Orientador: Ramon Kaneno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-04T01:09:50Z (GMT). No. of bitstreams: 1 Martins_PriscilaRaquel_M.pdf: 3935077 bytes, checksum: a342a73cd22f638b9ca5c0901503c580 (MD5) Previous issue date: 2004 / Resumo: A atividade imunomoduladora de cogumelos medicinais é atribuída principalmente às J}-glucanas. Neste estudo, avaliamos o efeito de peptidoglucanas extraídas do cogumelo Agaricus blazei (ATF) quanto à atividade candidacida, expressão de receptores de manose e produção de H2O2 e NO por macrófagos peritoneais murinos. Camundongos normais BALB/c receberam três inoculações intraperitoneais de solução salina (grupo controle) ou fração ATF (grupo ATF) e após 48 horas os macrófagos peritoneais foram coletados e ensaiados contra leveduras de Candida albicans. Nossos resultados indicam que o tratamento aumentou a atividade candidacida de macrófagos, produção de H2O2e expressão de receptores de manose, contudo, o tratamento não alterou a produção de NO. Nossos resultados sugerem que a fração ATF pode aumentar a resistência contra agentes infecciosos devido à estimulação da atividade microbicida de macrófagos / Abstract: Immunomodulatory activity of medicinal mushrooms is attributed to glucans. In the present study we avaluated the eifect of peptidoglycans of Agaricus blazei (ATF) on the candidacidal activity, the expression of mannose receptors (MR), production of H2O2 and NO by murine peritoneal macrophages. Normal BALB/c mice were i.p. treated with 3 inoculations of ATF (ATF group) or salt solution (control group) and after 48hr peritoneal macrophages were assayed against Candida albicans yest forms. Our results indicated that the treatment enhanced the candidacidal activity of peritoneal macrophages and increased the H2O2 production and MR expression. However ATF was not able to increase the spontaneous production of NO. The results suggest that ATF can enhance the host resistence against infectious agents due to the stimulation of the microbicidal activity of macrophages / Mestrado / Mestre em Farmacologia

Yeung Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 136-162). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT (ENGLISH) --- p.iii~v / ABSTRACT (CHINESE) --- p.vi~vii / TABLE OF CONTENTS --- p.viii~xiii / LIST OF TABLES --- p.xiv~xv / LIST OF FIGURES --- p.xvi~xviii / LIST OF ABBREVIATIONS --- p.xix~xx / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Different lipoproteins and their functions --- p.1 / Chapter 1.1.1 --- Chylomicrons --- p.4 / Chapter 1.1.2 --- VLDL --- p.4 / Chapter 1.1.3 --- LDL --- p.4 / Chapter 1.1.4 --- HDL --- p.5 / Chapter 1.2 --- Risk factors of coronary heart disease (CHD) --- p.5 / Chapter 1.2.1 --- Background information of CHD --- p.6 / Chapter 1.2.2 --- "Relationship between serum total cholesterol (TC), Low-density lipoprotein (LDL) cholesterol and CHD" --- p.7 / Chapter 1.2.3 --- High-density lipoprotein (HDL) cholesterol and CHD --- p.8 / Chapter 1.2.4 --- Triglyceride and CHD --- p.9 / Chapter 1.3 --- Cholesterol homeostasis --- p.10 / Chapter 1.3.1 --- Roles of HMG-CoA reductase in cholesterol biosynthesis --- p.13 / Chapter 1.3.2 --- Roles of cholesterol 7α-hydroxylase (CYP7A) in cholesterol catabolism…… --- p.15 / Chapter 1.3.3 --- Effects of Short-Chain Fatty Acid (SCFA) --- p.17 / Chapter 1.3.4 --- Related hormone --- p.18 / Chapter 1.4 --- Possible mechanisms of hypolipidemic agents --- p.19 / Chapter 1.4.1 --- Hypolipidemic functional foods --- p.20 / Chapter 1.4.2 --- Pharmacological drugs --- p.26 / Chapter 1.5 --- Edible and medicinal mushrooms --- p.28 / Chapter 1.5.1 --- General introduction --- p.28 / Chapter 1.5.2 --- Hypolipidemic agents from Fungi --- p.31 / Chapter 1.6 --- Animal model --- p.35 / Chapter 1.7 --- Objectives --- p.36 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.37 / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Mushroom samples and control --- p.37 / Chapter 2.1.1.1 --- Sample introduction --- p.37 / Chapter 2.1.1.2 --- Sample collection --- p.40 / Chapter 2.1.1.3 --- Sample preparation --- p.41 / Chapter 2.1.1.4 --- Moisture content --- p.45 / Chapter 2.1.2 --- Animal diets for different experiments --- p.45 / Chapter 2.1.2.1 --- Basal diet --- p.45 / Chapter 2.1.2.2 --- Diet for preliminary screening --- p.46 / Chapter 2.1.2.3 --- Diet for dosage experiment --- p.46 / Chapter 2.1.2.4 --- Diet for active ingredient experiments --- p.47 / Chapter 2.1.2.5 --- Diet for long-term feeding experiment --- p.47 / Chapter 2.1.3 --- Animal model --- p.49 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- Nutritional components of mushroom samples --- p.49 / Chapter 2.2.1.1 --- Crude protein content (Kjeldahl method) --- p.49 / Chapter 2.2.1.2 --- Total dietary fiber content --- p.50 / Chapter 2.2.1.3 --- Crude lipid content --- p.52 / Chapter 2.2.1.4 --- Ash content --- p.53 / Chapter 2.2.1.5 --- Moisture content --- p.53 / Chapter 2.2.2 --- Animal handling experiments --- p.54 / Chapter 2.2.2.1 --- Feeding experiment standards --- p.54 / Chapter 2.2.2.1.1 --- Feeding experiments of preliminary screening test --- p.54 / Chapter 2.2.2.1.2 --- Feeding experiments of dosage test --- p.55 / Chapter 2.2.2.1.3 --- Feeding experiments of solvent extracts from Agrocybe aegerita (Brig) Sing (AA) --- p.56 / Chapter 2.2.2.1.3.1 --- Fractionation of ethanol & water soluble components of AA --- p.56 / Chapter 2.2.2.1.3.2 --- Feeding experiments of ethanol & water soluble components of AA --- p.57 / Chapter 2.2.2.1.4 --- Feeding experiment of long-term test --- p.58 / Chapter 2.2.2.2 --- Blood sample collection --- p.58 / Chapter 2.2.2.3 --- Serum preparation --- p.58 / Chapter 2.2.2.4 --- Liver sample preparation --- p.58 / Chapter 2.2.2.5 --- Fecal sample preparation --- p.59 / Chapter 2.2.3 --- Determination of serum lipid profiles --- p.59 / Chapter 2.2.3.1 --- Serum total cholesterol (TC) assay --- p.59 / Chapter 2.2.3.2 --- Serum triglyceride (TG) assay --- p.60 / Chapter 2.2.3.3 --- Serum high-density lipoprotein (HDL) cholesterol assay --- p.61 / Chapter 2.2.3.3.1 --- Separation of HDL fraction --- p.61 / Chapter 2.2.3.3.2 --- HDL cholesterol (HDL-c) determination --- p.61 / Chapter 2.2.4 --- Determination of liver lipid profiles --- p.62 / Chapter 2.2.4.1 --- Liver total cholesterol (TC) level determination --- p.62 / Chapter 2.2.4.2 --- Determination of liver total lipid (TL) level --- p.64 / Chapter 2.2.5 --- Quantitative determination of fecal neutral & acidic sterols --- p.64 / Chapter 2.2.5.1 --- Separation of fecal neutral & acidic sterols --- p.64 / Chapter 2.2.5.2 --- Derivatisation of fecal neutral sterols --- p.65 / Chapter 2.2.5.3 --- Derivatisation of fecal acidic sterols --- p.65 / Chapter 2.2.5.4 --- Gas chromatographic analysis of fecal neutral & acidic sterols --- p.66 / Chapter 2.2.6 --- Assays of liver key enzymes in cholesterol metabolism --- p.67 / Chapter 2.2.6.1 --- Preparation of hepatic microsome --- p.67 / Chapter 2.2.6.2 --- Assay of HMG-CoA reductase activity --- p.68 / Chapter 2.2.6.3 --- Assay of CYP7A activity --- p.69 / Chapter 2.3 --- Data statistics --- p.71 / Chapter CHAPTER THREE: --- RESULTS AND DISCUSSION --- p.72 / Chapter 3.1 --- Preliminary screening of eleven mushrooms for their hypolipidemic effect in hyperlipidemic S.D. rats --- p.72 / Chapter 3.1.1 --- Body weight and food intake --- p.73 / Chapter 3.1.2 --- Effect of mushroom supplementation on serum lipid profiles --- p.75 / Chapter 3.1.2.1. --- Effect of mushroom supplementation on serum TC levels --- p.75 / Chapter 3.1.2.2. --- Effect of mushroom supplementation on serum TG levels --- p.77 / Chapter 3.1.2.3. --- Effect of mushroom supplementation on serum HDL levels --- p.79 / Chapter 3.1.2.4 --- Discussion of serum lipid profiles of S.D. rats fed M.S. diets in mushroom screening experiments --- p.83 / Chapter 3.1.3 --- Effect and discussion of mushroom supplementation on hepatic lipid profiles --- p.84 / Chapter 3.1.4 --- Effect and discussion of mushroom supplementation on fecal neutral sterol excretion --- p.87 / Chapter 3.1.5 --- Summary (mushroom screening experiments) --- p.90 / Chapter 3.2 --- Hypolipidemic effect of Agrocybe aegerita (Brig.) Sing (AA) in a dose response study in hyperlipidemic S.D. rats --- p.91 / Chapter 3.2.1 --- Nutritional composition of AA mushroom --- p.91 / Chapter 3.2.2 --- Body weight and food intake --- p.91 / Chapter 3.2.3 --- Effect of three different dosages of AA mushroom supplementation on blood lipid profiles of S.D. rats --- p.93 / Chapter 3.2.3.1 --- Effect of different dosages of AA mushroom supplementation diets on serum TC level --- p.93 / Chapter 3.2.3.2 --- Effect of different dosages of AA mushroom supplementation diets on serum TG level --- p.93 / Chapter 3.2.3.3 --- Effect of different dosages of AA mushroom supplementation diets on serum HDL level --- p.95 / Chapter 3.2.3.4 --- Discussion of different dosages of AA mushroom supplementation diets on serum lipid profiles --- p.97 / Chapter 3.2.4 --- Effect and discussion of three different dosages of AA mushroom supplementation on hepatic lipid profiles --- p.98 / Chapter 3.2.5 --- Effect and discussion of three different dosages of AA mushroom supplementation on fecal neutral & acidic sterol excretion --- p.101 / Chapter 3.2.6 --- Summary (dose response study) --- p.105 / Chapter 3.3 --- Hypolipidemic effect of ethanol extract (E.E.) & water extract (W.E.) from AA in hyperlipidemic S.D. rats --- p.106 / Chapter 3.3.1 --- Extraction yield --- p.106 / Chapter 3.3.2 --- Body weight & food intake --- p.106 / Chapter 3.3.3 --- Effect of AA extract supplementation on serum lipid profiles --- p.107 / Chapter 3.3.3.1 --- Effect of AA extract supplementation on serum TC level --- p.107 / Chapter 3.3.3.2 --- Effect of AA extract supplementation on serum TG level --- p.108 / Chapter 3.3.3.3 --- Effect of AA extract supplementation on serum HDL level --- p.109 / Chapter 3.3.4 --- Effect of AA extract supplementation on hepatic lipid profiles --- p.111 / Chapter 3.3.5 --- Effect of AA extract supplementation on fecal neutral & acidic sterols excretion --- p.111 / Chapter 3.3.6 --- Discussion (active fraction extract study) --- p.113 / Chapter 3.4 --- Long-term evaluation of the hypolipidemic effect of AA supplementation in normolipic S.D. rats --- p.116 / Chapter 3.4.1 --- Body weight & food intake --- p.116 / Chapter 3.4.2 --- Effect of long term AA supplementation on serum lipid profiles --- p.117 / Chapter 3.4.2.1 --- Effect of long term AA supplementation on serum TC level --- p.117 / Chapter 3.4.2.2 --- Effect of long term AA supplementation on serum TG level --- p.118 / Chapter 3.4.2.3 --- Effect of long term AA supplementation on serum HDL level --- p.119 / Chapter 3.4.3 --- Effect of long term AA supplementation on hepatic lipid profiles --- p.119 / Chapter 3.4.4 --- Effect of long term AA supplementation on fecal neutral & acidic sterols excretion --- p.121 / Chapter 3.4.5 --- Effect of long term AA supplementation on hepatic key enzymes of cholesterol metabolism ´ؤ HMG-CoA reductase and CYP7A --- p.123 / Chapter 3.4.5.1 --- Quantitation of hepatic microsomal protein --- p.123 / Chapter 3.4.5.2 --- Effect of long term AA supplementation on HMG-CoA reductase activity in S.D. rats --- p.124 / Chapter 3.4.5.3 --- Effect of long term AA supplementation on CYP7A activity in S.D. rats --- p.124 / Chapter 3.4.7 --- Discussion (long-term study) --- p.126 / Chapter CHAPTER FOUR: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.130 / References --- p.136

Hepatocellular carcinoma (HCC), the major form of primary liver cancer, is a common cause of cancer-related death worldwide. Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the pathogenesis of HCC. Inhibition of STAT3 signaling has been proposed as a promising strategy for treating HCC. Due to the limitations of conventional therapeutics, increasing attention has been paid to complementary and alternative medicines (CAM) including traditional Chinese medicine (TCM) for the management of HCC. Antrodia camphorata (AC), a medicinal mushroom, is historically used for treating HCC. Pharmacological data showed that extracts and constituents of AC are able to inhibit STAT3 activation. Natural AC is scarce, cultured AC mycelia are becoming alternatives. AC mycelia have been demonstrated to possess anti-HCC properties. We hypothesize that inhibition of the STAT3 signaling pathway contributes to the anti-HCC mechanisms of AC mycelia. To test our hypothesis, we evaluated the safety and investigated the anti-HCC effects of the ethyl acetate fraction of an ethanolic extract of AC mycelia (EEAC); and we further explored the involvement of STAT3 signaling in EEAC's anti-HCC effects. Acute and repeated dose 28-day oral toxicity studies showed that EEAC had no toxicity in rats. The maximum tolerable dose for acute oral toxicity and the no-observed-adverse effects level for repeated dose 28-day oral toxicity of EEAC were higher than 5,000 mg/kg body weight and 1,000 mg/kg body weight, respectively, in rats. In cultured cells, EEAC is less toxic in normal liver-derived cells than in HCC cells. In HepG2 and SMMC-7721 cells, EEAC reduced viability, induced apoptosis, and retarded migration and invasion. In SMMC-7721 cell-bearing mice, EEAC significantly suppressed tumor growth. EEAC inhibited cell proliferation, induced apoptosis and suppressed angiogenesis in tumors. Mechanistic studies showed that EEAC downregulated protein levels of phosphorylated and total STAT3 and JAK2 (an upstream kinase of STAT3) in HCC cells and tumors. In cultured HCC cells, EEAC lowered the protein level of nuclear STAT3, decreased the transcriptional activity of STAT3, and downregulated protein levels of STAT3 targeted molecules. Over-activation of STAT3 in HCC cells diminished the cytotoxic effects of EEAC. STAT3 can be activated by receptor tyrosine kinases (RTKs). Phospho-RTK array assays showed that EEAC significantly inhibited the tyrosine phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-β) in HepG2 cells. EEAC dose-dependently lowered mRNA levels of PDGF BB (a ligand of PDGFR-β) and protein levels of p-PDGFR-β and PDGFR-β in HCC cells. Activating PDGFR-β enhanced STAT3 activation, and inhibiting PDGFR-β blocked STAT3 activation in HCC cells. EEAC reversed PDGF BB induced STAT3 activation in HCC cells. Our data indicate that EEAC exerts anti-HCC effects, and inhibition of PDGFR-β/STAT3 signaling is, at least in part, responsible for these effects. In summary, we have demonstrated that EEAC exerts anti-HCC effects without significant toxicity in vitro and in vivo. We have also demonstrated that inhibition of PDGFR-β/STAT3 signaling contributes to the anti-HCC mechanisms of EEAC. Our findings provide a pharmacological basis for the development of EEAC as a modern anti-HCC agent and for the traditional use of AC in treating HCC. In addition, our data suggest that the PDGFR-β/STAT3 pathway plays a pathogenic role and presents a novel therapeutic target in HCC.

Lentinula edodes é um cogumelo comestível que possui qualidades nutricionais, terapêuticas e medicinais. Além disso, muitos estudos na área médica têm comprovado que o cogumelo possui efeito antibiótico sobre microrganismos patogênicos ao homem. Na área agrícola, alguns trabalhos realizados com o cogumelo demonstraram possíveis efeitos no controle de fitopatógenos. O presente trabalho teve como objetivo demonstrar a produção de substâncias antimicrobianas por L. edodes ativas sobre Colletotrichum sublineolum, agente causal da antracnose em sorgo, Alternaria solani, responsável pela pinta preta do tomateiro, Xanthomonas axonopodis pv. passiflorae, agente causal da mancha bacteriana em maracujazeiro e Tobacco mosaic virus (TMV), causador de mosaico foliar em fumo. Para os testes com C. sublineolum e A. solani foram utilizados extratos aquosos de L. edodes, obtidos a partir de basidiocarpos desidratados em pó, dos isolados LE JAB-K, LE 96/22, LE 96/17 e LE 95/01. Os resultados evidenciaram que o extrato aquoso de basidiocarpos do isolado LE 96/22 inibiu o crescimento micelial in vitro e a formação de apressórios por C. sublineolum. Já os extratos dos isolados LE JAB-K e LE 95/01 apresentaram efeito inibitório na germinação de conídios e na formação de apressórios do patógeno. Em contrapartida, os extratos aquosos de basidiocarpos dos diferentes isolados de L. edodes não apresentaram efeito inibitório na germinação dos conídios e no crescimento micelial de A. solani. Por sua vez, os extratos aquosos de basidiocarpos a 20% (v/v) e o filtrado do crescimento micelial de L. edodes, misturados à suspensão de X. axonopodis pv. passiflorae, exibiram redução na multiplicação bacteriana. Todos os extratos aquosos de basidiocarpos dos diferentes isolados testados na multiplicação da bactéria mostraram-se termolábeis, quando autoclavados a 121 °C por 20 min. Em experimentos com plantas de fumo, os extratos aquosos de basidiocarpos dos isolados LE 96/17 e LE 96/22 adicionados à suspensão contendo partículas do TMV reduziram significativamente a ocorrência de lesões locais nas folhas. O extrato aquoso do isolado LE 96/22 apresentou compostos antivirais de natureza termoestável. Finalmente, o extrato aquoso de basidiocarpos do isolado LE 96/22, o qual apresentou a maior atividade antimicrobiana, foi purificado parcialmente por cromatografia de troca aniônica (CTA). O pico V apresentou efeito inibitório no crescimento micelial de C. sublineolum. Por sua vez, a multiplicação de X. axonopodis pv. passiflorae foi inibida pelos picos IV, V e VII. Já os picos I, II e III, obtidos em CTA por gradiente linear de NaCl e o pico I obtido em CTA pelo método "step wise", reduziram significativamente a infectividade do TMV em plantas de fumo. Com base nesses resultados, evidencia-se a ação de preparações de L. edodes sobre fitopatógenos, o que demonstra o uso potencial do mesmo no controle de agentes causais de doenças infecciosas em plantas. / Lentinula edodes is an edible mushroom that has nutritious, therapeutical and medicinal qualities. Moreover, many studies in the medical area have shown that the mushroom exhibits antibiotic effects on pathogenic microorganism to the man. In the agricultural area, work carried out with the mushroom has demonstrated its possible effects to control phytopathogens. The objective of the present work was to demonstrate the productionof antimicrobial substances of L. edodes active on Colletotrichum sublineolum, causal agent of anthracnose in sorghum, Alternaria solani, responsible for the black spot of the tomato plants, X. axonopodis pv. passiflorae, causal agent of the bacterial spot in passion fruit plants and on Tobacco mosaic virus, causal agent of the mosaic in tobacco plants. For the test with C. sublineolum and A. solani aqueous extracts were obtained from dehydrated fruiting bodies from the shiitake isolates LE JAB-K, LE 96/22, LE 96/17 and LE 95/01. The results showed that the fruiting body aqueous extract from isolate LE 96/22 inhibited micelial growth and appressorium formation by C. sublineolum. The aqueous extracts of isolates LE JAB-K and LE 95/01 exhibited inhibitory effect on conidium germination and on formation of appressorium by the patogen. On the other hand, the extracts of the different isolates of L. edodes did not exhibit inhibitory effect on conidium germination and micelial growth of A. solani. The aqueous extracts of fruiting bodies at 20% (v/v) concentration and filtrate of the micelial growth of L. edodes, when mixed to the suspension of X. axonopodis pv. passiflorae, exhibited decreased on bacterial multiplication. All the aqueous extracts of fruiting bodies tested from the different isolates in the bacterial multiplication were thermobile, when heated at 121 °C for 20 min. In experiments with tobacco plants, the aqueous extracts of fruiting bodies of isolates LE 96/17 and LE 96/22 when added to the suspension of TMV reduced the amount of local lesions on the leaves. When the aqueous extracts of LE 96/22 were heated the antiviral nature was not lost. Finally, the aqueous extract of fruiting bodies from isolate LE 96/22 that presented major antimicrobial activity was partially purified by anion exchange chromatography (AEC). The peak V exhibited inhibitory effect on micelial growth of C. sublineolum. Multiplication of X. axonopodis pv. passiflorae was inhibited by peaks IV, V and VII. Regarding TMV infectivity, peaks I, II and III, obtained in CTA through linear gradient of NaCl, and peak I also obtained through CTA by the method "step wise", significantly reduced virus infectivity in tobacco plants. Based upon these results, it is shown that preparations of L. edodes can interfere whith phytopathogen multiplication, demonstrating its potential to control plant diseases.

All these results advance our understanding on how stimulatory agents can be used to increase the production of useful metabolites by submerged fermentation of mushroom mycelium and indicate its potential biotechnological applications. / By use of one- and two-dimensional gels in proteomic analysis, some functional mycelial proteins that were differentially expressed by the addition of Tween 80 were identified. The up-regulation of heat shock proteins might help to maintain cellular viability under environmental stress. A down-regulation of YALI0E34793p and an up-regulation of ATP citrate lyase isoform 2 might suppress the activity of TCA cycle and subsequently stimulated the EPS production. Up-regulation of fatty acid synthase alpha subunit FasA might promote the synthesis of long-chain fatty acids and their incorporation into the mycelial cell membranes. Up-regulation of mitogen-activated protein kinase might facilitate the signal transduction in these processes. / The BPS, a highly branched glucomannan produced by the addition of Tween 80 in the fermentation broth of PTR mycelium had similar carbohydrate and protein content, monosaccharide composition and glycosidic linkages except by having a significantly lower molecular weight when compared to those of the control. Both BPS, with and without addition of Tween 80, could significantly inhibit (p < 0.05) the in vitro growth of a chronic myelogenous leukemia cells K562 in a dose dependent manner, with an estimated IC50 value of 43.7 and 47.6 microg/mL, respectively. / The effects of different kinds of stimulatory agents including fatty acids, surfactants and organic solvents were compared. The optimum results were achieved when 3.0 g/L Tween 80 was added to the fermentation broth on the 5th day of the fermentation, to give a maximum increase of 51.3 and 41.8% (p < 0.05) in the yield of mycelial biomass and BPS production, respectively. / The underlying mechanisms by which Tween 80 could increase the mycelial growth and EPS production in PTR were investigated by three novel approaches including changes in the nutrient uptake by mycelium, the morphology of mycelial pellets, and the fatty acid composition in the mycelial cell membrane. Firstly, the addition of Tween 80 significantly increased the glucose consumption rate by the mycelium, implying that the efficiency of nutrient uptake from the fermentation broth was enhanced. Secondly the addition of Tween 80 could extend the growth period of the mycelium possibly by maintaining the intact structure of the mycelial pellets and preventing its disintegration caused by shear stress in the fermentation system. Thirdly, the addition of Tween 80 could increase the incorporation of oleic acid which was a constituent of Tween 80 itself into the mycelial cell membrane of PTR, altering its fatty acid composition and increase the cell membrane permeability. The first two results explained the enhancement in the mycelial growth and EPS production while the last one was related to the extracellular transport of EPS to the fermentation broth. / This study aimed at comparing the effectiveness in the use of stimulatory agents with different chemical structures for enhancing the production of mycelial biomass and exopolysaccharide (BPS) by submerged fermentation of an edible mushroom Pleurotus tuber-regium (PTR). The chemical characteristics and antitumor activity of the BPS produced with and without the addition of the most effective stimulatory agent (Tween 80 which is a permitted food additive) were also compared. The underlying mechanisms by which Tween 80 could exert its effect on the mushroom mycelium were investigated by using chemical methods and microscopic techniques as well as proteomic analysis. / Zhang, Bobo. / Adviser: Chi Keung Peter Cheung. / Source: Dissertation Abstracts International, Volume: 73-08, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 132-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Sandra J. Chapman. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 95-103). / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- History of zinc --- p.1 / Chapter 1.2 --- The biological role of zinc --- p.2 / Chapter 1.3 --- Zinc toxicosis --- p.6 / Chapter 1.4 --- Mechanisms of zinc uptake and transport in fungi --- p.7 / Chapter 1.5 --- Bioremediation --- p.11 / Chapter 1.6 --- Uptake of heavy metals by fruit bodies of edible mushrooms --- p.13 / Chapter 1.7 --- Mushroom fungi selected for study --- p.15 / Chapter 1.8 --- Purpose of study --- p.17 / Chapter 2. --- Materials and Methods --- p.18 / Chapter 2.1 --- Organisms --- p.18 / Chapter 2.2 --- Media --- p.18 / Chapter 2.3 --- Media chelation --- p.20 / Chapter 2.4 --- Chemicals --- p.20 / Chapter 2.5 --- Zinc content of fruit bodies grown on substrates containing different concentrations of zinc --- p.21 / Chapter 2.5.1 --- Substrate preparation for V. volvacea inoculum --- p.21 / Chapter 2.5.2 --- Cultivation of V. volvacea fruit bodies --- p.21 / Chapter 2.5.3 --- Cultivation of P.sajor-caju fruit bodies --- p.22 / Chapter 2.5.4 --- Cultivation of L. edodes fruit bodies --- p.23 / Chapter 2.5.5 --- Preparation of biological material for atomic absorption spectrophotometry --- p.24 / Chapter 2.6 --- Effect of different concentrations of zinc on the growth of six mushroom fungi --- p.25 / Chapter 2.6.1 --- Radial growth study --- p.25 / Chapter 2.6.2 --- Biomass study --- p.26 / Chapter 2.7 --- Microscopic studies of V. volvacea --- p.27 / Chapter 2.7.1 --- "Coomassie Blue preparation, staining of V.volvacea hyphae" --- p.27 / Chapter 2.7.2 --- Dithizone staining of V. volvacea hyphae --- p.27 / Chapter 2.7.3 --- Fluorescence microscopy --- p.28 / Chapter 2.7.4 --- Scanning electron microscopy --- p.28 / Chapter 2.8 --- Preparation and analysis of V. volvacea proteins using gel electrophoresis --- p.29 / Chapter 3. --- Results --- p.33 / Chapter 3.1 --- Zinc Uptake by Fruit Bodies --- p.33 / Chapter 3.1.1 --- Uptake of zinc by V. volvacea --- p.33 / Chapter 3.1.2 --- Uptake of zinc by P. sajor-caju --- p.33 / Chapter 3.1.3 --- Uptake of zinc by L. edodes --- p.34 / Chapter 3.1.4 --- Symptoms of zinc toxicity in L. edodes --- p.44 / Chapter 3.2 --- Growth studies --- p.49 / Chapter 3.2.1 --- Radial growth measurements --- p.49 / Chapter 3.2.2 --- Biomass measurements --- p.56 / Chapter 3.2.3 --- Morphological alterations due to zinc observed with light and electron microscopy --- p.63 / Chapter 3.3 --- V. volvacea staining studies --- p.73 / Chapter 3.3.1 --- Protein staining using Coomassie Blue --- p.73 / Chapter 3.3.2 --- Zinc staining by dithizone and fluorescence staining by DAPI --- p.75 / Chapter 3.4 --- V. volvacea protein profile comparisons after gel electrophoresis --- p.81 / Chapter 4. --- Discussion --- p.83 / Chapter 4.1 --- Zinc uptake by fruit bodies / Chapter 4.1.1 --- Uptake of zinc by V. volvacea and P. sajor-caju fruit bodies --- p.83 / Chapter 4.1.2 --- Accumulation of zinc by L. edodes fruit bodies and mechanism of toxicity --- p.84 / Chapter 4.2 --- Effects of zinc on growth --- p.88 / Chapter 4.3 --- V. volvacea mechanisms of tolerance --- p.89 / Chapter 4.4 --- Differences in protein profiles of V. volvacea grown on different concentrations of zinc --- p.93 / Chapter 5. --- References

Lo Kit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 193-208). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (Chinese version) --- p.v / CONTENT --- p.vii / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xviii / LIST OF ABBREVIATIONS --- p.xx / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- An introduction of natural antioxidants --- p.1 / Chapter 1.1.1 --- Definition of antioxidants --- p.1 / Chapter 1.1.2 --- Application of natural antioxidants in foods --- p.3 / Chapter 1.1.2.1 --- Oxidation of foods --- p.3 / Chapter 1.1.2.1.1 --- Autoxidation of food --- p.3 / Chapter 1.1.2.1.2 --- Photo-oxidation of food --- p.4 / Chapter 1.1.3 --- Free radicals and antioxidants --- p.6 / Chapter 1.1.3.1 --- Free radicals and reactive oxygen species --- p.6 / Chapter 1.1.3.1.1 --- Superoxide anion radical --- p.8 / Chapter 1.1.3.1.2 --- Hydrogen peroxide --- p.9 / Chapter 1.1.3.1.3 --- Hydroxyl radical --- p.10 / Chapter 1.1.3.1.4 --- Peroxyl radical --- p.12 / Chapter 1.1.3.1.5 --- Lipid peroxidation of cell membranes --- p.13 / Chapter 1.1.3.1.6 --- Oxidation of LDL and atherosclerosis --- p.16 / Chapter 1.1.4 --- Natural antioxidants and their mechanisms --- p.18 / Chapter 1.1.4.1 --- Carotenoids --- p.18 / Chapter 1.1.4.2 --- Phenolic compounds --- p.20 / Chapter 1.1.4.2.1 --- Flavonoids --- p.21 / Chapter 1.1.4.2.2 --- Phenolic acids --- p.22 / Chapter 1.1.4.3 --- Sterols --- p.24 / Chapter 1.1.4.4 --- Vitamins --- p.25 / Chapter 1.2 --- Antioxidant in mushrooms --- p.27 / Chapter 1.2.1 --- Antioxidant properties of mushrooms --- p.27 / Chapter 1.2.2 --- Characterization of mushroom phenolic antioxidants --- p.30 / Chapter 1.2.3 --- Biosynthesis of phenolic compounds in mushrooms or fungi --- p.33 / Chapter 1.3 --- Assays for evaluation of antioxidants --- p.35 / Chapter 1.3.1 --- Beta-carotene bleaching method --- p.35 / Chapter 1.3.2 --- Scavenging activity of DPPH radical --- p.36 / Chapter 1.3.3 --- Erythrocyte hemolysis --- p.36 / Chapter 1.3.4 --- Scavenging activity of ABTS ´Ø + radical cation --- p.37 / Chapter 1.3.5 --- Scavenging activity of hydroxyl radical --- p.37 / Chapter 1.3.6 --- Assay for lipid peroxidation of rat brain homogenate --- p.38 / Chapter 1.3.7 --- Inhibition of low-density lipoproteins (LDLs) oxidation --- p.39 / Chapter 1.4 --- Analysis of phenolic antioxidants --- p.41 / Chapter 1.4.1 --- Extraction of phenolic compounds --- p.41 / Chapter 1.4.2 --- Determination of total phenolic content --- p.43 / Chapter 1.4.3 --- Chromatographic fractionation of phenolic compounds --- p.44 / Chapter 1.4.4 --- Characterization of phenolic compounds --- p.45 / Chapter 1.4.4.1 --- Thin-layer chromatography (TLC) --- p.45 / Chapter 1.4.4.2 --- High performance liquid chromatography (HPLC) --- p.46 / Chapter 1.4.4.3 --- Liquid chromatography-Mass spectrometry (LC-MS) --- p.47 / Chapter 1.5 --- Objectives --- p.49 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.50 / Chapter 2.1 --- Sample preparation --- p.50 / Chapter 2.2 --- Sample extraction. --- p.51 / Chapter 2.2.1 --- Small-scale methanol and water extraction --- p.51 / Chapter 2.2.2 --- Large-scale methanol and water extraction and fractionation --- p.54 / Chapter 2.2.2.1 --- Large-scale methanol and water extraction --- p.54 / Chapter 2.2.2.2 --- Fractionation of crude extracts --- p.55 / Chapter 2.2.2.2.1 --- Fractionation of methanol crude extract --- p.55 / Chapter 2.2.2.2.2 --- Fractionation of water crude extract --- p.55 / Chapter 2.3 --- Fractionation by column chromatography --- p.58 / Chapter 2.4 --- Assays for measuring antioxidant activity --- p.60 / Chapter 2.4.1 --- Beta-carotene bleaching method --- p.60 / Chapter 2.4.2 --- Scavenging activity of DPPH radical --- p.62 / Chapter 2.4.3 --- Erythrocyte hemolysis --- p.63 / Chapter 2.4.4 --- Scavenging activity of ABTS ´Ø + radical cation --- p.64 / Chapter 2.4.5 --- Scavenging activity of hydroxyl radical --- p.65 / Chapter 2.4.6 --- Assay for lipid peroxidation of rat brain homogenate --- p.66 / Chapter 2.4.7 --- Inhibition of human low-density lipoproteins (LDLs) oxidation --- p.67 / Chapter 2.4.7.1 --- Isolation of human LDLs --- p.67 / Chapter 2.4.7.2 --- Calculation of density --- p.68 / Chapter 2.4.7.3 --- Lowry's method for determination of protein content --- p.69 / Chapter 2.4.7.4 --- Preparation of reagents --- p.69 / Chapter 2.4.7.5 --- Determination of thiobarbituric acid reactive substance (TBARS) --- p.70 / Chapter 2.5 --- Total phenolic content --- p.70 / Chapter 2.6 --- Total carbohydrate content --- p.71 / Chapter 2.7 --- Determination of protein content- the Biuret method --- p.71 / Chapter 2.8 --- Thin-layer chromatography (TLC) --- p.72 / Chapter 2.9 --- High performance liquid chromatography (HPLC) --- p.73 / Chapter 2.9.1 --- Analysis of subfractions of methanol crude extract --- p.73 / Chapter 2.9.2 --- Analysis of fractionated subfractions and subfractions of Pevf and Aa mushrooms --- p.74 / Chapter 2.10 --- Liquid chromatography- Mass spectrometry (LC-MS) --- p.74 / Chapter 2.10.1 --- Liquid chromatography --- p.74 / Chapter 2.10.2 --- Mass spectrometry --- p.75 / Chapter 2.11 --- Data analysis --- p.75 / Chapter CHAPTER 3: --- RESULTS AND DISCUSSION --- p.77 / Chapter 3.1 --- Small-scale extraction scheme --- p.77 / Chapter 3.1.1 --- Extraction yield --- p.77 / Chapter 3.1.2 --- Assays for measuring antioxidant activity --- p.80 / Chapter 3.1.2.1 --- Beta-carotene bleaching method --- p.80 / Chapter 3.1.2.2 --- Scavenging activity of DPPH radical --- p.91 / Chapter 3.1.2.3 --- Erythrocyte hemolysis --- p.98 / Chapter 3.1.2.4 --- Summary for small-scale extraction --- p.105 / Chapter 3.2 --- Large-scale extraction and fractionation scheme --- p.107 / Chapter 3.2.1 --- Extraction yield for crude extracts and subfractions --- p.107 / Chapter 3.2.2 --- Antioxidant activity of subfractions and crude extracts of Aa and Pevf mushrooms --- p.111 / Chapter 3.2.2.1 --- Scavenging activity of ABTS ´Ø + radical cation --- p.111 / Chapter 3.2.2.2 --- Scavenging activity of hydroxyl radical --- p.114 / Chapter 3.2.2.3 --- Assay for lipid peroxidation of rat brain homogenate --- p.125 / Chapter 3.2.2.4 --- Summary for large-scale extraction --- p.135 / Chapter 3.2.3 --- Chemical characterization of the crude extracts and their sub fractions of Aa and Pevf mushrooms --- p.13 8 / Chapter 3.2.3.1 --- Total phenolic content --- p.139 / Chapter 3.2.3.1.1 --- Total phenolic content of crude extract and their sub fractions of Aa and Pevf mushrooms --- p.139 / Chapter 3.2.3.1.2 --- Correlation between total phenolic content and antioxidant activity --- p.140 / Chapter 3.2.3.2 --- Total carbohydrate content --- p.144 / Chapter 3.2.3.2.1 --- Total carbohydrate content of water crude extract and their sub fractions of Aa and Pevf mushrooms --- p.144 / Chapter 3.2.3.2.2 --- Correlation between total carbohydrate content and antioxidant activity --- p.144 / Chapter 3.2.3.3 --- Determination of protein content- the Biuret method --- p.146 / Chapter 3.2.3.3.1 --- Protein content of water crude extract and their sub fractions of Aa and Pevf mushrooms --- p.146 / Chapter 3.2.3.3.2 --- Correlation between protein content and antioxidant activity --- p.147 / Chapter 3.2.3.4 --- Summary of correlation between chemical components and antioxidant activity --- p.148 / Chapter 3.3 --- Column fractionation of ethyl acetate and butanol subfractions of Aa mushroom --- p.151 / Chapter 3.3.1 --- Rf value in TLC and yield of fractionated subfractions --- p.151 / Chapter 3.3.2 --- Total phenolic content of fractionated subfractions --- p.153 / Chapter 3.3.3 --- Antioxidant activity of fractionated subfractions --- p.155 / Chapter 3.3.3.1 --- Scavenging activity of ABTS ´Ø + radical cation --- p.155 / Chapter 3.3.3.2 --- Scavenging activity of DPPH radical --- p.158 / Chapter 3.3.3.3 --- Inhibition of human low-density lipoprotein (LDL) oxidation --- p.163 / Chapter 3.4 --- Chromatographic characterization of the subfractions of methanol crude extract of Aa and Pevf mushrooms --- p.167 / Chapter 3.4.1 --- Thin-layer chromatography (TLC) --- p.167 / Chapter 3.4.2 --- High performance liquid chromatography (HPLC) --- p.172 / Chapter 3.5 --- Chromatographic and spectrometric characterization of the fractionated subfractions of the ethyl acetate and butanol subfractions of Aa --- p.177 / Chapter 3.5.1 --- High performance liquid chromatography (HPLC) --- p.177 / Chapter 3.5.2 --- Liquid chromatography- Mass spectrometry (LC-MS) --- p.186 / Chapter CHAPTER 4: --- CONCLUSION --- p.189 / REFERENCES --- p.193 / RELATED PUBLICATION --- p.209

Tjia Wai Mui. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 123-140). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Growth of L. edodes --- p.3 / Chapter 1.2.1 --- Life cycle of L. edodes --- p.3 / Chapter 1.2.2 --- Growth parameters of L. edodes --- p.6 / Chapter 1.2.2.1 --- Temperature --- p.6 / Chapter 1.2.2.2 --- Relative humidity --- p.7 / Chapter 1.2.2.3 --- Moisture content in substrate --- p.7 / Chapter 1.2.2.4 --- Light --- p.8 / Chapter 1.2.2.5 --- pH --- p.8 / Chapter 1.3 --- Cultivation of L. edodes --- p.9 / Chapter 1.3.1 --- History and development of artificial cultivation --- p.9 / Chapter 1.3.2 --- Use of forced fruiting --- p.11 / Chapter 1.4 --- Molecular studies of stress on fungi --- p.12 / Chapter 1.4.1 --- Studies of temperature stress in mushroom --- p.12 / Chapter 1.4.2 --- Studies of molecular chaperones in fungi --- p.13 / Chapter 1.4.2.1 --- Role of molecular chaperones --- p.13 / Chapter 1.4.2.2 --- Heat shock protein 70 (Hsp70) and their cochaperones --- p.13 / Chapter 1.4.2.3 --- Other chaperones --- p.15 / Chapter 1.4.2.4 --- Molecular chaperones and development --- p.16 / Chapter 1.5 --- Prospectus --- p.19 / Chapter Chapter Two --- The Effect of Stress on the Growth of L. edodes / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Strain and culture conditions --- p.24 / Chapter 2.2.2 --- Stress treatments --- p.24 / Chapter 2.2.3 --- Data collection --- p.25 / Chapter 2.2.4 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Reliability analysis --- p.27 / Chapter 2.3.2 --- Descriptive statistics --- p.28 / Chapter 2.3.3 --- Independent t-test (ANOVA) --- p.33 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter Three --- Sequence Analysis of selected Stress Genes / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- Isolation of stress genes --- p.40 / Chapter 3.2.1.1 --- Construction of primordial cDNA library --- p.40 / Chapter 3.2.1.2 --- Screening of cDNA clones --- p.40 / Chapter 3.2.2 --- Sequence analyses of stress genes --- p.41 / Chapter 3.2.2.1 --- Amplification and purification of cDNA insert --- p.41 / Chapter 3.2.2.2 --- Full length DNA cycle sequencing --- p.42 / Chapter 3.2.2.3 --- Sequence analyses --- p.43 / Chapter 3.2.3 --- Screening of LeSSA (Inducible HSP70) --- p.45 / Chapter 3.2.3.1 --- PCR screening of LeSSA by degenerate primers and LeSSB specific primers --- p.45 / Chapter 3.2.3.2 --- Screening of LeSSA from cDNA library by hybridization --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Sequence analyses --- p.51 / Chapter 3.3.1.1 --- LeSSB --- p.51 / Chapter 3.3.1.2 --- LeMge1 --- p.57 / Chapter 3.3.1.3 --- LeSTI1 --- p.62 / Chapter 3.3.1.4 --- LeTCP1β --- p.69 / Chapter 3.3.1.5 --- LeTCP1γ --- p.74 / Chapter 3.3.2 --- Failure of isolating LeSSA (Inducible HSP70) --- p.80 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Sequence analyses --- p.82 / Chapter 3.4.2 --- Screening of LeSSA --- p.84 / Chapter Chapter Four --- Characterization of stress genes upon different stresses / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.87 / Chapter 4.2.1 --- Strain and culture conditions --- p.87 / Chapter 4.2.2 --- Stress treatments --- p.87 / Chapter 4.2.3 --- Isolation of total RNAs --- p.87 / Chapter 4.2.4 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.88 / Chapter 4.2.4.1 --- Reverse transcription --- p.88 / Chapter 4.2.4.2 --- PCR amplification by specific primers of stress genes --- p.89 / Chapter 4.2.5 --- Northern blot analyses --- p.91 / Chapter 4.2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.91 / Chapter 4.2.5.2 --- Northern blotting --- p.91 / Chapter 4.2.5.3 --- Preparation of probes --- p.92 / Chapter 4.2.5.4 --- Hybridization and stringency washes --- p.93 / Chapter 4.2.6 --- Isolation of total protein --- p.94 / Chapter 4.2.7 --- Quantification of protein by Bradford method --- p.95 / Chapter 4.2.8 --- Western blot analyses --- p.95 / Chapter 4.2.8.1 --- Sodium dodecyl sulfate ´ؤ polyacrylamide gel electrophoresis (SDS-PAGE) --- p.95 / Chapter 4.2.8.2 --- Western blotting --- p.96 / Chapter 4.2.8.3 --- Immunodetection --- p.98 / Chapter 4.2.8.4 --- ECL detection --- p.98 / Chapter 4.3 --- Results --- p.99 / Chapter 4.3.1 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.99 / Chapter 4.3.2 --- Northern blot hybridization --- p.106 / Chapter 4.3.2.1 --- Establishing an internal control --- p.106 / Chapter 4.3.2.2 --- Dig-labelling of stress genes --- p.106 / Chapter 4.3.2.3 --- Northern blot hybridizaton of stress genes --- p.106 / Chapter 4.3.3 --- Western blot hybridization --- p.111 / Chapter 4.4 --- Discussions --- p.113 / Chapter Chapter Five --- General Discussions --- p.118 / References --- p.123

A 12-kDa ribonuclease was purified from Pleurotus sajor-caju . The ribonuclease inhibited fungi growth and two species of bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. It reduced the viability of hepatoma and leukemia cells and inhibited translation in a cell-free rabbit reticulocyte lysate system. / A 13-kDa lectin was isolated from Collybia veultipes. Its N-terminal sequence shows some similarity to other fungal immunomodulatory proteins. It stimulated [3H-methyl] thymidine uptake by mouse splenocytes and inhibited proliferation of leukemia cells. / A 14.4-kDa antifungal protein was purified from Agrocybe cylindracea . It exerted antifungal activity but lacked inhibitory activity against bacteria when tested up to 300 muM. It attenuated the activity of HIV-1 reverse transcriptase. / A 17-kDa hemolysin was purified from Pleurotus eryngii. It exhibited cytotoxicity toward leukemia cells but not toward fungi. It exhibited antibacterial activity against Bacillus species. / A 27.5-kDa antifungal protein, with an N-terminal sequence similar to heat shock protein and endoglucanase, was purified from Lentinula edodes. It inhibited fungal growth and exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells. / A 7-kDa ubiquitin-like protein was purified from Agrocybe cylindracea . It showed antiproliferative activity on leukemia and hepatoma cell lines, and enhanced nitric oxide production in murine peritoneal macrophages. / An 18-kDa lectin, with an N-terminal sequence similar to some lectins and fungal immunomodulatory proteins, was isolated from Ganoderma capense. It exhibited potent mitogenic activity toward mouse splenocytes, and antiproliferative activity toward leukemia and hepatoma cells. / Mushrooms produce a variety of proteins with interesting biological activities. They include lectins, antifungal proteins, ribonucleases, ubiquitin-like proteins, hemolysins and other peptides. / This study demonstrates that different types of defense proteins with diverse biological activities are produced by mushrooms. Some overlap is observed in the spectra of biological activities of the same type of defense proteins. The results of protein characterization provide crucial information for future genetic manipulation in agricultural and food industries. Studies of the in vitro action of the abovementioned defense proteins on fungi, bacteria, viral enzyme, immune cells and cancer cells indicate that the proteins are potentially exploitable drug agents. / Ngai Hung-kui. / "July 2005." / Adviser: Ng Tzi Bun. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0012. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 228-294). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Cheung Lai Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 153-161). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (Chinese version) --- p.v / CONTENTS --- p.vi / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xiii / LIST OF ABBREVIATIONS --- p.xv / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter 1.1 --- Free radical --- p.2 / Chapter 1.1.1 --- Definition --- p.2 / Chapter 1.1.2 --- Reaction mechanism --- p.3 / Chapter 1.1.3 --- Sources of oxygen reactive species --- p.4 / Chapter 1.1.3.1 --- Enzymes --- p.4 / Chapter 1.1.3.2 --- The auto-oxidation of small molecules --- p.4 / Chapter 1.1.3.3 --- Haem proteins --- p.5 / Chapter 1.1.3.4 --- Endoplasmic reticulum sources --- p.5 / Chapter 1.1.3.5 --- Mitochondrial sources --- p.5 / Chapter 1.1.3.6 --- Nucleus --- p.6 / Chapter 1.1.4 --- Lipid peroxidation --- p.6 / Chapter 1.1.4.1 --- Initiation of lipid peroxidation --- p.7 / Chapter 1.1.4.2 --- Propagation of lipid peroxidation --- p.8 / Chapter 1.1.4.3 --- Products of lipid peroxidation --- p.9 / Chapter 1.1.5 --- Human diseases associated with free radicals --- p.10 / Chapter 1.2 --- Antioxidants --- p.12 / Chapter 1.2.1 --- Definition --- p.12 / Chapter 1.2.2 --- Defence against free radical damage --- p.13 / Chapter 1.2.2.1 --- Catalytic free radical removal --- p.13 / Chapter 1.2.2.2 --- Free radical scavenging --- p.14 / Chapter 1.2.2.3 --- Removal of catalytic iron and copper ions --- p.14 / Chapter 1.2.3 --- Synthetic vs. natural antioxidant --- p.15 / Chapter 1.2.3.1 --- Synthetic antioxidants --- p.15 / Chapter 1.2.3.2 --- Natural antioxidants --- p.16 / Chapter 1.3 --- Measurement of antioxidant activity --- p.17 / Chapter 1.3.1 --- Loss of substrate --- p.17 / Chapter 1.3.1.1 --- Beta-carotene bleaching method --- p.17 / Chapter 1.3.2 --- Measurement of free radical scavenging --- p.17 / Chapter 1.3.2.1 --- "Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH´Ø)" --- p.17 / Chapter 1.3.2.2 --- Superoxide scavenging --- p.18 / Chapter 1.3.2.3 --- Hydrogen peroxide scavenging --- p.18 / Chapter 1.3.2.4 --- Hydroxyl radical scavenging --- p.19 / Chapter 1.3.2.5 --- Peroxyl radical --- p.19 / Chapter 1.3.3 --- Measurement of end product --- p.21 / Chapter 1.3.3.1 --- Diene conjugation --- p.21 / Chapter 1.3.3.2 --- Light emission --- p.21 / Chapter 1.3.3.3 --- The thiobarbituric acid (TBA) test --- p.22 / Chapter 1.3.4 --- Low-density lipoprotein oxidation --- p.22 / Chapter 1.4 --- Phenolic antioxidant --- p.24 / Chapter 1.4.1 --- Chemistry --- p.24 / Chapter 1.4.2 --- Mechanism of action of phenolic antioxidants --- p.25 / Chapter 1.4.3 --- Isolation and characterization --- p.25 / Chapter 1.4.3.1 --- Extraction --- p.25 / Chapter 1.4.3.2 --- Analysis of phenolic compounds --- p.27 / Chapter 1.4.3.2.1 --- Colorimetric method --- p.27 / Chapter 1.4.3.2.2 --- Enzymatic method --- p.28 / Chapter 1.4.3.2.3 --- Paper chromatography --- p.28 / Chapter 1.4.3.2.4 --- Thin-layer chromatography --- p.29 / Chapter 1.4.3.2.5 --- UV-Vis absorption spectroscopy --- p.29 / Chapter 1.4.3.2.6 --- High-performance liquid chromatography --- p.30 / Chapter 1.4.4 --- Natural sources of phenolic antioxidants --- p.31 / Chapter 1.4.4.1 --- Olive oil --- p.31 / Chapter 1.4.4.2 --- Berry --- p.32 / Chapter 1.4.4.3 --- Cherry --- p.32 / Chapter 1.4.4.4 --- Red wine --- p.32 / Chapter 1.4.4.5 --- Herb --- p.33 / Chapter 1.4.4.6 --- Vegetables --- p.33 / Chapter 1.5 --- Mushroom Sample --- p.34 / Chapter 1.5.1 --- Pleurotus tuber-regium --- p.34 / Chapter 1.5.2 --- Lentinus edodes --- p.34 / Chapter 1.5.3 --- Volvariella volvacea --- p.35 / Chapter 1.5.4 --- Antioxidants in fungi or mushroom --- p.37 / Chapter 1.5.5 --- Phenolic compounds in mushrooms --- p.39 / Chapter 1.6 --- Objectives --- p.42 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS --- p.43 / Chapter 2.1 --- Sample Collection --- p.43 / Chapter 2.2 --- Sample Preparation --- p.43 / Chapter 2.3 --- Moisture Content --- p.43 / Chapter 2.4 --- Solvent Extraction --- p.44 / Chapter 2.4.1 --- Scheme I (Aqueous extraction only) --- p.44 / Chapter 2.4.2 --- Scheme II (Methanol and water extraction) --- p.45 / Chapter 2.4.3 --- Scheme III (Differential solvent extraction) --- p.46 / Chapter 2.4.4 --- Scheme IV (Scaled-up extraction) --- p.47 / Chapter 2.5 --- Antioxidant activity assays --- p.50 / Chapter 2.5.1 --- Beta-carotene bleaching method --- p.50 / Chapter 2.5.2 --- "Scavenging activity on 1,1 -diphenyl-2-picrylhydrazyl radicals" --- p.51 / Chapter 2.5.3 --- Assay for erythrocyte hemolysis --- p.51 / Chapter 2.5.4 --- Assay of lipid peroxidation using rat brain --- p.52 / Chapter 2.5.5 --- LDL oxidation (TBARS) --- p.53 / Chapter 2.5.5.1 --- LDL Isolation --- p.53 / Chapter 2.5.5.2 --- Calculation of density --- p.54 / Chapter 2.5.5.3 --- Lowry Method for Protein Determination --- p.55 / Chapter 2.5.5.4 --- Reagents for TBARS assay --- p.55 / Chapter 2.5.5.5 --- TBARS formation --- p.56 / Chapter 2.6 --- Determination of total polyphenolic compounds --- p.56 / Chapter 2.7 --- Fractionation --- p.57 / Chapter 2.7.1 --- Fractionation of the methanol crude extracts obtained under reflux by solvent --- p.57 / Chapter 2.7.2 --- Fractionation of boiling water crude extracts by ultrafiltration --- p.57 / Chapter 2.8 --- Crude Protein Content (Kjeldahl method) --- p.58 / Chapter 2.9 --- Total carbohydrate content --- p.59 / Chapter 2.10 --- Thin-layer chromatography --- p.59 / Chapter 2.11 --- High performance liquid chromatography --- p.60 / Chapter 2.11.1 --- Analysis of methanol fractions --- p.60 / Chapter 2.11.2 --- Analysis of water fractions --- p.61 / Chapter 2.12 --- Liquid chromatography-Mass spectrometry --- p.61 / Chapter 2.12.1 --- Liquid chromatography --- p.61 / Chapter 2.12.2 --- Mass spectrometric analysis --- p.62 / Chapter 2.13 --- Data analysis --- p.62 / Chapter CHAPTER THREE: --- RESULTS AND DISCUSSION --- p.63 / Chapter 3.1 --- Mushroom sample --- p.63 / Chapter 3.2 --- Extraction scheme I --- p.65 / Chapter 3.2.1 --- Antioxidant activity --- p.65 / Chapter 3.2.1.1 --- Effect of extraction temperature --- p.65 / Chapter 3.2.1.2 --- Effect of concentration of extracts --- p.66 / Chapter 3.3 --- Extraction scheme II --- p.69 / Chapter 3.3.1 --- Antioxidant activity --- p.69 / Chapter 3.3.1.1 --- Effect of extraction temperature --- p.69 / Chapter 3.3.1.2 --- Effect of concentration of extracts --- p.72 / Chapter 3.3.1.3 --- Effect of solvent --- p.72 / Chapter 3.4 --- Extraction scheme III --- p.75 / Chapter 3.4.1 --- Extraction yield --- p.75 / Chapter 3.4.2 --- Total phenolic content --- p.76 / Chapter 3.4.3 --- Antioxidant activity --- p.80 / Chapter 3.4.3.1 --- Beta-carotene bleaching method --- p.80 / Chapter 3.4.3.1.1 --- Effect of extract concentration --- p.80 / Chapter 3.4.3.1.2 --- Relation between total phenolic content and antioxidant activity --- p.82 / Chapter 3.4.3.2 --- "Scavenging activity of 1,1 -diphenyl-2-picrylhydrazyl (DPPH) radical" --- p.85 / Chapter 3.4.3.3 --- Assay for erythrocyte hemolysis --- p.88 / Chapter 3.5 --- Extraction scheme IV --- p.91 / Chapter 3.5.1 --- Yield and Fractionation --- p.91 / Chapter 3.5.2 --- Chemical characterization of fractions --- p.93 / Chapter 3.5.2.1 --- Protein content --- p.93 / Chapter 3.5.2.2 --- Total carbohydrate content --- p.93 / Chapter 3.5.2.3 --- Total phenolic content --- p.94 / Chapter 3.5.3 --- Antioxidant activity --- p.99 / Chapter 3.5.3.1 --- Assay for lipid peroxidation of rat brain --- p.99 / Chapter 3.5.3.2 --- LDL oxidation --- p.118 / Chapter 3.5.4 --- Identification of antioxidant by chromatographic methods --- p.126 / Chapter 3.5.4.1 --- Thin-layer chromatography --- p.126 / Chapter 3.5.4.2 --- High-performance liquid chromatography --- p.132 / Chapter 3.5.4.3 --- Liquid chromatography-Mass spectrometry --- p.142 / Chapter CHAPTER FOUR: --- CONCLUSION --- p.148 / REFERENCES --- p.153 / RELATED PUBLICATION --- p.161