Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2023 / Plants are not only an important source of medicines, but also play a significant role in drug development for the treatment of diseases such as Tuberculosis (TB). TB is a pulmonary disease that is caused by Mycobacterium tuberculosis complex. The aim of the study is to identify and characterise antimycobacterial compounds from Dombeya rotundifolia. The plant was collected from the University of Limpopo, dried and ground into fine powder. Extraction was done using different solvents that differ in polarity. The plant was screened and analysed for phytochemicals. Three major phytochemicals were quantified using reagent assays and analysed using standard curves. The antioxidant activity of the plant was determined using 2,2-Diphenyl-1- picrylhydrazyl (DPPH) and ferric reducing power assay. The antibacterial activity of the plant extracts was tested against Mycobacterium smegmatis using bioautography and serial microplate broth dilution assay. The antibiofilm activity of the plant extracts were evaluated using crystal violet assay. The anti-inflammatory activity of the plant was determined using egg albumin protein denaturation assay. The cytotoxic effects of the extracts were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on THP-1 cell line. The antimycobacterial compounds were isolated and purified using bioassay-guided assay incorporated with column chromatography and preparative TLC. The isolated compounds were analysed and identified using nuclear magnetic resonance spectroscopy (NMR). The results obtained in this study showed that water was the best extractant, extracting 57.6 mg of the plant material, followed by methanol with 38.1 mg and hexane as the least extractant with 12.4 mg. In the phytochemical analysis of TLC plates, there was a separation of compounds in all the mobile systems, and a clear separation of compounds was observed in the BEA mobile system, followed by the EMW system. The plant has all major phytochemicals that are needed for drug development. The plant extracts had antioxidant activity, which was confirmed by the quantitative assays. The high inhibitory activity of the extracts was indicated by low MIC values that inhibited the bacterial growth. The butanol extract had the lowest MIC value (0.13 mg/mL). The plant extracts were able to prevent the formation of biofilm at different concentrations. The plant was proven to have anti-inflammatory properties by having a high inhibition capacity to prevent protein denaturation. The cytotoxicity results showed that the plant was toxic to the cells. The isolated antimycobacterial compounds were identified as Eicosanoic acid and Docosanoic. They have moderate activity with the MIC value of 0.25 mg/mL. The study indicated that the isolated compounds have antimycobacterial activity, which showed that the plant has a potential to treat TB or symptoms related to TB. However, further studies are needed to evaluate their toxic effects before use. / The National Research Foundation (NRF)

No description available.

The following study was conducted to investigate the antibacterial and feed additive potential of medicinal plants. Ethanol extracts of different medicinal plants including Curcuma longa (Turmeric), Zingiber officinale (Ginger), Piper nigrum (Black Pepper), Cinnamomum cassia (Cinnamon), Thymus vulgaris (Thyme), Laurus nobilis (Bay leaf), and Syzgium aromaticum (Clove) were tested using the disc diffusion method for their antimicrobial activity against the common poultry pathogens E. coli, S. typhimurium, E. faecium, and E. faecalis. Cinnamon extract (CE), at 130 mg/disk, exhibited antibacterial activity against E. coli, S. typhimurium, and E. faecalis. Thyme extract (TE), at 30 mg/disk, exhibited antibacterial activity against E. coli, E. faecium, and E. faecalis while the remaining medicinal plants extracts showed no activity. The minimum inhibitory concentration (MIC) of the cinnamon and thyme ranged from 31.25 to 250 mg/ml by the dilution method. From this in vitro antibacterial study, cinnamon and thyme were selected for a 21-d feeding trial in broilers to study their influence on feed consumption, body weight gain, and feed conversion. There were 6 dietary treatments groups: 1) negative control (NC) containing no plant extracts or antibiotic, 2) positive control (PC) containing BMD (bacitracin) at 50g/ton of feed, 3) Diet 1 plus low level of cinnamon extract (LCE) at 290 gm/100 kg of feed, 4) Diet 1 plus high level of cinnamon extract (HCE) at 580 gm/ 100 kg of feed, 5) Diet 1 plus low level of thyme extract (LTE) at 290 gm/100kg of feed, and 6) Diet 6 plus high level of thyme extract (HTE) at 580 gm/100 kg of feed. No significant changes in body weight gain were observed with the cinnamon extracts compared to the NC or PC at 7, 14, or 21 d. The HTE reduced body weight gain compare to the NC and PC at 7, 14, and 21 d (P < 0.02). No difference in feed efficiency was observed with any of the treatments except LCE which reduced feed efficiency compared to other treatments. No difference in feed consumption was found among any of the treatments. These results suggest that cinnamon and thyme have antibacterial activity in vitro, and thyme has an activity that reduces body weight. Since cinnamon caused no significant change in body weight gain compared to positive or negative controls, it warrants further study as a substitute for antibiotics in the diet. / Master of Science

Wong Chun-kwan. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 127-137). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.viii / Tables of Contents --- p.ix / List of Figures --- p.xv / List of Tables --- p.xxvi / Chapter Chapter 1: --- INTRODUCTION --- p.1 / Chapter Chapter 2: --- LITERATURE REVIEW --- p.8 / Chapter 2.1 --- Toxicology --- p.8 / Chapter 2.1.1 --- Acute toxicity test --- p.8 / Chapter 2.1.2 --- Biochemical Analysis --- p.9 / Chapter 2.1.3 --- Organ weights --- p.10 / Chapter 2.2 --- Histology --- p.11 / Chapter 2.2.1 --- Light Microscope --- p.11 / Chapter 2.2.2 --- Electron Microscopy --- p.11 / Chapter 2.3 --- Tissue injury --- p.12 / Chapter 2.3.1 --- Free-radical mechanisms --- p.12 / Chapter 2.3.2 --- Lipid peroxidation --- p.13 / Chapter 2.4 --- Carbon tetrachloride (CC14) --- p.14 / Chapter 2.4.1 --- Mechanisms of carbon tetrachloride toxicity --- p.15 / Chapter 2.5 --- Trichloroethylene (TCE) --- p.18 / Chapter 2.5.1 --- Mechanisms of trichloroethylene toxicity --- p.21 / Chapter 2.6 --- Dimethyl sulfoxide (DMSO) --- p.25 / Chapter 2.7 --- N-acetylcysteine (NAC) --- p.27 / Chapter Chapter 3: --- MATERIALS AND METHODS --- p.28 / Chapter 3.1 --- Materials --- p.28 / Chapter 3.2 --- Methods --- p.31 / Chapter 3.2.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.31 / Chapter 3.2.1.1 --- Preparation of aqueous extracts of seaweed --- p.31 / Chapter 3.2.1.2 --- Experimental protocol --- p.31 / Chapter 3.2.1.3 --- Biochemical assays --- p.32 / Chapter 3.2.1.4 --- Organ weights --- p.36 / Chapter 3.2.1.5 --- Histopathological examination --- p.36 / Chapter 3.2.1.6 --- Statistical analysis --- p.36 / Chapter 3.2.2 --- Curative and preventive tests of seaweed aqueous extracts against the CCl4-induced hepatotoxicity --- p.37 / Chapter 3.2.2.1 --- Preparation of aqueous extracts of seaweed --- p.37 / Chapter 3.2.2.2 --- Experimental protocol --- p.37 / Chapter 3.2.2.3 --- Biochemical assays --- p.39 / Chapter 3.2.2.4 --- Organ weights --- p.39 / Chapter 3.2.2.5 --- Histopathological examination --- p.40 / Chapter 3.2.2.6 --- Statistical analysis --- p.41 / Chapter 3.2.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.42 / Chapter 3.2.3.1 --- Experimental protocol --- p.42 / Chapter 3.2.3.2 --- Biochemical assays --- p.43 / Chapter 3.2.3.3 --- Organ weights --- p.43 / Chapter 3.2.3.4 --- Histopathological examination --- p.44 / Chapter 3.2.3.5 --- Statistical analysis --- p.44 / Chapter 3.2.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.44 / Chapter 3.2.4.1 --- Preparation of aqueous extracts of seaweed --- p.44 / Chapter 3.2.4.2 --- Experimental protocol --- p.45 / Chapter 3.2.4.3 --- Biochemical assays --- p.46 / Chapter 3.2.4.4 --- Organ weights --- p.46 / Chapter 3.2.4.5 --- Histopathological examination --- p.46 / Chapter 3.2.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE- induced poisoning in rats --- p.47 / Chapter 3.2.5.1 --- Experimental protocol --- p.47 / Chapter 3.2.5.2 --- Biochemical assays --- p.48 / Chapter 3.2.5.3 --- Organ weights --- p.48 / Chapter 3.2.5.4 --- Histopathological examination --- p.49 / Chapter 3.2.6 --- Hepatoprotective effect of seaweeds' methanol extract against CC14- and TCE-induced poisoning in rats --- p.49 / Chapter 3.2.6.1 --- Preparation of methanol extracts of seaweed --- p.49 / Chapter 3.2.6.2 --- Experimental protocol --- p.50 / Chapter 3.2.6.3 --- Biochemical assays --- p.52 / Chapter 3.2.6.4 --- Organ weights --- p.52 / Chapter 3.2.6.5 --- Histopathological examination --- p.53 / Chapter Chapter 4 --- RESULTS --- p.54 / Chapter 4.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.54 / Chapter 4.1.1 --- The biochemical assays of the serum transaminase activity --- p.54 / Chapter 4.1.2 --- The organ weight (Aqueous seaweed crude extracts) --- p.56 / Chapter 4.2 --- Curative and preventive tests of seaweed aqueous extracts against the CCl4-induced hepatotoxicity --- p.58 / Chapter 4.2.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.58 / Chapter 4.2.2 --- The organ weight (Curative) --- p.60 / Chapter 4.2.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.62 / Chapter 4.2.4 --- The organ weight (Preventive) --- p.64 / Chapter 4.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.66 / Chapter 4.3.1 --- Oral route --- p.66 / Chapter 4.3.1.1 --- One-time oral route --- p.66 / Chapter 4.3.1.2 --- Two-time oral route --- p.66 / Chapter 4.3.2 --- Intraperitoneal route --- p.66 / Chapter 4.3.3 --- Time course of the effective dose of 20% TCE in i.p. route --- p.67 / Chapter 4.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.12 / Chapter 4.4.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.72 / Chapter 4.4.2 --- The organ weight (Curative) --- p.74 / Chapter 4.4.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.76 / Chapter 4.4.4 --- The organ weight (Preventive) --- p.78 / Chapter 4.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE-induced poisoning in rats --- p.80 / Chapter 4.5.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.80 / Chapter 4.5.2 --- The organ weight (Curative) --- p.82 / Chapter 4.5.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.84 / Chapter 4.5.4 --- The organ weight (Preventive) --- p.86 / Chapter 4.6 --- Hepatoprotective effect of methanol extract of seaweed against CC14- and TCE-induced poisoning in rats --- p.88 / Chapter 4.6.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.88 / Chapter 4.6.2 --- The organ weight (Curative) --- p.89 / Chapter 4.7 --- Histopathological examinations --- p.90 / Chapter 4.7.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.91 / Chapter 4.7.2 --- Curative and preventive tests of seaweed aqueous extracts against the CC14-induced hepatotoxicity --- p.92 / Chapter 4.7.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.99 / Chapter 4.7.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.100 / Chapter 4.7.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE-induced poisoning in rats --- p.100 / Chapter 4.7.6 --- Hepatoprotective effect of methanol extract of seaweed against CC14- and TCE-induced poisoning in rats --- p.102 / Chapter Chapter 5 --- DISCUSSION --- p.106 / Chapter Chapter 6 --- CONCLUSION --- p.124 / REFERENCES --- p.127 / APPENDIX --- p.138

Wong Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 124-144). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer treatment and potential novel antitumor agents --- p.1 / Chapter 1.2 --- History of mushroom polysaccharides in medical uses --- p.1 / Chapter 1.3 --- Life cycle of mushroom --- p.3 / Chapter 1.4 --- Classification of antitumor mushroom polysaccharides --- p.5 / Chapter 1.4.1 --- (3-glucans --- p.5 / Chapter 1.4.2 --- Heteropolysaccharides --- p.7 / Chapter 1.4.3 --- Polysaccharide-protein complexes --- p.7 / Chapter 1.5 --- Structure-activity relationship of mushroom polysaccharides --- p.8 / Chapter 1.5.1 --- Lentinan as typical example --- p.9 / Chapter 1.5.2 --- Molecular weight --- p.10 / Chapter 1.5.3 --- Conformation --- p.10 / Chapter 1.5.4 --- Chemical modification --- p.11 / Chapter 1.5.5 --- Degree of branching --- p.13 / Chapter 1.6 --- Antitumor mushroom polysaccharides obtained from different developmental stages --- p.17 / Chapter 1.7 --- Mechanisms of in vitro antitumor activity of mushroom polysaccharides: cell cycle arrest and apoptotic induction --- p.20 / Chapter 1.7.1 --- Cell cycle regulation --- p.21 / Chapter 1.7.2 --- Induction of apoptosis --- p.24 / Chapter 1.8 --- The novel strategies for cancer treatment --- p.27 / Chapter 1.9 --- Literature Review on Pleurotus tuber-regium --- p.30 / Chapter 1.10 --- Objectives --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Assay kits --- p.35 / Chapter 2.1.2 --- Mushroom samples --- p.35 / Chapter 2.1.3 --- Cell lines and their subculture --- p.36 / Chapter 2.1.4 --- Antibodies --- p.37 / Chapter 2.2 --- Extraction of mushroom polysaccharides --- p.38 / Chapter 2.2.1 --- Hot-water extracts from mushroom fruiting body --- p.38 / Chapter 2.2.2 --- Hot-water extracts from mushroom mycelia --- p.38 / Chapter 2.2.3 --- Exo-polysaccharides from submerged fermentation medium --- p.39 / Chapter 2.3 --- Chemical and physio-chemical composition of PTR extracts --- p.41 / Chapter 2.3.1 --- Neutral monosaccharides --- p.41 / Chapter 2.3.1.1 --- Acid Depolymerization --- p.41 / Chapter 2.3.1.2 --- Neutral sugar derivatization --- p.42 / Chapter 2.3.1.3 --- Determination of neutral sugar composition by GC- --- p.43 / Chapter 2.3.2 --- Uronic acid (acidic monosaccharides) content --- p.45 / Chapter 2.3.3 --- Total carbohydrate content --- p.46 / Chapter 2.3.4 --- Protein content --- p.46 / Chapter 2.3.5 --- Molecular weight and the homogeneity --- p.47 / Chapter 2.4 --- In vitro growth inhibitory effects --- p.48 / Chapter 2.4.1 --- Trypan blue dye exclusion method --- p.48 / Chapter 2.4.2 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.49 / Chapter 2.5 --- In vitro cell proliferation assay --- p.50 / Chapter 2.6 --- Cell-cycle analysis --- p.51 / Chapter 2.7 --- Apoptotic determination --- p.52 / Chapter 2.8 --- Expression of proteins involved in apoptosis and cell-cycle --- p.52 / Chapter 2.8.1 --- Preparation of cell lysates --- p.53 / Chapter 2.8.2 --- Determination of protein concentrations --- p.53 / Chapter 2.8.3 --- Western blot --- p.54 / Chapter 2.9 --- Statistics --- p.57 / Chapter Chapter 3 --- Results and Discussion --- p.58 / Chapter 3.1 --- Yield of extract samples isolated from different developmental stages of PTR --- p.58 / Chapter 3.2 --- Chemical characteristics of hot-water extracts isolated from different stages of PTR --- p.60 / Chapter 3.2.1 --- The total carbohydrate and protein content of PTR extracts- --- p.60 / Chapter 3.2.2 --- The monosaccharide composition of PTR extracts --- p.62 / Chapter 3.3 --- Molecular weight distribution of PTR extracts --- p.64 / Chapter 3.4 --- Chemical characterization of PTR extracts --- p.69 / Chapter 3.5 --- Cytotoxic effect of PTR extracts on various cell line in vitro --- p.71 / Chapter 3.5.1 --- Effect of PTR extracts on HL-60 cell viability --- p.71 / Chapter 3.5.2 --- Effect of PTR extracts on K562 cell viability --- p.74 / Chapter 3.5.3 --- Effect of PTR extracts on MCF-7 cell proliferation --- p.76 / Chapter 3.5.4 --- Effect of PTR extracts on HepG2 cell proliferation --- p.76 / Chapter 3.5.5 --- Effect of PTR extracts on normal cell proliferation --- p.78 / Chapter 3.6 --- Effect of PTR extracts on the proliferation rate of various cell lines in vitro --- p.78 / Chapter 3.6.1 --- Effect of PTR extracts on HL-60 cell proliferation --- p.79 / Chapter 3.6.2 --- Effect of PTR extracts on K562 cell proliferation --- p.79 / Chapter 3.6.3 --- Effect of PTR extracts on MCF-7 cell proliferation --- p.80 / Chapter 3.6.4 --- Effect of PTR extracts on HepG2 cell proliferation --- p.80 / Chapter 3.6.5 --- Effect of PTR extracts on normal cell proliferation --- p.84 / Chapter 3.7 --- Summary of the cytotoxic and antiproliferative activities exhibited by PTR extracts --- p.84 / Chapter 3.8 --- Analysis of the effect of PTR extracts on the cell-cycle phases of HL-60 and K562 cells --- p.87 / Chapter 3.8.1 --- Effect of CEP on cell-cycle phases of HL-60 and K562 cells --- p.87 / Chapter 3.8.2 --- Effect of EDP on cell-cycle phases of HL-60 and K562 cells --- p.92 / Chapter 3.8.3 --- Effect of HWE1 on cell-cycle phases of HL-60 and K562 cells --- p.95 / Chapter 3.8.4 --- Effect of HWE2 on cell-cycle phases of HL-60 and K562 cells --- p.98 / Chapter 3.8.5 --- Effect of HWE3 on cell-cycle phases of HL-60 and K562 cells --- p.102 / Chapter 3.8.6 --- Summary --- p.105 / Chapter 3.9 --- The effect of PTR extracts on expression of cellular proteins involved in cell-cycle control and apoptotic pathway in HL-60 cells --- p.106 / Chapter 3.9.1 --- Expression of Bcl-2 and Bax proteins in HL-60 cells treated with PTR extracts --- p.106 / Chapter 3.9.2 --- Expression of cyclins and Cdks in HL-60 cells by PTR extracts --- p.115 / Chapter 3.9.3 --- The plausible antiproliferative mechanism(s) involved in PTR extracts on HL-60 cells --- p.117 / Chapter Chapter 4 --- Conclusions and Future works --- p.120 / Chapter 4.1 --- Conclusions --- p.120 / Chapter 4.2 --- Future works --- p.122 / References --- p.124 / Related Publications --- p.144

肝癌是造成癌症相關死亡的主要原因之一。而常規化療受耐藥性的發展和各種副作用的限制。由於無毒性和鲜明的生物药物能力，從蘑菇提取的代謝物在癌症治療中獲得更多的注意和关注。我們以前的研究已經證明來自平菇香菇多醣蛋白複合物的抗癌作用。本研究的目的是探討一種含有多醣蛋白複合物的秀珍菇（PP）熱水提取物在肝癌細胞中抗癌活性的分子機制。 / 我們的研究結果表明，用PP处理过的肝癌細胞，不僅顯著的显示出降低的體外腫瘤細胞的增殖和侵襲，也增強化療藥物順鉑的藥物敏感性。無論是口服和腹腔注射都顯著抑制移植免疫BALB / c裸小鼠的腫瘤生長。同时，PP也能在體外和體內实验顯著抑制PI3K/Akt信號通路在肝癌細胞。有趣的是，当过表达AKT时，Myr-AKT，PP的這種抑制癌细胞生长的效果有减弱的趋势，同时也反映在PP对癌细胞侵襲抑制的作用上。印跡和酶聯免疫吸附試驗結果表明，在PP处理过的肝癌細胞中，血管內皮生長因子（VEGF）的表達和分泌減少了。此外， rhVEGF的加入减弱了 PP对PI3K/Akt通路和肝癌细胞表型的抑製作用。 / 我們的研究結果表明，PP能在體外和體內试验中抑制肝癌細胞增殖，侵襲和耐藥性，通过抑制分泌血管內皮生長因子誘導PI3K/Akt的信號通路。這項研究表明了PP的潛在治療肝癌的治療意義。 / Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anti-cancer effects of polysaccharide-protein complexes derived from the Pleurotus mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anti-cancer activity of a hot water extract containing a polysaccharide-protein complex isolated from Pleurotus pulmonarius (PP) in liver cancer cells. / Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the in vitro cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells in vitro and in vivo, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. / Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells in vitro and in vivo, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. All these results suggest the potential therapeutic implication of PP in the treatment of human liver cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Wenwen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 83-99). / Abstracts also in Chinese. / Thesis Committee --- p.i / English Abstract --- p.ii / Chinese Abstract --- p.iv / Acknowledgements --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / Abbreviations --- p.x / Content page --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Mushroom as functional foods --- p.1 / Chapter 1.1.1 --- Introduction of functional food --- p.1 / Chapter 1.1.2 --- Functional food and cancer --- p.1 / Chapter 1.1.3 --- Edible Mushroom as functional food --- p.4 / Chapter 1.1.4 --- Pleurotus pulmonarius and its function --- p.7 / Chapter 1.2 --- Hepatocellular carcinoma --- p.9 / Chapter 1.2.1 --- Liver and hepatocellular carcinoma --- p.9 / Chapter 1.2.2 --- Carcinogenesis of liver cancer --- p.12 / Chapter 1.2.2.1 --- Hallmarks of cancer --- p.12 / Chapter 1.2.2.2 --- Cell cycle --- p.13 / Chapter 1.2.2.3 --- Apoptosis --- p.15 / Chapter 1.2.2.4 --- Angiogenesis --- p.17 / Chapter 1.2.2.5 --- Invasion and metastasis --- p.19 / Chapter 1.2.2.6 --- Drug resistance --- p.21 / Chapter 1.2.3 --- The role of PI3K/AKT pathway --- p.23 / Chapter 1.2.4 --- The role of growth factor Vascular endothelial growth factor (VEGF) in HCC --- p.25 / Chapter 1.3 --- Research objectives --- p.27 / Chapter 1.3.1 --- Hypothesis and objectives --- p.27 / Chapter 1.3.2 --- Experimental design --- p.28 / Chapter Chaper 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Mushroom Pleurotus pulmonarius --- p.29 / Chapter 2.1.2 --- Drugs and cell lines --- p.29 / Chapter 2.1.3 --- Antibodies list --- p.30 / Chapter 2.1.4 --- Animal models --- p.32 / Chapter 2.2 --- Sample preparation and structure investigation --- p.32 / Chapter 2.2.1 --- Polysaccharide extraction from mushroom --- p.32 / Chapter 2.2.2 --- Endotoxin test --- p.32 / Chapter 2.2.3 --- Determination of monosaccharide profile by gas chromatography and mass spectrometry (GC/MS) --- p.33 / Chapter 2.2.3.1 --- Sample preparation for gas chromatography analysis --- p.33 / Chapter 2.2.3.1.1 --- Acid depolymerisation --- p.33 / Chapter 2.2.3.1.2 --- Neutral sugar derivatization --- p.33 / Chapter 2.2.3.1.3 --- External monosaccharide standard preparation --- p.34 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC/MS) --- p.34 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method (Dubois, 1956) --- p.36 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method (Lowry et al.,1951) --- p.37 / Chapter 2.3 --- Biological assays --- p.38 / Chapter 2.3.1 --- In vitro assays --- p.38 / Chapter 2.3.1.1 --- MTT assay --- p.38 / Chapter 2.3.1.2 --- Colony formation assay --- p.38 / Chapter 2.3.1.3 --- Plasmid transfection --- p.39 / Chapter 2.3.1.4 --- In vitro cell invasion assay --- p.39 / Chapter 2.3.1.5 --- Cell cycle analysis --- p.39 / Chapter 2.3.1.6 --- Western blot analysis --- p.40 / Chapter 2.3.1.7 --- VEGF ELISA Kit --- p.42 / Chapter 2.3.2 --- In vivo assays --- p.43 / Chapter 2.3.2.1 --- Tumor xenograft nude mouse model --- p.43 / Chapter 2.3.2.2 --- Immunohistochemistry --- p.45 / Chapter 2.3.2.3 --- H&Estaining --- p.45 / Chapter 2.3.3 --- Statistical analysis --- p.45 / Chapter Chaper 3 --- Results and discussion --- p.46 / Chapter 3.1 --- The yield and chemical characteristic of PP --- p.46 / Chapter 3.1.1 --- The yield of PP from mushroom Pleurotus pulmonarius --- p.46 / Chapter 3.1.2 --- Total carbohydrate and protein content --- p.47 / Chapter 3.1.3 --- Monosaccharide composition by GC-MS --- p.48 / Chapter 3.2 --- Toxicity of the PP water by Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.2.1 --- Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.3 --- Effects of PP on the proliferation of liver cancer cell lines --- p.50 / Chapter 3.3.1 --- MTT assay --- p.50 / Chapter 3.3.2 --- Colony-formation assay --- p.51 / Chapter 3.3.3 --- Cytotoxic effects of PP against normal liver cell --- p.52 / Chapter 3.3.4 --- The anti-proliferative effect of PP on other cancer types --- p.53 / Chapter 3.3.5 --- Cell cycle analysis by flow cytometry of PP treated liver cancer cells --- p.54 / Chapter 3.3.6 --- Protein expression by western blot analysis of P treated liver cancer cells --- p.56 / Chapter 3.4 --- Anti-cancer effect of PP on liver cancer cells through inactivation of PI3K/AKT signaling pathway --- p.57 / Chapter 3.4.1 --- Effect of PP on inactivation of PI3K/AKT pathway --- p.57 / Chapter 3.4.2 --- The abrogated inhibitory effect of PP on Huh7 with overexpression of AKT. --- p.59 / Chapter 3.4.3 --- The abrogated inhibitory effect of PP on PI3K/AKT signal pathway with overexpression of the constitutively active form of AKT, Myr-AKT --- p.60 / Chapter 3.5 --- Inhibition of VEGF expression and secretion by PP --- p.62 / Chapter 3.5.1 --- ELISA result of PP on VEGF secretion --- p.62 / Chapter 3.5.2 --- The attenuated inhibitory effect of PP on cell proliferation with addition of rhVEGF --- p.63 / Chapter 3.5.3 --- The attenuated inhibitory effect of PP on PI3K/AKT signal pathway with addition of rhVEGF --- p.64 / Chapter 3.6 --- Effect of PP on enhancing the chemosensitivity of liver cancer cells to Cisplatin --- p.66 / Chapter 3.6.1 --- Synergistic effect of PP with cisplatin (DDP) in liver cancer cells --- p.66 / Chapter 3.6.2 --- The abrogated drug-resistant effect by PP by overexpression of the constitutively active form of AKT, Myr-AKT --- p.67 / Chapter 3.6.3 --- The abrogated drug-resistant effect of PP with addition of rhVEGF --- p.68 / Chapter 3.7 --- The anti-invasive potential of PP on liver cancer cells. --- p.69 / Chapter 3.7.1 --- Boyden chamber assay --- p.69 / Chapter 3.7.2 --- The attenuated anti-invasive effect of PP on liver cancer cells with overexpression of constitutively activated AKT --- p.71 / Chapter 3.7.3 --- The attenuated anti-invasive effect of PP on liver cancer cells with addition of rhVEGF --- p.72 / Chapter 3.8 --- The anti-tumor effect of PP in vivo --- p.73 / Chapter 3.8.1 --- The anti-tumor effect of PP by using tumor xenograft model --- p.73 / Chapter 3.8.2 --- Body weight of nude mice treated with PP --- p.75 / Chapter 3.8.3 --- Harmful effect of PP on nude mice --- p.76 / Chapter 3.8.4 --- Immunohistochemist analysis of mice tumor xenograft treated with PP --- p.77 / Chapter 3.8.5 --- Western blot anaylysis using the tumor tissues harvested from mice xenograftes treated with PP --- p.78 / Chapter Chapter 4 --- Conclusion and future Plan --- p.81 / Reference --- p.83 / Related Publication List --- p.100

Chau, Hiu Yan Anita. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 109-122). / Abstracts in English and Chinese. / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction on mushroom life cycle --- p.1 / Chapter 1.2 --- Introduction of mushroom sclerotium --- p.2 / Chapter 1.3 --- Different extraction methods of mushroom polysaccharides --- p.3 / Chapter 1.4 --- Bioactivities of mushroom polysaccharides and factors affecting their biological activities --- p.4 / Chapter 1.4.1 --- Molecular weight --- p.4 / Chapter 1.4.2 --- Linkages --- p.5 / Chapter 1.4.3 --- Branching rate --- p.5 / Chapter 1.4.4 --- Conformation --- p.6 / Chapter 1.5 --- Mechanisms for antitumor activites of mushrooms polysaccharides.… --- p.7 / Chapter 1.5.1 --- Cancer-preventing activity --- p.7 / Chapter 1.5.2 --- Immuno-enhancing activity (BRM) --- p.8 / Chapter 1.5.3 --- Direct tumor inhibition activity --- p.8 / Chapter 1.6 --- Cell cycle regulation and induction of apoptosis --- p.9 / Chapter 1.6.1 --- The cell cycle machinery --- p.9 / Chapter 1.6.2 --- Cell cycle arrest and regulation --- p.11 / Chapter 1.6.3 --- Apoptosis and regulation --- p.13 / Chapter 1.7 --- Literature review on Pleurotus tuber-regium --- p.16 / Chapter 1.7.1 --- Introduction of Pleurotus tuber-regium --- p.16 / Chapter 1.7.2 --- Antitumor effect of mushroom polysaccharides isolated from different developmental stages of Pleurotus tuber-regium --- p.17 / Chapter 1.7.2.1 --- Sclerotium --- p.17 / Chapter 1.7.2.2 --- Mycelium --- p.19 / Chapter 1.7.2.3 --- Culture medium --- p.19 / Chapter 1.7.2.4 --- Fruiting body --- p.20 / Chapter 1.8 --- Literature review on Size reduction process --- p.21 / Chapter 1.8.1 --- Introduction of micron technology --- p.21 / Chapter 1.8.1.1 --- Ball milling --- p.21 / Chapter 1.8.1.2 --- Jet milling --- p.22 / Chapter 1.8.1.3 --- High-pressure micronizing --- p.22 / Chapter 1.8.1.4 --- Oscillatory milling --- p.23 / Chapter 1.8.2 --- Effect of particle sizes on physicochemical properties and biological activities of plant materials --- p.23 / Chapter 1.8.2.1 --- Physicochemical properties --- p.24 / Chapter 1.8.2.2 --- Biochemical activities --- p.24 / Chapter 1.9 --- Objectives --- p.26 / Chapter Chapter 2. --- Materials and methods --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Mushroom sclerotia --- p.28 / Chapter 2.1.2 --- Micronisation --- p.29 / Chapter 2.1.3 --- Cell lines --- p.31 / Chapter 2.1.4 --- Antibodies --- p.33 / Chapter 2.1.5 --- Animal model --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Micronisation --- p.34 / Chapter 2.2.2 --- Hot water extraction for mushroom sclerotia --- p.35 / Chapter 2.2.3 --- Measurement of monosaccharide profile --- p.36 / Chapter 2.2.3.1 --- Acid deploymerisation --- p.36 / Chapter 2.2.3.2 --- Neutral sugar derivatization --- p.36 / Chapter 2.2.3.3 --- Gas chromatography (GC) --- p.37 / Chapter 2.2.4 --- Total sugar content by Phenol-sulphuric acid Method --- p.38 / Chapter 2.2.5 --- Acidic sugar content by measuring uronic acid content --- p.39 / Chapter 2.2.6 --- Protein content by Lowry-Folin Method --- p.40 / Chapter 2.2.7 --- Size exclusion chromatography by high pressure liquid chromatograhy (HPLC) --- p.41 / Chapter 2.2.8 --- In vitro antitumor assay --- p.41 / Chapter 2.2.8.1 --- Trypan blue exclusion assay --- p.42 / Chapter 2.2.8.2 --- MTT Assay --- p.42 / Chapter 2.2.9 --- Cell cycle analysis by Flow Cytometry --- p.43 / Chapter 2.2.10 --- Protein expression involved in apoptosis --- p.45 / Chapter 2.2.10.1 --- Cell lysates preparation --- p.45 / Chapter 2.2.10.2 --- Determination of protein concentrations --- p.46 / Chapter 2.2.10.3 --- Western blot --- p.46 / Chapter 2.2.11 --- In vivo antitumor assay --- p.50 / Chapter 2.2.11.1 --- BALB/c mice --- p.50 / Chapter 2.2.11.2 --- Athymic nude mice --- p.50 / Chapter 2.2.12 --- Statistical methods --- p.51 / Chapter Chapter 3 --- Results and Discussion --- p.52 / Chapter 3.1 --- Yield of hot water-soluble extracts from Pleurotus tuber-regium --- p.52 / Chapter 3.2 --- Chemical composition of hot water-soluble extracts from PTR --- p.56 / Chapter 3.2.1 --- Total carbohydrate content --- p.56 / Chapter 3.2.2 --- Uronic acid content --- p.57 / Chapter 3.2.3 --- Protein content --- p.58 / Chapter 3.3 --- Monosaccharide profiles of hot water-soluble extracts from PTR by gas chromatography (GC) --- p.61 / Chapter 3.4 --- Molecular weight profile of hot water-soluble extracts from PTR by size exclusion chromatography (SEC) --- p.64 / Chapter 3.5 --- Antitumor effects of mushroom sclerotial polysaccharides --- p.72 / Chapter 3.5.1 --- In vitro antiproliferation study --- p.72 / Chapter 3.5.1.1 --- In vitro antiproliferation study by HL-60 --- p.72 / Chapter 3.5.1.2 --- In vitro antiproliferation study by THP-1 --- p.75 / Chapter 3.5.1.3 --- In vitro antiproliferation study by MCF-7 --- p.77 / Chapter 3.5.1.4 --- In vitro antiproliferation study by K562 --- p.77 / Chapter 3.5.1.5 --- In vitro antiproliferation study by SI80 --- p.79 / Chapter 3.5.1.6 --- In vitro antiproliferation study by normal cells --- p.79 / Chapter 3.5.1.7 --- Dose-response relationship between hot water-soluble extract from PTR and tumor cell inhibition --- p.80 / Chapter 3.5.2 --- In vivo antitumor study --- p.83 / Chapter 3.5.2.1 --- BALB/c mice --- p.83 / Chapter 3.5.2.2 --- Athymic nude mice --- p.84 / Chapter 3.6 --- Flow cytometric analysis of tumor cells treated by various hot wter-soluble extracts from PTR --- p.88 / Chapter 3.6.1 --- Antiproliferative effect of various hot water-soluble extracts from 10PTR on HL-60 --- p.88 / Chapter 3.6.2 --- Antiproliferative effect of various hot water-soluble extracts from 10PTR on THP-1 --- p.93 / Chapter 3.7 --- Effects of various hot water-soluble extracts from 10PTR on expression of Bcl-2 and Bax proteins in HL-60 cells --- p.99 / Chapter 3.8 --- "Correlation between particle size, structure and antitumor activity of mushroom sclerotial extracts" --- p.101 / Chapter Chapter 4. --- Conclusions and Future Works --- p.105 / List of References --- p.109 / Related Publications --- p.123

Thesis (Ph. D. (Botany)) -- University of Limpopo, 2014 / Refer to document

Lim Sze Nee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 165-176). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xiii / List of Abbreviation --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Classification of algae --- p.2 / Chapter 1.2 --- Chemical and mineral composition of marine macroalgae --- p.4 / Chapter 1.3 --- Uses of marine macroalgae --- p.7 / Chapter 1.3.1 --- Food --- p.7 / Chapter 1.3.2 --- Industrial uses --- p.8 / Chapter 1.3.3 --- Agricultural uses --- p.9 / Chapter 1.3.3.1 --- Fertilizer --- p.9 / Chapter 1.3.3.2 --- Fodder --- p.9 / Chapter 1.3.4 --- Medicinal properties --- p.10 / Chapter 1.4 --- Pharmacological effects of marine macroalgae --- p.11 / Chapter 1.4.1 --- Antioxidant activity --- p.11 / Chapter 1.4.2 --- Hypotensive activity --- p.11 / Chapter 1.4.3 --- Antiviral activity --- p.12 / Chapter 1.4.4 --- Antimicrobial activity --- p.12 / Chapter 1.4.5 --- Antitumor activity --- p.13 / Chapter 1.4.6 --- Hypocholesterolemic activity --- p.14 / Chapter 1.5 --- Objectives --- p.14 / Chapter CHAPTER 2 --- Free Radical Scavenging and Antioxidative Activities of Marine Macroalgae --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Free radicals: definition and sources --- p.16 / Chapter 2.1.2 --- Free radical-induced damage --- p.16 / Chapter 2.1.2.1 --- Biological lipid peroxidation --- p.16 / Chapter 2.1.2.2 --- Lipid oxidation of foods --- p.18 / Chapter 2.1.3 --- Antioxidants --- p.19 / Chapter 2.1.3.1 --- Antioxidants --- p.19 / Chapter 2.1.3.2 --- Antioxidant mechanisms --- p.20 / Chapter 2.1.4 --- Synthetic antioxidants --- p.21 / Chapter 2.1.5 --- Natural antioxidants --- p.24 / Chapter 2.1.6 --- Objectives --- p.27 / Chapter 2.2 --- Methods and Materials --- p.28 / Chapter 2.2.1 --- Preparation of algae extracts --- p.28 / Chapter 2.2.2 --- Determination of free radical scavenging activities --- p.32 / Chapter 2.2.2.1 --- Superoxide anions scavenging activity --- p.32 / Chapter 2.2.3 --- Antioxidative activity using hemolysis assay --- p.33 / Chapter 2.2.3.1 --- Preparation of red blood cell (RBC) --- p.33 / Chapter 2.2.3.2 --- Hemolysis assay --- p.33 / Chapter 2.2.4 --- Lipid peroxidation assay --- p.34 / Chapter 2.2.4.1 --- Preparation of rat brain homogenates --- p.34 / Chapter 2.2.4.2 --- Measurement of lipid peroxidation --- p.34 / Chapter 2.2.5 --- Statistics --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Superoxide radical scavenging activity of algal extracts --- p.36 / Chapter 2.3.2 --- Effects of algae extracts on hemolysis assay --- p.41 / Chapter 2.3.3 --- Effects of algae extracts on lipid peroxidation --- p.44 / Chapter 2.4 --- Discussion --- p.50 / Chapter CHAPTER 3 --- Isolation of Antioxidative Phenolic Compounds from Sargassum siliquastrum --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.1.1 --- Phenolic compounds --- p.60 / Chapter 3.1.2 --- Major classes of phenolic compounds --- p.60 / Chapter 3.1.3 --- Functional aspects of phenolic compounds --- p.61 / Chapter 3.1.3.1 --- Functions of phenolic compounds in plants --- p.61 / Chapter 3.1.3.2 --- Biological and pharmacological activities --- p.64 / Chapter 3.1.3.3 --- Food industry --- p.65 / Chapter 3.1.4 --- Polyphenolic compounds in brown algae --- p.66 / Chapter 3.1.5 --- Objectives --- p.68 / Chapter 3.2 --- Methods and Materials --- p.69 / Chapter 3.2.1 --- Extraction and isolation of antioxidant components from S siliquastrum --- p.69 / Chapter 3.2.2 --- Thin-Layer chromatography --- p.70 / Chapter 3.2.3 --- Antioxidant activity --- p.71 / Chapter 3.2.4 --- Determination of total phenolics --- p.71 / Chapter 3.2.5 --- Infrared spectra --- p.72 / Chapter 3.2.6 --- Ultra-violet and visible (UV-vis) spectrophotometry --- p.72 / Chapter 3.2.7 --- Statistics --- p.73 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Identification of phenolic compounds from various solvent extracts of S. siliquastrum --- p.73 / Chapter 3.3.2 --- Isolation of dichloromethane fraction by liquid chromatography --- p.81 / Chapter 3.3.3 --- Phenolic content of isolated compounds --- p.86 / Chapter 3.3.4 --- IR and UV-vis spectra --- p.86 / Chapter 3.4 --- Discussion --- p.92 / Chapter 3.4.1 --- Antioxidative activities --- p.92 / Chapter 3.4.2 --- Relationship between phenolic contents and antioxidant activity --- p.95 / Chapter 3.4.3 --- Identification of antioxidant compounds --- p.97 / Chapter CHAPTER 4 --- Hypotensive Activities of Marine Algae in the Rat --- p.102 / Chapter 4.1 --- Introduction --- p.102 / Chapter 4.1.1 --- Basic principles of cardiovascular system --- p.102 / Chapter 4.1.2 --- Regulation of arterial pressure --- p.105 / Chapter 4.1.2.1 --- Short-term regulation of arterial pressure --- p.105 / Chapter 4.1.2.2 --- Long-term regulation of arterial pressure --- p.107 / Chapter 4.1.3 --- Hypertension --- p.108 / Chapter 4.1.3.1 --- Causes of hypertension --- p.109 / Chapter 4.1.3.2 --- Where do antihypertensive or hypotensive agents act? --- p.114 / Chapter 4.1.3.2.1 --- Sympathetic nervous system inhibitors --- p.115 / Chapter 4.1.3.2.2 --- Diuretics --- p.120 / Chapter 4.1.3.2.3 --- Vasodilators --- p.121 / Chapter 4.1.3.2.4 --- Calcium antagonist (Calcium channel blockers) --- p.121 / Chapter 4.1.3.2.5 --- Angiotensin-converting enzyme (ACE) inhibitors --- p.122 / Chapter 4.1.3.2.6 --- Antihypertensive drug combination --- p.122 / Chapter 4.1.4 --- The relationship between hypertension and free radicals --- p.123 / Chapter 4.1.5 --- Development of new antihypertensive agenrs --- p.124 / Chapter 4.2 --- Materials and methods --- p.125 / Chapter 4.2.1 --- Animal care --- p.125 / Chapter 4.2.2 --- Preparation of the blood pressure measurement in rats --- p.125 / Chapter 4.2.2.1 --- Effects of seaweed extracts on arterial blood pressure of rat --- p.126 / Chapter 4.2.2.1.1 --- Single-dose response curve --- p.126 / Chapter 4.2.2.1.2 --- Cumulative-dose response curve --- p.126 / Chapter 4.2.2.2 --- Pharmacological blocker studies --- p.128 / Chapter 4.2.3 --- Statistics --- p.131 / Chapter 4.3 --- Results --- p.131 / Chapter 4.3.1 --- Hypotensive effects of marine algal extracts --- p.131 / Chapter 4.3.2 --- Effects of pharmacological blockers on MAP --- p.135 / Chapter 4.4 --- Discussion --- p.150 / Chapter 4.4.1 --- Hypotensive effects of the marine algal extracts --- p.150 / Chapter 4.4.2 --- Pharmacological action of marine algal extracts --- p.152 / Chapter CHAPTER 5 --- Conclusion --- p.160 / REFERENCES --- p.165 / RELATED PUBLICATIONS --- p.177

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