University of Macau / Institute of Chinese Medical Sciences

Non‐alcoholic fatty liver diseases (NAFLD) is manifested in the absent of alcohol abuse. This disease is the major cause of liver failure and death among adults and children worldwide, including South Africa. Its increasing prevalence urges the need of therapeutic intervention. The main objectives of this study were to investigate the following: (1) The effect of 38.9% high fat diet (HFD)‐induced insulin resistance and fatty liver in male Wistar rats, (2) The efficacy of aqueous extracts from Sutherlandia frutescens leaves and Prunus africana bark and metformin in the treatment of HFDinduced insulin resistance and fatty liver. Male Wistar rats were fed on HFD (the HF group) or normal rat chow (the LF group) for 12 weeks. Even though the HFD‐fed rats had developed insulin resistance by week 12, fatty liver developed by week 16. After week 12, the HF group was divided into four groups of 6‐7 rats each and three of those groups were gavaged with either 0.125 mg P. africana extract/kg bwt/day (the HF+Pa group) or 50 mg S. frutescens extract kg bwt/day (the HF+Sf group) or 16 mg metformin/ kg bwt/day (HF+Met group), while kept on the same diet for an additional of 4 weeks, to investigate whether two medicinal plant extracts and metformin can prevent HFD to induce fatty liver or not. After 16 weeks, the liver histological images revealed that the HF group developed fatty liver in the form of both microsteatosis and macrosteatosis. Fatty liver was confirmed by significant increased liver total lipid (TL) and activities of glucose‐6‐phosphate dehydrogenase (cG6PD) and xanthine oxidase (XO), mitochondrial NADH oxidase (mNOX) and by a decrease (P<0.05) in the activities of the homogenate superoxide dismutase (hSOD) and mitochondrial complex II in the HF group, when compared to the LF group. Since the activities of mCS and cACL enzymes were not changed in the HF group, hence increased cG6PD activity in the HF group indicates that there was increased NADPH demand for lipid accumulation from activated NEFAs taken up by the liver from circulation and for maintenance of the NADPH‐dependent antioxidants and oxidants, respectively. The obtained data also show that mitochondria of the HFD‐fed rats adapted to an increase in energy availability, thereby compensation through decreasing complex II activity, to allow electron flux from β‐oxidation to respiratory chain in the HF group. Liver TL content was significantly decreased in the rats treated with metformin and P. africana extract, but not in the rats treated with S. frutescens when compared to the HF group (P < 0.05). However, the TL content remained >5% per liver weight in all treated groups. The present study demonstrates that these two plant extracts and metformin have different glucogenic and lipogenic effects from that presented by HFD alone when compared to the LFD alone. In conclusion, metformin and P. africana extract can attenuate HFD‐induced fatty liver without changing the dietary habits. Hence S. frutescens extract is less effective in the prevention of HFD‐induced fatty liver. A change in the dietary habits is recommended to be considered during the use of these three remedies in the treatment of HFD‐induced insulin resistance and fatty liver. All three treatments enhanced antioxidant capacity, and may improve insulin resistance and fatty liver mediated by the present HFD through different mechanism of actions in the liver.

Advanced pancreatic cancer (APC) has a poor prognosis due to the high degree of resistance after systemic chemotherapy. Celastrol (CSL), a quinone methyl triterpenoid monomer extracted from Tripterygium wilfordii Hook F, exhibits superior antitumor activity on pancreatic cancer (PC) both in vitro and in vivo. In addition, CLS counteracts multiple mechanisms involved in multi-drug resistance (MDR) of PC cells. However, CSL induced toxicity to normal tissues (e.g. liver) is the major impediment to its clinical application. Thus, it is desirable to seek strategy to facilitate CSL selectively targeting PC tissues and simultaneously reducing its exposure to healthy tissues (e.g. liver). Aptamers are single-stranded oligonucleotides which specifically recognize and bind to targets by distinct secondary or tertiary structures. Nucleolin, a protein overexpressed on the plasma membrane of PC cells than that of normal cells (e.g. liver cell), which shuttle between cell surface, cytoplasm and nucleus, work as a cell surface receptor. Nucleolin aptamer is an anti-proliferative G-rich oligonucleotide with high affinity and specificity to nucleolin, which has been proved to be safe in clinical research. Then, nucleolin aptamer, as a target moiety, provide an approach to facilitate CLS selectively targeting PC cells. Taken together, our hypothesis is that the nucleolin aptamer modification could facilitate the conjugated CSL selectively targeting pancreatic cancer cells to achieve higher antitumor activity and less liver toxicity. In our study, CSL was conjugated to nucleolin aptamer to form Nucleolin Aptamer-Celastrol Conjugate (NACC). A CRO Aptamer-Celastrol conjugate (CACC) was also synthesized as a control for comparison. The water solubility of NACC was significantly higher than that of CSL. Then, the molecular weight of NACC was detected by ESI mass sepectrum (MS). The anti-proliferative efficacy of NACC was higher than CSL in vitro. NACC could selectively bind to PANC-1 cells over normal liver cells. The cellular uptake of NACC by PANC-1 cell was stronger than CSL. Moreover, NACC could be taken up by PANC-1 cells mainly via macropinocytosis. Tissue distribution study revealed that NACC could selectively accumulate in pancreatic tumor tissue and reduce the distribution in liver in vivo. In addition, NACC demonstrated higher antitumor activity and less liver toxicity in vivo, compared with CSL and CACC. The above results revealed that the nucleolin aptamer modification could facilitate the conjugated CSL selectively targeting PC cells to achieve higher antitumor activity and less liver toxicity.

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

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

Finally, the neuroprotective effects of TGP against corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells, an in vitro experimental model of depression were studied. The results showed TGP treatment dose-dependently protected the cells against corticosterone-induced toxicity. The cytoprotection afforded by TGP treatment was shown to be associated with an enhanced antioxidant activity, and increased expressions of neurotrophins including brain-derived neurotrophic factor, nerve growth factor and neurotrophin-3. / Secondly, the antidepressant-like effect of TGP was evaluated by a rat model of depression induced by chronic unpredictable mild stress (CUMS). The results showed that a 5-week CUMS caused depression-like behavior in rats, as indicated by a significant decreases in sucrose consumption (assessed by sucrose preference test) and locomotor activity (assessed by open-field test), and an increase in immobility time (assessed by forced swim test). Intragastric administration of TGP during the five weeks of CUMS procedure significantly suppressed these behavioral changes induced by CUMS. / Taken together, the results confirmed the antidepressant-like effect of TGP. The antidepressive action of TGP may be mediated by the modulation of the hypothalamic-pituitary-adrenal axis function, the inhibition of oxidative stress, and the up-regulation of neurotrophins, thereby leading to the neuroprotective effects. / The antidepressant-like effect of TGP was firstly evaluated by the behavioral despair test, forced swim test and tail suspension test. The results showed that intragastric administration of TGP caused a significant reduction of immobility time in both forced swim and tail suspension tests in mice. TGP treatment also significantly reduced the duration of immobility time in the forced swim test in rats. / The root of Paeonia lactiflora Pall. (Family: Ranunculaceae), commonly known as peony, is a component herb of many traditional formulae for the treatment of depression-like disorders. Previous studies have demonstrated the antidepressive effect of peony extract in mouse models of depression. Total glycosides of peony (TGP) is regarded as the major active ingredients of peony. The present study aims to confirm the antidepressive potential of TGP and evaluate its action mechanisms. / Thirdly, the neuroprotective effects of TGP on CUMS-treated rats and its possible mechanisms were investigated. The results showed that treatment with TGP for 5 weeks produced neuroprotective effects on the hippocampus of CUMS-treated rats. This effect was associated with the attenuation of hypothalamic-pituitary-adrenal axis hyperactivation (characterized by a decreased serum corticosterone level and an increased hippocampal glucocorticoid receptor expression), an inhibition of oxidative stress, and up-regulation of neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3 in the hippocampus. / Mao, Qingqiu. / Adviser: Che Chun-Tao. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 158-186). / 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.

EGb761 consists of two major groups of substances, flavonoids and terpenoids. Using human neuroblastoma SH-SY5Y cells, the present study demonstrated that, EGb761 could block Abeta-42 (a 42-amino acid cytoxic form of beta amyloid protein)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways, possibly via its antioxidant and platelet activating factor (PAF) antagonizing activities. Two active constituents of EGb761, quercetin (a flavonoid) and ginkgolide B (a terpenoid) might contribute to the protective effects of EGb761. Quercetin but not ginkgolide B might be responsible for the antioxidant action of EGb761. Both compounds might be involved in the PAF antagonist activity of EGb761. / EGb761, a Ginkgo biloba extract, is a medicinal product for the treatment and prevention of cardiovascular and neuronal diseases, including Alzheimer's disease (AD). While considerable researches have documented its neuroprotective effects, its clinical effect is inconclusive and the precise neuroprotective mechanisms are not clearly known. / In conclusion, EGb761 may have beneficial effects in treatment and prevention of neurodegenerative diseases like AD. Its neuroprotective effects may be associated with constituent multiplicity, the dosage and BBB permeability. / The ability of EGb761 to cross the blood brain barrier (BBB) is unclear. In this study, the ability of EGb761 to cross the BBB was speculated through comparison of the effects of EGb761 on mitochondrial function between platelets and central nervous system in two animal models, the senescence accelerated prone 8 (SAMP8) mouse strain and ovariectomized rats. Mitochondrial function was evaluated as cytochrome c oxidase (COX) activity, mitochondrial ATP content and mitochondrial glutathione (GSH) content. SAMP8 mice have been widely used as a model of age-related cognitive decline with relevance to biochemical and genetic alterations in AD. Using two age groups (3-week-old and 40-week-old) of SAMP8 mice, this study found that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of old mice, but only showed protective effects on platelet mitochondria of young mice. Estrogen withdrawal was suggested to play a primary role in the onset of post-menopausal AD. Using ovariectomized middle-aged rats to mimic the post-menopausal pathophysiological changes, this study also demonstrated that, EGb761 protected against mitochondrial dysfunction in both platelets and hippocampi of ovariectomized rats. In contrast, in sham-operated rats, EGb761 increased mitochondrial GSH content in platelets but failed to show similar effect on hippocampi. These results suggested that the effects of EGb761 on the brain might be interfered by the BBB permeability. / The effective dosage of EGb761 in the brain remains undetermined. Using SH-SY5Y cells, this study demonstrated that low doses of EGb761 (50--100 mug/ml) inhibited hydrogen peroxide (H2O2)-induced cell apoptosis via inactivation of Alet, JNK and caspase 3 while high doses of EGb761 (250--500 flg/ml) enhanced H2O2 toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments suggested that the dosage effect of EGb761 on apoptotic signaling proteins might be correlated with regulation of the cell redox state. / Shi, Chun. / Adviser: Lee Ka Ho Kenneth. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 81-99). / 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.

Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata v ABSTRACT Infectious diarrhoea is the second largest single cause of mortality in children under the age of five globally. Bacteria are responsible for most diarrhoeal episodes especially in developing countries, and progressive increase in antimicrobial resistance has given rise to the need to investigate other sources of therapy such as medicinal plants. Ten plant extracts were analysed for their antimicrobial activities using the agar well diffusion and broth microdilution method. Their phytochemical contents were screened, and their effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess their antioxidant activities. Their toxicity profiles were evaluated using the XTT Cytotoxicity Assay. Water and methanol extracts of Adansonia digitata seeds and pulp showed no inhibition against all the test organisms, while water and methanol extracts of A. digitata leaves showed inhibition, with minimum inhibitory concentration (MIC) ranging from 0.39 to 6.25mg/ml. Water and methanol extracts of Garcinia livingstonei and Sclerocarya birrea barks showed good activity against all the test organisms, with MICs between 0.39 and 1.56 mg/ml. Alkaloids, phenols, flavonoids, saponins, tannins, and terpenoids were found in one or more of the plant extracts, and all the plant extracts demonstrated scavenging power against DPPH.The cytotoxicity of extracts of Garcinia livingstonei, and Sclerocarya birrea barks ranged between 105.9 μg/ml and 769.9 μg/ml. The results obtained in this study validate the traditional use of A. digitata leaves, G. livingstonei and S. birrea bark in treating bacteria causing diarrhoea. / Life Sciences / M. Sc. (Life Sciences)