銀屑病是一種慢性炎症性皮膚病，其發病率約佔全球1-3%的人口。銀屑病的病理特徵包括角質細胞增殖和分化異常，同時伴隨炎症反應，白細胞聚集於真皮和表皮以及血管擴張。證據顯示角質細胞能參與及延續免疫反應，以達致維持或促進該病的作用。研究亦建議角質細胞減少凋亡是引致銀屑病的一個特定現象；因此，長期以來誘導角質細胞凋亡就被用作為治療銀屑病的一種有效策略。 / 根據銀屑病的嚴重程度，治療方法可分為三級：外用藥物主要用於比較輕微的病患，而光療適合中等程度的病患；對於嚴重病例則可使用系統性治療或生物製劑。基於大約75%的銀屑病患者屬於輕微至中度病患，外用藥物是目前應用最為廣泛的治療方法。在中國銀屑病治療的歷史中曾經使用過中草藥，研究亦表明，其治療機制可能通過抑制角質細胞增殖和誘導角質細胞凋亡。比較研究也指出，傳統中藥比西藥的副作用相對較少，及具有較長的舒緩期和較低的復發率。 / 我們先前的研究發現，茜草根提取物能夠抑制一個和銀屑病相關的HaCaT角質細胞增殖。本研究證實，茜草根的乙酸乙酯提取物（EA）能誘導HaCaT細胞凋亡，其抑制角質細胞增殖的作用比茜草根的乙醇提取物（EE）更為有效，並可和一個流行於歐洲國家的重要外用銀屑病治療藥地蒽酚相比。另外，透過不同的檢測，包括形態學觀察，細胞凋亡雙染（磷脂結合蛋白V-碘化丙啶）分析，細胞週期分析，去氧核醣核酸斷裂測試，原位末端轉移酶標記技術，免疫熒光染色以及西方墨點法，我們發現一種在茜草中的化合物，1,4-二羥基-2-萘甲酸（DHNA）能通過死亡受體介導，線粒體介導或不依賴胱天蛋白酶的途徑導致HaCaT細胞凋亡。同時，在其中一種銀屑病動物模型，小鼠鼠尾鱗片表皮上的初步研究顯示DHNA亦可誘導角質細胞分化。此外，在細胞水平（存活率，釋放白细胞介素-1α）和動物上（Draize動物皮膚刺激性試驗）的實驗結果表明DHNA比地蒽酚的刺激性較小。 / 總括而言，本研究透過人類皮膚細胞和動物實驗說明EA和DHNA的細胞凋亡機制，以及DHNA對皮膚的潛在刺激性。這些結果顯示EA和DHNA有潛能發展成為安全及能有效治療銀屑病的替代藥物。EA和DHNA可在一個連續療程中結合使用，其中EA藥效媲美地蒽酚，應能迅速清除銀屑病皮損；而DHNA比地蒽酚的刺激性小，則比較適合應用在這個連續療程中後來的維護保養階段 / Psoriasis is a chronic inflammatory skin disorder that affects approximately 1-3% of the population worldwide. It is characterized by epidermal hyperplasia or abnormal differentiation, infiltration of leucocytes into the dermis and epidermis, dilation of blood vessels in dermis and inflammation. Evidence indicates keratinocytes contributed to the disease, and keratinocytes also participate in maintaining the chronically perpetuating immune response that sustains psoriasis. Decrease in keratinocytes apoptosis is suggested to be a specific pathogenic phenomenon, and induction of keratinocytes apoptosis have long been considered as an effective anti-psoriatic strategy. / Treatment of psoriasis is based on disease severity. Topical agents are predominantly for mild conditions; phototherapy for moderate conditions and systemic treatment or biological agents for severe cases. Topical treatment remains the most widely used method as an estimated 75% of psoriatic patients have mild to moderate disease. Chinese herbs have been used for the treatment of psoriasis in China, and studies showed their mechanism on treating psoriasis may through inhibition of keratinocyte proliferation and induction of apoptosis. Comparison studies also show that traditional Chinese medicine has relatively fewer side effects than western therapeutic agents, with a longer remission time and lower recurrence rate. / The extract of the root of Rubia cordifolia L. (Rubiae Radix et Rhizoma) was previously found to inhibit keratinocyte proliferation using a psoriasis-relevant HaCaT cells model. In this study, the ethyl acetate extract of the root of Rubia cordifolia L. (EA) was confirmed to induce apoptosis on HaCaT cell, and the antiproliferative effect of EA is more potent than the ethanol extract of the herb (EE) and is comparable to dithranol, an important and popular topical treatment for psoriasis among Europe countries. Besides, we identified one of the components in Rubia cordifolia L., 1,4-dihydroxy-2-naphthoic acid (DHNA), could induce HaCaT keratinocyte apoptosis through the death receptor and mitochondria mediated pathway as well as in a caspase independent manner using various assays such as morphological examination, annexin V-PI staining, cell cycle analysis, DNA fragmentation, TUNEL assay, immunofluorescence staining and Western blot analysis. Moreover, DHNA was found to induce keratinocyte differentiation in a preliminary study using the in vivo mouse tail model of psoriasis. Furthermore, results from in vitro (cell viability, IL-1α release) and in vivo (Draize animal skin irritation test) experiments suggested DHNA have less irritation problems than dithranol. / In summary, this study describes the apoptotic mechanism of EA and DHNA, as well as the irritation potential of DHNA using different human skin cells and animal model. These results suggest EA and DHNA have the potential to develop as safe and effective therapeutic alternative for the treatment of psoriasis. EA and DHNA can be used together in a sequential therapy, in which EA is effective in rapid clearing of psoriatic lesions as its potency is comparable to dithranol; whereas DHNA is better suited for the later maintenance therapy for its milder irritation effect compared with dithranol. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Mok, Chong Fai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 164-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English version) --- p.iv / Abstract (Chinese version) --- p.vi / List of Publication and Presentation --- p.viii / Acknowledgements --- p.ix / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Psoriasis --- p.1 / Chapter 1.1.1. --- Histological features --- p.2 / Chapter 1.1.2. --- Role of keratinocytes in psoriasis --- p.4 / Chapter 1.1.3. --- Decrease in skin cell apoptosis --- p.8 / Chapter 1.2. --- Treatment of psoriasis --- p.9 / Chapter 1.2.1. --- Conventional treatment --- p.9 / Chapter 1.2.1.1. --- Mild disease --- p.10 / Chapter 1.2.1.1.1. --- Corticosteroids --- p.10 / Chapter 1.2.1.1.2. --- Vitamin D₃ analogs --- p.11 / Chapter 1.2.1.1.3. --- Tazarotene --- p.11 / Chapter 1.2.1.1.4. --- Anthralin --- p.12 / Chapter 1.2.1.1.5. --- Coal tar --- p.12 / Chapter 1.2.1.2. --- Moderate disease --- p.12 / Chapter 1.2.1.2.1. --- Phototherapy --- p.12 / Chapter 1.2.1.3. --- Severe disease --- p.13 / Chapter 1.2.1.3.1. --- Retinoids --- p.13 / Chapter 1.2.1.3.2. --- Methotrexate --- p.14 / Chapter 1.2.1.3.3. --- Cyclosporine --- p.14 / Chapter 1.2.1.3.4. --- Fumaric acid --- p.15 / Chapter 1.2.1.3.5. --- Biological agents --- p.15 / Chapter 1.2.2. --- Alternative treatment --- p.16 / Chapter 1.2.2.1. --- Traditional Chinese Medicine (TCM) --- p.17 / Chapter 1.3. --- Aims and objectives of the present study --- p.19 / Chapter Chapter 2: --- Apoptotic Action of Ethyl Acetate Fraction of the Root of Rubia cordifolia L. (Rubiae Radix et Rhizoma) on HaCaT Human Keratinocytes --- p.21 / Chapter 2.1. --- Introduction --- p.21 / Chapter 2.1.1. --- Rubia cordifolia L. --- p.21 / Chapter 2.1.2. --- Apoptosis --- p.22 / Chapter 2.1.3. --- Study objectives --- p.28 / Chapter 2.2. --- Materials and Methods --- p.30 / Chapter 2.2.1. --- Sources of medicinal materials --- p.30 / Chapter 2.2.2. --- Preparation of extracts --- p.30 / Chapter 2.2.3. --- Reagents --- p.31 / Chapter 2.2.4. --- Cell culture --- p.31 / Chapter 2.2.5. --- Proliferation assay --- p.32 / Chapter 2.2.6. --- Fluorescent staining for morphological evaluation --- p.33 / Chapter 2.2.7. --- Annexin V/propidium iodide staining --- p.33 / Chapter 2.2.8. --- JC-1 staining --- p.34 / Chapter 2.2.9. --- Statistical analysis --- p.35 / Chapter 2.3. --- Results --- p.36 / Chapter 2.3.1. --- EA inhibits proliferation of human epidermal HaCaT keratinocytes --- p.36 / Chapter 2.3.2. --- Alteration of cellular morphology --- p.39 / Chapter 2.3.3. --- EA increases phosphatidylserine externalization in HaCaT cells --- p.41 / Chapter 2.3.4. --- EA decreases MMP --- p.45 / Chapter 2.4. --- Discussion --- p.47 / Chapter Chapter 3: --- Identification of Pure Compound for Possible Apoptotic Action on HaCaT Human Keratinocytes and Detailed Mechanistic Study --- p.51 / Chapter 3.1. --- Introduction --- p.51 / Chapter 3.1.1. --- Anthraquinone --- p.51 / Chapter 3.1.2. --- Study objectives --- p.52 / Chapter 3.2. --- Materials and Methods --- p.54 / Chapter 3.2.1. --- Reagents --- p.54 / Chapter 3.2.2. --- Cell culture --- p.54 / Chapter 3.2.3. --- Proliferation assay --- p.55 / Chapter 3.2.4. --- Fluorescent staining for morphological evaluation --- p.56 / Chapter 3.2.5. --- Annexin V/propidium iodide staining --- p.56 / Chapter 3.2.6. --- JC-1 staining --- p.56 / Chapter 3.2.7. --- Cell cycle analysis --- p.56 / Chapter 3.2.8. --- Detection of DNA fragmentation --- p.57 / Chapter 3.2.9. --- Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End Labeling (TUNEL) assay --- p.57 / Chapter 3.2.10. --- Western blot analysis --- p.58 / Chapter 3.2.11. --- Immunofluorescence staining --- p.59 / Chapter 3.2.12. --- Statistical analysis --- p.60 / Chapter 3.3. --- Results --- p.61 / Chapter 3.3.1. --- DHNA inhibits proliferation of human epidermal HaCaT Keratinocytes --- p.61 / Chapter 3.3.2. --- Alteration of cellular morphology --- p.70 / Chapter 3.3.3. --- DHNA increases phosphatidylserine externalization in HaCaT cells --- p.72 / Chapter 3.3.4. --- DHNA decreases MMP --- p.76 / Chapter 3.3.5. --- DHNA causes G0/G1 cell cycle arrest in HaCaT cells --- p.78 / Chapter 3.3.6. --- DHNA increases DNA fragmentation --- p.81 / Chapter 3.3.7. --- DHNA increases TUNEL positive cells in HaCaT cells --- p.83 / Chapter 3.3.8. --- Western blot analysis --- p.85 / Chapter 3.3.9. --- DHNA induced Fas aggregation in HaCaT cells --- p.88 / Chapter 3.3.10. --- Caspase inhibition assay --- p.90 / Chapter 3.3.11. --- DHNA induced caspase independent apoptosis in HaCaT cells --- p.93 / Chapter 3.3.12. --- Effects of DHNA on MAPK in HaCaT cells --- p.96 / Chapter 3.3.13. --- MAPK inhibition assay --- p.100 / Chapter 3.4. --- Discussion --- p.104 / Chapter Chapter 4: --- Anti-Psoriatic Effects of Topical 1,4-Dihydroxy-2-naphthoic acid Formulation on in vivo Mouse Tail Experiments --- p.111 / Chapter 4.1. --- Introduction --- p.111 / Chapter 4.1.1. --- Keratinocytes differentiation process --- p.111 / Chapter 4.1.2. --- Animal model for psoriasis --- p.114 / Chapter 4.1.3. --- Study objectives --- p.119 / Chapter 4.2. --- Materials and Methods --- p.122 / Chapter 4.2.1. --- Reagents --- p.122 / Chapter 4.2.2. --- Formulation and preparation of topical drug --- p.122 / Chapter 4.2.3. --- Mice for in vivo experiments --- p.123 / Chapter 4.2.4. --- Treatment with topical preparations --- p.124 / Chapter 4.2.5. --- Statistical analysis --- p.125 / Chapter 4.3. --- Results --- p.126 / Chapter 4.3.1. --- Tail skin appearance after topical treatment --- p.126 / Chapter 4.3.2. --- Histological examination and findings --- p.128 / Chapter 4.4. --- Discussion --- p.132 / Chapter Chapter 5: --- Prediction of Skin Irritation Potential of 1,4-Dihydroxy-2-naphthoic acid by in vitro and in vivo Experiments --- p.135 / Chapter 5.1. --- Introduction --- p.135 / Chapter 5.1.1. --- Skin irritation --- p.135 / Chapter 5.1.2. --- Viability test and IL-1α release --- p.136 / Chapter 5.1.3. --- Animal irritation test --- p.139 / Chapter 5.1.4. --- Study objectives --- p.139 / Chapter 5.2. --- Materials and Methods --- p.141 / Chapter 5.2.1. --- Reagents --- p.141 / Chapter 5.2.2. --- Cell culture --- p.141 / Chapter 5.2.3. --- Viability test --- p.141 / Chapter 5.2.4. --- IL-1α release assay --- p.142 / Chapter 5.2.5. --- Animal irritation test --- p.142 / Chapter 5.2.6. --- Statistical analysis --- p.143 / Chapter 5.3. --- Results --- p.144 / Chapter 5.3.1. --- Viability test --- p.144 / Chapter 5.3.2. --- IL-1α release assay --- p.144 / Chapter 5.3.3. --- Animal irritation test --- p.147 / Chapter 5.4. --- Discussion --- p.152 / Chapter Chapter 6: --- General Discussion and Conclusions --- p.155 / References --- p.164

Chan, Yuet Wa. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 217-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Lau, Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 223-237). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgments --- p.vi / Publication List --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xx / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.1.1 --- Cancer in Hong Kong --- p.1 / Chapter 1.1.2 --- Different types of cancer treatments and the side effects --- p.4 / Chapter 1.1.3 --- Alternative therapies for cancer treatment --- p.6 / Chapter 1.1.3.1 --- Successful examples of anti-cancer drugs from traditional Chinese herbs --- p.7 / Chapter 1.2 --- Anti-tumor study approaches --- p.11 / Chapter 1.2.1 --- Direct cytotoxic activities --- p.12 / Chapter 1.2.2 --- Immunomodulatory activities --- p.14 / Chapter 1.2.3 --- Anti-angiogenesis activities --- p.16 / Chapter 1.3 --- Objectives of our study --- p.20 / Chapter Chapter 2 --- Background of selected Chinese herbs in our study / Chapter 2.1 --- Search for anti-tumor Chinese herbs --- p.21 / Chapter 2.1.1 --- Chinese herbs commonly used for cancer treatment --- p.21 / Chapter 2.1.2 --- Literature Search --- p.21 / Chapter 2.2 --- Results --- p.22 / Chapter 2.2.1 --- Lists of Chinese herbs from various Chinese medicine practitioners --- p.22 / Chapter 2.2.2 --- Selected traditional Chinese herbs from literature search --- p.22 / Chapter 2.2.3 --- Selected Chinese herbs for our study --- p.27 / Chapter 2.3 --- Background information of the five selected Chinese herbs --- p.28 / Chapter 2.3.1 --- Fructus Bruceae (FB) --- p.28 / Chapter 2.3.1.1 --- Traditional uses --- p.28 / Chapter 2.3.1.2 --- Previous Studies of Fructus Bruceae --- p.28 / Chapter 2.3.1.3 --- Isolated compounds of FB --- p.31 / Chapter 2.3.2 --- Cortex Phellodendri Amurensis (PA) --- p.35 / Chapter 2.3.2.1 --- Traditional uses --- p.35 / Chapter 2.3.2.2 --- Previous studies of Cortex Phellodendri Amurensis --- p.35 / Chapter 2.3.2.3 --- Previous studies of Berberine --- p.38 / Chapter 2.3.3 --- Radix et Rhizoma Asteris (RA) --- p.39 / Chapter 2.3.3.1 --- Traditional uses --- p.39 / Chapter 2.3.3.2 --- Previous Studies of Radix et Rhizoma Asteris --- p.39 / Chapter 2.3.4 --- Semen Coicis (SC) --- p.41 / Chapter 2.3.4.1 --- Traditional uses --- p.41 / Chapter 2.3.4.2 --- Previous Studies of Semen Coicis --- p.41 / Chapter 2.3.5 --- Radix Scrophulariae (RS) --- p.43 / Chapter 2.3.5.1 --- Traditional uses --- p.43 / Chapter 2.3.5.2 --- Previous Studies of Radix Scrophulariae --- p.43 / Chapter 2.4 --- Authentication of selected Chinese herbs --- p.45 / Chapter 2.4.1 --- Sources --- p.45 / Chapter 2.4.2 --- Morphological characteristics of the Chinese herbs --- p.47 / Chapter 2.4.2.1 --- Fructus Bruceae --- p.47 / Chapter 2.4.2.2 --- Cortex Phellodendri Amurensis --- p.48 / Chapter 2.4.2.3 --- Radix et Rhizoma Asteris --- p.49 / Chapter 2.4.2.4 --- Semen Coicis --- p.50 / Chapter 2.4.2.5 --- Radix Scrophulariae --- p.51 / Chapter 2.5 --- Extraction of selected Chinese herbs --- p.52 / Chapter 2.5.1 --- Materials and methods --- p.52 / Chapter 2.5.1.1 --- Preparation of aqueous extracts of selected Chinese herbs --- p.52 / Chapter 2.5.2 --- Results --- p.53 / Chapter 2.5.2.1 --- Percentage yield of aqueous extract of selected Chinese herbs --- p.53 / Chapter 2.6 --- Discussion --- p.54 / Chapter Chapter 3 --- Direct cytotoxic effect of selected Chinese herbs / Chapter 3.1 --- Background --- p.55 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Cell cultures --- p.56 / Chapter 3.2.2 --- Determination of cell viability by MTT assay --- p.58 / Chapter 3.2.3 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.59 / Chapter 3.2.4 --- Preparation of etoposide for direct cytotoxic assay --- p.60 / Chapter 3.2.5 --- Statistical analysis --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Cytotoxic effects of five selected Chinese herbs on a panel of human cancer cell lines and human normal cell line --- p.62 / Chapter 3.3.2 --- Comparison of the cytotoxic effect of etoposide and the selected Chinese herbal extracts on a panel of human tumor cells --- p.72 / Chapter 3.3.3 --- Further investigations of the anti-tumor effect of PA --- p.75 / Chapter 3.3.3.1 --- Materials and methods --- p.75 / Chapter 3.3.3.1.1 --- Quantification of berberine chloride in PA aqueous extract using TLC --- p.75 / Chapter 3.3.3.1.2 --- Determination of cell viability by MTT assay --- p.76 / Chapter 3.3.3.2 --- Results --- p.76 / Chapter 3.3.3.2.1 --- Quantification of berberine chloride in PA aqueous extract using TLC --- p.76 / Chapter 3.3.3.2.2 --- Cytotoxic effect of berberine on a panel of human cancer cell lines --- p.78 / Chapter 3.4 --- Discussion --- p.80 / Chapter Chapter 4 --- Immunomodulatory effects of selected Chinese herbs / Chapter 4.1 --- Background --- p.84 / Chapter 4.2 --- Materials and methods --- p.87 / Chapter 4.2.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.87 / Chapter 4.2.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.88 / Chapter 4.2.3 --- Preparation of cell mitogens --- p.88 / Chapter 4.2.4 --- Statistical analysis --- p.89 / Chapter 4.3 --- Results --- p.89 / Chapter 4.3.1 --- Mitogenic activities of the selected herbal extracts on huPBMCs --- p.89 / Chapter 4.4 --- Further investigations of the mitogenic activities of SC and RA extracts --- p.96 / Chapter 4.4.1 --- Materials and methods --- p.96 / Chapter 4.4.1.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.96 / Chapter 4.4.1.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.96 / Chapter 4.4.2 --- Results --- p.97 / Chapter 4.4.2.1 --- Mitogenic effects of SC and RA aqueous extracts (in the presence of polymyxin B) --- p.97 / Chapter 4.5 --- Chemical characterization of RA aqueous extract --- p.100 / Chapter 4.5.1 --- Materials and methods --- p.100 / Chapter 4.5.1.1 --- Quantification of polysaccharide and carbohydrate contents in RA aqueous extract --- p.100 / Chapter 4.5.1.2 --- Quantification of protein content in RA aqueous extract --- p.101 / Chapter 4.5.2 --- Results --- p.103 / Chapter 4.5.2.1 --- Chemical characterization of RA aqueous extract --- p.103 / Chapter 4.6 --- Further investigations of the underlying mechanisms of the mitogenic activities of RA aqueous extract --- p.104 / Chapter 4.6.1 --- Materials and methods --- p.104 / Chapter 4.6.1.1 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.104 / Chapter 4.6.1.2 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.104 / Chapter 4.6.1.3 --- Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) --- p.105 / Chapter 4.6.1.4 --- Statistical analysis --- p.106 / Chapter 4.6.2 --- Results --- p.106 / Chapter 4.6.2.1 --- Effects of RA aqueous extract on productions of cytokinesin huPBMCs --- p.106 / Chapter 4.7 --- Discussion --- p.108 / Chapter Chapter 5 --- Anti-angiogenesis effects of selected Chinese herbs / Chapter 5.1 --- Background of in vivo zebrafish model --- p.112 / Chapter 5.2 --- Materials and methods --- p.117 / Chapter 5.2.1 --- Maintenance of zebrafish --- p.117 / Chapter 5.2.2 --- Collection of zebrafish embryos --- p.117 / Chapter 5.2.3 --- Zebrafish embryos treated with different herbal extracts --- p.117 / Chapter 5.2.4 --- Visual screens of zebrafish embryos using fluorescence microscopy --- p.118 / Chapter 5.2.5 --- Statistical analysis --- p.118 / Chapter 5.3 --- Results --- p.120 / Chapter 5.3.1 --- Anti-angiogenesis effect of SU5416 --- p.120 / Chapter 5.3.2 --- Anti-angiogenesis effects of selected herbal extracts on zebrafish model --- p.122 / Chapter 5.4 --- Discussion --- p.133 / Chapter Chapter 6 --- Further investigations on the anti-tumor effects of Fructus Bruceae and its sub-fractions / Chapter 6.1 --- Introduction --- p.136 / Chapter 6.2 --- Solvent partition of FB aqueous extract --- p.138 / Chapter 6.2.1 --- Materials and methods --- p.138 / Chapter 6.2.1.1 --- Solvent partition --- p.138 / Chapter 6.2.1.2 --- Thin layer chromatography of FB fractions --- p.138 / Chapter 6.2.2 --- Results --- p.139 / Chapter 6.2.2.1 --- Percentage yield of different fractions of FB aqueous extract --- p.139 / Chapter 6.2.2.2 --- Thin layer chromatography of FB fractions --- p.140 / Chapter 6.3 --- Investigations of the anti-tumor activities of FBW fraction --- p.141 / Chapter 6.3.1 --- Materials and methods --- p.141 / Chapter 6.3.1.1 --- Cell cultures --- p.141 / Chapter 6.3.1.2 --- Determination of cell viability by MTT assay --- p.141 / Chapter 6.3.1.3 --- Preparation of human peripheral blood mononuclear cells (huPBMCs) --- p.141 / Chapter 6.3.1.4 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.141 / Chapter 6.3.1.5 --- Statistical analysis --- p.141 / Chapter 6.3.2 --- Results --- p.142 / Chapter 6.3.2.1 --- Cytotoxic effects of FBW on a panel of human cancer cells and human normal cells --- p.142 / Chapter 6.3.2.2 --- Mitogenic activities of FBW fraction on huPBMCs --- p.145 / Chapter 6.4 --- Chemical characterizations of FB aqueous extract and FBW fraction --- p.147 / Chapter 6.4.1 --- Materials and methods --- p.147 / Chapter 6.4.2 --- Results --- p.147 / Chapter 6.5 --- Bioassay guided fractionation of FBW --- p.149 / Chapter 6.5.1 --- Fractionation using macroporous resin column (D101) --- p.149 / Chapter 6.5.2 --- Investigations of the anti-tumor effects of the sub-fractions of FBW --- p.151 / Chapter 6.5.2.1 --- Direct cytotoxic effects of FBW sub-fractions on NB-4 cells and human normal cells --- p.151 / Chapter 6.5.2.2 --- Immunomodulatory effects of FBW-DH sub-fraction --- p.154 / Chapter 6.5.3 --- Fractionation using ethanol precipitation --- p.155 / Chapter 6.5.3.1 --- Chemical characterization of sub-fractions of FBW-DH --- p.156 / Chapter 6.5.3.2 --- "Direct cytotoxic effects of 50P, 80P and 80S on NB-4 cells and human normal cells" --- p.159 / Chapter 6.5.3.2.1 --- DNA agarose gel electrophoresis --- p.163 / Chapter 6.5.3.2.2 --- Cell death detection ELISA --- p.166 / Chapter 6.5.3.2.3 --- ELISA of apoptotic related proteins --- p.168 / Chapter 6.5.3.2.4 --- Telomerase PCR ELISA --- p.176 / Chapter 6.5.3.3 --- "Immunomodulatory effects of 50P, 80P and 80S" --- p.178 / Chapter 6.5.3.3.1 --- Human Thl/Th2 cytokine cytometric bead array (CBA) --- p.180 / Chapter 6.5.3.3.2 --- Limulus Amebocyte Lysate assay --- p.183 / Chapter 6.5.3.4 --- "Anti-angiogenic effects of 50P, 80P and 80S" --- p.184 / Chapter 6.5.3.5 --- Liquid chromatography mass spectrometry (LCMS) analysis of 50P --- p.192 / Chapter 6.6 --- Discussion --- p.194 / Chapter Chapter 7 --- General discussions and conclusions / Chapter 7.1 --- Anti-tumor activities of five selected Chinese herbs --- p.202 / Chapter 7.2 --- Significance of the present study --- p.213 / Chapter 7.3 --- Limitations of our study --- p.214 / Chapter 7.4 --- Future work --- p.215 / Appendices / Appendix I Phenol-sulphuric acid spectrophotometric assay --- p.216 / Appendix II Bradford assay --- p.217 / Appendix III Calibration curves of cytokines in CBA assay --- p.218 / Appendix IV Endotoxin standard curve --- p.220 / Appendix V LCMS data of two chemical markers of FB --- p.221 / Bibliography --- p.223

Li Mi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 125-136). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Publications --- p.vi / Acknowledgement --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Pyrrolizidine alkaloids --- p.1 / Chapter 1.1.1 --- Distribution and plant sources --- p.1 / Chapter 1.1.2 --- Structures and nomenclature --- p.3 / Chapter 1.2 --- PA-containing Chinese medicinal herbs --- p.6 / Chapter 1.3 --- PA-induced toxicity。 --- p.7 / Chapter 1.3.1 --- Acute toxicity and chronic toxicity --- p.7 / Chapter 1.3.2 --- Genotoxicity --- p.8 / Chapter 1.3.3 --- Tumorigenicity --- p.8 / Chapter 1.3.4 --- Hepatotoxicity --- p.8 / Chapter 1.3.5 --- Mechanism of toxic effects --- p.9 / Chapter 1.3.5.1 --- Metabolic pathways --- p.10 / Chapter 1.3.5.2 --- Liver tissue-bound pyrroles --- p.16 / Chapter 1.3.5.3 --- Metabolizing enzymes --- p.17 / Chapter 1.3.5.3.1 --- Phase I metabolizing enzymes --- p.17 / Chapter 1.3.5.3.2 --- Phase II metabolizing enzymes --- p.18 / Chapter 1.3.5.4 --- Species and gender specificity toward toxicity --- p.19 / Chapter 1.3.5.5 --- Structure-activity relationships --- p.20 / Chapter 1.4 --- Prevention of PAs-induced toxicity --- p.23 / Chapter 1.4.1 --- Significance of prevention in humans --- p.23 / Chapter 1.4.2 --- Regulations toward preventing toxicity induced by PAs --- p.24 / Chapter 1.5 --- Aim of the present study --- p.25 / Chapter Chapter 2 --- Qualitative and Quantitative Analysis of PA-containing Chinese Medicinal Herbs --- p.26 / Chapter 2.1 --- Materials and equipments --- p.27 / Chapter 2.1.1 --- Herbal materials --- p.27 / Chapter 2.1.2 --- Chemicals and solvents --- p.27 / Chapter 2.1.3 --- Equipment and instrumentation --- p.27 / Chapter 2.2 --- Preparation of herbal extracts。 --- p.28 / Chapter 2.2.1 --- Crude herbal extract --- p.28 / Chapter 2.2.2 --- Total pyrrolizidine alkaloid extract --- p.28 / Chapter 2.3 --- Qualitative and quantitative analysis of Ligularia hodgsonii --- p.29 / Chapter 2.3.1 --- Methods --- p.29 / Chapter 2.3.1.1 --- HPLC-UV condition --- p.29 / Chapter 2.3.1.2 --- HPLC-MS condition --- p.29 / Chapter 2.3.1.3 --- Calibration curve for clivorine --- p.29 / Chapter 2.3.1.4 --- Recovery test --- p.30 / Chapter 2.3.1.5 --- Sample test --- p.30 / Chapter 2.3.2 --- Results and discussions --- p.30 / Chapter 2.3.2.1 --- Qualitative analysis of PAs in Ligularia hodgsonii --- p.30 / Chapter 2.3.2.2 --- Calibration curve for clivorine --- p.35 / Chapter 2.3.2.3 --- Result of the recovery test --- p.37 / Chapter 2.3.2.4 --- Quantification of PAs in Ligularia hodgsonii --- p.37 / Chapter 2.4 --- Qualitative and quantitative analysis of Tussilago farfara --- p.37 / Chapter 2.4.1 --- Methods --- p.38 / Chapter 2.4.1.1 --- HPLC-UV condition --- p.38 / Chapter 2.4.1.2 --- HPLC-MS condition --- p.39 / Chapter 2.4.1.3 --- Calibration curve for senkirkine --- p.39 / Chapter 2.4.1.4 --- Recovery test --- p.39 / Chapter 2.4.1.5 --- Sample test --- p.39 / Chapter 2.4.2 --- Results and discussions --- p.40 / Chapter 2.4.2.1 --- Qualitative analysis of senkirkine in Tussilago farfara --- p.40 / Chapter 2.4.2.2 --- Calibration curve for senkirkine --- p.42 / Chapter 2.4.2.3 --- Result of the recovery test --- p.42 / Chapter 2.4.2.4 --- Quantification of senkirkine in Tussilago farfara --- p.43 / Chapter 2.5 --- Qualitative and quantitative analysis of Gynura segetum --- p.43 / Chapter 2.5.1 --- Methods --- p.43 / Chapter 2.5.1.1 --- HPLC-UV condition --- p.43 / Chapter 2.5.1.2 --- HPLC-MS condition --- p.44 / Chapter 2.5.1.3 --- Calibration curves for senecionine and seneciphylline --- p.44 / Chapter 2.5.1.4 --- Recovery test… --- p.44 / Chapter 2.5.1.5 --- Sample test --- p.44 / Chapter 2.5.2 --- Results and discussions。 --- p.45 / Chapter 2.5.2.1 --- Qualitative analysis of PAs in Gynura segetum --- p.45 / Chapter 2.5.2.2 --- Calibration curves for senecionine and seneciphylline --- p.49 / Chapter 2.5.2.3 --- Result of the recovery test --- p.49 / Chapter 2.5.2.4 --- Quantification of PAs in Gynura segetum --- p.49 / Chapter 2.6 --- Qualitative and quantitative analysis of Crotalaria sessiliflora --- p.50 / Chapter 2.6.1 --- Methods --- p.50 / Chapter 2.6.1.1 --- HPLC-UV condition --- p.50 / Chapter 2.6.1.2 --- HPLC-MS condition --- p.50 / Chapter 2.6.1.3 --- Calibration curve --- p.51 / Chapter 2.6.1.4 --- Recovery test --- p.51 / Chapter 2.6.1.5 --- Sample test --- p.51 / Chapter 2.6.2 --- Results and discussions --- p.52 / Chapter 2.6.2.1 --- Qualitative analysis of monocrotaline in Crotalaria sessiliflora --- p.52 / Chapter 2.6.2.2 --- Calibration curve for monocrotaline --- p.54 / Chapter 2.6.2.3 --- Result of the recovery test --- p.54 / Chapter 2.6.2.4 --- Quantification of PAs in Crotalaria sessiliflora --- p.55 / Chapter 2.7 --- Qualitative and quantitative analysis of Senecio scandens --- p.55 / Chapter 2.7.1 --- Methods --- p.55 / Chapter 2.7.1.1 --- HPLC-UV condition --- p.55 / Chapter 2.7.1.2 --- HPLC-MS condition --- p.56 / Chapter 2.7.1.3 --- Sample test --- p.56 / Chapter 2.7.2 --- Results and discussions --- p.56 / Chapter 2.7.2.1 --- Qualitative analysis of PAs in Senecio scandens --- p.56 / Chapter 2.7.2.2 --- Quantification of PAs in Senecio scandens --- p.59 / Chapter Chapter 3 --- Hepatotoxicity Induced by PA-containing Chinese Medicinal Herbs --- p.60 / Chapter 3.1 --- Materials and methods --- p.62 / Chapter 3.1.1 --- Reagents --- p.62 / Chapter 3.1.2 --- Animal models --- p.62 / Chapter 3.1.3 --- Determination of the serum ALT activity --- p.64 / Chapter 3.1.4 --- Determination of hepatic GSH level --- p.68 / Chapter 3.1.5 --- Quantitation of liver tissue-bound pyrroles --- p.69 / Chapter 3.1.6 --- Histological assessment of liver morphological changes --- p.70 / Chapter 3.1.7 --- Assessment of hepatocytes apoptosis --- p.71 / Chapter 3.1.8 --- Statistical analysis --- p.72 / Chapter 3.2 --- Results and discussion --- p.72 / Chapter 3.2.1 --- Calibration curves --- p.72 / Chapter 3.2.1.1 --- Calibration curve for the determination of serum ALT activity --- p.72 / Chapter 3.2.1.2 --- Calibration curve of determination of hepatic GSH level --- p.73 / Chapter 3.2.2 --- Hepatotoxicity Study of Crotalaria sessiliflora --- p.74 / Chapter 3.2.2.1 --- Hepatotoxicity at 24 hrs after treatment --- p.74 / Chapter 3.2.2.1.1 --- Correlation between dosage of monocrotaline in Crotalaria sessiliflora and amount of liver tissue-bound pyrroles --- p.74 / Chapter 3.2.2.1.2 --- Effects of Crotalaria sessiliflora on the serum ALT activity --- p.79 / Chapter 3.2.2.1.3 --- The correlation between the elevated level of ALT activity and apoptosis of liver cells --- p.85 / Chapter 3.2.2.1.4 --- Effects of Crotalaria sessiliflora on the hepatic GSH level --- p.86 / Chapter 3.2.2.1.5 --- Histological changes of liver sections --- p.89 / Chapter 3.2.2.2 --- Hepatotoxicity within 4 days after administration --- p.92 / Chapter 3.2.2.3 --- Sub-acute hepatotoxicity within 14 days after administration --- p.93 / Chapter 3.2.2.4 --- Conclusion in hepatotoxicity study of Crotalaria sessiliflora --- p.99 / Chapter 3.2.3 --- Hepatotoxicity Study of Gynura segetum --- p.102 / Chapter 3.2.3.1 --- Correlation between the dosage of PAs present in Gynura segetum and the amount of liver tissue-bound pyrroles --- p.102 / Chapter 3.2.3.2 --- "Effects of Gynura segetum on serum ALT activity, hepatic GSH level and morphological changes of liver" --- p.104 / Chapter 3.2.4 --- Hepatotoxicity Study of Ligularia hodgsonii --- p.108 / Chapter 3.2.4.1 --- Correlation between the dosage of PAs present in Ligularia hodgsonii and the formation of liver tissue-bound pyrroles --- p.108 / Chapter 3.2.4.2 --- Effects of Ligularia hodgsonii on serum ALT activity and hepatic GSH level --- p.111 / Chapter 3.2.5 --- Hepatotoxicity Study of Tussilago farfara --- p.113 / Chapter 3.2.6 --- Hepatotoxicity Study of PA-containing medicinal herbs --- p.115 / Chapter 3.2.6.1 --- Correlation between formation of liver tissue-bound pyrroles and elevated serum ALT level --- p.115 / Chapter 3.2.6.2 --- Correlation between dosage of PAs and amount of liver tissue-bound pyrroles --- p.117 / Chapter 3.2.7 --- Test of Liver Tissue-bound Pyrroles as a biomarker using Senecionis scandentis --- p.118 / Chapter 3.2.8 --- Conclusions --- p.120 / Chapter Chapter 4 --- General Conclusions --- p.121 / Chapter 4.1 --- Qualitative and quantitative analysis of five PA-containing medicinal herbs --- p.121 / Chapter 4.2 --- Hepatotoxicity induced by PA-containing medicinal herbs in rats --- p.122 / Chapter 4.3 --- The correlation between hepatotoxicity induced by PA-containing medicinal herbs and the formation of liver tissue-bound pyrroles --- p.123 / Chapter 4.4 --- Threshold of the amount of liver tissue-bound pyrroles related to the hepatotoxicity induced by PA-containing medicinal herbs --- p.123 / References --- p.125

by Cheng Yuk Kwan, Anna. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 104-112). / Acknowledgements / Table of Contents --- p.i / Abbreviations --- p.iv / Abstract --- p.vi / List of figures --- p.ix / List of tables --- p.xi / Chapter Chapter One: --- Introduction / Chapter 1.1 --- Objective and scope of the project --- p.1 / Chapter 1.2 --- Literature review of Mimosa pudica / Chapter 1.2.1 --- Morphology of Mimosa pudica --- p.3 / Chapter 1.2.2 --- Chemistry of Mimosa pudica --- p.5 / Chapter 1.2.3 --- Uses in traditional medicine --- p.5 / Chapter 1.2.4 --- Clinical and pharmacological studies of Mimosa pudica --- p.6 / Chapter 1.2.5 --- Toxicology of Mimosa pudica --- p.8 / Chapter 1.2.6 --- Characteristics and toxicology of mimosine --- p.9 / Chapter 1.3 --- Immunomodulation / Chapter 1.3.1 --- Overview of the immune system --- p.11 / Chapter 1.3.2 --- Strategies on the study of immunomodulation of Mimosa pudica --- p.13 / Chapter 1.4 --- Toxicology / Chapter 1.4.1 --- Principles of the toxicological assays / Chapter 1.4.1.1 --- LD50 --- p.17 / Chapter 1.4.1.2 --- Enzyme assays --- p.18 / Chapter 1.4.1.3 --- Subacute toxicity test --- p.24 / Chapter 1.4.1.4 --- Reproductive toxicity test --- p.25 / Chapter Chapter Two: --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Mimosa pudica --- p.27 / Chapter 2.1.2 --- Animals --- p.27 / Chapter 2.1.3 --- Chemicals --- p.28 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Extraction of Mimosa pudica --- p.32 / Chapter 2.2.2 --- Assays for the immunomodulatory effects of Mimosa pudica / Chapter 2.2.2.1 --- Cell preparation / Chapter a) --- Splenocytes --- p.35 / Chapter b) --- Thymocytes --- p.35 / Chapter c) --- Macrophages --- p.36 / Chapter 2.2.2.2 --- Splenocyte proliferation --- p.37 / Chapter 2.2.2.3 --- Thymocyte proliferation --- p.38 / Chapter 2.2.2.4 --- Phagocytic activity of macrophages --- p.39 / Chapter 2.2.2.5 --- Release of IL-1 by macrophages --- p.40 / Chapter 2.2.2.6 --- Plaque forming cells --- p.41 / Chapter 2.2.2.7 --- Restoration on splenocyte blastogenesis of old mice --- p.42 / Chapter 2.2.3 --- Assays for the toxicity of Mimosa pudica / Chapter 2.2.3.1 --- LD50 --- p.43 / Chapter 2.2.3.2 --- Enzyme assays --- p.43 / Chapter 2.2.3.3 --- Subacute toxicity --- p.43 / Chapter 2.2.3.4 --- Reproductive toxicity --- p.44 / Chapter 2.2.4 --- Statistical analysis --- p.44 / Chapter Chapter Three: --- Results / Chapter 3.1 --- Immunomodulatory effects of Mimosa pudica / Chapter 3.1.1 --- In vitro study on the lymphocyte proliferation / Chapter 3.1.1.1 --- Splenocyte proliferation --- p.45 / Chapter 3.1.1.2 --- Thymocyte proliferation --- p.50 / Chapter 3.1.2 --- In vivo study on the lymphocyte proliferation --- p.53 / Chapter 3.1.3 --- Phagocytic activity of macrophages --- p.58 / Chapter 3.1.4 --- Release of IL-1 by macrophages --- p.64 / Chapter 3.1.5 --- Plaque forming cells --- p.67 / Chapter 3.1.6 --- Restoration on splenocyte blastogenesis of old mice --- p.69 / Chapter 3.2 --- Toxicity of Mimosa pudica / Chapter 3.2.1 --- LD50 --- p.72 / Chapter 3.2.2 --- Enzyme assays --- p.75 / Chapter 3.2.3 --- Subacute toxicity --- p.80 / Chapter 3.2.4 --- Reproductive toxicity --- p.85 / Chapter Chapter Four: --- General discussion on the immunomodulatory effects and toxicity of Mimosa pudica / Chapter 4.1 --- Immunomodulatory effects of Mimosa pudica --- p.88 / Chapter 4.2 --- Toxicity of Mimosa pudica --- p.95 / Chapter Chapter Five: --- Concluding remarks --- p.99 / References --- p.104 / Appendix --- p.113

Cheung, Wing Shing David. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Law, Wai Tak. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 107-123). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Wound healing --- p.1 / Chapter 1.1.1 --- Physiology of wound healing --- p.1 / Chapter 1.1.2 --- Three phases of wound healing --- p.3 / Chapter 1.1.3 --- Angiogenesis in wound healing --- p.10 / Chapter 1.2 --- Delayed wound healing --- p.11 / Chapter 1.2.1 --- Chronic ulcers --- p.11 / Chapter 1.2.2 --- Examples of ulcers --- p.12 / Chapter 1.3 --- Traditional Chinese medicine (TCM) --- p.16 / Chapter 1.3.1 --- Principles of TCM --- p.16 / Chapter 1.3.2 --- TCM and chronic ulcers --- p.16 / Chapter 1.4 --- Objectives of study --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Selection of traditional Chinese herbs --- p.21 / Chapter 2.2 --- Authentication of TCM --- p.22 / Chapter 2.3 --- Preparation of TCM --- p.23 / Chapter 2.4 --- In vitro studies on the effects of TCM on wound healing --- p.23 / Chapter 2.4.1 --- Angiogenesis study by using human umbilical vein endothelial cell (HUVEC) --- p.25 / Chapter 2.4.2 --- Granulation study by using human fibroblast cell line (CRL) --- p.32 / Chapter 2.4.3 --- Preparation of cell culture conditions --- p.35 / Chapter 2.5 --- In vivo study on the effects of TCM on wound healing by using diabetic mice --- p.38 / Chapter 2.5.1 --- Diabetic mice model --- p.38 / Chapter 2.5.2 --- Diabetic mice wound induction --- p.41 / Chapter 2.5.3 --- "Measurement of body weight, blood glucose level and ulcer area" --- p.43 / Chapter Chapter 3 --- Results / Chapter 3.1 --- The percentage yield of each herbs --- p.48 / Chapter 3.2 --- pH value of all the effective treatment concentration --- p.49 / Chapter 3.3 --- Selection of traditional Chinese herbs --- p.53 / Chapter 3.4 --- Effect of selected TCM on the proliferation of HUVEC --- p.55 / Chapter 3.5 --- Effect of selected TCM on the migration of HUVEC --- p.61 / Chapter 3.6 --- Effect of selected TCM on the proliferation of CRL --- p.63 / Chapter 3.7 --- "Effect of Radix Rehmanniae (selected TCM) on the change in body weight, blood glucose level and ulcer area" --- p.66 / Chapter Chapter 4 --- Discussions --- p.75 / Chapter Chapter 5 --- How does my study contribute towards the modernisation of Chinese medicine? --- p.100 / References --- p.107 / Appendix --- p.124

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.