 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.4.2 --- Quantitation of protein by Bradford method --- p.29 / Chapter 2.4.3 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.4.4 --- Western blot analysis --- p.31 / Chapter 2.5 --- Reagents and buffers --- p.32 / Chapter 2.5.1 --- Reagents for competent cell preparation --- p.32 / Chapter 2.5.2 --- Reagents provided by QIAGEN Plasmid Maxipreps kit --- p.33 / Chapter 2.5.3 --- Reagents for SDS-PAGE --- p.34 / Chapter 2.5.4 --- Reagents and buffers for Western Blotting --- p.35 / Chapter 2.5.5 --- Cell lines --- p.36 / Chapter 2.5.6 --- Antibodies --- p.37 / Chapter 2.5.7 --- Plasmids --- p.37 / Chapter 2.5.8 --- Other Chemicals --- p.38 / Chapter Chapter 3 --- The effect of Danshen on GSK-3 induced hyperposphorylation of tau in Cos7 cells / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.1.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.39 / Chapter 3.1.2 --- Structure of GSK-3 --- p.39 / Chapter 3.1.3 --- The importance of GSK-3 in AD --- p.39 / Chapter 3.2 --- Materials and Methods --- p.41 / Chapter 3.2.1 --- Transfection of Gsk-3 and tau into Cos7 monkey kidney cells --- p.43 / Chapter 3.2.2 --- Extraction of total proteins from culture cells --- p.44 / Chapter 3.2.3 --- Quantitation of protein by Bradford method --- p.44 / Chapter 3.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.5 --- Western blot analysis --- p.44 / Chapter 3.3 --- Results --- p.45 / Chapter 3.3.1 --- Toxicity test on Cos7 cells --- p.45 / Chapter 3.3.2 --- The effect of ethanol extract of Danshen on GSK-3 β induced tau phosphorylation --- p.45 / Chapter 3.3.3 --- The effect of aqueous extract of Danshen on GSK-3 β induced tau phosphorylation --- p.48 / Chapter 3.3.4 --- The effect of Protocatechualdehyde on GSK-3β induced tau phosphorylation --- p.48 / Chapter 3.3.5 --- The effect of Salvianolic acid B on GSK-3β induced tau phosphorylation --- p.49 / Chapter 3.4 --- Discussion --- p.60 / Chapter Chapter 4 --- Cdk5 induced hyperposphorylation of tau in CHO cells / Chapter 4.1 --- Introduction --- p.63 / Chapter 4.1.1 --- Cyclin dependent kinase 5 (Cdk5) --- p.63 / Chapter 4.1.2 --- Structure of Cdk5 --- p.63 / Chapter 4.1.3 --- Neurological functions of Cdk5 --- p.64 / Chapter 4.2 --- Materials and Methods --- p.66 / Chapter 4.2.1 --- Transfection of p35 and tau into CHO cells --- p.66 / Chapter 4.2.2 --- Extraction of total proteins from culture cells --- p.67 / Chapter 4.2.3 --- Quantitation of protein by Bradford method --- p.67 / Chapter 4.2.4 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.67 / Chapter 4.2.5 --- Western blot analysis --- p.67 / Chapter 4.3 --- Results --- p.68 / Chapter 4.3.1 --- Toxicity test on CHO cells --- p.68 / Chapter 4.3.2 --- Tau transfection in Cdk5/p35 and TauON3R transiently transfected in CHO cells --- p.68 / Chapter 4.3.3 --- Effect of roscovitine treatment on the transiently tau and p35 transfection in CHO cells --- p.74 / Chapter 4.3.4 --- "Effects of aqueous active components of Danshen, PCAH and SAB on the transiently tau and p35 transfection in CHO cells " --- p.74 / Chapter 4.4 --- Discussion --- p.79 / Chapter Chapter 5 --- Antioxidant effect of Danshen and its active components on lipid peroxidation / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.1.1 --- Red-blood-cell hemolysis model --- p.82 / Chapter 5.2 --- Materials and methods --- p.84 / Chapter 5.2.1 --- Red-blood-cell hemolysis model --- p.84 / Chapter 5.2.2 --- Materials --- p.85 / Chapter 5.2.2.1 --- Animals --- p.85 / Chapter 5.2.2.2 --- Chemicals --- p.85 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Aqueous and ethanol extracts of Danshen --- p.86 / Chapter 5.3.2 --- Active components ´ؤ Protocatechualdehyde and Salvianolic acid B --- p.87 / Chapter 5.4 --- Discussion --- p.91 / Chapter Chapter 6 --- General discussion and Outlook / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Proposed study in the future --- p.95 / Chapter 6.2.1 --- In vitro kinase assay using gamma32 P ATP and substrate with or without TCM --- p.95 / Chapter 6.2.2 --- Use of neuroblastoma cells (SHSY-5Y) to study the effect of Danshen and its active components on tau phosphorylation --- p.95 / Chapter 6.2.3 --- Thiobarbituric acid-reacting substances (TBARS) assay --- p.96 / Chapter 6.2.4 --- In vitro phosphatase kinase assay --- p.96

published_or_final_version / abstract / toc / Paediatrics / Master / Master of Philosophy

Background. Danshen, the dried root of Salvia miltiorrhiza Bge, is used for treating coronary heart disease. In China, numerous pharmaceutical dosage forms of Danshen are commercially available. Although the pharmacological effects of different components of Danshen are well identified, its absorption as well as pharmacokinetics studies are still insufficient and inconsistent. The current study aims to: (1) screen for the major absorbable active components of Danshen; (2) interpret the absorption mechanism and pharmacokinetics characteristics of the identified components; (3) develop an improved oral drug delivery system for the identified components of Danshen. / Conclusion. Both danshensu and SAB have limited intestinal permeability and oral bioavailabilities. Our results demonstrated the usefulness of sodium caprate as a potential absorption enhancer for danshensu and SAB in Danshen product. / Methods. Six major active components in commercially available Danshen products were identified and quantified. In vitro human Caco-2 cell monolayer model, rat in situ intestinal perfusion model as well as rat in vivo pharmacokinetic model were used to investigate the intestinal absorption and pharmacokinetics profiles of the identified Danshen components. Effect of the absorption enhancer on the oral absorption and bioavailabilities of the studied Danshen components was further evaluated. / Results. Danshensu, salvianolic acid B (SAB) and protocatechuic aldehyde (PCA) were identified as the major components in Danshen products. Investigations using in vitro, in situ and in vivo model found that both danshensu and SAB had poor permeabilities and low bioavailabilities (Danshensu: 11.09%; SAB: 3.90%), which may be due to their absorption via the paracellular transport pathways. Studies of PCA suggested that it may have a intestinal first pass metabolism with an oral bioavailability of only 18.02%. It was found that the permeabilities of both danshensu and SAB were significantly increased upon addition of sodium caprate, a paracellular absorption enhancer. The oral bioavailabilities of both danshensu and SAB in pure compound form as well as Danshen extract form were also increased in the presence of sodium caprate in rats. / Zhou, Limin. / Advisers: Zuo Zhong; Moses S.S. Chow. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3457. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Danshen, the dried root of Salvia miltiorrhiza Bunge, is a herb listed in the Chinese Pharmacopoeia for the treatment of cardiovascular and cerebrovascular diseases. Danshen has been reported to have antiplatelet, cardioprotective, anti-inflammatory, hepatoprotective, and anti-HIV effects in preclinical studies. However, exaggerated anticoagulation and bleeding complications have also been observed during concurrent use of Danshen and warfarin in patients, although the mechanism(s) of the herb-drug interaction, pharmacodynamic and/or pharmacokinetic interactions, remained uncertain. Characterization of the cytochrome P450 isoforms responsible for the metabolism(s) of drugs and herbal constituents is important for the identification of potential drug-drug or drug-herb interactions. The present study investigated the effects of Danshen on the metabolism of probe substrates of specific CYP isoforms including CYP1A2, CYP3A and CYP2C9, the isoforms that are responsible for the metabolism of warfarin to assess the potential interactions of Danshen with drugs that utilize these isoforms for their biotransformation. / Firstly, the effects of Danshen and its tanshinone components on CYP1A2 activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract and the aqueous extract from Danshen root, and individual tanshinones inhibited phenacetin O-deethylation (CYP1A2) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone were competitive CYP1A2 inhibitors. Acute, sub-chronic and chronic pretreatments of formulated Danshen extract decreased the clearance (CL) of caffeine, with a concomitant increase in the area under concentration-time curve (AUC), and prolongation of the plasma half-life (T 1/2). These results suggested that Danshen inhibited rat CYP1A2 activity and altered the pharmacokinetics of the CYP1A2 probe substrates in vivo. / In conclusion, these results confirmed that Danshen-inhibited CYP activity, especially CYP1A2, then CYP2C9/11 (CYP2C9 in human, CYP2C11 in rats) in vitro. In vivo studies confirmed the clearance of the probe substrates was also decreased when co-administered with Danshen. Given that CYP1A2, CYP2C9 and CYP3A4 are responsible for the metabolism and disposition of a large number of drugs currently used in man, the concomitant use of Danshen with drugs which are substrates of CYP1A2, 2C9 and 3A4, especially CYP1A2, must be met with great caution. / Secondly, the effects of Danshen and its tanshinone components on CYP3A activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract from Danshen root, and tanshinones inhibited testosterone 6beta-hydroxylation (CYP3A) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, and cryptotanshinone were competitive CYP3A inhibitors, whereas dihydrotanshinone was a noncompetitive CYP3A inhibitor. In vivo studies showed the pretreatments of formulated Danshen extract did not significantly change the pharmacokinetics of midazolam. / The effects of Danshen and its tanshinones on human CYP1A2 (phenacetin O-deethylation), CYP3A4 (testosterone 6beta-hydroxylation), and CYP2C9 (tolbutamide 4-hydroxylation) activities were also investigated in vitro using pooled human liver microsomes and human CYP isoforms. The ethanolic fraction of Danshen root was more effective than water-soluble fraction in inhibiting human CYP1A2, CYP3A4 and CYP2C9 activities. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, and cryptotanshinone were competitive inhibitors of CYP1A2, CYP3A4 and CYP2C9 with varying effectiveness. Dihydrotanshinone was not a competitive inhibitor of CYP1A2 and CYP2C9, but a noncompetitive CYP3A4 inhibitor. CYP1A2 was most affected and CYP3A4 was least affected by Danshen and tanshinones. Compared with the results obtained from rat and human, rat is a good animal model for predicting Danshen-drug interactions in humans, especially drugs which are substrates of CYP1A2. / Thirdly, the effects of Danshen and its tanshinone components on CYP2C11 activity were investigated in vitro and in vivo in the rat. Formulated Danshen extract, the ethanolic extract from Danshen root, and tanshinones inhibited testosterone 2alpha-hydroxylation (CYP2C11) activity in vitro. Enzyme kinetic studies showed that tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone were competitive CYP2C11 inhibitors. Sub-chronic pretreatment of formulated Danshen extract increased the AUC, T1/2 but decreased CL of tolbutamide. These results suggested that Danshen inhibited the CYP2C activity in the rat. In conclusion, these results confirmed the possible mechanism is enzyme inhibition, involved in the interaction of Danshen and warfarin previously observed in rats. / Wang, Xin. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4699. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 284-302). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

丹参是一种著名的传统中药，富含丹酚酸和丹参酮。其中，丹参酮的潜在抗肿瘤作用近年来引起众多关注。本研究评价了主要的丹参酮及其衍生物对结肠癌细胞的细胞毒性。结果显示DHTS具有最强的抗结肠癌活性和显著的肿瘤特异性细胞毒性，其细胞毒性主要由于凋亡诱导而不是引起坏死。初步的构效关系分析提示丹参酮母环结构中的A环和B环增加的离域性有助于提高其对结肠癌细胞的细胞毒性，而非二维结构及较小的D环也是进行结构改造的可能方向。 / 基于以上发现，本研究进一步探讨了DHTS的体内外抗肿瘤活性及内在机制。本研究发现DHTS的促凋亡活性并不依赖于p53的表达，而依赖于caspase活性及线粒体介导的细胞质中氧自由基 ROS及钙离子的聚集。DHTS可引起浓度及时间依赖caspase-9/-3/-7的活化而并未显著引起caspase-8的活化，这一现象发生于同样以浓度及时间依赖方式进行的线粒体中cytochrome c及AIF转位之后。在DHTS诱导的结肠癌细胞凋亡中，cytochrome c及caspase介导的凋亡通路及AIF介导的凋亡通路均被激活并显示出两条通路之间的交叉调控。 / 此外，线粒体在DHTS的促凋亡活性中的作用在本研究中被深入探讨。本研究发现线粒体可能是DHTS的一个直接靶点, 而氧化磷酸化复合体III则更可能是其作用位点。DHTS可以引起迅速而明显的线粒体功能障碍，随之引起细胞质中大量的氧自由基及钙离子聚集，诱导凋亡的产生。 / 与体外结果一致，本研究证实了DHTS对免疫缺陷小鼠中的结肠癌移植廇也具有明显的抗肿瘤作用。与溶媒对照组比较，DHTS治疗组中移植廇的增长显著被减缓，在治疗终点时的廇体积与重量也显著被降低。TUNEL检测确认DHTS诱导移植廇中癌细胞的显著凋亡。免疫荧光检测也发现DHTS诱导caspase-3及caspase-7在移植廇中癌细胞的明显活化。 / 综上所述，本研究提供了丹参酮抗结肠癌活性的一些初步构效关系的信息，为提高丹参酮抗结肠癌活性的结构改造提供一定的参考。更重要的是，本研究证明了DHTS的体内外抗结肠癌活性并探讨了其作用机制及可能靶点，为DHTS作为新的应用于抗结肠癌药物或辅助治疗用药提供了临床前研究证据。 / Salvia miltiorrhiza Bunge, also known as Danshen, rich in phenolic acid and tanshinones, has been widely used to treat various kinds of diseases including heart diseases and hepatitis in China with minimal side effects. Among the tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone I are the major bioactive constituents in this herb. In this study, the anti-colon cancer potential of five tanshinones and six derivatives of tanshinone IIA were evaluated in several colon cancer cell lines. It was found that apoptosis but not necrosis contributed significantly to the cytotoxicity of the tanshinones. Dihydrotanshinone I (DHTS) was confirmed to be the most potent and selective anti-cancer compound among the tanshinones tested in this study. Preliminary SAR (structure activity relationship) of tanshinones reveals that the increase of delocalizability of A and B rings in the chemical structure of the tanshinones enhances their cytotoxicity on cancer cells, while compounds with a non-planar and small sized D ring region are better choices for anti-cancer effect. / The underlying mechanisms of the anti-colon cancer activity of DHTS were further studied. It was found that apoptosis induced by DHTS was p53 independent but caspase dependent, which was closely related to intracellular accumulation of ROS (reactive oxidant stress) and calcium mediated by mitochondria. A concentration- and time-dependent activation of caspase-9,-3,-7 but not caspase-8 by DHTS in HCT116 cells was detected after the translocation of cytochrome c and AIF (apoptosis inducing factor) from mitochondria. In this process, the crosstalk between the caspase-dependent and caspase-independent pathways was firstly shown in the apoptotic mechanism of DHTS. To this end, the release of cytochrome c happened first and the translocation of apoptosis inducing factor (AIF) was prevented by a pan caspase inhibitor. In the meantime, the release of cytochrome c and activation of caspase-9 and PARP (poly-ADP-ribose polymerase) cleavage were decreased after AIF knockdown. Especially, mitochondrion was suggested to be the direct target of DHTS and OXPHOS complex III but not OXPHOS complex I was probably the acting site of DHTS. / In accordance with the results obtained in vitro, the potential anti-colon cancer activity of DHTS was also observed in nude mice with xenograft tumors and the compound did not produce any observable systemic toxicity. DHTS efficiently delayed tumor growth by decreasing the tumor size and weight through the induction of apoptosis in cancer cells but not by inhibition of cell proliferation. In the same tissues, a distinct activation of caspase-3 and caspase-7 in tumor cells was also detected by immunofluorescence assay. / Collectively, the present study provides preliminary information about the SAR of the anti-colon cancer activity for tanshinones. It also confirms that DHTS is a promising compound for anti-cancer action both in vitro and in vivo. In addition, this study gives us a better understanding regarding the mechanisms of how DHTS induces apoptosis in cancer cells. All these findings could provide solid pre-clinical evidence to propel the development and application of DHTS and perhaps its derivatives as novel therapeutic or adjuvant agents for the treatment of colon cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 122-132). / Abstracts also in Chinese. / Wang, Lin.

Chan Yin Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ABSTRACT --- p.III / ACKNOWLEDGEMENT --- p.X / TABLE OF CONTENTS --- p.XI / ABBREVIATIONS --- p.XV / LIST OF FIGURES --- p.XVII / LIST OF TABLES --- p.XXI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction to Cardiovascular Disease and Atherosclerosis --- p.1 / Chapter 1.1.1 --- Cardiovascular Disease --- p.1 / Chapter 1.1.2 --- A therosclerosis --- p.3 / Chapter 1.1.2.1 --- Structure of Arteries --- p.4 / Chapter 1.1.2.2 --- Pathophysiology of Atherosclerosis --- p.5 / Chapter 1.1.2.3 --- Endothelial Dysfunction --- p.8 / Chapter 1.1.3 --- Current Western Therapies --- p.11 / Chapter 1.1.3.1 --- Surgery --- p.11 / Chapter 1.1.3.2 --- Western Medications --- p.13 / Chapter 1.1.4 --- Traditional Chinese Medicine --- p.17 / Chapter 1.1.4.1 --- Long History --- p.17 / Chapter 1.1.4.2 --- As Alternative Medicine --- p.18 / Chapter 1.1.4.3 --- Modernization of Chinese Medicine --- p.19 / Chapter 1.2 --- Introduction and Selection of Chinese Medicine --- p.20 / Chapter 1.2.1 --- Selection ofTCM Formulation from Pharmacopoeia --- p.20 / Chapter 1.2.1.1 --- Compound Formulation --- p.20 / Chapter 1.2.2 --- Introduction to the Herbal Medicines --- p.21 / Chapter 1.2.2.1 --- Danshen (Salvia miltiorrhiza) --- p.21 / Chapter 1.2.2.2 --- Gegen (Puerariae thomsonii and Puerariae lobata) --- p.22 / Chapter 1.2.2.3 --- Yanhu (Corydalis yanhusuo) and its Exclusion --- p.24 / Chapter 1.2.3 --- Source and Authentication of the Herbal Medicines --- p.25 / Chapter CHAPTER 2 --- OPTIMIZATION OF DANSHEN-GEGEN FORMULA --- p.26 / Chapter 2.1 --- Project History --- p.26 / Chapter 2.2 --- aims for the present study --- p.27 / Chapter 2.3 --- Methods and Materials --- p.30 / Chapter 2.3.1 --- Extracts --- p.30 / Chapter 2.3.2 --- Extraction Process --- p.31 / Chapter 2.3.3 --- In vitro Antioxidation Model --- p.33 / Chapter 2.3.4 --- Ex vivo Vasodilation Model --- p.35 / Chapter 2.3.5 --- Statistical Analysis --- p.38 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Vasodilation Results --- p.39 / Chapter 2.4.2 --- Antioxidation Results --- p.43 / Chapter 2.5 --- Discussion --- p.46 / Chapter 2.6 --- Further Modification of the Formula --- p.49 / Chapter 2.6.1 --- Extracts --- p.49 / Chapter 2.6.2 --- Results --- p.49 / Chapter 2.7 --- discussion --- p.52 / Chapter CHAPTER 3 --- MARKER CHEMICAL CONTENTS OF HERBAL EXTRACTS AND THEIR PHARMACOLOGICAL PROPERTIES --- p.56 / Chapter 3.1 --- HPLC Analysis of Marker Contents --- p.56 / Chapter 3.1.1 --- Methods --- p.57 / Chapter 3.1.2 --- Results --- p.58 / Chapter 3.1.2.1 --- HPLC Chromatograms --- p.59 / Chapter 3.1.2.2 --- Content Percentage of Marker Compounds --- p.63 / Chapter 3.1.3 --- Discussion --- p.64 / Chapter 3.2 --- Studies on Marker Compounds --- p.65 / Chapter 3.2.1 --- Introduction --- p.65 / Chapter 3.2.2 --- Methods and Materials --- p.67 / Chapter 3.2.2.1 --- Source of Pure Compounds --- p.67 / Chapter 3.2.2.2 --- Purification and Identification of SAB --- p.68 / Chapter 3.2.2.3 --- Vasodilation model --- p.70 / Chapter 3.2.2.4 --- Antioxidation Model --- p.71 / Chapter 3.2.2.5 --- Structures of Pure Compounds --- p.72 / Chapter 3.2.3 --- Results --- p.73 / Chapter 3.2.3.1 --- Vasodilation Results --- p.73 / Chapter 3.2.3.2 --- Antioxidation Results --- p.76 / Chapter 3.3 --- Discussion --- p.79 / Chapter 3.4 --- Synergistic Effect Study --- p.85 / Chapter 3.4.1 --- Introduction --- p.85 / Chapter 3.4.2 --- Methods --- p.85 / Chapter 3.4.3 --- Results --- p.86 / Chapter 3.4.4 --- Discussion --- p.88 / Chapter 3.5 --- STUDY ON 3'-HYDROXYPlIERARIN AND 3'-METHOXYPUERARIN PURIFIED FROM YFGE --- p.90 / Chapter 3.5.1 --- 3 '-hydroxypuerarin and 3'-methoxypuerarin --- p.90 / Chapter 3.5.2 --- Methods and Materials --- p.91 / Chapter 3.5.2.1 --- Purification by HPLC semi-preparation --- p.91 / Chapter 3.5.2.2 --- Bioassays --- p.93 / Chapter 3.5.3 --- Results --- p.94 / Chapter 3.5.3.1 --- Vasodilation Study --- p.94 / Chapter 3.5.3.2 --- Antioxidative Effect of Yege --- p.95 / Chapter 3.5.4 --- Discussion / Chapter CHAPTER 4 --- MECHANISTIC STUDY --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.1.1 --- Nitric Oxide-mediated Vasodilation --- p.99 / Chapter 4.1.2 --- Prostacyclin-mediated Vasodilation --- p.100 / Chapter 4.1.3 --- EDHF-mediated Vasodilation --- p.101 / Chapter 4.1.4 --- Endothelium-dependent and -independent Vasodilations --- p.103 / Chapter 4.2 --- Methods and Materials --- p.104 / Chapter 4.3 --- Results --- p.107 / Chapter 4.3.1 --- Danshen-Gegen Formula (DY80) --- p.107 / Chapter 4.3.2 --- Salvianolic acid B --- p.112 / Chapter 4.3.3 --- Daidzein --- p.117 / Chapter 4.4 --- Discussion --- p.121 / Chapter CHAPTER 5 --- STUDY ON LIPID PEROXIDATION AND UPTAKE BY MACROPHAGES --- p.128 / Chapter 5.1 --- Study of DY 80 and SAB on Copper-ion induced Low Density Lipoprotein Oxidation --- p.128 / Chapter 5.1.1 --- Pathologic Role of oxidized Low Density Lipoprotein --- p.128 / Chapter 5.1.2 --- Antioxidants in Low Density Lipoprotein and Role of Transition Metals --- p.129 / Chapter 5.1.3 --- Methods and Materials --- p.130 / Chapter 5.1.4 --- Results --- p.131 / Chapter 5.1.5 --- Discussion --- p.133 / Chapter 5.2 --- Study of Scavenger Receptor Regulation in Macrophages --- p.135 / Chapter 5.2.1 --- Introduction --- p.135 / Chapter 5.2.2 --- Methods and Materials --- p.136 / Chapter 5.2.3 --- Results --- p.139 / Chapter 5.2.4 --- Discussions --- p.140 / Chapter CHAPTER 6 --- General Discussion --- p.143 / REFERENCES --- p.147

心臟病目前仍然是最普遍的威脅人類生命安全的三大病因之一。同西藥相比, 傳統中醫藥具有多靶點，協同作用及小副作用等特性。在中藥歷史中， 丹參和葛根這兩種草藥經常出現在中藥方劑用於治療心血管相關的疾病，已有幾千年的歷史。 我們實驗室發現了一個丹參葛根湯劑具有保護動脈粥樣硬化病人心臟功能的作用，並且可以使收縮的大鼠大動脈舒張的作用。 本研究主要通過舒張豬冠狀動脈，提高大鼠對抗過氧化和離子擾動能力以及提高血管增生四個方面探討丹參葛根複方水提物 (質量比7:3) (DG配方)對血管的作用以提供其治療心血管疾病的藥理基礎。 / 在本研究的第一部， 我們主要探討了DG配方對缺血再灌注損傷的心臟及其心肌細胞的保護作用。我們發現DG配方明顯抑制了心臟損傷相關的肌酸激酶和乳酸脫氫的釋放。同時DG配方顯著促進了再灌注後冠狀動脈內血流量速度和收縮力度的恢復。這些結果說明DG配方可以保護缺血再灌注心臟並且有效促進其功能恢復。我們還觀察了長期給大鼠用DG配方14天後其心臟在缺血再灌注中的表現。類似於再灌注時給藥的結果，DG配方同樣抑制了損傷酶的釋放並且有效促進了冠狀動脈內血流量速度和收縮力度的恢復。 / 同時，在缺氧再灌注離體細胞模型中，我們發現DG配方明顯抑制了缺氧再灌注損傷帶來的細胞死亡。流式細胞儀分析結果表明，藥物處理組中的凋亡類的細胞明顯比對照組中少主要通過抑制促凋亡的caspase3表達明以及促進抗凋亡的Bcl2表達升高。DG配方減少了心肌細胞內細胞色素c從線粒體中釋放明顯以及抑制了線粒體去極化。這說明DG配方也保護了線粒體的膜的完整性，從而確保線粒體功能進而保證細胞的能量系統穩定。最有意思的是DG配方可以直接抑制缺氧再灌注相關的兩條通路, 它不僅抑制活性氧化物質的釋放, 同時也抑制了再灌注後鈣離子的累積。總之，DG配方以抗氧化和抗離子擾動的方式保護了在缺血缺氧再灌注損傷中心臟和心肌細胞的結構和功能。 / 第二部分的研究是關於DG配方對從豬心臟上分離的左冠狀動脈前室間支 (左前降支) 血管的作用及其內在的機制，我們的結果表明對由U46619引起的冠狀動脈血管收縮DG配方表現了濃度依賴的舒血管作用。而該作用並非依賴于內皮細胞及其釋放的舒張血管因數一氧化氮和前列腺素類似物環素和大部分的鉀離子通道。其中只有內向整合鉀離子通道部分參與了舒血管的過程。肌球蛋白輕鏈的磷酸化明顯被DG配方抑制，但是RhoA 的活性並沒有受其影響。鈣離子引發的血管收縮則被DG配方濃度依賴性的受到抑制。這部分的研究證明瞭DG配方主要通過類似鈣離子通道拮抗劑作用抑制鈣離子進入到血管平滑肌細胞減少肌球蛋白磷酸化達到舒張血管的作用。結果說明DG配方可以作為一種安全的藥物用於治療心血管疾病特別是高血壓和心絞痛。 / 本研究的第三部分是關於DG配方的促血管增生的作用。我們發現DG配方可以明顯促進斑馬魚的腸下動脈的出芽並且促進血管增生相關基因的表達，血管內皮細胞生長因數及其受體的mRNA表達。內皮細胞是血管增生的基礎。所以我們利用人源微血管內皮細胞檢測了DG配方在細胞的增生，遷移，分化和形成血管方面的影響以解釋它在斑馬魚中促進血管增生的作用機理。結果發現，DG配方明顯促進了該種內皮細胞的增殖，遷移和形成管狀結構。 / 綜上所述，DG配方可以通過舒張血管，抗氧化，抗離子紊亂和促進血管增生提供心血管保護功能。DG配方通過螯合活性氧化物質和抑制鈣離子的累積保護了因缺血再灌注引起的心臟損傷，說明DG配方可以作為手術的輔助藥物減少心臟病人在缺血再灌注過程中受到的損傷。它以拮抗L型鈣離子通道方式減少鈣離子進入到血管平滑肌細胞來舒張收縮的冠狀動脈血管。說明DG配方可以用於治療高血壓和心絞痛等心臟病。另外DG配方也可以促進血管增生，可用于心肌梗死病人促進其心臟血管系統重建，本研究對於未來臨床實驗具有重要的參考價值。 / Coronary heart diseases (CHD) are one of the most prevalent causes of premature death all over the world. In contrast to western medicine, traditional Chinese medicine (TCM) has shown the benefit of multi-targeting and synergism to treat CHD. Two kinds of Chinese herbs, Danshen (Radix Salviae Miltiorrhiza) (D) and Gegen (Radix Puerariae Lobatae) (G) always present on the TCM formula for treating heart disease. We found a useful formula of Danshen and Gegen decoction with weight ratio of 7:3 (DG) exerting properties of improving the heart function in patient with atheroslcerosis and providing vasodiation and antioxidant protection on the rat cardiovascular system. The present study was designed to evaluate the effects of DG on the vascular activity by its properties on antioxidant and anti-ion stunning to inhibiting the ischemia and reperfusion injury, vasodilation effect on pig coronary artery and angiogenesis effect on zebrafish model. / In the first part of the study, we explored protective effect of DG on rat hearts and cardiomyocytes after ischemia-reperfusion and hypoxia-reoxygenation injury. Comparing to control group, the release of creatine kinase (CK) and lactate dehydrogenase (LDH) significantly decreased in the DG treated groups in a dose-dependent manner. The recovery percentage of coronary flow and contractile force in the DG was higher than that in the control group. These results suggested that DG dose-dependently improved the heart function after ischemia and reperfusion injury in a dose-dependent manner. We also examined chronic effect of DG (14 days pretreatment) on rat heart with ischemia and reperfusion injury. DG induced rat heart with high potential to deal with I/R injury, less damaged enzymes release and high recovery percentage of heart function recovery. / In the cell hypoxia and reoxygenation model, DG significantly inhibited the cell death after H/R treatment with bcl2 expression increase and caspase3 expression decrease. DG also reversed the H/R-induced mitochondrial depolarization and inhibited cytochrome c diffusing out of mitochondria, which confirmed DG anti-apoptosis activity. DG also was found to significantly decrease the intracellular calcium accumulation and reactive oxygen species release within H9c2. / In the second part of present study, results revealed that DG elicited a concentration-dependent relaxation of U46619-preconstricted porcine coronary artery. DG-induced relaxation responses were not altered by the presence of endothelium-related dilator inhibitors, most potassium channel blockers, GMP and AMP pathway inhibitors and endothelium removal. Ba²⁺ (an inward rectifier K⁺ channel blocker) slightly attenuated DG-induced relaxation. The protein expression of phosphorylated myosin light chain (MLC) was inhibited by DG in a concentration-dependent manner whereas the activity of RhoA was not modified. Ca²⁺-induced contraction of coronary artery was inhibited by DG in a concentration-dependent fashion. DG acted as an antagonist of calcium channel inducing the porcine artery dilation. / The third part of the present study is about the pro-angiogenic effect of DG. We found that DG dose-dependently induced zebrafish sub-intestinal vessel sprouting and increased the mRNA expression of vascular endothelial growth factor (VEGF) and its receptors. To explore the underlying mechanism, we also examined the proangiogenic effect of DG on the angiogenesis of endothelial cells. The results showed that DG induced the HMEC-1 proliferation, migration and forming tube. / In conclusion, we found that DG could provide cardiac and cardiovascular protection by its multiple targets. It prevented heart injuries after ischemia or hypoxia and reperfusion through scavenging ROS and inhibiting calcium accumulation. Moreover, it mainly acts as an antagonist of L-type calcium channel to relax the contracted LAD vessel. It also exerted property of inducing angiogenesis in vivo and in vitro. Therefore, DG would be useful for treating coronary artery disease depending on its multiple targets. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Hu, Fan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Cardiovascular system and coronary artery diseases --- p.1 / Chapter 1.1.1 --- The cardiovascular system --- p.1 / Chapter 1.1.2 --- Contraction and relaxation of the vascular myocyte in arteries --- p.4 / Chapter 1.1.2.1 --- Ultrastructure of the vascular myocyte --- p.4 / Chapter 1.1.2.2 --- Contraction mechanisms of vascular myocyte --- p.5 / Chapter 1.1.2.3 --- Relaxation mechanisms of vascular myocyte --- p.7 / Chapter 1.1.3 --- Chronic coronary heart disease --- p.9 / Chapter 1.2 --- The way to treat chronic CAD --- p.11 / Chapter 1.2.1 --- Angiogenesis --- p.11 / Chapter 1.2.2 --- Clinical surgery for treating CAD --- p.13 / Chapter 1.2.2.1 --- Three common surgeries for treating CAD --- p.13 / Chapter 1.2.2.2 --- Ischemia and reperfusion (I/R) injury in surgeries --- p.15 / Chapter 1.2.3 --- Drugs for treating CAD --- p.19 / Chapter 1.2.3.1 --- Western medicine therapy in CAD --- p.19 / Chapter 1.2.3.2 --- Traditional Chinese Medicine treatment in CAD --- p.20 / Chapter 1.3 --- Aims of studies --- p.28 / Chapter 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Solutions and Materials --- p.29 / Chapter 2.1.1 --- Solutions --- p.29 / Chapter 2.1.2 --- Chemicals and enzymes --- p.36 / Chapter 2.2 --- Methods --- p.38 / Chapter 2.2.1 --- Herbal preparation --- p.38 / Chapter 2.2.2 --- Identification and quantification of chemical markers in Danshen and Gegen decoction (DG) --- p.38 / Chapter 2.2.3 --- Assay development for the determination of the DG marker compounds in rat plasma --- p.40 / Chapter 2.2.4 --- Isolation of pig left anterior descending coronary artery --- p.44 / Chapter 2.2.5 --- Isometric tension measurement --- p.45 / Chapter 2.2.6 --- Langendorff related experiment --- p.50 / Chapter 2.2.7 --- Cell culture of H9c2 cells --- p.54 / Chapter 2.2.8 --- Cell viability assay (MTT assay) --- p.56 / Chapter 2.2.9 --- Cell proliferation measurement --- p.57 / Chapter 2.2.10 --- Hypoxia and reperfusion cell model (H9c2) --- p.58 / Chapter 2.2.11 --- Determination of cell apoptosis with Annexin VFITC and PI double staining --- p.59 / Chapter 2.2.12 --- Measurement of mitochondria depolarization --- p.61 / Chapter 2.2.13 --- Measurement of ROS release --- p.63 / Chapter 2.2.14 --- Measurement of calcium localization in H9c2 cells by fluo4 dye and confocal microscopy --- p.64 / Chapter 2.2.15 --- Extraction of proteins from tissue, cell and subcellular fractions --- p.65 / Chapter 2.2.16 --- Western blot assay --- p.67 / Chapter 2.2.17 --- Human microvascular endothelial cells (HMEC1) cell culture --- p.68 / Chapter 2.2.18 --- Cell cycle analysis by PI staining --- p.69 / Chapter 2.2.19 --- Scratch assay for HMEC1cells migration --- p.70 / Chapter 2.2.20 --- Tube formation assay --- p.71 / Chapter 2.2.21 --- Vessel sprouting of Zebrafish --- p.72 / Chapter 2.2.22 --- Real time PCR --- p.74 / Chapter 2.2.23 --- Statistical analysis --- p.76 / Chapter 3 --- Chapter 3 Cardiac protection of Danshen and Gegen decoction in hypoxia/ischemia and reperfusion induced injury --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- Cytotoxicity of DG --- p.81 / Chapter 3.2.2 --- The morphology alteration of H9c2 after H/R treatment --- p.83 / Chapter 3.2.3 --- Effect on H H9c2 cell survival after H/R treatment --- p.84 / Chapter 3.2.4 --- Effect on membrane skeleton of H9c2 cells with H/R injury --- p.86 / Chapter 3.2.5 --- Effect on the apoptosis in H9c2 cells induced by H/R injury --- p.88 / Chapter 3.2.6 --- Effect on cytochrome c release from mitochondria of damaged H9c2 cells --- p.92 / Chapter 3.2.7 --- Effect on mitochondria depolarization of H9c2 after H/R treatment --- p.94 / Chapter 3.2.8 --- Effect on reactive oxidant species (ROS) release --- p.96 / Chapter 3.2.9 --- Effect on calcium accumulation within H9c2 in the reperfusion phase --- p.98 / Chapter 3.2.10 --- Effect on heart functions of rat hearts with I/R injury (acute effect) --- p.101 / Chapter 3.2.11 --- Effect on heart function in rats with I/R injury (chronic effect) --- p.107 / Chapter 3.3 --- Discussion --- p.113 / Chapter 4 --- Chapter 4 Vasodilation effects of Danshen and Gegen decoction in porcine coronary artery and its underlying mechanism --- p.118 / Chapter 4.1 --- Introduction --- p.118 / Chapter 4.2 --- Results --- p.121 / Chapter 4.2.1 --- Investigations of endothelium dependent and independent mechanisms --- p.121 / Chapter 4.2.2 --- Effects on cAMP and cGMP pathway --- p.121 / Chapter 4.2.3 --- Effects on potassium channel opening --- p.121 / Chapter 4.2.4 --- Effects on calcium induced contraction and calcium sensitization --- p.122 / Chapter 4.2.5 --- Effects on MLC phosphorylations --- p.123 / Chapter 4.3 --- Discussion --- p.132 / Chapter 5 --- Chapter 5 In vitro and in vivo angiogenic effects of DG --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.140 / Chapter 5.2.1 --- Effect on subintestinal vessels sprouting in the zebrafish embryo --- p.140 / Chapter 5.2.2 --- Effect on the transcription and expression of VEGFA and VEGF receptors -- Flt1 and KDR/Flk2 --- p.143 / Chapter 5.2.3 --- Effect on HMEC1 proliferation --- p.145 / Chapter 5.2.4 --- Effect on cell cycle of HMEC1 --- p.148 / Chapter 5.2.5 --- Effect on cell migration of HMEC1 --- p.151 / Chapter 5.2.6 --- Effect on tube formation of HMEC1 --- p.154 / Chapter 5.3 --- Discussion --- p.157 / Chapter 6 --- Chapter 6 Conclusions and future work --- p.160 / Chapter 6.1 --- Cardiac protection of DG in the I/R and H/R injury --- p.160 / Chapter 6.2 --- Vasodilation effect of DG on the porcine coronary artery --- p.165 / Chapter 6.3 --- Angiogenic effect of DG in vivo and in vitro --- p.167 / Chapter 6.4 --- Overall conclusion of the study --- p.169 / Chapter 7 --- References --- p.170

Cheung Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 164-175). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Publications based on the work in this thesis --- p.vii / Table of content --- p.viii / Abbreviations --- p.xii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Traditional Chinese Medicine --- p.1 / Chapter 1.1.1 --- Danshen --- p.2 / Chapter 1.1.2 --- Chemical constituents --- p.5 / Chapter 1.1.3 --- Pharmacological effects --- p.7 / Chapter 1.1.3.1 --- On blood vessels --- p.7 / Chapter 1.1.3.2 --- On blood pressure --- p.8 / Chapter 1.1.3.3 --- On heart --- p.8 / Chapter 1.1.3.4 --- On myocardial ischaemia and reperfusion --- p.9 / Chapter 1.1.3.5 --- On platelet activity --- p.10 / Chapter 1.1.3.6 --- Other actions --- p.11 / Chapter 1.1.4 --- Clinical studies --- p.12 / Chapter 1.2 --- The Vascular System --- p.13 / Chapter 1.2.1 --- The circulation network --- p.13 / Chapter 1.2.2 --- Physiology of blood vessels --- p.13 / Chapter 1.2.3 --- Control of vascular lone --- p.14 / Chapter 1.3 --- Mechanisms of Vasodilatation --- p.16 / Chapter 1.3.1 --- Endothelium derived relaxant factors (EDRFs) --- p.16 / Chapter 1.3.1.1 --- Nitric oxide (NO) --- p.16 / Chapter 1.3.1.2 --- Prostacyclin (PGI：) --- p.17 / Chapter 1.3.1.3 --- Endotheliun-derived hyperpolarization factors (EDHFs) --- p.18 / Chapter 1.3.1.3.1 --- Epoxyeicosatrienoic acids (EETs) --- p.19 / Chapter 1.3.1.3.2 --- Potassium ion (IC) --- p.20 / Chapter 1.3.1.3.3 --- Gap junction --- p.20 / Chapter 1.3.2 --- Signal transduction pathways --- p.21 / Chapter 1.3.2.1 --- Guanylyl cyclase-cGMP pathway --- p.21 / Chapter 1.3.2.2 --- Adenylyl cyclase-cAMP pathway --- p.22 / Chapter 1.3.3 --- Ion channels in vascular smooth muscle cell --- p.24 / Chapter 1.3.3.1 --- Potassium channels (K+ channels) --- p.24 / Chapter 1.3.3.2 --- Calcium channels (Ca2+ channels) --- p.24 / Chapter 1.3.3.3 --- Chloride channel (Cl channel) --- p.25 / Chapter 1.3.4 --- Receptor-operated mechanisms --- p.27 / Chapter 1.3.4.1 --- Muscarinic receptors --- p.27 / Chapter 1.3.4.2 --- Adrenoceptors --- p.27 / Chapter 1.3.4.3 --- Histamine receptors --- p.28 / Chapter 1.3.4.4 --- CGRP receptors --- p.29 / Chapter 1.3.4.5 --- Tachykinin receptors --- p.30 / Chapter 1.4 --- Aims of the studies --- p.31 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Extraction of Water and Lipid-solubie Fractions from Danshen --- p.32 / Chapter 2.1.1 --- Preparation of water-soluble and lipid-soluble fractions --- p.33 / Chapter 2.2 --- Experiments on Rat Knee Joint --- p.35 / Chapter 2.2.1 --- Animals --- p.35 / Chapter 2.2.2 --- Materials --- p.35 / Chapter 2.2.3 --- Preparatory protocols --- p.37 / Chapter 2.2.3.1 --- Anaesthesia of animals --- p.37 / Chapter 2.2.3.2 --- Cannulation of trachea --- p.37 / Chapter 2.2.3.3 --- Cannulation of carotid artery --- p.38 / Chapter 2.2.3.4 --- Blood pressure measurement --- p.38 / Chapter 2.2.4 --- Measurement of knee joint blood flow --- p.39 / Chapter 2.2.4.1 --- Preparation for measurement of knee joint blood flow --- p.41 / Chapter 2.2.5 --- Experimental protocols --- p.41 / Chapter 2.2.5.1 --- Danshen on knee joint blood flow --- p.41 / Chapter 2.2.5.2 --- Antagonists on Danshen --- p.41 / Chapter 2.2.5.3 --- Positive controls --- p.43 / Chapter 2.2.6 --- Image analysis --- p.44 / Chapter 2.2.7 --- Data analysis --- p.44 / Chapter 2.3 --- Experiments on Rat Femoral Artery --- p.45 / Chapter 2.3.1 --- Animals --- p.45 / Chapter 2.3.2 --- Materials --- p.45 / Chapter 2.3.2.1 --- Chemicals --- p.45 / Chapter 2.3.2.2 --- Physiological salt solution --- p.48 / Chapter 2.3.3 --- Preparatory protocols --- p.48 / Chapter 2.3.3.1 --- Small vessel myograph --- p.48 / Chapter 2.3.3.2 --- Isolation and mounting of tissue --- p.49 / Chapter 2.3.4 --- Experimental protocols --- p.50 / Chapter 2.3.4.1 --- Studies on the vasodilator response to Danshen --- p.50 / Chapter 2.3.4.2 --- Studies of antagonists on Danshen --- p.50 / Chapter 2.3.4.2.1 --- Endothelium-dependent mechanisms --- p.51 / Chapter 2.3.4.2.2 --- Endothelium-independent mechanisms --- p.54 / Chapter 2.3.4.2.3 --- K+ channel blockers --- p.54 / Chapter 2.3.4.2.4 --- Positive controls --- p.55 / Chapter 2.3.4.3 --- Danshen on Ca2+-induced contraction --- p.56 / Chapter 2.3.5 --- Data analysis --- p.57 / Chapter CHAPTER 3 --- RESULTS --- p.58 / Chapter 3.1 --- Danshen on Rat Knee Joint Blood Flow --- p.58 / Chapter 3.1.1 --- Topical administration of Danshen --- p.58 / Chapter 3.1.2 --- Antagonists on Danshen --- p.59 / Chapter 3.1.2.1 --- Muscarinic receptor antagonist --- p.59 / Chapter 3.1.2.2 --- β-adrenoceptor antagonist --- p.60 / Chapter 3.1.2.3 --- Histamine receptor antagonists --- p.60 / Chapter 3.1.2.4 --- Nitric oxide synthase inhibitor --- p.61 / Chapter 3.1.2.5 --- Cyclo-oxygenase inhibitors --- p.62 / Chapter 3.1.2.6 --- CGRPi receptor antagonist --- p.62 / Chapter 3.1.2.7 --- NK1 receptor antagonist --- p.63 / Chapter 3.1.2.8 --- Potassium channel inhibitor --- p.64 / Chapter 3.1.2.9 --- "Combination of cyclo-oxygenase inhibitor, nitric oxide synthase inhibitor and CGRP1 receptor antagonist" --- p.64 / Chapter 3.1.3 --- Antagonists on water-soluble fraction of Danshen --- p.91 / Chapter 3.1.3.1 --- Nitric oxide synthase inhibitor --- p.91 / Chapter 3.1.3.2 --- Cyclo-oxygenase inhibitors --- p.91 / Chapter 3.1.3.3 --- CGRP1 receptor antagonist --- p.92 / Chapter 3.1.3.4 --- NK1 receptor antagonist --- p.92 / Chapter 3.1.3.5 --- Potassium channel inhibitor --- p.92 / Chapter 3.2 --- Danshen on Rat Femoral Artery --- p.99 / Chapter 3.2.1 --- Danshen on precontracted arterial ring --- p.99 / Chapter 3.2.2 --- Endothelium-dependent mechanisms --- p.106 / Chapter 3.2.3 --- Endothelium-independent mechanisms --- p.114 / Chapter 3.2.4 --- K+ channel blockers --- p.119 / Chapter 3.2.4.1 --- Effect on Danshen --- p.119 / Chapter 3.2.4.2 --- Effect on water-soluble and lipid-soluble fractions of Danshen --- p.121 / Chapter 3.2.4.3 --- Effect on Danshensu --- p.122 / Chapter 3.2.5 --- Danshen on Ca2+-induced contractions --- p.133 / Chapter CHAPTER 4 --- DISCUSSION --- p.138 / Chapter 4.1 --- In Vivo Studies of Danshen on Rat Knee Joint Blood Flow --- p.139 / Chapter 4.2 --- In Vitro Studies of Danshen on Isolated Rat Femoral Artery --- p.148 / Chapter 4.2.1 --- Comparisons of the use of different precontractors --- p.148 / Chapter 4.2.2 --- Investigations on endothelium-dependent mechanisms --- p.151 / Chapter 4.2.3 --- Investigations on endothelium-independent mechanisms --- p.152 / Chapter 4.2.4 --- Effects of K+ channel blockers --- p.154 / Chapter 4.2.5 --- Inhibition of Ca2+ influx in vascular smooth muscle --- p.157 / Chapter 4.3 --- Comparisons of Results from In Vivo and In Vitro Studies --- p.159 / Chapter 4.4 --- Future Studies --- p.161 / Chapter 4.5 --- Conclusion --- p.162 / REFERENCES --- p.164

Yam Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-160). / Abstracts in English and Chinese. / Abstract English --- p.i / Chinese --- p.iii / Acknowledgments --- p.v / Table of contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Chinese Medicine and Western Medication --- p.1 / Chapter 1.2 --- Chinese Medicine and Compound Formula --- p.2 / Chapter 1.3 --- Cardiovascular disease (CVD) and atherosclerosis --- p.6 / Chapter 1.4 --- General Research Objectives --- p.19 / Chapter Chapter 2 --- Establishment of compound formulation and Extract Preparation --- p.21 / Chapter 2.1 --- Formulation searched from Chinese Pharmacopoeia --- p.21 / Chapter 2.2 --- Aqueous extract preparation --- p.25 / Chapter 2.2.1 --- Materials and Methods --- p.25 / Chapter 2.2.2 --- Discussion --- p.27 / Chapter Chapter 3 --- Vasodilation study --- p.28 / Chapter 3.1 --- Vascular Smooth Muscle Contraction and Relaxation --- p.28 / Chapter 3.2 --- Endothelium and Vasodilation --- p.30 / Chapter 3.3 --- Vasodilation in organ bath --- p.32 / Chapter 3.3.1 --- Materials and Methods --- p.32 / Chapter 3.3.2 --- Results --- p.35 / Chapter 3.3.3 --- Discussion --- p.40 / Chapter 3.4 --- Endothelium dependent vasodilation --- p.40 / Chapter 3.4.1 --- Materials and Methods --- p.43 / Chapter 3.4.2 --- Results --- p.45 / Chapter 3.4.3 --- Discussion --- p.54 / Chapter 3.5 --- Adrenoceptor and vasodilation --- p.55 / Chapter 3.5.1 --- Materials and Methods --- p.57 / Chapter 3.5.2 --- Results --- p.58 / Chapter 3.5.3 --- Discussion --- p.62 / Chapter 3.6 --- Potassium Channels and Vasodilation --- p.63 / Chapter 3.6.1 --- Materials and Methods --- p.65 / Chapter 3.6.2 --- Results --- p.67 / Chapter 3.6.3 --- Discussion and Summary --- p.77 / Chapter 3.7 --- Potential active components from Fenge and Danshen --- p.82 / Chapter 3.7.1 --- Materials and Methods --- p.82 / Chapter 3.7.2 --- Results --- p.83 / Chapter 3.7.3 --- Discussion --- p.87 / Chapter Chapter 4 --- Comparison of Fenge and Yege --- p.88 / Chapter 4.1 --- Vasodilative effects of Fenge and Yege --- p.89 / Chapter 4.1.1 --- Materials and Methods --- p.89 / Chapter 4.1.2 --- Results --- p.89 / Chapter 4.1.3 --- Discussion --- p.101 / Chapter 4.2 --- The comparison of antioxidative effect between Yege and Fenge --- p.104 / Chapter 4.2.1 --- Red blood cell hemolysis model --- p.106 / Chapter 4.2.1.1 --- Materials and Methods --- p.106 / Chapter 4.2.1.2 --- Results --- p.108 / Chapter 4.2.1.3 --- Discussion --- p.110 / Chapter 4.2.2 --- Ischemia-reperfusion on Langendroff --- p.112 / Chapter 4.2.2.1 --- Materials and Methods --- p.114 / Chapter 4.2.2.2 --- Results --- p.117 / Chapter 4.2.2.3 --- Discussion --- p.125 / Chapter Chapter 5 --- Comparison of Chemical Profiles of Fenge and Yege --- p.127 / Chapter 5.1 --- The application of HPLC --- p.127 / Chapter 5.2 --- HPLC standardization --- p.129 / Chapter 5.2.1 --- Materials and Methods --- p.132 / Chapter 5.2.2 --- Results --- p.133 / Chapter 5.2.3 --- Discussion --- p.144 / Chapter Chapter 6 --- "Summaries, Discussion and prospects" --- p.146 / Chapter 6.1 --- Summaries and Discussion --- p.146 / Chapter 6.2 --- Prospects --- p.148 / Chapter 6.2.1 --- "Cardiovascular tonic effect of pure compounds, extracts with difference solvents and their vasodilative mechanism." --- p.148 / Chapter 6.2.2 --- Macrophage Foam Cell and Atherosclerosis --- p.149 / Chapter 6.2.3 --- The D:F (7:3) and D:Y (7:3) compound formulae capsule with GMP --- p.152 / References --- p.154