University of Macau / Institute of Chinese Medical Sciences

Includes bibliographical references and index.

Bi-directional transport studies in SimBioDASRTM and Caco-2 cells were employed to examine the transport profiles of Buph, Ligs and SenA. Apical to basolateral (A-B) transport studies of the tested compounds revealed high intestinal permeability and predicted human absorption of over 98%. Permeability ratio of B-A/A-B of Buph (0.7-1.3) and Ligs (0.8-1.2) indicated that they were transported by passive transcellular and paracellular pathways while the low B-A/A-B ratio of SenA may imply possible involvement of other transport mechanisms. One metabolite (M-1) generated from hydration of Buph was observed in Caco-2 cells and the fraction of metabolism was 12.5% (A-B). / In conclusion, Buph, Ligs and SenA were predicted to have good intestinal absorptions in human and rat. However, extensive hepatic and intestinal first-pass metabolism of Buph in rat and human were found to cause its low oral bioavailability. On the other hand, certain degree of hepatic first-pass metabolism of Ligs and SenA may account for the partial loss of drugs via oral administration to rat. Therefore, other routes of delivery, such as sublingual administration, are worth to be considered to improve the therapeutic effects of chuanxiong. / In the rat SPIP, permeability calculated from the appearance of Buph in mesenteric blood (Pblood) was 6.0+/-1.7 x 10-4 cm/s while the fraction of formation of M-1 was about 7.1%. Together with the in vitro results, it is proposed that first-pass metabolism of Buph was present in human and rat small intestine. Moreover, Ligs and SenA had high Pblood values of 4.2+/-1.2 x 10-3 cm/s and 3.8+/-2.8 x 10-3 cm/s, respectively, indicated that they were highly permeable across rat intestinal mucosa. No metabolism of Ligs was observed. But several metabolites of SenA were detected despite they were not quantified in the present study. / In vitro metabolic studies of Buph demonstrated that major metabolite M-1, which was also found in Caco-2 cells and SPIP, formed mainly in intestine and liver cytosol in rat and human. The intrinsic clearance (Vmax/Km) of Buph was extensive and similar in both organs, and its extent in human was comparable to that in rat. The sum of the estimated in vivo extraction ratio of Buph by liver (48.3%) and intestine (55.0%) was higher the loss via oral administration to rat (77%). On the other hand, several metabolites of Ligs and SenA were found in rat and human liver microsome but not in intestinal preparations. The estimated in vivo extraction ratio by liver of rat was 47.3% (Ligs) and 22.9% (SenA), respectively, which were less than the corresponding loss via oral administration to rat (Ligs: 92.2% and SenA: 97.7%), suggesting that first-pass effect other than metabolism of these two compounds in intestine also contributed to their low oral bioavailability. / Rhizoma chuanxiong is commonly prescribed orally for improving blood circulation and treating cardiovascular disorders in China. Like other traditional Chinese medicines, chuanxiong has been used for thousands of years in China but its chemical basis, pharmacological effects and pharmacokinetic fates of the active ingredients, especially absorption, are poorly understood. Recently, seventeen compounds such as 3-butylidenephthalide (Buph), Z-ligustilide (Ligs), senkyunolide A (SenA), vanillin (Vani), ferulic acid (Fera), senkyunolide I (SenI), senkyunolide H (SenH), coniferyl ferulate (ConFer), sedanolide (Sdan), riligustilide (Rili) and levistolide A (LevA) have been isolated and recognized as the main constituents of chuanxiong by our research team (Li et al., 2003). Moreover, it has been demonstrated that Buph, Ligs and SenA are bioactive components of chuanxiong for vasodilatation and anti-thromboembolism (Chan, 2005) though their oral bioavailability in rat are very low (2.3, 7.8 and 23% respectively) (Yan, 2005). Therefore, the present study aims at investigating the intestinal permeability of the major ingredients of chuanxiong and characterizing the intestinal absorption and first-pass metabolism of Buph, Ligs and SenA by in vitro Caco-2 cell monolayers, SimBioDASRTM , in situ single-pass intestinal perfusion (SPIP) in rat and in vitro metabolism using rat and human intestine and liver subcellular fractions respectively. / Using the in vitro cell monolayers of SimBioDAS RTM, the intestinal permeability of major components of chuanxiong ranged from 12.2+/-1.6 x 10-6 cm/s to 70.6+/-9.6 x 10-6 cm/s with a rank order of Fera < Buph < Ligs < Sdan < SenH < SenI < SenA < Vani. They were predicted to have over 70% absorption in human. However, ConFer, Rili and LevA were estimated to have poor human oral absorption. / Ko Nga Ling. / "September 2005." / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1577. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 215-239). / 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.

by Fung Yin Lee, Annie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 181-189). / ABSTRACT --- p.i / LIST OF ABBREVIATIONS --- p.iv / ACKNOWLEDGEMENT --- p.v / TABLE OF CONTENTS --- p.vi / LIST OF FIGURES --- p.ix / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.4 / Chapter I. --- A. Arterial pressure --- p.4 / Chapter B. --- Regulation of arterial pressure --- p.7 / Chapter II. --- Hypertension --- p.14 / Chapter III. --- Treatment of hypertension --- p.29 / Chapter IV. --- Plants and their effects on blood pressure --- p.48 / Chapter V. --- Characteristics of the three plants being studied --- p.50 / MATERIALS AND METHODS --- p.55 / Chapter A. --- Preparative procedures --- p.55 / Chapter 1. --- Preparation of plant extracts --- p.55 / Chapter 2. --- Animal preparation for invivo blood pressure measurement --- p.56 / Chapter 3. --- Preparation of right atria for in vitro studies --- p.56 / Chapter 4. --- Preparation of artery strips for in vitro studies --- p.57 / Chapter 5. --- Preparation for diuretic studies --- p.58 / Chapter B. --- Experiments done --- p.60 / Chapter 1. --- Cumulative dose response of individual plant extract --- p.60 / Chapter 2. --- Combination of plant extracts --- p.60 / Chapter 3. --- Pharmacological antagonists studies --- p.64 / Chapter a. --- Autonomic ganglion transmission --- p.64 / Chapter b. --- Alpha adrenergic activity --- p.64 / Chapter c. --- Beta adrenergic activity --- p.65 / Chapter d. --- Cholinergic activity --- p.65 / Chapter e. --- Histaminergic activity --- p.65 / Chapter f. --- Serotoninergic activity --- p.65 / Chapter 4. --- Urinary and sodium excretionin water loaded rats --- p.66 / Chapter 5. --- Studies on chronotropic and inotropic effects on isolated right atrium --- p.66 / Chapter a. --- Effect of individual plant extract --- p.66 / Chapter b. --- Effect of combination of plant extracts --- p.66 / Chapter 6. --- Effect of plant extract on contractile responses of rat tail artery strips --- p.70 / Chapter a. --- Effect of individual plant extract --- p.70 / Chapter b. --- Effect of combination of plant extracts --- p.70 / Chapter 7. --- Effect of acute oral feeding of plant extracts on blood pressure of rats --- p.71 / Chapter C. --- Statistics --- p.71 / RESULTS / Chapter A. --- Preparation of plant extracts --- p.72 / Chapter B. --- Effect of plant extracts on blood pressure changes --- p.72 / Chapter 1. --- Individual plant extract --- p.72 / Chapter 2. --- Combination of two plant extracts --- p.73 / Chapter 3. --- Combination of three plant extracts --- p.76 / Chapter C. --- Pharmacological antagonist studies --- p.79 / Chapter 1. --- Autonomic ganglion transmission --- p.79 / Chapter 2. --- Alpha adrenergic activity --- p.79 / Chapter 3. --- Beta adrenergic activity --- p.81 / Chapter 4. --- Cholinergic activity --- p.82 / Chapter 5. --- Histaminergic activity --- p.83 / Chapter 6. --- Serotoninergic activity --- p.84 / Chapter D. --- Urinary and sodium excretion in water loaded rats --- p.85 / Chapter E. --- Chronotropic and inotropic studies of isolated right atrium --- p.88 / Chapter 1. --- Effect of individual plant extract --- p.88 / Chapter 2. --- Effect of combination of plant extracts --- p.89 / Chapter F. --- Effect of plant extracts on contractile responses of rat tail artery strips --- p.101 / Chapter G. --- Effect of acute oral feeding of plant extracts on MAP of rats --- p.102 / DISCUSSION --- p.156 / Chapter A. --- Comment on preparation of plant extracts --- p.156 / Chapter B. --- The hypotensive effects of the plant extracts --- p.157 / Chapter C. --- The mechanism of action --- p.159 / Chapter D. --- The renal effect of plant extracts --- p.161 / Chapter E. --- The interaction of the hypotensive effect of plant extracts --- p.164 / Chapter F. --- In vitro studies --- p.167 / Chapter G. --- The oral effect of the plant extracts --- p.174 / SUMMARY --- p.176 / CONCLUSION --- p.179 / REFERENCES --- p.181 / APPENDIX --- p.190 / "Appendix I To study the hypotensive effects of trypsin treated green bean, rue and kelp" --- p.191 / "Appendix II To study the hypotensive effects of ether treated green bean, rue and kelp" --- p.194

by Yau Ming Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-154). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgements --- p.ix / Abstract --- p.x / 槪論 --- p.xi / List of abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species (ROS) --- p.2 / Chapter 1.1.1 --- Hydrogen peroxide --- p.2 / Chapter 1.1.2 --- Superoxide anion --- p.3 / Chapter 1.1.3 --- Hydroxyl radical --- p.3 / Chapter 1.1.4 --- Carbon centered radical --- p.4 / Chapter 1.1.5 --- Nitric oxide --- p.4 / Chapter 1.2 --- Physiological roles of ROS --- p.5 / Chapter 1.2.1 --- Signal transduction --- p.5 / Chapter 1.2.2 --- Phagocytic activity --- p.6 / Chapter 1.3 --- Defense systems against ROS --- p.7 / Chapter 1.3.1 --- Endogenous antioxidant enzymes --- p.8 / Chapter 1.3.1.1 --- Catalase --- p.8 / Chapter 1.3.1.2 --- Superoxide dismutase --- p.8 / Chapter 1.3.1.3 --- Selenium-glutathione peroxidase --- p.9 / Chapter 1.3.1.4 --- Glutathione reductase --- p.10 / Chapter 1.3.1.5 --- Glutathione-S-transferases --- p.10 / Chapter 1.3.2 --- Non-enzymatic antioxidants --- p.12 / Chapter 1.3.2.1 --- Vitamin E (tocopherols and tocotrienols) --- p.12 / Chapter 1.3.2.2 --- Vitamin C (L-ascorbic acid) --- p.13 / Chapter 1.3.2.3 --- Glutathione --- p.14 / Chapter 1.3.2.4 --- Flavonoids and polyphenols --- p.15 / Chapter 1.3.2.5 --- Uric acid --- p.16 / Chapter 1.4 --- Roles of ROS in pathogenesis --- p.16 / Chapter 1.4.1 --- Liver diseases --- p.17 / Chapter 1.4.2 --- Genital malfunctioning --- p.19 / Chapter 1.5 --- "The traditional Chinese medicinal formula, Wu-zi-yan-zong-wan" --- p.19 / Chapter 1.5.1 --- Pharmacology of individual herbal components --- p.20 / Chapter 1.5.1.1 --- Semen Cuscuta --- p.20 / Chapter 1.5.1.2 --- Fructus Lycii --- p.21 / Chapter 1.5.1.3 --- Fructus Schisandrae --- p.21 / Chapter 1.5.1.4 --- Fructus Rubi --- p.22 / Chapter 1.5.1.5 --- Semen Plantaginis --- p.22 / Chapter 1.5.2 --- Effect of Wu-zi-yan-zong-wan on infertility --- p.23 / Chapter 1.5.3 --- Effect of Wu-zi-yan-zong-wan on liver disease --- p.23 / Chapter 1.6 --- Objectives of the present study --- p.24 / Chapter Chapter 2 --- Antioxidant Activity of Aqueous Extracts of the Herbal Components of Wu-zi-yan-zong-wan in in vitro Free Radical Generating Systems --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Application of in vitro ROS generating systems --- p.27 / Chapter 2.1.1.1 --- Superoxide generation --- p.27 / Chapter 2.1.1.2 --- Hydroxyl radical generation system --- p.28 / Chapter 2.1.1.3 --- "2,2'-Azobis(2-amidinopropane) dihydrochloride- induced hemolysis" --- p.28 / Chapter 2.1.1.4 --- Bleomycin-iron-dependent DNA damage --- p.28 / Chapter 2.1.2 --- Objective --- p.29 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.2 --- Preparation of aqueous herbal extracts --- p.30 / Chapter 2.2.3 --- Superoxide-scavenging assay --- p.30 / Chapter 2.2.4 --- Microsome lipid peroxidation inhibition assay --- p.31 / Chapter 2.2.5 --- "2,2'-Azobis(2-amidinopropane) dihydrochloride-induced hemolysis inhibition assay" --- p.32 / Chapter 2.2.6 --- Bleomycin-iron-dependent DNA damage inhibition assay --- p.32 / Chapter 2.2.7 --- Statistical analysis --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Extraction yield --- p.34 / Chapter 2.3.2 --- Free radical scavenging activity of Wu-zi-yan-zong-wan extract --- p.34 / Chapter 2.3.3 --- Free radical scavenging activity of FR extract --- p.37 / Chapter 2.3.3.1 --- Superoxide-scavenging activity --- p.37 / Chapter 2.3.3.2 --- Effect on hydroxyl radical-induced lipid peroxidation --- p.37 / Chapter 2.3.3.3 --- Effect on AAPH-induced hemolysis --- p.40 / Chapter 2.3.3.4 --- Effect on bleomycin-iron-dependent DNA damage --- p.40 / Chapter 2.3.4 --- Pro-oxidant activity of FR extract --- p.40 / Chapter 2.3.5 --- Free radical scavenging activity of the remaining herbal extracts --- p.44 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3 --- Effect of Aqueous Extract of the Herbal Components of Wu- zi-yan-zong-wan on tert-Butyl Hydroperoxide-Induced Oxidative Damage in Primary Rat Hepatocyte --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.1.1 --- Primary rat hepatocyte as pharmacological model --- p.52 / Chapter 3.1.2 --- tert-Butyl hydroperoxide as an oxidative stress inducer --- p.53 / Chapter 3.1.3 --- Detection of ROS --- p.54 / Chapter 3.1.4 --- Objective --- p.55 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Primary rat hepatocyte isolation --- p.56 / Chapter 3.2.2.1 --- Liver perfusion --- p.56 / Chapter 3.2.2.2 --- Collagen pre-coated plates preparation --- p.57 / Chapter 3.2.2.3 --- Hepatocyte culture --- p.58 / Chapter 3.2.3 --- Drug treatment and oxidative stress induction --- p.58 / Chapter 3.2.4 --- Cytotoxicity assessment --- p.58 / Chapter 3.2.4.1 --- Lactate dehydrogenase leakage measurement --- p.59 / Chapter 3.2.4.2 --- MTT assay --- p.59 / Chapter 3.2.5 --- Cellular GSH content determination --- p.59 / Chapter 3.2.6 --- Protein determination by Lowry's method --- p.60 / Chapter 3.2.7 --- MDA measurement --- p.60 / Chapter 3.2.8 --- GSSG measurement --- p.61 / Chapter 3.2.9 --- ROS measurement with fluorescent dye --- p.61 / Chapter 3.2.10 --- "Vitamin C, vitamin E and butylated hydroxytoluene treatment" --- p.62 / Chapter 3.2.11 --- Antioxidant enzyme activity measurement --- p.62 / Chapter 3.2.11.1 --- Catalase activity measurement --- p.62 / Chapter 3.2.11.2 --- Superoxide dismutase activity measurement --- p.63 / Chapter 3.2.11.3 --- Glutathione peroxidase activity measurement --- p.63 / Chapter 3.2.11.4 --- Glutathione-S-transferases activity measurement --- p.63 / Chapter 3.2.11.5 --- Glutathione reductase activity measurement --- p.64 / Chapter 3.2.12 --- Statistical analysis --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Cytotoxicity of FR extract on rat hepatocyte --- p.65 / Chapter 3.3.2 --- Effect of tBHP and FR extract on hepatocyte viability --- p.65 / Chapter 3.3.3 --- Time-dependent effect of FR extract on tBHP-induced cytotoxicity --- p.69 / Chapter 3.3.4 --- Effect of tBHP and FR extract on hepatocyte GSH content --- p.69 / Chapter 3.3.5 --- Effect of tBHP and FR extract on GSSG formation in hepatocyte --- p.72 / Chapter 3.3.6 --- Effect of tBHP and FR extract on MDA formation in hepatocyte --- p.72 / Chapter 3.3.7 --- ROS-scavenging activity of FR extract in hepatocyte --- p.77 / Chapter 3.3.8 --- Effect of FR extract on antioxidant enzymes activities --- p.77 / Chapter 3.3.9 --- Comparison between typical antioxidants --- p.77 / Chapter 3.3.10 --- Effect of WZ and remaining herbal extracts on tBHP-induced oxidative damage in hepatocyte --- p.81 / Chapter 3.4 --- Discussion --- p.84 / Chapter Chapter 4 --- Effect of Aqueous Extract of Wu-zi-yan-zong-wan and Fructus Rubi on tert-Buty Hydroperoxide Induced Oxidative Damage in Mouse Model --- p.91 / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Materials and methods --- p.93 / Chapter 4.2.1 --- Materials --- p.93 / Chapter 4.2.2 --- Animal treatments --- p.93 / Chapter 4.2.3 --- Serum preparation --- p.94 / Chapter 4.2.4 --- Marker enzyme measurement --- p.94 / Chapter 4.2.5 --- Liver MDA and GSH determination --- p.95 / Chapter 4.2.6 --- Statistical analysis --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Effect of tBHP and FR extract on mouse serum ALT and AST activities --- p.97 / Chapter 4.3.2 --- Effect of tBHP and FR extract on mouse liver MDA and GSH content --- p.97 / Chapter 4.3.3 --- Effect of WZ extract on tBHP-induced increase in serum ALT and AST activities --- p.97 / Chapter 4.4 --- Discussion --- p.102 / Chapter Chapter 5 --- Characterization of the Active Antioxidant Principlein Aqueous Extract of FR --- p.105 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and methods --- p.107 / Chapter 5.2.1 --- Materials --- p.107 / Chapter 5.2.2 --- Chemical/physical treatments on FR extract --- p.107 / Chapter 5.2.3 --- Digestion with enzymes --- p.108 / Chapter 5.2.4 --- Antioxidant activity determination --- p.109 / Chapter 5.2.5 --- Chemical composition determination --- p.109 / Chapter 5.2.5.1 --- Uronic acid determination --- p.109 / Chapter 5.2.5.2 --- Hexose determination --- p.109 / Chapter 5.2.5.3 --- Tannin determination --- p.110 / Chapter 5.2.5.4 --- Protein determination --- p.110 / Chapter 5.2.6 --- Column chromatography --- p.110 / Chapter 5.2.6.1 --- Polyamide CC6 resin column chromatography --- p.111 / Chapter 5.2.6.2 --- Sephadex LH-20 gel column chromatography --- p.111 / Chapter 5.2.7 --- Antioxidant activity of commercially available tannin --- p.111 / Chapter 5.2.8 --- Bovine serum albumin precipitation --- p.112 / Chapter 5.2.9 --- Statistical analysis --- p.112 / Chapter 5.3 --- Results --- p.113 / Chapter 5.3.1 --- Effect of chemical/physical treatments on antioxidant activity of FR extract --- p.113 / Chapter 5.3.2 --- Effect of enzyme digestions on antioxidant activity of FR extract --- p.113 / Chapter 5.3.3 --- Chemical composition of FR extract --- p.118 / Chapter 5.3.4 --- Polyamide CC6 resin column chromatography --- p.118 / Chapter 5.3.5 --- Sephadex LH-20 gel column chromatography --- p.118 / Chapter 5.3.6 --- Antioxidant activity of commercially available tannin --- p.123 / Chapter 5.3.7 --- Effect of BSA precipitation on superoxide-scavenging activity --- p.123 / Chapter 5.4 --- Discussion --- p.127 / Conclusion --- p.131 / References --- p.132

Leung Lai-Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 110-113). / Abstracts in English and Chinese. / Abstract English --- p.i / Chinese --- p.iii / Acknowledgments --- p.v / Table of contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Establishment of compound formulation --- p.4 / Chapter 2.1 --- Formulation research --- p.4 / Chapter 2.2 --- Aqueous extract preparation --- p.6 / Chapter 2.2.1 --- Materials and Methods --- p.6 / Chapter 2.2.2 --- Results --- p.7 / Chapter 2.2.3 --- Discussion --- p.9 / Chapter 2.3 --- Preliminary test --- p.10 / Chapter 2.3.1 --- Materials and Methods --- p.10 / Chapter 2.3.2 --- Results --- p.12 / Chapter 2.3.3 --- Discussion --- p.14 / Chapter Chapter 3 --- Quality Control --- p.15 / Chapter 3.1 --- HPLC standardization --- p.16 / Chapter 3.2 --- Materials and Methods --- p.19 / Chapter 3.3 --- Results --- p.20 / Chapter 3.4 --- Discussion --- p.28 / Chapter Chapter 4 --- Antioxidant study --- p.29 / Chapter 4.1 --- Red blood cell hemolysis model --- p.30 / Chapter 4.1.1 --- Materials and Methods --- p.30 / Chapter 4.1.2 --- Results --- p.30 / Chapter 4.1.3 --- Discussion --- p.32 / Chapter 4.2 --- Ischemia-Reperfusion on Langendorff --- p.33 / Chapter 4.2.1 --- Materials and Methods --- p.34 / Chapter 4.2.2 --- Results --- p.37 / Chapter 4.2.3 --- Discussion --- p.60 / Chapter Chapter 5 --- Vasodilation study --- p.61 / Chapter 5.1 --- Vasodilation in organ bath --- p.63 / Chapter 5.1.1 --- Materials and Methods --- p.63 / Chapter 5.1.2 --- Results --- p.65 / Chapter 5.1.3 --- Discussion --- p.79 / Chapter 5.2 --- Endothelium dependent vasodilation --- p.80 / Chapter 5.2.1 --- Materials and Methods --- p.80 / Chapter 5.2.2 --- Results --- p.83 / Chapter 5.2.3 --- Discussion --- p.95 / Chapter Chapter 6 --- Anti-platelet study --- p.96 / Chapter 6.1 --- CFU-MK plasma clot colony assay --- p.97 / Chapter 6.2 --- Materials and Methods --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.4 --- Discussion --- p.103 / Chapter Chapter 7 --- Discussions and prospects --- p.104 / Chapter 7.1 --- Discussions --- p.104 / Chapter 7.2 --- Prospects --- p.107 / Chapter 7.2.1 --- TCM capsule with GMP --- p.107 / Chapter 7.2.2 --- Clinical Trial of the capsule --- p.109 / References --- p.110

by Lam Fung Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 187-202). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xiii / List of Tables --- p.xvii / List of Figures --- p.xviii / Publication --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of Diabetes Mellitus --- p.1 / Chapter 1.2 --- Definition of Diabetes Mellitus --- p.1 / Chapter 1.3 --- Glucose Homeostasis and Diabetes Mellitus --- p.2 / Chapter 1.4 --- Classification of Diabetes Mellitus --- p.6 / Chapter 1.4.1 --- Type 1 Diabetes Mellitus --- p.6 / Chapter 1.4.2 --- Type 2 Diabetes Mellitus --- p.7 / Chapter 1.4.3 --- Gestational Diabetes Mellitus --- p.8 / Chapter 1.4.4 --- Other specific types --- p.8 / Chapter 1.5 --- Diagnostic Criteria of Diabetes Mellitus --- p.9 / Chapter 1.6 --- Complications of Diabetes Mellitus --- p.11 / Chapter 1.7 --- Pharmacological Treatment of Diabetes --- p.12 / Chapter 1.7.1 --- Treatment for type 1 diabetes mellitus --- p.12 / Chapter 1.7.2 --- Treatment for Type 2 diabetes mellitus --- p.13 / Chapter 1.7.2.1 --- Sulfonylureas --- p.14 / Chapter 1.7.1.2 --- Meglitinides --- p.15 / Chapter 1.7.1.3 --- Biguanides --- p.15 / Chapter 1.7.1.4 --- Thazolidinediones --- p.16 / Chapter 1.7.1.5 --- α-Glucosidase inhibitor --- p.16 / Chapter 1.8 --- Diabetes and Traditional Chinese Medicine --- p.17 / Chapter 1.9 --- Objective of this project --- p.18 / Chapter Chapter 2 --- "Botanical, Preparation and Authentication of Traditional Chinese Herbs" --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Herbal Materials --- p.22 / Chapter 2.3 --- Authentication of Herbal Material --- p.30 / Chapter 2.4 --- Extraction Method --- p.32 / Chapter 2.4.1 --- Material and Methods --- p.32 / Chapter 2.4.2 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.32 / Chapter Chapter 3 --- In vitro Studies on Selected Traditional Chinese Herbs --- p.35 / Chapter 3.1. --- Introduction --- p.35 / Chapter 3.2 --- Hepatic Gluconeogenesis Studies --- p.36 / Chapter 3.2.1 --- Introduction --- p.36 / Chapter 3.2.2 --- Material and Methods --- p.41 / Chapter 3.2.2.1 --- Cell Culture of H4IIE --- p.41 / Chapter 3.2.2.2 --- Glucose Production Assay --- p.42 / Chapter 3.2.2.3 --- Bicinchoninic Acid (BCA) Protein Assay --- p.43 / Chapter 3.2.3 --- Results --- p.44 / Chapter 3.3 --- Intestinal Glucose Absorption Studies --- p.46 / Chapter 3.3.1 --- Introduction --- p.46 / Chapter 3.3.2 --- Material and Methods --- p.48 / Chapter 3.3.2.1 --- Preparation of BBMV --- p.48 / Chapter 3.3.2.1.1 --- Chemicals --- p.48 / Chapter 3.3.2.1.2 --- Method --- p.48 / Chapter 3.3.2.2 --- Preparation of Herbal Extracts --- p.50 / Chapter 3.3.2.3 --- BBMV Glucose Uptake Assay --- p.51 / Chapter 3.3.2.4 --- Bicinchoninic Acid (BCA) Protein Assay --- p.54 / Chapter 3.3.3 --- Results --- p.54 / Chapter 3.4 --- Fibroblast Glucose Uptake Studies --- p.57 / Chapter 3.4.1 --- Introduction --- p.57 / Chapter 3.4.2 --- Material and Methods --- p.58 / Chapter 3.4.2.1 --- Cell Culture of Hs68 --- p.58 / Chapter 3.4.2.2 --- 2-Deoxy-D-glucose Uptake Assay --- p.59 / Chapter 3.4.2.3 --- Bicinchoninic Acid (BCA) Protein Assay --- p.60 / Chapter 3.4.3 --- Results --- p.60 / Chapter 3.5 --- Adipocyte Glucose Uptake Studies --- p.63 / Chapter 3.5.1 --- Introduction --- p.63 / Chapter 3.5.2 --- Material and Methods --- p.65 / Chapter 3.5.2.1 --- Cell Culture of 3T3-L1 --- p.65 / Chapter 3.5.2.2 --- Differentiation of 3T3-L1 --- p.65 / Chapter 3.5.2.3 --- 2-Deoxy-D-glucose Uptake Assay --- p.66 / Chapter 3.5.2.4 --- Bicinchoninic Acid (BCA) Protein Assay --- p.68 / Chapter 3.5.3 --- Results --- p.69 / Chapter 3.6 --- Glucose Transporter Type 4 (GLUT4) Expression Studies --- p.71 / Chapter 3.6.1 --- Introduction --- p.71 / Chapter 3.6.2 --- Material and Methods --- p.48 / Chapter 3.6.2.1 --- Cell Culture of 3T3-L1 --- p.71 / Chapter 3.6.2.2 --- Differentiation of 3T3-L1 --- p.71 / Chapter 3.6.2.3 --- GLUT4 Expression Assay --- p.72 / Chapter 3.6.2.4 --- Preparation of RNA --- p.72 / Chapter 3.6.2.5 --- RT-PCR --- p.73 / Chapter 3.6.2.6 --- PCR Analysis on GLUT4 Expression --- p.74 / Chapter 3.6.2.7 --- Real-time PCR --- p.75 / Chapter 3.6.3 --- Results --- p.77 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Discussion of Hepatic Gluconeogenesis Studies --- p.81 / Chapter 3.7.2 --- Discussion of Intestinal Glucose Absorption Studies --- p.82 / Chapter 3.7.3 --- Discussion of Fibroblast Glucose Uptake Studies --- p.83 / Chapter 3.7.4 --- Discussion of Adipocyte Glucose Uptake Studies --- p.84 / Chapter 3.7.5 --- Discussion of Glucose Transporter Type 4 (GLUT4) Expression Studies --- p.86 / Chapter 3.7.6 --- Conclusion --- p.87 / Chapter Chapter 4 --- Purification of Cortex Moutan --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.1.1 --- Phytochemical Studies of Cortex Moutan --- p.90 / Chapter 4.2 --- Organic Extraction of Cortex Moutan --- p.93 / Chapter 4.2.1 --- Extraction Material and Methods --- p.93 / Chapter 4.2.2. --- Results --- p.93 / Chapter 4.3 --- BBMV Glucose Uptake Assay with Cortex Moutan Organic Extract (CM-C and CM-D) --- p.96 / Chapter 4.3.1 --- Material and Methods --- p.48 / Chapter 4.3.2 --- Results --- p.96 / Chapter 4.4 --- Fractionation of CM-C and CM-D --- p.98 / Chapter 4.4.1 --- Material and Methods --- p.98 / Chapter 4.4.1.1 --- Chemicals --- p.98 / Chapter 4.4.1.2 --- Methods --- p.98 / Chapter 4.4.2 --- Results --- p.100 / Chapter 4.5 --- BBMV Glucose Uptake Assay of CM-C and CM-D Sub-fractions --- p.105 / Chapter 4.5.1 --- Results --- p.105 / Chapter 4.6 --- Sulfonylation of CM-D1 --- p.107 / Chapter 4.6.1 --- Material and Methods --- p.107 / Chapter 4.6.1.1 --- Chemicals --- p.107 / Chapter 4.6.1.2 --- Methods --- p.107 / Chapter 4.6.2 --- Structure Elucidation of CM-D1s --- p.108 / Chapter 4.6.2.1 --- 1H-NMR Analysis --- p.108 / Chapter 4.6.3 --- BBMV Glucose Uptake Assay of CM-D1s --- p.108 / Chapter 4.6.4 --- Results --- p.108 / Chapter 4.7 --- "Structural Elucidation of CM-D3, CM-D4 and CM-D5" --- p.112 / Chapter 4.7.1 --- Material and Methods --- p.112 / Chapter 4.7.1.1 --- Mass Spectrometry --- p.112 / Chapter 4.7.1.2 --- 1H-NMR Analysis --- p.112 / Chapter 4.7.2 --- Results --- p.113 / Chapter 4.8 --- "BBMV Glucose Uptake Assay of Acetovallione, CM-D3，CM-D4 and CM-D5" --- p.116 / Chapter 4.8.1 --- Results --- p.116 / Chapter 4.9 --- Synthesis of CM-D3s --- p.118 / Chapter 4.9.1 --- Material and Methods --- p.118 / Chapter 4.9.1.1 --- Chemicals --- p.118 / Chapter 4.9.1.2 --- Methods --- p.118 / Chapter 4.9.2 --- Structure Elucidation of synthesized product --- p.119 / Chapter 4.9.3 --- Results --- p.119 / Chapter 4.10 --- Discussion --- p.121 / Chapter Chapter 5 --- In vivo Studies on Selected Herbs --- p.123 / Chapter 5.1 --- Introduction --- p.123 / Chapter 5.1.1 --- Diabetic Animal Models --- p.123 / Chapter 5.1.2 --- Neonatal Streptozotocin-induced Diabetic Rat Model --- p.125 / Chapter 5.2 --- Oral Glucose Tolerance Test (OGTT) --- p.126 / Chapter 5.2.1 --- Animal --- p.126 / Chapter 5.2.2 --- Rat Induction Material and Methods --- p.126 / Chapter 5.2.3 --- Testing Method for diabetic condition of rats --- p.127 / Chapter 5.3.4 --- Results --- p.128 / Chapter 5.3 --- Basal Glycaemia Test --- p.138 / Chapter 5.3.1 --- Animal --- p.138 / Chapter 5.3.2 --- Rat Induction Material and Methods --- p.138 / Chapter 5.3.3 --- Testing Method --- p.138 / Chapter 5.3.4 --- Results --- p.140 / Chapter 5.4 --- Discussion --- p.143 / Chapter Chapter 6 --- General Discussion --- p.147 / Chapter 6.1 --- Introduction --- p.147 / Chapter 6.2 --- Summary of Research Findings --- p.151 / Chapter 6.3 --- Result Interpretation --- p.152 / Chapter 6.3.1 --- Result Interpretation of In Vitro Studies --- p.152 / Chapter 6.3.2 --- Result Interpretation of Cortex Moutan Purification --- p.154 / Chapter 6.3.3 --- Result Interpretation of In Vivo Studies --- p.157 / Chapter 6.4 --- Limitations and Improvements --- p.161 / Chapter 6.5 --- Future Directions --- p.163 / Chapter 6.6 --- Conclusions --- p.169 / Appendices --- p.170 / References --- p.187

丹參和葛根為我國民間常用的傳統藥材, 常用於心血管疾病的治療。本試驗主要研究丹參葛根複方(DG, 7:3)對大鼠基底動脈的舒張作用 及腦保護作用。 / 上述所有藥物對U46619預收縮的基底動脈環呈現濃度依賴性的舒張作用。一氧化氮合酶抑制劑L-NAME以及鳥苷酸環化酶抑制劑ODG部分抑制葛根素的舒張作用。在另一組去內皮試驗中, 腺苷酸環化酶抑制劑SQ22536, 鳥苷酸環化酶抑制劑ODG, 大電導鈣離子依賴型鉀通道抑制劑Iberiotoxin以及電壓門控型鉀通道抑制劑4-AP對所有藥物的舒張作用沒有影響。然而， ATP型鉀通道抑制劑格列本脲能夠抑制丹參葛根複方，丹參，葛根，丹參素，葛根素，大豆苷以及大豆苷元的最大舒張反應。內向整流型鉀通道抑制劑氯化鋇則降低丹參酚酸B和大豆苷元的最大反應值。非選擇性鉀通道抑制劑乙基氯化銨以及所有鉀通道抑制劑的混合物顯著抑制上述所有藥物的舒張作用。除了葛根素之外，所有的藥物動度依賴性的抑制氯化鈣所引起的血管收縮。 / 體內研究發現大鼠經歷10分鐘雙側頸總動脈夾閉合並低血壓，及24小時的複灌後，與假手術組動物相比，腦血流量顯著降低，氧化性損傷明顯可見。連續7天口服丹參葛根複方（0.3g/kg 和 3g/kg）, 丹參 (3g/kg)，或者葛根 (3g/kg)對血壓沒有影響。但是，高劑量的丹參葛根複方 (3g/kg) 能夠提高超氧化物歧化酶和過氧化氫酶的活性，抑制丙二醛和一氧化氮的產生。3g/kg的葛根可以提高超氧化物歧化酶的活性，3g/kg的丹參則能抑制一氧化氮的產生。在大鼠中動脈阻塞模型中，連續7天口服丹參葛根複方（3g/kg）能明顯降低腦部的梗死率，同時改善大鼠的神經行為學。 / 總體來說，研究發現丹參葛根複方，丹參，葛根，丹參酚酸B，大豆苷以及大豆苷元的血管舒張作用是通過開平滑肌細胞的通鉀離子通道以及抑制鈣離子內流而實現的。然而葛根素的血管舒張作用是內皮依賴性的，通過產生一氧化氮，開放平滑肌細胞的鉀離子通道而實現的。丹參葛根複方能起到一定的腦保護作用。總而言之，研究表明上述藥物可能會對阻塞性腦血管病的人群有益處。 / Danshen and gegen are used in traditional Chinese medicine for the treatment of cardiovascular diseases. In this study, the relaxant actions of a danshen and gegen formulation (DG; ratio 7:3) and its constituents were investigated on rat-isolated cerebral basilar artery. In addition, the neuroprotective effect of DG was explored in rats subjected to global and focal ischaemia. / DG and all its constituents produced concentration-dependent relaxation of the artery rings precontracted by U46619. Removal of the endothelium had no effect on their vasodilator actions except the maximum response (I[subscript max]) to puerarin was inhibited by 42%. The nitric oxide synthase (NOS) inhibitor L-NAME and guanylyl cyclase (GC) inhibitor ODQ but not the cyclo-oxygenase (COX) inhibitor flurbiprofen produced partial inhibition on the puerarin-induced effect. In a set of endothelium-denuded artery rings, adenylyl cyclase (AC) inhibitor SQ22536, GC inhibitor ODQ, KV channel inhibitor 4-aminopyridine (4-AP) and BK[subscript Ca) channel inhibitor iberiotoxin had no influence on their vasodilator actions. However, pretreatment with K[subscript ATP] channel inhibitor glibenclamide reduced Imax to DG, danshen, gegen, danshensu, puerarin, daidzein and daidzin. K[subscript IR] inhibitor barium chloride (BaCl₂) reduced II[subscript max] to salvianolic acid B and daidzein. The non-selective K⁺ channel inhibitor tetraethylammonium (TEA), or a combination of all the K⁺ channel inhibitors produced significant partial inhibitions on all the agents’ actions. Electrophysiological studies on smooth muscle cells isolated from rat basilar artery also confirmed that DG, danshen, gegen danshensu, puerarin, daidzein and daidzin elevated K[subscript ATP] currents. In addition, DG and all its constituents, except puerarin, produced concentration-dependent inhibition on CaCl₂-induced vasoconstrictions. These findings were confirmed by con-focal microscopy studies. / In vivo study on a rat model of global ischaemia showed that challenging the rats with 10 min bilateral common carotid artery occlusion combined with hypotension, and followed by 24 h reperfusion produced significant decrease in cerebral blood flow and oxidative damage compared to sham-operated animals. Administration of DG (0.3 g/kg and 3 g/kg, p.o.), danshen (3 g/kg, p.o.) or gegen (3 g/kg, p.o.) for 7 days had no effect on blood pressure. However, the 7 days treatment with DG (3 g/kg) restored superoxide dismutase (SOD) and catalase (CAT) activities, suppressed the production of maleic dialdehyde (MDA), and inhibited the production of nitric oxide (NO). In addition, gegen (3 g/kg) restored SOD enzyme activity, whereas, danshen (3 g/kg) inhibited NO production. In addition, treatment with DG (3 g/kg) showed a significant reduction in infarct weight and improved the neurological deficit in a rat model of focal cerebral ischaemia induced by middle cerebral artery occlusion (MCAO). / In conclusion, the vasorelaxant actions of DG, danshen, gegen, salvianolic acid B, danshensu, daidzein and daidzin were found to involve the opening of K⁺ channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving NO production and an endothelium-independent pathway mediated by the opening of K⁺ channels. DG may have some cerebro-protective effects. Overall, the present studies showed that DG and its constituents could be beneficial to patients with obstructive cerebrovascular diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Deng, Yan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 164-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.v / 摘要 --- p.iviii / ACKNOWLEDGEMENTS --- p.x / PUBLICATIONS BASED ON THIS THESIS --- p.xii / ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Chinese Medicines in treatment of cerebrovascular diseases --- p.2 / Chapter 1.2 --- Danshen --- p.4 / Chapter 1.2.1 --- Chemical constituents --- p.4 / Chapter 1.2.1.1 --- Hydrophilic compounds of danshen --- p.4 / Chapter 1.2.1.2 --- Lipophilic compounds of danshen --- p.5 / Chapter 1.2.1.3 --- Other compounds --- p.5 / Chapter 1.2.2 --- Pharmacological activities --- p.5 / Chapter 1.2.2.1 --- Vascular protection --- p.5 / Chapter 1.2.2.2 --- Anti-tumour --- p.7 / Chapter 1.2.2.3 --- Treatment of liver diseases --- p.8 / Chapter 1.2.2.4 --- Treatment of drug addiction --- p.9 / Chapter 1.2.2.5 --- Treatment of kidney diseases --- p.10 / Chapter 1.2.3 --- Pharmacokinetics --- p.10 / Chapter 1.3 --- Gegen --- p.12 / Chapter 1.3.1 --- Chemical constituents --- p.12 / Chapter 1.3.2 --- Pharmacology --- p.13 / Chapter 1.3.2.1 --- Vascular effects --- p.13 / Chapter 1.3.2.2 --- Anti-diabetes --- p.14 / Chapter 1.3.2.3 --- Anti-hypercholesterolaemia --- p.15 / Chapter 1.3.2.4 --- Anti-inflammation --- p.16 / Chapter 1.3.2.5 --- Anti-platelet aggregation --- p.17 / Chapter 1.3.3 --- Pharmacokinetics --- p.17 / Chapter 1.4 --- Danshen and gegen formulation --- p.19 / Chapter 1.5 --- Mechanisms of vasodilatation --- p.22 / Chapter 1.5.1 --- Endothelium derived relaxant factors (EDRFs) --- p.22 / Chapter 1.5.1.1 --- Nitric oxide (NO) --- p.22 / Chapter 1.5.1.2 --- Prostacyclin (PGI₂) --- p.23 / Chapter 1.5.1.3 --- Endothelium-derived hyperpolarizating factors (EDHFs)- --- p.23 / Chapter 1.5.2 --- Signal transduction pathways --- p.24 / Chapter 1.5.2.1 --- Guanylyl cyclase-cGMP pathway --- p.24 / Chapter 1.5.2.2 --- Adenylyl cyclase-cAMP pathway --- p.24 / Chapter 1.5.3 --- Ion channels --- p.25 / Chapter 1.5.3.1 --- Potassium channels (K⁺ channels) --- p.25 / Chapter 1.5.3.2 --- Calcium channel (Ca²⁺ channels) --- p.25 / Chapter 1.6 --- Aims of study --- p.27 / Chapter CHAPTER 2 --- Materials and method --- p.28 / Chapter 2.1 --- Herbal preparation --- p.28 / Chapter 2.1.1 --- DG, danshen and gegen preparation --- p.28 / Chapter 2.1.2 --- Identification and quantification of chemical markers in DG water extract --- p.29 / Chapter 2.2 --- Experiments on rat basilar artery --- p.30 / Chapter 2.2.1 --- Animals --- p.30 / Chapter 2.2.2 --- Chemicals --- p.30 / Chapter 2.2.3 --- Isolation and mounting of blood vessels --- p.33 / Chapter 2.2.4 --- Protocols --- p.34 / Chapter 2.2.4.1 --- Effects on U46619-precontracted tone --- p.34 / Chapter 2.2.4.2 --- Endothelium-dependent mechanism --- p.34 / Chapter 2.2.4.3 --- Endothelium-independent mechanism --- p.35 / Chapter 2.2.4.4 --- Calcium channels --- p.36 / Chapter 2.2.4.5 --- Positive control --- p.36 / Chapter 2.2.5 --- Statistical analysis --- p.37 / Chapter 2.3 --- Experiments on rat cerebral basilar artery smooth muscle cells K[subscript ATP] channals --- p.38 / Chapter 2.3.1 --- Animals --- p.38 / Chapter 2.3.2 --- Chemicals --- p.38 / Chapter 2.3.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.40 / Chapter 2.3.4 --- Whole cell patch-clamp electrophysiology --- p.40 / Chapter 2.3.5 --- Statistical analysis --- p.44 / Chapter 2.4 --- Experiments on rat cerebral basilar artery smooth muscle cells calcium channels --- p.45 / Chapter 2.4.1 --- Animals --- p.45 / Chapter 2.4.2 --- Chemicals --- p.45 / Chapter 2.4.3 --- Isolation of rat cerebral vascular smooth muscle cells --- p.47 / Chapter 2.4.4 --- Dye loading and determination of [Ca²⁺]i --- p.47 / Chapter 2.4.5 --- Statistical analysis --- p.48 / Chapter 2.5 --- In vivo study of global ischaemia --- p.49 / Chapter 2.5.1 --- Animals --- p.49 / Chapter 2.5.2 --- Drugs and chemicals --- p.49 / Chapter 2.5.3 --- Experimental protocols for global ischaemia --- p.49 / Chapter 2.5.4 --- Induction of global ischaemia --- p.50 / Chapter 2.5.5 --- Blood pressure measurement --- p.52 / Chapter 2.5.6 --- Measurement of cerebral blood flow --- p.52 / Chapter 2.5.7 --- Biochemical assessment --- p.53 / Chapter 2.5.7.1. --- Dissection and homogenization --- p.53 / Chapter 2.5.7.2. --- Measurement of malondialdehyde (MDA) --- p.53 / Chapter 2.5.7.3. --- Estimation of nitrite --- p.53 / Chapter 2.5.7.4 --- Superoxide dismutase activity (SOD) --- p.54 / Chapter 2.5.7.5 --- Reduced glutathione (GSH) --- p.54 / Chapter 2.5.7.6 --- Catalase (CAT) --- p.55 / Chapter 2.5.7.7 --- NOS activity --- p.55 / Chapter 2.5.7.8 --- Protein --- p.56 / Chapter 2.5.8 --- Statistical analysis --- p.56 / Chapter 2.6 --- In vivo study of focal ischaemia --- p.57 / Chapter 2.6.1 --- Animals --- p.57 / Chapter 2.6.2 --- Drugs and chemicals --- p.57 / Chapter 2.6.3 --- Experimental protocols for global ischaemia --- p.57 / Chapter 2.6.4 --- Focal cerebral ischaemia-reperfusion model --- p.57 / Chapter 2.6.5 --- Assessment of neurobehavioural changes --- p.59 / Chapter 2.6.6 --- Assessment of cerebral infarction --- p.60 / Chapter 2.6.7 --- Statistical analysis --- p.60 / Chapter CHAPTER 3 --- Results --- p.61 / Chapter 3.1 --- Identification and quantification of chemical markers in DG water extract --- p.61 / Chapter 3.2 --- Effects of DG and its constituents on rat cerebral basilar artery --- p.64 / Chapter 3.2.1 --- Investigations on endothelium-dependent mechanisms --- p.64 / Chapter 3.2.2 --- Investigations on endothelium-independent mechanisms --- p.68 / Chapter 3.2.3 --- Positive control --- p.86 / Chapter 3.2.3 --- Investigations on calcium channels --- p.88 / Chapter 3.3 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cells --- p.91 / Chapter 3.3.1 --- Effects of water crude-extracts of DG, danshen, and gegen on K[subscript ATP] channels --- p.91 / Chapter 3.3.2 --- Effects of active constituents of danshen hydrophilic fraction on K[subscript ATP] channels --- p.100 / Chapter 3.3.3 --- Effects of the major isoflavonoids of gegen on K[subscript ATP] channels --- p.105 / Chapter 3.4 --- Effects of DG and its constituents on calcium channels of basilar artery smooth muscle cells --- p.112 / Chapter 3.5 --- Effects of DG, danshen and gegen on rat global ischaemia --- p.117 / Chapter 3.5.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.117 / Chapter 3.5.2 --- Effects of DG, danshen and gegen on lipid peroxidation --- p.120 / Chapter 3.5.3 --- Effects of DG, danshen and gegen on SOD activity --- p.120 / Chapter 3.5.4 --- Effects of DG, danshen and gegen on CAT activity --- p.120 / Chapter 3.5.5 --- Effects of DG, danshen and gegen on reduced GSH level --- p.121 / Chapter 3.5.6 --- Effects of DG, danshen and gegen on NOS system --- p.126 / Chapter 3.6 --- Effect of DG on rat focal ischaemia --- p.129 / Chapter 3.6.1 --- Effect of DG on cerebral infarction --- p.129 / Chapter 3.6.2 --- Effect of DG on neurological deficits --- p.129 / Chapter CHAPTER 4 --- Discussion --- p.132 / Chapter 4.1 --- Studies of DG and its constituents on rat cerebral basilar artery --- p.133 / Chapter 4.1.1 --- Constituents of DG on U46619-precontracted tone --- p.133 / Chapter 4.1.2 --- Investigations on endothelium-dependent mechanisms --- p.133 / Chapter 4.1.3 --- Investigations on endothelium-independent mechanisms --- p.136 / Chapter 4.1.4 --- Investigations on calcium channels --- p.139 / Chapter 4.2 --- Effects of DG and its constituents on rat cerebral basilar artery smooth muscle cell K[subscript ATP] channels --- p.143 / Chapter 4.3 --- Effects of DG and its constituents on calcium influx in rat basilar artery smooth muscle cells --- p.147 / Chapter 4.4 --- Effects of DG, danshen and gegen on rat transient global ischaemia --- p.150 / Chapter 4.4.1 --- Effects of DG, danshen and gegen on rats’ blood pressure and cerebral blood flow --- p.150 / Chapter 4.4.2 --- Effects of DG, danshen and gegen on lipid peroxidation, SOD and CAT activity, and GSH level --- p.152 / Chapter 4.4.3 --- Effects of DG, danshen and gegen on NOS system --- p.155 / Chapter 4.5 --- Effects of DG on rat focal ischaemia --- p.157 / Chapter 4.6 --- Further studies --- p.160 / Chapter 4.7 --- Conclusion --- p.162 / REFERENCES --- p.164

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.

Angiogenesis is a process of new blood vessels sprouting from the pre-existing vasculature, and mediated by multiple angiogenic and anti-angiogenic factors. Disturbance of the balance often leads to development of neovascular diseases. Neovascularization affecting the eye is a common cause of visual impairment and even blindness, particularly when corneal or choroidal neovascularization (NV) is involved. While there are effective treatment modes for ocular neovascularization, they are expensive and only inhibit disease progress. Since herbal medicine has been applied for anti-angiogenesis and anti-carcinogenesis therapies, we investigate the anti-angiogenic effect of selected herbal molecules: isoliquiritigenin (ISL), a flavonoid from licorice; epigallocatechin gallate (EGCG), a polyphenol from green tea; and resveratrol (Rst), a polyphenol phytoalexin derived from grapes. / In conclusion, by in vitro and in vivo studies, we showed that ISL, EGCG and Rst contributed to anti-angiogenesis via different biological mechanisms. We propose that these three herbal molecules (ISL, EGCG and Rst) are candidate anti-angiogenic agents for the treatment of ocular angiogenesis diseases. Their distribution profiles and pharmacokinetic properties should be investigated. / Results showed that sub-toxic levels of ISL (10 microM), EGCG (50 microM) and Rst (10microM) effectively suppressed endothelial cell proliferation and migration in the scratch-wound assay. Treatment with ISL was found to significantly up-regulate PEDF, which is known as a potent angiostatic factor. EGCG and Rst downregulate VEGF signaling cascade by suppressing Akt and FAK activation and affecting MMP-2, MMP-9 expression. In vivo angiogenesis assays further showed the suppressive effect of ISL, EGCG and Rst on neovascularization in three different animal models. Application of ISL at 1 microM showed the suppressive effect on chick CAM assay, corneal NV and choroidal NV assays consistently, the most effective dosage was close to 10 microM. EGCG at 1 microM showed the effect to reduce chick CAM vessel formation and corneal NV, and at 10 microM (the lowest tested concentration) to suppress choroidal NV in mice. Variable effects were observed in Rst treatment. Rst at 10 microM prohibited vessel growth in chick CAM, and 1 microM suppressed corneal NV formation and 2 microM deterred choroidal NV development. / This thesis contains two major parts. The first in vitro cell-based analysis investigated the toxicity of these herbal chemicals and their effect on endothelial cell growth and migration. The expression profile of vascular endothelial growth factor (VEGF) signaling cascade events, including Akt and focal adhesion kinase (FAK) activation, VEGF, pigment epithelium-derived factor (PEDF) and matrix metalloproteinases (MMPs) were examined by Western blotting. Then three in vivo models were established to study the effect of these herbal chemicals on angiogenesis. They were (1) developmental angiogenesis in chick chorioallantoic membrane (CAM), (2) pathological angiogenesis in silver nitrate cauterization-induced corneal neovascularization in BALB/c mice and, (3) laser photocoagulation-induced choroidal neovascularization in C57BL/6 mice. Changes of vascularization were determined by qualification of vessel number changes on the edge of gelatin sponge in 24 hours (chick CAM assay), measurement of vascularized area, live imaging of vessel leakage (fundus fluorescence angiography, FFA) and immunochemistry using antibodies specific for endothelial cells (corneal & choroidal NV assays) respectively. / Liu, Huanming. / Adviser: Chi Pui Pang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 155-180). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.