by Wong Wah Yau. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 219-228.

by Lai Wing Hong Kevin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 63-74). / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction --- p.3 / Chapter 1.1 --- Advantages of Antisense Oligonucleotides Inhibition --- p.4 / Chapter 1.2 --- The Uses of Antisense Oligonucleotide in Leukemic Therapy --- p.5 / Chapter 1.3 --- Oncogenes in the Pathogenesis of Leukemia --- p.6 / Chapter 1.4 --- Apoptosis and Apoptosis-Related Genes --- p.9 / Chapter 1.5 --- Protooncogene bcl-2 --- p.10 / Chapter 1.6 --- Bcl-2 Homologues --- p.11 / Chapter 1.7 --- Regulation of Apoptosis by Other Genes --- p.13 / Chapter 1.8 --- Promyelocytic Leukemia HL-60 Cell Line --- p.15 / Chapter 1.9 --- Aim of Project --- p.16 / Chapter Chapter 2 --- Chemical Synthesis of DNA Oligonucleotides / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.3 --- Results --- p.24 / Chapter 2.4 --- Discussion --- p.26 / Chapter Chapter 3 --- The Apoptotic Effects of TPA and Ouabain on the Promyelocytic Leukemic HL-60 cell line / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.33 / Chapter 3.3 --- Results --- p.40 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4 --- Effect of Antisense Oligonucleotides on TPA-Induced Apoptosisin Leukemic HL-60 cells / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Materials and Methods --- p.49 / Chapter 4.3 --- Results --- p.52 / Chapter 4.4 --- Discussion --- p.54 / Chapter Chapter 5 --- General Discussion --- p.57 / References --- p.63

by Wang He-Xiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 161-179). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / LIST OF CONTENTS --- p.v / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1. --- General Introduction --- p.1 / Chapter CHAPTER 2. --- Literature Review --- p.5 / Chapter 2.1. --- Lectins --- p.5 / Chapter 2.1.1. --- Aspects of lectins --- p.5 / Chapter 2.1.2. --- Isolation and purification of lectins --- p.8 / Chapter 2.1.3. --- Characteristics of lectins --- p.9 / Chapter 2.1.4. --- Effects of lectins on biological activities --- p.10 / Chapter 2.1.4.1. --- The role of lectins in plant defence --- p.11 / Chapter 2.1.4.2. --- The specificity of some legume lectins --- p.13 / Chapter 2.1.4.3. --- Some properties of animal lectins --- p.14 / Chapter 2.1.4.4. --- Hypotensive activity of the lectins --- p.18 / Chapter 2.1.4.5. --- Lectins in immunology --- p.20 / Chapter 2.2. --- Mushroom Lectins and Polysaccharides --- p.24 / Chapter 2.2.1. --- General aspects of mushroom lectins and polysaccharides --- p.24 / Chapter 2.2.2. --- Mushroom lectins --- p.25 / Chapter 2.2.2.1. --- Hericium erinaceum lectin --- p.26 / Chapter 2.2.2.2. --- Lactarius deterrimus lectin --- p.26 / Chapter 2.2.2.3. --- Laetiporus sulfureus lectin --- p.27 / Chapter 2.2.2.4. --- Grifola frondosa lectin --- p.28 / Chapter 2.2.2.5. --- Volvariella volvacea lectin --- p.28 / Chapter 2.2.2.6. --- Flammulina veltipes lectin --- p.29 / Chapter 2.2.2.7. --- Ischnoderma resinosum agglutinin --- p.31 / Chapter 2.2.2.8. --- Lectins from Agaricus spp --- p.31 / Chapter 2.2.3. --- Mushroom polysaccharides --- p.34 / Chapter 2.2.3.1. --- Lentinan --- p.35 / Chapter 2.2.3.2. --- "PSK (trade name, Krestin)" --- p.35 / Chapter 2.2.3.3. --- PSP (Polysaccharopeptide) --- p.37 / Chapter 2.2.3.4. --- PSPC (polysaccharide-peptide complex) --- p.38 / Chapter CHAPTER 3. --- Isolation and Characterization of Two Distinct Lectins from the Cultured Mycelium of the Edible Mushroom Tricholoma mongolicum --- p.44 / Chapter 3.1. --- Introduction --- p.44 / Chapter 3.2. --- Materials and Methods --- p.45 / Chapter 3.2.1. --- Strain and culture condition --- p.45 / Chapter 3.2.2. --- Extraction --- p.46 / Chapter 3.2.3. --- Purification --- p.46 / Chapter 3.2.4. --- Hemagglutination activity --- p.47 / Chapter 3.2.5. --- Test of hemagglutination inhibition by various carbohydrates --- p.47 / Chapter 3.2.6. --- MW estimation by gel filtration and SDS- PAGE --- p.48 / Chapter 3.2.7. --- Glycoprotein staining with PAS reagent --- p.49 / Chapter 3.2.8. --- Carbohydrate content --- p.49 / Chapter 3.2.9. --- Thermal stability --- p.49 / Chapter 3.2.10. --- pH stability --- p.49 / Chapter 3.2.11. --- Effect of cations --- p.50 / Chapter 3.2.12. --- Amino acid analysis --- p.50 / Chapter 3.2.13. --- Antiproliferative activity of lectins --- p.50 / Chapter 3.2.14. --- Statistics --- p.51 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Extraction and purification --- p.51 / Chapter 3.3.2. --- General characteristics of lectins --- p.52 / Chapter 3.3.3. --- Antiproliferative activity of lectins --- p.54 / Chapter 3.4. --- Discussion --- p.55 / Chapter 3.5. --- Summary --- p.58 / Chapter CHAPTER 4. --- The Immunomodulatory and Antitumor Activities of Lectins from the Mushroom Tricholoma mongolicum --- p.79 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Materials and Methods --- p.81 / Chapter 4.2.1. --- Lectins --- p.81 / Chapter 4.2.2. --- Animals --- p.81 / Chapter 4.2.3. --- Assay for antitumor activity --- p.81 / Chapter 4.2.4. --- Assessment of tumor growth and host survival after lectin treatment --- p.82 / Chapter 4.2.5. --- Mitogenic activity of lectins --- p.82 / Chapter 4.2.6. --- Production of nitrite ions in response to lectin treatment --- p.83 / Chapter 4.2.7. --- Preparation of concanavalin A-stimulated lymphokines --- p.84 / Chapter 4.2.8. --- Assay for macrophage activating factor --- p.85 / Chapter 4.2.9. --- Production of tumor necrosis factor (TNF) --- p.86 / Chapter 4.2.10. --- Bioassay for tumor necrosis factor --- p.86 / Chapter 4.2.11. --- Statistics --- p.87 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- Antitumor activity --- p.87 / Chapter 4.3.2. --- Assessment of tumor growth and host survival --- p.87 / Chapter 4.3.3. --- Mitogenic activity --- p.88 / Chapter 4.3.4. --- Production of nitrite ions --- p.89 / Chapter 4.3.5. --- Production of macrophage activating factor --- p.89 / Chapter 4.3.6. --- Tumor necrosis factor assay --- p.90 / Chapter 4.4. --- Discussion --- p.90 / Chapter 4.5. --- Summary --- p.94 / Chapter CHAPTER 5. --- Hypotensive and Vasorelaxing Activities of a Lectin (TML-1) from the Edible Mushroom Tricholoma mongolicum --- p.109 / Chapter 5.1. --- Introduction --- p.109 / Chapter 5.2. --- Materials and Methods --- p.111 / Chapter 5.2.1. --- Animals --- p.111 / Chapter 5.2.2. --- In vivo blood pressure measurement in rats --- p.112 / Chapter 5.2.3. --- Study employing blockade of autonomic ganglion transmission --- p.113 / Chapter 5.2.4. --- Study employing alpha-adrenergic blockade --- p.113 / Chapter 5.2.5. --- Study employing beta-adrenergic blockade --- p.114 / Chapter 5.2.6. --- Study employing cholinergic blockade --- p.114 / Chapter 5.2.7. --- Study employing histaminergic blockade --- p.114 / Chapter 5.2.8. --- Study employing inhibitor of the renin- angiotensin system --- p.115 / Chapter 5.2.9. --- Preparation of right atrium for in vitro studies --- p.115 / Chapter 5.2.10. --- Preparation of aorta for in vitro studies --- p.116 / Chapter 5.2.11. --- Adenosine receptor binding assays --- p.116 / Chapter 5.2.12. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.118 / Chapter 5.2.13. --- Statistics --- p.118 / Chapter 5.3. --- Results --- p.118 / Chapter 5.3.1. --- Blood pressure changes in vivo --- p.118 / Chapter 5.3.2. --- Pharmacological studies using receptor antagonists --- p.119 / Chapter 5.3.3. --- Adenosine receptor binding assay --- p.119 / Chapter 5.3.4. --- Effects on the right atrium in vitro --- p.120 / Chapter 5.3.5. --- Effect of TML-1 on vascular relaxation --- p.120 / Chapter 5.3.6. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.120 / Chapter 5.4. --- Discussion --- p.120 / Chapter 5.5. --- Summary --- p.123 / Chapter CHAPTER 6. --- A Polysaccharide-Peptide Complex with Immunoenhancing and Antitumor Activities from Cultured Mycelia of the Mushroom Tricholoma mongolicum --- p.134 / Chapter 6.1. --- Introduction --- p.134 / Chapter 6.2. --- Materials and Methods --- p.135 / Chapter 6.2.1. --- Extraction --- p.135 / Chapter 6.2.2. --- Purification --- p.135 / Chapter 6.2.3. --- PSP for purpose of comparison --- p.136 / Chapter 6.2.4. --- Polysaccharide and protein contents --- p.136 / Chapter 6.2.5. --- MW determination of F1 using gel filtration --- p.136 / Chapter 6.2.6. --- Animals --- p.136 / Chapter 6.2.7. --- Antiproliferative activity assay --- p.137 / Chapter 6.2.8. --- Mitogenic activity --- p.137 / Chapter 6.2.9. --- Production of nitrite ions --- p.138 / Chapter 6.2.10. --- Macrophage activating factor assay --- p.138 / Chapter 6.2.11. --- Antitumor activity assay --- p.139 / Chapter 6.2.12. --- Statistics --- p.139 / Chapter 6.3. --- Results --- p.140 / Chapter 6.3.1. --- Purification of polysaccharide-peptide complex --- p.140 / Chapter 6.3.2. --- Antiproliferative activity --- p.140 / Chapter 6.3.3. --- Mitogenic activity in vitro --- p.140 / Chapter 6.3.4. --- Molecular weight of Fl --- p.141 / Chapter 6.3.5. --- Mitogenic activity in vivo --- p.141 / Chapter 6.3.6. --- Production of nitrite ions --- p.141 / Chapter 6.3.7. --- Production of macrophage activating factor --- p.141 / Chapter 6.3.8. --- Antitumor activity in vivo --- p.142 / Chapter 6.4. --- Discussion --- p.142 / Chapter 6.5. --- Summary --- p.144 / GENERAL DISCUSSION --- p.155 / CONCLUSIONS --- p.158 / REFERENCES

Song Jing-mei. / Thesis submitted in: December 1998. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 141-156). / Abstract also in Chinese. / Title page --- p.i / Acknowledgments --- p.ii / Table of contents --- p.iii / Abbreviations --- p.viii / Abstract --- p.x / 摘要 --- p.xii / Chapter Chapter1 --- Introduction --- p.1 / Chapter Chapter2 --- Evaluation of Antiulcer Effect Exhibited by Hippophae rhamnoides Using Different Ulcer Models / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.1.1 --- Ethanol-induced gastric lesions --- p.24 / Chapter 2.1.2 --- NSAID-induced gastric lesions --- p.24 / Chapter 2.1.3 --- Stress-induced gastric lesions --- p.25 / Chapter 2.1.4 --- Pylorus ligation-induced gastric lesions --- p.25 / Chapter 2.1.5 --- Acetic acid-induced chronic gastric ulcer --- p.26 / Chapter 2.1.6 --- Necrotizing agent-induced lesion model --- p.27 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Plant materials --- p.28 / Chapter 2.2.2 --- Identification of the plant --- p.28 / Chapter 2.2.3 --- Preparation of crude extract for animal studies --- p.28 / Chapter 2.2.4 --- Experimental animals --- p.31 / Chapter 2.2.5 --- Ethanol-induced gastric mucosal lesions --- p.31 / Chapter 2.2.6 --- Acidified aspirin-induced gastric lesions --- p.32 / Chapter 2.2.7 --- Water immersion plus restraint-induced stress lesion model --- p.32 / Chapter 2.2.8 --- Pylorus ligation-induced gastric lesions --- p.32 / Chapter 2.2.9 --- Acetic acid-induced chronic gastric ulcer --- p.34 / Chapter 2.2.10 --- Necrotizing agent-induced gastric lesions --- p.34 / Chapter 2.2.11 --- Test of acute toxicity of Hippophae --- p.35 / Chapter 2.2.12 --- Statistical analysis --- p.35 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Effect of Hr extract on ethanol-induced gastric lesions --- p.36 / Chapter 2.3.2 --- Effect of Hr extract on aspirin-induced gastric damage --- p.39 / Chapter 2.3.3 --- Effect of Hr extract on stress-induced gastric lesions --- p.40 / Chapter 2.3.4 --- Effect of Hr extract on pylorus ligation-induced gastric injury --- p.43 / Chapter 2.3.5 --- Effect of Hr extract on acetic acid-induced chronic ulcer --- p.48 / Chapter 2.3.6 --- Effect of Hr extract on necrotizing agent-induced gastric damage --- p.54 / Chapter 2.3.7 --- Test of acute toxicity of Hr --- p.55 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- Cytoprotective effect of Hr against ethanol-induced lesions --- p.56 / Chapter 2.4.2 --- Preventive effect of Hr on NSAIDs-induced gastric lesions --- p.57 / Chapter 2.4.3 --- Inhibitory effect of Hr on stress-induced lesions --- p.58 / Chapter 2.4.4 --- Inhibitory effect of Hr extract on pylorus ligation-induced gastric lesions --- p.59 / Chapter 2.4.5 --- Healing effect of Hr extract on acetic acid-induced gastric ulcer --- p.60 / Chapter 2.4.6 --- Protective effect of Hr extract on necrotizing agent-induced gastric damage --- p.61 / Chapter 2.4.7 --- Summary --- p.61 / Chapter Chapter3 --- Study on Cytoprotective Effect of Hippophae rhamnoides on Ethanol-induced Gastric Damage / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Materials and Methods --- p.65 / Chapter 3.2.1 --- Chemicals and Instruments --- p.65 / Chapter 3.2.2 --- Test on effect of different concentrations of ethanol on gastric mucosa --- p.67 / Chapter 3.2.3 --- Examination of the gastric protective effect of Hr extract by different routes of administration --- p.68 / Chapter 3.2.4 --- Study on relationship between gastric protective effect of Hr extract and endogenous PGs --- p.68 / Chapter 3.2.5 --- Measurement of gastric mucosal blood flow (GMBF) --- p.69 / Chapter 3.2.6 --- Measurement of gastric secretion and acidity in gastric juice --- p.70 / Chapter 3.2.7 --- Measurement of gastric gastric emptying rate --- p.70 / Chapter 3.2.8 --- Measurement of pepsin content in gastric juice --- p.71 / Chapter 3.2.9 --- Measurement of protein content in gastric juice --- p.73 / Chapter 3.2.10 --- Measurement of mucus content on gastric wall --- p.75 / Chapter 3.2.11 --- Measurement of GSH content in gastric mucosa --- p.77 / Chapter 3.2.12 --- Measurement of PGE2 content in gastric mucosa --- p.79 / Chapter 3.2.13 --- Determination of protein content in gastric mucosa --- p.81 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Test on gastric lesions induced by different concentrations of ethanol --- p.83 / Chapter 3.3.2 --- Effect of Hr extract on ethanol-induced gastric damage by different routes of administration --- p.83 / Chapter 3.3.3 --- Effect of Hr extract on GMBF and output of gastric acid --- p.85 / Chapter 3.3.4 --- Effect of Hr extract on gastric emptying rate --- p.87 / Chapter 3.3.5 --- Effect of Hr extract on gastric mucus --- p.88 / Chapter 3.3.6 --- Effect of Hr extract on gastric GSH content --- p.89 / Chapter 3.3.7 --- Influence of Hr extract on endogenous prostanglandin-E2 --- p.90 / Chapter 3.3.8 --- Antagonistic effect of indomethacin on the gastric protection of Hr extract --- p.91 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Formation of gastric lesions induced by ethanol at different concentrations --- p.92 / Chapter 3.4.2 --- Different routes of administration --- p.92 / Chapter 3.4.3 --- "Role of GMBF, gastric acidity and acid output in the formation of gastric lesions" --- p.93 / Chapter 3.4.4 --- Effect of Hr extract on gastric motility --- p.95 / Chapter 3.4.5 --- Effect of Hr extract on gastric mucus --- p.96 / Chapter 3.4.6 --- Effect of Hr extract on gastric GSH content --- p.96 / Chapter 3.4.7 --- Effect of Hr extract on endogenous prostaglandins --- p.98 / Chapter 3.4.8 --- Summary --- p.99 / Chapter Chapter 4 --- Study on plant constituents of Hippophae rhamnoides / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials and Methods --- p.100 / Chapter 4.2.1 --- Plant Materials --- p.100 / Chapter 4.2.2 --- Plant Extraction --- p.101 / Chapter 4.2.3 --- Fractionation of hexane-extract by column chromatography --- p.103 / Chapter 4.2.4 --- Phytochemical identification and analyses of vitamin content in Hr extract --- p.104 / Chapter 4.2.4.1 --- Identification of vitamin A and vitamin C in the Hr extract by TLC --- p.104 / Chapter 4.2.4.2 --- Identification of α-tocopherol and γ-tocopherol by HPLC --- p.105 / Chapter 4.2.4.3 --- Analyses of the content of α-tocopherol in the Hr extract --- p.108 / Chapter 4.2.4.4 --- Identification and analysis of fatty acid in the Hr fractions --- p.111 / Chapter 4.2.4.4.1 --- Esterification of fatty acids --- p.111 / Chapter 4.2.4.4.2 --- Isolation and identification of FAME by GC-MS --- p.111 / Chapter 4.2.4.5 --- Quantitative analysis of composition and relative content of fatty acid in the Hr fractions --- p.112 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Phytochemical analysis and identification --- p.114 / Chapter 4.3.1.1 --- Identification of vitamin A --- p.114 / Chapter 4.3.1.2 --- Identification of vitamin C --- p.115 / Chapter 4.3.1.3 --- Identification of α-tocopherol and γ-tocopherol --- p.116 / Chapter 4.3.1.4 --- Quantitative analysis of α-tocopherol content in the Hr extract --- p.117 / Chapter 4.3.1.5 --- Identification of fatty acid composition --- p.117 / Chapter 4.3.1.6 --- Analysis of relative content of fatty acids in the Hr extract --- p.122 / Chapter 4.3.1.7 --- Study on phytosterols of Hr --- p.124 / Chapter 4.3.2 --- Examination of antiulcer effect of Hr fractions against ethanol-induced gastric lesions --- p.124 / Chapter 4.3.2.1 --- Effect of different extracts of Hr seed on ethanol-induced gastric lesions --- p.125 / Chapter 4.3.2.2 --- Effect of fractions of hexane-extract of Hr on gastric lesions induced by ethanol --- p.126 / Chapter 4.3.2.3 --- Effect of Hr components on gastric lesions induced by different ulcer models --- p.127 / Chapter 4.3.2.3.1 --- Effect of Hr components on ethanol-induced lesions --- p.127 / Chapter 4.3.2.3.2 --- Effect of Hr components against stress-induced gastric lesions --- p.128 / Chapter 4.3.2.3.3 --- Effect of β-sitosterol against gastric lesions induced by pylorus ligation --- p.129 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Role of fatty acids in the stomach protection --- p.130 / Chapter 4.4.2 --- Role of vitamins in the gastric protection --- p.133 / Chapter 4.4.3 --- Role of plant terpenoids in the stomach --- p.134 / Chapter 4.4.4 --- Summary --- p.135 / Chapter Chapter 5 --- General discussion --- p.136 / References --- p.141

Chan Chi Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 100-109). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Abbreviations --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- "Free radicals, oxidative stress and antioxidants" --- p.2 / Chapter 1.1.1 --- Free radical and ROS --- p.2 / Chapter 1.1.2 --- Oxidative stress --- p.6 / Chapter 1.1.3 --- Antioxidants --- p.8 / Chapter 1.2 --- Plant as a source of antioxidants --- p.13 / Chapter 1.2.1 --- Common food sources of antioxidants --- p.13 / Chapter 1.2.2 --- Legume seeds as antioxidant sources --- p.15 / Chapter 1.3 --- Methods used to evaluate the antioxidant activity --- p.16 / Chapter 1.3.1 --- β-carotene bleaching method --- p.17 / Chapter 1.3.2 --- DPPH. scavenging method --- p.17 / Chapter 1.3.3 --- High-performance liquid chromatograph (HPLC) --- p.18 / Chapter 1.3.4 --- Single cell gel electrophoresis (SCGE) --- p.20 / Chapter 1.4 --- Objectives of the study --- p.22 / Chapter 2 --- Materials and Methods --- p.23 / Chapter 2.1 --- Plant materials and chemicals --- p.23 / Chapter 2.2 --- Sample preparation --- p.23 / Chapter 2.3 --- Determination of antioxidant activity using β-carotene bleaching method --- p.24 / Chapter 2.4 --- Evaluation of free radical scavenging ability --- p.26 / Chapter 2.5 --- HPLC separation of seed extract --- p.27 / Chapter 2.6 --- Evaluation of protective effects of legumes on the DNA damage using the comet assay --- p.28 / Chapter 2.6.1 --- Preparation of reagents --- p.28 / Chapter 2.6.2 --- Blood sample --- p.29 / Chapter 2.6.3 --- Hydrogen peroxide (H2O2) treatment --- p.29 / Chapter 2.6.4 --- Ethidium bromide-acridine orange (Et-Ac) viability determination --- p.31 / Chapter 2.6.5 --- Slide preparation --- p.31 / Chapter 2.6.6 --- Alkaline comet assay --- p.31 / Chapter 2.6.7 --- Quantification of DNA damage --- p.33 / Chapter 2.6.8 --- Statistical analysis --- p.33 / Chapter 3 --- Results --- p.40 / Chapter 3.1 --- General description of 24 selected legume seeds --- p.40 / Chapter 3.1 --- Determination of antioxidant activity using β-carotene bleaching method --- p.40 / Chapter 3.2 --- Evaluation of free radical scavenging ability --- p.43 / Chapter 3.3 --- Evaluation of protective effects of legumes on the DNA damage using the comet assay --- p.45 / Chapter 3.3.1 --- Optimal assay conditions --- p.46 / Chapter 3.3.2 --- Protective effects of seed extract and vitamin C --- p.47 / Chapter 3.3.3 --- Effects of heat treatment on the protective effect --- p.48 / Chapter 4 --- Discussion --- p.84 / Chapter 4.1 --- Methanolic extraction --- p.84 / Chapter 4.2 --- Antioxidant activities determined with β-carotene bleaching method and DPPH' scavenging method --- p.84 / Chapter 4.3 --- Evaluation of protective effects of legumes on the DNA damage using the comet assay --- p.93 / Chapter 4.3.1 --- H202-mediated DNA damage --- p.93 / Chapter 4.3.2 --- Protective effects of seed extracts and vitamin C --- p.94 / Chapter 5 --- Conclusion --- p.98 / References --- p.100

Lau Kit-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Declaration --- p.vi / Table of contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xv / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Tea and Ku-Ding-Cha --- p.1 / Chapter 1.2 --- Ligustrum robustum / Chapter 1.2.1 --- The plant --- p.4 / Chapter 1.2.2 --- Chemical constituents --- p.4 / Chapter 1.2.3 --- Pharmacological activities --- p.4 / Chapter 1.2.4 --- Toxicity --- p.5 / Chapter 1.3 --- Objectives and scope of the project --- p.7 / Chapter Chapter Two: --- Antioxidative effect / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Oxidants and antioxidants --- p.8 / Chapter 2.1.2 --- In vitro antioxidative tests / Chapter 2.1.2.1 --- PMS-NADH system --- p.19 / Chapter 2.1.2.2 --- Fe3+/ascorbate/H202 system --- p.19 / Chapter 2.1.2.3 --- Red-blood-cell hemolysis model --- p.20 / Chapter 2.2 --- Objectives --- p.22 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Materials / Chapter 2.3.1.1 --- Guizhou Ku-Ding-Cha --- p.23 / Chapter 2.3.1.2 --- Other tea leaves --- p.23 / Chapter 2.3.1.3 --- Animals --- p.23 / Chapter 2.3.1.4 --- Chemicals --- p.24 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Aqueous extraction of L. robustum and other tea leaves --- p.25 / Chapter 2.3.2.2 --- Ethanol extraction of L. robustum and fraction separations --- p.25 / Chapter 2.3.2.3 --- Activity-guided purification of L. robustum --- p.26 / Chapter 2.3.2.4 --- Assays for testing antioxidative effect / Chapter 2.3.2.4.1 --- PMS-NADH system --- p.28 / Chapter 2.3.2.4.2 --- Fe3+/ascorbate/H202 system --- p.28 / Chapter 2.3.2.4.3 --- Red-blood-cell hemolysis model --- p.29 / Chapter 2.3.2.5 --- Statistical analysis --- p.29 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Ligustrum robustum and other tea leaves --- p.30 / Chapter 2.4.2 --- Ethanol extract of L. robustum --- p.48 / Chapter 2.4.3 --- Water-soluble and water-insoluble fractions --- p.52 / Chapter 2.4.4 --- "Fractions B1, B2 and B3" --- p.56 / Chapter 2.4.5 --- Sub-fractions B2-1 to B2-16 --- p.61 / Chapter 2.4.6 --- Pure compounds --- p.66 / Chapter 2.4.7 --- Changes in antioxidant effects --- p.72 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Antioxidant potency of L. robustum --- p.76 / Chapter 2.5.2 --- Effects of extraction methods on antioxidant activities --- p.78 / Chapter 2.5.3 --- Active antioxidant components of L. robustum --- p.78 / Chapter 2.5.4 --- Structure-activity relationship of glycosides and flavonoid --- p.80 / Chapter 2.5.5 --- Antioxidant mechanism of L. robustum --- p.81 / Chapter 2.5.6 --- Prospects for further investigation --- p.82 / Chapter Chapter Three: --- Anti-inflammatory effect / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Mechanisms and mediators of inflammation --- p.83 / Chapter 3.1.2 --- In vivo anti-inflammatory assays / Chapter 3.1.2.1 --- Acetic acid-induced vascular permeability test --- p.94 / Chapter 3.1.2.2 --- Croton oil-induced ear edema test --- p.94 / Chapter 3.2 --- Objective --- p.96 / Chapter 3.3 --- Materials and Methods / Chapter 3.3.1 --- Materials / Chapter 3.3.1.1 --- Animals --- p.97 / Chapter 3.3.1.2 --- Chemicals --- p.97 / Chapter 3.3.2 --- Methods / Chapter 3.3.2.1 --- Assays for testing anti-inflammatory effect / Chapter 3.3.2.1.1 --- Acetic acid-induced vascular permeability test --- p.98 / Chapter 3.3.2.1.2 --- Croton oil-induced ear edema test --- p.98 / Chapter 3.3.2.2 --- Statistical analysis --- p.99 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Acetic acid-induced vascular permeability test --- p.100 / Chapter 3.4.2 --- Croton oil-induced ear edema test --- p.100 / Chapter 3.5 --- Discussion --- p.103 / Chapter Chapter Four: --- Hepato-protective effect / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Liver structures and functions --- p.105 / Chapter 4.1.2 --- Carbon tetrachloride-induced liver injury --- p.112 / Chapter 4.1.2.1 --- Mechanisms --- p.112 / Chapter 4.1.2.2 --- Hepatic cytotoxicity --- p.112 / Chapter 4.1.2.3 --- Diagnostic methods / Chapter 4.1.2.3.1 --- Liver weight --- p.114 / Chapter 4.1.2.3.2 --- Lipid peroxidation --- p.114 / Chapter 4.1.2.3.3 --- Serum enzyme levels --- p.114 / Chapter 4.1.2.3.4 --- Histopathological observation --- p.115 / Chapter 4.2 --- Objectives --- p.116 / Chapter 4.3 --- Materials and Methods / Chapter 4.3.1 --- Materials / Chapter 4.3.1.1 --- Animals --- p.117 / Chapter 4.3.1.2 --- Chemicals --- p.117 / Chapter 4.3.2 --- Methods / Chapter 4.3.2.1 --- Carbon tetrachloride-induced acute liver injury --- p.118 / Chapter 4.3.2.2 --- Statistical analysis --- p.120 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Preventive effect / Chapter 4.4.1.1 --- Liver weight --- p.121 / Chapter 4.4.1.2 --- Malondialdehyde content --- p.121 / Chapter 4.4.1.3 --- Serum aminotransferse levels --- p.121 / Chapter 4.4.1.4 --- Histopathological observations --- p.122 / Chapter 4.4.2 --- Curative effect / Chapter 4.4.2.1 --- Liver weight --- p.126 / Chapter 4.4.2.2 --- Malondialdehyde content --- p.126 / Chapter 4.4.2.3 --- Serum aminotransferse levels --- p.126 / Chapter 4.4.2.4 --- Histopathological observations --- p.126 / Chapter 4.5 --- Discussion --- p.130 / Chapter Chapter Five: --- Prospects for product development --- p.134 / Chapter Chapter Six: --- Conclusion --- p.136 / Appendices / Appendix A. Procedure for determining the activity of aspartate aminotransferase (AST) --- p.139 / Appendix B. Procedure for determining the activity of alanine aminotransferase (ALT) --- p.140 / Appendix C. Procedure for preparing a calibration curve for the measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities --- p.141 / Appendix D. Procedure for tissue preparation for light microscopic study --- p.143 / References --- p.144

by Chung Lung Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 149-157). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstracts --- p.v / List of figures --- p.ix / List of tables --- p.xii / Contents --- p.xiii / Contents / General Introduction --- p.1 / Chapter CHAPTER ONE --- CISPLATIN RESISTANCE MECHANISMS / Chapter 1.1 --- INTRODUCTION --- p.3 / Chapter 1.1.1 --- History of Cisplatin as An Anticancer Drug --- p.3 / Chapter 1.1.2 --- Active Mechanisms of Cisplatin --- p.8 / Chapter 1.1.3 --- Formation of DNA Adducts --- p.8 / Chapter 1.1.4 --- Cisplatin Resistance Mechanisms --- p.9 / Chapter 1.1.4.1 --- Intracellular Accumulation of Cisplatin --- p.11 / Chapter 1.1.4.2 --- Glutathione-S-transferase and Glutathion --- p.12 / Chapter 1.1.4.3 --- Metallothionein --- p.16 / Chapter 1.1.4.4 --- Cell Cycle Perturbation --- p.16 / Chapter 1.1.4.5 --- P-glycoprotein --- p.17 / Chapter 1.1.4.6 --- Multidrug Resistant Protein --- p.19 / Chapter 1.1.4.7 --- Topoisomerase II --- p.20 / Chapter 1.1.4.8 --- DNA Repair --- p.22 / Chapter 1.1.4.9 --- Induction of Programme Cell Death --- p.23 / Chapter 1.2 --- OBJECTIVES --- p.27 / Chapter 1.3 --- MATERIALS AND METHODS / Chapter 1.3.1 --- Materials --- p.28 / Chapter 1.3.2 --- Methods --- p.31 / Chapter 1.3.2.1 --- Cell Lines --- p.31 / Chapter 1.3.2.2 --- Drug Sensitivity Assay --- p.31 / Chapter 1.3.2.3 --- Platinum Uptake --- p.32 / Chapter 1.3.2.4 --- Cell Cycle Analysis --- p.32 / Chapter 1.3.2.5 --- Western Blot Analysis --- p.33 / Chapter 1.3.2.6 --- Glutathione Content Determination --- p.36 / Chapter 1.3.2.7 --- DNA Fragmentation --- p.36 / Chapter 1.3.2.8 --- JC-1 Staining --- p.37 / Chapter 1.3.2.9 --- HE and DCF Staining --- p.38 / Chapter 1.3.2.10 --- Quantitative RT-PCR --- p.38 / Chapter 1.4 --- RESULTS / Chapter 1.4.1 --- Cisplatin Sensitivity of A431 Cells by MTT Assay --- p.40 / Chapter 1.4.2 --- Cross-resistance to Anti-cancer Drugs --- p.40 / Chapter 1.4.3 --- Quantitation of Cisplatin Accumulation in A431 Cells by AAS --- p.44 / Chapter 1.4.4 --- Drug Detoxification Agent --- p.45 / Chapter 1.4.5 --- Detection of Cell Cycle Arrest by Flow Cytometer --- p.47 / Chapter 1.4.6 --- Expression of Drug Resistance Related Genes --- p.48 / Chapter 1.4.7 --- Detection of Apoptosis by DNA Fragmentation --- p.50 / Chapter 1.4.8 --- Role of Mitochondria and Reactive Oxygen Species by Flow Cytometer --- p.52 / Chapter 1.4.9 --- Detection of Apoptotic mRNA Level by Quantitative RT-PCR --- p.57 / Chapter 1.4.10 --- Detection of Apoptotic Protein Level by Western Blot Analysis --- p.57 / Chapter 1.5 --- DISCUSSIONS --- p.59 / Chapter CHAPTER TWO: --- THE INTERACTION BETWEEN DRUG RESISTANCE MECHANISMS AND GROWTH FACTOR SIGNALLING PATHWAY / Chapter 2.1 --- INTRODUCTION --- p.63 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.64 / Chapter 2.1.2 --- Growth Factor Signal Transduction Pathway --- p.69 / Chapter 2.1.3 --- Biological Effect of EGF --- p.69 / Chapter 2.1.3.1 --- Modification of Drug Sensitivity by EGF --- p.71 / Chapter 2.2 --- OBJECTIVES --- p.74 / Chapter 2.3 --- MATERIALS AND METHODS / Chapter 2.3.1 --- Materials --- p.75 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.76 / Chapter 2.3.2.2 --- Drug Sensitivity Assay --- p.77 / Chapter 2.3.2.3 --- Northern Blot Analysis --- p.77 / Chapter 2.3.2.4 --- Southern Blot Analysis --- p.78 / Chapter 2.3.2.5 --- Others --- p.78 / Chapter 2.4 --- RESULTS / Chapter 2.4.1 --- Sensitivity to EGF --- p.79 / Chapter 2.4.2 --- EGFR Expression Levels --- p.80 / Chapter 2.4.3 --- EGF Induced Protein Phosphorylation Pattern --- p.84 / Chapter 2.4.4 --- Effect of EGF on A431 Cells --- p.86 / Chapter 2.4.5 --- Response of Cells to Agents Targeting on EGF Signalling Pathway --- p.91 / Chapter 2.4.6 --- Response of Cells to Other Growth Factors --- p.97 / Chapter 2.4.7 --- Sensitivity of Cells to Different Anti-cancer Drugs --- p.99 / Chapter 2.4.8 --- Drug Resistance Mechanisms --- p.103 / Chapter 2.4.9 --- 5-Fluorouracil Sensitivity in A431 Cells --- p.108 / Chapter 2.4.10 --- Cisplatin Sensitivity in A431 Cells --- p.113 / Chapter 2.5 --- DISCUSSIONS --- p.117 / Chapter CHAPTER THREE: --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENE IN A431 CELLS BY DIFFERENTIAL DISPLAY / Chapter 3.1 --- INTRODUCTION --- p.122 / Chapter 3.2 --- MATERIALS AND METHODS / Chapter 3.2.1 --- Materials --- p.128 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Identification of Differentially Expressed Genes by RT-PCR / Chapter 3.2.2.2 --- Cloning of a Differentially Expressed cDNAs --- p.129 / Chapter 3.2.2.3 --- Screening and Sequencing of cDNA Inserts --- p.130 / Chapter 3.2.2.4 --- Rapid Amplification of cDNA Ends (RACE) --- p.131 / Chapter 3.2.2.5 --- Amplifcation Reaction --- p.131 / Chapter 3.2.2.6 --- Cloning and Sequencing of the RACE Fragment --- p.132 / Chapter 3.3 --- RESULTS / Chapter 3.3.1 --- Identification of novel cDNA by mRNA differential display --- p.133 / Chapter 3.4 --- DISCUSSIONS --- p.145 / General Conclusion --- p.147 / References --- p.149

by Chan Hung Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 143-151). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Abstract (Chinese version) --- p.v / Abbreviations --- p.viii / Content --- p.ix / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1. --- Free radical and free radical-mediated antitumor drugs --- p.1 / Chapter 2. --- Mitomycin C (MC) / Chapter 2.1 --- Drug actions of MC --- p.2 / Chapter 2.2 --- Adverse side effects of MC --- p.5 / Chapter 3. --- Doxorubicin (DOX) / Chapter 3.1 --- Drug actions of DOX --- p.7 / Chapter 3.2 --- Adverse side effects of DOX --- p.8 / Chapter 4. --- Antioxidants --- p.14 / Chapter 5. --- Effects of exogenous ATP on the antitumor activity of Doxorubicin and Mitomycin C / Chapter 5.1 --- Glutathione (GSH) and related enzymes --- p.17 / Chapter 5.2 --- Glutathione (GSH) and Anticancer Quinones --- p.19 / Chapter 5.3 --- Glutathione and the cardiac toxicity of the anticancer drugs --- p.20 / Chapter 5.4 --- Glutathione depletion in tumor cells by exogenous ATP --- p.21 / Chapter 6. --- Aim of research --- p.24 / Chapter CHAPTER TWO --- THE EFFECT OF ANTIOXIDANTS ON DOXORUBICIN- OR MITOMYCIN C-INDUCED CYTOTOXICITY ON HUMAN TUMOR AND NORMAL CELL LINES / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.3 --- Results --- p.36 / Chapter 2.4 --- Discussion --- p.60 / Chapter CHAPTER THREE --- STUDY OF CARDIOPROTECTIVE EFFECTS OF ANTIOXIDANTS AGAINST DOXORUBICIN- OR MITOMYCIN C-INDUCED TOXICITY BY LANGENDORFF PERFUEED ISOLATED RAT HEART MODEL / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Materials and Methods --- p.67 / Chapter 3.3 --- Results --- p.75 / Chapter 3.4 --- Discussion --- p.76 / Chapter CHAPTER FOUR --- THE EFFECT OF ANTIOXIDANTS DURING CHEMOTHERAPY OF DOXORUBICIN OR MITOMYCIN C IN TUMOR-BEARING MICE / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.3 --- Results --- p.83 / Chapter 4.4 --- Discussion --- p.93 / Chapter CHAPTER FIVE --- HISTOLOGICAL STUDY AND LIPID PEROXIDATION STUDY OF PROTECTIVE EFFECT OF ANTIOXIDANTS IN TUMOR-BEARING MICE TREATED WITH DOXORUBICIN OR MITOMYCIN C / Chapter 5.1 --- Introduction --- p.95 / Chapter 5.2 --- Materials and Methods --- p.98 / Chapter 5.3 --- Results --- p.103 / Chapter 5.4 --- Discussion --- p.117 / Chapter CHAPTER SIX --- EFFECT OF EXOGENOUS ATP ON THE ANTITUMOR ACTIVITY OF DOXORUBICIN AND MITOMYCIN C ON CULTURED HUMAN HEPATOMA CELLS / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.124 / Chapter 6.3 --- Results --- p.126 / Chapter 6.4 --- Discussion --- p.136 / Chapter CHAPTER SEVEN --- CONCLUSION / Chapter 7.1 --- Conclusion --- p.139 / Chapter 7.2 --- Future perspective --- p.141 / Bibliography --- p.142

丹參為雙子葉植物唇形科鼠尾草族植物的乾燥根及根莖。在中國，丹參為廣泛用於治療心血管疾病的藥用植物，而在西方，丹參也常作為一種輔助性藥物。《中國藥典2010版》收錄了35個以上含有丹參的複方或者方劑。在這些複方中，採用了富含丹酚酸和丹參酮的丹參水提物、乙醇提取物或兩者的混合物。丹參提取物具有較強的抗氧化作用，被認為在化學預防和化療的輔助治療中有一定用途。作為主要的丹參水溶性成分，熱敏感的丹酚酸在提取與加熱過程中可能會降解為其他丹酚酸。丹參水提取物的化學組成可能會在不同熱水提取溫度下有所不同，進而影響其藥理活性。在本研究中，通過加熱回流提取和在不同溫度下的微波提取（MAE-W）獲得了6種丹參水提取物，並對這些提取物進行化學成分和藥理分析，考察它們的抗氧化、抗凋亡和血管舒張作用。在這些提取物中，第三輪的微波提取物（100 oC）含有最多的丹酚酸和丹參酮，在1，1-二苯基-2-三硝基苯肼（DPPH）法和鐵還原/抗氧化能力（FRAP）法中具有最強的抗氧化活性，在2,2'-偶氮二(2-脒基丙烷)二鹽酸鹽（AAPH）誘導人血紅細胞的溶血實驗和過氧化氫誘導大鼠心肌細胞H9c2凋亡實驗中還顯示了最強的抑制作用，對大鼠腦基底動脈有最強的鬆弛效應。這些丹參水提取物的抗氧化作用與它們的血管舒張效應呈一定的線性關係（回歸係數r = 0.895 - 0.977）。通過多元線性回歸分析發現，丹參素可以作為丹參水提物的抗氧化和血管舒張功能的顯著性標記物，而丹參酮IIA則是抑制過氧化氫誘導大鼠心肌H9c2細胞凋亡的標記物。 / 作為丹參中主要的脂溶性成分，丹參酮在不同的腫瘤細胞系和荷瘤小鼠模型中展示了抗癌潛力。這些丹參酮的抗癌機制包括細胞週期阻滯，觸發半胱天冬酶（Caspase）依賴的內源性和外源性的凋亡途徑和絲裂原激活的蛋白激酶（MAPK）信號通路等。丹參新酮（miltirone）是從丹參中分離得到的松香烷型二萜醌類化合物，具有多種的藥理活性，如抗氧化，抗焦慮和抗腫瘤等。本研究評估了丹參新酮在人肝癌HepG2細胞系和P-糖蛋白（P-gp）過表達的阿霉素耐藥HepG2細胞系（R-HepG2）中的凋亡作用及其機制。丹參新酮在HepG2細胞中顯示了細胞毒性（EC₅₀值為7.06 微摩），而丹參新酮在抑制HepG2和R-HepG2細胞增殖中的濃度依賴性沒有顯著性差異。丹參新酮（1.56 - 6.25微摩）與阿霉素（DOX）對R-HepG2細胞的增殖具有協同效應，在達到50的生長抑制時，它們的聯合用藥指數為0.3至0.5。流式細胞術分析表明，丹參新酮降低了R-HepG2細胞中P-gp介導的阿霉素外排，分子對接研究表明該效果是通過抑制P-gp的藥物結合位點。在非壞死濃度（25微摩或以下），丹參新酮在HepG2和R-HepG2細胞中活化了Caspase依賴的凋亡途徑，誘導產生活性氧（ROS）和氧化應激，且觸發ROS介導的包括p38 MAPK，應激活化蛋白激酶/c-Jun氨基末端激酶（SAPK / JNK）以及細胞外調節激酶1和2在內的MAPK信號通路。綜上所述，在R-HepG2中丹參新酮是P-gp和細胞增殖的雙重抑制劑，顯示了其在治療肝癌（HCC）的潛力。 / 為了增加藥物開發的成功率，在藥物發現的早期階段應考察新化學實體（NCEs）的蛋白結合率，清除率，藥動學參數，以及藥物代謝相互作用等體內代謝參數。以往的研究已經顯示了從丹參中分離得到的四種主要丹參酮對人和大鼠的細胞色素P450酶介導的探針底物的代謝具有不同程度的抑制作用，需要注意丹參和其他藥物間的相互作用。本研究的另一目的是在人類肝微粒體中探討丹參新酮與探針底物間的細胞色素P450酶介導的代謝相關的相互作用。人肝微粒體孵育實驗結果表明丹參新酮對CYP1A2（IC₅₀值為 1.73微摩）和CYP2C9（IC₅₀值為8.61微摩）有中等強度的抑制，對CYP2D6（IC₅₀值為30.20微摩）和CYP3A4（IC₅₀值為33.88微摩）有弱的抑制。酶動力學和分子對接研究的結果進一步表明，丹參新酮為CYP1A2（Ki值為3.17微摩）的中等強度混合型抑制劑，是CYP2C9（Ki值為1.48微摩）的中等強度競爭型抑制劑，也是CYP2D6（Ki值為24.25微摩）和CYP3A4（Ki值為35.09微摩）的弱的混合型抑制劑。這些結果表明，應考慮丹參新酮與CYP1A2和CYP2C9代謝的藥物間的相互作用，但是可認為其與CYP2D6及CYP3A4代謝的藥物間幾乎不存在相互作用。 / 總之，本研究考察了不同提取方法對丹參提取物成份及其藥效的影響，確定了不同用途的丹參提取物的質控標記物。本研究還考察了丹參新酮體外抗肝癌的能力及其藥物代謝相互作用為基礎的類藥性,為其進一步的體內試驗提供了依據。 / Danshen, the dried root and rhizome of Salvia miltiorrhiza Bg. (Fam. Labiatae), is a widely used medicinal plant for the treatment of cardiovascular diseases in China and also a complementary medicine in the West. Danshen is indexed in the Pharmacopoeia of People’s Republic of China (2010 Edition), with more than 35 formulations and concoctions containing Danshen water-extracts, ethanolic extracts or their combination which are rich in phenolic acids and tanshinones with various contents. Danshen extracts have been considered for the use as an adjunct in chemoprevention and chemotherapy due to their strong antioxidant effects. Phenolic acids, the major water-soluble components in Danshen, are thermosensitive and may degrade to other phenolic acids during extractions upon heating. The chemical profiles of Danshen water-extracts may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts obtained. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were prepared for evaluation of their composition and pharmacological effects such as antioxidant, anti-apoptosis and vascular relaxation. Among these extracts obtained, the third-round MAE-W (100 °C) product, which was the last round product obtained by extracting the same crude material three times, had the highest contents of phenolic acids and tanshinones, with the strongest antioxidant activity estimated by 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing / antioxidant potential (FRAP) assay. This extract also possessed the strongest inhibitory effects on 2, 2'-azobis-2-amidino-propane (AAPH)-induced haemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r = 0.895 to 0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of water-extracts. / Tanshinones, the major lipid-soluble components isolated from Danshen, have been reported for their anti-cancer potential in various cell lines and tumor-bearing mice models. Their anti-cancer mechanisms are also well-studied, mainly through cell cycle arrest, caspase-dependent apoptotic pathways and mitogen activated protein kinase (MAPK) signaling pathways. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. This study evaluated the apoptotic effect of miltirone and the underlying mechanisms in a human hepatoma HepG2 cell line and its p-glycoprotein (P-gp)-overexpressed doxorubicin-resistant counterpart (R-HepG2). Miltirone showed similar cytotoxicity in HepG2 (EC₅₀ = 7.06 μM) and R-HepG2 (EC₅₀ = 12.0 μM), demonstrated synergistic effects (1.56 - 6.25 μM) with doxorubicin (DOX) on the growth inhibition of R-HepG2 (synergism: 0.3 < CI < 0.5 at 50 % inhibition). Flow cytometric analysis showed that miltirone decreased P-gp-mediated DOX efflux in R-HepG2, and molecular docking studies illustrated that this effect was through inhibition on the active site of P-gp. At non-necrotic concentrations (25 μM or below), miltirone activated caspase-dependent apoptotic pathways, and induced the generation of reactive oxygen species (ROS) and oxidative stress which triggered ROS-mediated MAPK signaling pathways, including p38 MAPK, stress-activated protein kinase / c-Jun N-terminal kinase (SAPK/JNK) and extracellular regulated kinase 1/2, in both HepG2 and R-HepG2 cells. It is therefore concluded that miltirone is a dual inhibitor on P-gp and cell proliferation in R-HepG2 cells, with potential for the treatment of human hepatocellular carcinoma (HCC). / In order to improve the successful rates in drug development, the in vivo metabolic parameters of new chemical entities (NCEs), such as protein bindings, clearance rate, pharmacokinetic parameters and metabolism-based drug-drug interactions, should be considered at the early stage of drug discovery. Previous studies have shown that major tanshinones isolated from Danshen inhibited the metabolism of model probe substrates of human and rat CYP450 enzymes, with potential in causing herb-drug interactions. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC₅₀ = 1.73 μM) and CYP2C9 (IC₅₀ = 8.61 μM), and weak inhibition on CYP2D6 (IC₅₀ = 30.20 μM) and CYP3A4 (IC₅₀ = 33.88 μM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (Ki = 1.48 μM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with Ki values of 3.17 μM, 24.25 μM and 35.09 μM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of human CYP450 isoforms. These findings suggested that miltirone may have potential drug-drug interactions with CYP1A2- and CYP2C9-metabolized drugs, and to a lesser extent with CYP2D6- and CYP3A4-metabolized drugs. / In conclusion, this study investigated the effects of Danshen water-extracts produced by different extraction methods on the chemical compositions and pharmacological activities, and consequently confirmed the biomarkers for the quality control of Danshen water-extracts for different medicinal uses. This study also demonstrated the anti-cancer potential of miltirone for HCC in vitro and the metabolism-based drug-drug interactions for its drug-likeness, which may provide useful and promising data for in vivo anti-cancer study of miltirone and further pre-clinical studies. / 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, Xuelin / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.v / Publications based on the work in this thesis --- p.viii / Acknowledgements --- p.x / Abbreviations --- p.xii / Table of Contents --- p.xv / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and carcinogenesis --- p.1 / Chapter 1.2 --- Reactive oxygen species and tumor progression & metastasis --- p.2 / Chapter 1.3 --- Antioxidant enzymes in chemoprevention and chemotherapy --- p.3 / Chapter 1.3.1 --- Glutathione and Glutathione reductase --- p.5 / Chapter 1.3.2 --- Glutathione Peroxidase --- p.5 / Chapter 1.3.3 --- Glutathione S-transferases --- p.6 / Chapter 1.3.4 --- NAD(P)H: quinone reductase 1 --- p.7 / Chapter 1.3.5 --- Heme oxygenase-1 --- p.8 / Chapter 1.3.6 --- Thioredoxin reductase --- p.9 / Chapter 1.3.7 --- Superoxide Dismutase --- p.10 / Chapter 1.3.8 --- Catalase --- p.11 / Chapter 1.4 --- Medicinal uses of Danshen --- p.12 / Chapter 1.5 --- Analysis of Danshen and its components --- p.14 / Chapter 1.6 --- Antioxidant effects of Danshen extract and its bioactive compounds in chemoprevention and chemotherapy-related disease --- p.19 / Chapter 1.7 --- Anti-cancer effects of tanshinones isolated from Danshen --- p.21 / Chapter 1.7.1 --- Tanshinone IIA --- p.22 / Chapter 1.7.2 --- Tanshinone I --- p.26 / Chapter 1.7.3 --- Cryptotanshinone --- p.27 / Chapter 1.7.4 --- Dihydrotanshinone --- p.27 / Chapter 1.8 --- Metabolism / disposition of Danshen and its major active ingredients --- p.28 / Chapter 1.9 --- Herb-drug interactions with Danshen --- p.31 / Chapter 1.10 --- Effects of Danshen (and its major active ingredients) on model probe substrates of CYP isoforms --- p.33 / Chapter 1.11 --- CYPs induction by Danshen and its active components --- p.38 / Chapter 1.12 --- Effects of Danshen / active ingredients on drug transporter proteins --- p.40 / Chapter 1.13 --- CYP450 inhibition screening for new chemical entity --- p.42 / Chapter 1.14 --- Molecular docking analysis --- p.44 / Chapter 1.15 --- The Aim of this study --- p.45 / Chapter Chapter 2 --- Quantitative and qualitative studies to evaluate the efficiency of different heat water-extractions --- p.48 / Chapter 2.1 --- Introduction --- p.48 / Chapter 2.2 --- Materials and methods --- p.51 / Chapter 2.2.1 --- Materials and apparatus --- p.51 / Chapter 2.2.2 --- Extraction procedures --- p.51 / Chapter 2.2.3 --- HPLC analysis --- p.54 / Chapter 2.2.4 --- DPPH assay and FRAP assay --- p.54 / Chapter 2.2.5 --- Inhibition of 2,2'-azobis-2-amidinopropane (AAPH)-induced haemolysis in human red blood cells --- p.55 / Chapter 2.2.6 --- Protective effects on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells --- p.56 / Chapter 2.2.7 --- Vasodilation effects on rat basilar artery --- p.57 / Chapter 2.2.8 --- Statistical analysis --- p.58 / Chapter 2.3 --- Results and Discussion --- p.59 / Chapter 2.3.1 --- Chemical profiles analyzed by HPLC analysis --- p.59 / Chapter 2.3.2 --- DPPH assay and FRAP assay --- p.63 / Chapter 2.3.3 --- Inhibition of AAPH-induced haemolysis --- p.65 / Chapter 2.3.4 --- Protective effects on hydrogen peroxide-induced apoptosis --- p.69 / Chapter 2.3.5 --- Vasodilation effects on rat basilar artery --- p.71 / Chapter 2.3.6 --- Multiple linear regression analysis --- p.76 / Chapter Chapter 3 --- Effects of miltirone on cell proliferation in a hepatoma HepG2 cell line and its doxorubicin-resistant counterpart --- p.83 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Chemicals --- p.87 / Chapter 3.2.2 --- Cell culture --- p.87 / Chapter 3.2.3 --- Cell viability test --- p.88 / Chapter 3.2.4 --- Drug-efflux study by flow cytometry --- p.89 / Chapter 3.2.5 --- Molecular docking study and Ligand-based prediction --- p.90 / Chapter 3.2.6 --- Measurement of ROS generation by confocal microscopy and flow cytometry --- p.91 / Chapter 3.2.7 --- GSH and GSSG determination for oxidative stress --- p.93 / Chapter 3.2.8 --- Apoptosis-related proteins expression detected by Western blotting analysis --- p.94 / Chapter 3.2.9 --- Data analysis --- p.96 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- Cytotoxicity in hepatoma cells --- p.97 / Chapter 3.3.2 --- Drug-efflux study by flow cytometry --- p.104 / Chapter 3.3.3 --- Molecular docking study and Ligand-based prediction --- p.108 / Chapter 3.3.4 --- ROS generation --- p.113 / Chapter 3.3.5 --- Determination of GSH/GSSG ratio --- p.117 / Chapter 3.3.6 --- Caspase-dependent apoptosis. --- p.121 / Chapter 3.3.7 --- Phosphorylation of MAPKs --- p.126 / Chapter 3.4 --- Discussion --- p.134 / Chapter Chapter 4 --- Enzyme kinetic and molecular docking studies of miltirone on major human cytochrome P450 isozymes inhibitions --- p.139 / Chapter 4.1 --- Introduction --- p.139 / Chapter 4.2 --- Material and Methods --- p.141 / Chapter 4.2.1 --- Materials and Reagents --- p.141 / Chapter 4.2.2 --- Incubation conditions --- p.142 / Chapter 4.2.3 --- Samples preparation --- p.143 / Chapter 4.2.4 --- HPLC analysis --- p.143 / Chapter 4.2.5 --- CYP inhibition and enzymatic kinetic study --- p.144 / Chapter 4.2.6 --- Molecular docking analysis --- p.145 / Chapter 4.2.7 --- Data analysis --- p.146 / Chapter 4.3 --- Results --- p.148 / Chapter 4.3.1 --- CYP inhibition and enzymatic kinetic study --- p.148 / Chapter 4.3.2 --- Molecular docking study of miltirone --- p.167 / Chapter 4.4 --- Discussions --- p.184 / Chapter Chapter 5 --- General discussion --- p.188 / References --- p.195

何首烏為寥科多年生藤本植物，而中藥何首烏則為該植物的乾燥塊根。常用於治療白髮及與老年化相關的疾病。然而，何首烏的治療機理卻少有報導。本文對何首烏的粗提物及其主要生物活性成份2,3,5,4' -四羥基二萃乙烯-2-0-ß-D-葡萄糖苷(THSG) 在老鼠及人類的黑色素細胞中影響黑色素生成機理及細胞毒性進行了深入研究。 / 利用高效液相層析連接質譜儀的測定， THSG 在水及乙醇粗提物中含量分別為0.064 及0.75 的百分比。由於THSG 在水及乙醇粗提物中佔有一定份量，所以它對粗提物所產生的生物及生化反應有著重要的影響。在低於細胞毒範圍的劑量內，何首烏粗提物及THSG 能降低老鼠黑色素細胞株melan-a 的左旋多巴(L-DOPA)的轉化反應。在細胞毒性檢測中，水粗提物及THSG 在<100 μg/ml的劑量下均未有對至少5 種黑色素細胞株及黑色素瘤細胞造成傷害。然而，乙醇粗提物的細胞毒卻是水粗提物或THSG 的3-4 倍。 / 在無細胞系統中， THSG 在可逆轉的情況下抑制酪氨酸酶將左旋多巴轉化成黑色素。在細胞系統中，它也能阻止由蛋白激醋A (PKA)引發的黑色素生成反應。THSG 在老鼠及人類黑色素細胞中的中位值抑制率(lC₅₀)分別為123.0 μM及61.5 μM。 THSG 抑制酪氮酸醋的能力展現在黑色素細胞/角質細胞的共培養比在單黑色素細胞培養中更明顯。 / 調控酪氯酸臨可以在脫氧核糖核酸(DNA)轉錄及翻譯後修飾兩方面達成。在DNA 轉錄中，小眼球相關轉錄因數(MITF)的減少導致酪氨酸酶的表達隨著THSG 濃度而減少。翻譯後酪氮酸臨主要依靠蛋白激臨C-ß (PKC-ß)使其磷酸化，從而增加酪氯酸酶/酪氯酸酶相關蛋白-1(TRP-1 )組成複合蛋白。然而THSG 均減少蛋白激酶 C-ß的表達及酪氮酸酪/酪氮酸醋相關蛋白-1 所組成的複合蛋白。 另一方面， THSG 卻沒有影響酪氯酸酶蛋白在內質網/高爾基氏複合體內的糖基化及內涵體與溶酶體間的運輸。 / 總而言之，本文首次展示何首烏粗提物及THSG 在單細胞培養及共培養細胞的系統下抑制黑色素生成。THSG 能在可逆轉的抑制機制下阻止酪氯酸酶作出反應。而在PKA 引發的黑色素生成反應中， THSG 也能在DNA 轉錄及翻譯後修飾等過程中減低酪氯酸酶的活性。 / Radix Polygoni Multiflori, the dried root of Polygonum multiflorum (PM), is well documented for its clinical effects in treating various diseases associated with aging and hair graying, but the evidence based-mechanisms remain largely unknown. In this study, PM was extracted with water and 70% ethanol and a major constituent, 2,3,5,4'-tetrahydroxystilbene-2-0-I3-D-glucoside (THSG) of about 0.064% and 0.75%, respectively, were found in these extracts as analyzed by high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The melanogenic properties and cytotoxicity of the two extracts and THSG were evaluated using murine and human melanocytes. / Both water and ethanol extracts of PM and THSG showed a dose-dependent anti-melanogenic activity in an in vitro murine melan-a melanocyte assay for reduction of L-DOPA conversion by tyrosinase. Of at least 5 melanoma and melanocyte cell lines tested, both water PM extract and THSG were relatively safe, which at doses <100 μg/ml did not demonstrate any significant cytotoxic effects. On the other hand, ethanol PM extract was about 3-4 folds more cytotoxic. / Tyrosinase is the rate-limiting enzyme for melanogenesis. In a cell-free kinetic analysis, THSG inhibited tyrosinase activity in a reversible and non-competitive manner. At the cellular level, this inhibition is mediated through a PKA-dependent melanogenic pathway, as well as in a dose-dependent manner, with IC₅₀ = 123.0 μM and 61.5 μM for murine and human melanocytes, respectively. Tyrosinase was much more sensitive to the inhibitory effect of THSG in the melanocytelkeratinocyte co-culture system than in the melanocyte mono-culture system. / Functional tyrosinase is regulated at both transcriptional and post-translational modification levels. At the transcription level, THSG reduced expression of microphthalmia-associated transcription factor (MITF) esulted in a down-regulation of tyrosinase expression. At the post-translational modification level, THSG inhibited expression of PKC-β which is responsible for tyrosinase phosphorylation, and enhanced tyrosinase/TRP-1 complex formation. On the hand, THSG did not affect glycosylation of tyrosinase nor its trafficking from ER/Golgi to endosomal/ lysosomal compartments. / Taken our results together, the anti-melanogenic property of PM extracts and THSG were firstly demonstrated in both mono- and co-culture system using murine or human melanocytes and keratinocytes. THSG is a reversible and competitive inhibitor, which lowered the tyrosinase activity at both transcription and post-translational modification levels via PKA-mediated melanogenesis. / 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. / Cheung, Wing Ki. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 160-174). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter 1 / General introduction / Chapter 1.1 --- Anatomy and Physiology of the Skin --- p.5 / Chapter 1.1.1 --- Epidermis --- p.5 / Chapter 1.1.1.1 --- Stratum basale --- p.6 / Chapter 1.1.1.2 --- Stratum spinosum --- p.6 / Chapter 1.1.1.3 --- stratum granulosum --- p.7 / Chapter 1.1.1.4 --- Stratum corneum --- p.7 / Chapter 1.1.2 --- Dermis --- p.8 / Chapter 1.2 --- Melanogenesis of the Skin --- p.9 / Chapter 1.2.1 --- History of melanogenesis study --- p.9 / Chapter 1.2.2 --- Today's melanogenesis study --- p.10 / Chapter 1.3 --- Hyperpigmentary Disorders --- p.14 / Chapter 1.3.1 --- Malasma --- p.14 / Chapter 1.3.2 --- Lentigines --- p.15 / Chapter 1.3.2.1 --- Lentigo simplex --- p.16 / Chapter 1.3.2.2 --- Lentigo senilis etActinicus --- p.16 / Chapter 1.3.3 --- Post-inflammatory hyperpigmentation --- p.17 / Chapter 1.4 --- Current Available Treatment for Hyperpigmentation --- p.18 / Chapter 1.4.1 --- Topical treatment and their strategies --- p.18 / Chapter 1.4.1.1 --- Inhibition of tyrosinase activity --- p.18 / Chapter 1.4.1.2 --- Antioxidation --- p.21 / Chapter 1.4.1.3 --- Melanosome transfer inhibition --- p.22 / Chapter 1.4.1.4 --- Stimulation of desquamation --- p.22 / Chapter 1.4.2 --- Laser treatment and their strategies --- p.23 / Chapter 1.4.3 --- Sunscreen --- p.24 / Chapter 1.5 --- Theories and the Treatment of Hyperpigmentation with Chinese Herbal Medicine --- p.25 / Chapter 1.6 --- Testing Systems --- p.26 / Chapter 1.7 --- Aims and Objectives of Study --- p.27 / Chapter 2 / Chemical Properties of THSG / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.1.1 --- Radix Polygoni multiflori --- p.30 / Chapter 2.1.2 --- 2,3,5,4'-tetrahydroxystilbene glucoside (THSG) --- p.31 / Chapter 2.1.2.1 --- Stilbene --- p.32 / Chapter 2.1.2.2 --- Chemical properties of THSG --- p.34 / Chapter 2.1.3 --- Objectives --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Plant materials --- p.36 / Chapter 2.2.2 --- Extraction --- p.36 / Chapter 2.2.3 --- High performance liquid chromatography (HPLC) analysis --- p.36 / Chapter 2.2.4 --- Enzymatic hydrolysis of THSG and salicin --- p.37 / Chapter 2.2.5 --- HPLC/MS analysis --- p.38 / Chapter 2.2.6 --- Benedict's test --- p.38 / Chapter 2.2.7 --- Enzymatic oxidation --- p.38 / Chapter 2.2.8 --- Thin layer chromatography (TLC) analysis --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- The THSG content in water and alcohol extracts of PM --- p.40 / Chapter 2.3.2 --- The stability of THSG against oxidation --- p.41 / Chapter 2.3.3 --- Enzymatic hydrolysis ofTHSG --- p.42 / Chapter 2.4 --- Discussion --- p.46 / Chapter 3 / The Melanogenic inhibitory mechanisms of Radix Polygonum multiflorum (PM) extracts and THSG in murine melanocyte / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.1.1 --- Murine melanocyte --- p.47 / Chapter 3.1.2 --- Melanogenesis --- p.48 / Chapter 3.1.2.1 --- Factors affecting Melanogenesis --- Oxidation --- p.49 / Chapter 3.1.2.2 --- Factors affecting Melanogenesis --- UV radiation --- p.50 / Chapter 3.1.2.3 --- Factors affecting melanogenesis---- Cellular regulation --- p.51 / Chapter 3.1.2.3.1 --- The regulation of transcription, translation, and post-translational modification of tyrosianse --- p.51 / Chapter 3.1.2.3.2 --- PKA-, PKC-, and PKG- melangenic pathways --- p.56 / Chapter 3.1.3 --- Kinetic analysis of tyrosinase --- p.57 / Chapter 3.1.4 --- Objectives --- p.58 / Chapter 3.2 --- Materials and Methods --- p.59 / Chapter 3.2.1 --- Cell culture --- p.59 / Chapter 3.2.2 --- Kinetic analysis of tyrosinase activity inhibition --- p.60 / Chapter 3.2.3 --- SRB assay --- p.61 / Chapter 3.2.4 --- L -DOPA conversion assay --- p.62 / Chapter 3.2.5 --- Melanin production measurement --- p.62 / Chapter 3.2.6 --- ROS detection by flow cytometer --- p.63 / Chapter 3.2.7 --- In-situ tyrosinase activity assay --- p.63 / Chapter 3.2.8 --- Western blotting (WB) analysis --- p.64 / Chapter 3.2.9 --- Immunofluorescence microscopy --- p.66 / Chapter 3.2.10 --- Glycosylation analysis --- p.67 / Chapter 3.2.11 --- Co-Immunoprecipitation --- p.68 / Chapter 3.2.12 --- Radix Polygonum Multiflorum and THSG metabolite collection from rat serum --- p.69 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Enzyme kinetic study of the catalysis of L-DOPA by murine melanocyte lysate --- p.71 / Chapter 3.3.2 --- Inhibitory effect of crude PM preparations and THSG on tyrosinase activity and melanin synthesis in murine melan-a melanocytes --- p.74 / Chapter 3.3.3 --- Effect of THSG on H₂0₂- induced oxidation --- p.77 / Chapter 3.3.4 --- THSG inhibits PKA-induced melanogenesis --- p.78 / Chapter 3.3.5 --- Reduction of in situ tyrosinase activity in PKA-induced melanogenesis --- p.82 / Chapter 3.3.6 --- Alternation of melanogenic proteins --- p.85 / Chapter 3.3.7 --- THSG does not alter the tyrosinase trafficking in ER and Golgi --- p.89 / Chapter 3.3.8 --- THSG does not alter the tyrosinase trafficking in endosomal lysosomal compartments --- p.93 / Chapter 3.3.9 --- Glycosylation analysis --- p.95 / Chapter 3.3.10 --- Reduction of interaction between tyrosinase and TRP-1 to form heterodimeric complexes --- p.97 / Chapter 3.3.11 --- The metabolite of PM water extract and THSG maintained the in vitro tyrosinase activity --- p.101 / Chapter 3.4 --- Discussion --- p.103 / Chapter 4 / The Inhibitory Effect of THSG on Melanogenesis in Monolayer Culture of Human Melanocytes and in Co-culture of Melanocyte-Keratinocyte / Chapter 4.1 --- Introduction --- p.110 / Chapter 4.1.1 --- Human melanocyte --- p.110 / Chapter 4.1.1.1 --- The origin and the development of melanocyte --- p.110 / Chapter 4.1.1.2 --- Morphology, body site distribution and histological location --- p.111 / Chapter 4.1.1.3 --- In vitro growth of human melanocyte --- p.112 / Chapter 4.1.1.3.1 --- Lifespan vs. culture conditions --- p.113 / Chapter 4.1.1.3.2 --- Lifespan vs. donor age and skin type --- p.114 / Chapter 4.1.1.4 --- Modulation of pigmentation in response to stress --- p.114 / Chapter 4.1.1.5 --- Difference between human and murine TRPs --- p.115 / Chapter 4.1.2 --- Keratinocyte-Melanocyte interaction --- p.117 / Chapter 4.1.2.1 --- Release of melanogenic factors --- p.117 / Chapter 4.1.2.2 --- Release of survival and proliferating factors --- p.118 / Chapter 4.1.2.3 --- Melanosome transfer determines the cutaneous pigmentation --- p.118 / Chapter 4.1.2.3.1 --- Molecular events during melanosome transfer --- p.119 / Chapter 4.1.2.4 --- Others --- p.121 / Chapter 4.1.3 --- Objectives --- p.121 / Chapter 4.2 --- Materials and Methods --- p.122 / Chapter 4.2.1 --- Cell Culture --- p.122 / Chapter 4.2.1.1 --- Human melanocytes isolation and cultivation --- p.122 / Chapter 4.2.1.2 --- Immortalized keratinocytes - HaCaT cells --- p.123 / Chapter 4.2.1.3 --- Co-culture of melanocytes and HaCaT cells --- p.124 / Chapter 4.2.1.3.1 --- Monolayer co-culture --- p.124 / Chapter 4.2.1.3.2 --- Two-layer co-culture --- p.124 / Chapter 4.2.2 --- SRB assay --- p.125 / Chapter 4.2.3 --- L-DOPA conversion assay --- p.125 / Chapter 4.2.4 --- Western blotting (WB) analysis --- p.125 / Chapter 4.2.5 --- Light microscopy and immunofluorescent microscopy --- p.126 / Chapter 4.2.6 --- cAMP immunoassay --- p.126 / Chapter 4.3 --- Results --- p.128 / Chapter 4.3.1 --- The isolation and purification of human melanocytes --- p.128 / Chapter 4.3.2 --- Aging vs. Tyrosinase activity and melanin content --- p.131 / Chapter 4.3.3 --- Inhibitory effect of THSG in tyrosinase activity in human melanocyte --- p.133 / Chapter 4.3.4 --- Alternation of melanogenic proteins --- p.135 / Chapter 4.3.5 --- Sensitization of melanocytes to THSG treatment in co-culture system --- p.138 / Chapter 4.3.6 --- Induction of melanocyte dendricity in co-culture system --- p.140 / Chapter 4.3.7 --- THSG inhibited cAMP induction by forskolin and paracrine factors from keratinocytes --- p.141 / Chapter 4.4 --- Discussion --- p.143 / Discussion / Chapter 5.1 --- Discussion --- p.149 / References --- p.160