胰腺癌是一種致死率極高的惡性疾病,在全世界所有的癌症中死亡率排列第八, 在美國排列第四。 很多因素造成了胰腺癌較差的預後,其中包括: 早期檢出率極低; 較少胰腺癌患者的腫瘤適宜手術切除;高轉移率;以及對傳統放療和化療具有較高抗性等。 因此,發展新的治療藥物迫在眉睫。 / 近年來, 植物藥以及從這些植物藥裡分離出的天然化合物, 單獨使用或者與傳統化療藥物合併使用時, 都顯示出對不同類型的癌症具有較好療效。植物藥毛萼香茶菜(唇形科)含有豐富的具有抗癌活性的二萜類化合物。其中毛萼乙素(EriB) 是一個擁有最好抗癌活性的對映-貝殼杉烷型二萜化合物。 基於此背景, 本研究的目標為:利用胰腺癌體外體內模型, 研究EriB的抗胰腺癌活性以及誘導胰腺癌細胞凋亡的機理。 / 體外實驗中, EriB對四種胰腺癌細胞株都顯示了顯著的細胞毒活性,其活性與化療藥物喜樹堿類似。其中, EriB對胰腺癌細胞株CAPAN-2活性最強, 半數致死濃度IC₅₀為0.73 μM。細胞凋亡特徵:細胞核凝聚, 磷脂醯絲氨酸外翻, DNA梯狀條帶以及片斷化,在EriB誘導的胰腺癌細胞株CAPAN-2中出現。此外, EriB還造成癌細胞在細胞週期G2/M期的阻滯。機理研究發現, EriB是通過啟動絲裂原活化蛋白激酶(MAPK), caspase及 p53信號通路來誘導細胞凋亡和細胞週期阻滯的。抗凋亡蛋白與促凋亡蛋白比率(bcl-2/bak)的減少也可能對啟動細胞凋亡內途徑發揮一定作用。除此以外, EriB對癌細胞的細胞毒活性及致凋亡作用依賴于活性氧分子(ROS)的產生。在對細胞進行抗氧化劑預處理的實驗中發現, 只有含巰基基團的抗氧化劑能夠有效的阻斷EriB對癌細胞的活性。進一步實驗證明, EriB對細胞內兩個抗氧化系統: 谷胱甘肽系統及硫氧還蛋白系統的抑制作用導致了ROS在癌細胞中的積聚。同時,ROS的產生啟動了MAPK,熱休克蛋白70以及caspase信號通路,卻抑制了NFκB通路。 / 動物體內實驗證實, 每天對胰腺癌細胞移植瘤裸鼠進行腹腔注射EriB(2.5 毫克/千克),能有效的抑制腫瘤生長, 並且對心臟,肝臟和腎臟沒有引起顯著毒性。 對腫瘤組織的分析表明, 給藥組(EriB)比溶劑對照組出現更多的細胞凋亡, 並產生較多的ROS積聚。 / 綜上所述, 本項研究首次闡述了EriB具有顯著的體內外抗胰腺癌活性。機理研究證明, EriB抑制胰腺癌細胞內兩個含巰基基團的抗氧化系統, 從而導致ROS在細胞中積聚, 並啟動(或抑制)了包括MAPK, p53, caspase和NFB在內的信號通路, 最終導致癌細胞死亡。 此外, 動物體內研究證明EriB的抗腫瘤生長活性和低毒性, 令該化合物具有潛力進一步研究發展成為抗胰腺癌的新藥物。 / Pancreatic cancer is the fourth and eighth leading cause of cancer-related deaths in the U.S. and worldwide, respectively. Its poor prognosis is attributed to its late diagnosis, limitation to surgical resection, aggressive local invasion, and early metastases, as well as high resistance to chemotherapy and radiotherapy. Therefore, a search for an alternative to therapeutic agents is in desperate need. / In recent years, herbal medicines or natural compounds isolated from herbs either used alone or in combination with conventional anti-cancer agents have been shown to have beneficial effects on various cancers. In this context, the Chinese herb Isodon eriocalyx (Dunn.) Hara (family Lamiaceae) is a well-known source of anti-cancer diterpenoids, the most potent one being Eriocalyxin B (EriB, an ent-kauranoid). Therefore, the aims of the present study are to investigate the anti-tumor activities of EriB in human pancreatic adenocarcinoma cells and tumor-bearing mouse model, as well as the underlying mechanisms. / Our results showed that EriB exhibited significant cytotoxic effects on four pancreatic adenocarcinoma cell lines, with potencies being comparable to that of chemotherapeutic agent camptothecin. EriB had the most potent cytotoxicity in CAPAN-2 cells with IC₅₀ = 0.73 μM. The hallmark features of apoptosis, such as nuclear condensation, translocation of phosphatidylserine, DNA laddering, and DNA fragmentation were observed in EriB-treated CAPAN-2 cells. On the other hand, EriB also induced G2/M phase cell cycle arrest. Mechanistic studies revealed that EriB induced apoptosis and cell cycle arrest through the activation of MAPKs (p38, ERK1/2), caspase cascade, and p53/p21/cdk1-cyclinB1 signaling pathways. A decrease in the ratio of anti-apoptotic to pro-apoptotic proteins (bcl-2/bak) also contributed to the activation of intrinsic apoptotic pathway. Further investigation showed that EriB-induced cytotoxic and apoptotic effects were dependent on reactive oxygen species (ROS) production. Such demonstrated effects could be inhibited by pre-treatment with thiol-containing antioxidants. Furthermore, EriB induced ROS was mediated via the inhibition of two main antioxidant systems, namely glutathione and thioredoxin systems. EriB-mediated ROS activated multiple targets or signal pathways, including MAPK, heat shock protein (Hsp) 70, and caspase cascade, while inhibiting the NFκB pathway. / On the other hand, in vivo study demonstrated that daily intraperitoneal administration of EriB (2.5mg/kg/day) in human pancreatic tumor xenografts BALB/c nude mice significantly inhibited tumor growth, but without having toxicity in the heart, liver and kidney. In addition, EriB treatments induced in vivo cell apoptosis and superoxide production as observed in tumor tissues. / In conclusion, the present study reports for the first time that EriB has possessed anti-proliferative activities in pancreatic cancer cells. The anti-proliferative effects of EriB on CAPAN-2 cells could be attributable to the regulation of cellular apoptosis and cell cycle arrest. The inhibitory effects of EriB on two antioxidant systems result in the accumulation of ROS, which in turn activate MAPK, p53, Hsp70 and caspase cascade, while inhibiting NFB pathway and finally leading to pancreatic cancer cell death. Meanwhile, in vivo study further confirms the anti-tumor properties of EriB, suggesting that EriB could be considered as a potential chemotherapeutic agent for patients with pancreatic cancer. / 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. / Li, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 207-230). / Abstracts also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Publications --- p.vi / Acknowledgements --- p.vii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- The pancreas --- p.2 / Chapter 1.1.1 --- Anatomy of the pancreas --- p.2 / Chapter 1.1.2 --- Histology of the pancreas --- p.4 / Chapter 1.1.3 --- Exocrine pancreas --- p.5 / Chapter 1.1.3.1 --- Structure of secretory acini, ducts and stroma in pancreas --- p.5 / Chapter 1.1.3.2 --- Functions of exocrine pancreas --- p.6 / Chapter 1.1.4 --- Endocrine pancreas --- p.9 / Chapter 1.1.4.1 --- Structure of islets cells --- p.10 / Chapter 1.1.4.2 --- Functions of endocrine pancreas --- p.10 / Chapter 1.1.5 --- Disorders of the pancreas --- p.11 / Chapter 1.2 --- Pancreatic cancer --- p.14 / Chapter 1.2.1 --- Epidemiology --- p.14 / Chapter 1.2.2 --- The risks and causes of pancreatic cancer --- p.15 / Chapter 1.2.3 --- Signs and symptoms of pancreatic cancer --- p.18 / Chapter 1.2.4 --- Types of pancreatic cancer --- p.19 / Chapter 1.2.5 --- Diagnosis of pancreatic cancer --- p.21 / Chapter 1.2.6 --- Staging of pancreatic cancer --- p.27 / Chapter 1.3 --- Treatments for pancreatic cancer --- p.29 / Chapter 1.3.1 --- Surgery --- p.29 / Chapter 1.3.2 --- Chemotherapy --- p.30 / Chapter 1.3.2.1 --- 5-fluorouracil (5-FU) --- p.32 / Chapter 1.3.2.2 --- Gemcitabine (Gem) --- p.33 / Chapter 1.3.2.3 --- Other cytotoxic agents --- p.34 / Chapter 1.3.3 --- Radiotherapy --- p.35 / Chapter 1.3.4 --- Target therapies --- p.37 / Chapter 1.3.4.1 --- Antiangiogenic therapy --- p.37 / Chapter 1.3.4.2 --- Epidermal growth factor receptor (EGFR) signaling inhibitors --- p.39 / Chapter 1.3.4.3 --- Hedgehog and Notch signaling pathways inhibitors --- p.41 / Chapter 1.3.5 --- Gene therapy --- p.42 / Chapter 1.3.6 --- Immunotherapy --- p.45 / Chapter 1.3.7 --- Combination therapies --- p.46 / Chapter 1.4 --- Molecular targets for pancreatic cancer chemotherapy --- p.49 / Chapter 1.4.1 --- Therapies-induced apoptosis --- p.49 / Chapter 1.4.1.1 --- Caspase cascade and bcl-2 Family --- p.49 / Chapter 1.4.1.2 --- Role of mitogen-activated protein kinases (MAPKs) in apoptosis --- p.50 / Chapter 1.4.2 --- Nuclear factor-κB activation in pancreatic cancer --- p.50 / Chapter 1.4.3 --- The PI3K and AKT pathway --- p.51 / Chapter 1.4.4 --- JAK/STAT pathway --- p.51 / Chapter 1.4.5 --- Other molecular targets --- p.52 / Chapter 1.5 --- Herbal medicine as an alternative treatment for cancer treatment --- p.53 / Chapter 1.5.1 --- Herbal medicines for different types of cancer treatment --- p.53 / Chapter 1.5.2 --- Herbal medicines for pancreatic cancer treatment --- p.59 / Chapter 1.6 --- Introduction of Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.1 --- Background of Isodon genus and Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.2 --- Diterpenoids from Isodon species and their activities --- p.62 / Chapter 1.6.3 --- The potential anti-cancer activity of Eriocalyxin B, a diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara --- p.62 / Chapter 1.7 --- Aims and objectives of this study --- p.66 / Chapter Chapter 2 --- Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways --- p.67 / Chapter 2.1 --- Introduction --- p.68 / Chapter 2.2 --- Materials and methods --- p.71 / Chapter 2.2.1 --- Preparation and quality control of Eriocalyxin B --- p.71 / Chapter 2.2.2 --- Materials --- p.72 / Chapter 2.2.3 --- Cell culture --- p.72 / Chapter 2.2.4 --- Preparation of human peripheral blood mononuclear cells (PBMC) --- p.73 / Chapter 2.2.5 --- Cytotoxicity assay --- p.75 / Chapter 2.2.6 --- Hoechst 33258 staining for morphological evaluation --- p.76 / Chapter 2.2.7 --- DNA fragmentation detection by DNA ladder --- p.76 / Chapter 2.2.8 --- Cell death detection ELISA --- p.77 / Chapter 2.2.9 --- Apoptosis detection by flow cytometry --- p.78 / Chapter 2.2.10 --- Cell cycle analysis by flow cytometry --- p.78 / Chapter 2.2.11 --- Western blot analysis --- p.79 / Chapter 2.2.12 --- Statistical analysis --- p.80 / Chapter 2.3 --- Results --- p.81 / Chapter 2.3.1 --- EriB induces cytotoxic effect in human pancreatic cancer cells --- p.81 / Chapter 2.3.2 --- EriB induces apoptosis in CAPAN-2 cells --- p.85 / Chapter 2.3.3 --- Activation of pro-apoptotic caspases in EriB-treated CAPAN-2 cells --- p.89 / Chapter 2.3.4 --- Modulation of bcl-2/bak ratio in EriB-treated CAPAN-2 cells --- p.92 / Chapter 2.3.5 --- EriB causes G2/M cell cycle arrest --- p.94 / Chapter 2.3.6 --- EriB modulates expression of G2/M cell cycle regulatory proteins through activation of the p53 pathway --- p.96 / Chapter 2.4 --- Discussion --- p.99 / Chapter Chapter 3 --- Eriocalyxin B induces apoptosis in pancreatic cancer CAPAN-2 cells via mediation of reactive oxygen species --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and methods --- p.113 / Chapter 3.2.1 --- Materials --- p.113 / Chapter 3.2.2 --- Cell culture and MTT assay --- p.113 / Chapter 3.2.3 --- Apoptosis detection by flow cytometry --- p.114 / Chapter 3.2.4 --- Reactive oxygen species (ROS) detection by flow cytometry --- p.114 / Chapter 3.2.5 --- Glutathione assessment --- p.115 / Chapter 3.2.6 --- Glutathione peroxidase (GPx) activity detection --- p.116 / Chapter 3.2.7 --- Thioredoxin reductase (TrxR) activity detection --- p.116 / Chapter 3.2.8 --- Nuclear and cytosolic fractionation --- p.117 / Chapter 3.2.9 --- Western blot analysis --- p.117 / Chapter 3.2.10 --- Electrophoretic mobility shift assay --- p.119 / Chapter 3.2.11 --- Statistical analysis --- p.119 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Thiol-containing antioxidants inhibits EriB-induced cytotoxic effects --- p.120 / Chapter 3.3.2 --- Thiol-containing antioxidants inhibits EriB-induced apoptotic effects --- p.122 / Chapter 3.3.3 --- Effects of EriB on hydrogen peroxide production --- p.125 / Chapter 3.3.4 --- EriB depletes glutathione level and suppresses GPx activity --- p.128 / Chapter 3.3.5 --- EriB inhibits thioredoxin system and activates ASK1 --- p.130 / Chapter 3.3.6 --- EriB increases Hsp70 and cleaved-PARP expression through ROS --- p.134 / Chapter 3.3.7 --- EriB inhibits NFkB pathway in CAPAN-2 cells --- p.137 / Chapter 3.4 --- Discussion --- p.142 / Chapter Chapter 4 --- In vivo study of the anti-tumor efficacy of Eriocalyxin B in human pancreatic tumor xenograft model --- p.149 / Chapter 4.1 --- Introduction --- p.150 / Chapter 4.2 --- Materials and methods --- p.154 / Chapter 4.2.1 --- Establishment of a subcutaneous pancreatic cancer xenograft model --- p.154 / Chapter 4.2.2 --- Evaluation of the effects of EriB on tumor growth --- p.155 / Chapter 4.2.2.1 --- Pilot study for EriB and camptothecin treatment --- p.155 / Chapter 4.2.2.2 --- Confirmation study of effective dose of EriB --- p.156 / Chapter 4.2.2.3 --- Dose-comparison study of CPT-11 --- p.156 / Chapter 4.2.2.4 --- Comparison study of EriB and CPT-11 treatments --- p..157 / Chapter 4.2.3 --- Measurement of plasma-specific enzyme levels --- p.157 / Chapter 4.2.4 --- Assays of terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) --- p..158 / Chapter 4.2.5 --- Histological evaluation --- p.159 / Chapter 4.2.6 --- Detection of superoxide by DHE staining --- p.159 / Chapter 4.2.7 --- Establishment of an orthotopic model (SW1990) of pancreatic cancer and detection of the plasma biomarker CA19-9 --- p.160 / Chapter 4.2.7.1 --- Detection of CA19-9 expression by immunofluorescent staining and western blot --- p.161 / Chapter 4.2.7.2 --- Establishment of an orthotopic pancreatic cancer xenograft model by SW1990 cells --- p.162 / Chapter 4.2.8 --- Statistical analysis --- p.164 / Chapter 4.3 --- Results --- p.165 / Chapter 4.3.1 --- EriB inhibits the growth of CAPAN-2 human pancreatic tumor xenografts --- p.165 / Chapter 4.3.2 --- EriB treatments induce cell apoptosis in tumor tissues --- p.173 / Chapter 4.3.3 --- Toxicity tests for EriB --- p.175 / Chapter 4.3.3.1 --- Plasma enzyme levels after EriB treatments --- p.175 / Chapter 4.3.3.2 --- No apparent alterations in histology of the heart, liver and kidney tissues --- p..176 / Chapter 4.3.4tEriB induces superoxide production in the tumor tissues --- p.178 / Chapter 4.3.5 --- Successful establishment of an orthotopic xenograft model --- p.180 / Chapter 4.4 --- Discussion --- p.184 / Chapter Chapter 5 --- General Discussion --- p.188 / Chapter 5.1 --- Discussion --- p.189 / Chapter 5.2 --- Conclusion --- p.204 / Chapter 5.3 --- Limitations of the study --- p.205 / Chapter 5.4 --- Future work --- p.206 / Chapter Chapter 6 --- References --- p.207

Wang, Yanli. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 90-104). / In English with some Chinese characters; abstract also in Chinese. / Acknowledgement --- p.I / Abstract --- p.III / 摘要 --- p.V / Table of Content --- p.VII / List of Figures --- p.XIII / List of Tables --- p.XV / Abbreviations --- p.XVI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Traditional Chinese Medicine (TCM) --- p.1 / Chapter 1.2. --- The development history and present situation of Traditional Chinese Medicine --- p.2 / Chapter 1.3. --- Modernization of Traditional Chinese Medicine --- p.3 / Chapter 1.4. --- Authentication of Traditional Chinese Medicines --- p.4 / Chapter 1.5. --- Methods for authentication of Traditional Chinese Medicine --- p.5 / Chapter 1.5.1. --- Morphological and histological methods --- p.5 / Chapter 1.5.2. --- Chemical methods --- p.6 / Chapter 1.5.3. --- Molecular methods --- p.6 / Chapter 1.6. --- DNA regions suitable for molecular authentication of Traditional Chinese Medicine --- p.8 / Chapter 1.6.1. --- The chloroplast genome --- p.8 / Chapter 1.6.2. --- Nuclear sequences --- p.9 / Chapter 1.6.3. --- Mitochondrial genome --- p.12 / Chapter 1.7. --- Herb Tu Si Zi --- p.12 / Chapter 1.7.1. --- The identity of Traditional Chinese Medicine Tu Si Zi --- p.12 / Chapter 1.7.2. --- The medicinal values of Tu Si Zi --- p.13 / Chapter 1.7.3. --- Local substitutes of Tu Si Zi --- p.14 / Chapter 1.7.4. --- The need for molecular authentication of Tu Si Zi --- p.15 / Chapter 1.8. --- Traditional Chinese Medicinal herbs from Isodon --- p.15 / Chapter 1.8.1. --- The genus Isodon --- p.15 / Chapter 1.8.2. --- Xi Huang Cao --- p.16 / Chapter 1.8.2.1. --- Identity of Xi Huang Cao --- p.16 / Chapter 1.8.2.2. --- Medicinal values of Xi Huang Cao --- p.17 / Chapter 1.8.2.3. --- Confusions of herb Xi Huang Cao --- p.17 / Chapter 1.8.3. --- Dong Ling Cao --- p.18 / Chapter 1.8.3.1. --- Identity of Dong Ling Cao --- p.18 / Chapter 1.8.3.2. --- Medicinal values of Dong Ling Cao --- p.18 / Chapter 1.8.4. --- The molecular authentication of two Isodon herbs --- p.19 / Chapter 1.9. --- Fagaropsis and Luvunga --- p.20 / Chapter 1.9.1. --- The classification of Rutaceae --- p.20 / Chapter 1.9.2. --- Controversial taxonomic issues with Fagaropsis and Luvunga --- p.21 / Chapter 1.9.3. --- The need of phylogenetic studies of genus Fagaropsis and Luvunga --- p.23 / Chapter Chapter 2. --- Objectives --- p.24 / Chapter Chapter 3. --- Materials and Methods --- p.25 / Chapter 3.1. --- Samples used in this study --- p.25 / Chapter 3.1.1. --- Tu Si Zi (Dodder seeds) --- p.25 / Chapter 3.1.2. --- Isodon herbs --- p.28 / Chapter 3.1.3. --- Fagaropsis and Luvunga --- p.31 / Chapter 3.2. --- Methods --- p.34 / Chapter 3.2.1. --- Sample preparation --- p.34 / Chapter 3.2.2. --- Total DNA extraction --- p.34 / Chapter 3.2.2.1. --- Cetyltriethylammonium bromide extraction --- p.34 / Chapter 3.2.2.2. --- Commercial kit extraction --- p.36 / Chapter 3.2.3. --- DNA amplification --- p.38 / Chapter 3.2.3.1. --- psbA-trnH intergenic spacer --- p.39 / Chapter 3.2.3.2. --- trnL-trnF region --- p.39 / Chapter 3.2.3.3. --- ITS region --- p.42 / Chapter 3.2.4. --- Agarose gel electrophoresis --- p.43 / Chapter 3.2.5. --- Purification of PCR product --- p.44 / Chapter 3.2.6. --- Cloning --- p.46 / Chapter 3.2.6.1. --- Ligation --- p.46 / Chapter 3.2.6.2. --- Transformation --- p.46 / Chapter 3.2.6.3. --- Cell cultivation --- p.47 / Chapter 3.2.6.4. --- Plasmid extraction --- p.47 / Chapter 3.2.6.5. --- Insert confirmation --- p.49 / Chapter 3.2.7. --- DNA sequencing --- p.49 / Chapter 3.2.7.1. --- Cycle sequencing --- p.49 / Chapter 3.2.7.2. --- Purification of cycle sequencing product --- p.50 / Chapter 3.2.7.3. --- DNA analysis --- p.50 / Chapter 3.2.8. --- Sequence analysis and phylogeny construction --- p.51 / Chapter Chapter 4. --- Tu Si Zi (Dodder Seeds) - Results and Discussion --- p.52 / Chapter 4.1. --- Results --- p.52 / Chapter 4.1.1. --- Dendrogram constructed using psbA-trnH intergenic spacer --- p.52 / Chapter 4.1.2. --- Dendrogram constructed using trnL-trnF region --- p.53 / Chapter 4.1.3. --- Dendrogram constructed with the combination of psbA-trnH and trnL-trnF region --- p.59 / Chapter 4.2 --- Discussion --- p.60 / Chapter 4.2.1. --- Identification of DNA markers for Cuscuta species --- p.60 / Chapter 4.2.2. --- Molecular authentication of dodder seeds --- p.60 / Chapter Chapter 5. --- Isodon herbs - Results and Discussion --- p.64 / Chapter 5.1. --- Results --- p.64 / Chapter 5.1.1. --- Dendrogram constructed with internal transcribed spacer 1 --- p.64 / Chapter 5.1.2. --- Dendrogram established with internal transcribed spacer 2 --- p.65 / Chapter 5.1.3. --- Dendrogram established with the whole internal transcribed spacer region --- p.66 / Chapter 5.2. --- Discussion --- p.73 / Chapter 5.2.1. --- ITS region performing as DNA marker for Dong Ling Cao --- p.73 / Chapter 5.2.2. --- The identify of TCM materials of Xi Huang Cao --- p.73 / Chapter Chapter 6. --- Fagaropsis and Luvunga - Results and Discussion --- p.75 / Chapter 6.1. --- Results --- p.75 / Chapter 6.1.1. --- Phylogenetic tree constructed with internal transcribed spacer 1 --- p.76 / Chapter 6.1.2. --- Phylogenetic tree constructed with trnL-trnF region --- p.76 / Chapter 6.1.3. --- Phylogenetic tree constructed with combined of trnL-trnF region and ITS-1 region --- p.77 / Chapter 6.1.4. --- The location of Fagaropsis and Luvunga in 3 different phylogenetic trees --- p.78 / Chapter 6.2. --- Discussion --- p.85 / Chapter 6.2.1. --- Fagaropsis 一 a member of the ´بProto-Rutaceae´ة group --- p.85 / Chapter 6.2.2. --- Luvunga 一 a member of Aurantioideae --- p.86 / Chapter 6.2.3. --- DNA sequencing providing a useful methodology in plant phylogenetic studies --- p.87 / Chapter Chapter 7. --- Conclusions --- p.89 / References --- p.90 / Appendix 1. Sequence alignment ofpsbA-trnH intergenic spacer of dodder --- p.105 / Appendix 2. Sequence alignment of trnL-trnF region of dodder samples --- p.108 / Appendix 3. Sequence alignment of ITS region of Isodon herbs and specimens --- p.117 / Appendix 4. Sequence alignment of ITS-1 region of Rutaceae species --- p.124 / Appendix 5. Sequence alignment of trnL-trnF region of Rutaceae species --- p.129

Wong, Ying Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 73-78). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / 摘要 --- p.IV / List of Tables --- p.VI / List of Figures --- p.VII / List of Abbreviations --- p.VIII / Chapter Chapter 1 --- Introduction --- p.3 / Chapter 1.1 --- Chemical Constituents of Rhodiola Genus --- p.5 / Chapter 1.1.1 --- Phenylethyl Derivatives --- p.5 / Chapter 1.1.2 --- Phenylpropanoids --- p.8 / Chapter 1.1.3 --- Phenolic Derivatives --- p.11 / Chapter 1.1.4 --- Flavonoids --- p.14 / Chapter 1.1.4.1 --- Flavone and Flavone Glycosides --- p.15 / Chapter 1.1.4.2 --- Flavonols and Their Glycosides --- p.17 / Chapter 1.1.4.3 --- Flavan-3-ol Derivatives --- p.23 / Chapter 1.1.5 --- Monoterpenoids --- p.26 / Chapter 1.1.6 --- Triterpenes --- p.30 / Chapter 1.1.7 --- Miscellaneous Compounds --- p.32 / Chapter 1.2 --- Biological Activities of Rhodiola Genus --- p.33 / Chapter 1.2.1 --- Anti-oxidative Effect --- p.34 / Chapter 1.2.1.1 --- Protective Effect on Ischemia and Reperfusion --- p.34 / Chapter 1.2.1.2 --- Anti-Aging Effect --- p.35 / Chapter 1.2.2 --- Learning and Memory --- p.36 / Chapter 1.2.3. --- Immune Response --- p.37 / Chapter 1.2.4 --- Anti-cancer Effect --- p.38 / Chapter 1.3 --- Objective --- p.39 / Chapter Chapter 2 --- Experimental --- p.40 / Chapter 2.1 --- General Experimental Procedures --- p.40 / Chapter 2.2 --- Plant Materials --- p.40 / Chapter 2.3 --- Extraction and Isolation --- p.41 / Chapter 2.3.1 --- Isolation and Purification of the Ethyl Acetate (E.A.) Fraction --- p.41 / Chapter 2.3.2 --- Isolation and Purification of the Butanol Fraction --- p.44 / Chapter 2.4 --- Characterization of the Isolated Compounds --- p.46 / Chapter 2.4.1 --- β-Sitosterol (1) --- p.46 / Chapter 2.4.2 --- Tyrosol (2) --- p.46 / Chapter 2.4.3 --- trans-Hydroxycinnamic acid (3) --- p.47 / Chapter 2.4.4 --- Geranyl-β-glucopyranoside (4) --- p.47 / Chapter 2.4.5 --- Neryl-β-glucopyranoside (5) --- p.48 / Chapter 2.4.6 --- Hexyl β-Glucopyranoside (6) --- p.48 / Chapter 2.4.7 --- Gallic Acid (7) --- p.49 / Chapter 2.4.8 --- Epigallocatechin-3-Gallate (8) --- p.49 / Chapter 2.4.9 --- Rhodiolgin (9) --- p.50 / Chapter 2.4.10 --- lsolariciresinol-9-β-Glucopyranoside (10) --- p.51 / Chapter 2.4.11 --- Rhodiooctanoside (11) --- p.52 / Chapter 2.4.12 --- Sacranoside B (12) --- p.52 / Chapter Chapter 3 --- Results and Discussion --- p.53 / Chapter 3.1 --- Structural Determination of the Isolated Compounds --- p.53 / Chapter 3.1.1 --- Identification of β-sitosterol (1) --- p.53 / Chapter 3.1.2 --- Identification of Tyrosol (2) --- p.54 / Chapter 3.1.3 --- Identification of trans-Hydroxycinnamic Acid (3) --- p.55 / Chapter 3.1.4 --- Identification of Geranyl-jS-glucopyranoside (4) --- p.56 / Chapter 3.1.5 --- Identification of Neryl-β-glucopyranoside (5) --- p.58 / Chapter 3.1.6 --- Identification of Hexyl β-Glucopyranoside (6) --- p.59 / Chapter 3.1.7 --- Identification of Gallic Acid (7) --- p.60 / Chapter 3.1.8 --- Identification of (-)-Epigallocatechin 3-Gallate (8) --- p.61 / Chapter 3.1.9 --- Identification of Rhodiolgin (9) --- p.63 / Chapter 3.1.10 --- Identification of lsolariciresinol-9-β-glucopyranoside (10) --- p.65 / Chapter 3.1.11 --- Identification of Rhodiooctanoside (11) --- p.67 / Chapter 3.1.12 --- Identification of Sacranoside B (12) --- p.69 / Chapter Chapter 4 --- Conclusion --- p.70 / References --- p.73

Shum Ka Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 174-182). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of content --- p.viii / List of figures --- p.xvi / List of tables --- p.xxii / Abbreviations --- p.xxv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Importance of authentication of Traditional Chinese Medicines --- p.1 / Chapter 1.1.1 --- Confusing nomenclatures --- p.1 / Chapter 1.1.2 --- Similar morphologies of different medicinal materials --- p.2 / Chapter 1.1.3 --- Toxicities of medicinal materials --- p.2 / Chapter 1.1.4 --- Conservation of natural products --- p.2 / Chapter 1.2 --- TCM listed in the Pharmacopoeia of People's Republic of China --- p.3 / Chapter 1.3 --- Overview of mis-use and intoxication of TCM --- p.4 / Chapter 1.4 --- Ordinances regulating Chinese medicines as natural products --- p.7 / Chapter 1.4.1 --- Laws governing Chinese medicine --- p.7 / Chapter 1.4.2 --- Laws governing endangered species --- p.8 / Chapter 1.5 --- Current technologies in the authentication of Traditional Chinese Medicines and their limitations --- p.9 / Chapter 1.6 --- Historical applications of Radix Aconiti --- p.12 / Chapter 1.7 --- Modern applications of Radix Aconiti --- p.16 / Chapter 1.8 --- Research on Radix Aconiti and its chemical components --- p.17 / Chapter 1.8.1 --- Chemistry --- p.17 / Chapter 1.8.2 --- Pharmacology --- p.19 / Chapter 1.8.3 --- Molecular interaction --- p.22 / Chapter 1.9 --- Brief review on the systematics and phylogeny of Aconitum --- p.23 / Chapter 1.10 --- Historical applications of Radix Aucklandiae and related materials --- p.25 / Chapter 1.11 --- Modern applications of Radix Aucklandiae and related material --- p.27 / Chapter 1.12 --- Research on Aucklandiae and related material and their chemical components --- p.28 / Chapter 1.12.1 --- Chemistry --- p.28 / Chapter 1.12.2 --- Pharmacology --- p.29 / Chapter 1.13 --- Brief review on the systematics and phylogeny of Aucklandia and related medicinal species --- p.31 / Chapter 1.14 --- Authentication by DNA sequencing --- p.33 / Chapter 1.14.1 --- Introduction --- p.33 / Chapter 1.14.2 --- Criteria of sequence markers --- p.36 / Chapter 1.14.3 --- Model used to process polymorphism in DNA sequences --- p.37 / Chapter 1.15 --- Screening for novel markers --- p.38 / Chapter 1.15.1 --- Reason for screening novel markers --- p.38 / Chapter 1.15.2 --- Basic principle --- p.39 / Chapter 1.16 --- Introduction to gas chromatography- mass spectrometry --- p.40 / Chapter 1.16.1 --- Basic principles and components of GC-MS --- p.41 / Chapter 1.16.2 --- Advantages and limitations of GC-MS --- p.42 / Chapter 1.16.3 --- Usage of GC-MS on natural product analysis --- p.43 / Chapter 1.16.4 --- Chemometric analysis --- p.44 / Chapter 1.17 --- Objectives --- p.46 / Chapter Chapter 2. --- Materials and Methods --- p.47 / Chapter 2.1 --- Plant samples --- p.47 / Chapter 2.1.1 --- Samples of Aconitum --- p.47 / Chapter 2.1.2 --- Samples of Aucklandia and related species --- p.51 / Chapter 2.2 --- DNA extraction method --- p.58 / Chapter 2.2.1 --- Reagents --- p.58 / Chapter 2.2.2 --- Methods --- p.59 / Chapter 2.3 --- Chemical extraction methods --- p.61 / Chapter 2.4 --- Chemical standard extraction and purification method --- p.62 / Chapter 2.5 --- DNA sequencing --- p.63 / Chapter 2.5.1 --- Reagents --- p.63 / Chapter 2.5.2 --- Methods --- p.65 / Chapter 2.6 --- Genomic subtraction --- p.70 / Chapter 2.7 --- Search for species-specific markers from the subtraction library --- p.74 / Chapter 2.8 --- Gas chromatography- mass spectrometry --- p.74 / Chapter 2.9 --- GC-MS chemometric analysis --- p.75 / Chapter Chapter 3. --- Authentication of Aconitum by DNA Sequencing --- p.76 / Chapter 3.1 --- Introduction --- p.76 / Chapter 3.2 --- Methods --- p.77 / Chapter 3.3 --- Results - 5S spacer --- p.77 / Chapter 3.3.1 --- Sequence information --- p.77 / Chapter 3.3.2 --- Sequence similarity --- p.78 / Chapter 3.3.3 --- Phylogram study --- p.81 / Chapter 3.4 --- Results -psbA-trnH --- p.85 / Chapter 3.4.1 --- Sequence information --- p.85 / Chapter 3.4.2 --- Sequence similarity --- p.85 / Chapter 3.4.3 --- Phylogram study --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Overview of nuclear ribosomal 5S spacer --- p.91 / Chapter 3.5.2 --- Extensive polymorphism of 5S spacer --- p.91 / Chapter 3.5.3 --- Distribution of samples in the phylograms constructed by 5S spacer --- p.93 / Chapter 3.5.4 --- Utility of 5S spacer for authentication --- p.94 / Chapter 3.5.5 --- Overview of psbA-trnH spacer --- p.94 / Chapter 3.5.6 --- Distribution of samples in the phylograms constructed by psbA-trnH spacer --- p.95 / Chapter 3.5.7 --- A distinctive region of inversion --- p.96 / Chapter 3.5.8 --- Utility of psbA-trnH for authentication --- p.97 / Chapter Chapter 4. --- Screening for Novel Markers for Authentication of Aconitum --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Methods --- p.99 / Chapter 4.3 --- Results - subtracted clones --- p.99 / Chapter 4.4 --- Results - SSH6 --- p.104 / Chapter 4.4.1 --- Sequence information --- p.104 / Chapter 4.4.2 --- Sequence similarity --- p.105 / Chapter 4.5 --- Results-SSH15 --- p.107 / Chapter 4.5.1 --- Sequence information --- p.107 / Chapter 4.5.2 --- Sequence similarity --- p.107 / Chapter 4.5.3 --- Phylogram study --- p.109 / Chapter 4.6 --- Results-SSH45 --- p.113 / Chapter 4.6.1 --- Sequence information --- p.113 / Chapter 4.6.2 --- Sequence similarity --- p.113 / Chapter 4.6.3 --- Phylogram study --- p.115 / Chapter 4.7 --- Discussion --- p.119 / Chapter 4.7.1 --- Utility of subtraction in screening markers --- p.119 / Chapter 4.7.2 --- SSH6 --- p.121 / Chapter 4.7.3 --- SSH15 --- p.122 / Chapter 4.7.4 --- SSH45 --- p.123 / Chapter 4.7.5 --- Hybridization in Aconitum --- p.124 / Chapter 4.7.6 --- Inferring species identities of samples from the market --- p.126 / Chapter 4.8 --- Conclusion --- p.128 / Chapter Chapter 5. --- Assessment of Aucklandia lappa and Related Species by GC-MS --- p.129 / Chapter 5.1 --- Introduction --- p.129 / Chapter 5.2 --- Methods --- p.130 / Chapter 5.3 --- Results --- p.130 / Chapter 5.3.1 --- Extraction of essential oil --- p.130 / Chapter 5.3.2 --- GC-MS analysis --- p.131 / Chapter 5.3.3 --- Peak alignment and hierarchical cluster analysis --- p.133 / Chapter 5.3.4 --- Purification of chemical markers from Aucklandia lappa --- p.148 / Chapter 5.3.5 --- Standardization of the purified chemical markers --- p.148 / Chapter 5.3.6 --- Quantitative analysis of chemical markers --- p.152 / Chapter 5.4 --- Discussion --- p.154 / Chapter 5.4.1 --- Analysis of chemical composition --- p.154 / Chapter 5.4.2 --- A comparison on chemometric methods --- p.154 / Chapter 5.4.3 --- Similarity of chemical profiles --- p.156 / Chapter 5.4.4 --- Dendrogram analysis --- p.157 / Chapter 5.4.5 --- Utility of GC-MS in authentication of A. lappa and related species --- p.159 / Chapter 5.4.6 --- Limitations --- p.159 / Chapter 5.4.7 --- Comparison with molecular data --- p.161 / Chapter 5.4.8 --- Contents of dehydrocostuslactone and costunolide --- p.163 / Chapter 5.4.9 --- Locality study --- p.164 / Chapter 5.5 --- Conclusion --- p.165 / Chapter Chapter 6. --- General Discussion --- p.167 / Chapter 6.1 --- DNA sequencing --- p.168 / Chapter 6.2 --- Genomic subtraction --- p.169 / Chapter 6.3 --- Future work on molecular authentication --- p.170 / Chapter 6.4 --- Future work on authentication of Aconitum --- p.170 / Chapter 6.5 --- Gas chromatography- mass spectrometry --- p.171 / Chapter 6.6 --- Future work on authentication by GC-MS --- p.172 / Chapter 6.7 --- Future work on authentication of Aucklandia lappa and related species … --- p.173 / References --- p.174 / Appendix A. Sequence Alignment of 5S Spacer from Aconitum Species --- p.183 / Appendix B. Sequence Alignment of psbA- trnH Spacer from Aconitum Species --- p.188 / Appendix C. Sequences of Subtracted Clones from Aconitum --- p.191 / Appendix D. Sequence Alignment of SSH6 from Aconitum Species --- p.194 / Appendix E. Sequence Alignment of SSH15 from Aconitum Species --- p.195 / Appendix F. Sequence Alignment of SSH45 from Aconitum Species --- p.200 / Appendix G. Gas Chromatograms of Essential Oil Extracts of Aucklandia lappa and Related Species --- p.202

Yip Pui Ying. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-184). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1 / Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5 / Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8 / Chapter 1.3.1. --- DNA sequencing --- p.9 / Chapter 1.3.2. --- DNA fingerprinting --- p.14 / Chapter 1.3.3. --- Nucleic acid hybridization --- p.19 / Chapter 1.4. --- Objectives --- p.20 / Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22 / Chapter 2.1. --- Introduction --- p.22 / Chapter 2.2. --- Materials and Methods --- p.22 / Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22 / Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22 / Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33 / Chapter 2.2.2. --- Extraction of total DNA --- p.35 / Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35 / Chapter 2.2.4. --- Purification of PCR products --- p.38 / Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38 / Chapter 2.2.5.1. --- Polishing --- p.38 / Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38 / Chapter 2.2.5.3. --- Transformation --- p.40 / Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40 / Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41 / Chapter 2.2.6. --- Sequencing of ITS regions --- p.41 / Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41 / Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41 / Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42 / Chapter 2.2.7. --- Sequence analysis and alignment --- p.42 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Extraction of total DNA --- p.42 / Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44 / Chapter 2.3.2.1. --- Interspecific ITS study --- p.44 / Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46 / Chapter 2.3.3. --- Sequence analysis and alignment --- p.47 / Chapter 2.3.3.1. --- Interspecific ITS study --- p.47 / Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60 / Chapter 2.4.2. --- The results agreed with previously published works --- p.60 / Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60 / Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60 / Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61 / Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Extraction of total DNA --- p.62 / Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.3.2 --- Fingerprint analysis --- p.69 / Chapter 3.4 --- Discussion --- p.85 / Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85 / Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85 / Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86 / Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88 / Chapter 3.4.5 --- Explanation of results --- p.88 / Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89 / Chapter 3.4.7 --- Further studies --- p.90 / Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Samples for microarray study --- p.92 / Chapter 4.2.2 --- Extraction of total DNA --- p.95 / Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95 / Chapter 4.2.4 --- Preparation of labeled probe --- p.95 / Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97 / Chapter 4.2.6 --- Preparation of slides --- p.103 / Chapter 4.2.7 --- Hybridization and washing --- p.104 / Chapter 4.2.8 --- Scanning and data analysis --- p.105 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- DNA extraction --- p.105 / Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107 / Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112 / Chapter 4.3.4 --- Preparation of slides --- p.112 / Chapter 4.3.5 --- Scanning and data analysis --- p.116 / Chapter 4.4 --- Discussion --- p.134 / Chapter 4.4.1 --- Implications --- p.134 / Chapter 4.4.2 --- Applying the findings --- p.134 / Chapter 4.4.3 --- Ways to maximize specificity --- p.137 / Chapter 4.4.4 --- Optimisation --- p.138 / Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138 / Chapter Chapter Five --- General Discussion and Summary --- p.140 / Chapter 5.1. --- Objectives of this study --- p.140 / Chapter 5.2. --- rDNA ITS sequencing --- p.140 / Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140 / Chapter 5.2.2. --- Implications from the results --- p.140 / Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141 / Chapter 5.3. --- AP-PCR fingerprinting --- p.141 / Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141 / Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142 / Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143 / Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143 / Chapter 5.4.2. --- Implications from the results --- p.143 / Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144 / Chapter 5.5. --- Overall summary --- p.144 / Chapter 5.6. --- Future studies --- p.146 / References --- p.147 / Appendix --- p.185

Lam Rosanna Yen Yen. / Thesis submitted in: September 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 142-154). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese Abstract --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species --- p.1 / Chapter 1.1.1 --- Intracellular sources of ROS --- p.1 / Chapter 1.1.2 --- Extracellular sources of ROS --- p.2 / Chapter 1.1.3 --- Superoxide anion radicals --- p.2 / Chapter 1.1.4 --- Hydrogen peroxide --- p.3 / Chapter 1.1.5 --- Hydroxyl radicals --- p.3 / Chapter 1.1.6 --- Singlet oxygen --- p.4 / Chapter 1.1.7 --- Peroxyl radicals and peroxides --- p.4 / Chapter 1.1.8 --- Damage of cellular structures by ROS --- p.5 / Chapter 1.2 --- Antioxidative defence in the body --- p.6 / Chapter 1.2.1 --- Antioxidant proteins --- p.6 / Chapter 1.2.2 --- Antioxidant enzymes --- p.6 / Chapter 1.2.3 --- Antioxidant compounds --- p.7 / Chapter 1.2.3.1 --- Vitamin E --- p.8 / Chapter 1.2.3.2 --- Vitamin C --- p.9 / Chapter 1.2.3.3 --- Glutathione --- p.9 / Chapter 1.2.3.4 --- Urate --- p.9 / Chapter 1.2.3.4.1 --- Purine metabolism --- p.10 / Chapter 1.2.3.4.2 --- Xanthine oxidase --- p.12 / Chapter 1.2.4 --- Oxidative stress and antioxidant defence mechanisms in RBC --- p.12 / Chapter 1.2.5 --- Oxidative stress and antioxidant defence mechanisms in LDL --- p.16 / Chapter 1.3 --- Human diseases originated from pro-oxidant conditions --- p.16 / Chapter 1.3.1 --- Atherosclerosis --- p.17 / Chapter 1.3.2 --- Ischemia /reperfusion injury --- p.17 / Chapter 1.3.3 --- Glucose-6-phosphate dehydrogenase deficiency --- p.18 / Chapter 1.3.4 --- DNA mutation --- p.18 / Chapter 1.3.5 --- Other pro-oxidant state related diseases --- p.19 / Chapter 1.4 --- Hyperuricemia and gout: diseases originated from an extreme antioxidant condition --- p.19 / Chapter 1.4.1 --- Inhibition of XOD as a treatment method for hyperuricemia --- p.20 / Chapter 1.4.2 --- Relationship between ROS injury and hyperuricemia --- p.22 / Chapter 1.5 --- Antioxidants in human nutrition --- p.23 / Chapter 1.6 --- Chinese medicinal therapeutics --- p.23 / Chapter 1.6.1 --- Rhubarb --- p.25 / Chapter 1.6.2 --- Aloe --- p.26 / Chapter 1.6.3 --- Ginger --- p.27 / Chapter 1.6.4 --- Objectives of the project --- p.30 / Chapter 1.6.5 --- Strategies applied to achieve the objectives of the present project --- p.30 / Chapter Chapter 2 --- Materials and methods --- p.31 / Chapter 2.1 --- XOD inhibition assay --- p.31 / Chapter 2.1.1 --- Assay development --- p.31 / Chapter 2.1.2 --- Dose-dependent study --- p.32 / Chapter 2.1.3 --- Reversibility of the enzyme inhibition --- p.32 / Chapter 2.1.4 --- Lineweaver-Burk plots --- p.33 / Chapter 2.2 --- Lipid peroxidation inhibition assay of mouse liver microsomes --- p.34 / Chapter 2.2.1 --- Preparation of mouse liver microsomes --- p.34 / Chapter 2.2.2 --- Basis of assay --- p.34 / Chapter 2.2.3 --- Assay procedures --- p.35 / Chapter 2.3 --- AAPH-induced hemolysis inhibition assay --- p.36 / Chapter 2.3.1 --- Preparation of RBC --- p.36 / Chapter 2.3.2 --- Basis of assay --- p.36 / Chapter 2.3.3 --- Assay procedures --- p.37 / Chapter 2.4 --- Lipid peroxidation inhibition assay of RBC membrane --- p.38 / Chapter 2.4.1 --- Preparation of RBC membrane --- p.38 / Chapter 2.4.2 --- Basis of assay --- p.39 / Chapter 2.4.3 --- Assay procedures --- p.40 / Chapter 2.5 --- ATPase protection assay --- p.41 / Chapter 2.5.1 --- Preparation of RBC membrane --- p.41 / Chapter 2.5.2 --- Preparation of malachite green (MG) reagent --- p.41 / Chapter 2.5.3 --- Basis of assay --- p.41 / Chapter 2.5.4 --- Assay procedures --- p.42 / Chapter 2.5.5 --- Determination of ATPase activities --- p.43 / Chapter 2.5.6 --- Assay buffers --- p.43 / Chapter 2.6 --- Sulfhydryl group protection assay --- p.44 / Chapter 2.6.1 --- Preparation of RBC membrane --- p.44 / Chapter 2.6.2 --- Basis of assay --- p.45 / Chapter 2.6.3 --- Assay procedures --- p.45 / Chapter 2.7 --- Lipid peroxidation inhibition assay of LDL by the AAPH method --- p.46 / Chapter 2.7.1 --- Basis of assay --- p.46 / Chapter 2.7.2 --- Assay procedures --- p.46 / Chapter 2.8 --- Lipid peroxidation inhibition assay of LDL by the hemin method --- p.47 / Chapter 2.8.1 --- Basis of assay --- p.47 / Chapter 2.8.2 --- Assay procedures --- p.47 / Chapter 2.9 --- Protein assay --- p.48 / Chapter 2.10 --- Statistical analysis --- p.48 / Chapter 2.11 --- Test compounds --- p.48 / Chapter Chapter 3 --- Xanthine oxidase inhibition assay: results and discussion --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Results --- p.54 / Chapter 3.3 --- Discussion --- p.59 / Chapter Chapter 4 --- Lipid peroxidation inhibition in mouse liver microsomes: results and discussion --- p.64 / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- Results --- p.64 / Chapter 4.3 --- Discussion --- p.69 / Chapter Chapter 5 --- Assays on protection of RBC from oxidative damage: results and discussion --- p.71 / Chapter 5.1 --- Introduction --- p.71 / Chapter 5.2 --- Results --- p.75 / Chapter 5.2.1 --- AAPH-induced hemolysis inhibition assay --- p.75 / Chapter 5.2.2 --- Lipid peroxidation inhibition assay of RBC membranes --- p.82 / Chapter 5.2.3 --- Ca2+-ATPase protection assay --- p.88 / Chapter 5.2.4 --- Na+/K+-ATPase protection assay --- p.95 / Chapter 5.2.5 --- Sulfhydryl group protection assay --- p.100 / Chapter 5.3 --- Discussion --- p.110 / Chapter 5.3.1 --- AAPH-induced hemolysis inhibition assay --- p.110 / Chapter 5.3.2 --- Lipid peroxidation inhibition assay of RBC membranes --- p.111 / Chapter 5.3.3 --- Ca2+-ATPase protection assay --- p.113 / Chapter 5.3.4 --- Na+/K+-ATPase protection assay --- p.114 / Chapter 5.3.5 --- Sulfhydryl group protection assay --- p.115 / Chapter 5.3.6 --- Chapter summary --- p.117 / Chapter Chapter 6 --- Lipid peroxidation inhibition assay of LDL: results and discussion --- p.118 / Chapter 6.1 --- Introduction --- p.118 / Chapter 6.2 --- Results --- p.118 / Chapter 6.3 --- Discussion --- p.134 / Chapter Chapter 7 --- General discussion --- p.137 / References --- p.142

Yu, Wai Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 79-88). / Abstracts in English and Chinese. / Acknowledgements --- p.IV / Abstract --- p.V / 論文摘要 --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2.1 --- Risk factors causing HCC --- p.3 / Chapter 1.2.2 --- Molecular mechanism of HCC --- p.7 / Chapter 1.2.3 --- Treatment of HCC --- p.8 / Chapter 1.3 --- Tetrandrine (Tet) - A Natural Compound Derived from Traditional Chinese Medicine (TCM) --- p.10 / Chapter 1.3.1 --- Traditional Chinese Medicine (TCM) --- p.10 / Chapter 1.3.2 --- Tetrandrine (Tet) --- p.12 / Chapter 1.4 --- Molecular View of Apoptosis --- p.14 / Chapter 1.4.1 --- Overview of apoptosis --- p.14 / Chapter 1.4.2 --- Caspase cascade --- p.15 / Chapter 1.4.3 --- Bcl-2 protein family --- p.18 / Chapter 1.4.4 --- The role of mitochondria in apoptosis --- p.20 / Chapter 1.5 --- Anti-cancer Agents Inducing Apoptosis Are New Targets --- p.22 / Chapter 1.6 --- Aim of Study --- p.26 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Cell Culture And Treatment --- p.27 / Chapter 2.1.1 --- Cell lines used --- p.27 / Chapter 2.1.2 --- Tetrandrine (Tet) --- p.28 / Chapter 2.1.3 --- Chemicals and reagents 2 --- p.83 / Chapter 2.1.4 --- Solution preparation --- p.29 / Chapter 2.1.5 --- Procedures --- p.30 / Chapter 2.2 --- Cell viability --- p.32 / Chapter 2.2.1 --- Chemicals and reagents . --- p.32 / Chapter 2.2.2 --- Solution preparation --- p.32 / Chapter 2.2.3 --- Procedures --- p.32 / Chapter 2.3 --- Apoptosis detection --- p.34 / Chapter 2.3.1 --- Chemicals and reagents --- p.34 / Chapter 2.3.2 --- Solution preparation --- p.35 / Chapter 2.3.3 --- Procedures --- p.36 / Chapter 2.4 --- Gene expression in tet-induced apoptotic cells --- p.39 / Chapter 2.4.1 --- Chemicals and reagents --- p.39 / Chapter 2.4.2 --- Solution preparation --- p.40 / Chapter 2.4.3 --- Procedures --- p.40 / Chapter 2.5 --- Protein expression in tet-induced apoptotic cells --- p.44 / Chapter 2.5.1 --- Chemicals and reagents --- p.44 / Chapter 2.5.2 --- Solution preparation --- p.45 / Chapter 2.5.3 --- Procedures --- p.48 / Chapter 2.6 --- Cell cycle analysis of tet-treated cells --- p.54 / Chapter 2.5.1 --- Chemicals and reagents --- p.54 / Chapter 2.5.2 --- Solution preparation --- p.54 / Chapter 2.5.3 --- Procedures --- p.54 / Chapter Chapter 3 --- Result --- p.56 / Chapter Chapter 4 --- Discussion --- p.70 / Chapter 4.1 --- Dose- and Time- Dependent Inhibitory Effects of Tet were found on HuH-7 And JHH-4 Cell Lines --- p.70 / Chapter 4.2 --- Tet Is More Selective Towards Liver Cancer Cells --- p.71 / Chapter 4.3 --- The Cell Death in HuH-7 Cells Induced by Tet is Mediated Through Apoptosis --- p.72 / Chapter 4.4 --- Hepatocellular Carcinoma (HCC)Tet Induces G1 Phase Cell Cycle Arrest as Part of Its Mechanism in Inducing Apoptosis in HuH-7 Cells --- p.73 / Chapter 4.5 --- Tet Could Probably Induce G1 Phase Cell Cycle Arrest in JHH-4 Cells --- p.75 / Chapter 4.6 --- "Tet-induced Apoptosis Involves the Intrinsic, Caspase-Dependent Pathway in Both the HuH-7 and JHH-4 Cell Lines" --- p.75 / Chapter 4.7 --- Proteins in Bcl-2 Family are Involved in the Inhibitory Mechanism of Tet --- p.77 / Reference --- p.79

by Wai Ching Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-159). / Abstracts in English and Chinese. / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / List of Abbreviations --- p.viii / Table of contents --- p.x / List of Tables --- p.xiv / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.4 / Chapter 2.1 --- Plant lectins --- p.4 / Chapter 2.1.1 --- Introduction --- p.4 / Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4 / Chapter 2.2 --- Monocot mannose-binding lectins --- p.6 / Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6 / Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7 / Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10 / Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11 / Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12 / Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13 / Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14 / Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15 / Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15 / Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17 / Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18 / Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20 / Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20 / Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22 / Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24 / Chapter 2.5.1 --- The facts of rice --- p.24 / Chapter 2.5.2 --- Rice storage proteins --- p.25 / Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28 / Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29 / Chapter 2.6 --- Protein trafficking in plants --- p.30 / Chapter 2.6.1 --- Golgi-dependent pathways --- p.30 / Chapter 2.6.2 --- Golgi-independent pathway --- p.32 / Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Chemcials --- p.35 / Chapter 3.3 --- Bacterial strains --- p.35 / Chapter 3.4 --- Cloning of POL cDNA --- p.36 / Chapter 3.4.1 --- Plant materials --- p.36 / Chapter 3.4.2 --- RNA extraction --- p.36 / Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36 / Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38 / Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39 / Chapter 3.5 --- Analysis of POL protein --- p.40 / Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40 / Chapter 3.5.2 --- Western blot analysis --- p.41 / Chapter 3.6 --- Chimeric gene construction --- p.42 / Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44 / Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48 / Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55 / Chapter 3.7 --- Expression of transgenes in rice --- p.55 / Chapter 3.7.1 --- Plant materials --- p.55 / Chapter 3.7.2 --- Agrobacterium transformation --- p.55 / Chapter 3.7.3 --- Callus induction --- p.56 / Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56 / Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56 / Chapter 3.7.6 --- Isolation of genomic DNA --- p.58 / Chapter 3.7.7 --- Southern blot analysis --- p.58 / Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59 / Chapter 3.7.9 --- Extraction of seed total RNA --- p.59 / Chapter 3.7.10 --- Northern blot analysis --- p.60 / Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60 / Chapter 3.7.12 --- Western blot analysis --- p.61 / Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61 / Chapter 3.8.1 --- Protein extraction --- p.61 / Chapter 3.8.2 --- CPE reduction assay --- p.62 / Chapter 3.9 --- Confocal immunofluorescence --- p.63 / Chapter 3.9.1 --- Preparation of sections --- p.63 / Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63 / Chapter 3.9.3 --- Image collection --- p.64 / Chapter Chapter 4 --- Results --- p.65 / Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65 / Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65 / Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67 / Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68 / Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75 / Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77 / Chapter 4.3 --- Constitutional expression of POL in rice --- p.79 / Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80 / Chapter 4.3.2 --- Southern blot analysis --- p.82 / Chapter 4.3.3 --- Northern blot analysis --- p.84 / Chapter 4.3.4 --- Western blot analysis --- p.85 / Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87 / Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88 / Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89 / Chapter 4.4.2 --- Southern blot analysis --- p.92 / Chapter 4.4.3 --- Northern blot analysis --- p.96 / Chapter 4.4.4 --- Western blot analysis --- p.101 / Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117 / Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122 / Chapter 4.6 --- Confocal immunofluorescence studies --- p.125 / Chapter Chapter 5 --- Discussion --- p.134 / Chapter 5.1 --- Cloning of POL cDNA --- p.134 / Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136 / Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138 / Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146 / Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148 / Chapter 5.6 --- Future prospects --- p.151 / Chapter Chapter 6 --- Conclusion --- p.153