Lee Kin-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 187-203). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / CHINESE ABSTRACT --- p.ix / TABLE OF CONTENTS --- p.xii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Neuroblastoma-An Overview --- p.1 / Chapter 1.1.1 --- Epidemiology of Neuroblastoma --- p.1 / Chapter 1.1.2 --- Clinical Presentations of Neuroblastoma --- p.2 / Chapter 1.1.3 --- Diagnosis of Neuroblastoma --- p.3 / Chapter 1.1.4 --- Staging of Neuroblastoma --- p.5 / Chapter 1.1.5 --- Prognostic Considerations of Neuroblastoma --- p.6 / Chapter 1.1.5.1 --- Age --- p.6 / Chapter 1.1.5.2 --- Primary Tumor Site --- p.6 / Chapter 1.1.5.3 --- Tumor Histopathology --- p.7 / Chapter 1.1.5.4 --- Serum Markers --- p.9 / Chapter 1.1.5.5 --- Genetic Markers --- p.9 / Chapter 1.1.6 --- Standard Treatment Modalities for Neuroblastoma --- p.11 / Chapter 1.1.6.1 --- Surgery --- p.11 / Chapter 1.1.6.2 --- Chemotherapy --- p.12 / Chapter 1.1.6.3 --- Radiotherapy --- p.13 / Chapter 1.1.7 --- Differentiation of Neuroblastoma In Vivo and In Vitro --- p.14 / Chapter 1.1.8 --- Differentiation Therapy of Neuroblastoma --- p.16 / Chapter 1.2 --- Glycyrrhizin ´ؤ the Major Active Component of Licorice --- p.17 / Chapter 1.2.1 --- Chemistry of Licorice --- p.17 / Chapter 1.2.2 --- Metabolism of Glycyrrhizin --- p.21 / Chapter 1.2.3 --- Pharmacological Effects of Glycyrrhizin and 18β-Glycyrrhetinic Acid --- p.22 / Chapter 1.2.3.1 --- Anti-inflammatory Effect --- p.22 / Chapter 1.2.3.2 --- Hepatoprotective Effect --- p.23 / Chapter 1.2.3.3 --- Anti-carcinogenic and Anti-tumor Effects --- p.25 / Chapter 1.2.3.4 --- Anti-viral Effect --- p.27 / Chapter 1.2.3.5 --- Immunomodulatory Effect --- p.29 / Chapter 1.2.3.6 --- Mineralocorticoid Effect --- p.30 / Chapter 1.2.4 --- Pharmacokinetics of Glycyrrhizin and Glycyrrhetinic Acid --- p.32 / Chapter 1.2.5 --- Health Hazards of Glycyrrhizin and Glycyrrhetinic Acid --- p.33 / Chapter 1.3 --- Aims and Scopes of This Study --- p.36 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Cell Lines --- p.39 / Chapter 2.1.2 --- "Cell Culture Media, Buffers and Other Reagents" --- p.39 / Chapter 2.1.3 --- Drugs and Chemicals --- p.43 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.44 / Chapter 2.1.5 --- Reagents for Neutral Red Assay --- p.45 / Chapter 2.1.6 --- Reagents for Clonogenic Assay --- p.45 / Chapter 2.1.7 --- Reagents and Buffers for Immunocytochemistry --- p.46 / Chapter 2.1.8 --- Reagents for DNA Extraction --- p.48 / Chapter 2.1.9 --- Reagent for DNA Staining --- p.49 / Chapter 2.1.10 --- Reagents and Buffers for Flow Cytometry --- p.49 / Chapter 2.1.11 --- Reagents for Total RNA Isolation --- p.50 / Chapter 2.1.12 --- Reagents and Buffers for RT-PCR --- p.50 / Chapter 2.1.13 --- Reagents and Buffers for Gel Electrophoresis --- p.55 / Chapter 2.1.14 --- Reagents and Buffers for Western Blot Analysis --- p.56 / Chapter 2.2 --- Methods --- p.62 / Chapter 2.2.1 --- Cell Culture Methodology --- p.62 / Chapter 2.2.2 --- Determination of Cell Proliferation --- p.62 / Chapter 2.2.3 --- Determination of Cell Viability by Trypan Blue Exclusion Test --- p.64 / Chapter 2.2.4 --- Limiting Dilution Assay --- p.65 / Chapter 2.2.5 --- Clonogenic Assay --- p.65 / Chapter 2.2.6 --- Measurement of Apoptosis by DNA Fragmentation Analysis --- p.66 / Chapter 2.2.7 --- Assessment of Apoptosis by Hoechst 33342 Staining --- p.67 / Chapter 2.2.8 --- Cell Morphological Study --- p.67 / Chapter 2.2.9 --- Immunocytochemistry --- p.68 / Chapter 2.2.10 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.69 / Chapter 2.2.11 --- Gene Expression Study --- p.70 / Chapter 2.2.12 --- Protein Expression Study --- p.73 / Chapter 2.2.13 --- Statistical Analysis --- p.76 / Chapter CHAPTER 3: --- ANTI-PROLIFERATIVE EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID ON NEUROBLASTOMA CELLS --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results --- p.79 / Chapter 3.2.1 --- Differential Anti-proliferative Effect of Glycyrrhizin and 18β- Glycyrrhetinic Acid on Various Neuroblastoma Cell Lines In Vitro --- p.79 / Chapter 3.2.2 --- Effect of 18P-Glycyrrhetinic Acid on the Clonogenicity of the Murine Neuroblastoma BU-1 Cells In Vitro --- p.91 / Chapter 3.2.3 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.93 / Chapter 3.2.4 --- Cytotoxic Effect of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells In Vitro --- p.100 / Chapter 3.2.5 --- Inability of Glycyrrhizin and 18β-Glycyrrhetinic Acid to Induce DNA Fragmentation in the Neuroblastoma BU-1 Cells --- p.102 / Chapter 3.3 --- Discussion --- p.107 / Chapter CHAPTER 4: --- DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID ON NEUROBLASTOMA CELLS --- p.112 / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Results --- p.114 / Chapter 4.2.1 --- Morphological Changes in Glycyrrhizin and 18β-Glycyrrhetinic Acid-treated Neuroblastoma BU-1 Cells --- p.114 / Chapter 4.2.2 --- Immunocytochemistry of Glycyrrhizin and 18β-Glycyrrhetinic Acid-treated Neuroblastoma BU-1 Cells --- p.118 / Chapter 4.2.3 --- Effect of 18β-Glycyrrhetinic Acid on the Expression of Proto-oncogenes in Neuroblastoma BU-1 Cells --- p.124 / Chapter 4.2.4 --- Effect of 18β-Glycyrrhetinic Acid on the Expression of Differentiation-Related Genes in Neuroblastoma BU-1 Cells --- p.127 / Chapter 4.3 --- Discussion --- p.130 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI-PROLIFERATIVE AND DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID --- p.136 / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Effects of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Cell Cycle Kinetics of Neuroblastoma BU-1 Cells In Vitro --- p.139 / Chapter 5.2.2 --- Modulatory Effects of 18β-Glycyrrhetinic Acid on the Expression of Cell Cycle Regulatory Genes and Proteins --- p.145 / Chapter 5.2.3 --- "Combined Effects of Glycyrrhizin, 18β-Glycyrrhetinic Acid and All-Trans Retinoic Acid on the Proliferation of Neuroblastoma BU-1 Cells In Vitro" --- p.149 / Chapter 5.2.4 --- "Combined Effects of Glycyrrhizin, 18β-Glycyrrhetinic Acid and All-Trans Retinoic Acid on the Differentiation of Neuroblastoma BU-1 Cells In Vitro" --- p.153 / Chapter 5.2.5 --- Modulatory Effect of 18β-Glycyrrhetinic Acid on the Expression of PKC Isoforms in Neuroblastoma BU-1 Cells --- p.156 / Chapter 5.2.6 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative and Differentiation-Inducing Effects of 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.158 / Chapter 5.2.7 --- The Possible Involvement of Protein Kinase A in the Anti-proliferative and Differentiation-Inducing Effects of 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.165 / Chapter 5.3 --- Discussion --- p.173 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.182 / REFERENCES --- p.187

Leung Yuet Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 147-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-16T19:51:10Z No. of bitstreams: 1 paulohenriquediasdecarvalho.pdf: 3125617 bytes, checksum: 3e951b5c5e73d3862e1ed9b6e9e28451 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T14:26:36Z (GMT) No. of bitstreams: 1 paulohenriquediasdecarvalho.pdf: 3125617 bytes, checksum: 3e951b5c5e73d3862e1ed9b6e9e28451 (MD5) / Made available in DSpace on 2016-07-13T14:26:36Z (GMT). No. of bitstreams: 1 paulohenriquediasdecarvalho.pdf: 3125617 bytes, checksum: 3e951b5c5e73d3862e1ed9b6e9e28451 (MD5) Previous issue date: 2013-07-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A descoberta de protótipos naturais associada às metodologias de isolamento e identificação química de constituintes a partir de novas fontes botânicas, bem como da avaliação das atividades farmacológicas e toxicológicas dessas moléculas demonstram grandes perspectivas para o desenvolvimento racional de novos fármacos. Tendo em vista a alta incidência de doenças do fígado, no Brasil e no mundo, e que existem poucos medicamentos eficazes e capazes de reverter ou reduzir a progressão destas, o isolamento e a identificação de substâncias com potencial hepatoprotetor é, hoje, uma área promissora na busca de novas substâncias bioativas. Tradicionalmente, as raízes de Glycyrrhiza sp., conhecidas como licorice, são usadas na medicina alternativa com inúmeras finalidades, dentre elas hepatoprotetora. Entretanto, até o momento, não existem relatos desta atividade vinculada à licochalcona A, uma das substâncias majoritárias nas raízes de Glycyrrhiza inflata. No presente trabalho avaliaram-se as atividades da licochalcona A em ensaios de viabilidade celular das linhagens de fibroblásto (NIH/3T3) e carcinoma hepatocelular humano (HepG-2), adesão celular de HepG-2 e em modelo de lesão hepática induzida por ligação do ducto biliar (BDL) em ratos Wistar. Além disso, desenvolveu-se uma metodologia para determinação da licochalcona A em CLAE-UV, utilizando coluna C18, fase móvel em gradiente de água acidificada (0,1% H3PO4) e metanol (50:50 – 20:80 v/v), fluxo de 1,1 mL/min e comprimento de onda para detecção em 372 nm. A licochalcona A isolada a partir do extrato seco das raízes de G. inflata foi identificada por RMN 1H e 13C. O isolamento apresentou-se satisfatório, bem como a metodologia proposta para quantificação desta substância por CLAE-UV, que apresentou excelente linearidade, precisão e exatidão. Nos experimentos in vitro, a licochalcona A não demonstrou redução significativa na viabilidade das células da linhagem HepG-2 (IC50 > 200 μM) e da NIH/3T3 (IC50 > 100 μM), bem como no experimento de adesão das células HepG-2 (IC50 > 200 μM) (p>0,05). Estes dados corroboram com aqueles encontrados no experimento in vivo, no qual a licochalcona A (50 mg/Kg) também não apresentou toxicidade ao fígado, já que os resultados encontrados não foram significativamente diferentes aos do grupo controle (p>0,05). Contudo, ela também não demonstrou capacidade de promover ou reduzir os danos hepáticos causado pelo BDL, no tempo de tratamento do estudo realizado (48 h). / The natural prototypes discovery associated with methods of chemical constituents isolation and identification from new botanical sources, as well as the evaluation of pharmacological and toxicological activities of these molecules show great prospects for the new drugs rational development. In view of the high incidence of liver disease in Brazil and the world, and there are few effective drugs and able to reverse or slow the progression of these disease, the substances isolation and identification with potential hepatoprotective today is a promising area in search for new bioactive substances. Traditionally, the roots of Glycyrrhiza sp., known as licorice, are used in alternative medicine with numerous purposes, among them hepatoprotective. However, to date, there are no reports of this activity linked to licochalcona A, one majority of the substances in the roots of Glycyrrhiza inflata. In the present study evaluated the activities of licochalcone A in cell viability assays of strains fibroblast (NIH/3T3) and human hepatocellular carcinoma (HepG-2), cell adhesion HepG-2 and model of liver injury induced by bile duct ligation (BDL) in Wistar rats. In addition, we developed a methodology for determining the licochalcone A quantitative HPLC-UV, using C18 column and a mobile phase gradient of acidified water (0.1% H3PO4) and methanol (50:50 - 20:80 v/v), flow rate of 1.1 mL/min and detection wavelength at 372 nm. The licochalcone A isolated from the dried extract of the roots of G. inflata was identified by 1H and 13C NMR. The isolation had to be satisfactory, as well as the proposed methodology for quantification of this substance by HPLC-UV, which showed excellent linearity, reproducibility and accuracy. In in vitro experiments, licochalcone A showed no significant reduction in the viability of the cell line HepG-2 (IC50 > 200 μM) and NIH/3T3 (IC50 > 100 μM), as well as in cell adhesion HepG-2 experiments (IC50 > 200 μM) (p> 0.05). These data corroborate those found in the in vivo experiment, in which the licochalcone A (50 mg/kg) also showed no toxicity to the liver, since the results were not significantly different to the control group (p>0.05). Nevertheless, it has not shown the ability to promote or reduce liver damage caused by BDL, at the treatment time of the study (48 h).

肺癌是導致男性死亡的最常見原因以及是排在乳腺癌和結腸癌之後的導致女性死亡的第三大原因。雖然肺癌如此嚴重，但是如今治疗肺癌仍然是一个挑战。現今對肺癌的治療主要集中在化學治療和靶點藥物治療，但是由於這些治療有著很大的副作用和低治愈率，尋找其他的醫學替代方法十分迫切。甘草是其中最常用的中藥，它常常用作食品工業中的甜味劑。以往的研究表明，甘草具有多種的生物活性。但是甘草提取物對於肺癌的治療卻是十分匱乏的。 / 本論文主要目的是評價甘草提取物以及其中的有效成份對非小型肺癌細胞株A549 的影響，以及其作用的機理。我們的數據表明，甘草的乙酸乙酯（EAL）成份比甘草的乙醇提取物有著比較強的抑制癌細胞的作用。另外，對甘草的五個單體進行的測試中發現lico-3 是最具有抑制肺癌作用的。利用高效液相色譜法對甘草活性成份分析表明，lico-3 是EAL中的其中一個單體。 / 乳酸脫氫酶滲漏（LDH）的檢測結果以及异硫氰酸荧光素-碘化丙啶（FITC-PI）雙染的結果表明，EAL 能夠引起肺癌細胞的凋亡現象而非壞死現象。實驗結果表明由EAL引起的A549細胞凋亡是跟Bcl-2家族及Caspase家族有關係，同時EAL還能夠抑制Akt途徑從而導致細胞的死亡。 / 致肺癌細胞死亡的原因進行進一步研究表明，EAL還能夠引起抑制細胞週期的運作，停留在G2/M 時期。這可能是由於EAL引發了p53與p21的上調作用從而抑制了細胞的生長與增殖。 / 實驗結果說明了EAL引起的肺癌細胞株A549的凋亡作用是跟多重細胞通路有關， 同時表明了EAL是具有抗擊肺癌作用的潛能，能夠作為治療肺癌的藥物。 / Lung cancer is the most common cause of cancer death in men and third in women followed by breast cancer and colon cancer, yet treatment of lung cancer remains a challenge. Current treatments including chemotherapy and targeted drug treatment come with side-effects and low successful rate. Alternative medicine for treatment of lung cancer is warranted. Glycyrrhiza uralensis (Gan-Cao), commonly called “licorice, is one of the most commonly used herbs in traditional Chinese medicine (TCM). It is also used as flavoring and sweetening agents in many of food products. Previous studies have indicated that licorice exhibits a variety of biological activities. However, anticancer effects of licorice extract on lung cancer remain unclear. / In this study, we evaluated effects of licorice extract and its chemical components on human lung cancer cell line A549, and studied its mode of action. Our results showed the ethyl acetate fraction of licorice (EAL) was more effective in inhibition of A549 cell growth followed by ETL (IC₅₀: 50μg/mL). Moreover, among the five compounds tested, lico-3 was more potent compound. The HPLC analysis of the active fraction indicated that lico-3 was one of the compounds distributed in the EA fraction. / The results of LDH assay and FITC-PI co-staining method suggested low concentration of EAL can trigger apoptosis but not necrosis. The experimental findings show that EAL induce apoptosis in A549 cell lines involved in Bcl-2 family and caspase cascade. Also, EAL can arrest the Akt survival pathway in A549. Furthermore, the results indicate that EAL triggered G2/M phase arrest. The studies suggest EAL can up-regulate p53 and p21 to promote cell cycle arrest resulting in inhibition of proliferation. / Experimental results indicate that EAL is involved in multiple signal pathways to induce lung cancer cell death. The result suggests EAL is a potential candidate for lung cancer therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Yanling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-110). / Abstracts in Chinese. / Abstract --- p.III / 論文摘要 --- p.V / Acknowledgement --- p.VII / List of Contents --- p.VIII / List of Figures --- p.X / List of Tables --- p.XI / List of Abbreviations --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Lung cancer --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.2 / Chapter 1.1.3 --- Types of lung cancer --- p.4 / Chapter 1.1.4 --- Stages and treatment of lung cancer --- p.5 / Chapter 1.1.5 --- Chemotherapy for lung cancer treatment --- p.8 / Chapter 1.2 --- Traditional Chinese Medicines --- p.11 / Chapter 1.2.1 --- Overview --- p.11 / Chapter 1.2.2 --- Licorice --- p.14 / Chapter 1.2.3 --- Chemical study of licorice --- p.16 / Chapter 1.2.4 --- Pharmacological activities of licorice --- p.16 / Chapter 1.3 --- Molecular mechanism of apoptosis --- p.21 / Chapter 1.3.1 --- Overview --- p.21 / Chapter 1.3.2 --- Bcl2 family --- p.21 / Chapter 1.3.3 --- Caspase pathway --- p.23 / Chapter 1.3.4 --- Akt pathway --- p.24 / Chapter 1.3.5 --- p53 protein --- p.26 / Chapter 1.3.6 --- Apoptosis and cancer --- p.27 / Chapter 1.4 --- Cell cycle --- p.29 / Chapter 1.4.1 --- Overview --- p.29 / Chapter 1.4.2 --- Cell cycle and p53 --- p.29 / Chapter 1.4.3 --- Cell cycle and cancer --- p.30 / Chapter 1.5 --- Aims of study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Cell culture and treatment --- p.33 / Chapter 2.1.1 --- Cell line --- p.33 / Chapter 2.1.2 --- Chemicals and reagents --- p.34 / Chapter 2.1.3 --- Preparation of solutions --- p.34 / Chapter 2.2 --- Preparation of Licorice sample --- p.35 / Chapter 2.3 --- HPLC analysis --- p.35 / Chapter 2.3.1 --- Chemical and materials --- p.35 / Chapter 2.3.2 --- Instrumentation --- p.36 / Chapter 2.3.3 --- Preparation of Standard solutions --- p.36 / Chapter 2.3.4 --- Preparation of samples --- p.37 / Chapter 2.3.5 --- HPLC conditions --- p.37 / Chapter 2.3.6 --- Method validation --- p.37 / Chapter 2.4 --- Cell viable assay --- p.38 / Chapter 2.4.1 --- Samples preparation --- p.39 / Chapter 2.4.2 --- Procedure --- p.39 / Chapter 2.5 --- LDH assay --- p.40 / Chapter 2.5.1 --- Reagent preparation --- p.40 / Chapter 2.5.2 --- Procedure --- p.41 / Chapter 2.6 --- Annexin V assay --- p.41 / Chapter 2.6.1 --- Reagent --- p.42 / Chapter 2.6.2 --- Procedure --- p.42 / Chapter 2.7 --- Cell cycle study --- p.43 / Chapter 2.7.1 --- Chemicals and reagent --- p.43 / Chapter 2.7.2 --- Procedure --- p.44 / Chapter 2.8 --- Caspase3/7 Assay --- p.44 / Chapter 2.8.1 --- Reagent preparation --- p.45 / Chapter 2.8.2 --- Procedure --- p.46 / Chapter 2.9 --- Western blotting --- p.46 / Chapter 2.9.1 --- Reagent and antibodies --- p.46 / Chapter 2.9.2 --- Procedure --- p.50 / Chapter 2.9.3 --- Determination of protein concentration --- p.51 / Chapter 2.10 --- Data analysis --- p.51 / Chapter Chapter 3 --- Results --- p.52 / Chapter 3.1 --- Chromatographic conditions and HPLC identity conformation --- p.52 / Chapter 3.1.1 --- Linearity, limits of detection and quantification --- p.56 / Chapter 3.1.2 --- Reproducibility --- p.56 / Chapter 3.1.3 --- Analysis of ethyl acetate of licorice (EAL) using the validated method --- p.56 / Chapter 3.2 --- Licorice induces apoptosis in nonsmall cell lung carcinoma --- p.61 / Chapter 3.2.1 --- Cell viability assay --- p.61 / Chapter 3.2.2 --- LDH leakage assay --- p.71 / Chapter 3.2.3 --- Annexin V and PI staining --- p.73 / Chapter 3.3 --- Protein expression in EALinduced apoptotic cells --- p.75 / Chapter 3.3.1 --- Bcl2 family --- p.75 / Chapter 3.3.2 --- Activation of caspases by EAL treatment --- p.77 / Chapter 3.4 --- EAL could block Akt survival pathway --- p.79 / Chapter 3.5 --- EAL induces cell cycle arrest in nonsmall cell lung carcinoma --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Chemical analysis of licorice --- p.85 / Chapter 4.2 --- Licorice induced apoptosis but not necrosis on lung cancer cell A549 --- p.86 / Chapter 4.2.1 --- Licorice exhibits specific cytotoxicity to different cancer cells in vitro --- p.86 / Chapter 4.2.2 --- EAL induces cell death via apoptosis but not necrosis --- p.87 / Chapter 4.3 --- Growth inhibition by EAL inducing apoptosis --- p.89 / Chapter 4.3.1 --- EAL induces apoptotic cell death through modification of Bcl2 family --- p.89 / Chapter 4.3.2 --- EAL activate the caspase proteins --- p.90 / Chapter 4.4 --- Growth inhibition by EAL inducing survival pathway arrest --- p.92 / Chapter 4.5 --- Growth inhibition by EAL inducing cellcycle arrest --- p.94 / Chapter 4.6 --- General discussion --- p.96 / Reference --- p.99

A nanotecnologia é aplicada em praticamente todos os setores da ciência, incluindo a área cosmética. Nanoemulsões apresentam-se mais estáveis do que macroemulsões, possuem boa espalhabilidade e facilitam a penetração de ativos na pele. O óleo de babaçu (Orbignya oleifera) é utilizado no tratamento de várias afecções da pele, devido às propriedades anti-inflamatória, cicatrizante e antiséptica. O extrato de sementes de Areca catechu inibe a produção de elastase e colagenase, enzimas responsáveis pela flacidez e perda de elasticidade da pele no envelhecimento. Além disso, possui ação antioxidante, anti-hialuronidase, estimulante da proliferação de fibroblastos e inibidor da melanogênese. Outro extrato muito utilizado em cosméticos é o de raízes de Glycyrrhiza glabra que possui atividade antioxidante, anti-inflamatória, adstringente, bactericida e inibidor da tirosinase. Já o extrato de portulaca (Portulaca oleracea), possui propriedades anti-alérgica, anti-inflamatória, anti-irritante e cicatrizante. Nesta pesquisa, desenvolveu-se nanoemulsões contendo óleo de babaçu adicionadas ou não de extratos vegetais e avaliou-se a estabilidade físico-química e propriedades biológicas das mesmas, como potencial antioxidante, anti-irritante, anti-inflamatório, influência na hidratação, valor de pH e oleosidade cutânea. A ordem de adição dos componentes, temperatura, velocidade e tempo de agitação foram críticos na obtenção das nanoemulsões. No estudo de estabilidade acelerada, a 45°C, observou-se alteração significativa dos valores de diâmetro dos glóbulos, pH e condutividade elétrica no final de 120 dias, além da ocorrência simultânea dos processos de Ostwald ripening e coalescência nas duas nanoemulsões. Os extratos de areca, alcaçuz e a formulação aditivada apresentaram atividade antioxidante pelos métodos DPPH e xantina oxidase. Nos testes in vitro de irritação, as formulações foram classificadas como ligeiramente irritantes e não-irritantes pelos métodos HET-CAM e RBC, respectivamente. Por meio dos testes in vivo, observou-se que ambas as formulações apresentaram atividade anti-inflamatória, hidratante, aumento da oleosidade e nenhuma alteração no valor de pH cutâneo. Os resultados obtidos sugerem a segurança e eficácia do uso destas nanoemulsões após exposição solar. / Nanotechnology is applied in all science fields, including the cosmetic. Nanoemulsions are more stable than macroemulsions, have good spreadability and facilitate the active skin penetration. Babassu oil (Orbignya oleifera) is used to treat various skin disorders due to anti-inflammatory, healing and antiseptic properties. Areca catechu seed extract inhibits the collagenase and elastase production, enzymes responsible for sagging and loss of skin elasticity in aging. It also has antioxidant, anti-hyaluronidase, and melanogenesis inhibitor properties. Another extract often used in cosmetics is the Glycyrrhiza glabra roots extract. It has antioxidant, anti-inflammatory, adstringent, antibacterial and tyrosinase inhibitor activities. Yet the portulaca (Portulaca oleracea) extract is anti-allergic, anti-inflammatory, anti-irritant and healing. In this research, babassu nanoemulsions with or without extracts were developed and evaluated for physicochemical stability and biological properties as antioxidant, anti-irritant, anti-inflammatory, hydration, pH value and oily skin. The components addition order, temperature, stirring and time speed were critical in obtaining the nanoemulsions. In accelerated stability tests at 45°C, there was significant change in the droplets size, pH and electrical conductivity values at the end of 120 days. In addition, the Ostwald ripening and coalescence processes occurred simultaneous in nanoemulsions. The areca, licorice and the formulation containing this extracts showed antioxidant activity by DPPH and xanthine oxidase methods. In vitro tests of irritation, the formulations were classified as slightly irritating and non-irritating by HET-CAM and RBC methods, respectively. Both nanoemulsions showed anti-inflammatory, moisturizing characteristic, increased oiliness and none changing in the pH skin value. The results obtained suggest the safety and efficacy of these nanoemulsions after sun exposure.

A nanotecnologia é aplicada em praticamente todos os setores da ciência, incluindo a área cosmética. Nanoemulsões apresentam-se mais estáveis do que macroemulsões, possuem boa espalhabilidade e facilitam a penetração de ativos na pele. O óleo de babaçu (Orbignya oleifera) é utilizado no tratamento de várias afecções da pele, devido às propriedades anti-inflamatória, cicatrizante e antiséptica. O extrato de sementes de Areca catechu inibe a produção de elastase e colagenase, enzimas responsáveis pela flacidez e perda de elasticidade da pele no envelhecimento. Além disso, possui ação antioxidante, anti-hialuronidase, estimulante da proliferação de fibroblastos e inibidor da melanogênese. Outro extrato muito utilizado em cosméticos é o de raízes de Glycyrrhiza glabra que possui atividade antioxidante, anti-inflamatória, adstringente, bactericida e inibidor da tirosinase. Já o extrato de portulaca (Portulaca oleracea), possui propriedades anti-alérgica, anti-inflamatória, anti-irritante e cicatrizante. Nesta pesquisa, desenvolveu-se nanoemulsões contendo óleo de babaçu adicionadas ou não de extratos vegetais e avaliou-se a estabilidade físico-química e propriedades biológicas das mesmas, como potencial antioxidante, anti-irritante, anti-inflamatório, influência na hidratação, valor de pH e oleosidade cutânea. A ordem de adição dos componentes, temperatura, velocidade e tempo de agitação foram críticos na obtenção das nanoemulsões. No estudo de estabilidade acelerada, a 45°C, observou-se alteração significativa dos valores de diâmetro dos glóbulos, pH e condutividade elétrica no final de 120 dias, além da ocorrência simultânea dos processos de Ostwald ripening e coalescência nas duas nanoemulsões. Os extratos de areca, alcaçuz e a formulação aditivada apresentaram atividade antioxidante pelos métodos DPPH e xantina oxidase. Nos testes in vitro de irritação, as formulações foram classificadas como ligeiramente irritantes e não-irritantes pelos métodos HET-CAM e RBC, respectivamente. Por meio dos testes in vivo, observou-se que ambas as formulações apresentaram atividade anti-inflamatória, hidratante, aumento da oleosidade e nenhuma alteração no valor de pH cutâneo. Os resultados obtidos sugerem a segurança e eficácia do uso destas nanoemulsões após exposição solar. / Nanotechnology is applied in all science fields, including the cosmetic. Nanoemulsions are more stable than macroemulsions, have good spreadability and facilitate the active skin penetration. Babassu oil (Orbignya oleifera) is used to treat various skin disorders due to anti-inflammatory, healing and antiseptic properties. Areca catechu seed extract inhibits the collagenase and elastase production, enzymes responsible for sagging and loss of skin elasticity in aging. It also has antioxidant, anti-hyaluronidase, and melanogenesis inhibitor properties. Another extract often used in cosmetics is the Glycyrrhiza glabra roots extract. It has antioxidant, anti-inflammatory, adstringent, antibacterial and tyrosinase inhibitor activities. Yet the portulaca (Portulaca oleracea) extract is anti-allergic, anti-inflammatory, anti-irritant and healing. In this research, babassu nanoemulsions with or without extracts were developed and evaluated for physicochemical stability and biological properties as antioxidant, anti-irritant, anti-inflammatory, hydration, pH value and oily skin. The components addition order, temperature, stirring and time speed were critical in obtaining the nanoemulsions. In accelerated stability tests at 45°C, there was significant change in the droplets size, pH and electrical conductivity values at the end of 120 days. In addition, the Ostwald ripening and coalescence processes occurred simultaneous in nanoemulsions. The areca, licorice and the formulation containing this extracts showed antioxidant activity by DPPH and xanthine oxidase methods. In vitro tests of irritation, the formulations were classified as slightly irritating and non-irritating by HET-CAM and RBC methods, respectively. Both nanoemulsions showed anti-inflammatory, moisturizing characteristic, increased oiliness and none changing in the pH skin value. The results obtained suggest the safety and efficacy of these nanoemulsions after sun exposure.

Tsang Yuen-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 200-218). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.vii / CHINESE ABSTRACT --- p.xi / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.4 / Chapter 1.2 --- Leukemia --- p.5 / Chapter 1.2.1 --- Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification of Leukemia --- p.7 / Chapter 1.2.3 --- Etiology and Symptoms of Leukemia --- p.9 / Chapter 1.2.4 --- Therapeutic Strategies for Leukemia --- p.10 / Chapter 1.2.4.1 --- Conventional Therapies --- p.10 / Chapter 1.2.4.2 --- Differentiation Therapy and Induction of Apoptosis in Leukemia --- p.11 / Chapter 1.2.5 --- Regulation of Apoptosis and Cell Cycle Progression --- p.12 / Chapter 1.2.5.1 --- Apoptosis --- p.12 / Chapter 1.2.5.2 --- Cell Cycle --- p.13 / Chapter 1.2.5.3 --- Disregulation of Apoptosis and Cell Cycle Contribute to the Development of Leukemia --- p.14 / Chapter 1.3 --- Licorice --- p.16 / Chapter 1.3.1 --- Chemistry of Licorice --- p.16 / Chapter 1.3.2 --- Pharmacological Activities of Glycyrrhizin and 18-β Glycyrrhetinic Acid --- p.22 / Chapter 1.3.2.1 --- Mineralocorticoid Activity --- p.22 / Chapter 1.3.2.2 --- Anti-inflammatory Effect --- p.23 / Chapter 1.3.2.3 --- Anti-allergic Effect --- p.24 / Chapter 1.3.2.4 --- Enhancement of Immune Response --- p.24 / Chapter 1.3.2.5 --- Anti-hepatotoxic Effects --- p.26 / Chapter 1.3.2.6 --- Anti-viral Activity --- p.27 / Chapter 1.3.2.7 --- Anti-carcinogenic and Anti-tumor Effects --- p.28 / Chapter 1.3.3 --- Other Biological Activities of Licorice --- p.30 / Chapter 1.3.4 --- A 96-kDa Glycyrrhizin-Binding Protein (gb96) --- p.31 / Chapter 1.3.5 --- Evaluation of Health Hazard --- p.32 / Chapter 1.4 --- Aims and Scopes of This Research --- p.34 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Animals --- p.37 / Chapter 2.1.2 --- Cell Lines --- p.37 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Reagents" --- p.39 / Chapter 2.1.4 --- Recombinant Cytokines --- p.42 / Chapter 2.1.5 --- [methyl-3H] Thymidine (3H-TdR) --- p.43 / Chapter 2.1.6 --- Liquid Scintillation Cocktail --- p.44 / Chapter 2.1.7 --- Reagents and Buffers for Flow Cytometry --- p.44 / Chapter 2.1.8 --- Monoclonal Antibodies --- p.45 / Chapter 2.1.9 --- Reagents for DNA Extraction --- p.47 / Chapter 2.1.10 --- Reagents for Total RNA Isolation --- p.48 / Chapter 2.1.11 --- Reagents and Buffers for RT-PCR Study --- p.49 / Chapter 2.1.12 --- Reagents and Buffers for Gel Electrophoresis --- p.55 / Chapter 2.1.13 --- Reagents and Buffers for Western Blot Analysis --- p.56 / Chapter 2.2 --- Methods --- p.63 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.63 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Primary Mouse Cells" --- p.63 / Chapter 2.2.3 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.64 / Chapter 2.2.4 --- Determination of Cell Viability --- p.65 / Chapter 2.2.5 --- Cell Morphology Study --- p.66 / Chapter 2.2.6 --- Apoptosis Study by DNA Fragmentation --- p.66 / Chapter 2.2.7 --- Flow Cytometric Analysis --- p.67 / Chapter 2.2.8 --- Cell Cycle/DNA Content Evaluation --- p.68 / Chapter 2.2.9 --- Gene Expression Study --- p.69 / Chapter 2.2.10 --- Protein Expression Study --- p.72 / Chapter 2.2.11 --- Statistical Analysis --- p.75 / Chapter CHAPTER 3: --- THE ANTI-TUMOR EFFECTS OF GLYCYRRHIZIN AND 18-β GLYCYRRHETINIC ACID ON VARIOUS LEUKEMIC CELL LINES / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Results --- p.78 / Chapter 3.2.1 --- The Growth Inhibitory Effects of Glycyrrhizin on Various Leukemic Cell Lines --- p.78 / Chapter 3.2.1.1 --- Differential Anti-proliferative Effects of Glycyrrhizin on Various Leukemic Cell Lines In Vitro --- p.78 / Chapter 3.2.1.2 --- Effects of Glycyrrhizin on the Viability of Various Leukemic Cell Lines and Normal Hematopoietic Cells In Vitro --- p.89 / Chapter 3.2.1.3 --- Induction of DNA Fragmentation in Leukemia Cells by Glycyrrhizin --- p.94 / Chapter 3.2.1.4 --- Effect of Glycyrrhizin on the Cell Cycle Kinetics of HL-60 Cells In Vitro --- p.97 / Chapter 3.2.1.5 --- Effect of Glycyrrhizin on the Cell Cycle Kinetics of JCS Cells In Vitro --- p.100 / Chapter 3.2.1.6 --- Effect of Glycyrrhizin on the In Vivo Tumorigenicity of the Murine Myeloid Leukemia JCS Cells --- p.103 / Chapter 3.2.2 --- The Growth Inhibitory Effects of 18-β Glycyrrhetinic Acid on Various Leukemic Cells Lines --- p.105 / Chapter 3.2.2.1 --- Differential Anti-proliferative Effect of 18-β Glycyrrhetinic Acid on Various Leukemic Cell Lines In Vitro --- p.105 / Chapter 3.2.2.2 --- Effects of 18-β Glycyrrhetinic Acid on the Viability of Various Leukemic Cell Lines and Normal Hematopoietic Cells In Vitro --- p.115 / Chapter 3.2.2.3 --- Induction of DNA Fragmentation in Leukemia Cells by 18-β Glycyrrhetinic Acid --- p.120 / Chapter 3.2.2.4 --- Effect of 18-β Glycyrrhetinic Acid on the Cell Cycle Kinetics of HL-60 Cells In Vitro --- p.123 / Chapter 3.2.2.5 --- Effect of 18-β Glycyrrhetinic Acid on the Cell Cycle Kinetics of JCS Cells In Vitro --- p.126 / Chapter 3.2.2.6 --- Effect of 18-β Glycyrrhetinic acid on the In Vivo Tumorigenicity of the Murine Myeloid Leukemia JCS Cells --- p.129 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- THE DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18-β GLYCYRRHETINIC ACID ON MURINE MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Results --- p.138 / Chapter 4.2.1 --- Morphological Changes in Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.138 / Chapter 4.2.2 --- Surface Antigen Immunophenotyping of Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.141 / Chapter 4.2.3 --- Endocytic Activity of Glycyrrhizin or 18-β Glycyrrhetinic Acid-treated JCS Cells --- p.155 / Chapter 4.3 --- Discussion --- p.158 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI LEUKEMIC ACTIVITIES OF GLYCYRRHIZIN AND 18-P GLYCYRRHETINIC ACID / Chapter 5.1 --- Introduction --- p.161 / Chapter 5.2 --- Results --- p.164 / Chapter 5.2.1 --- Combining Effect of Glycyrrhizin or 18-β Glycyrrhetinic Acid with Hematopoietic Cytokines in Modulating the Proliferation of the Murine Myeloid Leukemia JCS Cells --- p.164 / Chapter 5.2.1.1 --- Combining Effect of Glycyrrhizin and IL-lα on the Proliferation of JCS Cells --- p.164 / Chapter 5.2.1.2 --- Combining Effects of Glycyrrhizin with IFN-γ or TNF-α on the Proliferation of JCS Cells --- p.166 / Chapter 5.2.1.3 --- Combining Effect of 18-β Glycyrrhetinic Acid and IL-lα on the Proliferation of JCS Cells --- p.169 / Chapter 5.2.1.4 --- Combining Effects of 18-β Glycyrrhetinic Acid with IFN-γ or TNF-α on the Proliferation of JCS Cells --- p.169 / Chapter 5.2.2 --- Elucidation of the Molecular Mechanisms of Glycyrrhizin or 18-β Glycyrrhetinic Acid on Leukemic Cell Differentiation and Growth Arrest --- p.173 / Chapter 5.2.2.1 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of Cytokine Genes in the Leukemia JCS Cells --- p.173 / Chapter 5.2.2.2 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of PKC Isoforms in the Leukemia JCS Cells --- p.176 / Chapter 5.2.2.3 --- Modulatory Effects of Glycyrrhizin and 18-β Glycyrrhetinic Acid on the Expression of Growth- regulatory Genes in the Leukemia JCS Cells --- p.180 / Chapter 5.2.2.4 --- Modulatory Effects of 18-β Glycyrrhetinic Acid on the Expression of Apoptosis-related Genes in the Leukemia JCS Cells --- p.183 / Chapter 5.2.2.5 --- Modulatory Effects of 18-β Glycyrrhetinic Acid on the Expression of Growth-regulatory and Apoptosis-related Proteins in JCS Cells --- p.185 / Chapter 5.3 --- Discussion --- p.187 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.194 / REFERENCES --- p.200

Lee Kai Woo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Thesis Committee --- p.iii / Abstracts --- p.v / 論文槪要 --- p.vii / List of figures --- p.viii / List of abbreviations --- p.ix / Chapter Chapter one --- Introduction --- p.1 / Chapter 1.1 --- Drug metabolism and UGTs --- p.1 / Chapter 1.2 --- Natural substrates of UGTs --- p.4 / Chapter 1.3 --- Functions of UGT isoforms: roles of UGT polymorphisms --- p.6 / Chapter 1.4 --- Evolution of the UGT1 gene locus in vertebrates --- p.8 / Chapter 1.5 --- Multiple Variable First Exons: A Mechanism for Cell- and Tissue-Specific Gene regulation --- p.13 / Chapter 1.6 --- Evolutionary Origin of the Variable and Constant Genomic Organization --- p.14 / Chapter 1.7 --- Variable and Constant Genomic Organizations Exist in Mammalian UGTs --- p.20 / Chapter 1.8 --- The history of recombinant UGT expression --- p.20 / Chapter 1.9 --- UGT1A8 --- p.21 / Chapter 1.10 --- Licorice and its active component --- p.24 / Chapter 1.11 --- Enzyme induction in the liver --- p.25 / Chapter 1 12 --- Objectives --- p.28 / Chapter Chapter two --- Methods and Materials --- p.29 / Chapter 2.1 --- UGT1A8 induction studies --- p.30 / Chapter 2.1.1 --- Drug preparation --- p.30 / Chapter 2.1.2 --- Cell viability study with Neutral Red Assay Rat treatment --- p.30 / Chapter 2.1.3 --- Cell treatment --- p.31 / Chapter 2.1.4 --- Rat treatment --- p.31 / Chapter 2.1.5 --- RNA extraction from rat liver and cell culture --- p.31 / Chapter 2.1.6 --- Quantization of RNA --- p.32 / Chapter 2.1.7 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.1.8 --- Northern hybridization --- p.33 / Chapter 2.1.9 --- Probe for Northern Blotting --- p.34 / Chapter 2.1.10 --- Agarose Gel analysis and Northern Blot analysis --- p.34 / Chapter 2.2 --- Recombinant expression of UGT1A8 in E.coli JM109 --- p.35 / Chapter 2.2.1 --- cDNA synthesis --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction --- p.35 / Chapter 2.2.3 --- Agarose gel electrophoresis for DNA --- p.35 / Chapter 2.2.4 --- "Amplification of target gene, UGT1A8" --- p.36 / Chapter 2.2.5 --- Restriction enzyme digestion of plasmid and insert --- p.36 / Chapter 2.2.6 --- Ligation of plasmid and insert DNA --- p.37 / Chapter 2.2.7 --- Amplification of target plasmid --- p.37 / Chapter 2.2.8 --- Screening of target plasmid --- p.37 / Chapter 2.2.9 --- DNA sequencing --- p.38 / Chapter 2.2.10 --- Transformation of protein expression host --- p.38 / Chapter 2.2.11 --- Confirmation of transformation of protein expression host --- p.38 / Chapter 2.2.12 --- Protein expression --- p.39 / Chapter 2.2.13 --- Protein purification --- p.39 / Chapter 2.2.14 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.15 --- Confirmation of the protein --- p.40 / Chapter 2.3 --- Characterization of recombinant UGT1A8 --- p.41 / Chapter 2.3.1 --- UGT assay --- p.41 / Chapter 2.4 --- Routine experiment methods --- p.41 / Chapter 2.4.1 --- Determination of protein --- p.41 / Chapter 2.4.2 --- Nucleic acid purification --- p.42 / Chapter 2.4.3 --- Preparation of chemically competent bacterial cells --- p.42 / Chapter 2.4.4 --- Colony PCR --- p.43 / Chapter 2.4.5 --- Plasmid rescue by alkaline lysis --- p.44 / Chapter 2.4.6 --- Charging of His-tagged column --- p.44 / Chapter 2.4.7 --- Washing of His-tagged column --- p.45 / Chapter Chapter three --- Results --- p.46 / Chapter 3.1 --- UGT1A8 Expression in clone9 and H4IIE after treatment with licorice and 18 β glycyrrhentinic acid --- p.46 / Chapter 3.2 --- UGT1A8 induction in wistar and j/j rats after treatment --- p.63 / Chapter 3.3 --- Construction of pRset-UGT 1A8 Vector --- p.70 / Chapter 3.4 --- Purification of recombinant UGT1A8 --- p.75 / Chapter 3.5 --- Screening of substrate of the purified enzyme --- p.77 / Chapter Chapter four --- Discussion --- p.78 / Chapter 4.1 --- Effects of licorice and 18βglycyrrhetinic acid in the induction of UGT1A8 in different cell lines --- p.78 / Chapter 4.2 --- Comparison of wistar and j/j rats in the induction of UGT1A8 --- p.79 / Chapter 4.3 --- Comparison of licorice and 18(3 glycyrrhetinic acid in the induction of UGT1A8 in rats --- p.81 / Chapter 4.4 --- Comparison of in vivo and in vitro of drug treatment --- p.81 / Chapter 4.5 --- Expression of UGT1A7 after drug treatment in vitro --- p.82 / Chapter 4.6 --- Protein expression and purification --- p.83 / Chapter 4.7 --- Substrates of UGT1A8 --- p.83 / Chapter Chapter Five --- Conclusions --- p.86 / References --- p.90 / Appendix --- p.105

Made available in DSpace on 2014-06-11T19:27:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-14Bitstream added on 2014-06-13T19:56:08Z : No. of bitstreams: 1 oliveira_jr_me_sjc.pdf: 1011850 bytes, checksum: 0914206b09a47699d72d4dc7cfb6450f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A proposta deste estudo foi avaliar a atividade antimicrobiana e citotoxicidade dos extratos vegetais de Equisetum arvense L. (cavalinha), Glycyrrhiza glabra L. (alcaçuz), Punica granatum L. (romã) e Stryphnodendron barbatimam Mart. (barbatimão). A atividade antimicrobiana dos extratos vegetais foi analisada em cepas de Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Candida albicans, Candida glabrata e Candida tropicalis, sendo uma ATCC e nove clínicas, através do teste de microdiluição em caldo, contudo para se determinar a Concentração Microbicida Mínima (CMM) dos extratos foram feitas semeaduras do conteúdo dos poços das placas de microdiluição. A citotoxicidade das CMM dos extratos naturais foi verificada pelo método colorimétrico MTT e pela quantificação da produção de citocinas IL-1β e TNF-α pelo teste ELISA, para tanto, foram utilizadas culturas celulares de macrófagos de camundongos (RAW 264.7). Os resultados foram analisados estatisticamente pelo teste ANOVA complementado por Tukey, com significância de 5%. Para o extrato de cavalinha, romã e barbatimão foi determinada como CMM efetiva para todas as cepas a concentração de 50 mg/mL e para o extrato de alcaçuz 100 mg/mL. Quanto à citotoxicidade destas concentrações, pelo método MTT, foi observada redução significativa da viabilidade celular (p≤0,05), em relação ao grupo controle, para 48% no grupo tratado com cavalinha, 79% para alcaçuz, 76% para romã e 86% para barbatimão. Em relação à quantificação de IL-1β, os grupos tratados com extratos de alcaçuz e cavalinha exibiram semelhanças ao grupo controle (p≥0,05) com produção de 1,99 e 1,32 pg/mL respectivamente, contudo o grupo tratado com extrato de barbatimão produziu 3,98 pg/mL (p<0,05) e o grupo que recebeu tratamento com extrato de romã a produção foi de 7,72 pg/mL (p<0,01), diferindo... / The objective of this study was to verify the antimicrobial activity and cytotoxicity of Equisetum arvense L. (horsetail), Glycyrrhiza glabra L. (licorice), Punica granatum L. (pomegranate) and Stryphnodendron barbatimam Mart. (barbatimam) glycolic extracts. The antimicrobial activity of the extracts was analyzed in Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Candida albicans, Candida glabrata and Candida tropicalis strains, being one ATCC and nine clinics through the method of microdilution in broth according to NCCLS. It was realized sowing of the contents from the well of microdilution plate, after incubation, for determination of MMC of the natural extracts. The cytotoxicity of the extracts was evaluated for MTT assay, having as an experimental model cultivation of mice macrophages (RAW 264.7), and through the ELISA test, for cytokyne quantification IL-1β and TNF-α produced by the cellular cultivation after a period of 24 hours exposed to the extracts MMC. The results were subordinated to statistical analisys (ANOVA and Turkey test), with p≤0,05. Related to the antimicrobial activity, the natural extracts of horsetail, pomegranate and barbatimam eliminated all the strains to the concentration of 50 mg/mL and the natural extract of licorice promoted the eliimination of 100 mg/mL. Regarding to the cellular viability of the active MMC for the 100% of strains, it was observed meaningful reduction (p<0,01) in all the natural extracts compared to the control group, and, as a result, horsetail exhibited an average of 48%; licorice, 79%; pomegranate, 76%; e barbatimam, 86%. As the production of cytokyne the pomegranate extract presented production of IL-1β (7,72 pg/mL) significantly superior to the other groups, including the control (p<0,05). The barbatimam extract exhibited an intermediate average value of IL-1β (3,98 pg/mL), being statistically... (Complete abstract click electronic access below)

Introduction: Dark spots appearing on the skin caused by hyperpigmentation results from the action of tyrosinase, an enzyme whose activity leads to the production of the skin pigment melanin. Extracts of the plant Glycyrrhiza glabra, also known as liquorice, are commonly used to treat a range of conditions including skin hyperpigmentation. This study aimed at isolating and identifying compounds in extracts from South African liquorice root and assaying these compounds as to their antioxidant activity, their ability to inhibit the tyrosinase enzyme and their level of cytotoxicity. Methods: The ability of plant extracts to scavenge free radicals was tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS) and the ferric ion reducing power (FRAP) tests. The polyphenolic content of extract fractions was determined and extract compounds were identified using UHPLC-QToF-20 MS. In vitro anti-tyrosinase activity was also investigated as well as cytotoxicity in HepG2 liver and SK-MEL-1 melanoma cells using the MTT cell viability assay. Results: Of the four fractions prepared from the 70% methanolic extract of liquorice root, fraction 3 (F3) showed increased polyphenolic content and antioxidant properties with IC50 of 56.1 ± 6.32, 39.14 ± 1.1 and 66.34 ± 1.4 μg/ml against DPPH, ABTS and FRAP, respectively. The anti-tyrosinase activity of this fraction showed an IC50 of 358.54 μg/ml compared to Kojic acid (0.75 mM) used as the control. In addition, this fraction showed reduced liver toxicity as a higher percentage cell viability was noted in the HepG2 cells compared to the SK-MEL-1 skin melanoma cells. However, both cell types showed higher percentage viability compared to acetaminophen that was used as cytotoxic control. The LC-MS analysis revealed the presence of a wide variety of compounds including 4-azido-3-benzyl-coumarin, ferulic acid, glycyrrhizin, quercitrin, cirsilineol, gentioflavine and 4'',6,7-trihydroxyisoflavone. The literature indicates the use of these compounds regarding antioxidant and anti-tyrosinase activity. Significantly, cularidine was identified in this study, a compound not previously reported in studies involving liquorice root. Conclusion: The results from this study concur with previous reports as to the anti-tyrosinase and antioxidant activities associated with liquorice roots, activities perhaps due to the relatively high polyphenolic content in extracts from South African liquorice root. / Life and Consumer Sciences / M. Sc. (Life Sciences)