No description available.

Three unusual amino acids, y-amino-n-butyric acid, pipecolic acid, and 4-hydroxypipecolic acid, and an uncommon dipeptide, y-glutamyltyrosine, have been isolated and characterized from the seeds of members of the genus Sophora. Structural proof of these compounds was carried out by paper chromatography, thin-layer chromatography, column chromatography on amino acid analyzer, infrared, nuclear magnetic resonance, mass spectrometry, and C, H, N analysis. The presence and absence of these compounds was used as a criterion for the classification of 23 species of the genus Sophora. A phylogenetic classification which seems to follow the morphological taxonomy of this genus was carried out on the basis of seeds that contained pipecolic acid, those which did not contain pipecolic acid, and plants which contained both pipecolic acid and 4-hydroxypipecolic acids. Another chemical classification was also introduced based on the presence and absence of y-amino-n-butyric acid and y-glutamyltyrosine.

by Tsang Kit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 179-188). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ABSTRACT IN CHINESE (摘要） --- p.iii / ACKNOWLEDGEMENTS --- p.v / CONTENTS --- p.vi / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiv / ABBREVIATIONS --- p.xvi / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.2 / Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.2 / Chapter 1.1.2 --- Therapies for Hepatocellular Carcinoma --- p.4 / Chapter 1.2 --- Multidrug Resistance of Tumor Cells --- p.8 / Chapter 1.3 --- Therapeutic Potential of Traditional Chinese Medicine on Human Hepatoma --- p.10 / Chapter 1.4 --- Sophora flavescens Ait --- p.13 / Chapter 1.5 --- Biological Activities of Sophorae Radix --- p.15 / Chapter 1.5.1 --- Antitumor Activities --- p.16 / Chapter 1.5.2 --- "Antibacterial, Antimalarial and Antiviral Activities" --- p.17 / Chapter 1.6 --- Objectives and Significance of Study --- p.19 / Chapter 1.6.1 --- Bioassay-guided Isolation of Active Compounds from Sophora flavescens --- p.19 / Chapter 1.6.2 --- Action Mechanisms of the Bioactive Compounds Isolated from Sophora flavescens --- p.20 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.21 / Chapter 2.1 --- Cell Culture --- p.22 / Chapter 2.1.1 --- Cell Lines --- p.22 / Chapter 2.1.2 --- Cell Culture Media --- p.24 / Chapter 2.2 --- Isolation of Bioactive Compounds from Sophora flavescens --- p.25 / Chapter 2.3 --- MTT assay --- p.27 / Chapter 2.4 --- Cell Cycle Analysis --- p.28 / Chapter 2.5 --- Detection of Phosphatidylserine Externalization with Annexin V-FITC and PI --- p.29 / Chapter 2.6 --- DNA Fragmentation Assay --- p.30 / Chapter 2.7 --- Western Blot Analysis --- p.32 / Chapter 2.7.1 --- Extraction of Total Cellular Protein --- p.32 / Chapter 2.7.2 --- Extraction of Cytosolic Protein --- p.32 / Chapter 2.7.3 --- Determination of Protein Concentration --- p.33 / Chapter 2.7.4 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.35 / Chapter 2.7.5 --- Electroblotting of Protein --- p.36 / Chapter 2.7.6 --- Probing of Proteins with Antibodies --- p.37 / Chapter 2.7.7 --- Enhanced Chemiluminescence (ECL) Assay --- p.39 / Chapter 2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent dye --- p.39 / Chapter 2.9 --- cDNA Microarray Analysis --- p.40 / Chapter 2.9.1 --- Isolation of Total RNA --- p.40 / Chapter 2.9.2 --- Microarray Hybridization and Analysis --- p.41 / Chapter 2.9.3 --- Validation of Candidate Genes --- p.44 / Chapter 2.9.3.1 --- Determination of RNA Concentration --- p.44 / Chapter 2.9.3.2 --- First-Strand cDNA Synthesis --- p.44 / Chapter 2.9.3.3 --- Reverse-Transcription Polymerase Chain Reaction (RT-PCR) of Candidate Genes --- p.45 / Chapter 2.10 --- Two-Dimensional Polyacrylamide Gel Electrophoretic Analysis (2D-PAGE) --- p.47 / Chapter 2.10.1 --- Extraction of Total Cellular Protein for 2-D Gel Electrophoresis --- p.47 / Chapter 2.10.2 --- Determination of Protein Concentration --- p.47 / Chapter 2.10.3 --- First-Dimension Isoelectric Focusing (IEF) --- p.49 / Chapter 2.10.4 --- Second-Dimension SDS-PAGE --- p.49 / Chapter 2.10.5 --- Visualization of 2-D Gel by Silver Staining --- p.50 / Chapter 2.10.6 --- Identification of Differentially Expressed Proteins with Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) --- p.51 / Chapter 2.11 --- Statistical Analysis --- p.53 / Chapter CHAPTER THREE: --- BIOASSAY-GUIDED ISOLATION AND CHARACTERISATION OF BIOACTIVE COMPOUNDS FROM SOPHORA FLAVESCENS --- p.54 / Chapter 3.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.55 / Chapter 3.2 --- Structure Identification of the Bioactive Compounds Isolated from Sophora flavescens --- p.64 / Chapter 3.3 --- In Vitro Anti-tumor Effect of the Bioactive Compounds Isolated from Sophora flavescens --- p.71 / Chapter CHAPTER FOUR: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G IN THE INDUCTION OF APOPTOSIS IN HEPATOCELLULAR CARCINOMA CELLS --- p.76 / Chapter 4.1 --- In Vitro Anti-tumor Effect of Sophoraflavanone G --- p.77 / Chapter 4.2 --- Cell Cycle Analysis of Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.81 / Chapter 4.3 --- Induction of Apoptosis in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.88 / Chapter 4.3.1 --- Induction of Phosphatidylserine Externalization in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.89 / Chapter 4.3.2 --- Induction of DNA Fragmentation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.94 / Chapter 4.3.3 --- Induction of Caspase-3 activation in Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.97 / Chapter 4.4 --- Underlying Mechanisms of Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.102 / Chapter 4.4.1 --- Involvement of Death Receptor Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.103 / Chapter 4.4.2 --- Involvement of Bid protein in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.105 / Chapter 4.4.3 --- Involvement of Mitochondrial Pathway in Sophoraflavanone G- induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.108 / Chapter 4.4.4 --- Induction of Mitochondrial Membrane Depolarization in Human Hepatocellular Carcinoma Cells by Sophoraflavanone G --- p.112 / Chapter 4.4.5 --- Involvement of Caspase-independent Pathway in Sophoraflavanone G-induced Apoptosis in Human Hepatocellular Carcinoma Cells --- p.116 / Chapter CHAPTER FIVE: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HUMAN HEPATOCELLULAR CARCINOMA CELLS BY USING cDNA MICROARRAY ANALYSIS --- p.119 / Chapter 5.1 --- Identification of Differentially Expressed Genes in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by cDNA Microarray Analyasis --- p.120 / Chapter CHAPTER SIX: --- MECHANISTIC STUDY OF SOPHORAFLAVANONE G ON HEPATOCELLULAR CARCINOMA CELLS BY USING TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS --- p.136 / Chapter 6.1 --- Identification of Differentially Expressed Proteins in Sophoraflavanone G- treated Human Hepatocellular Carcinoma Cells by Two-Dimensional Polyacrylamide Gel Electrophoresis --- p.137 / Chapter CHAPTER SEVEN: --- DISCUSSION --- p.150 / Chapter 7.1 --- Bioassay-guided Isolation of Bioactive Compounds from Sophora flavescens --- p.151 / Chapter 7.2 --- Induction of Apoptosis in Human Hepatocellular Carcinoma cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.154 / Chapter 7.3 --- Differential Gene Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells --- p.161 / Chapter 7.4 --- Differential Protein Expression Induced by Sophoraflavanone G in Human Hepatocellular Carcinoma Cells and Multidrug Human Hepatocellular Carcinoma Cells --- p.164 / Chapter 7.5 --- Toxicity of Sophoraflavanone G against Normal Liver Cells --- p.170 / Chapter CHAPTER EIGHT: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.173 / Chapter 8.1 --- Conclusion --- p.174 / Chapter 8.2 --- Future Prospects --- p.176 / REFERENCES --- p.179

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biologia Vegetal. / Made available in DSpace on 2012-10-20T23:27:21Z (GMT). No. of bitstreams: 1 197538.pdf: 4940827 bytes, checksum: 89d93569b1e2049d3ee18bd316698a8e (MD5) / O gênero Sophora é composto por ervas perenes, arbustos e árvores, com aproximadamente 45-50 espécies, largamente distribuídas, principalmente na Eurásia e América do Norte. Sophora tomentosa ocorre no litoral de todas as regiões tropicais do mundo. No Brasil, pode-se encontrála do Nordeste ao Sul. É considerada espécie típica de dunas móveis e semi-fixas. Possui inflorescências com flores amarelas e sementes com dispersão autocórica e hidrocórica. Este trabalho objetiva estudar a ecologia reprodutiva de S. tomentosa e foi realizado na restinga da Praia da Joaquina na Ilha de Santa Catarina, Florianópolis, SC. S. tomentosa possui uma floração longa, ocorrendo entre os meses de outubro e maio, com um pico em outubro e novembro e outro menor em março. A frutificação começa logo após o início do período de floração e se estende até o mês de agosto, com um pico nos meses de outubro, novembro e dezembro e outro menor em março e abril. A antese é diurna, não havendo um horário definido para a abertura da flor. Cada inflorescência abre de 2 a 5 flores novas por dia, durando de 4 a 5 dias. Das espécies de abelhas que visitam as flores de S. tomentosa, Pseudocentron sp. (Megachilidae) apresenta características de um polinizador eficiente. As abelhas Xylocopa (Megaxylocopa) brasilionorum

A herbal formula (SL) comprising edible Sophorae Flos and Lonicerae Japonicae Flos was used to treat melanoma in ancient China. In current Chinese medicine practice, the two ingredient herbs of SL are commonly prescribed by Traditional Chinese medicine (TCM) doctors for treating melanoma. However, there is no modern clinical or experimental evidence about the anti-melanoma actions of this formula. Signal transducer and activator of the transcription (STAT3), which is constitutively activated in melanoma, has been proposed as one of the anti-melanoma targets. Some natural compounds in SL have been shown to assault cancers including melanoma via inhibiting STAT3 signaling. In this study, we investigated the anti-melanoma effects and explored STAT3 signaling-related mechanism of action of SL. We also identified bioactive components responsible for SL's anti-melanoma effects. Our in vitro and in vivo studies showed that SLE, an ethanolic extract of SL, induced apoptosis, inhibited proliferation, migration and invasion in melanoma cells, inhibited melanoma growth, angiogenesis and prolonged host survival in melanoma-bearing mice. SLE significantly suppressed the activation of STAT3 and its upstream kinase Src in both mouse melanoma tissues and cultured melanoma cells. In melanoma cells, we also found that SLE restrained STAT3 nuclear localization and inhibited the expression of STAT3-regulated genes related to melanoma growth, metastasis and angiogenesis. Overactivation of STAT3 in A375 human melanoma cells diminished the anti-proliferative, pro-apoptotic and anti-invasive effects of SLE. RNA-seq and small RNA sequencing analyses showed that SLE altered both the gene expression profile and miRNA signature in B16F10 melanoma tissues. Based on the RNA-seq data, we further validated that SLE inhibited the IL-17-IL-6-STAT3 axis in melanoma. Verification assays for the candidate miRNAs suggested that the significantly upregulated miR-205-5p is a possible target of SLE. Enforced miR-205 expression has been shown to suppress EMT in melanoma cells. In this study, we demonstrated that SLE inhibited melanoma cell EMT, and miR-205-5p knockdown diminished this effect of SLE. In addition, we computationally demonstrated that luteolin, a naturally occurring edible flavone abundant in Lonicerae Japonicae Flos, could directly bind to Src kinase domain. Experimentally, we verified that luteolin inhibited the Src/STAT3 signaling in both melanoma cells and tissues. In addition to inhibit STAT3 activation, luteolin promoted ubiquitin-proteasome pathway-mediated degradation of STAT3. Luteolin also exerted evident in vitro and in vivo anti-melanoma effects, and overactivation of STAT3 diminished its anti-melanoma effects. In conclusion, we demonstrated that SLE exerted in vivo and in vitro anti-melanoma effects, and inhibition of Src/STAT3 signaling and elevation of miR-205-5p expression contributed to these effects. Luteolin was identified to be one of the active components responsible for the inhibitory effects of SLE on STAT3 signaling and the anti-melanoma effects of SLE. This study provides a pharmacological and chemical basis for the traditional use of the formula SL in treating melanoma, and suggests that SLE and SLE-derived compounds have the potential to be developed as modern alternative and/or complimentary agents for melanoma management.

Cheung Sao Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves xxiv-xxxvii). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Abstract in Chinese --- p.viii / List of Figures and Tables --- p.x / List of Abbreviations --- p.xix / Table of Content --- p.xxiii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Human Liver Cancer --- p.1 / Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.1 / Chapter 1.1.2 --- Causes and Symptoms of Hepatocellular Carcinoma --- p.4 / Chapter 1.1.3 --- Treatment Options for Hepatocellular Carcinoma --- p.4 / Chapter 1.1.4 --- Multi-drug Resistance --- p.5 / Chapter 1.1.4.1 --- Mechanisms of Multi-drug Resistance --- p.5 / Chapter 1.2 --- Traditional Chinese Medicine --- p.10 / Chapter 1.2.1 --- Sophora flavescens and Radix Sophorae --- p.10 / Chapter 1.2.2 --- Flavonoid and its Sub-classification --- p.13 / Chapter 1.2.3 --- Flavonoid and Human Health --- p.15 / Chapter 1.3 --- Cell Death --- p.17 / Chapter 1.3.1 --- Necrosis --- p.17 / Chapter 1.3.2 --- Apoptosis --- p.17 / Chapter 1.3.3 --- Signaling Pathways in Apoptosis --- p.18 / Chapter 1.3.3.1 --- Extrinsic (Death Receptor-mediated) Pathway --- p.20 / Chapter 1.3.3.2 --- Intrinsic (Mitochondrial) Pathway --- p.21 / Chapter 1.3.3.3 --- Cysteine Aspartatic Acid Proteases --- p.21 / Chapter 1.4 --- Research Objective (s) --- p.22 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Cell Lines --- p.23 / Chapter 2.1.1.1 --- HepG2 --- p.24 / Chapter 2.1.1.2 --- RHepG2 --- p.24 / Chapter 2.1.1.3 --- WRL-68 --- p.25 / Chapter 2.1.2 --- Culture Media --- p.26 / Chapter 2.1.2.1 --- Rosewell Park Memorial Institute( RPMl) 1640 Medium --- p.26 / Chapter 2.1.2.2 --- Dulbecco's Modified Eagle's Medium (DMEM) --- p.26 / Chapter 2.1.3 --- Animals --- p.27 / Chapter 2.2 --- Traditional Chinese Medicines and Conventional Anti-cancer Drugs --- p.27 / Chapter 2.3 --- Antibodies --- p.29 / Chapter 2.4 --- Chemicals --- p.30 / Chapter 2.5 --- Reagents and Buffers --- p.34 / Chapter 2.5.1 --- Reagents for Silica Gel Column Chromatography --- p.34 / Chapter 2.5.2 --- Buffers for Common Use --- p.34 / Chapter 2.5.3 --- Reagents for Cell Viability Assay --- p.35 / Chapter 2.5.4 --- Reagents and Buffers for Typical Apoptosis Experiments --- p.35 / Chapter 2.5.4.1 --- Cell Cycle Analysis --- p.35 / Chapter 2.5.4.2 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.35 / Chapter 2.5.4.3 --- DNA Fragmentation Detection --- p.35 / Chapter 2.5.5 --- Reagents and Buffers for Western Blot Study --- p.36 / Chapter 2.5.5.1 --- Whole-cell Protein Extraction --- p.38 / Chapter 2.5.5.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.38 / Chapter 2.5.6 --- Reagents and Buffers for Mitochondrial Transmembrane Potential Depolarization Measurement --- p.39 / Chapter 2.5.7 --- Reagents and Buffers for in vivo Animal Study --- p.39 / Chapter 2.5.8 --- Reagents and Buffers for Two-Dimensional Gel Electrophoresis --- p.40 / Chapter 2.5.8.1 --- Sample Preparation --- p.40 / Chapter 2.5.8.2 --- First Dimension Gel Electrophoresis - Isoelectric Focusing (IEF) --- p.40 / Chapter 2.5.8.3 --- Second Dimension Gel 日ectrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter 2.5.8.4 --- Silver Staining --- p.41 / Chapter 2.5.9 --- Reagents for Mass Spectrometry Preparation --- p.42 / Chapter 2.5.9.1 --- Destaining --- p.42 / Chapter 2.5.9.2 --- Trypsin Digestion --- p.42 / Chapter 2.5.9.3 --- Desalting of Peptide Mixture --- p.43 / Chapter 2.5.10 --- Reagents and Buffers for Real-Time PCR --- p.43 / Chapter 2.6 --- Methods --- p.44 / Chapter 2.6.1 --- Isolation of Bioactive Constituents by Silica Gel Column Chromatography --- p.44 / Chapter 2.6.2 --- Cell Viability Assay --- p.45 / Chapter 2.6.3 --- Typical Apoptosis Experiments --- p.45 / Chapter 2.6.3.1 --- Cell Cycle Analysis --- p.46 / Chapter 2.6.3.2 --- Annexin V-FITC/ PI Staining Experiment --- p.47 / Chapter 2.6.3.3 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.48 / Chapter 2.6.3.4 --- DNA Fragmentation Reaction --- p.48 / Chapter 2.6.4 --- Western Blot Study --- p.49 / Chapter 2.6.4.1 --- Whole-cell Protein Extraction --- p.49 / Chapter 2.6.4.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.50 / Chapter 2.6.5 --- Caspase Activity Determination --- p.54 / Chapter 2.6.6 --- Mitochondrial Transmembrane Potential Depolarization Measurement --- p.55 / Chapter 2.6.7 --- in vivo Animal Study --- p.56 / Chapter 2.6.8 --- Two-Dimensional Gel Electrophoresis --- p.58 / Chapter 2.6.8.1 --- Sample Preparation --- p.58 / Chapter 2.6.8.2 --- First Dimension Electrophoresis - Isoelectric Focusing (IEF) --- p.59 / Chapter 2.6.8.3 --- Second Dimension Electrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.60 / Chapter 2.6.8.4 --- Silver Staining --- p.61 / Chapter 2.6.9 --- Mass Spectrometry Preparation --- p.63 / Chapter 2.6.9.1 --- Destaining and Trypsin Digestion --- p.63 / Chapter 2.6.9.2 --- Peptide Extraction --- p.63 / Chapter 2.6.9.3 --- Desalting of Peptide Mixture --- p.64 / Chapter 2.6.10 --- Real-Time PCR --- p.65 / Chapter 2.6.11 --- Cellular Glutathione Level Detection --- p.69 / Chapter 2.7 --- Statistical Analysis --- p.70 / Chapter Chapter 3 --- RESULTS AND DISCUSSIONS - CYTOTOXICITY OF FLAVONOIDS ISOLATED FROM RADIX SOPHORAE --- p.72 / Chapter 3.1 --- Screening of Cytotoxic Flavonoids from Radix Sophorae --- p.72 / Chapter 3.2 --- Cytotoxicity of Leachianone A on Human Hepatoma Cell Lines --- p.74 / Chapter 3.3 --- Cytotoxicity of Leachianone A on Human Normal Liver Cell Line --- p.77 / Chapter 3.4 --- Cytotoxicity of Sophoraflavone J on Human Hepatoma Cell Line --- p.79 / Chapter 3.5 --- Cytotoxicity of Sophoraflavone J on Human Normal Liver Cell Line --- p.79 / Chapter 3.6 --- Cytotoxicities of Cisplatin and Taxol on Human Hepatoma as well as Normal Liver Cell Lines --- p.81 / Chapter 3.7 --- Conclusion --- p.86 / Chapter Chapter 4 --- "RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF LEACHIANONE A-INDUCED CELL DEATH IN HEPATOMA CELLS, HepG2 and RHepG2" --- p.88 / Chapter 4.1 --- Promotion of Cell Cycle Arrest --- p.88 / Chapter 4.2 --- Induction of Apoptosis as Evidenced by Phosphatidylserine Externalization and DNA Fragmentation --- p.93 / Chapter 4.2.1 --- Occurrence of Phosphatidylserine Externalization --- p.94 / Chapter 4.2.2 --- DNA Fragmentation Detection --- p.99 / Chapter 4.2.2.1 --- Terminal Deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.99 / Chapter 4.2.2.2 --- DNA Laddering Pattern in Agarose Gel Electrophoresis --- p.103 / Chapter 4.3 --- Recruitment of Multiple Signaling Pathways in Leachianone A-induced Apoptosis --- p.105 / Chapter 4.3.1 --- "Activation of Caspases-3, -8, and -9" --- p.105 / Chapter 4.3.2 --- Altered Expressions of Bcl-2 Family Proteins --- p.112 / Chapter 4.3.3 --- Loss of Mitochondrial Membrane Potential --- p.115 / Chapter 4.4 --- in vivo Tumor Growth Inhibition in HepG2-bearing Nude Mice --- p.121 / Chapter 4.5 --- Conclusion --- p.127 / Chapter Chapter 5 --- RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF SOPHORAFLAVONE J-INDUCED CELL DEATH IN HEPATOMA CELLS HepG2 --- p.132 / Chapter 5.1 --- Execution of Cellular Apoptosis --- p.133 / Chapter 5.2 --- Involvement of Multiple Signaling Pathways in Sophoraflavone J-induced Apoptosis --- p.138 / Chapter 5.3 --- Differential Proteomes of Control and Sophoraflavone J-treated HepG2 Cells --- p.148 / Chapter 5.4 --- Conclusion --- p.167 / Chapter Chapter 6 --- OVERALL CONCLUSION AND FUTURE PERSPECTIVES --- p.169 / References --- p.xxiv