鉤藤是眾多用於治療神經性退行性疾病的傳統中藥複方的組成成份之一。文獻研究發現鉤藤提取物能夠顯著抑制β澱粉樣蛋白纖維的形成和拆卸預製β澱粉樣蛋白纖維。然而鉤藤作用於老年性癡呆模型的實驗研究還未見報道。本課題的研究目的是探討鉤藤提取物對認知功能的改善作用，從而篩選出鉤藤抗老年性癡呆的有效化學成份及探討鉤藤抗老年性癡呆有效化學成份的神經保護作用及其作用機理。 / 首先我們探討了70%乙醇鉤藤提取物對D-半乳糖引起小鼠認知功能障礙的改善作用。水迷宮試驗結果顯示鉤藤提取物(200 和400毫克/千克)能顯著改善D-半乳糖處理小鼠的空間學習和記憶能力。此外，鉤藤提取物(200 和400毫克/千克)還顯著提高D-半乳糖處理小鼠腦組織中乙醯膽鹼和還原型穀胱甘肽的含量，以及超氧化物歧化酶和過氧化氫酶的活性，同時也能降低D-半乳糖處理小鼠腦組織中乙醯膽鹼酯酶的活性和丙二醛的含量。以上研究結果表明鉤藤提取物能改善D-半乳糖處理小鼠認知功能障礙的作用可能是通過抑制腦組織中乙醯膽鹼酯酶的活性和提高腦組織的氧化能力而達成的。 / 其次，我們選用β澱粉樣蛋白引致PC12細胞神經毒性的體外細胞模型來跟蹤篩選出鉤藤提取物中抗老年性癡呆的有效活性成分。結果顯示從鉤藤提取物中分離出六個生物鹼，分別為柯諾辛堿，柯諾辛堿B，去氫鉤藤堿，異鉤藤堿，異去氫鉤藤堿和鉤藤堿。在這六個生物鹼中，只有鉤藤堿和異鉤藤堿具有顯著降低β澱粉樣蛋白導致PC12細胞的死亡，而異鉤藤堿是鉤藤提取物中對β澱粉樣蛋白所致的PC12細胞損傷有最強的保護作用。 / 在明確異鉤藤堿是鉤藤提取物中抗老年性癡呆的主要有效成分的研究基礎上，我們應用β澱粉樣蛋白所致PC12細胞的神經毒性的體外實驗模型來探討異鉤藤堿的神經保護作用及其作用機理。實驗結果顯示異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用呈良好的量效關係。異鉤藤堿對β澱粉樣蛋白引起PC12細胞的神經毒性的保護作用是通過抑制細胞內鈣離子的超載，氧化應激，tau蛋白的過度磷酸化和線粒體細胞凋亡。 此外，異鉤藤堿還顯著抑制3β糖原合成酶激酶的活性，同時啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿對β澱粉樣蛋白所致的PC12細胞的神經毒性的保護作用與PI3K/Akt/GSK3信號通路相關密切相關。 / 最後，我們進一步探討了異鉤藤堿對β澱粉樣蛋白致大鼠認知功能障礙的改善作用及其作用機理。研究結果表明異鉤藤堿(20和40毫克/千克/天)能顯著改善β澱粉樣蛋白所致的大鼠認知功能障礙（用水迷宮試驗來評價）及明顯增加海馬CA1區錐體細胞數目。同時，異鉤藤堿能顯著抑制β澱粉樣蛋白導致大鼠海馬的氧化應激，神經元凋亡以及tau蛋白過度磷酸化。此外，異鉤藤堿能顯著抑制3β糖原合成酶激酶的活性，啟動磷酸化磷脂醯肌醇3-激酶底物Akt，提示異鉤藤堿改善β澱粉樣蛋白導致大鼠認知功能障礙的作用機理與PI3K/Akt/GSK3信號通路相關。 / 綜上所述，鉤藤和異鉤藤堿具有顯著的抗老年癡呆的作用。異鉤藤堿的神經保護作用與其抑制β澱粉樣蛋白導致PC12細胞和大鼠海馬的氧化應激，神經元凋亡以及tau蛋白的過度磷酸化有關。異鉤藤堿神經保護的作用機理與PI3K/Akt/GSK3信號通路密切相關。以上研究結果提示異鉤藤堿具有很好的進一步開發成新的抗老年性癡呆製劑的應用前景。 / The stem with hooks of Uncaria rhynchophylla (Ramulus Uncariae cum Uncis) is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. Previous studies have demonstrated that the extract of U. rhynchophylla inhibited beta-amyloid (Aβ) fibril formation and disassemble preformed Aβ fibrils. However, scientific evidence concerning the efficacy of U. rhynchophylla in Alzheimer’s disease (AD) experimental models is lacking. The present study aimed at investigating the cognition-improving effect of U. rhynchophylla, identifying the active anti-AD chemical constituents and elucidating the underlying mechanisms of neuroprotective action. / Firstly, we investigated whether 70% aqueous ethanol extract of U. rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The results showed that EUR (200 or 400 mg/kg) significantly improved spatial learning and memory function in D-gal-treated mice as assessed by the Morris water maze test. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione, and the activities of superoxide dismutase and catalase, while it decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR was able to ameliorate cognitive deficits induced by D-gal in mice, and the observed pharmacological action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of the brain tissues. / Secondly, we tried to identify the active ingredients of U. rhynchophylla by a bioassay-guided fractionation approach using beta-amyloid (Aβ)-induced neurotoxicity in rat pheochromocytoma (PC12) cells, a well established cellular model of AD. As a result of this work, six alkaloids, namely corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline were isolated from the extract of U. rhynchophylla. Among them, only rhynchophylline and isorhynchophylline could significantly decrease Aβ-induced cell death in PC12 cells. Moreover, isorhynchophylline (IRN) was found to be the most active ingredient responsible for the protective action of U. rhynchophylla against Aβ₂₅₋₃₅-induced cell death. / Thirdly, the neuroprotective effects and its action mechanism of IRN against Aβ₂₅₋₃₅-induced neurotoxicity in PC12 cells, an in vitro experimental model of AD, were examined. The results showed that treatment with IRN dose-dependently protected PC12 cells against Aβ₂₅₋₃₅-induced neurotoxicity. The neuroprotective effect of IRN may be mediated, at least in part, by inhibiting the intracellular calcium overloading, oxidative stress, tau protein hyperphosphorylation and mitochondrial cellular apoptosis induced by Aβ₂₅₋₃₅. Moreover, IRN also inhibited the activity of glycogen synthase kinase (GSK)-3β, an important kinase responsible for tau protein hyperphosphorylation in the development of AD; and activated the phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the neuroprotective action of IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway. / Finally, the ameliorating effect on cognitive deficits of IRN and its underlying mechanism of action in Aβ₂₅₋₃₅-treated rats were investigated. The results showed that oral administration of IRN with two different doses (20 or 40 mg/kg) for 21 days significantly ameliorated cognitive impairments and suppressed the oxidative stress, neuronal apoptosis, and tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats. In addition, IRN also inhibited the activity of GSK-3β, and activated phosphorylation of phosphatidylinositol 3-kinase (PI3K) substrate Akt, suggesting that the amelioration of cognitive deficits by IRN is associated with inhibition of GSK-3β activity and activation of PI3K/Akt signaling pathway. / Taken together, these results confirmed the anti-AD effects of U. rhynchophylla and IRN. The neuroprotective action of IRN may be mediated via inhibition of oxidative stress, neuronal apoptosis and hyperphosphorylation tau protein induced by Aβ₂₅₋₃₅ in vitro and in vivo. The neuroprotective action of IRN is associated with the inhibition of GSK-3β activity and the activation of PI3K/Akt signaling pathway. These experimental findings render IRN a promising candidate worthy of further development into anti-AD pharmaceutical agents. / 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. / Detailed summary in vernacular field only. / Xian, Yanfang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 242-278). / Abstracts also in Chinese. / Abstract (English) --- p.I / 摘要 --- p.IV / Publications --- p.VII / Acknowledgements --- p.IX / Table of Contents --- p.X / List of Figures --- p.XXI / List of Tables --- p.XXVI / List of Abbreviation --- p.XXVII / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Alzheimer’s Disease --- p.2 / Chapter 1.1.1 --- Symptoms --- p.2 / Chapter 1.1.2 --- Epidemiology --- p.4 / Chapter 1.1.3 --- Pathology --- p.5 / Chapter 1.1.4 --- Risk factors --- p.6 / Chapter 1.2 --- Pathogenesis of AD --- p.10 / Chapter 1.2.1 --- Neurotransmitter dysfunction --- p.10 / Chapter 1.2.1.1 --- Cholinergic system dysfunction --- p.10 / Chapter 1.2.1.2 --- Glutamatergic system dysfunction --- p.11 / Chapter 1.2.2 --- Hippocampus atrophy --- p.15 / Chapter 1.2.3 --- “Amyloid Cascade hypothesis --- p.18 / Chapter 1.2.4 --- Increased oxidative stress --- p.21 / Chapter 1.2.5 --- Increased neuronal apoptosis --- p.23 / Chapter 1.2.6 --- Mitochondrial dysfunction --- p.27 / Chapter 1.2.7 --- Calcium dysregulation --- p.31 / Chapter 1.2.8 --- Increased tau protein hyperphosphorylation --- p.34 / Chapter 1.2.9 --- GSK3 hypothesis of AD --- p.37 / Chapter 1.3 --- Animal Models of AD --- p.41 / Chapter 1.3.1 --- Non-transgenic animal models of AD --- p.42 / Chapter 1.3.1.1 --- Spontaneous models --- p.42 / Chapter 1.3.1.2 --- Scopolamine-induced rodent models --- p.43 / Chapter 1.3.1.3 --- Aluminum-induced rodent models --- p.44 / Chapter 1.3.1.4 --- D-galactose-induced rodent models --- p.45 / Chapter 1.3.1.5 --- Aβ infusion rodent models --- p.46 / Chapter 1.3.2 --- Transgenic animal models of AD --- p.48 / Chapter 1.3.2.1 --- Transgenic rodent models for AD --- p.49 / Chapter 1.3.2.2 --- AD models in D. rerio --- p.53 / Chapter 1.3.2.3 --- AD models in D. melanogaster --- p.54 / Chapter 1.3.2.4 --- AD models in C. elegans --- p.54 / Chapter 1.4 --- Treatments for AD --- p.55 / Chapter 1.4.1 --- Current symptomatic treatments --- p.56 / Chapter 1.4.1.1 --- AChEIs --- p.56 / Chapter 1.4.1.2 --- NMDA antagonist --- p.57 / Chapter 1.4.2 --- Disease-modifying approaches --- p.61 / Chapter 1.4.2.1 --- Amyloid-directed therapies --- p.61 / Chapter 1.4.2.2 --- Tau-directed therapies --- p.61 / Chapter 1.4.2.3 --- Anti-oxidant agents --- p.62 / Chapter 1.4.2.4 --- NSAIDs --- p.63 / Chapter 1.4.2.5 --- Estrogen replacement therapy (ERT) --- p.64 / Chapter 1.4.3 --- Herbal medicines --- p.67 / Chapter 1.5 --- Uncaria rhynchophylla --- p.69 / Chapter 1.5.1 --- Chemical constituents --- p.69 / Chapter 1.5.2 --- Alkaloids --- p.72 / Chapter 1.6 --- Pharmacological Activities of Uncaria rhynchophylla and Its Alkaloids --- p.75 / Chapter 1.6.1 --- Effects on cardiovascular system --- p.75 / Chapter 1.6.2 --- Effects on central nervous system --- p.77 / Chapter 1.6.3 --- Antioxidant activities --- p.79 / Chapter 1.6.4 --- Anti-inflammatory and analgesic effects --- p.80 / Chapter 1.6.5 --- Effects on platelet aggregation and thrombosis --- p.81 / Chapter 1.6.6 --- Other pharmacological effects --- p.81 / Chapter 1.7 --- Hypothesis and Objectives of the Present Study --- p.83 / Chapter Chapter Two --- Uncaria rhynchophylla Ameliorates Cognitive Deficits Induced by D-galactose in Mice / Chapter 2.1 --- Introduction --- p.86 / Chapter 2.2 --- Materials and Methods --- p.88 / Chapter 2.2.1 --- Drugs and chemical reagents --- p.88 / Chapter 2.2.2 --- Plant materials and extraction --- p.89 / Chapter 2.2.3 --- Animals --- p.90 / Chapter 2.2.4 --- Experimental design and drugs treatment --- p.90 / Chapter 2.2.5 --- Morris water maze test --- p.91 / Chapter 2.2.6 --- Preparation of brain tissue samples --- p.92 / Chapter 2.2.7 --- Measurement of intracellular ROS level --- p.92 / Chapter 2.2.8 --- Assay of MDA level --- p.92 / Chapter 2.2.9 --- Assay of GSH level --- p.93 / Chapter 2.2.10 --- Measurement of SOD activity --- p.93 / Chapter 2.2.11 --- Measurement of CAT activity --- p.94 / Chapter 2.2.12 --- Assay of Ach level --- p.94 / Chapter 2.2.13 --- Measurement of AChE activity --- p.95 / Chapter 2.2.14 --- Statistical analysis --- p.95 / Chapter 2.3 --- Results --- p.95 / Chapter 2.3.1 --- Quality determination of EUR --- p.95 / Chapter 2.3.2 --- Effects of EUR on Morris water maze in D-gal-treated mice --- p.97 / Chapter 2.3.3 --- Effects of EUR on the level of intracellular ROS in the brains of D-gal-treated mice --- p.101 / Chapter 2.3.4 --- Effects of EUR on the levels of GSH and MDA in the brains of D-gal-treated mice --- p.103 / Chapter 2.3.5 --- Effects of EUR on the activities of SOD and CAT in the brains of D-gal-treated mice --- p.105 / Chapter 2.3.6 --- Effects of EUR on the level of ACh and the activity of AChE in the brains of D-gal-treated mice --- p.107 / Chapter 2.4 --- Discussion --- p.109 / Chapter Chapter Three --- Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla Against Beta-Amyloid-Induced Neurotoxicity / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.2 --- Materials and Methods --- p.114 / Chapter 3.2.2 --- Drugs and chemical reagents --- p.114 / Chapter 3.2.2 --- Preparation of aggregated Aβ₂₅₋₃₅ --- p.115 / Chapter 3.2.3 --- Extraction, fractionation, isolation and identification processes --- p.115 / Chapter 3.2.4 --- Cell culture and drug treatment --- p.119 / Chapter 3.2.5 --- Cell viability assay --- p.119 / Chapter 3.2.6 --- Statistical analysis --- p.120 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Isolation and structural determination of the isolated compounds --- p.120 / Chapter 3.3.2 --- Effects of different fractions and isolated compounds on Aβ₂₅₋₃₅-induced cells death in PC12 cells --- p.122 / Chapter 3.4 --- Discussion --- p.126 / Chapter Chapter Four --- Neuroprotective Effects of Isorhynchophylline Against Beta-Amyloid-Induced Neurotoxicity in PC12 Cells and Its Possible Mechanisms / Chapter 4.1 --- Introduction --- p.130 / Chapter 4.2 --- Materials and Methods --- p.131 / Chapter 4.2.1 --- Drugs and chemical reagents --- p.131 / Chapter 4.2.2 --- Cell culture and drugs treatment --- p.134 / Chapter 4.2.3 --- Cell viability assay --- p.134 / Chapter 4.2.4 --- Lactate dehydrogenase (LDH) activity assay --- p.135 / Chapter 4.2.5 --- Measurement of intracellular ROS production --- p.135 / Chapter 4.2.6 --- Malondialdehyde (MDA) and glutathione (GSH) assay --- p.136 / Chapter 4.2.7 --- Measurement of SOD activity --- p.137 / Chapter 4.2.8 --- Measurement of CAT activity --- p.137 / Chapter 4.2.9 --- Measurement of intracellular calcium concentration --- p.138 / Chapter 4.2.10 --- Measurement of mitochondrial membrane potential --- p.139 / Chapter 4.2.11 --- Quantification of DNA fragmentation --- p.139 / Chapter 4.2.12 --- Cytochrome c assay --- p.140 / Chapter 4.2.13 --- Western blotting analysis --- p.140 / Chapter 4.2.14 --- Real time-polymerase chain reaction (RT-PCR) analysis --- p.141 / Chapter 4.2.15 --- Statistical analysis --- p.142 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- Effects of IRN on Aβ₂₅₋₃₅-induced cytotoxicity in PC12 cells --- p.143 / Chapter 4.3.2 --- Effects of IRN on the level of intracellular ROS in Aβ₂₅₋₃₅-treated PC12 cells --- p.145 / Chapter 4.3.3 --- Effects of IRN on the levels of GSH and MDA in Aβ₂₅₋₃₅-treated PC12 cells --- p.147 / Chapter 4.3.4 --- Effects of IRN on the activities of SOD and CAT in Aβ₂₅₋₃₅-treated PC12 cells --- p.149 / Chapter 4.3.5 --- Effects of IRN on intracellular calcium level in Aβ₂₅₋₃₅-treated PC12 Cells --- p.151 / Chapter 4.3.6 --- Effects of IRN on MMP in Aβ₂₅₋₃₅-treated PC12 cells --- p.153 / Chapter 4.3.7 --- Effects of IRN on DNA fragmentation in Aβ₂₅₋₃₅-treated PC12 cells --- p.155 / Chapter 4.3.8 --- Effects of IRN on the release of cytochrome c in Aβ₂₅₋₃₅-treated PC12 cells --- p.157 / Chapter 4.3.9 --- Effects of IRN on the protein and mRNA levels of the ratio of Bcl-2/Bax in Aβ₂₅₋₃₅-treated PC12 cells --- p.159 / Chapter 4.3.10 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in Aβ₂₅₋₃₅-treated PC12 cells --- p.162 / Chapter 4.3.11 --- Effects of IRN on the protein of pro-caspase-8 and mRNA levels of the full length of caspase-8 in Aβ₂₅₋₃₅-treated PC12 cells --- p.165 / Chapter 4.3.12 --- Effects of IRN on tau protein hyperphosphorylation in Aβ₂₅₋₃₅-treated PC12 Cells --- p.168 / Chapter 4.3.13 --- Effects of IRN on Aβ₂₅₋₃₅-induced activation of GSK-3β in PC12 cells --- p.170 / Chapter 4.3.14 --- Effects of IRN on Aβ₂₅₋₃₅-induced inactivation of PI3K/Akt pathway --- p.173 / Chapter 4.4 --- Discussion --- p.177 / Chapter Chapter Five --- Isorhynchophylline Treatment Improves Cognitive Deficits Induced by Beta-Amyloid in Rats: Involvement of PI3K/Akt Signaling Pathway / Chapter 5.1 --- Introduction --- p.186 / Chapter 5.2 --- Materials and Methods --- p.187 / Chapter 5.2.1 --- Drugs and chemical reagents --- p.187 / Chapter 5.2.2 --- Animals --- p.188 / Chapter 5.2.3 --- Aβ₂₅₋₃₅ injections --- p.188 / Chapter 5.2.4 --- Experimental design and drugs treatment --- p.189 / Chapter 5.2.5 --- Morris water maze test --- p.190 / Chapter 5.2.6 --- Nissl’s staining for neurons --- p.193 / Chapter 5.2.7 --- Preparation of brain tissue samples --- p.193 / Chapter 5.2.8 --- Measurement of intracellular ROS level --- p.194 / Chapter 5.2.9 --- Assay of MDA level --- p.194 / Chapter 5.2.10 --- Assay of GSH level --- p.195 / Chapter 5.2.11 --- Measurement of SOD activity --- p.195 / Chapter 5.2.12 --- Measurement of CAT activity --- p.195 / Chapter 5.2.13 --- Cytochrome c assay --- p.196 / Chapter 5.2.14 --- Western blotting analysis --- p.196 / Chapter 5.2.15 --- RT-PCR analysis --- p.197 / Chapter 5.2.16 --- Statistical analysis --- p.198 / Chapter 5.3 --- Results --- p.199 / Chapter 5.3.1 --- IRN treatment rescued behavioral impairment in the Morris water maze test --- p.199 / Chapter 5.3.2 --- Effects of IRN on the number of pyramidal neuronal cells in the hippocampal CA1 region of Aβ₂₅₋₃₅-treated rats --- p.203 / Chapter 5.3.3 --- Effects of IRN on the intracellular ROS level in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.205 / Chapter 5.3.4 --- Effects of IRN on the levels of GSH and MDA in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.207 / Chapter 5.3.5 --- Effects of IRN on the activities of SOD and CAT in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.209 / Chapter 5.3.6 --- Effects of IRN on cytochrome c in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.211 / Chapter 5.3.7 --- Effects of IRN on the protein and mRNA level of the ratio of Bcl-2/Bax in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.213 / Chapter 5.3.8 --- Effects of IRN on the protein and mRNA levels of cleaved caspase-3 and caspase-9 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.216 / Chapter 5.3.9 --- Effects of IRN on the protein and mRNA levels of caspase-8 in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.219 / Chapter 5.3.10 --- Effects of IRN on the tau protein hyperphosphorylation in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.222 / Chapter 5.3.11 --- Effects of IRN on the activation of GSK-3β in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.224 / Chapter 5.3.12 --- Effects of IRN on the PI3K/Akt pathway in the hippocampus of Aβ₂₅₋₃₅-treated rats --- p.226 / Chapter 5.4 --- Discussion --- p.228 / Chapter Chapter Six --- General Discussion and Future Perspectives / Chapter 6.1 --- General Discussion and Conclusions --- p.237 / Chapter 6.2 --- Future Perspectives --- p.243 / References by Alphabetical Order --- p.246

重症肌無力是由自身抗體在神經肌肉接頭特異性的結合乙酰膽鹼受體和肌肉特異性激酶引起的一種獲得性免疫性疾病。疾病的主要症狀是骨骼肌的軟弱無力和易疲勞性。這一症狀在運動後尤為顯著，休息後會有所緩解。重症肌無力在世界範圍的發病率是百萬分之三到三十。由於近年來患者的數量在不斷增加，重症肌無力引起了醫學界的廣泛關注。但是，目前的診斷和治療措施還不能完全滿足臨床病人的需要。在本課題研究中，我們希望運用代謝組學的手段建立一種新的更加有效可靠的方法用於重症肌無力的診斷。同時，我們希望在代謝物的水平上來闡釋強肌健力方（一種中藥復方）對重症肌無力的治療作用。 / 本研究所用樣本來自42個重症肌無力病人和16個健康志願者。樣本由廣州中醫藥大學第一附屬醫院於二零零七年到二零零八年收集所得。診斷後，病人每日口服一定劑量的強肌健力方接受治療，連續服藥兩個月。分別在服藥前和治療後對病人抽血採樣。進一步分離血清後，樣品進行質譜分析。多元統計學方法如主成分分析，正交偏最小二乘和正交偏最小二乘判別分析等用於質譜數據的分析。 / 通過和健康者比較分析，我們在重症肌無力病人的血液中找到142個顯著改變的離子。其中，14個離子得到鑒定，包括：γ-氨基丁酸，2-哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰基甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，糞甾烷酸，植物鞘氨醇-1-磷酸，鵝去氧膽酸甘氨酸結合物，輔酶Q4和甘氨酸膽。基於以上142個離子建立的數學診斷模型在診斷重症肌無力時表現出很高的靈敏度和特異性，分別高達92.8%和83.3%。強肌健力方能夠逆轉由重症肌無力引起的特異性代謝變化，將病人體內被改變的代謝網絡恢復正常，特別是大部分的代謝標誌物在治療後都恢復到了相對正常水平，包括：γ-氨基丁酸，哌啶酸，鳥氨酸，5,8-十四碳二羧酸，精胺，己酰肉毒鹼，N-油酰甘氨酸，鞘氨醇-1-磷酸，聯原膽酸，輔酶Q4和甘氨酸膽。 / 本研究揭示了基於液質聯用的代謝組學方法適用於探索重症肌無力的代謝標誌物，並提供了一種可用於診斷重症肌無力的新方法。同時，本研究證實強肌健力方適用於重症肌無力的治療，且無明顯副作用。 / Myasthenia gravis (MG) is an acquired autoimmune disease caused by specific autoantibodies against acetylcholine receptors (AChRs) and muscle-specific kinase (MuSK) proteins at the neuromuscular junctions. The disease is characterized by weakness and fatigability of the voluntary muscles that gets worse with exertion and improves with rest. The global incidence rate of MG is about 3-30 cases per million per year. In recent years, the worldwide prevalence rate of MG is increasing as a result of increased awareness. However, current diagnostic measures and treatments are not conclusive and satisfactory for MG. In this study, a mass spectrometry-based metabolomic strategy was applied to develop a novel and reliable diagnostic measure for MG on the basis of metabolic analysis, and to explore the therapeutic effect of Qiangji Jianli Fang (QJF, a newly developed Chinese medicine formula) on MG at the metabolite level. / Total 42 MG patients (13 males and 29 females) and 16 volunteers (5 males and 11 females) were recruited at the First Affiliated Hospital of Guangzhou University of Chinese Medicine between March 2007 and March 2008. The patients took QJF once per day for 2 months. Peripheral blood from patients was collected at diagnosis and after 2-month treatment, respectively. Sera prepared from the blood samples were monitored by the liquid chromatography Fourier transform mass spectrometry (LC-FTMS). Mass spectral data were analyzed by multivariate statistical analyses, including principal component analysis (PCA), orthogonal partial least squares (OPLS), and orthogonal partial least squares discriminant analysis (OPLS-DA). / By comparing analysis with the healthy volunteers, 142 significantly changed ions from serum metabolic profile of MG patients were picked out as the potential biomarkers of MG. Among of them, 14 ions were temporarily identified. They were gamma-aminobutyric acid (GABA), pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, sphingosine-1-phosphate (S1P), bisnorcholic acid, coprocholic acid, phytosphingosine-1-P, chenodeoxycholylglycine, coenzyme Q4, and cholylglycine. The developed OPLS-DA diagnostic model based on the 142 special ions showed a high sensitivity (92.8%) and specificity (83.3%) in detecting MG. QJF showed a powerful action on MG by recovering the holistic serum metabolic profile from the disease level to the normal level. Especially, the levels of GABA, pipecolic acid, ornithine, 5,8-tetradecadienoic acid, spermine, hexanoylcarnitine, N-oleoyl glycine, S1P, bisnorcholic acid, coenzyme Q4, and cholylglycine in MG patients were regulated to a relatively normal level after QJF treatment. / My results first indicated that the LC-FTMS-based metabolomics was a useful tool in biomarkers exploration of MG, and it was potentially applicable as a new diagnostic approach for MG. Also, my results demonstrated that QJF was a good optional choice for the treatment of MG, with no reported side effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Yonghai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 113-129). / Abstract also in Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.vi / Acknowledgements --- p.viii / Table of contents --- p.ix / Abbreviations --- p.xiv / List of Tables --- p.xviii / List of Figures --- p.xix / Chapter 1: Introduction --- p.1 / Chapter 1.1 --- Myasthenia gravis --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.2 / Chapter 1.1.3 --- Clinical features --- p.2 / Chapter 1.1.4 --- Clinical classification --- p.4 / Chapter 1.1.5 --- Pathophysiology --- p.5 / Chapter 1.1.6 --- Diagnosis --- p.9 / Chapter 1.1.6.1 --- Physical examination --- p.9 / Chapter 1.1.6.2 --- Blood test --- p.10 / Chapter 1.1.6.3 --- Electrodiagnostic test --- p.10 / Chapter 1.1.6.4 --- Edrophonium test --- p.11 / Chapter 1.1.6.5 --- Imaging --- p.11 / Chapter 1.1.6.6 --- Pulmonary function test --- p.11 / Chapter 1.1.7 --- Treatment --- p.12 / Chapter 1.1.7.1 --- Medication --- p.12 / Chapter 1.1.7.2 --- Thymectomy --- p.12 / Chapter 1.1.7.3 --- Plasmapheresis and intravenous immunoglobulin --- p.13 / Chapter 1.2 --- Qiangji Jianli Fang --- p.14 / Chapter 1.2.1 --- Huang qi --- p.15 / Chapter 1.2.2 --- Dang shen --- p.16 / Chapter 1.2.3 --- Bai shu --- p.16 / Chapter 1.2.4 --- Dang gui --- p.17 / Chapter 1.2.5 --- Sheng ma --- p.17 / Chapter 1.2.6 --- Chai hu --- p.18 / Chapter 1.2.7 --- Chen pi --- p.18 / Chapter 1.2.8 --- Gan cao --- p.19 / Chapter 1.3 --- Metabolomics --- p.19 / Chapter 1.3.1 --- What’s metabolomics? --- p.20 / Chapter 1.3.1.1 --- Metabolites --- p.20 / Chapter 1.3.1.2 --- Metabolome --- p.21 / Chapter 1.3.1.3 --- Two terms: metabolomics and metabonomics --- p.21 / Chapter 1.3.2 --- How metabolomics works? --- p.22 / Chapter 1.3.2.1 --- Sample preparation --- p.22 / Chapter 1.3.2.1.1 --- Quenching --- p.23 / Chapter 1.3.2.1.2 --- Separating metabolites --- p.24 / Chapter 1.3.2.1.3 --- Sample concentration --- p.24 / Chapter 1.3.2.2 --- Analytical technologies (Sample analysis) --- p.25 / Chapter 1.3.2.3 --- Data analysis --- p.26 / Chapter 1.3.2.4 --- Database --- p.28 / Chapter 1.3.3 --- Why metabolomics? --- p.29 / Chapter 1.3.4 --- Metabolomics for human diseases --- p.30 / Chapter 1.3.5 --- Metabolomics for Traditional Chinese Medicine --- p.32 / Chapter 1.4 --- Objectives and significances of the present study --- p.34 / Chapter Chapter 2 --- Metabolic biomarkers of myasthenia gravis --- p.36 / Chapter 2.1 --- Introduction --- p.36 / Chapter 2.2 --- Materials and methods --- p.40 / Chapter 2.2.1 --- Chemicals --- p.40 / Chapter 2.2.2 --- Patients --- p.40 / Chapter 2.2.3 --- Volunteers --- p.42 / Chapter 2.2.4 --- Blood collection --- p.43 / Chapter 2.2.5 --- QC samples --- p.43 / Chapter 2.2.6 --- Sample processing --- p.43 / Chapter 2.2.7 --- Liquid chromatography-mass spectrometry --- p.44 / Chapter 2.2.8 --- Data analysis --- p.45 / Chapter 2.2.9 --- Metabolite identification --- p.45 / Chapter 2.3 --- Results --- p.46 / Chapter 2.3.1 --- Method validation --- p.46 / Chapter 2.3.2 --- An overall comparative analysis between 28 patients and 10 volunteers --- p.48 / Chapter 2.3.3 --- Classification of MG --- p.53 / Chapter 2.3.4 --- Comparative analysis of the metabolic changes in early- and late-stage MG patients respectively --- p.54 / Chapter 2.3.5 --- Biomarker identification --- p.56 / Chapter 2.4 --- Discussion --- p.58 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3 --- A novel diagnostic approach for myasthenia gravis --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals --- p.68 / Chapter 3.2.2 --- Patients and Volunteers --- p.69 / Chapter 3.2.2.1 --- Training set for establishment of diagnostic model --- p.69 / Chapter 3.2.2.2 --- Test set for evaluation of diagnostic model --- p.69 / Chapter 3.2.3 --- QC samples --- p.70 / Chapter 3.2.4 --- Sample processing --- p.71 / Chapter 3.2.5 --- Chromatography --- p.71 / Chapter 3.2.6 --- Mass spectrometry --- p.72 / Chapter 3.2.7 --- Data analysis --- p.72 / Chapter 3.3 --- Results --- p.72 / Chapter 3.3.1 --- Method validation --- p.73 / Chapter 3.3.2 --- Alterations in serum metabolic profile under MG --- p.74 / Chapter 3.3.3 --- Prediction of MG based on biomarkers --- p.74 / Chapter 3.3.4 --- Establishment of diagnostic model on the basis of metabolic profile --- p.77 / Chapter 3.3.5 --- Prediction of MG with diagnostic model --- p.79 / Chapter 3.4 --- Discussion --- p.80 / Chapter 3.5 --- Conclusion --- p.83 / Chapter Chapter 4 --- Qiangji Jianli Fang treatment for myasthenia gravis --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Chemicals --- p.88 / Chapter 4.2.2 --- Herbs --- p.88 / Chapter 4.2.3 --- Participants --- p.88 / Chapter 4.2.4 --- QC samples --- p.90 / Chapter 4.2.5 --- Sample processing --- p.90 / Chapter 4.2.6 --- Liquid chromatography-mass spectrometry --- p.90 / Chapter 4.2.7 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Method validation --- p.91 / Chapter 4.3.2 --- Symptomatic examination after QJF treatment --- p.92 / Chapter 4.3.3 --- Holistic metabolic responses to QJF treatment --- p.93 / Chapter 4.3.4 --- MG biomarkers changes after QJF treatment --- p.95 / Chapter 4.3.5 --- Drug-related biomarkers of QJF --- p.97 / Chapter 4.4 --- Discussion --- p.100 / Chapter 4.5 --- Conclusion --- p.103 / Chapter Chapter 5 --- Conclusions --- p.104 / Chapter Chapter 6 --- Perspectives --- p.107 / Chapter 6.1 --- Experimental autoimmune myasthenia gravis model --- p.107 / Chapter 6.2 --- Chemical composition of Qiangji Jianli Fang --- p.111 / References --- p.113 / Appendices --- p.130

For cardiotonic actions, DF caused a transient increase in contractility and a transient decrease in contraction rate in an isolated rat heart perfusion system. The positive inotropic effect and the negative chronotropic effect were generated by the dose-dependent inhibitions of Na+/K +-ATPase and Ca2+-ATPase respectively in rat heart homogenate. In both assays, Danshen exhibited more potent inhibitions than DF, while Fenge showed negligible inhibitory actions. / In vivo study on Spontaneously Hypertensive Rats (SHR) showed that DF could not restore the established high blood pressure to the normal level. Earlier DF treatment attenuated, but could not prevent, hypertension development. In aorta, DF improved endothelium-dependent vasodilation by potentiating acetylcholine-induced relaxation and basal nitric oxide (NO) production, and inhibiting endothelial Ca2+ATPases. Relaxation of vascular smooth muscle cells (VSMC) towards NO donors was also enhanced. For anti-oxidation, upon DF treatment, mRNA levels of superoxide dismutase (SOD), extracellular superoxide dismutase (ecSOD), catalase and glutathione peroxidase (GPx) were elevated in heart and aorta. However, studies on SOD and catalase demonstrated insignificant changes in the protein expression levels in both organs. For vasodilation, mRNA level of endothelial nitric oxide synthase (eNOS) in the aorta was upregulated, but no change on eNOS and phosphorylated eNOS (peNOS) proteins were detected. A parallel study showed that DF did not cause hypotension or improve antioxidant defense in normotensive Wistar Kyoto rats (WKY). These findings suggest the use of the Danshen and Fenge 7:3 (w/w) formulation on the comprehensive cardiovascular protection. / Previously established Danshen and Fenge 7:3 (w/w) formulation (DF) was shown to exhibit antioxidative activity by preventing oxidant-induced red blood cell hemolysis and H9c2 rat myoblast cell death in a dose-dependent manner, in which Danshen was demonstrated to be a more potent antioxidant than DF. Fenge showed no antioxidative property. The effect of in vivo ischemia-reperfusion was mimicked by the hypoxia-reoxygenation model of primary culture of neonatal rat heart cardiomyocytes. Danshen could protect cardiomyocytes against hypoxiareoxygenation damage. / Reactive oxygen species attack on cardiovascular system can lead to atherosclerosis and finally cardiac ischemia. Reperfusion, allowing the restoration of blood flow in treating atherosclerosis, in turn generates free radicals which irreversibly damage cardiomyocytes and endothelial cells. Endothelial cell damage eventually leads to hypertension. Radix Salviae Miltiorrhizae (Danshen) and Radix Puerariae Thomsonii (Fenge) have long been used together to treat various heart diseases in China. This project was focused on the antioxidative, cardiotonic and vasodilative effects of the aqueous extracts of Danshen and Fenge. / Lam Hung Ming. / "September 2006." / Adviser: Miu Yee Mary Waye. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1381. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 218-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Woo Ka-yan. / "September 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

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

Two South African medicinal plants, Strophanthus speciosus and Eucomis montana, were investigated phytochemically. From Strophanthus speciosus a cardenolide, neritaloside, was isolated, whilst Eucomis montana yielded three homoisoflavanones, 3,9- dihydroeucomin, 4'-demethyl-3,9-dihydroeucomin, and 4'-demethyl-5-0-methyl-3,9- dihydroeucomin. The structures were elucidated on the basis of spectroscopic data. The homoisoflavanones were screened for anti-inflammatory activity using a chemiluminescent luminol assay, modified for microplate usage. All of the homoisoflavanones exhibited good inhibition of chemiluminescence, with IC50 values for 3,9-dihydroeucomin, 4'-demethyl-3,9-dihydroeucomin, and 4'-demethyl-5-0-methyl-3,9- dihydroeucomin being 14mg/mL, 7mg/mL, and 13mg/mL respectively. The IC50 value of 4'-demethyl-3,9-dihydroeucomin compared favourably with the NSAID control (meloxicam), which had an IC50 of 6mg/mL. Neritaloside was not screened for biological activity as the yield of 14.4mg was insufficient for the muscle-relaxant screen for which it was intended. An assay for antioxidant/free radical scavenging activity was also performed. All the compounds had excellent antioxidant/free radical scavenging activity, with percentage inhibition of the reaction being 92%, 96%, and 94% for 3,9-dihydroeucomin, 4'-demethyl- 3,9-dihydroeucomin, and 4'-demethyl-5-0-methyl-3,9-dihydroeucomin respectively at a concentration of 10mg/mL. However, the control compounds, diclofenac and meloxicam, also exhibited strong activity, with the result that the precise mode of anti-inflammatory activity could not be unequivocally determined. The results from the biological screenings thus provided a rational scientific basis for the indigenous ethnomedicinal use of Eucomis species in the treatment of rheumatism, inflammation and pain. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2006.

In this thesis I synthesized different aspects related to diversity, distribution, uses and conservation of medicinal plants in Nepal and also have attempted to recommend guidelines for sustainability of two highly used alpine plant species. The over-harvesting or human induced activities are not the only problem for biodiversity but recently invasion of alien species has also emerged as serious problem in Nepal. I thus also attempted to analyze the effect of invasive species on community composition in the last paper. The first two papers deal with diversity, distribution, uses and harvesting. Paper I showed that medicinal plants in Nepal have unimodal relationship with elevation and the maximum total species richness is at 1000 m. Paper II which deals with the uses of medicinal plants in the Humla region, west Nepal showed that there are 161 medicinal plant species belonging to 61 families and 106 genera used for treating 72 human and 7 veterinary ailments. Medicinal plants in Humla were mostly collected in wild. This induces a serious threat to diversity of the medicinal plants and it is therefore necessary to develop proper management guidelines for their harvesting in wild and/or their domestication. Rheum australe, an endemic plant to west Himalayan region, is widely used plant in traditional...

Aims. The objectives of this study were (i) to isolate and characterize by conventional column chromatography, structurally diverse triterpenes from G. lucidum to serve as chemical markers; (ii) to develop a high performance liquid chromatography (HPLC) method for the quality control and/or standardization of Lingzhi-containing products; (iii) to utilize and optimize operating conditions for the newer extraction technology: supercritical fluid extraction (SFE), in order to maximize yields of bioactive triterpenes, and to reduce time and costs. / Background. The dried fruiting body of Ganoderma lucidum, commonly known as Lingzhi, has been used extensively as a traditional Chinese medicine (TCM) for many centuries not only in China, but also in other countries such as Japan and Korea. In recent years, Lingzhi has also become a popular health supplement in many Western countries. The chemical composition of Lingzhi is complex, but it has been well documented that the lipophilic triterpenoid class of compounds possess a range of biological effects that include antitumor, immunomodulatory, cardiovascular, respiratory and antihepatotoxic activity. A major drawback in TCM research has been the lack of authentic chemical standards, and efficient methods for the extraction and analysis of bioactive fractions and/or single components. Conventional extraction methods for G. lucidum are time-consuming and laborious, and often result in low yields of useful chemical constituents. / Conclusion. This study enabled the development of a method for the simultaneous analysis of structurally diverse triterpenes with remarkably different chromatographic profiles. The isolated triterpenes, as chemical markers, and the HPLC method can readily be used for quality control and/or standardization purposes in evaluating Lingzhi-containing products. Optimization of operating conditions for SFE facilitated the rapid and selective extraction of acidic triterpenes from raw G. lucidum in significantly higher yields. / Methods. Raw material of G. lucidum was extracted with 80% ethanol; subjected to repeated column chromatography to purify triterpenes; and characterized by NMR (1H and 13C) and mass spectroscopy. Isolated lipophilic triterpenes were qualitatively and quantitatively analyzed by reversed-phase HPLC using an ODS column (150 x 4.6 mm) and PDA detection at 256 nm. The assay was validated over appropriate concentration ranges and benzophenone was used as an internal standard. Supercritical fluid extraction of G. lucidum was carried out using a commercial supercritical fluid extractor system Thar, SFE-1000M. Briefly, the raw powder of G. lucidum was soaked in ethanol containing 10% aqueous ammonia for 30 minutes prior to extraction. Extractions were performed at temperatures of 40, 50 and 60°C; and pressures of 200, 250, 300 and 350 bar; 5% ethanol was used as the co-solvent; and the flow rate of CO 2 was set at 20 g/min. / Results. Eight compounds were isolated and identified from G. lucidum: four triterpenes; namely, lucidenic acid N, ganoderic acid B, ganodermanontriol, and ganodermadiol; two steroids; ergosterol-7, 22-dien-3beta-ol and ergosterol peroxide; and two fatty acids, oleic acid and tetracosanoic acid. The four triterpenes were utilized as chemical markers, and the developed HPLC method was able to simultaneously analyze the structurally diverse components with good resolution. The total analysis run time was 110 min, and retention times (tR) were 14.88, 18.96, 63.88 and 90.73 min respectively, and the eluting system was a mixture of three solvents, methanol (A), acetonitrile (B) and 2% acetic acid solution (C): 0-22 min, 5% A, 25% B and 70% C; 22-85 min, gradient elution, the ratio changed gradually to 5% A, 80% B and 15% C; 85-110 min, gradient elution, the ratio changed gradually to 5% A, 85% B and 10% C. The validated HPLC method and the isolated chemical markers were effectively applied to determine the triterpenoid contents in a variety of commercial Lingzhi products. Supercritical fluid extraction conditions of: pressure 300 bar and temperature 50°C, gave the highest yields of triterpene-containing extracts. HPLC analysis of the SFE extracts showed predominantly acidic triterpenes such as lucidenic acid N and ganoderic acid B. / Hong Xin. / "December 2006." / Advisers: Ho Yee Ping; Albert H. L. Chow. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5968. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 149-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.