The topic of this thesis is forest park's Bažantnice mapping. The thesis suggests the way of aplication of botanic terrain education. Botany and dendrology is one of the main educations question on elementary and secondary school. Park Bažantnice can be used in science lesson for its hight potential. The thesis i s divided into two parts. The first of them summarizes differents types of the terrain education and focuses on didactic using of the park Bažantnice. The second one deals with the station mapping and describes way of data colecting and adapting. Accompanying pictures and generated maps are included in appendiced at the back of the thesis. Keywords: dendrology, biology, botany, education, park, Bažantnice, Kladno, mapping, herbs, woody plants, terrain education

Os antioxidantes são substâncias capazes de inibir a oxidação lipídica, responsável pela produção de compostos de cor, aroma e sabor indesejáveis. Na busca por produtos naturais associada à crescente preocupação com a saúde do consumidor, à vista dos possíveis riscos que o uso irregular dos antioxidantes sintéticos pode acarretar ao homem, torna-se essencial verificar a viabilidade do emprego de compostos naturais, com poder antioxidante, em um óleo cuja suscetibilidade à oxidação é reconhecidamente elevada. Com esse objetivo, o presente trabalho avaliou a proteção antioxidante oferecida por extratos hidroalcoólicos de especiarias ao óleo de soja. Os resultados físico-químicos do teor de compostos fenólicos totais, do sequestro do radical livre DPPH (1,1-difenil-2-picrilidrazil), Trolox, da estabilidade oxidativa, através de teste acelerado em estufa e em Rancimat, e da qualidade oxidativa, como o índice de peróxido e a absortividade na faixa do UV, foram base para a seleção dos extratos de alecrim, sálvia, tomilho e orégano, os quais apresentaram maior potencial antioxidante. Os extratos de alecrim, sálvia e tomilho, adicionados ao óleo de soja, foram igualmente eficientes em sua ação antioxidante no teste acelerado em estufa. O óleo, adicionado destes extratos, foi avaliado sensorialmente para verificar o mais preferido pelos consumidores quanto às características de cor, aroma e sabor. Houve uma preferência pelo óleo de soja adicionado de extrato de alecrim. / Antioxidants are compounds able of inhibiting lipid oxidation, responsible for the production of compounds of undesirable color, aroma and flavor. The search for natural products associated with the growing concern over the health of the consumer, in view of the possible risks that the irregular use of synthetic antioxidants can lead to man becomes essential to checking the feasibility of employing natural compounds with antioxidant power, in oil whose susceptibility to oxidation is high. With this purpose, the present study aimed to evaluate the antioxidant protection offered by hydroalcoholic extracts of spices to soybean oil. Analytical results of total phenol compounds, content of kidnapping free radical DPPH (1,1-diphenyl-2-picrilidrazil) substances, Trolox, oxidative stability, through accelerated test in Schaal oven and Rancimat tests, oxidative status, as the acid value and peroxide and absorptivity in the UV range, had been the basis for the selection of extracts of rosemary, sage, thyme and oregano, which presented the highest antioxidant potential. Rosemary, sage and thyme extracts added to soybean oil were similarly efficient as antioxidant in the Schaal Oven Test. Oil added of these extracts were sensorially evaluated according to their colour and flavor. Based on these sensorial characteristics, consumers preferred soybean oil added of rosemary extract.

Finally, the hepatoprotection of the 50%EtWZ was evaluated using rat model. The results indicated that the 50%EtWZ possessed potent hepatoprotective activities. The protective effect of the extract against hepatotoxicity induced by long-term treatment with ethanol might be attributed to its inhibitory action on oxidative stress. Although multiple factors could be involved in the inhibition of oxidative injury in the liver, the inhibition of CYP2E1 pathway and the enhanced GSH-related antioxidant capacity might be responsible for the protective effect. In addition, the 50%EtWZ also produced anti-inflammatory effect partly by interfering Toll-Like-Receptor-4 (TLR-4)-mediated signal pathway and reducing the production of Tumor Necrosis Factor-alpha (INF-alpha) in Kupffer cells during long-term ethanol exposure. / First, in order to determine which kind of extract possesses the strongest hepatoprotective effect on ethanol-induced cytotoxicity, various extracts were screened for cytochrome P450 2E1 isoenzyme (CYP2E1) inhibitory activity using the fluorogenic CYP2E1 substrate and HepG2 cells overexpressing human CYP2E1. The results showed that all extracts (aqueous, 50% ethanol, and 90% ethanol) of WZ produced inhibitory effect on CYP2E1. The 50% ethanol extract of WZ (50%EtWZ) displayed a stronger CYP2E1 inhibition than the aqueous and 90% ethanol extracts. The aqueous extract and 50%EtWZ showed protective effect against ethanol-induced cytotoxicity at concentrations equivalent to 100 and 1000 mug raw herb/ml. At the same concentration of 100 1.1g/ml, the 50%EtWZ exhibited a more potent protective effect. Higher degree of cytotoxicity was found in the 90% ethanol extract of WZ. Thus, 50%EtWZ was chosen for further study. / In summary, all data suggest that the inhibition of CYP2E1 pathway and the inhibition of oxidative stress by the 50%EtWZ, together with the anti-inflammatory effect on Kupffer cells, may contribute to its hepatoprotection against chronic ethanol-induced liver injury. / Second, the chemical components of the 50%EtWZ were analyzed by chromatographic fingerprints. The fingerprint revealed six hepatoprotective compounds including schisandrin B, schisandrin, deoxyschisandrin, betaine, hyperin, and quercitrin in the formula. / Third, the protective mechanism of the 50%EtWZ was investigated in E47 cells model. The 50%EtWZ protected against CYP2E1-dependent toxicity and oxidative stress induced by ethanol. The mechanism of protection involved the decrease of reactive oxygen species production and the inhibition of lipid peroxidation. The hepataprotection was associated with the maintenance of mitochondrial GSH. Pre-treating E47 cells with the 50%EtWZ significantly inhibited the expression of CYP2E1. Therefore, the protective effect of the 50%EtWZ was most likely attributed to its antioxidant activities and the inhibition of CYP2E1. In addition, the 50%EtWZ prevented ethanol-induced apoptosis and protected against oxidative damage to mitochondria which are critical for maintenance of cell viability. / Wu-Zi-Yan-Zong-Wan (WZ), a traditional medicinal formula, is used for treatment of male sexual dysfunctions. In this study, the hepatoprotection afforded by Wu-Zi-Yan-Zong-Wan treatment and its biochemical mechanism involved against chronic alcohol-induced injury were investigated. / Chen, Mengli. / "May 2008." / Adviser: Che Chun Tao. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1609. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 157-179). / 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.

Angiogenesis is a process of new blood vessels sprouting from the pre-existing vasculature, and mediated by multiple angiogenic and anti-angiogenic factors. Disturbance of the balance often leads to development of neovascular diseases. Neovascularization affecting the eye is a common cause of visual impairment and even blindness, particularly when corneal or choroidal neovascularization (NV) is involved. While there are effective treatment modes for ocular neovascularization, they are expensive and only inhibit disease progress. Since herbal medicine has been applied for anti-angiogenesis and anti-carcinogenesis therapies, we investigate the anti-angiogenic effect of selected herbal molecules: isoliquiritigenin (ISL), a flavonoid from licorice; epigallocatechin gallate (EGCG), a polyphenol from green tea; and resveratrol (Rst), a polyphenol phytoalexin derived from grapes. / In conclusion, by in vitro and in vivo studies, we showed that ISL, EGCG and Rst contributed to anti-angiogenesis via different biological mechanisms. We propose that these three herbal molecules (ISL, EGCG and Rst) are candidate anti-angiogenic agents for the treatment of ocular angiogenesis diseases. Their distribution profiles and pharmacokinetic properties should be investigated. / Results showed that sub-toxic levels of ISL (10 microM), EGCG (50 microM) and Rst (10microM) effectively suppressed endothelial cell proliferation and migration in the scratch-wound assay. Treatment with ISL was found to significantly up-regulate PEDF, which is known as a potent angiostatic factor. EGCG and Rst downregulate VEGF signaling cascade by suppressing Akt and FAK activation and affecting MMP-2, MMP-9 expression. In vivo angiogenesis assays further showed the suppressive effect of ISL, EGCG and Rst on neovascularization in three different animal models. Application of ISL at 1 microM showed the suppressive effect on chick CAM assay, corneal NV and choroidal NV assays consistently, the most effective dosage was close to 10 microM. EGCG at 1 microM showed the effect to reduce chick CAM vessel formation and corneal NV, and at 10 microM (the lowest tested concentration) to suppress choroidal NV in mice. Variable effects were observed in Rst treatment. Rst at 10 microM prohibited vessel growth in chick CAM, and 1 microM suppressed corneal NV formation and 2 microM deterred choroidal NV development. / This thesis contains two major parts. The first in vitro cell-based analysis investigated the toxicity of these herbal chemicals and their effect on endothelial cell growth and migration. The expression profile of vascular endothelial growth factor (VEGF) signaling cascade events, including Akt and focal adhesion kinase (FAK) activation, VEGF, pigment epithelium-derived factor (PEDF) and matrix metalloproteinases (MMPs) were examined by Western blotting. Then three in vivo models were established to study the effect of these herbal chemicals on angiogenesis. They were (1) developmental angiogenesis in chick chorioallantoic membrane (CAM), (2) pathological angiogenesis in silver nitrate cauterization-induced corneal neovascularization in BALB/c mice and, (3) laser photocoagulation-induced choroidal neovascularization in C57BL/6 mice. Changes of vascularization were determined by qualification of vessel number changes on the edge of gelatin sponge in 24 hours (chick CAM assay), measurement of vascularized area, live imaging of vessel leakage (fundus fluorescence angiography, FFA) and immunochemistry using antibodies specific for endothelial cells (corneal & choroidal NV assays) respectively. / Liu, Huanming. / Adviser: Chi Pui Pang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 155-180). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wang, Xiaoan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Table of Contents --- p.I / Acknowledgements --- p.VI / Publications --- p.VII / Abstract (in English) --- p.VIII / Abstract (in Chinese) --- p.X / List of Figures --- p.XII / List of Tables --- p.XVI / List of Abbreviations --- p.XVII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Overview of oseltamivir --- p.1 / Chapter 1.1.1 --- General description of oseltamivir --- p.1 / Chapter 1.1.2 --- Pharmacological activities of oseltamivir --- p.3 / Chapter 1.1.3 --- Pharmacokinetics of oseltamivir --- p.3 / Chapter 1.1.3.1 --- Absorption of oseltamivir --- p.4 / Chapter 1.1.3.2 --- Distribution of oseltamivir --- p.5 / Chapter 1.1.3.3 --- Metabolism of oseltamivir --- p.6 / Chapter 1.1.3.4 --- Elimination of oseltamivir --- p.8 / Chapter 1.1.4 --- Side effects and toxicities of oseltamivir --- p.9 / Chapter 1.2 --- Overview of Chinese medicine formulae CMF1 (Yinqiaosan and Sangjuyin) --- p.9 / Chapter 1.2.1 --- Background and clinical use of CMF1 --- p.9 / Chapter 1.2.2 --- Quality control of CMF1 by manufacturer --- p.11 / Chapter 1.2.3 --- Major active components of CMF1 --- p.12 / Chapter 1.3 --- Previous studies on herb-drug interactions between O and CMF1 --- p.18 / Chapter 1.4 --- Rationale of the current study --- p.19 / Chapter 1.5 --- objectives --- p.19 / Chapter Chapter Two. --- Identification and quantification of major marker compounds in Yinqiaosan and Sangiuyin products --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Chemicals --- p.23 / Chapter 2.2.2 --- Instruments --- p.24 / Chapter 2.2.3 --- Chromatographic conditions --- p.24 / Chapter 2.2.4 --- Preparation of standard solutions --- p.25 / Chapter 2.2.5 --- Calibration curves --- p.26 / Chapter 2.2.6 --- Validation of the assay method --- p.26 / Chapter 2.2.7 --- Sample preparations for Yinqiaosan and Sangjuyin products --- p.27 / Chapter 2.2.7.1 --- Sample extraction from Yinqiaosan or Sangjuyin granules --- p.27 / Chapter 2.2.7.2 --- Sample extraction from Yinqiaosan or Sangjuyin tablets --- p.27 / Chapter 2.2.7.3 --- Sample extraction recoveries --- p.27 / Chapter 2.3 --- Results and discussions --- p.28 / Chapter 2.3.1 --- Chromatography --- p.28 / Chapter 2.3.2 --- Linearity and sensitivity --- p.33 / Chapter 2.3.3 --- Accuracy and precision --- p.33 / Chapter 2.3.4 --- Stability --- p.36 / Chapter 2.3.5 --- Contents of identified active components in commercial available Yinqiaosan or Sangjuyin products and CMF1 --- p.36 / Chapter 2.3.6 --- Sample extraction recovery --- p.40 / Chapter 2.4 --- Conclusion --- p.43 / Chapter Chapter Three. --- Effect of CMF1/CMF1 components on the metabolism of oseltamivir and related mechanistic studies --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and methods --- p.47 / Chapter 3.2.1 --- Materials --- p.47 / Chapter 3.2.2 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.48 / Chapter 3.2.3 --- Inhibition of metabolism of O by CMFl/CMFl components --- p.49 / Chapter 3.2.3.1 --- In vitro inhibition of metabolism of O in rat plasma --- p.49 / Chapter 3.2.3.2 --- In vitro inhibition of metabolism of O in rat liver microsome (RLM) --- p.49 / Chapter 3.2.4 --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.50 / Chapter 3.2.5 --- Sample preparation and LC/MS/MS analysis --- p.50 / Chapter 3.2.6 --- Data analyses --- p.52 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.53 / Chapter 3.3.2 --- Inhibition of metabolism of O by CMF1/CMF1 components --- p.53 / Chapter 3.3.2.1 --- Enzyme inhibition of metabolism of O by CMFl/CMF1 components in rat plasma --- p.53 / Chapter 3.3.2.2 --- Enzyme inhibition of metabolism of O by CMF1/CMF1 components in rat liver microsome (RLM) --- p.58 / Chapter 3.3.2.3 --- Selection of potent enzyme inhibitor from CMF1 --- p.60 / Chapter 3.3.4. --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.61 / Chapter 3.4 --- Discussions --- p.63 / Chapter 3.5 --- Conclusion --- p.74 / Chapter Chapter Four. --- Effect of CMFl/CMFl components on the absorption of oseltamivir and related mechanistic studies --- p.75 / Chapter 4.1 --- Introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Materials --- p.79 / Chapter 4.2.2 --- PAMPA permeation model --- p.80 / Chapter 4.2.2.1 --- Permeation of O and OC in PAMPA --- p.80 / Chapter 4.2.2.2 --- Sample preparation and LC/MS/MS analysis --- p.81 / Chapter 4.2.2.3 --- Data analysis --- p.81 / Chapter 4.2.3 --- Absorption of O in presence of CMF/CMFl components in Caco-2 and MDCK cell monolayer models --- p.82 / Chapter 4.2.3.1 --- Cell culture --- p.82 / Chapter 4.2.3.2 --- Preparation of loading solutions to the cell models --- p.83 / Chapter 4.2.3.3 --- Stability of O in transport buffer --- p.84 / Chapter 4.2.3.4 --- Cytotoxicity tests of O and CMFl/CMFl components --- p.84 / Chapter 4.2.3.5 --- Transport study in Caco-2 and MDCK monolayer model --- p.85 / Chapter 4.2.3.6 --- Sample preparation and LC/MS/MS analysis --- p.86 / Chapter 4.2.3.7 --- Data analysis --- p.87 / Chapter 4.2.4 --- Absorption of O in presence of CMF 1 in rat in situ single pass intestinal perfusion model --- p.88 / Chapter 4.2.4.1 --- Preparation of perfusion solutions --- p.88 / Chapter 4.2.4.2 --- Stabilities of O and arctigenin in perfusate --- p.88 / Chapter 4.2.4.3 --- Rat in situ single pass intestinal perfusion of O in presence and absence of CMFl and relevant inhibitors --- p.89 / Chapter 4.2.4.4 --- Sample preparation and LC/MS/MS analysis --- p.90 / Chapter 4.2.4.5 --- Data analysis --- p.90 / Chapter 4.3 --- Resul ts --- p.91 / Chapter 4.3.1 --- Permeation of O and OC in PAMPA --- p.91 / Chapter 4.3.2 --- Absorption of O in presence of CMF/CMF1 components in Caco-2 and MDCK cell monolayer models --- p.92 / Chapter 4.3.2.1 --- Stabilities of O in transport buffer --- p.92 / Chapter 4.3.2.2 --- Cytotoxicity tests of O and CMF1/CMF1 components in transport buffer --- p.93 / Chapter 4.3.2.3 --- Proof of O as a substrate of P-gp by Caco-2 cell model --- p.95 / Chapter 4.3.2.4 --- Effect of CMF 1 on the absorption transport of o in Caco-2 cell mode --- p.98 / Chapter 4.3.2.5 --- Effect of CMF1 components on the absorption transport of o in Caco-2 cell model --- p.102 / Chapter 4.3.2.6 --- Effect of arctigenin on bi-directional transport of o in Caco- 2 cell model --- p.106 / Chapter 4.3.2.7 --- Proof of O as a substrate of P-gp by MDCK transfected cell lines --- p.108 / Chapter 4.3.2.8 --- Bi-directional transport of O in MDCK-MDR1 cell model --- p.111 / Chapter 4.3.2.9 --- Effect of CMF 1 on the absorption transport of O in MDCK-MDR1 cell model --- p.112 / Chapter 4.3.3 --- Absorption of O in presence of CMF1 in rat in situ single pass intestinal perfusion model --- p.113 / Chapter 4.3.3.1 --- Stabilities of O and arctigenin in the perfusion buffer --- p.113 / Chapter 4.3.3.2 --- Intestinal absorption of O in presence and absence of CMF1 in rat in situ intestinal perfusion model --- p.114 / Chapter 4.4 --- Discussions --- p.116 / Chapter 4.5 --- Conclusion --- p.124 / Chapter Chapter Five. --- Preliminary evaluation of antiviral activity of CMFl/CMFl components --- p.125 / Chapter 5.1 --- Introduction --- p.125 / Chapter 5.2 --- Materials and methods --- p.128 / Chapter 5.2.1 --- Materials and animals --- p.128 / Chapter 5.2.2 --- Animal treatment --- p.129 / Chapter 5.2.3 --- Plasma sample collection and preparation --- p.130 / Chapter 5.2.4 --- Evaluation of antiviral activities of CMFl/ CMFl components --- p.130 / Chapter 5.2.4.1 --- Plaque reduction assay --- p.131 / Chapter 5.2.4.2 --- Optimization of plasma sample dilution ratio --- p.131 / Chapter 5.2.5 --- Data analyses --- p.133 / Chapter 5.3 --- Results and discussions --- p.135 / Chapter 5.3.1 --- Ex vivo evaluation of antiviral activity of CMF1 --- p.135 / Chapter 5.3.2 --- In vitro evaluation of antiviral activity of CMF1 major marker compounds --- p.139 / Chapter 5.4 --- Conclusion --- p.141 / Chapter Chapter Six. --- Overall conclusion --- p.142 / References --- p.145

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

黑色素生成是為了保護皮膚細胞免受紫外光傷害的一個生化過程。在這過程中，黑色素在人類表皮底層的黑色素細胞的黑色素體內產生。該過程可以被基因，荷爾蒙或環境因素所影響。黑色素的製造量是依賴速度限制酶酪氨酸酶的活性，黑色素體的數量和大小，黑色素體通過黑色素細胞的偽足傳送致角質細胞的速度及黑色素體在角質細胞內的分佈。這些細胞過程會受皮膚顏色或紫外光曝光量的變化而影響。當黑色素的產生超過黑色素的降解，黑色素沉著毛病便出現。根據不同的皮膚類型，年齡組別及累積紫外光曝光程度而引發雀斑或黃褐斑的形成。很多治療方法市面上能夠提供，它們包括人工合成化粧品，激光，整容手術等。這些治療方法通常會產生副作用及蘊藏高風險。因此從天然物質裏尋找治療藥物便成了美容學的一個新的研究方向。在這研究裏，十種草本植物就從自古以來用作治療黑色素沉著毛病的傅統中藥中被挑選出來。那些草本植物被四種擁有不同極性的溶劑提取。小鼠黑色素細胞被用以篩選提取物的降黑色素能力。結果發現當歸的正己烷及二氯甲烷的提取物有正面效用。當歸的化學成份4-乙基間苯二酚、4-乙基苯酚及1-十四烷醇也能降低小鼠黑色素細胞的黑色素量。數種生化技術繼而被應用作研究有效化學物的藥理。他們包括西方墨點法、環磷酸腺苷測試、蛋白激酶A活性測試及酪氨酸酶活性測試。 / Melanogenesis is a biochemical process designated for protecting skin cells from ultraviolet (UV)-induced damage. During the process, melanin is produced in the melanosomes of the melanocytes located at the basal epidermis of human. The process could be affected by genetic, hormonal or environmental factors. Amount of melanin synthesized depending on the activity of the rate-limiting enzyme tyrosinase, number and size of melanosomes, the transfer rate of melanosomes to keratinocytes through the melanocyte dendritic projections and the distribution pattern of melanosomes within keratinocytes. These cellular processes are influenced by variations in skin color or UV exposure amount. When melanin synthesis exceeds melanin degradation, hyperpigmentation disorder arises. This lead to the formation of freckles or chloasma according to different skin types, age groups and degree of cumulative UV exposure. A number of treatments are commercially available, they include applying synthetic cosmetics, laser, plastic surgery, etc. These treatments usually produce side-effects and possess high risk. Therefore, searching for therapeutic agents from natural compounds has become a new research direction in cosmetology. In this study, ten herbs were chosen from traditional Chinese medicine (TCM) which had been applied for treating hyperpigmentation. The herbs were extracted by four solvents with different polarity. The extracts were screened for their hypopigmentary ability by using melan-a cells. It was found that the hexane and dichloromethane extracts of Angelica sinensis possessed positive effects. 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol, the chemical constituents of A. sinensis, also attenuated melanin amount in melan-a cells. Moreover, several biochemical techniques were utilized to study the pharmaceutical mechanisms of the potent compounds. They include Western blot, cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay and tyrosinase activity assay. / Detailed summary in vernacular field only. / Lam, Rosanna Yen Yen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Chinese Abstract --- p.iii / Acknowledgements --- p.iv / List of Publications --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Demand of cosmetics --- p.1 / Chapter 1.2 --- Skin structures and functions --- p.1 / Chapter 1.2.1 --- Epidermis --- p.3 / Chapter 1.2.1.1 --- Stratum corneum --- p.4 / Chapter 1.2.1.2 --- Stratum granulosum --- p.4 / Chapter 1.2.1.3 --- Stratum spinosum --- p.4 / Chapter 1.2.1.4 --- Stratum basale --- p.4 / Chapter 1.2.2 --- Dermis --- p.5 / Chapter 1.2.3 --- Hypodermis --- p.5 / Chapter 1.3 --- Sun irradiation --- p.5 / Chapter 1.4 --- Variety of skin types --- p.6 / Chapter 1.5 --- Biochemical reactions within melanocyte --- p.7 / Chapter 1.6 --- Pigmentation disorder --- p.14 / Chapter 1.7 --- From the view of traditional Chinese medicine --- p.16 / Chapter 1.8 --- Treatments available for hyperpigmentation --- p.18 / Chapter 1.9 --- Aims of study and application of strategies --- p.19 / Chapter Chapter 2 --- Investigation of the inhibitory effect of herbal extracts and their constituent compounds on melanin synthesis --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Materials --- p.21 / Chapter 2.2.2 --- Herbal extraction --- p.23 / Chapter 2.2.3 --- Cell culture --- p.25 / Chapter 2.2.4 --- Growth curve and melanin production curve --- p.25 / Chapter 2.2.5 --- SRB assay --- p.26 / Chapter 2.2.6 --- Calibration curve for SRB assay --- p.27 / Chapter 2.2.7 --- Measurement of melanin production --- p.27 / Chapter 2.2.8 --- Calibration curve for melanin production assay --- p.28 / Chapter 2.2.9 --- Statistical analysis --- p.28 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- Growth curve and melanin production curve for assay development --- p.29 / Chapter 2.3.2 --- Calibration curves of SRB assay and melanin production assay --- p.32 / Chapter 2.3.3 --- Hypopigmentary effect of 40 herbal extracts --- p.35 / Chapter 2.3.4 --- Hypopigmentary effects of chemical components of A. sinensis --- p.41 / Chapter 2.4 --- Discussion --- p.49 / Chapter Chapter 3 --- Study of the effect of potential compounds on melanogenic protein expression by Western blot --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Cell culture --- p.56 / Chapter 3.2.3 --- Preparation of cell lysates --- p.57 / Chapter 3.2.4 --- Protein assay --- p.57 / Chapter 3.2.5 --- SDS-PAGE and membrane transfer --- p.58 / Chapter 3.2.6 --- Washing of blotted antibodies and film exposure --- p.59 / Chapter 3.3 --- Results --- p.61 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- Study of the effect of potential compounds on melanogenic gene expression by RT-PCR --- p.76 / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.76 / Chapter 4.2.1 --- Materials --- p.77 / Chapter 4.2.2 --- Cell culture --- p.77 / Chapter 4.2.3 --- RNA extraction --- p.78 / Chapter 4.2.4 --- cDNA synthesis --- p.78 / Chapter 4.2.5 --- PCR --- p.80 / Chapter 4.3 --- Results --- p.83 / Chapter 4.4 --- Discussion --- p.85 / Chapter Chapter 5 --- Study of the effect of potential compounds on cAMP level by EIA --- p.85 / Chapter 5.1 --- Introduction --- p.86 / Chapter 5.2 --- Materials and methods --- p.86 / Chapter 5.2.1 --- Materials --- p.86 / Chapter 5.2.2 --- Cell culture --- p.86 / Chapter 5.2.3 --- Preparation of cell lysates --- p.86 / Chapter 5.2.4 --- Protein assay --- p.87 / Chapter 5.2.5 --- The cAMP assay --- p.88 / Chapter 5.2.6 --- Preparation of cAMP calibration curve --- p.88 / Chapter 5.2.7 --- Calculation --- p.89 / Chapter 5.2.8 --- Statistical analysis --- p.89 / Chapter 5.3 --- Results --- p.90 / Chapter 5.4 --- Discussion --- p.94 / Chapter Chapter 6 --- Study of the effect of potential compounds on PKA activity by PKA activity assay --- p.96 / Chapter 6.1 --- Introduction --- p.96 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Materials --- p.97 / Chapter 6.2.2 --- Cell culture --- p.97 / Chapter 6.2.3 --- Preparation of cell lysates --- p.98 / Chapter 6.2.4 --- Protein assay --- p.98 / Chapter 6.2.5 --- The PKA kinase activity assay --- p.100 / Chapter 6.2.6 --- Calculation --- p.100 / Chapter 6.2.7 --- Statistical analysis --- p.100 / Chapter 6.3 --- Results --- p.101 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter 7 --- Study of the effect of potential compounds on tyrosinase activity by enzyme inhibition assay --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Materials --- p.108 / Chapter 7.2.2 --- Assay development for mushroom tyrosinase --- p.109 / Chapter 7.2.3 --- Mushroom tyrosinase inhibition assay --- p.109 / Chapter 7.2.4 --- Cell culture --- p.110 / Chapter 7.2.5 --- Preparation of cellular tyrosinase --- p.110 / Chapter 7.2.6 --- Protein assay --- p.111 / Chapter 7.2.7 --- Cellular tyrosinase inhibition assay --- p.111 / Chapter 7.2.8 --- Calculation --- p.112 / Chapter 7.2.9 --- Statistical analysis --- p.112 / Chapter 7.3 --- Results --- p.113 / Chapter 7.4 --- Discussion --- p.120 / Chapter Chapter 8 --- General discussion --- p.123 / References --- p.127

Thesis (M.Sc. (Biochemistry )) --University of Limpopo, 2007 / Refer to document / Medical Research Council (MRC), National Research Foundation (NRF) and University of Limpopo

This thesis set out to examine the soils, plants, lichens, invertebrates and common skinks along a gradient of habitat modification. This gradient was represented by ten study sites, of varying management histories, in a mid altitude tall tussock (Chionochloa spp.) grassland in eastern Central Otago. One extreme of the gradient was represented by intact tall tussock plants with a high density of inter tussock vegetation. The other extreme was represented by intensively managed exotic pasture. Native plants, invertebrates and reptiles were present along the entire gradient of modification and there was a clear threshold in the diversity of native plants along the gradient. This threshold was reached after the management practice of ploughing was applied to a site. Sites that had not been ploughed were found to be similar in their native plant diversity, and all unploughed sites had significantly higher native plant diversity than unploughed sites. There was considerable variation in the abundance of individual plant and Coleoptera species along the modification gradient. The most intensively modified sites had the lowest plant diversity, the lowest abundance and diversity of Orthoptera, as well as the lowest abundance of common skinks. Sites with low levels of modification contained a higher abundance of common skinks, and, native forbs, lichens and bryophytes than the more intensively modified sites. With regard to individual plant and Coleoptera species recorded, there were generally not clear thresholds for their presence and absence along the modification gradient. Rather, there was a gradual turnover for most species along the gradient such that the plant and Coleoptera community at opposing ends of the gradient had less species in common sites of similar modification levels. A high proportion of the Coleoptera species observed were present in all sites, suggesting that disturbance has selected for an adaptive generalist life trait, and that the species remaining comprise the resilient portion of the Coleoptera fauna. The relationship between Coleoptera communities and the gradient of modification was scale dependent, with the strongest relationship being observed at the largest scale measured. The hypothesis that faunal diversity and abundance would be best predicted by the structural diversity of the vegetation rather than by plant species diversity was tested. Structural diversity was strongly correlated (p < 0.01) with Coleoptera diversity, and Coleoptera abundance whereas plant species diversity was not. However, plant species diversity was a stronger correlate of common skink abundance than plant structural diversity. The structural diversity of the vegetation has the potential to be measured remotely and could be a correlate for faunal diversity and abundance when undertaking landscape scale studies. This thesis demonstrates that native biota exists within the agricultural environment of the mid-altitude tall tussock (Chionochloa rigida) grasslands of eastern Central Otago despite intensive modification in some areas. The findings suggest that it is possible to integrate the objectives of agriculture and the conservation of some faunal groups especially for the more resilient species. However, this study also indicates that relatively unmodified sites are valuable reservoirs of biodiversity in the mid altitude zone and it is recommended that the limited number of lightly modified sites that remain in the mid altitude zone be conserved.

University of Macau / Institute of Chinese Medical Sciences