Dissertations / Theses on the topic 'Mushroom extract'

Hyperglycemia is associated with a higher risk for the development of cardiovascular diseases such as atherosclerosis and hypertension. Hyperglycemia-induced generation of reactive oxygen species and the endothelial dysfunction largely account for this phenomenon. Ergothioneine is a naturally occurring amino acid that is abundantly found in mushroom. Numerous benefits have been found associated with ergothioneine such as cation chelating, regulation of gene expression, improvement in immunity and bioenergetics, and of most concern its antioxidative property. The aim of this study was to investigate whether mushroom extract and synthetic ergothioneine can exert protective effect on endothelial cells against oxidative stress. Human umbilical vein endothelial cells served as the cell model. Pyrogallol, hydrogen peroxide and high glucose were used to create the oxidative stress condition in endothelial cells. Biochemical assay was used to measure the viability of the cells. It was found that only the mushroom extract could significantly reduce the cell death induced by pyrogallol. Both the mushroom extract and synthetic ergothioneine significantly decreased the cell death induced by high glucose. However, neither mushroom extract nor synthetic ergothioneine have any positive effect on hydrogen peroxide-induced cell death. These results indicated that mushroom extract and synthetic ergothioneine did exert certain level of protective effect on endothelial cells. However, this protective effect is relatively weak. Besides, it is still unclear if antioxidation is the sole mechanism accounting for the cytoprotective effect of ergothioneine. Further investigation is required to examine if other mechanisms are also involved.<br>published_or_final_version<br>Pharmacology and Pharmacy<br>Master<br>Master of Medical Sciences

碩士<br>靜宜大學<br>應用化學系<br>90<br>Tyrosinase or polyphenol oxidase (EC1.14.18.1) is a copper-containing enzyme that is widespread in nature. It is responsible for not only melanization in animals but also browning in plants. In recent years, there is an increasing interest in finding natural tyrosinase inhibitors from plants and microorganisms. The tyrosinase inhibitors should have broad applications, especially in medicinal and cosmetics products in relation to hyperpigmentation. This study was aimed to evaluate the tyrosinase inhibitory activity of water or 50% ethanolic extracts from Graptopetalum paraguayense. The results were also compared with those obtained from the well-known tyrosinase inhibitors, benzoic acid and kojic acid. The results showed that the water or 50% ethanolic extracts of Graptopetalum paraguayense had tyrosinase inhibitory activity on the L-Dopa oxidation by mushroom tyrosinase. It was also found that the inhibitory activity increased with the concentrations of extracts to a certain extent and then leveled off as the concentration further increased. The 50% ethanolic extracts of Graptopetalum paraguayense had higher tyrosinase inhibitory activity than the water extracts, as evidenced by the lower half-inhibition concentration (IC50) needed for this oxidation. As compared to the well-known tyrosinase inhibitors, the Graptopetalum paraguayense extracts showed less inhibitory activity than benzoic acid and kojic acid. The inhibition was also a function of pH, becoming less as the pH was increased. In addition, the inhibition kinetics analyzed by a Dixon plots found that the Graptopetalum paraguayense extract to be a competitive inhibitor for this oxidation. The results of this study implied that Graptopetalum paraguayense had tyrosinase inhibitory activity.

碩士<br>中國文化大學<br>生活應用科學系<br>101<br>Abstract The main purpose of this study was to investigate the effect of needle mushroom extract addition on quality of raw ground pork during cold storage. The extracts were prepared with 70% ethanol or hot water. The ethanol (E) or hot water (W) needle mushroom extracts were added respectively in raw ground pork by the concentrations of 0、1、3、5% (v/w). The meat samples were stored at 4±1℃and drawn at 2 days interval (0, 2nd, 4th, 6th, and 8th day) for evaluation of pH, Hunter color values, color difference (ΔE), met-myoglobin formation percentage (Met-Mb%), 2-thiobarbituric acid reacting substances (TBARS) values, and total plate counts. The results indicated that among the ethanol extract treatments, the pork meat samples treated with 5% (E-5) showed a small decrease in the pH value. E-5 samples exhibited higher L (lightness) and a (redness) values significantly (p < 0.05) than the other samples during storage. In the E-5 treatment, slight increases in the Met-Mb% and TBARS values were observed during the storage period, yet, were lower than the control, E-1 and E-3 treated samples (p < 0.05). For the experiments of hot water extract, addition to pork meat at a level of 3% (W-3) was effective to suppress metMb formation as well as lipid oxidation during storage. The antioxidant effects of E-5 and W-3 were further compared to ascorbic acid (AA) and tocopherol (Toc). The data showed that the groups of E-5, W-3 and AA stabilized the pH values compared with the control and Toc groups. Regard to color, E-5 had high values in brightness and yellowness, however, Toc had a high value in redness. During the storage, AA (1.52) showed a minimal ΔE value, followed by Toc (1.75), W-3 (2.08) and E-5 (2.57), which were significantly lower than the control group (4.96) (p < 0.05). The Met-Mb% of Toc (28.38%), E-5 (39.88%), W-3 (40.76%), and AA (45.76%) were significantly lower than control (71.07%) (p < 0.05). The TBARS values (mg/kg) of Toc (2.06), AA (2.31), W-3 (2.44), and E-5 (2.47) were significantly lower than control (2.96) (p < 0.05). The total plate count (log CFU/g) of E-5 (10.10), Toc (10.11) were significantly lower than the W-3 (10.26), AA (10.36) and control (10.44). In conclusion, the raw ground pork added with 5% ethanol or 3% hot water needle mushroom extracts had shown the effects of the stabilization of pH, preservation of color, inhibition of lipid oxidation and met-myoglobin formation effectively at 4℃ storage. Key words: needle mushroom, extract, antioxidant, ground pork

碩士<br>慈濟科技大學<br>放射醫學科學研究所<br>106<br>Abstract Brazilian mushrooms are well-known traditional medicinal mushrooms, and studies have shown that they have certain effects on cellular immunity, anticancer activity, and radiation protection. The purpose of this study is to assess the potential and possible mechanism of Brazilian mushroom extract (BM) and polysaccharide (BMP) on wound healing after radiation injury. In this study, mouse fibroblasts (NIH-3T3) were used to model the model of cancer patients with local irradiation and wound healing after surgery. In wound healing, many factors may lead to impaired wound healing that slows down the rate of repair, delays healing, and even does not heal. The experimental conditions were divided into six groups, including the control group, Brazilian mushroom extract group, Brazil mushroom polysaccharide group, radiation control group (5 Gy), radiation plus Brazil mushroom extract group, and radiation plus Brazilian mushroom polysaccharide group. Brazilian mushroom polysaccharides are high-molecular polysaccharides, which can induce many physiological reactions and regulate physiological functions. The results also show that the Brazilian mushroom extract and polysaccharides have the ability to promote wound healing in cells, confirming accelerated healing by ERK and NF-κB. The polysaccharides inhibit the production of ROS after irradiated by cells. At the same time, the polysaccharides also prevented the G2/M phase arrest of fibroblasts in NIH-3T3 mice after radiation injury. Extracts and polysaccharides both reduced apoptosis and increased cell migration rate after radiation injury. From the results, The Brazilian mushroom extract and polysaccharide stimulated ERK and NF-κB secretion in fibroblasts of NIH-3T3 mice to induce cell activation to increase the rate of inflammation, proliferation, and migration of cells to accelerate wound healing. Presumably related to its Ras and MMP-9 pathway.

碩士<br>南台科技大學<br>生物科技系<br>97<br>In the study, we are looking for construct many kinds of non-genetic deficient DM 2 animal models, which closely simulates the metabolic abnormalities of the human disease and is also cost-effective compared with the genetic models currently available. It is suggested that disease models prior to serving with renal fibrosis, which may be used for studies on type 2 diabetic nephropathy animal models. The experimental mice were orally administrated high-fat diet (HFD) for consecutive 8 weeks, following by 7 consecutive days of streptozotocin (STZ, 50mg/Kg, ip) plus nicotinamide (NA, 200mg/Kg, ip), eventually which almost caused hyperglycemia and hyperinsulina occurred on HFD-controlled C57BL/6 mice (fasting blood glucose over 150 mg/dl, and ACR over 2 g/mg, alternatively). Inonotus obliquus is a white rot fungus, which belongs to the family Hymenochaetaceae of Basidiomycetes. Recently, there have been reports hat melanin complexes from sclerotia contain strong anti-oxidants. In our study, we detect the effect of hyperglycemia via the toxicity of diabetic nephropathy-prone, which produced by hyperglycemia from the damage of LLC-PK1 renal tubular cells. It suggested that the effectiveness of Inonotus obliquus needed to be conducted for the treatment of type 2 diabetic nephropathy. The preparation of experimental animals started with high fat diet (HFD) administration for 8 weeks followed by intraperitoneal injection of streptozotocin (STZ 50 mg/Kg) and nicotinamide (NA, 200 mg/kg). Once fasting hyperglycemia (over 150mg/dl, fasting period), hyperinsulinemia (10 unit), and positive microalbuminuria (ACR, 2 mg/g Creatinine) occured gradually on HFD-controlled C57BL/6 mice, the candidates were divided into 4 groups, (1) saline, (2) Innonotus oblique (IO), (3) rosiglitazone (R.S), and (4) Cordyceps sinensis (CS). Treatment with oral dosing ethanol extracts of Innonotus oblique and Cordyceps sinensis of 1000 mg/kg/day and 300 mg/kg/day for 8 weeks. The result MTT assay will claim the glucotoxicity, respectively in renal proximal tubular cell line（LLC-PK1） in vitro in a concentration-dependent AGE. We found that the Innonotus oblique also improve the glucose tolerance and insulin resistance with increasing TGF-β and NFκB expression kindey of high fat diet-induced diabetic nephropathy mice by western blotting. Immunohistochemical evaluation showed weak TGF-β staining in the glomeruli of native and I.O group, but in the control group the signal was markedly stronger. In conclusion, our data demonstrated that the alcoholic extract from Inonotus obliquus treatment has shown a potential anti-hyperglycemic effect, and significantly inhibited the early stage and late-stage renal fibrosis of glucolipotoxicity-induced nephrofibrosis in high fat diet-induced diabetic nephropathy mice.

Dissertação de mestrado em Genética Molecular<br>Mushrooms and their compounds are widely appreciated, not only for their nutritional but also for their medicinal properties. In fact, the search for various bioactive properties in different mushroom extracts or in the compounds isolated from those mushroom has been the focus of attention from the scientific community working in the area of natural products. The present work has focused on the study of the antitumor and immunomodulatory properties of extracts from three different mushrooms. Thus, the first aim of the present work was to gain insight into the mechanism of action of a methanolic extract of Cordyceps militaris in the non-small cell lung cancer cell line NCI-H460, since this extract had previously been shown to have tumour cell growth inhibitory activity in this cell line in particular. In addition, the second aim was to study the immunomodulatory activity of the Suillus luteus polysachararidic (PLS) extract and of the Morchella esculenta phenolic extract, using the monocytic THP-1 cell line which differentiates into machrophages upon stimulation. The response of NCI-H460 cells to the methanolic extract of C. militaris was studied regarding its effect on cellular viability, proliferation, cell cycle profile, apoptosis and DNA damage. Results showed that treatment with the methanolic extract of C. militaris caused a decrease in NCI-H460 cellular proliferation, a cell cycle arrest at G0/G1 and an increase in apoptosis. Interestingly, treatment with the extract was shown to increase the cellular levels of p53 and p21. Moreover, this study also showed evidence of cellular DNA damage caused by this extract, since increased levels of P-H2A.X and 53BP1 foci/cell were observed. Overall, this part of the work suggested that the methanolic extract of C. militaris affected NCI-H460 cellular viability through a mechanism which involved DNA damage and p53 activation. In addition, preliminary experiments were carried out to gain insight into the immunomodulatory potential of a PLS extract of S. luteus and of a phenolic extract of M. esculenta in THP-1 cells. Results showed that neither of the extracts presented cytotoxicity towards THP-1 cells or induced THP-1 monocytes differentiation into macrophages. Interestingly, the PLS extract of S. luteus caused a dose-dependent increase in the metabolic activity of THP-1 monocytes, probably due to increased proliferation. These preliminary results need be further confirmed and continued in future work. Overall, the work carried out in this thesis further supports the potential of mushrooms extracts in the search for bioactive compounds.<br>Os cogumelos e seus compostos são muito apreciados, não só pelas suas propriedades nutricionais, mas também pelas medicinais. De facto, a procura de propriedades bioativas em diferentes extratos de cogumelos ou em compostos isolados desses cogumelos, tem sido um foco de interesse da comunidade científica que trabalha na área de produtos naturais. O presente trabalho visou o estudo de propriedades antitumorais e imunomoduladoras de extratos de três cogumelos diferentes. Assim sendo, como primeiro objetivo deste trabalho pretendeu-se analisar o mecanismo de ação de um extrato metanólico de Cordyceps militaris na linha celular NCI-H460 (de cancro do pulmão de não pequenas células), uma vez que tinha sido previamente demonstrado que este extrato inibe o crescimento celular, destas células em particular. Além deste, um segundo objetivo visou o estudo da atividade imunomoduladora de um extrato polisacarídico (PLS) de Suillus luteus e de um fenólico de Morchella esculenta, usando a linha celular monocítica THP-1 que se diferencia em macrófagos, após estimulação. Foi estudada a resposta das células NCI-H460 ao tratamento com o extrato metanólico de C. militaris relativamente ao efeito na viabilidade celular, proliferação, perfil do ciclo celular, apoptose e no dano no DNA. Os resultados demonstraram que o tratamento com o extrato metanólico de C. militaris nas células NCI-H460 diminuiu a proliferação celular, bloqueou o ciclo celular nas fases G0/G1 e induziu apoptose. De particular interesse foi o facto de este extrato causar um aumento dos níveis de p53 e p21. Para além disso, este estudo também mostrou ainda evidências de danos no DNA causados por este extrato, dada a observação de aumento quer nos níveis de P-H2A.X como no número de 53BP1 foci/célula. Em geral, esta parte do trabalho sugeriu que o extrato metanólico de C. militaris diminuiu a viabilidade celular das células NCI-H460 por um mecanismo que envolve a ativação de danos no DNA e de p53. Neste trabalho, foram realizadas experiências preliminares para averiguar o potencial imunomodulador de um extrato PLS de S. luteus e de um extrato fenólico de M. esculenta em células THP-1. Os resultados demonstraram que nenhum dos extratos analisados apresentou citotoxicidade para células THP-1 ou induziu a diferenciação desta linha celular de monócitos em macrófagos. De particular interesse foi o facto de se verificar que o extrato PLS do S. luteus causou um aumento (dependente da dose) da atividade metabólica de monócitos THP-1, provavelmente devido ao aumento da proliferação celular. Estes resultados preliminares terão ainda de ser confirmados num trabalho futuro, assim como o potencial imunomodulador destes extratos. De um modo geral, o trabalho realizado nesta tese contribui para a descrição do potencial dos extratos de cogumelos na busca de compostos bioativos.

碩士<br>中國文化大學<br>生活應用科學系<br>100<br>The study is designed to explore the antioxidant properties of mushroom extracts and the effects of extraction solvents; in addition, to investigate the contents and profiles of total phenolics, including both soluble free and bound forms, by applying solvent extraction, alkaline hydrolysis, and liquid-liquid extraction methods. Five mushrooms (Hypsizygus marmoreu, Flammulina velutipes collybia velutipes , Pleurotus eryngii, Pleurotus citrinopileatus, Lentinus edodes) were investigated and five antioxidant capacities (DPPH scavenging activity, reducing power, ferrous chelating effect, superoxide anion scavenging activity and trolox equivalent antioxidant capacity (TEAC)) were measured. The results showed significant differences (p < 0.05) in the chemical compositions of the samples. Among five antioxidant capacities, mushroom extracts possessed only DPPH scavenging activity, reducing power, and TEAC, which activities were related to the varieties of mushroom. To these three antioxidant activities, Pleurotus citrinopileatus and Pleurotus eryngii were the highest. Pleurotus citrinopileatus had the highest total phenolic content (7.36 mg gallic acid equiv/g), followed by Pleurotus eryngii, Lentinus edodes, Hypsizygus marmoreu and Flammulina velutipes Collybia velutipes. The major portion of phenolics in mushrooms existed in the free form (51% in Hypsizygus marmoreu, 75% in Flammulina velutipes collybia velutipes, 70% in Pleurotus eryngii, 56% Pleurotus citrinopileatus, 69% in Lentinus edodes). The higher content of total phenolics and antioxidant activity were obtained from extraction with 80% acetone with comparision to 100% methanol. Key words: mushrooms, antioxidant, phenolics

Lai, Tsz Ching.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.<br>Includes bibliographical references (leaves 121-136).<br>Abstracts in English and Chinese.<br>Acknowledgements<br>Abstract<br>摘要<br>Content<br>List of tables<br>List of figures<br>List of abbreviations<br>Chapter Chapter 1: --- Introduction --- p.1<br>Chapter 1.1 --- Introduction of food market trends in Hong Kong and mushroom productivity in the world --- p.1<br>Chapter 1.1.1 --- Agrocybe aegerita --- p.1<br>Chapter 1.1.2 --- Pleurotus spp --- p.2<br>Chapter 1.1.3 --- Pholiota nameko --- p.3<br>Chapter 1.2 --- Objectives --- p.5<br>Chapter Chapter 2: --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.6<br>Chapter 2.1 --- Introduction --- p.6<br>Chapter 2.1.1 --- Reactive oxygen species (ROS) --- p.6<br>Chapter 2.1.1.1 --- Definition of ROS --- p.6<br>Chapter 2.1.1.2 --- Sources of ROS --- p.6<br>Chapter 2.1.1.2.1 --- Endogenous sources of ROS --- p.6<br>Chapter 2.1.1.2.2 --- Exogenous sources of ROS --- p.8<br>Chapter 2.1.1.3 --- Damaging effects of ROS --- p.8<br>Chapter 2.1.2 --- Antioxidants --- p.10<br>Chapter 2.1.2.1 --- Mechanism of action --- p.10<br>Chapter 2.1.2.2 --- Sources of antioxidants --- p.11<br>Chapter 2.1.2.2.1 --- Dietary antioxidants --- p.11<br>Chapter 2.1.2.2.2 --- Antioxidants in edible mushrooms --- p.12<br>Chapter 2.1.2.2.3 --- Phenolic compounds in mushrooms --- p.13<br>Chapter 2.2 --- Materials and Methods --- p.16<br>Chapter 2.2.1 --- Materials --- p.16<br>Chapter 2.2.1.1 --- Mushroom fruiting bodies --- p.16<br>Chapter 2.2.2 --- Principles of Methods and Experimental Protocols --- p.17<br>Chapter 2.2.2.1 --- Sample preparation --- p.17<br>Chapter 2.2.2.2 --- Evaluation of antioxidant capacity --- p.18<br>Chapter 2.2.2.2.1 --- DPPH radical scavenging activity --- p.18<br>Chapter 2.2.2.2.2 --- Superoxide anion scavenging activity --- p.19<br>Chapter 2.2.2.2.3 --- Hydroxyl radical scavenging activity --- p.20<br>Chapter 2.2.2.2.4 --- Hydrogen peroxide scavenging activity --- p.22<br>Chapter 2.2.2.3 --- Determination of phenolic compounds --- p.24<br>Chapter 2.2.2.3.1 --- Total phenolic content --- p.24<br>Chapter 2.2.2.3.2 --- Identification of phenolic acids --- p.25<br>Chapter 2.2.3 --- Statistical analysis --- p.27<br>Chapter 2.3 --- Results and Discussion --- p.28<br>Chapter 2.3.1 --- Extraction yield --- p.28<br>Chapter 2.3.2 --- Evaluation of antioxidant capacity --- p.29<br>Chapter 2.3.2.1 --- DPPH radical scavenging activity --- p.29<br>Chapter 2.3.2.2 --- Superoxide anion scavenging activity --- p.31<br>Chapter 2.3.2.3 --- Hydroxyl radical scavenging activity --- p.33<br>Chapter 2.3.2.4 --- Hydrogen peroxide scavenging activity --- p.35<br>Chapter 2.3.2.5 --- Comparison of the effective concentrations (EC50) of mushroom water extracts in different antioxidant assays --- p.37<br>Chapter 2.3.3 --- Determination of phenolic compounds --- p.38<br>Chapter 2.3.3.1 --- Total phenolic content --- p.38<br>Chapter 2.3.3.2 --- Identification of phenolic acids --- p.39<br>Chapter 2.4 --- Summary --- p.45<br>Chapter Chapter 3: --- Anti-angiogenic properties of the Aa water extract --- p.46<br>Chapter 3.1 --- Introduction --- p.46<br>Chapter 3.1.1 --- Angiogenesis --- p.46<br>Chapter 3.1.1.1 --- Process of angiogenesis --- p.46<br>Chapter 3.1.1.2 --- Regulations of angiogenesis --- p.47<br>Chapter 3.1.1.2.1 --- Fibroblast growth factor (bFGF) --- p.47<br>Chapter 3.1.1.2.2 --- Vascular endothelial growth factor (VEGF) --- p.48<br>Chapter 3.1.2 --- Tumor angiogenesis --- p.49<br>Chapter 3.1.2.1 --- ROS generation in tumor cells --- p.50<br>Chapter 3.1.2.2 --- Hydrogen peroxide and VEGF --- p.51<br>Chapter 3.1.2.3 --- Previous studies on tumor angiogenesis --- p.52<br>Chapter 3.1.2.3.1 --- ROS and endothelial cells proliferation --- p.52<br>Chapter 3.1.2.3.2 --- VEGF and endothelial cells functions --- p.53<br>Chapter 3.1.3 --- Use of antioxidants in cancer treatment --- p.53<br>Chapter 3.1.3.1 --- Antioxidant use of cancer therapy --- p.53<br>Chapter 3.1.3.2 --- Antioxidant and endothelial cells functions --- p.54<br>Chapter 3.1.3.3 --- Anti-angiogenic effects of polyphenols --- p.56<br>Chapter 3.1.3.3.1 --- Phenolic acids --- p.56<br>Chapter 3.1.3.3.2 --- Tea catechin --- p.57<br>Chapter 3.1.3.3.3 --- Resveratrol --- p.57<br>Chapter 3.1.3.3.4 --- Genistein --- p.58<br>Chapter 3.2 --- Principles of Methods and Experimental Protocols --- p.60<br>Chapter 3.2.1 --- Sample preparation --- p.60<br>Chapter 3.2.2 --- Toxicity of the Aa water extract --- p.60<br>Chapter 3.2.2.1 --- Limulus amebocyte lysate (LAL) test --- p.60<br>Chapter 3.2.2.2 --- Toxicity towards normal cells --- p.61<br>Chapter 3.2.2.2.1 --- Cell line and its subculture --- p.61<br>Chapter 3.2.2.2.2 --- Colorimetric (MTT) assay --- p.62<br>Chapter 3.2.3 --- Effect of the Aa water extract on cancer cells --- p.63<br>Chapter 3.2.3.1 --- Cell line and its subculture --- p.63<br>Chapter 3.2.3.2 --- Redox status --- p.63<br>Chapter 3.2.3.3 --- VEGF secretion --- p.65<br>Chapter 3.2.4 --- In vitro cell culture anti-angioenesis analysis --- p.66<br>Chapter 3.2.4.1 --- Cell line and its subculture --- p.66<br>Chapter 3.2.4.2 --- Endothelial cells proliferation --- p.67<br>Chapter 3.2.4.3 --- Endothelial cells migration --- p.68<br>Chapter 3.2.4.3.1 --- Wound healing assay --- p.68<br>Chapter 3.2.4.3.2 --- Transwell culture insert assay --- p.69<br>Chapter 3.2.4.4 --- Endothelial cells tubule formation --- p.71<br>Chapter 3.2.5 --- In vitro organ culture anti-angiogenesis analysis --- p.72<br>Chapter 3.2.5.1 --- Aortic ring assay --- p.72<br>Chapter 3.2.6 --- Statistical analysis --- p.74<br>Chapter 3.3 --- Results and Discussions --- p.75<br>Chapter 3.3.1 --- Toxicity of the Aa water extract --- p.75<br>Chapter 3.3.1.1 --- Limulus amebocyte lysate (LAL) test --- p.75<br>Chapter 3.3.1.2 --- Toxicity towards normal cells --- p.75<br>Chapter 3.3.2 --- Effect of the Aa water extract on cancer cells --- p.77<br>Chapter 3.3.2.1 --- Redox status --- p.77<br>Chapter 3.3.2.2 --- VEGF secretion --- p.79<br>Chapter 3.3.2.3 --- Relationship between intracellular ROS and VEGF secretion detected --- p.80<br>Chapter 3.3.3 --- Effect of the Aa water extract on angiogenesis --- p.82<br>Chapter 3.3.3.1 --- Endothelial cells proliferation --- p.82<br>Chapter 3.3.3.2 --- Endothelial cells migration --- p.84<br>Chapter 3.3.3.2.1 --- Wound healing assay --- p.84<br>Chapter 3.3.3.2.2 --- Transwell culture insert assay --- p.87<br>Chapter 3.3.3.3 --- Endothelial cells tubule formation --- p.90<br>Chapter 3.3.3.4 --- Aortic ring assay --- p.97<br>Chapter 3.3.4 --- Effect of phenolic acids on endothelial cells --- p.101<br>Chapter 3.3.4.1 --- Endothelial cells proliferation --- p.101<br>Chapter 3.3.4.2 --- Endothelial cells migration --- p.102<br>Chapter 3.3.4.2.1 --- Wound healing assay --- p.102<br>Chapter 3.3.4.2.2 --- Transwell culture insert assay --- p.105<br>Chapter 3.3.4.3 --- Endothelial cells tubule formation --- p.106<br>Chapter 3.3.4.4 --- Aortic ring assay --- p.112<br>Chapter 3.4 --- Summary --- p.116<br>Chapter Chapter 4 --- Conclusions and future works --- p.118<br>References --- p.121

碩士<br>東海大學<br>化學工程與材料工程學系<br>98<br>The purpose of this study was to investigate the effect of the extracts with different solvents and autolysis treatment of mycelia of medicinal mushroom (P. cinnabarinus, G. lucidum, A. camphorate and C. cinereus）on ACE inhibitory activity and expected to develop a new healthy food for regulating blood pressure. The mycelia were produced by submerged cultures or solid-state fermentation using different substrate. Based on the results, when cold water was used for extraction, higher ACE inhibitor activities were obtained, which were 69.4%、57.2%、26.8% and 15.2% for P. cinnabarinus, G. lucidum, A. camphorate and C. cinereus, respectively. Moreover, the IC5 values were determined as 2.45、4.13、9.28 and 13.5 mg/ml. The maximum ACE inhibitor activity was obtained when the mycelium was extracted using water. The ethanol extract showed no ACE inhibitor activity. Pearl barley, oats and buckwheat were used as substrate for solid-state fermentation. When pearl barley was used as a substrate, the mycelium extracts of P. cinnabarinus, A. camphorate and G. lucidum had the higher inhibitor activity, however oats was a more suitable substrate for the culture of C. cinereus.. Concerning the autolysis of P. cinnabarinus , the optimum condition was determined to be a temperature equal to 50℃ and pH 4. When commercial proteases (papain, bromelain and alcalas) were employed for the mycelia hydrolysis of P. cinnabrinusn, alcalase was demonstrated to achieve the highest ACE inhibitor activity of 82.3%.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2011<br>Lectins are among a large number of proteins produced by mushrooms. Mushroom lectins with important biological functions have been isolated and studied. However, none of the studies were reported on lectins isolated from mushrooms indigenous to southern Africa. A galactose-specific lectin from one of the common mushroom species in southern Africa, Schizophyllum commune, was isolated and characterized. Initially, twenty mushroom samples were collected and their crude extracts screened for lectin activities. Assays involved in the screening procedures included heamagglutination, carbohydrate inhibition, enzyme linked glycoprotein (ELGA) and various stability assays. Four different mushroom samples exhibited positive lectin activities with varying stabilities towards thermal treatment and susceptibility to proteolytic degradation. Further screening assays resulted in ZHR1 being selected for identification and purification of the lectin. This was due to its ability to agglutinate rabbit erythrocytes. In addition to its activity being destroyed after 3 hours of treatment with trypsin-NIPAAM conjugate, the activity of this lectin was also completely destroyed after an hour incubation in boiling water. In contrast to other mushroom extracts assayed, heamagglutination activity of the crude extract of ZHR1 was not inhibited by glycoproteins only but also by the sugars such as galactose, lactose and mannose. ZHR1 was identified as S. commune. S. commune lectin (ScL) was purified using affinity chromatography on a fetuin-agarose column and further purified using gel-filtration chromatography on Biogel P-100 column. ScL was characterized as a glycosylated, galactose-specific dimeric lectin with a molecular weight of approximately 32 and 33 kDa. ScL is a thermolabile lectin which loses its activity as early as 5 minutes after being incubated at 60°C. Anti-ScL antibodies, to be employed in screening for the presence of ScL in the protein extracts, were developed in the rabbits and their interaction with ScL was detected using the double immunodiffusion assays whereas their specificity towards the lectin was detected using Western blot. ScL is one of the first mushroom lectins to be isolated and studied in southern African region. If the lectin is found to be exhibiting important biological functions, ScL can be of commercial importance in the region.

by Joyce Chui Kwan Ho.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.<br>Includes bibliographical references (leaves 61-75).<br>Abstracts in English and Chinese.<br>Acknowledgments --- p.i<br>List of Abbreviations --- p.iii<br>Abstract<br>English --- p.1<br>Chinese --- p.2<br>Chapter Chapter 1 --- General Introduction<br>Chapter 1.1 --- Tumor Formation --- p.3<br>Chapter 1.2 --- Anti-tumor Pathways --- p.4<br>Chapter 1.3 --- Aim of Project --- p.13<br>Chapter Chapter 2 --- The In Vivo effect of Polysaccharopeptide<br>Chapter 2.1 --- Introduction --- p.15<br>Chapter 2.2 --- Materials and Methods --- p.17<br>Chapter 2.3 --- Results --- p.18<br>Chapter 2.4 --- Discussion --- p.19<br>Chapter Chapter 3 --- Cytotoxicity<br>Chapter 3.1 --- Introduction --- p.23<br>Chapter 3.2 --- Materials and Methods --- p.26<br>Chapter 3.3 --- Results --- p.28<br>Chapter 3.4 --- Discussion --- p.28<br>Chapter Chapter 4 --- Anti-angiogenic Effect<br>Chapter 4.1 --- Introduction --- p.30<br>Chapter 4.2 --- Materials and Methods --- p.35<br>Chapter 4.3 --- Results --- p.39<br>Chapter 4.4 --- Discussion --- p.42<br>Chapter Chapter 5 --- Immunomodulation<br>Chapter 5.1 --- Introduction --- p.45<br>Chapter 5.2 --- Materials and Methods --- p.47<br>Chapter 5.3 --- Results --- p.50<br>Chapter 5.4 --- Discussion --- p.52<br>Chapter Chapter 6 --- General Discussion --- p.57<br>References --- p.61

碩士<br>國立中興大學<br>生命科學院碩士在職專班<br>99<br>Asthma is a chronic airway inflammation disease caused by the TH1/ TH2 cytokines secretion imbalance. One of the mechanisms is IL-5 secreted by TH2 cells activating eosinophils infiltration to airway inflammation site, and to release more pro-inflammatory mediators aggravating symptoms. In clinical, steroids are often used to improve inflammation, but long term or improper dependence on drugs would follow some complications. According to this,we proposed that there may exist some ingredients in the extracts of mushroom which have beneficial effect of immunoregulation on murine airway inflammation. Our data shown that mushroom extracts D and E could significantly reduce airway hyperresponsiveness (AHR) to methacholine in airway inflammatory mice as low as the AHR of mice fed with prednisolone. the eosinophil percentage in BALF significantly suppressed by mushroom extracts .the airway inflammatory mice administered with mushroom extracts,can significant increase IFN-γ production of ConA、OVA splenocytes. In conclusion, according to our study design, the proper effect on improving airway inflammatory mice was fed with mushroom extracts after OVA-immunization.

碩士<br>國立屏東科技大學<br>食品科學系<br>93<br>The antioxidant activities and protection to liver cells during ethanol-induced cytotoxicity by hexane extracts from three species of commercially available edible mushrooms, namely, Agaricus bisporus, Lentinula edodes and Pleurotus eryngii were evaluated. The scavenging effect of hexane extracts from edible mushrooms on α,α-diphenyl-β-picrylhydrazyl radicals were 77.59, 66.10 and 63.06 % for Agaricus bisporus, Lentinula edodes and Pleurotus eryngii, respectively. At 10 % of hexane extract, the induction period (Rancimat, 20 L/h, 120℃) of the soybean oil were in the order Pleurotus eryngii (31.3 hrs) > Lentinula edodes (8 hrs) > Agaricus bisporus (4.44 hrs). The antioxidative properties of 10 % hexane extract in winterized lard showed a similar trend. Hexane extracts from three commercial edible mushrooms at three different concentrations (1, 5, 10 mg/mL) toward the 6 % alchol treated tumor cell lines were tested. The cell proliferation rate of HepG2 cells treated by hexane extracts from edible mushrooms at 10 mg/mL were 98, 49 and 86 % for Agaricus bisporus, Lentinula edodes and Pleurotus eryngii, respectively. The content of hexane-extracted lipophilic compounds from edible mushrooms was 0.28, 0.57 and 0.73 % for Agaricus bisporus, Lentinula edodes and Pleurotus eryngii, respectively. Contents of total phenols were 7.463, 8.585 and 7.191 mg/g for Agaricus bisporus, Lentinula edodes and Pleurotus eryngii, respectively. Gas chromatography-mass spectrometry were used to characterize the phytosterols in these mushrooms. Ergosterol was present in all the mushrooms, and its contents were in the order Pleurotus eryngii (52.20 %) > Agaricus bisporus (0.06 %) > Lentinula edodes (0.02 %). Results of other chemical composition analysis of the extracts were as follows: iodine value, 113, 108 and 101 I2 g/100 g oil for Agaricus bisporus, Lentinula edodes and Pleurotus eryngii, respectively. The fatty acid was found to contain high levels of polyunsaturated fatty acids and less monounsaturated fatty acids. The hexane extract exhibit significant antioxidative activities and protective effect on alchol-treated liver cells.

碩士<br>亞洲大學<br>生物科技學系碩士班<br>96<br>Abstract In living organisms, melanin can be formed by enzymatic reaction of tyrosinase with certain substrates such as L-3,4-dihydroxyphenylalanine (L-dopa). The objective of this study was to determine the inhibitory effects of seed and peel ethanol extracts from kyoho grape and red globe grape on tyrosinase activity. Ethanol extracts of grape cells derived from Vitis vinifera L.Gamayreaux callus and red globe grape seed were also tested to evaluate the potential of using grape as the source for skin care. The activity of tyrosinase was defined as the reactive rate to hydrolyze L-dopa into dopachrome, which can be detected at OD475. The results indicated that both the extracts from grape seed and peel showed inhibitory effect on the activity of mushroom tyrosinase. The inhibitory influence was not significant between the extracts from kyoho grape seed and peel; however, the influence was significantly higher in grape seed extract than grape peel extract in red globe grape. Therefore, ethanol extract from kyoho grape was considered to have higher inhibitory effect toward mushroom tyrosinase than ethanol extract from red globe grape. Ethanol extracts from the grape cells showed slight influence on mushroom tyrosinase activity, the inhibition rate was only 9-31%. Analysis of enzyme kinetics revealed that all the extracts showed mixed-type inhibition toward mushroom tyrosinase activity.

The food industry has shown a growing interest in the so-called functional foods due to increase in consumers’ demand for foods with pharmaceutical and nutraceutical functions, from the natural origin to avert various side effects from the traditional drugs for cardiovascular diseases (CVD), etc. Thus, this work aims to formulate products using probiotics and prebiotics together (synbiotic), which have cholesterol-lowering and antihypertensive properties. This study deals with the evaluation of curd and human fecal microbial isolates for their probiotic, in vitro hypocholesterolemic and hypotensive properties. The isolates having probiotic potential were then utilized to formulate synbiotic functional foods (soymilk-fortified tea curd, and shelf-stable dry functional foods) in combination with prebiotic rich soymilk and extracts of best compatible mushrooms, respectively. Then the effect of storage on their biochemical properties and sensory attributes was studied. The best probiotic potential was depicted by isolate CI1 Enterococcus faecium),HF1 and FS3 (both Lactobacillus plantarum strains). They also exhibited in vitro cholesterol reduction, and angiotensin-converting enzyme (ACE)inhibition thereby can be used to formulate therapeutic products having both hypocholesterolemic and hypotensive properties. CI1 and HF1were also found to be compatible with soymilk and P. florida mushroom extract. Hence, these two isolates were utilized for soymilk fortified tea curd formulation. Tea curd is a functional food providing the health benefits of tea polyphenols and the bioactive peptides mainly ACE inhibitory) produced by probiotic fermentation. The tea polyphenols showed varying degrees of stability during storage. Soymilk fortification significantly increased the viability of probiotics in the curd plus it also increased the ACE inhibition. P. florida extract had highest β- glucan and very high inulin content in addition to high amount of eritadenine a hypocholesterolemic and hypotensive compound reported exclusively in edible mushrooms), and it showed the highest compatibility with CI1, which was proven by the prebiotic activity score analysis. The main aim of the study was to formulate dry shelf stable synbiotic foods which do not require refrigeration for storage and transport, hence being highly economical. Synbiotic microencapsulation protects the probiotic bacteria in food products as well as en route colon. Two types of drying processes,lyophilization and spray drying, were utilized and compared for better preservation of probiotics’ viability as well as organoleptic properties of food matrices used for incorporation and storage. The synbiotic microcapsules incorporated food formulations were found to be very promising both in terms of viable cell load as well as sensory acceptability after long-term storage for ≥15 weeks. While spray dried synbiotic powder of CI1 and P. florida when incorporated into food formulations proved to be more acceptable and favorable due to long-term storage stability (even after 15 weeks of storage). This process may be employed in future for efficient production and commercialization of several dry shelf stable synbiotic functional foods by varying the sources of prebiotics, the dry food matrix used and the type of health-promoting probiotic bacteria.

碩士<br>國立嘉義大學<br>食品科學系碩士班<br>93<br>The purpose of this study was to isolate and purify tyrosinase inhibitor from the edible mushrooms including Pleurotus eryngii and Agrocybe aegerita. After fractionation and isolation by HPLC (C18 column) from the water extract of Pl. eryngii fruiting body, one compound being volatile and UV nonabsorbent exhibited potent tyrosinase inhibitory activity. Due to the compound was toxic to a small fish, it was suspected to contain a cyanogenic group. The compound was also present in the submerged-cultivated of Pl. eryngii. For safety consideration a glass distilling “Robean” tube (rolled-peanut shape) was designed and applied for recovery of the broth vapor. When AgNO3 solution was used as an absorbent to interact with the vapor, a white precipitate was formed and identified as AgCN by FT-Raman analysis. When AgNO3 was substituted by KOH for distillation, after evaporated to dryness, dissolved in D2O and followed by C13-NMR analysis, a KCN spectrum was achieved. This confirmed HCN production in the broth by Pl. eryngii. When a sodium picrate solution (1.4 %) was used as an absorbent and various authentic KCN solutions (ranged from 0.04 to 1.2 ppm) were applied for distillation and followed by absorbance determination at 510 nm, a linear dose-dependent relationship (R2 = 0.9985) was obtained. When the KCN solutions reacted directly with picrate solution for absorbance determination, a linear relationship (R2 = 0.9994) was also achieved and indicated that loss or incomplete evaporation of HCN during distillation could be ignored. When the distillation method was applied for HCN quantification of the fruiting bodies of Pl. eryngii collected from local markets, 67.3% of the products contained HCN less than 1.0 ppm, 17.3% between 1.0 and 2.0 ppm and 15.4% higher than 2.0 ppm. When the fruiting body was sliced and cooked in water at 95 oC for 6 min, 89.1 % of the original HCN was lost. When five Pl. eryngii strains (BCRC 36037, BCRC 36163, RCPE-1, RCPE-2 and RCPE-3) were respectively submerged-cultivated with YMB and YMB supplemented with 2.5% glycine for 16 days, HCN content was higher in the latter than the former for each strain. When the culture of BCRC 36037 was submerged-cultivated with YMB supplemented with various amount of glycine (0%, 0.1%, 2.5% and 5%), HCN production increased with an increase of glycine supplementation. There was no difference of HCN production as affected by supplementation of glycine before and after sterilization. After isolation and purification by HPLC (C18 column) from 60% methanol extract of Agrocybe aegerita, the tyrosinase inhibitor was identified as 5-Hydroxy-tryptophan (5-HTP) by NMR and L.C. Mass. Condensation reaction of 5-HTP with acetaldehyde to form 1-methyl-6-hydroxy-2,3,4,9-tetrahydro-1H-β-carboline-3-carboxylic acid was observed.

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

Guo, Cuixia.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.<br>Includes bibliographical references (leaves 75-85).<br>Abstracts in English and Chinese.<br>Thesis committee --- p.ii<br>Abstract --- p.iii<br>摘要 --- p.iv<br>Acknowledgment --- p.v<br>List of Tables --- p.vi<br>List of Figures --- p.vii<br>List of Abbreviations --- p.viii<br>Chapter 1. --- Introduction --- p.1<br>Chapter 1.1 --- Introduction to immune system --- p.1<br>Chapter 1.2 --- Immune effecter cells --- p.1<br>Chapter 1.2.1 --- Macrophage --- p.1<br>Chapter 1.2.2 --- Dendritic Cells (DCs) --- p.5<br>Chapter 1.3 --- Immunomodulatory and antitumor activities of mushrooms --- p.8<br>Chapter 1.3.1 --- Introduction to mushroom --- p.11<br>Chapter 1.3.2 --- Mushroom polysaccharides --- p.11<br>Chapter 1.3.3 --- Mushroom β-glucan --- p.14<br>Chapter 1.4 --- The receptors for polysaccharides associated with immune effecter cells --- p.16<br>Chapter 1.4.1 --- CR3 --- p.16<br>Chapter 1.4.2 --- Dectin-1 --- p.18<br>Chapter 1.4.3 --- TLR2 --- p.19<br>Chapter 1.5 --- Nuclear factor-kappa B (NF-kB) activation --- p.19<br>Chapter 1.6 --- Previous studies on mushroom sclerotium --- p.20<br>Chapter 1.6.1 --- Pleurotus tuber-regium (PT) --- p.20<br>Chapter 1.6.2 --- Polyporus rhinocerus (PR) --- p.21<br>Chapter 1.7 --- Objectives --- p.21<br>Chapter 2. --- Materials and Methods --- p.23<br>Chapter 2.1 --- Materials --- p.23<br>Chapter 2.1.1 --- Mushroom sclerotia --- p.23<br>Chapter 2.1.2 --- Animal --- p.23<br>Chapter 2.1.3 --- Cell lines --- p.24<br>Chapter 2.2 --- Methods --- p.24<br>Chapter 2.2.1 --- Hot water extraction --- p.24<br>Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.25<br>Chapter 2.2.2.1 --- Acid depolymerization --- p.25<br>Chapter 2.2.2.2 --- Neutral sugar derivatization --- p.25<br>Chapter 2.2.2.3 --- Gas chromatography (GC) --- p.26<br>Chapter 2.2.3 --- Determination of molecular weight by size exclusion chromatography (SEC) --- p.27<br>Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method --- p.28<br>Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method --- p.28<br>Chapter 2.2.6 --- Detection of endotoxin --- p.29<br>Chapter 2.2.7 --- Immunomodulatory activities induced in RAW264.7 cell line and murine peritoneal macrophages (PMs) --- p.30<br>Chapter 2.2.7.1 --- Isolation of murine peritoneal macrophages (PMs) --- p.30<br>Chapter 2.2.7.2 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.30<br>Chapter 2.2.7.3 --- Phagocytic uptake --- p.31<br>Chapter 2.2.7.4 --- Reactive Oxygen Species (ROS) generation --- p.32<br>Chapter 2.2.7.5 --- Nitric Oxide (NO) production --- p.32<br>Chapter 2.2.7.6 --- Inducible Nitric Oxide Synthase (iNOS) expression --- p.32<br>Chapter 2.2.7.6.1 --- Cell lysates preparation --- p.33<br>Chapter 2.2.7.6.2 --- Determination of protein concentrations --- p.33<br>Chapter 2.2.7.6.3 --- Western blot --- p.34<br>Chapter 2.2.7.7 --- Tumor Necrosis Factor-alpha (TNF-α) production --- p.36<br>Chapter 2.2.8 --- DC cell marker determination --- p.37<br>Chapter 2.2.9 --- Nuclear factor kappa B (NF-kB) activation --- p.37<br>Chapter 2.2.10 --- Determination of the expression of existing cell surface β-glucan receptors --- p.37<br>Chapter 2.2.11 --- Statistical methods --- p.38<br>Chapter 3. --- Results --- p.39<br>Chapter 3.1 --- Yield and chemical composition of mushroom sclerotial polysaccharides --- p.39<br>Chapter 3.2 --- Endotoxin examination --- p.41<br>Chapter 3.3 --- Monosaccharide profiles of PTW and PRW by GC --- p.41<br>Chapter 3.4 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.43<br>Chapter 3.5 --- Immunomodulatory activities induced in RAW264.7 cells and murine peritoneal macrophages (PMs) --- p.46<br>Chapter 3.5.1 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.46<br>Chapter 3.5.2 --- Phagocytic uptake --- p.49<br>Chapter 3.5.3 --- ROS generation --- p.53<br>Chapter 3.5.4 --- NO production --- p.56<br>Chapter 3.5.5 --- iNOS expression --- p.59<br>Chapter 3.5.6 --- TNF-α production --- p.60<br>Chapter 3.5.7 --- Morphological changes of cells --- p.62<br>Chapter 3.5.8 --- DC cell marker determination --- p.64<br>Chapter 3.6 --- Receptors expression on RAW 264.7 cells and PMs --- p.66<br>Chapter 3.7 --- NF-kB activation --- p.68<br>Chapter 3.8 --- Discussion --- p.70<br>Chapter 4. --- Conclusions and Future Works --- p.73<br>Chapter 5. --- References --- p.75

Lai Kin Ming Connie.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 121-141).<br>Abstracts in English and Chinese.<br>Chapter Chapter 1. --- Introduction --- p.1<br>Chapter 1.1 --- Introduction on growth cycle of mushroom --- p.1<br>Chapter 1.2 --- Literature review of mushroom biological activities --- p.3<br>Chapter 1.2.1 --- Various bioactivities of mushroom --- p.3<br>Chapter 1.2.2 --- Components responsible for various bioactivities of mushrooms --- p.3<br>Chapter 1.3 --- Mushroom polysaccharides and polysaccharide-protein complexes --- p.5<br>Chapter 1.3.1 --- Polysaccharides important for antitumor effects --- p.5<br>Chapter 1.3.2 --- Polysaccharide-protein complexes important for antitumor effects --- p.7<br>Chapter 1.4 --- Structure-function relationship of antitumor activities of polysaccharides --- p.8<br>Chapter 1.4.1 --- Effect of molecular mass --- p.8<br>Chapter 1.4.2 --- Effect of linkages --- p.9<br>Chapter 1.4.3 --- Effect of degree of branching --- p.9<br>Chapter 1.4.4 --- Effect of conformation --- p.10<br>Chapter 1.5 --- Immunomodulatory effects of mushroom polysaccharides and polysaccharide-protein complexes --- p.11<br>Chapter 1.5.1 --- Immunomodulatory effects of polysaccharides --- p.11<br>Chapter 1.5.1.1 --- Bioactive polysaccharides in Lentinus edodes --- p.11<br>Chapter 1.5.1.2 --- Bioactive polysaccharides in Ganoderma lucidum --- p.12<br>Chapter 1.5.2 --- Immunomodulatory effects of polysaccharide-protein complexes --- p.12<br>Chapter 1.5.2.1 --- Bioactive polysaccharide-protein complexes in Trametes versicolor --- p.13<br>Chapter 1.5.3 --- Immunotherapeutic effects of mushroom polysaccharides --- p.14<br>Chapter 1.6 --- Cell cycle and apoptosis --- p.14<br>Chapter 1.6.1 --- Introduction of cell cycle --- p.14<br>Chapter 1.6.2 --- Cell cycle regulation --- p.15<br>Chapter 1.6.3 --- Antitumor effects through apoptotic gene regulation --- p.17<br>Chapter 1.7 --- Mushroom sclerotium with antitumor activity --- p.20<br>Chapter 1.7.1 --- Literature review on Pleurotus tuber-regium --- p.20<br>Chapter 1.7.2 --- Literature review on Poria cocos --- p.22<br>Chapter 1.7.3 --- Literature review on Polyporus rhinocerus --- p.23<br>Chapter 1.8 --- Objectives --- p.23<br>Chapter Chapter 2. --- Materials and Methods --- p.25<br>Chapter 2.1 --- Materials --- p.25<br>Chapter 2.1.1 --- Mushroom sclerotia --- p.25<br>Chapter 2.1.2 --- Animal Model --- p.25<br>Chapter 2.1.3 --- Cell lines --- p.27<br>Chapter 2.2 --- Methods --- p.28<br>Chapter 2.2.1 --- Extraction Scheme for mushroom sclerotia --- p.28<br>Chapter 2.2.1.1 --- Hot water extraction only --- p.28<br>Chapter 2.2.1.2 --- Sequential extraction scheme --- p.28<br>Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.31<br>Chapter 2.2.2.1 --- Acid Depolymerisation --- p.31<br>Chapter 2.2.2.2 --- Neutral Sugar Derivatization --- p.31<br>Chapter 2.2.2.3 --- Gas Chromatography (GC) --- p.32<br>Chapter 2.2.3 --- High Pressure Liquid Chromatography (HPLC) --- p.33<br>Chapter 2.2.3.1 --- Size exclusion chromatography --- p.33<br>Chapter 2.2.3.2 --- Anion exchange chromatography --- p.34<br>Chapter 2.2.4 --- Linkage analysis by methylation --- p.34<br>Chapter 2.2.4.1 --- Preparation of partially methylated polysaccharides --- p.34<br>Chapter 2.2.4.2 --- Preparation of partially methylated alditol acetates (PMAAs) --- p.37<br>Chapter 2.2.4.3 --- Gas chromatography-Mass spectrometry (GC-MS) analysis --- p.37<br>Chapter 2.2.5 --- Determination of total sugar by phenol-sulphuric acid Method --- p.38<br>Chapter 2.2.6 --- Determination of acidic sugars by measurement of uronic acid content --- p.39<br>Chapter 2.2.7 --- Determination of protein content by Lowry-Folin method --- p.40<br>Chapter 2.2.8 --- Chemical modification by carboxymethylation --- p.41<br>Chapter 2.2.9 --- In vitro antitumor assay --- p.41<br>Chapter 2.2.9.1 --- Trypan blue exclusion assay --- p.42<br>Chapter 2.2.9.2 --- MTT Assay --- p.42<br>Chapter 2.2.10 --- Cell cycle analysis by Flow Cytometry --- p.43<br>Chapter 2.2.11 --- In vivo antitumor and immunomodulatory assay --- p.44<br>Chapter 2.2.11.1 --- Measurement on tumor growth --- p.44<br>Chapter 2.2.11.2 --- Blood sampling for immunostimulatory effects --- p.45<br>Chapter 2.2.12 --- Mouse Cytokine Array --- p.45<br>Chapter 2.2.13 --- Quantification of Mouse IL-13 by ELISA --- p.46<br>Chapter 2.2.14 --- Enumeration of peritoneal cells --- p.47<br>Chapter 2.2.15 --- Enumeration of splenocytes --- p.49<br>Chapter 2.2.16 --- Statistical methods --- p.50<br>Chapter Chapter 3. --- Results and Discussion --- p.51<br>Chapter 3.1 --- Yield of crude mushroom sclerotial extracts --- p.51<br>Chapter 3.2 --- Chemical composition of crude mushroom sclerotial extracts --- p.57<br>Chapter 3.2.1 --- Total carbohydrate content --- p.57<br>Chapter 3.2.2 --- Uronic acid content --- p.58<br>Chapter 3.2.3 --- Soluble protein content --- p.58<br>Chapter 3.3 --- Monosaccharide profiles of mushroom sclerotial extracts by GC --- p.60<br>Chapter 3.4 --- Chromatographic analyses of mushroom sclerotial extracts --- p.65<br>Chapter 3.4.1 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.65<br>Chapter 3.4.2 --- Charge distribution by ion exchange chromatography (IEC) --- p.73<br>Chapter 3.5 --- Antitumor effects of mushroom sclerotial extracts from hot water extraction alone --- p.73<br>Chapter 3.5.1 --- In vitro antiproliferative study by HL-60 --- p.73<br>Chapter 3.5.2 --- In vitro antiproliferative study by MCF-7 --- p.74<br>Chapter 3.5.3 --- In vivo antitumor study by BALB/c mice --- p.75<br>Chapter 3.6 --- Antitumor effects of extracts from sequential extraction scheme --- p.76<br>Chapter 3.6.1 --- In vitro antiproliferative study by HL-60 --- p.76<br>Chapter 3.6.2 --- In vitro antiproliferative study by MCF-7 --- p.78<br>Chapter 3.6.3 --- In vivo antitumor study by BALB/c mice --- p.80<br>Chapter 3.7 --- Comparison of in vitro and in vivo activities of mushroom sclerotial extracts --- p.82<br>Chapter 3.8 --- Dose-response relationship of hot water extract from PR on cancer cell lines --- p.85<br>Chapter 3.8.1 --- In vitro dose-response antiproliferation of PR-W and PR-HWE on HL-60 --- p.85<br>Chapter 3.8.2 --- In vitro dose-response antiproliferation of PR-W on K562 and S180 --- p.88<br>Chapter 3.8.3 --- In vivo dose-response relationship of PR-W on S180 --- p.91<br>Chapter 3.9 --- Flow cytometric analysis of PR-W on cancer cell lines --- p.92<br>Chapter 3.9.1 --- Antiproliferative effect of PR-W on HL-60 --- p.92<br>Chapter 3.9.2 --- Antiproliferative effect of PR-W on K562 --- p.95<br>Chapter 3.9.3 --- Proposed mechanisms of cell cycle arrest by PR-W --- p.98<br>Chapter 3.10 --- Host-mediated antitumor mechanism of PR-W --- p.100<br>Chapter 3.10.1 --- Mouse cytokine array --- p.100<br>Chapter 3.10.2 --- Quantification of IL-13 by ELISA --- p.105<br>Chapter 3.10.3 --- Immunostimulatory effects of PR-W on mice --- p.109<br>Chapter 3.11 --- Correlation between antitumor activity and structure of mushroom sclerotial extract from hot water extraction alone --- p.114<br>Chapter Chapter 4. --- Conclusions and Future works --- p.118<br>List of References --- p.121<br>Related Publications --- p.142

碩士<br>大葉大學<br>生物資源學系碩士班<br>102<br>In Taiwan, mushroom cultivation is very prevalent, there is about 110,000 metric tons of fresh mushroom and have output value of more than 7.3 billion every year. However, it arising from sawdust waste about 230,000 tons after mushroom cultivation. These sawdust wastes still contain a plenty nutrient for mushroom, if arbitrarily discarded when without any treatment, will cause enormous influence on the environment. Therefore, the purpose of this experiment is based on the re-use of sawdust for the Flammulina velutipes and Pleurotus eryngii, extract from these two sawdusts of mushroom which can promote the mushroom mycelium growth and enhance fruit body production. In this study, the hot water extracts and ethanol extract of F. velutipes and P. eryngii sawdust waste, were added to the PDA plate and PDB medium, for the mycelium growth of Lentinula edodes F, Auricularia polytricha A1, Pleurotus sajor-caju K113 and Pleurotus eryngii q, and added to the sawdust medium to cultivate P. sajor-caju K113 and P. eryngii q. The results indicated that most of the extracts added to PDA medium, could promote the promotion of the mycelium growth, which the mycelial growth rate of the extracts of P-P1, AP-P1, PSC-P1 and WEFV-SSW for L. edodes F, A. polytricha A1, P. sajor-caju K113 and P. eryngii q were 1.09, 1.54, 1.29 and 1.26 times more than that of the control, respectively. In the liquid culture, the P-P1, AP-P1 and WEFV-SSW were added to PDB medium, respective, also could promote the mycelial growth, which the mycelial growth rate for L. edodes F, A. polytricha A1 and P. eryngii q were 2.14, 2.92 and 1.26 times more than that of the control. In the sawdust cultivation, the extract had no promotion effects on the mycelial growth or enhance the production of fruiting body. In addition, using of P. eryngii sawdust waste to cultivate P. sajor-caju K113 was showed the same result with the control. In future, utilization of the extracts from F. velutipes and P. eryngii sawdust wastes will have potential as mycelium growth-pormoting agent a potential in the mycelium growth of mushroom. According to the characteristics of the F. velutipe and P. eryngii sawdust wastes can be reuse as the culture medium for the cultivation of other mushrooms which can save the costs of mushroom cultivation and increase the convenience of use of substrate.

碩士<br>大仁科技大學<br>製藥科技研究所<br>95<br>Tyrosinase ( EC 1.14.18.1) is a copper-containing enzyme that is widespread in nature. It is responsible for not only melanization in animal but also browning in plants. In recent years, there is an increasing interest in finding natural tyrosinase inhibitors from plants and microorganisms. The tyrosinase inhibitors should have broad applications, especially in medicinal and cosmetics products in relation to hyperpigmentation. This research was focused on investigating the effects of red mold rice extracts on mushroom tyrosinase and melanoma cell for the antioxidant activity and inhibitory activity. Since tyrosinase is a rate-limiting enzyme in the melanogensis process, we first used rice through fermentation on the Monascus purpureus TJ-5, and the fermentated rice was then extracted by using various extraction media. In addition, the antioxidant activity and the inhibitory activity of the extracts on mushroom tyrosinase activity in the presence of L-dopa were determined.The result showed that three extracts all exhibited antioxidant activity and inhibitory activity on the activity of the mushroom tyrosinase. The highest antioxidant activity was found to be in the water extract. The inhibition activity of tyrosinase was found to be 12.23% when the kojic acid was at 1000 μg/mL. The tyrosinase inhibition from water extract, ethanol extract and ethyl acetate extract was found to be 78.53%, 63.59% and 81.33%, respectively when the red mold rice extracts was at 100 μg/mL. The optimum temperature of the inhibition activity of tyrosinase was found to be in the range between 45℃ and 55℃. We also found that the Cu2+ ion exhibited reductive ability on the red mold rice extracts. Furthermore, we also investigated the red mold rice extracts in mouse melanoma B16-F0 cell on tyrosinase activeness suppression. Results showed that red mold rice extracts did not have the suppression effect in the water extract. However, the red mold rice in the ethanol and ethyl acetate extracts did show very good suppression effect and the suppression effect was found to be dose dependent. In addition, We further determined the proliferation rate and apoptosis of red mold rice extracts in mouse melanoma B16-F0 cell by using tetrazolium salt (MTT), tyrpan blue exclusion, DNA fragment, morphological change and western blotting assays. It showed that the red mold rice in both ethanol and ethyl acetate extracts significantly inhibited the melanoma cell’s proliferation rate and their IC50 values were found to be 66.5 and 63.2 μg/mL, respectivly . Morphological changes were observed, cell revealed evident characteristics of apoptosis including cell blebbing and formation of apoptotic bodies. DNA displayed a characteristic inter-nucleosomal ladder of DNA fragment. Furthermore, in cell treated with red mold rice extracts for 30 hours, the level of Bax, caspase-9 and caspase 3 increased, comparing to the control group. Based on the results, we could make sure the red mold rice on all the extracts has highest antioxidant activity. However, the ethanol and ethyl acetate extracts showed better inhibitory activity on mushroom tyrosinase and induction of apoptosis in melanoma cells. Therefore, we expect the red mold rice extracts possess great potential on the development of the skin-whitening and melanoma-cytotoxic agents.

碩士<br>元智大學<br>生物科技與工程研究所<br>98<br>Tyrosinase is the main enzyme in the formation of melanin, currently, most researches use tyrosine inhibitors to inhibit the function of this enzyme so as to reduce the production of melanin to achieve whitening effect. Therefore, the whitening agents of skin care products available in the market mostly based on the mechanism of the inhibition of Tyrosinase activity in the formation process of melanin. Many effective ingredients in Ganoderma lucidum and other mushrooms species such as Antrodia cinnamomea, Agaricus blazei Murrill, Hericium erinaceus , Cordyceps militaris have been used in health foods for many years, and many studies have already confirmed that through a appropriate separation, the effective ingredient of Ganoderma Lucidum,Antrodia cinnamomea, Agaricus blazei Murrill, Hericium erinaceum , Cordyceps militaris can be purified. Natural products or herbal extracts adding to whitening maintenance products for skin care products has been the demands of the mainstream in recent years, many well-known cosmetic companies are also committed to the development and application of whitening natural materials. This research aims to study Ganoderma lucidum and other edible mushrooms using culture mycelium of biotechnology fermentation, through the use of 75% ethanol, 50% ethanol and water, to conduct the extraction and test the tyrosinase inhibition activity of the extracts as well as screen mushrooms with tyrosinase inhibition effect. Preliminary results showed that the aglycon of 75% ethanol extracts , 50% ethanol extratcts, and 5% H2O extracts of Ganoderma lucidum at the concentration of 1000 ppm were up to 78.2%, 74.4% and 79.0%, respectively; And the aglycon of 75% ethanol extracts , 50% ethanol extratcts, and 5% H2O extracts of Ganoderma lucidum at the concentration of 100ppm were up to 40.3%, 26.7% and 36.7%, respectively; Compared to the extracts of other edible mushrooms of antrodia cinnamomea, Agaricus blazei Murrill, Hericium erinaceum , Cordyceps militaris, Ganoderma lucidum extracts were with higher activity in inhibiting the function of tyrosinase, and with no cell toxicity to HS68 human fibroblast cells. Thus, the extracts of Ganoderma lucidum may be a natural raw material with whitening effect.

碩士<br>實踐大學<br>食品營養研究所<br>92<br>Mushrooms have been known as a medicine in traditional Chinese medicine. From previously studies, there are many result showed that the mushrooms extracts could enhanced immunity response both on in vivo and in vitro model. The aim of this study, we try to investigate whether long term oral administration Ganoderma lucidum, Antrodia camphorata, Shiitake mushroom, and Phellinus linteus can effect the immunity for young mice. Besides, we also would like to explore the inhibition of those mushrooms extract on hepatitis B virus (HBV) with HepES-2 cells, and do those extracts have antioxidant activity. In animal model, the 3 to 4 weeks old male BLAB/c mice were divided into 4 groups oral administered 4 kinds of mushrooms G. lucidum, A. camphorata, S. mushroom and P. linteus at different dose age, 0 mg /Kg bw /day, 20 mg /Kg bw /day, 100 mg /Kg bw /day, and 250 mg /Kg bw /day, respectively for 8 weeks. MTT assay was used for detection of the lymphocyte proliferation rate. The flow cytometry analysis was used to estimate T, B, and NK cell number. The ELISA assay was used to determine cytokine (IL-2, IL-4, IFN- γ) secretion. The result showed that long term oral administration G. lucidum in BLAB/c mice could increased T, NK cell numbers (by using flow cytometry), IL-2 and IL-4 secretion (by using ELISA) when compared with control group. The lymphocyte proliferation rate was no significant difference in MTT assay, but T, NK cell numbers (by using flow cytometry) and IFN-γsecretion (by using ELISA) had significant increase with long term oral administration of A. camphorata when compared with control group in BLAB/c mice . On the other hand, long term oral administration S. mushroom in BLAB/c mice, the lymphocyte proliferation rate had significant increase in MTT assay, T, B, NK cell numbers (by using flow cytometry), IL-2, IL-4 and IFN-γsecretion (by using ELISA) also had significant increase when compared with control group. The lymphocyte proliferation rate was significant increase in MTT assay, T, B cell numbers (by using flow cytometry), IL-2, IL-4 and IFN-γsecretion (by using ELISA) also had significant increase with long term oral administration of P. linteus when compared with control group in BLAB/c mice. In this study, we conclude that long term oral administration G. lucidum and A. camphorata might enhanced T and NK cell activation, proliferation after antigen stimulation, and long term oral administration S. mushroom and P. linteus might enhanced T、B and NK cell activation, proliferation after antigen stimulation. Thus, long term oral administration mushrooms in mice could enhance the immunity of health host when they exposed in antigen. HepES-2 cells, which derived from HepG2 cells, transfected with HBV genome, and HepES-2 cells has been examined with constant HBeAg and HBsAg expression. The hot water extract of those mushrooms were used to treat with HepES-2 cells, and MTT assay was used to detect the cytotoxcity of those mushroom extracts. HepES-2 cells were plated at a density of 5×104 cells with suitable concentration of mushrooms extract, which caused less than 15 % HepES-2 cells death. The secretion of HBeAg and HBsAg from HepES-2 cells were determined via ELISA assay. DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay was used for evaluation of antioxidant activity of those mushroom extracts. The result showed that G. lucidum, A. camphorata, and S. mushroom extracts had significant cytotoxcity for HepES-2 cells start from the concentration of 125μg/ml treatment. Compared with those in sense control, all mushrooms have significant decrease secretion of HBeAg from HepES-2 cells, but only G. lucidum and S. mushroom have significant decrease secretion of HBsAg from HepES-2 cells. Besides, those extract of mushrooms scavenged the stable free radical DPPH resulting in a increase of does-dependently (250 ~ 2000μg/ml). Thus G. lucidum and S. mushroom extract possesses a significant anti-HBV activity and perhaps can develop into adjuvant or therapeutic drug of HBV infection patients in the future.

碩士<br>國立臺灣大學<br>食品科技研究所<br>95<br>Flammulina velutipes, a popular edible mushroom contain many biological active substances including immuno-modulating β-D-glucans, fungal immuno-modulating proteins (FIP) and polyphenol oxidase (PPO) inhibitor. The molecular-weight distribution of the glucans, FIP and the PPO inhibitor are following the order from large to small. In this study we successfully used hollow-fiber ultra-filtration technique to enrich these three substances into fractions in succession. The sequence of molecular-weight cut-off (MWCO) values of ultra-filtration membrane was 100, 10 and 3kDa following the separation order. The results indicated that retentate of 100kDa MWCO was mainly β-D-glucans, proteins were dispersed in 10-100kDa MWCO range and the polyphenol oxidase inhibitor was in fraction of molecular weight smaller than 3kDa. To understand the efficiency of each separation steps, we used ethanol precipitate fractionation to collect (1, 3)-β-D-glucans as control. The hot-water extracts containing 6.8% of (1, 3)- β-D- glucans was used in this study. The results indicated that (1, 3)-β-D-glucans recovery of using 40% ethanol precipitation and 76% ethanol precipitation were 85.0% and 95.1%, respectively. The ultra-filtration separation using 10kDa MWCO could recover 28.7% of solid and 95.1% (1, 3)-β-D-glucans in hot-water extracts, respectively. A method combining freeze-thaw cycle has been developed to recover (1, 3)-β-D-glucans which is large in molecular weight and having highly tendency to aggregate. The combination method could recover 3.8% of solid and 17.0% of (1, 3)-β-D-glucans in hot-water extracts, respectively. The content of (1, 3)-β-D- glucans was enriched 4.5 folds. The polyphenol oxidase inhibitors were concentrated in the fraction which permeated the MWCO of 3kDa membrane. The fraction could significant inhibited enzymatic browning of apple puree. The effective concentration was 12.5 mg/mL comparing to the effective concentration 18.75mg/mL for hot-water extract.

Ng Yuk Fan.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 145-162).<br>Abstracts in English and Chinese.<br>Thesis Committee: --- p.i<br>Acknowledgements --- p.ii<br>Abstract --- p.iii<br>摘要 --- p.v<br>Content --- p.vii<br>List of Tables --- p.xiii<br>List of Figures --- p.xvi<br>Abbreviations --- p.xviii<br>Chapter Chapter 1: --- Introduction --- p.1<br>Chapter 1.1 --- Antioxidants --- p.1<br>Chapter 1.1.1 --- Definition and mode of actions of antioxidants --- p.1<br>Chapter 1.1.2 --- Synthetic antioxidants --- p.2<br>Chapter 1.1.3 --- Natural antioxidants --- p.3<br>Chapter 1.2 --- Changes of antioxidant activity in food processing --- p.4<br>Chapter 1.2.1 --- Blanching --- p.4<br>Chapter 1.2.2 --- Drying --- p.5<br>Chapter 1.2.3 --- Microwave and Infrared energy --- p.7<br>Chapter 1.2.4 --- Freezing --- p.8<br>Chapter 1.3 --- Lipid oxidation and antioxidant --- p.8<br>Chapter 1.3.1 --- Free radicals --- p.8<br>Chapter 1.3.1.1 --- Superoxide --- p.10<br>Chapter 1.3.1.2 --- Hydrogen peroxide --- p.11<br>Chapter 1.3.1.3 --- Hydroxyl radical --- p.13<br>Chapter 1.3.2 --- Mechanism of lipid oxidation --- p.14<br>Chapter 1.3.3 --- Oxidation of low-density-liporoproteins (LDLs) and coronary heart disease --- p.15<br>Chapter 1.3.4 --- Role of antioxidants in inhibiting lipid oxidation --- p.16<br>Chapter 1.4 --- Hypocholesterolemic and antioxidant activity of phenolics --- p.19<br>Chapter 1.5 --- Medicinal properties of mushrooms --- p.21<br>Chapter 1.5.1 --- Background information of mushrooms --- p.21<br>Chapter 1.5.2 --- Phenolics in mushrooms --- p.22<br>Chapter 1.5.3 --- Hypocholesterolemic effect in mushroom --- p.23<br>Chapter 1.5.4 --- Previous studies in Agrocybe aegerita --- p.25<br>Chapter 1.6 --- Animal model for hypocholesteroliemic study --- p.27<br>Chapter 1.6.1 --- General requirements --- p.27<br>Chapter 1.6.2 --- Hamster model --- p.27<br>Chapter 1.7 --- Principles of assays that involved in antioxidant activity --- p.30<br>Chapter 1.7.1 --- ABTS + radical cation scavenging activity --- p.30<br>Chapter 1.7.2 --- Beta carotene bleaching method --- p.31<br>Chapter 1.7.3 --- Ferric reducing antioxidant power (FRAP) --- p.31<br>Chapter 1.7.4 --- Scavenging activity of hydroxyl radical --- p.32<br>Chapter 1.7.5 --- Inhibition of low-density lipoproteins (LDLs) oxidation --- p.33<br>Chapter 1.7.6 --- Total phenolic content determination --- p.33<br>Chapter 1.8 --- Principles of assays in hypocholesterolemic study --- p.34<br>Chapter 1.8.1 --- HDL-Cholesterol determination --- p.34<br>Chapter 1.8.2 --- Total cholesterol determination --- p.34<br>Chapter 1.8.3 --- Determination of plasma total triglyceride --- p.35<br>Chapter 1.9 --- Objectives --- p.36<br>Chapter Chapter 2: --- Materials and Methods --- p.37<br>Chapter 2.1 --- Sample preparation --- p.37<br>Chapter 2.2 --- Proximate Analysis of FAa and DAa --- p.38<br>Chapter 2.2.1 --- Determination of crude protein --- p.38<br>Chapter 2.2.2 --- Determination of ash --- p.39<br>Chapter 2.2.3 --- Total dietary fiber --- p.39<br>Chapter 2.2.4 --- Determination of fat --- p.41<br>Chapter 2.2.5 --- Moisture content --- p.42<br>Chapter 2.3 --- Sample extraction --- p.42<br>Chapter 2.3.1 --- Small-scale extraction --- p.42<br>Chapter 2.3.2 --- Large-scale extraction --- p.43<br>Chapter 2.4 --- Total phenolic content of DAa and FAa extract --- p.44<br>Chapter 2.5 --- Chemical assays for in vitro antioxidative properties determination --- p.45<br>Chapter 2.5.1 --- Hydroxyl free radical scavenging activity --- p.45<br>Chapter 2.5.2 --- Beta-carotene bleaching method --- p.46<br>Chapter 2.5.3 --- Inhibition of human low-density-lipoproteins (LDLs) oxidation --- p.47<br>Chapter 2.5.4 --- Scavenging activity of ABTS+radical cation --- p.50<br>Chapter 2.6 --- In vivo tests for antioxidative and hypocholesterolemic effect of DAa --- p.51<br>Chapter 2.6.1 --- Feeding experiments --- p.51<br>Chapter 2.6.2 --- Collection of plasma --- p.52<br>Chapter 2.6.3 --- Liver sample preparation --- p.52<br>Chapter 2.6.4 --- Determination of in vivo antioxidative effect --- p.54<br>Chapter 2.6.4.1 --- FRPA assay --- p.54<br>Chapter 2.6.4.2 --- ABTS + radical cation scavenging activity --- p.55<br>Chapter 2.6.5 --- Determination of plasma lipid profiles --- p.55<br>Chapter 2.6.5.1 --- Plasma total cholesterol (TC) --- p.55<br>Chapter 2.6.5.2 --- Plasma total triglyceride (TG) --- p.56<br>Chapter 2.6.5.3 --- Plasma high density lipoprotein cholesterol (HDL-C) determination --- p.57<br>Chapter 2.6.5.4 --- Hepatic cholesterol determination by gas chromatography analysis --- p.57<br>Chapter 2.7 --- Statistical analysis --- p.59<br>Chapter Chapter 3: --- Results and discussion --- p.61<br>Chapter 3.1 --- Proximate analysis --- p.61<br>Chapter 3.2 --- Small-scale extraction scheme --- p.63<br>Chapter 3.2.1 --- Extraction yield --- p.63<br>Chapter 3.2.2 --- Antioxidant assays --- p.65<br>Chapter 3.2.2.1 --- Hydroxyl free radical scavenging activity --- p.65<br>Chapter 3.2.2.2 --- Beta-carotene bleaching method --- p.68<br>Chapter 3.2.2.3 --- The formation of TBARS in human LDL oxidation --- p.75<br>Chapter 3.2.2.4 --- Total phenolic content (TPC) in DAa and FAa ethanolic and water extracts --- p.81<br>Chapter 3.2.2.5 --- Correlation between total phenolic content and antioxidant activity of mushroom extracts --- p.84<br>Chapter 3.2.2.6 --- Comparison of antioxidant activity and TPC in DAa and FAa ethanolic and water extracts in the small-scale extraction scheme --- p.88<br>Chapter 3.3 --- Large-scale extraction scheme --- p.91<br>Chapter 3.3.1 --- Extraction yield --- p.91<br>Chapter 3.3.2 --- Antioxidant assays --- p.91<br>Chapter 3.3.2.1 --- Hydroxyl free radical scavenging activity --- p.91<br>Chapter 3.3.2.2 --- Beta-carotene bleaching method --- p.94<br>Chapter 3.3.2.3 --- ABTS + radical cation scavenging activity --- p.96<br>Chapter 3.3.2.4 --- Formation of TBARS in human LDL oxidation in the DAa_E_l and Daa_W_1 --- p.97<br>Chapter 3.3.2.5 --- Total phenolic content (TPC) of DAa_E_l and DAa_W_l --- p.97<br>Chapter 3.3.2.6 --- Correlation between total phenolic content and antioxidant activity --- p.101<br>Chapter 3.3.2.7 --- Summary of large-scale extraction scheme --- p.103<br>Chapter 3.4 --- In vivo antioxidant activity and hypocholesterolemic effect of DAa studied by animal model --- p.104<br>Chapter 3.4.1 --- Effect of DAa´ؤE_1 and DAa_W_l on body weight and food intake --- p.105<br>Chapter 3.4.2 --- Effect of DAa一E´ؤ1 and DAa_W_l on plasma total cholesterol (TC) in hamsters --- p.108<br>Chapter 3.4.3 --- Effect of DAa´ؤE_1 and DAa W l on plasma total triglycerides (TG) in hamsters --- p.114<br>Chapter 3.4.4 --- Effect of DAa_E_l and DAa_W_l on plasma high-density-lipoprotein cholesterol (HDL-C) in hamsters --- p.119<br>Chapter 3.4.5 --- Effect of DAa_E_l and DAa一W_1 on hepatic cholesterol (HC) profile in hamsters --- p.124<br>Chapter 3.4.6 --- Effect of DAa_E_l and DAa W l on ferric reducing antioxidant power (FRAP) in hamsters (FRAP) --- p.128<br>Chapter 3.4.7 --- Effect of DAa_E_l and DAa_W_l on ABTS + cation radical scavenging activity --- p.131<br>Chapter 3.4.8 --- The antioxidant activity and hypocholesterolemic effect of DAa extracts --- p.134<br>Chapter 3.4.9 --- Summary of in vivo antioxidant activity and hypocholesterolemic effect of DAa studied by animal model --- p.140<br>Chapter Chapter 4: --- Conclusions --- p.142<br>References --- p.145

碩士<br>國立中興大學<br>食品暨應用生物科技學系所<br>106<br>As the population ages, the proportion of people suffering from osteoporosis will continue to increase, especially among postmenopausal women. Studies have pointed out that Antrodia camphorata fruit body can slow down the occurrence of osteoporosis. Antrodia salmonea and Antrodia camphorata belong to the same genus, and their physiologically active components are similar, but no study has been conducted to investigate the relationship between them and osteoporosis. In this study, the ovariectomized mice were used as an in vivo test to evaluate the effects of 70% ethanol extracts of Antrodia camphorata and Antrodia salmonea on postmenopausal osteoporosis. The bone parameters of the femur were determined by micro-computed tomography (micro-CT). Ovariectomized mice had significantly higher body weight and histopathological alterations of the liver were found to have diffuse fatty infiltration vesicles due to lack of estrogen, resulting in endocrine disorders and body fat accumulation. In addition, the relative weight of the uterus is significantly lower and atrophy of the uterine glands was found in histopathological alterations, which is also related to the lack of estrogen. In terms of bone quality assessment, the results of trabecular bone parameters showed that feeding high-dose of Antrodia camphorata mycelia ethanol extract to ovariectomized mice had the ability to delay bone loss. The bone density of trabecular bone and cortical bone were also significantly higher than those of ovariectomized mice, indicating that the ethanol extract of Antrodia camphorata has the potential to slow down the occurrence of osteoporosis. Studies have shown that probiotics have the ability of regulating intestinal flora, improving lactose intolerance, and obesity. Probiotics can promote the growth of probiotics in the intestine. The inflammatory response is the host''s immune defense against pathogens, but excessive secretion of pro-inflammatory cytokines can cause damage to tissues and organs. The mushroom’s carbohydrates which can not be digested by the intestinal can promote the growth of lactic acid bacteria in the intestine, with the potential to develop as probiotics. Studies have found that a variety of medicinal mushroom polysaccharides have the ability to regulate immunity, reduce pro-inflammatory cytokine production, and achieve anti-inflammatory properties. Therefore, this study first fermented four medicinal mushroom polysaccharides with Lactobacillus plantarum to investigate whether the mushroom polysaccharides has the potential as a probiotics and the fermentation broth was administered to mice-macrophages to investigate the ability of the fermentation broth to anti-inflammation. The experimental results showed that the growth of Lactobacillus plantarum was stable after 12 hours culture, and the amount of Lactobacillus plantarum was significantly higher with the addition of 1% mushroom polysaccharide. Although the survival rate of Antrodia salmonea polysaccharides group was significantly decreased in acid tolerance test, it still had 94.47%. The survival rate of the other groups did not get down significantly during the culture. The bile tolerance test showed that the survival rate of mushroom polysaccharide group did not decrease as the culture time increased. It was shown that adding mushroom polysaccharides in Lactobacillus plantarum culture medium had good effects of acid tolerance and bile tolerance. In the inflammatory response, the fermentation broth without sugar or adding mushroom polysaccharides and fructooligosaccharides have different effects on the pro-inflammatory cytokines secretion, but they could promote the secretion of anti-inflammatory cytokines. In the ratio of pro-inflammatory cytokines/anti-inflammatory cytokines, the ratios of all the experimental groups were significantly reduced, showing that the co-culture of inflamed macrophage cells with fermentation broth tends to have the good ability of immune regulation. The cells were prone to anti-inflammatory reactions and had immune regulation ability.

by Li Chi Yeung.<br>Thesis submitted in: November 2004.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.<br>Includes bibliographical references (leaves 151-176).<br>Abstracts in English and Chinese.<br>Thesis Committee --- p.i<br>Acknowledgement --- p.ii<br>Abstract (English Version) --- p.iii<br>Abstract (Chinese Version) --- p.v<br>Content Page --- p.vii<br>List of Tables --- p.xiii<br>List of Figures --- p.xv<br>Abbreviation --- p.xvii<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Diabetes Mellitus --- p.1<br>Chapter 1.1.1 --- Epidemiology --- p.1<br>Chapter 1.1.2 --- Economic Impact --- p.3<br>Chapter 1.2 --- "Digestion, Absorption and Metabolism of Carbohydrates" --- p.4<br>Chapter 1.2.1 --- Carbohydrate Digestion --- p.4<br>Chapter 1.2.2 --- Carbohydrate Absorption --- p.6<br>Chapter 1.2.3 --- Insulin Secretion --- p.6<br>Chapter 1.3 --- Pathophysiology of Diabetes Mellitus --- p.7<br>Chapter 1.3.1 --- Insulin-Dependent Diabetes Mellitus (lDDM) --- p.7<br>Chapter 1.3.1.1 --- Genetics --- p.8<br>Chapter 1.3.1.2 --- Autoimmunity --- p.9<br>Chapter 1.3.2 --- Non-Insulin-Dependent Diabetes Mellitus (NlDDM) --- p.11<br>Chapter 1.3.2.1 --- Insulin Resistance --- p.11<br>Chapter 1.3.2.2 --- Impaired Insulin Secretion --- p.14<br>Chapter 1.4 --- Management of Diabetes Mellitus --- p.15<br>Chapter 1.4.1 --- Sulfonylureas --- p.15<br>Chapter 1.4.2 --- Biguanides --- p.16<br>Chapter 1.4.3 --- Problems Encountered in the Management of Diabetes --- p.16<br>Chapter 1.4.4 --- Role of Dietary Fiber in the Management of Diabetes Mellitus --- p.18<br>Chapter 1.4.4.1 --- Dietary Fiber and Gastric Emptying Time --- p.19<br>Chapter 1.4.4.2 --- Dietary Fiber and Glucose Absorption in Small Intestine --- p.20<br>Chapter 1.4.5 --- Other Natural Products used for Diabetes Treatment…… --- p.22<br>Chapter 1.5 --- Mushrooms --- p.22<br>Chapter 1.5.1 --- The Definition of Mushrooms --- p.23<br>Chapter 1.5.2 --- Nutritional Values of Mushrooms --- p.24<br>Chapter 1.5.3 --- Production of Mushrooms --- p.25<br>Chapter 1.6 --- Medicinal (Antidiabetic) Properties of Mushrooms --- p.28<br>Chapter 1.6.1 --- Ganoderma lucidum --- p.29<br>Chapter 1.6.2 --- Tremella aurantia --- p.33<br>Chapter 1.6.3 --- Auricularia auricula --- p.36<br>Chapter 1.6.4 --- Grifola frondosa --- p.37<br>Chapter 1.7 --- Medicinal Uses of Hericium erinaceus --- p.39<br>Chapter 1.7.1 --- HeLa Cell Proliferation Inhibitors --- p.39<br>Chapter 1.7.2 --- Induction of Growth of Nerve Cells --- p.42<br>Chapter 1.7.3 --- Antitumour Activity --- p.42<br>Chapter 1.7.4 --- Antidiabetic Effect --- p.43<br>Chapter 1.8 --- Objectives --- p.45<br>Chapter Chapter 2 --- Materials and Methods --- p.46<br>Chapter 2.1 --- Extraction of Polysaccharides from the Fruiting Body of H. erinaceus --- p.46<br>Chapter 2.1.1 --- Small-scale Extraction --- p.46<br>Chapter 2.1.2 --- Large-scale Extraction --- p.47<br>Chapter 2.2 --- Physico-Chemical Characterization of HE-polysaccharides --- p.52<br>Chapter 2.2.1 --- Carbohydrate Content: Phenol-Sulfuric Acid Method --- p.52<br>Chapter 2.2.2 --- Protein Content: Lowry Assay --- p.52<br>Chapter 2.2.3 --- Uronic Acid Content --- p.53<br>Chapter 2.2.4 --- Molecular Weight Determination by High Pressure Liquid Chromatography (HPLC) --- p.55<br>Chapter 2.2.5 --- Determination of Monosaccharide Composition of Non-Starch Polysaccharides by Gas Chromatography (GC) --- p.56<br>Chapter 2.2.5.1 --- Acid Depolymerisation --- p.56<br>Chapter 2.2.5.2 --- Neutral Sugar Derivatisation --- p.56<br>Chapter 2.2.5.3 --- Determination of Neutral Sugar Composition by Gas Chromatography (GC) --- p.57<br>Chapter 2.2.6 --- Structural Study of Polysaccharides by Methylation --- p.59<br>Chapter 2.2.6.1 --- Preparation of dry Dimethyl Sulfoxide (DMSO) --- p.59<br>Chapter 2.2.6.2 --- Preparation of Methylsulfinyl Methyl Sodium (CH3SOCH2-Na+) from the dry DMSO and Sodium Hydride --- p.59<br>Chapter 2.2.6.3 --- Methylation Procedure --- p.60<br>Chapter 2.2.6.4 --- Preparation of Partially Methylated Alditol Acetates (PMAAs) --- p.61<br>Chapter 2.2.6.5 --- Analysis of the PMAAs by GC --- p.62<br>Chapter 2.2.7 --- The Measurement of Viscosity --- p.62<br>Chapter 2.3 --- In vitro Hypoglycemic Tests of HE-Polysaccharides --- p.64<br>Chapter 2.3.1 --- Glucose Dialysis Retardation Index (GDRl) --- p.64<br>Chapter 2.3.1.1 --- Experimental Setup --- p.64<br>Chapter 2.3.1.2 --- Measurement of Glucose in the Dialysate --- p.65<br>Chapter 2.3.2 --- Inhibition of Amylolysis --- p.66<br>Chapter 2.3.2.1 --- Experimental Setup --- p.66<br>Chapter 2.3.2.2 --- Measurement of Maltose in the Dialysate --- p.66<br>Chapter 2.4 --- In vivo Hypoglycemic Evaluation of HE-Polysaccharides --- p.67<br>Chapter 2.4.1 --- Oral Glucose Tolerance Test (OGTT) --- p.67<br>Chapter 2.4.2 --- Induction of Type l Diabetes in Normal BALB/c Mice --- p.69<br>Chapter 2.4.2.1 --- lnduction Protocol --- p.69<br>Chapter 2.4.2.2 --- Measurement of Plasma Glucose Level --- p.70<br>Chapter 2.4.3 --- Hypoglycemic Test on Normal and Diabetic BALB/c Mice --- p.71<br>Chapter 2.4.4 --- Measurement of Insulin Level by Enzyme-Linked Immunoadsorbent Assay (ELlSA) --- p.72<br>Chapter 2.4.4.1 --- Plasma Samples used in ELlSA --- p.72<br>Chapter 2.4.4.2 --- Assay Procedure --- p.73<br>Chapter 2.5 --- Statistical Evaluation --- p.74<br>Chapter Chapter 3 --- Results and Discussion --- p.75<br>Chapter 3.1 --- Yield of Polysaccharides extracted from H. erinaceus --- p.75<br>Chapter 3.2 --- Physico-chemical Properties of HE Polysaccharides --- p.79<br>Chapter 3.2.1 --- "Carbohydrate, Protein and Uronic Acid Content" --- p.79<br>Chapter 3.2.2 --- Monosaccharide Compositions --- p.83<br>Chapter 3.2.3 --- Molecular Weight of the HE polysaccharides --- p.85<br>Chapter 3.2.4 --- Structure of HE polysaccharides --- p.90<br>Chapter 3.2.5 --- Conclusion for the Physico-chemical Properties of HE-Polysaccharides --- p.96<br>Chapter 3.2.6 --- Viscosity of HE Polysaccharides --- p.99<br>Chapter 3.3 --- In vitro Study of the Hypoglycemic Effect of HE-Polysaccharides --- p.101<br>Chapter 3.3.1 --- Glucose Dialysis Retardation Index (GDRl) --- p.101<br>Chapter 3.3.2 --- Inhibition of α-Amylase Activity --- p.105<br>Chapter 3.4 --- In vivo Hypoglycemic Evaluation of HE-Polysaccharides --- p.109<br>Chapter 3.4.1 --- In vivo Oral Glucose Tolerance Test (OGTT) in Normal Mice --- p.109<br>Chapter 3.4.1.1 --- Oral Glucose Tolerance Test --- p.109<br>Chapter 3.4.1.2 --- Effect of Change of Viscosity of HE Polysaccharide in the Gl Tract of Mice --- p.114<br>Chapter 3.4.2 --- Establishment of a Diabetic Murine Model --- p.120<br>Chapter 3.4.3 --- Hypoglycemic Activity of HE-polysaccharides in Normal Mice --- p.123<br>Chapter 3.4.4 --- Hypoglycemic Activity of HE-polysaccharides in Diabetic Mice --- p.126<br>Chapter 3.4.5 --- Change of Plasma Insulin Level in the Hypoglycemic Test --- p.132<br>Chapter 3.4.6 --- Comparison of Hypoglycemic Activity of HE-Polysaccharides in Normal and Diabetic mice --- p.139<br>Chapter 3.4.6.1 --- Severity of Diabetic Conditions lnduced --- p.139<br>Chapter 3.4.6.2 --- Change in Insulin Secretion --- p.140<br>Chapter 3.4.6.3 --- Glucose Transporter --- p.140<br>Chapter 3.5 --- Other Factors that Affect in vivo Hypoglycemic Activity of the HE-polysaccharides --- p.141<br>Chapter 3.5.1 --- Route of Administration: Oral Feeding and Intraperitoneal Injection --- p.141<br>Chapter 3.5.2 --- Molecular Mechanisms of Hypoglycemic Activity --- p.142<br>Chapter 3.5.3 --- Glucose Toxicity --- p.144<br>Chapter 3.5.3.1 --- Insulin Resistance --- p.144<br>Chapter 3.5.3.2 --- Impaired Insulin Secretion --- p.145<br>Chapter Chapter 4 --- Conclusions and Future Works --- p.147<br>References --- p.151

Mestrado em cooperação com a Universidade de Salamanca<br>In the present study, the ethanolic extracts of fourteen wild edible mushrooms were investigated for their anti-inflammatory potential in LPS (Lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified parent molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their synthesised glucuronated and methylated derivatives obtained by chemical synthesis were evaluated for the same bioactivity, in order to establish a structure-activity relationship, and to understand the contribution of the compounds to the activity of the extracts. The extract Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential, presenting also the highest concentration in cinnamic acid, which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, especially the compound CoA-M1, that exhibited a very similar activity to the one showed by dexamethasone used as anti-inflammatory standard; by contrast, the p-hydroxybenzoic derivatives revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.