by Chan Ping Tim Timothy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 129-141). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.VI / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- History of tea --- p.1 / Chapter 1.2 --- Botany and agriculture of tea --- p.1 / Chapter 1.3 --- Classification of tea --- p.2 / Chapter 1.4 --- Composition of tea --- p.4 / Chapter 1.5 --- Tea processing --- p.8 / Chapter 1.5.1 --- Manufacture of green tea --- p.8 / Chapter 1.5.2 --- Manufacture of black tea --- p.8 / Chapter 1.5.3 --- Manufacture of oolong tea --- p.10 / Chapter 1.6 --- Pharmacological effects of tea catechins --- p.13 / Chapter 1.6.1 --- Antioxidative activity --- p.13 / Chapter 1.6.2 --- Hypolipidemic activity --- p.14 / Chapter 1.6.3 --- Antimutagenic activity --- p.15 / Chapter 1.6.4 --- Anticarcinogenic activity --- p.15 / Chapter 1.6.5 --- Antibacterial activity --- p.16 / Chapter CHAPTER 2 --- ANTIOXIDATIVE ACTIVITIES OF TEA ETHANOL EXTRACTS AND GTC ON OXIDATION OF CANOLA OIL --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1 --- Lipid oxidation in food --- p.18 / Chapter 2.1.2 --- Phenolic antioxidants --- p.19 / Chapter 2.1.2.1 --- Major phenolic antioxidants used in food --- p.19 / Chapter 2.1.2.2 --- Mechanism of action of phenolic antioxidants --- p.20 / Chapter 2.1.2.3 --- BHA and its safety --- p.22 / Chapter 2.1.2.4 --- BHT and its safety --- p.24 / Chapter 2.1.3 --- Natural antioxidants --- p.24 / Chapter 2.2 --- Objectives --- p.26 / Chapter 2.3 --- Materials --- p.28 / Chapter 2.4 --- Methods --- p.28 / Chapter 2.4.1 --- GTC extraction --- p.28 / Chapter 2.4.2 --- "HPLC analysis of GTC," --- p.29 / Chapter 2.4.3 --- Isolation and purification of individual epicatechin isomers --- p.30 / Chapter 2.4.4 --- Ethanol extraction of tea --- p.30 / Chapter 2.4.5 --- Effect of tea ethanol extracts on oxygen consumption of canola --- p.31 / Chapter 2.4.6 --- Effect of GTC on oxygen consumption of canola oil --- p.32 / Chapter 2.4.7 --- Fatty acid analysis --- p.32 / Chapter 2.4.8 --- Thermal loss of BHT --- p.33 / Chapter 2.4.9 --- Thermal loss of GTC --- p.33 / Chapter 2.4.10 --- Statistics --- p.35 / Chapter 2.5 --- Results --- p.37 / Chapter 2.5.1 --- Antioxidative activities of tea ethanol extracts --- p.37 / Chapter 2.5.2 --- The yield and composition of GTC from jasmine tea --- p.51 / Chapter 2.5.3 --- Antioxidative activity of GTC --- p.55 / Chapter 2.5.4 --- Antioxidative activities of individual epicatechin isomers --- p.55 / Chapter 2.5.5 --- Thermal loss of GTC --- p.60 / Chapter 2.6 --- Discussion --- p.62 / Chapter 2.6.1 --- Contribution of catechins to the antioxidative effects of tea ethanol extracts --- p.62 / Chapter 2.6.2 --- Antioxidaitve activities of different types of teas --- p.62 / Chapter 2.6.3 --- Proposed mechanisms for the relative activity of epicatechin isomers --- p.63 / Chapter 2.6.4 --- Loss of BHT via volatilization --- p.66 / Chapter 2.6.5 --- Potential of tea catechins as food antioxidants --- p.67 / Chapter 2.6.5.1 --- Safety of GTC --- p.67 / Chapter 2.6.5.2 --- Solubility of GTC --- p.68 / Chapter 2.6.5.3 --- Effects of GTC on food quality --- p.68 / Chapter CHAPTER 3 --- INHIBITORY EFFECTS OF GTC AND EPICATECHIN ISOMERS ON IN VITRO CU2+-MEDIATED LDL OXIDATION --- p.70 / Chapter 3.1 --- Introduction --- p.70 / Chapter 3.1.1 --- Mechanisms of LDL oxidation --- p.71 / Chapter 3.1.1.1 --- Nature and sources of oxidants underlying LDL oxidation --- p.71 / Chapter 3.1.1.2 --- Structural changes of ox-LDL --- p.72 / Chapter 3.1.2 --- Biological effects of ox-LDL --- p.74 / Chapter 3.1.3 --- Antioxidants and atherosclerosis --- p.76 / Chapter 3.2 --- Objectives --- p.78 / Chapter 3.3 --- Materials and methods --- p.79 / Chapter 3.3.1 --- LDL isolation --- p.79 / Chapter 3.3.2 --- LDL oxidation --- p.79 / Chapter 3.3.3 --- Thiobarbituric acid-reactive substance (TBARS) assay --- p.80 / Chapter 3.3.4 --- Lipid analysis --- p.80 / Chapter 3.3.5 --- Statistics --- p.81 / Chapter 3.4 --- Results --- p.82 / Chapter 3.4.1 --- Protective effects of GTC against LDL oxidation --- p.82 / Chapter 3.4.2 --- Varying protective effects of individual epicatechin isomers --- p.82 / Chapter 3.4.3 --- Protective effects of GTC against oxidative degradation of PUFAs in LDL --- p.86 / Chapter 3.5 --- Discussion --- p.88 / Chapter 3.5.1 --- Tea catechins as anti-atherogenic agents --- p.88 / Chapter 3.5.2 --- Mechanisms of the protective effects of tea catechins against Cu2+-induced LDL oxidation --- p.88 / Chapter 3.5.3 --- Relative antioxidative activities of epicatchin isomers --- p.89 / Chapter 3.5.4 --- Absorption of tea catechins --- p.90 / Chapter 3.5.5 --- Pro-oxidant activities of tea catechins --- p.91 / Chapter CHAPTER 4 --- HYPOLIPIDEMIC ACTIVITY OF GTC --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.1.1 --- High serum cholesterol as a risk factor of CHD --- p.93 / Chapter 4.1.2 --- Serum TG and CHD --- p.94 / Chapter 4.1.3 --- Hypolipidemic effect of tea --- p.95 / Chapter 4.1.4 --- Hamster as an animal model of cholesterol metabolism --- p.96 / Chapter 4.2 --- Objectives --- p.97 / Chapter 4.3 --- Materials and methods --- p.98 / Chapter 4.3.1 --- Animals --- p.98 / Chapter 4.3.2 --- Experiment 1 --- p.98 / Chapter 4.3.3 --- Experiment 2 --- p.100 / Chapter 4.3.4 --- Experiment 3 --- p.101 / Chapter 4.3.5 --- "Serum lipid, lipoprotein and apolipoprotein determinations" --- p.101 / Chapter 4.3.6 --- Lipid analysis of liver and carcass --- p.102 / Chapter 4.3.7 --- Analysis of fecal lipid content --- p.102 / Chapter 4.3.8 --- Determination of hepatic cholesterol content --- p.103 / Chapter 4.3.9 --- Assay of fatty acid synthase activity --- p.105 / Chapter 4.3.10 --- Statistics --- p.105 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Growth and food intake --- p.106 / Chapter 4.4.2 --- Effects of different levels of dietary GTC on serum TG and cholesterol --- p.106 / Chapter 4.4.3 --- Time course study of the hypolipidemic effects of dietary GTC --- p.109 / Chapter 4.4.4 --- Effects of GTWE on serum lipid and apolipoprotein profiles --- p.113 / Chapter 4.4.5 --- "Effects of dietary GTC on hepatic TG, FFA and cholesterol contents" --- p.113 / Chapter 4.4.6 --- "Effects of dietary GTC on carcass TG, FFA and cholesterol contents" --- p.118 / Chapter 4.4.7 --- Effects of dietary GTC on fatty acid synthase activity --- p.118 / Chapter 4.4.8 --- Effects of dietary GTC on fecal lipids content --- p.118 / Chapter 4.5 --- Discussion --- p.120 / Chapter 4.5.1 --- Hypolipidemic effect of GTC --- p.120 / Chapter 4.5.2 --- Effects of GTC on serum apolipoproteins --- p.120 / Chapter 4.5.3 --- Implication of GTC intake in humans --- p.121 / Chapter 4.5.4 --- Mechanisms for the hypolipidemic activity of GTC --- p.122 / Chapter 4.5.5 --- Reduction in hepatic TG and FFA contents in GTC-fed hamsters --- p.123 / Chapter 4.5.6 --- Suppression of body lipid accumulation by dietary GTC --- p.124 / Chapter 4.5.7 --- Mechanisms for the hypocholesterolemic activity of GTC --- p.124 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.126 / REFERENCES --- p.129

Os radicais livres são espécies químicas que reagem rapidamente com diversos compostos e alvos celulares, por possuírem tempo de meia vida muito curto e serem espécies altamente instáveis. A formação destes compostos constitui uma ação contínua e fisiológica, cumprindo funções biológicas essenciais as quais ocorrem pela perda ou adição de um único elétron a um composto não radicalar. Estas reações podem ocorrer em processos bioquímicos do sistema imune ou químicos, causando prejuízo às células através da destruição de componentes, como proteínas, lipídios, açúcares e nucleotídeos. Sabe-se que existem compostos que são efetivos contra tais espécies, prevenindo os danos provocados pelo estresse oxidativo. O objetivo deste trabalho foi estudar compostos heterocíclicos que possuam nitrogênio em sua estrutura (azóis), que figuram na literatura como moléculas exemplares de compostos de aplicação farmacológica de amplo espectro. Dentre estes compostos foram analisados os derivados de pirazóis (26 compostos), tiazóis (7 compostos) e 1 isoxazol (3,5-dimetilisoxazol). Estes 34 compostos foram avaliados pela metodologia ORAC (Capacidade de Absorção de Radicais Oxigênio) a fim de determinar e/ou de avaliar seu potencial antioxidante. A escolha do método ORAC se deu pelo fato das moléculas estudadas apresentarem características hidrofílicas e lipofílicas, além de ser um método validado pela literatura, disponível e de ampla aplicação. O método ORAC avalia a capacidade antioxidante da amostra, medindo sua habilidade de proteger a fluoresceína (FL) da oxidação pelo AAPH no meio reacional. O AAPH é um gerador de radicais livres que a 37°C retira hidrogênio do meio, promovendo a redução da fluorescência da fluoresceína em λ medido pelo tempo. Seis compostos apresentaram atividade antioxidante de boa à moderada: 3,5-dimetil-1H-pirazol (2.382 µmol eq.Trolox/g); 3-fenil-5-(4-fluorfenil)-1-tiocarbamoil-4,5-diidro-1H-pirazol (6.354 µmol eq.Trolox/g); 3-fenil-5-(2-metoxifenil)-1-tiocarbamoil-4,5-diidro-1H-pirazol (8.739 µmol eq.Trolox/g); 5-(2,4-diclorofenil)-3-fenil-1-tiocarbamoil-4,5-diidro-1H-pirazol (6.022,226 µmol eq.Trolox/g); 2-[5-(4-metoxifenil)-3-fenil-4,5-diidro-1H-pirazol-1-il]-4-feniltiazol (3.135 µmol eq.Trolox/g); e finalmente 2-[5-(3-nitrofenil)-3-fenil-4,5-diidro-1H-pirazol-1-il]-4-feniltiazol (2.700 µmol eq.Trolox/g). Os experimentos com o método ORAC para os azóis estudados apresentaram reprodutibilidade na execução experimental e demonstraram ser uma alternativa viável para estudos de moléculas sintéticas de potencial antioxidante. / Free radicals are chemical species that react rapidly with various compounds and target cells, as they have a very short half life and are highly unstable. The formation of these compounds consists of a continuous, physiological action, which includes essential biological functions and occurs through the loss or addition of a single electron to a non-radical compound. These reactions may occur in biochemical processes of the immune system, or by chemical reactions, causing damage to the cells through the destruction of components such as proteins, lipids, sugars and nucleotides. It is known that compounds exist which are effective against these species, preventing damage caused by oxidative stress. The object of this work was to study heterocyclic compounds that have nitrogen in their structure (azoles), which appear in the literature as exemplary molecules of compounds with a wide spectrum of pharmacological applications. Of these compounds, derivatives of pyrazoles (26 compounds), thiazoles (7 compounds) and 1 isoxazole (3,5-dimethylisoxazole) were analyzed. These 34 compounds were evaluated by the ORAC (Oxygen Radicals Absorption Capacity) in order to determine and/or evaluate its antioxidant potential. The choice of ORAC method is based on the fact that the molecules studied have hydrophilic and lipophilic characteristics, as well as a method validated by the literature, which is available and widely used. The ORAC method evaluates the antioxidant capacity of the sample, measuring its ability to protect the fluorescence (FL) of the oxidation by the AAPH in the reaction medium. AAPH is a generator of free radicals which, at 37°C, removes hydrogen from the medium, promoting the reduction of fluorescence from fluorescein in λ measured by time. Six compounds present good to moderate antioxidant activity: 3,5-dimethyl-1H-pyrazole (2.382 µmol eq.Trolox/g); 3-phenyl-5-(4-fluorphenyl)-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (6.354 µmol eq.Trolox/g); 3-phenyl-5-(2-methoxyphenyl)-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (8.739 µmol eq.Trolox/g); 5-(2,4-diclorophenyl)-3-phenyl-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (6.022,226 µmol eq.Trolox/g); 2-[5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-il]-4-phenylthiazole (3.135 µmol eq.Trolox/g); and finally, 2-[5-(3-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-il]- 4-phenylthiazole (2.700 µmol eq.Trolox/g). Experiments with the ORAC method for the azoles studied present reproducibility in the experimental execution, and have proven to be a viable alternative for studies of synthetic molecules with antioxidant potential.

Ho, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-124). / Abstracts in English and Chinese. / Thesis committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Abbreviations --- p.x / Content --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species (ROS) --- p.1 / Chapter 1.1.1 --- Definition and examples --- p.1 / Chapter 1.1.2 --- Generation of ROS in biological systems --- p.2 / Chapter 1.1.3 --- Features of specif ic ROS --- p.3 / Chapter 1.1.3.1 --- Superoxide anion --- p.3 / Chapter 1.1.3.2 --- Peroxyl radical --- p.4 / Chapter 1.1.3.3 --- Hydrogen peroxide --- p.4 / Chapter 1.1.3.4 --- Hydroxyl radical --- p.5 / Chapter 1.1.4 --- Damaging effects of ROS on biomolecules --- p.5 / Chapter 1.1.4.1 --- Lipid peroxidation --- p.6 / Chapter 1.1.4.2 --- DNA damage --- p.8 / Chapter 1.1.4.3 --- Protein oxidation --- p.9 / Chapter 1.2 --- Antioxidants --- p.11 / Chapter 1.2.1 --- Introduction --- p.11 / Chapter 1.2.2 --- Mode of action --- p.11 / Chapter 1.2.3 --- Endogenous Antioxidants --- p.12 / Chapter 1.2.3.1 --- Antioxidant enzymes --- p.12 / Chapter 1.2.3.2 --- Antioxidant compounds --- p.15 / Chapter 1.2.4 --- Exogenous antioxidants --- p.16 / Chapter 1.3 --- Oxidative stress --- p.17 / Chapter 1.3.1 --- Balance between ROS and antioxidants --- p.17 / Chapter 1.3.2 --- Diseases associated with oxidative stress --- p.18 / Chapter 1.4 --- Previous studies on edible mushroom antioxidants --- p.19 / Chapter 1.4.1 --- Previous studies on Agrocybe aegerita --- p.20 / Chapter 1.5 --- Cell culture models for antioxidant research --- p.21 / Chapter 1.6 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Mushroom fruiting bodies --- p.24 / Chapter 2.1.2 --- Cell lines and their subcultures --- p.24 / Chapter 2.2 --- Principle of Methods and Procedures --- p.26 / Chapter 2.2.1 --- Sample preparation and extraction --- p.26 / Chapter 2.2.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.28 / Chapter 2.2.2.1 --- ABTS + scavenging activity --- p.28 / Chapter 2.2.2.2 --- Hydroxyl radical scavenging activity --- p.30 / Chapter 2.2.2.3 --- Hydrogen peroxide scavenging activity --- p.32 / Chapter 2.2.3 --- Total phenolic content --- p.34 / Chapter 2.2.4 --- Cytotoxicity of hydrogen peroxide --- p.36 / Chapter 2.2.5 --- Cytoprotectivity of mushroom extracts --- p.36 / Chapter 2.2.6 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.37 / Chapter 2.2.7 --- Lactate dehydrogenase (LDH) assay --- p.39 / Chapter 2.2.8 --- Total cellular protein loss --- p.40 / Chapter 2.2.9 --- Comet assay (Single cell gel electrophresis assay) --- p.41 / Chapter 2.2.10 --- Thiobarbituric Acid Reactive Substances (TBARS) assay ..… --- p.44 / Chapter 2.2.11 --- Preparation of cell lysate for evaluating cellular antioxidant defense system --- p.45 / Chapter 2.2.12 --- Total Glutathione level --- p.46 / Chapter 2.2.13 --- Enzyme activity --- p.49 / Chapter 2.2.13.1 --- Catalase (CAT) --- p.49 / Chapter 2.2.13.2 --- Glutathione peroxidases (GPx) --- p.51 / Chapter 2.2.13.3 --- Glutathione Reductase (GR) --- p.53 / Chapter 2.2.13.4 --- Superoxide dismutase (SOD) --- p.54 / Chapter 2.2.14 --- Determination of protein --- p.56 / Chapter 2.2.15 --- Statistical analysis --- p.56 / Chapter Chapter 3 --- Results and discussions --- p.57 / Chapter 3.1 --- Extraction yield --- p.57 / Chapter 3.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.60 / Chapter 3.2.1 --- ABTS + scavenging activity --- p.60 / Chapter 3.2.2 --- Hydroxyl radicals scavenging activity --- p.61 / Chapter 3.2.3 --- Hydrogen peroxide scavenging activity --- p.64 / Chapter 3.3 --- Total phenolic content --- p.67 / Chapter 3.4 --- Cytotoxicity of hydrogen peroxide --- p.69 / Chapter 3.4.1 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.71 / Chapter 3.4.2 --- Lactate dehydrogenase (LDH) assay --- p.72 / Chapter 3.4.3 --- Total cellular protein loss --- p.73 / Chapter 3.4.4 --- Residual hydrogen peroxide level --- p.76 / Chapter 3.4.5 --- Lipid peroxidation --- p.77 / Chapter 3.4.6 --- DNA damage --- p.79 / Chapter 3.5 --- Cytotoxicity of extracts --- p.85 / Chapter 3.6 --- Protection of H2()2-induced oxidative damage in HDFa cells --- p.88 / Chapter 3.6.1 --- Protective effect of mushroom water extracts --- p.88 / Chapter 3.6.2 --- Protective effect of CfAa on H2()2-incluced damage to HDFa --- p.93 / Chapter 3.6.3 --- Protective effect of CfAa on DNA damage in HDFa cells --- p.96 / Chapter 3.7 --- Modulation of cellular antioxidant defense system by CfAa --- p.99 / Chapter 3.7.1 --- Intracellular total glutathione --- p.100 / Chapter 3.7.2 --- Enzyme activities --- p.102 / Chapter 3.8 --- Speculation on the possible components in CfAa --- p.108 / Chapter Chapter 4 --- Conclusion and further works --- p.109 / References --- p.111

Lam, Ming Yiu. / "September 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 149-169). / Abstracts in English and Chinese. / Thesis / Assessment Committee --- p.ii / Abstract (English) --- p.iii / Abstract (Chinese) --- p.vi / Acknowledgements --- p.viii / Table of contents --- p.x / List of figures --- p.xvi / List of tables --- p.xix / Abbreviations --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cerebral stroke --- p.1 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Risk factors and symptoms --- p.5 / Chapter 1.3.1 --- Non-modifiable risks --- p.5 / Chapter 1.3.2 --- Modifiable risks --- p.6 / Chapter 1.3.3 --- Symptoms --- p.9 / Chapter 1.4 --- Mechanisms of cell injury --- p.10 / Chapter 1.4.1 --- Energy failure and loss of ionic homeostasis --- p.10 / Chapter 1.4.2 --- Excitotoxicity and calcium-modulated cell damage --- p.11 / Chapter 1.4.3 --- Oxidative stress --- p.13 / Chapter 1.4.4 --- Inflammation --- p.16 / Chapter 1.4.5 --- Apoptosis --- p.18 / Chapter 1.5 --- Current treatment of ischemia --- p.19 / Chapter 1.6 --- Chinese herbal medicine --- p.21 / Chapter 1.6.1 --- Traditional Chinese medicine theory on stroke --- p.21 / Chapter 1.6.2 --- Danshen --- p.22 / Chapter 1.6.3 --- Gegen --- p.25 / Chapter 1.6.4 --- Danshen-Gegen formula --- p.28 / Chapter 1.7 --- Aim of study --- p.30 / Chapter Chapter 2 --- General methodology --- p.31 / Chapter 2.1 --- Induction of transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO) --- p.31 / Chapter 2.1.1 --- Intraluminal filament production --- p.32 / Chapter 2.1.2 --- Cerebral blood flow measurement by laser Doppler flowmetry --- p.33 / Chapter 2.1.3 --- Middle cerebral artery occlusion --- p.35 / Chapter 2.2 --- Neurological scoring --- p.38 / Chapter 2.3 --- Brain infarction measurement by triphenyltetrazolium chloride (TTC) staining --- p.40 / Chapter 2.4 --- Statistical analyses --- p.42 / Chapter Chapter 3 --- Preparation of herbal medicine --- p.43 / Chapter 3.1 --- Authentication of Chinese herbs --- p.43 / Chapter 3.1.1 --- Morphological authentication --- p.43 / Chapter 3.1.2 --- Chemical authentication using thin layer chromatography --- p.44 / Chapter 3.1.2.1 --- Danshen --- p.44 / Chapter 3.1.2.2 --- Gegen --- p.48 / Chapter 3.2 --- Danshen-Gegen (DG) extract preparation --- p.50 / Chapter 3.3 --- Chemical analysis of DG extract --- p.51 / Chapter 3.3.1 --- TLC --- p.51 / Chapter 3.3.2 --- HPLC --- p.54 / Chapter 3.4 --- Conclusion --- p.57 / Chapter Chapter 4 --- Protective effect of DG extract on cerebral ischemia --- p.58 / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Different models of ischemia --- p.58 / Chapter 4.1.2 --- Anti-oxidative enzymes in cerebral ischemia --- p.61 / Chapter 4.1.2.1 --- Superoxide dismutase (SOD) --- p.61 / Chapter 4.1.2.2 --- Catalase --- p.62 / Chapter 4.1.2.3 --- Glutathione peroxidase (GPX) --- p.62 / Chapter 4.2 --- Materials and methods --- p.64 / Chapter 4.2.1 --- "DG extract treatment, neurological deficit and brain infarction" --- p.64 / Chapter 4.2.2 --- Anti-oxidative enzymes activity determination --- p.65 / Chapter 4.2.2.1 --- Treatment with DG extract and induction of cerebral ischemia --- p.65 / Chapter 4.2.2.2 --- Extraction of enzymes from the brain --- p.66 / Chapter 4.2.2.3 --- Determination of protein concentration --- p.66 / Chapter 4.2.2.4 --- Assay kits --- p.67 / Chapter 4.3 --- Results --- p.70 / Chapter 4.4 --- Discussion --- p.80 / Chapter 4.4.1 --- Neurological score and percentage brain infarction --- p.80 / Chapter 4.4.2 --- Anti-oxidative enzyme induction --- p.82 / Chapter Chapter 5 --- Behavioral assessment using the shuttle box avoidance test on rats suffering from cerebral ischemia: effect of DG extract treatment --- p.86 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.1.1 --- Behavioral tests --- p.86 / Chapter 5.1.2 --- Theory of the test --- p.90 / Chapter 5.2 --- Materials and methods --- p.91 / Chapter 5.2.1 --- DG extract treatment --- p.91 / Chapter 5.2.2 --- Shuttle box training and MCAO --- p.92 / Chapter 5.2.3 --- Shuttle box testing --- p.96 / Chapter 5.2.4 --- Neurological score and brain infarction --- p.96 / Chapter 5.3 --- Results --- p.97 / Chapter 5.3.1 --- Shuttle box performance --- p.97 / Chapter 5.3.2 --- Neurological score --- p.105 / Chapter 5.3.3 --- Brain infarction --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter 5.4.1 --- The shuttle box protocol --- p.112 / Chapter 5.4.2 --- Shuttle box performance --- p.114 / Chapter 5.4.2.1 --- Pretreatment groups --- p.114 / Chapter 5.4.2.2 --- Pre + post treatment groups --- p.115 / Chapter 5.4.2.3 --- Comparison of pretreatment and pre + post treatment groups --- p.116 / Chapter 5.4.3 --- Neurological score --- p.117 / Chapter 5.4.4 --- Brain infarction --- p.118 / Chapter 5.4.5 --- Conclusion --- p.119 / Chapter Chapter 6 --- Anti-blood coagulation effect of DG extract --- p.121 / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.2 --- Materials and methods --- p.125 / Chapter 6.2.1 --- Treatment with DG extract and warfarin --- p.125 / Chapter 6.2.2 --- Tail bleeding time and volume --- p.126 / Chapter 6.2.3 --- Prothrombin time --- p.127 / Chapter 6.2.4 --- Platelet aggregation --- p.127 / Chapter 6.3 --- Results --- p.128 / Chapter 6.4 --- Discussion --- p.138 / Chapter Chapter 7 --- General discussion --- p.141 / Chapter 7.1 --- General discussion and conclusion --- p.141 / Chapter 7.2 --- Clinical significance of the study --- p.145 / Chapter 7.3 --- Limitations of the study --- p.146 / Chapter 7.4 --- Future work --- p.147 / References --- p.149 / Publications --- p.170

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xi, 148 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Endothelial cells (EC) exposed to oscillatory shear stress (OS) experience oxidative stress as a signature of atherosclerosis. Conversely, unidirectional laminar shear stress (LS) reduces reactive oxygen species (ROS) levels and inflammatory responses. Peroxiredoxins (PRX) are antioxidant enzymes that reduce hydrogen peroxide, but have yet to be investigated in response to shear stress. We hypothesized that LS, compared to OS, promotes increased expression of PRX, which in turn influences the balance of ROS in EC. In this study, we identified all six PRX family members in bovine aortic endothelial cells (BAEC). Furthermore, we revealed that PRX are regulated by shear stress in EC. When compared to OS and static culture (ST), exposure to chronic LS upregulated PRX1 levels intracellularly. LS also upregulated PRX5 relative to ST, but not OS. In addition, PRX exhibited broad subcellular localization in BAEC, but these patterns did not change in response to shear stress. To establish the functional importance of PRX1 in shear stress-dependent redox balance, we next examined the role of PRX1 in LS-mediated hydrogen peroxide regulation. Here, Amplex Red assay was used to measure ROS levels in BAEC. Depletion of PRX1 using siRNA resulted in significantly higher ROS levels following LS, OS, and ST, while PRX5 depletion did not. These findings indicated that chronic exposure to LS upregulates PRX1 expression to keep ROS levels low in EC. To identify the pathway by which atheroprotective LS stimulates PRX1 protein production, we also undertook gene expression studies. We discovered that LS upregulates Prdx1 gene in a time-dependent manner compared to OS or ST. However, this increase in expression was not due to stabilization of Prdx1 mRNA. In addition, Prdx1 promoter analysis revealed a Nrf2 transcription factor binding site 160bp upstream of the gene. Nrf2 overexpression promoted basal PRX1 protein production, while Nrf2 depletion reduced Prdx1 mRNA following exposure to LS. Collectively, our work illustrated that LS affects PRX1 by inducing the Prdx1 gene, in part via the transcription factor Nrf2. Moreover, this discovery of PRX1 as a mechanosensitive antioxidant may contribute important insights into endothelial cell biology and provide a novel therapeutic target for vascular diseases.

Thesis (Ph.D.). / Written for the School of Dietetics and Human Nutrition. Title from title page of PDF (viewed 2008/07/23). Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains x, 178 p. : ill. Includes abstract. Includes bibliographical references.

A ingestão excessiva de fluoreto por um longo período de tempo pode resultar em fluorose, que pode causar manifestações dentárias e esqueléticas. Danos metabólicos, funcionais e estruturais causados pela fluorose crônica tem sido relatados em vários tecidos. O objetivo deste estudo foi avaliar os efeitos do fluoreto administrado na água de beber, da administração de fluoreto na água de beber na defesa antioxidante de ratos. Quatro grupos de ratos wistar foram usados (n=10/grupo). Os animais receberam água de beber contendo 0 (controle), 5, 15 ou 50 ppm de fluoreto durante 60 dias. Eles foram eutanasiados e os tecidos (fígado, rins e coração) e plasma foram coletados e homogenizados. Superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa reduzida (GSH), substâncias antioxidantes totais (SAT), substâncias reativas ao ácido tiobarbitúrico (TBARS), hidroperóxido de lipídios (HL) e fluoreto foram análisadas. Dados foram analisados por ANOVA e teste de Tukey ou Kruskal-Wallis e teste de Dunn (p<0,05). Nos rins, SOD, GPx, GSH e SAT diminuiram e fluoreto e HL aumentaram significantivamente. No fígado, CAT e TBARS diminuiram, SOD, HL e SAT aumentaram significativamente. No coração, GPx aumentou significativamente. No plasma, SOD e HL diminuiram significativamente. Em resumo, esses resultados mostram que a administração crônica de fluoreto altera o sistema antioxidante de ratos. Nosso dados sugerem que a exposição em níveis elevados de fluoreto, a conversão do ânion superóxido em água nos rins parecem ocorrer principalmente através da SOD e CAT, com baixa participação do sistema glutationa, diferindo do que parece ocorrer no fígado. / Excessive fluoride intake over a long period of time could result in fluorosis, which can lead to dental and skeletal manifestations. Metabolic, functional and structural damages caused by chronic fluorosis have been reported in many tissues. The aim of this study was to evaluate the effect of fluoride, administered in drinking water, in the antioxidant defense of rats. Four groups of Wistar rats were included (n=10/group). The animals received drinking water containing 0 (control), 5, 15 or 50 ppm of fluoride during 60 days. They were euthanized and the tissues (liver, kidney and heart) and plasma were collected and homogenized. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), antioxidants, thiobarbituric acid reactive substances (TBARS), lipid hydroperoxide (LH), and fluoride were analyzed. Data were analyzed by ANOVA and Tukeys test or Kruskal-Wallis and Dunns tests (p<0.05). In the kidney SOD, GPx, GSH and SAT decreased and fluoride and LH increased significantly. In the liver, CAT and TBARS decreased and fluoride, SOD, LH and SAT increased significantly. In the heart, GPx increased significantly. In the plasma, SOD and LH decreased significantly. In summary, these results show that chronic fluoride administration alters the antioxidant system of the rats. Our data suggest that upon exposure to high levels of fluoride, the conversion of the superoxide anion to water in the kidney seems to occur mainly through the SOD and CAT, with a low participation of the glutathione system, differing from what seems to occur in the liver.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2015 / Antioxidants play an important role in living organisms to control level of free radicals and other reactive molecules in the body to reduce oxidative damage. Synthetic antioxidant compounds are used in food industries as food additives to boost our immune systems. These compounds are associated with a number of critical side effects including liver damage and carcinogenesis. Scientists are also concerned about microorganisms that have developed resistant genes against current antibiotics used in hospitals. The aim of the study was to isolate and characterize bioactive compounds from Ricinus communis leaves with activity against Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Consequently, medicinal plants are studied and considered for their efficacy and safety, because they possess bioactive compounds with various biological activities. Leaves of R. communis were collected at the University of Limpopo, Turfloop campus in Limpopo province, South Africa. The leaves were dried and milled to a fine powder. A number of trial extraction methods were employed using various solvents of different polarities on a fine powder leaves to identify the best extraction method. Plant extracts were analyzed by thin layer chromatography (TLC) developed in four mobile phases. To detect separated phytochemical compounds, TLC plates were sprayed with vanillin- sulphuric acid in methanol and heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 2, 2–diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates. Quantitative antioxidant activity was determined by measuring percentages scavenging activity of DPPH and 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical molecules by plant extracts. Antibacterial activity of all extracts was quantified by a serial microbroth dilution method while bioautography was used in qualitative analysis of the active compounds. Cytotoxicity effect of R. communis extracts was evaluated using tetrazolium-based calorimetric assay on human Caucasian skin fibroblast (Bud-8) cell line. Anti-inflammatory activity was assessed using phagoburst kit on Raw 264.7 macrophages cell line. Pure compounds were subjected to nuclear magnetic resonance spectroscopy for 1H, 13C and DEPT experiments to elucidate structures of compounds. 2 During extraction process, methanol was the best extractant, extracting greater amount of extracts than any of the other solvents. Serial exhaustive extraction method was selected as the best extraction method for extracting compounds from ground plant materials. In quantitative antioxidant assays, chloroform and methanol extracts had highest percentage scavenging activity against DPPH free radicals compared to other extracts and vitamin C. Methanol extract had the highest percentage scavenging activity of ABTS free radicals and minimum percentage scavenging activity was in hexane extract. Acetone, ethyl acetate and ethanol extracts showed strong antioxidant activity against DPPH free radicals in qualitative antioxidant assay on TLC plates. In quantitative antibacterial assay, crude extracts showed lowest minimum inhibitory concentration value of 0.13 mg/ml against all tested organisms and the highest was 1.05 mg/ml. Hexane extracts revealed potent antibacterial activity against all tested microorganisms on bioautograms. Hexane and acetone extracts also revealed anti-inflammatory activity and have ability to reduce oxidative stress. In cytotoxicity effect of plant extracts, Methanol extracts had lethal concentration for 50% of the cells (Lc50) of 784 μg/ml on Human Caucasian skin fibroblast (Bud-8) cell line while hexane extracts had Lc50 of 629 μg/ml. Plant extracts with high Lc50 are low toxic to normal cell line and preferable to work with for drug development. Bioassay-guided fractionations results in successful isolation of three antioxidant and two antibacterial compounds from R. communis using column chromatography. Isolated compounds were tested for their biological activities using qualitative DPPH assay on TLC plates for antioxidant activity and bioautography for antibacterial activity. Antioxidant compounds showed strong antioxidant activity after spraying with DPPH in methanol and antibacterial compounds showed less activity compared to the crude extracts. The study suggests the use of crude extracts to fight against pathogenic microorganisms compared to pure compounds. Compound 4 was successful identified as the mixture of stigmasterol and β-sitosterol. The present study recommends the use of R. communis leaves as the potential source of antioxidant, antibacterial and anti-inflammatory compounds. The study serves as a scientific proof for use of this plant in traditional medicine for treatment of various ailments.