Li, Yuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-132). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / LIST OF ABBREVATIONS --- p.vii / TABLE OF CONTENTS --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Theories of aging --- p.2 / Chapter 1.2.1 --- Free radical theory of aging --- p.3 / Chapter 1.2.2 --- Free radicals reactions and their effects --- p.4 / Chapter 1.2.2.1 --- Lipid peroxidation --- p.6 / Chapter 1.2.2.2 --- Protein oxidation --- p.6 / Chapter 1.2.2.3 --- DNA oxidation --- p.8 / Chapter 1.2.2.4 --- Carbohydrate oxidation --- p.8 / Chapter 1.2.3 --- Antioxidant defense systems --- p.9 / Chapter 1.2.3.1 --- Superoxide dismutase (SOD) --- p.9 / Chapter 1.2.3.2 --- Catalase --- p.11 / Chapter 1.2.3.3 --- Glutathione peroxidase (GPx) and reductase --- p.11 / Chapter 1.3 --- Models on aging study --- p.12 / Chapter 1.3.1 --- Non-mammalian model systems --- p.12 / Chapter 1.3.1.1 --- History of Drosophila on longevity study --- p.14 / Chapter 1.3.1.2 --- Advantages of Drosophila model --- p.14 / Chapter 1.3.2 --- Mammalian model systems --- p.15 / Chapter 1.4 --- Antioxidant effects on aging process --- p.16 / Chapter 1.4.1 --- Antioxidant capacity of tea --- p.16 / Chapter 1.4.2 --- Antioxidant capacity of vegetables --- p.19 / Chapter 1.5 --- Dietary fat on longevity --- p.21 / Chapter Chapter 2 --- Anti-aging: Effects of Green Tea Catechins on Life Span and Antioxidant Enzymes of Drosophila melanogaster / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Gbjectives --- p.27 / Chapter 2.3 --- Materials and methods --- p.28 / Chapter 2.3.1 --- Materials --- p.28 / Chapter 2.3.2 --- Preparation of GTCs --- p.28 / Chapter 2.3.3 --- Feeding experiment --- p.30 / Chapter 2.3.3.1 --- Dose determination --- p.31 / Chapter 2.3.3.2 --- Paraquat treatment --- p.31 / Chapter 2.3.3.3 --- Hydrogen peroxide (H202) treatment --- p.33 / Chapter 2.3.3.4 --- Enzyme activity assay --- p.34 / Chapter 2.3.4 --- Enzyme analysis --- p.34 / Chapter 2.3.4.1 --- SOD activity --- p.34 / Chapter 2.3.4.2 --- Catalase activity --- p.35 / Chapter 2.3.4.3 --- Lipid hydorperoxies (LOOHs) formation --- p.36 / Chapter 2.3.4.4 --- Determination of protein concentration --- p.37 / Chapter 2.3.5 --- Semi-quantitative RT-PCR assay --- p.37 / Chapter 2.3.5.1 --- RNA isolation --- p.37 / Chapter 2.3.5.2 --- Primer sequences --- p.38 / Chapter 2.3.5.3 --- Reverse Transcriptase PCR --- p.40 / Chapter 2.3.6 --- Statistics --- p.40 / Chapter 2.4 --- Results --- p.42 / Chapter 2.4.1 --- Dose determination --- p.42 / Chapter 2.4.2 --- Paraquat treatment --- p.45 / Chapter 2.4.3 --- H202 treatment --- p.48 / Chapter 2.4.4 --- SOD activity --- p.51 / Chapter 2.4.5 --- Catalase activity --- p.51 / Chapter 2.4.6 --- LOOHs formation --- p.53 / Chapter 2.4.7 --- Semi-quantitative RT-PCR assay --- p.53 / Chapter 2.5 --- Discussion --- p.55 / Chapter Chapter 3 --- Anti-aging: Effects of Vegetable Extracts on Life Span and Antioxidant Enzymes of Drosophila melanogaster / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Objectives --- p.64 / Chapter 3.3 --- Materials and methods --- p.65 / Chapter 3.3.1 --- Materials --- p.65 / Chapter 3.3.2 --- Preparation of vegetables extracts --- p.65 / Chapter 3.3.3 --- Feeding experiment --- p.65 / Chapter 3.3.3.1 --- Dose determination --- p.66 / Chapter 3.3.3.2 --- Paraquat and hydrogen peroxide (H2O2) treatment --- p.67 / Chapter 3.3.4 --- Lipid hydroperoxide (LOOHs) formation --- p.67 / Chapter 3.3.5 --- Enzyme analysis --- p.67 / Chapter 3.3.6 --- Semi-quantitative RT-PCR assay --- p.67 / Chapter 3.3.7 --- Statistics --- p.68 / Chapter 3.4 --- Results --- p.69 / Chapter 3.4.1 --- Dose determination --- p.69 / Chapter 3.4.2 --- Antioxidant effect among different vegetables --- p.69 / Chapter 3.4.3 --- SOD activity --- p.76 / Chapter 3.4.4 --- Catalase activity --- p.76 / Chapter 3.4.5 --- LOOHs formation --- p.78 / Chapter 3.4.6 --- Semi-quantitative RT-PCR assay --- p.78 / Chapter 3.5 --- Discussion --- p.80 / Chapter Chapter 4 --- Anti-aging Activity of Greeen Tea Catechins and Broccoli Extracts in Drosophila melanogaster Fed a High Fat Diet / Chapter 4.1 --- Introduction --- p.85 / Chapter 4.2 --- Objectives --- p.87 / Chapter 4.3 --- Materials and methods --- p.88 / Chapter 4.3.1 --- Materials --- p.88 / Chapter 4.3.2 --- Preparation of free fatty acid --- p.88 / Chapter 4.3.3 --- Prepartion of GTCs and BEs --- p.89 / Chapter 4.3.4 --- Feeding experiment --- p.89 / Chapter 4.3.5 --- LOOHs formation --- p.90 / Chapter 4.3.6 --- Enzyme analysis --- p.91 / Chapter 4.3.7 --- Semi-quantitative RT-PCR assay --- p.91 / Chapter 4.3.8 --- Statistics --- p.91 / Chapter 4.4 --- Results --- p.92 / Chapter 4.4.1 --- Dose determination --- p.92 / Chapter 4.4.2 --- Effects of GTCs or BEs on life span with lard consumption --- p.95 / Chapter 4.4.3 --- LOOHs formation --- p.101 / Chapter 4.4.4 --- SOD activity --- p.103 / Chapter 4.4.5 --- Catalase activity --- p.107 / Chapter 4.4.6 --- Semi-quantitative RT-PCR assay --- p.110 / Chapter 4.5 --- Discussion --- p.112 / References --- p.121

published_or_final_version / Biological Sciences / Master / Master of Philosophy

A evolução das resinas compostas fez com que esses materiais passassem a ter uma durabilidade maior e características estéticas muito boas, mas o risco de cárie recorrente é ainda um problema a ser resolvido. Na tentativa de solucionar esse problema, estudos vêm sendo conduzidos na tentativa de se formularem resinas compostas contendo agentes antibacterianos, como é o caso da incorporação de clorexidina (CHX). Outro fato que impede a longevidade deste material é a degradação de matriz de colágeno por proteases ativadas por pH ácido. Para tentar contornar esse problema, a adição de clorexidina, assim como Epigallocatechin gallate (EGCg), clássicos antibacterianos e inibidores de proteases da matriz , como as metaloproteinases da matriz (MMP) a resinas, poderia melhorar a eficácia destes materiais como substitutos de dentina em procedimentos restauradores, aumentando a longevidade do tratamento restaurador, mediante preservação das propriedades mecânicas do material. Assim, o objetivo desse estudo é avaliar o poder de inibição de resinas experimentais contendo inibidores conhecidos de proteases da matriz sobre gelatinases e colagenase. Para isso, copolímeros experimentais foram preparados combinando Bis-GMA com o diluente TEGDMA (70/30 mol%). Com exceção do copolímero placebo (sem drogas), EGCg ou CHX foram incorporados a 1% em peso isoladamente ou em combinação, a 0,5% em peso cada. Amostras contendo EGCg, CHX ou EGCg e CHX concentradas 10X foram obtidas do armazenamento de espécimes polimerizados da resina experimental em água deionizada (1 mL) após o período de 24h a 37°C e sua posterior concentração. O efeito da ação dos inibidores foi checado por zimografia e confirmado por um ensaio enzimático específico para colagenases e gelatinases. Os dados passaram por teste de homogeneidade (Bartlett) e normalidade (Kolmogorov-Smirnov) e foram avaliados por ANOVA a 2 critérios, seguido pelo teste de Bonferroni para comparações individuais (p<0,05). Os resultados do presente estudo, mostraram que, in vitro, a liberação de EGCg e CHX incorporados em resinas é capaz de reduzir a atividade gelatinolítica das MMPs -2 e -9, bem como a atividade da colagenase bacteriana, sugerindo um efeito potencial no aumento da longevidade de restaurações de resinas. Com isso, podemos afirmar que a liberação de ativos de resinas experimentais é possível e que esses ativos são capazes de inibir as MMPs, assim sugerindo um novo substituto para dentina em procedimentos restauradores. / The evolution of composite resins made these materials to have a greater durability and very good esthetics characteristics, but the risk of recurrent caries is still a problem to be solved. In the attempt to solve this problem, studies are being conducted with the purpose to formulate composite resins containing antibacterial agents, such as chlorhexidine (CHX). Another fact that prevents the longevity of this material is the degradation of the collagen matrix by the proteases activated by acidic pH. In order to solve this problem, the addition of chlorhexidine and/or Epigallocatechin gallate (EGCg), classical antibacterial agents and inhibitors of matrix proteases, such as matrix metalloproteinases (MMP) in resins, could improve the efficacy of these materials as dentin substitutes in restorative procedures, increasing the longevity of the restorative treatment, while preserving the mechanical properties of the material. Thus, the aim of this study is to evaluate the ability of experimental resins containing known matrix protease inhibitors on the inhibition of gelatinases and collagenase. For this purpose, experimental copolymers were prepared combining Bis-GMA with the diluent TEGDMA (70/30 mol%). Except for the placebo copolymer (drug free), EGCg or CHX were incorporated at 1% in weight, isolated or in combination (0.5% in weight each). Samples containing EGCg, CHX or EGCg and CHX concentrated 10X were obtained after storage of polymerized specimens of the experimental resin in deionized water (1 mL) after the period of 24 h, at 37°C and after that were concentra. The effect of the action of the inhibitors was checked by zymography and confirmed by an enzymatic test specific for collagenases and gelatinases. The data passed in the tests of homogeneity (Bartlett test) and normality (Kolmogorov-Smirnov test), and were evaluated by 2-way ANOVA, followed by Bonferroni test for individual comparisons (p<0.05). The results of this study showed that the in vitro release of EGCG and CHX incorporated in resins was able to reduce the gelatinolytic activity of MMPs-2 and -9 and bacterial collagenase activity, suggesting a potential effect in increasing the longevity of resin restorations. It can be concluded that the release of drugs from experimental resins is possible and that these drugs are able to inhibit MMPs, thereby suggesting a new substitute for dentin in restorative procedures.

A busca por substâncias imunomoduladoras oriundas de plantas medicinais é crescente devido à grande variedade de doenças relacionadas às respostas imunológicas desordenadas, como as autoimunidades e o câncer. Neste sentido, sabe-se que a Paullinia cupana, popularmente conhecida como guaraná, é uma planta medicinal que apresenta entre outros efeitos, propriedades quimiopreventiva e antitumoral em camundongos. Entretanto, até o presente estudo, não havia relatos na literatura a respeito dos efeitos desta planta sobre o sistema imune, conhecido por desempenhar papel fundamental não só no controle de infecções, mas também no combate ao câncer. Assim, o presente estudo avaliou os efeitos do pó das sementes de guaraná e de seus principais compostos ativos, cafeína e catequina, sobre o sistema imune de camundongos C57BL/6 através de protocolos preconizados pela Organization for Economic Cooperation and Development (OECD) com modificações. Os resultados obtidos mostraram que o guaraná, devido à ação majoritária da catequina, mas com a participação da cafeína também, diminui as respostas imunes, celular e humoral, verificada através da diminuição da expansão de linfócitos T frente a um antígeno específico, redução da resposta de hipersensibilidade do tipo tardia e título de anticorpos, diminuição dos pesos relativos do baço e timo e celularidade deste último, além de aumento de células imaturas no timo. Desta forma, os resultados aqui expostos demonstram, pela primeira vez, que a ingestão de guaraná reduz a imunidade adaptativa através da diminuição das respostas celular e humoral. Assim, futuros estudos deverão ser realizados, no sentido de se verificar a possibilidade do uso desta planta ou de seus princípios ativos no controle de doenças inflamatórias e autoimunes, caracterizadas por uma resposta exacerbada do sistema imune. / Seeking for immunomodulatory compounds derived from medicinal plants is increasing due to the huge variety of diseases related to disordered immune responses, such as autoimmunity and cancer. Therefore, it is known that the Paullinia cupana, popularly known as guarana, is a medicinal plant that has among other effects, chemopreventive and antitumor properties in mice. However, until the present moment, there were no reports in the literature about the effects of this plant on the immune system, which is known to play key role not only in the infectious control, but also in combat cancer. Thus, this study evaluated the effects of guarana seed powder and its major active principles, caffeine and catechin, on the immune system of C57BL/6 mice through protocols recommended by the Organization for Economic Cooperation and Development (OECD) with modifications. The results showed that guarana, due to the majority action of catechin, but also with the participation of caffeine, decreases the cellular and humoral immune responses, as indicated by the decreased expansion of antigen-specific T lymphocyte, reduced response delayed-type hypersensitivity and titer of antibodies, reduction in relative weights of thymus and spleen cellularity, besides increase of immature cells in the thymus. Thus, the results showed here demonstrate, for the first time, that the intake of guarana decreases the adaptive immunity by reducing cellular and humoral responses. Thus, futures studies should be conducted in order to better evaluate the use of this plant and/or its main active principle in the treatment of inflammatory and autoimmune diseases, which are characterized by exarcebated immune response.

Zhang Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-159). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract (in Chinese) --- p.IV / Publications --- p.V / List of Abbreviations --- p.VI / List of Tables --- p.VII / List of Figures --- p.VIII / Table of Contents --- p.XIII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.2 --- Tea --- p.4 / Chapter 1.2.1 --- Composition of green tea catechins (GTC) --- p.4 / Chapter 1.2.2 --- Pharmacological activity --- p.6 / Chapter 1.2.2.1 --- Anticarcinogenic activity --- p.6 / Chapter 1.2.2.2 --- Antioxidative activity --- p.7 / Chapter 1.2.2.3 --- Radical scavenge --- p.7 / Chapter 1.2.2.4 --- Cardiovascular activity --- p.8 / Chapter 1.2.3 --- Pharmacokinetics of GTC --- p.8 / Chapter 1.2.3.1 --- Absorption --- p.10 / Chapter 1.2.3.2 --- Distribution --- p.11 / Chapter 1.2.3.3 --- Elimination --- p.11 / Chapter 1.2.3.4 --- Metabolism --- p.12 / Chapter 1.2.3.4.1 --- Metabolism in the small intestine --- p.12 / Chapter 1.2.3.4.2 --- Metabolism in the liver --- p.13 / Chapter 1.2.3.5 --- Summary of the pharmacokinetics of GTC --- p.13 / Chapter 1.3 --- Hawthorn --- p.14 / Chapter 1.3.1 --- Composition of hawthorn --- p.14 / Chapter 1.3.2 --- Pharmacological activity --- p.16 / Chapter 1.3.2.1 --- Inotonic activity --- p.16 / Chapter 1.3.2.2 --- Antiarrhythmic activity --- p.17 / Chapter 1.3.2.3 --- Hypolipidemic activity --- p.17 / Chapter 1.3.2.4 --- Antihypertensive activity --- p.18 / Chapter 1.3.2.5 --- Antioxidative activity --- p.18 / Chapter 1.3.3 --- Pharmacokinetics of HF --- p.18 / Chapter 1.3.3.1 --- Absorption --- p.19 / Chapter 1.3.3.2 --- Distribution and elimination --- p.21 / Chapter 1.3.3.3 --- Summary of pharmacokinetic of HF --- p.22 / Chapter 1.4 --- Mechanisms of intestinal absorption --- p.22 / Chapter 1.4.1 --- Passive transcellular transport --- p.23 / Chapter 1.4.2 --- Paracellular transport --- p.23 / Chapter 1.4.3 --- Carrier-mediated transport --- p.23 / Chapter 1.5 --- ABC transporters --- p.24 / Chapter 1.5.1 --- Cellular location and tissue distribution --- p.25 / Chapter 1.5.2 --- Substrates and inhibitors of ABC transporters --- p.26 / Chapter 1.6 --- Oral absorption models --- p.31 / Chapter 1.6.1 --- Ussing chamber --- p.31 / Chapter 1.6.2 --- In situ intestinal perfusion model --- p.33 / Chapter 1.6.3 --- Cell culture model --- p.34 / Chapter 1.7 --- Aims of the study --- p.36 / Chapter Chapter Two. --- Transport mechanism of green tea catechins --- p.37 / Chapter 2.1 --- Introduction --- p.37 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Materials for cell culture --- p.38 / Chapter 2.2.3 --- Instruments --- p.39 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Analytical methods --- p.39 / Chapter 2.3.1.1 --- Analytical methods for validation of Caco-2 model --- p.39 / Chapter 2.3.1.1.1 --- Fluorescence analysis of lucifer yellow --- p.39 / Chapter 2.3.1.1.2 --- HPLC analysis of propranolol --- p.39 / Chapter 2.3.1.1.3 --- HPLC analysis of verapamil --- p.40 / Chapter 2.3.1.1.4 --- HPLC analysis of quinidine --- p.40 / Chapter 2.3.1.2 --- Analytical methods for samples contained GTC --- p.41 / Chapter 2.3.1.2.1 --- HPLC analysis for each GTC --- p.41 / Chapter 2.3.1.2.2 --- Preparation of calibration curves for each GTC --- p.42 / Chapter 2.3.1.2.3 --- HPLC/MS analysis of samples containing mixtures of four GTC --- p.42 / Chapter 2.3.1.2.4 --- Preparation of calibration curves for samples containing GTC mixture --- p.43 / Chapter 2.3.1.2.5 --- Validation of the HPLC methods --- p.43 / Chapter 2.3.1.3 --- Identification of metabolites with HPLC/MS --- p.44 / Chapter 2.3.2 --- Determination of stability profile of GTC in phosphate buffer --- p.44 / Chapter 2.3.3 --- Cell culture --- p.45 / Chapter 2.3.4 --- Validation of Caco-2 cell monolayer model --- p.46 / Chapter 2.3.4.1 --- Integrity of Caco-2 cell monolayer at pH 6.0 --- p.46 / Chapter 2.3.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.46 / Chapter 2.3.4.3 --- Validation of the existence of P-glycoprotein (P-gp) transporterin Caco-2 monolayer model --- p.46 / Chapter 2.3.4.4 --- Cytotoxicity test --- p.47 / Chapter 2.3.5 --- Transport study of GTC using Caco-2 cell monolayer model --- p.48 / Chapter 2.3.5.1 --- Bi-directional transport experiment --- p.48 / Chapter 2.3.5.2 --- Preparation of different dosing formulations of GTC --- p.48 / Chapter 2.3.5.2.1 --- Preparation of individual pure GTC solutions --- p.48 / Chapter 2.3.5.2.2 --- Preparation of cocktail 1 solution --- p.49 / Chapter 2.3.5.2.3 --- Preparation of green tea extract solution --- p.49 / Chapter 2.3.5.2.4 --- Preparation of cocktail 2 solution --- p.50 / Chapter 2.3.5.3 --- Sample treatment --- p.50 / Chapter 2.3.5.3.1 --- Samples for direct analysis --- p.50 / Chapter 2.3.5.3.2 --- Samples for enzymatic hydrolysis treatment --- p.51 / Chapter 2.3.5.4 --- Further investigation of the transport mechanism of GTC --- p.51 / Chapter 2.3.5.4.1 --- Inhibition transport of EC and EGC --- p.51 / Chapter 2.3.5.4.2 --- Transport mechanism of metabolites of EC and EGC --- p.52 / Chapter 2.3.5.4.3 --- Metabolic competition between EGC and the other GTC --- p.52 / Chapter 2.3.6 --- Calculation --- p.53 / Chapter 2.3.7 --- Data analysis --- p.54 / Chapter 2.4 --- Results --- p.55 / Chapter 2.4.1 --- Validation of the HPLC methods --- p.55 / Chapter 2.4.2 --- Stability of the GTC --- p.55 / Chapter 2.4.3 --- Extract of green tea leaves --- p.55 / Chapter 2.4.4 --- Validation of Caco-2 model --- p.59 / Chapter 2.4.4.1 --- Integrity of Caco-2 cell monolayer --- p.59 / Chapter 2.4.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.59 / Chapter 2.4.4.3 --- Validation of P-glycoprotein --- p.60 / Chapter 2.4.4.4 --- Cytotoxicity test --- p.61 / Chapter 2.4.5 --- Transport study of GTC --- p.63 / Chapter 2.4.5.1 --- Bi-directional transport of individual pure GTC --- p.63 / Chapter 2.4.5.2 --- Bi-directional transport of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.1 --- Absorption transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.2 --- Secretion transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.3 --- Identification of metabolites of each GTC formed during the transport in Caco-2 cell model --- p.71 / Chapter 2.4.6 --- Further investigation of the transport mechanism of GTC --- p.82 / Chapter 2.4.6.1 --- Inhibition transport of EC and EGC --- p.82 / Chapter 2.4.6.2 --- Transport mechanism of metabolites of EC and EGC --- p.82 / Chapter 2.4.6.3 --- Metabolic competition between EGC and the other GTC --- p.85 / Chapter 2.4.6.4 --- Contribution of GTC on the metabolism of EGC --- p.89 / Chapter 2.5 --- Discussion --- p.92 / Chapter 2.5.1 --- Stability of the four GTC --- p.92 / Chapter 2.5.2 --- Validation of Caco-2 cell model --- p.92 / Chapter 2.5.3 --- Bi-directional transport of GTC --- p.93 / Chapter 2.5.4 --- Structure related efflux --- p.97 / Chapter 2.5.5 --- Metabolism of GTC --- p.98 / Chapter 2.5.6 --- Relationship between metabolism and efflux transport of GTC --- p.99 / Chapter 2.5.7 --- Bi-directional transport of GTC in different dosing formulations …… --- p.100 / Chapter 2.5.7.1 --- Absorption transport profile of different dosing formulations --- p.100 / Chapter 2.5.7.2 --- Secretion transport profile of different dosing formulations --- p.101 / Chapter 2.6 --- Conclusion --- p.105 / Chapter Chapter Three. --- Transport mechanism of hawthorn flavonoids --- p.106 / Chapter 3.1 --- Introduction --- p.106 / Chapter 3.2 --- Materials --- p.107 / Chapter 3.2.1 --- Chemicals --- p.107 / Chapter 3.2.2 --- Materials for cell culture --- p.108 / Chapter 3.2.3 --- Instruments --- p.108 / Chapter 3.3 --- Methods --- p.109 / Chapter 3.3.1 --- Analytical methods for HF --- p.109 / Chapter 3.3.1.1 --- Analytical methods of individual pure compound of HF --- p.109 / Chapter 3.3.1.1.1 --- HPLC analysis of HP and IQ --- p.109 / Chapter 3.3.1.1.2 --- HPLC analysis of EC --- p.109 / Chapter 3.3.1.2 --- Preparation of calibration curves for individual pure HF --- p.109 / Chapter 3.3.1.3 --- HPLC/MS analysis of three HF in mixture --- p.110 / Chapter 3.3.1.4 --- Preparation of the calibration curves of three HF in mixture --- p.111 / Chapter 3.3.1.5 --- Validation of HPLC methods --- p.111 / Chapter 3.3.2 --- Analytical methods for identification of metabolites with HPLC/MS --- p.111 / Chapter 3.3.3 --- Cell culture --- p.112 / Chapter 3.3.4 --- Cytotoxicity test --- p.113 / Chapter 3.3.5 --- Transport studies of HF using Caco-2 monolayer model --- p.113 / Chapter 3.3.5.1 --- Bi-directional transport experiment --- p.113 / Chapter 3.3.5.2 --- Preparation of loading solutions in different dosing formulations of HF for Caco-2 cell model --- p.114 / Chapter 3.3.5.2.1 --- Preparation of individual pure HF solutions --- p.114 / Chapter 3.3.5.2.2 --- Preparation of cocktail 1 solution --- p.114 / Chapter 3.3.5.2.3 --- Preparation of hawthorn extract solution --- p.114 / Chapter 3.3.5.2.4 --- Preparation of cocktail 2 solution --- p.114 / Chapter 3.3.5.3 --- Sample treatment --- p.115 / Chapter 3.3.5.4 --- Further study of the transport mechanism of HF --- p.115 / Chapter 3.3.5.4.1 --- "Inhibition transport of EC, IQ, and HP" --- p.115 / Chapter 3.3.5.4.2 --- "Transport mechanisms of the metabolites of EC, HP, IQ" --- p.116 / Chapter 3.3.6 --- Calculation --- p.116 / Chapter 3.3.7 --- Data analysis --- p.117 / Chapter 3.4 --- Results --- p.118 / Chapter 3.4.1 --- Validation of the HPLC methods --- p.118 / Chapter 3.4.2 --- Cytotoxicity test --- p.118 / Chapter 3.4.3 --- Transport study of HF --- p.122 / Chapter 3.4.3.1 --- Bi-directional transport of individual pure HF --- p.122 / Chapter 3.4.3.2 --- Bi-directional transport of the HF in different formulations --- p.123 / Chapter 3.4.3.2.1 --- Absorption transport of different formulations of HF --- p.123 / Chapter 3.4.3.2.2 --- Secretion transport of different dosing forms --- p.123 / Chapter 3.4.3.3 --- Identification of metabolites of each HF formed during their transport in Caco-2 model --- p.126 / Chapter 3.4.4 --- Further study on the transport mechanism --- p.136 / Chapter 3.4.4.1 --- "Inhibition transport of EC, HP, IQ" --- p.136 / Chapter 3.4.4.2 --- Transport mechanism of metabolites of HF --- p.136 / Chapter 3.4.4.3 --- Transport profiles of HF metabolites upon the loading of different dosing formulations of HF --- p.138 / Chapter 3.5 --- Discussion --- p.140 / Chapter 3.5.1 --- Bi-directional transport of each HF --- p.140 / Chapter 3.5.2 --- Bi-directional transport of HF in different formulations --- p.141 / Chapter 3.6 --- Conclusion --- p.142 / Chapter Chapter Four. --- Limitations of the current study --- p.143 / Chapter Chapter Five. --- Overall conclusions --- p.146 / References --- p.148 / Appendices --- p.160

A busca por substâncias imunomoduladoras oriundas de plantas medicinais é crescente devido à grande variedade de doenças relacionadas às respostas imunológicas desordenadas, como as autoimunidades e o câncer. Neste sentido, sabe-se que a Paullinia cupana, popularmente conhecida como guaraná, é uma planta medicinal que apresenta entre outros efeitos, propriedades quimiopreventiva e antitumoral em camundongos. Entretanto, até o presente estudo, não havia relatos na literatura a respeito dos efeitos desta planta sobre o sistema imune, conhecido por desempenhar papel fundamental não só no controle de infecções, mas também no combate ao câncer. Assim, o presente estudo avaliou os efeitos do pó das sementes de guaraná e de seus principais compostos ativos, cafeína e catequina, sobre o sistema imune de camundongos C57BL/6 através de protocolos preconizados pela Organization for Economic Cooperation and Development (OECD) com modificações. Os resultados obtidos mostraram que o guaraná, devido à ação majoritária da catequina, mas com a participação da cafeína também, diminui as respostas imunes, celular e humoral, verificada através da diminuição da expansão de linfócitos T frente a um antígeno específico, redução da resposta de hipersensibilidade do tipo tardia e título de anticorpos, diminuição dos pesos relativos do baço e timo e celularidade deste último, além de aumento de células imaturas no timo. Desta forma, os resultados aqui expostos demonstram, pela primeira vez, que a ingestão de guaraná reduz a imunidade adaptativa através da diminuição das respostas celular e humoral. Assim, futuros estudos deverão ser realizados, no sentido de se verificar a possibilidade do uso desta planta ou de seus princípios ativos no controle de doenças inflamatórias e autoimunes, caracterizadas por uma resposta exacerbada do sistema imune. / Seeking for immunomodulatory compounds derived from medicinal plants is increasing due to the huge variety of diseases related to disordered immune responses, such as autoimmunity and cancer. Therefore, it is known that the Paullinia cupana, popularly known as guarana, is a medicinal plant that has among other effects, chemopreventive and antitumor properties in mice. However, until the present moment, there were no reports in the literature about the effects of this plant on the immune system, which is known to play key role not only in the infectious control, but also in combat cancer. Thus, this study evaluated the effects of guarana seed powder and its major active principles, caffeine and catechin, on the immune system of C57BL/6 mice through protocols recommended by the Organization for Economic Cooperation and Development (OECD) with modifications. The results showed that guarana, due to the majority action of catechin, but also with the participation of caffeine, decreases the cellular and humoral immune responses, as indicated by the decreased expansion of antigen-specific T lymphocyte, reduced response delayed-type hypersensitivity and titer of antibodies, reduction in relative weights of thymus and spleen cellularity, besides increase of immature cells in the thymus. Thus, the results showed here demonstrate, for the first time, that the intake of guarana decreases the adaptive immunity by reducing cellular and humoral responses. Thus, futures studies should be conducted in order to better evaluate the use of this plant and/or its main active principle in the treatment of inflammatory and autoimmune diseases, which are characterized by exarcebated immune response.

Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions. The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention. The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.

published_or_final_version / Public Health / Master / Master of Public Health

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 91-101). Also available on the World Wide Web.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominika Sobolová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of diploma thesis: Pharmacokinetics of flavanols This work is focusing on summarizing available information about the fate of flavanols in the organism. It is necessary to know the pharmacokinetics to explain their biological effects. In contrast to other flavonoids, they occur in the form of aglycones in plant foods. Galoylation, polymerization and optical isomerism have an important influence on the pharmacokinetics. Partial absorption of monomers begins in the small intestine after oral ingestion. In addition to the liver, the extensive metabolic changes take place even in the enterocytes. The resulting metabolites enter the circulation or they are effluxed back into the intestinal lumen, especially in the case of (epi)catechin sulfates. Epicatechin and catechin are present almost exclusively as glucuronides, sulfates or methylated compounds in the plasma. On the contrary, free unconjugated forms prevail within the gallates. The extent of their absorption is lower. They are excreted via biliary excretion, while other catechins are quickly eliminated by the kidneys in urine. The bioavailability of the parent...