Antioxidative activity of aqueous extracts from the herbal components of the traditional Chinese medicinal formula Wu-zi-yan-zong-wan.

Abstract

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