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

Background and aims: Treatment of acute fulminant liver damage arising as a result of various origins (ischemia-reperfusion injury, toxic shock, an infectious cause or cholestasis) still remains a major clinical problem. We currently do not have available clinically proven, pharmacologically effective and universal compound for the treatment of acute liver injury. The main aim of my research work was, therefore, to test the potential hepatoprotective effect of selected cytoprotective drugs and try to find out or suggest their mechanism of action, which we have examined in the systems for the intracellular gaseous signaling molecules NO and CO, where the key enzymes for their formation are NOS / HO respectively. My PhD study had two main directions: 1) Experimental study of the relationship between HO / CO and NOS / NO systems in the environment of hepatotoxic substances on isolated primary rat hepatocytes and in rat model, 2) Evaluation of ameliorative effect of selected substances in the hepatotoxicity models and to test the relationship of this effect on changes in some parameters of cytotoxicity / cytoprotection, antioxidant parameters, gene expression of mRNA for selected genes and histological changes in the state of cells / tissues / organs. Methods: We measured urea, bilirubin and liver...