Rozprawy doktorskie na temat „POLYSACCHARIDES MUSHROOM”

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Mushrooms have been valued as a functional food and dietary supplement for humanity, due to the presence of substances with high nutritional value and/or therapeutic. Among the mushrooms recognized for its therapeutic properties we have is the leafy Grifola, popularly known as “Maitake”, which has important pharmacological actions such as anti-tumor, immunomodulatory, antiinflammatory, antioxidant, and others, which have been mainly related to its carbohydrates. Nevertheless, this study aimed to review the chemistry of the polysaccharides from mushroom of the two lines ("white" and "gray", represented as Gfb and Gfc, respectively) of G. frondosa leafy that have been cultivated in Brazil. From these type it was obtained branched -glucans containing links of type (13) (16) and heteropolysaccharides formed mainly by galactose (heterogalactan) or mannose (heteromannan). The heterogalactan composed mainly of fucose (15.7%), mannose (20.5%) and galactose (63.8%) was denominated fucomannogalactan (FMG-Gfb). It contains a main chain consisting of units of α-D-Galp and 3-Me-O-α-D-Galp connected (16), which are present partially substituted by O-2 disaccharide 3-O- α-D-Manp-α-L-Fucp to a lesser extent with terminal non-reducing α-L-Fucp and α-D-Manp. The heteromannan composed of fucose (6.9%), xylose (33.1%) and mannose (60.0%), a fucoxylomannan (FXM-Gfc) was found to be composed of a backbone of units α-D-Manp (13) connected, and a part of them substituted by O-3 side chains made of fucose and xylose. The results of this study suggest that there are no significant differences between the carbohydrate of both strains, showing that white strain it provide the therapeutic effects attributed to carbohydrates of another strain.
Os cogumelos têm sido valorizados como alimento funcional e suplemento alimentar para a humanidade, devido à presença de substâncias com alto valor nutricional e/ou medicinal. Dentre os cogumelos reconhecidos pelas suas propriedades terapêuticas encontra-se o Grifola frondosa, popularmente conhecido como Maitake, o qual possui importantes ações farmacológicas como antitumoral, imunomoduladora, anti-inflamatória, antioxidante, entre outras, as quais têm sido relacionadas, principalmente, aos seus carboidratos. Diante do exposto, o presente trabalho teve como objetivo a avaliação química dos polissacarídeos dos basidiomas das duas linhagens (branca e cinza, representadas como Gfb e Gfc, respectivamente) de G. frondosa que vem sendo cultivadas no Brasil. A partir destes foram obtidas as -glucanas ramificadas contendo ligações do tipo (13) (16) e os heteropolissacarídeos formados principalmente por galactose (heterogalactana) ou manose (heteromanana). A heterogalactana constituída, principalmente, por fucose (15,7%), manose (20,5%) e galactose (63,8%), foi denominada de fucomanogalactana (FMG-Gfb). Esta contém uma cadeia principal formada por unidades de α-D-Galp e de 3-O-Me-α-D-Galp ligadas (16), as quais se apresentam parcialmente substituídas em O-2 pelo dissacarídeo 3-O-α-DManp- α-L-Fucp e em menor proporção com terminais não redutores de α-LFucp e α-D-Manp. A heteromanana, composta por fucose (6,9%), xilose (33,1%) e manose (60,0%), ou seja, uma fucoxilomanana (FXM-Gfc) mostrou ser constituída por uma cadeia principal composta por unidades de α-D-Manp (13) ligadas, sendo uma parte destas substituídas em O-3 por cadeias laterais formadas por fucose e xilose. Os resultados obtidos neste estudo sugerem que não há diferenças significativas entre os carboidratos de ambas as linhagens, evidenciando que a linhagem branca deve apresentar os efeitos terapêuticos atribuídos aos carboidratos da outra linhagem.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Polymers of mushroom cellular wall are recognized for presenting a lot of biological activities such as anti-inflammatory, antioxidant and anti-tumoral action. Polysaccharides from mushrooms of different molecular mass obtained mushrooms can activate leucocytes, stimulate fagocitic, citotoxic and antimicrobial activity including oxygen reactive species production. In this study were investigated chemical characteristics, in vitro antioxidant activity and anti-inflammatory action in an acute inflammation model of the polysaccharides extracted from Tylopilus ballouii. Results showed that were mainly extracted polysaccharides and that it primarily consisted of mannose and galactose with variable amounts of xylose and fucose. Infrared analysis showed a possible interation between this polysaccharides and proteins. In addition, molecular mass was about 140KDa. Antioxidant activity was tested by superoxide and hydroxyl radical scavenging assay, total antioxidant activity and lipid peroxidation assay. For superoxide and hydroxyl radical generation inhibition, polysaccharides have an IC50 of 2.36 and 0.36 mg/mL, respectively. Lipid peroxidation assay results showed that polysaccharides from Tylopilus ballouii present an IC50 of 3.42 mg/mL. Futhermore, anti-inflammatory assay showed that polysaccharides cause an paw edema decreasing in 32.8, 42 and 56% in 30, 50 and 70 mg/Kg dose, respectively. Thus, these results can indicate a possible use for these polysaccharides from Tylopilus ballouii as an anti-inflammatory and antioxidant.
Pol?meros da parede celular de fungos s?o conhecidos por possu?rem muitas atividades biol?gicas como suas a??es antiinflamat?rias, antioxidante e antitumoral. Polissacar?deos de diferentes pesos moleculares obtidos de cogumelos podem ativar os leuc?citos, estimular a atividade fagoc?tica, citot?xica e antimicrobiana, incluindo a produ??o de esp?cies reativas de oxig?nio. No presente estudo, foi investigada a caracter?stica qu?mica dos polissacar?deos extra?dos de Tylopilus ballouii sua atividade antioxidante in vitro e a sua atividade antiinflamat?ria no modelo de inflama??o aguda. Os resultados mostraram que foram extra?dos predominantemente polissacar?deos e esses consistiram primariamente de manose e galactose e possui quantidades vari?veis de xilose e fucose. As an?lises de infravermelho mostraram a poss?vel intera??o entre estes polissacar?deos e prote?nas. Al?m disso, seu peso molecular ? de cerca de 140 kDa. A atividade antioxidante foi testada com rela??o ao seq?estro sobre os radicais super?xido e hidroxila, atividade antioxidante total e peroxida??o lip?dica. Com rela??o ? inibi??o da forma??o dos radicais super?xido e hidroxila, os polissacar?deos atingiram um IC50 de 1,64 e 1,25 mg/ml, respectivamente. Os resultados do ensaio de peroxida??o lip?dica mostraram que os polissacar?deos de Tylopilus ballouii apresentam um IC50 de 1,65 mg/ml. Al?m disso, a atividade antiinflamat?ria mostrou que eles agem reduzindo o edema em 32,8, 42 e 56% nas doses de 30, 50 e 70 mg/kg, respectivamente. Assim, estes resultados podem indicar o poss?vel uso dos polissacar?deos de Tylopilus ballouii como antiinflamat?rio e antioxidante.

Medicinal mushrooms are therapeutic agents in traditional folk medicines. Previous studies have shown that a number of biologically active compounds in medicinal mushrooms contributed therapeutic functions against many diseases. These compounds include mainly large molecular weight (MW) compounds such as polysaccharides, dietary fibre and lipids. Mushroom polysaccharides have attracted great attention in food and pharmacology fields because of their biological activities. Polysaccharides vary in molecular weight, degree of branching and conformational structure. It has been reported that fine structure, molecular weight, and conformation of polysaccharide influence biological activities. The incidence and prevalence of inflammatory bowel disease (IBD) have been increasing worldwide, which is characterized by chronic inflammation of the gastrointestinal tract but without satisfactory treatment. Although there are many studies for the immuno-pharmacological activity of mushroom polysaccharides, their intestinal anti-inflammatory property has not been investigated sufficiently. Therefore, it is very important to elucidate whether there is the relationship among the MW, structure and anti-inflammatory activity of polysaccharide in IBD. Firstly, an exopolysaccharide from a mycelial culture of S. commune was obtained by isolation and purification using DEAE-52 cellulose and Sephadex G-150 column chromatography. The structure, conformation and chemical properties were investigated, including elemental compositions, MW, monosaccharide compositions, fourier transform infrared spectrum, thermogram analysis, nuclear magnetic resonance (NMR) spectrum, circular dichroism (CD) study, methylation analysis, and scanning electron microscope (SEM). The findings indicate that the exopolysaccharide is a homogeneous protein-bound heteropolysaccharide carrying molecular weight of 2900 kDa with a β-type glycosidic linkage. It belongs to a kind of β-(13)-D-glucans consisting of a backbone of β-(13)-linked glucose residues branched with (14) and (16)-β-D-glucopyranosyl residues on main-chain residues. The elemental analysis of this exopolysaccharide discover the element compositions as: C, 25.84%; H, 5.45%; and N, 0.65%. The total carbohydrate, protein and uronic acid contents of exopolysaccharide is 89.0%, 2.20% and 7.52%, respectively. In addition, lipopolysaccharide (LPS) was not detected in the exopolysaccharide. Glucose is the main monosaccharide structural unit in this exopolysaccharide, the content is 57.5%. The degradation temperature of exopolysaccharide is 278.9°C from the thermogram analysis curve. This exopolysaccharide looks like thin film with smooth and glittering surface in SEM photography. It is clear from these images that the exopolysaccharide is linear in structure and branched and coiled in aqueous solution. With these extraction, the preliminary anti-inflammatory activity of S. commune exopolysaccharide was conducted by inhibiting the production of nitric oxide (NO), activity of inducible nitric oxide synthase (iNOS) and activity of 5-lipoxygenase (5-LOX) from RAW 264.7 macrophages. The results showed that exopolysaccharide significantly inhibit LPS-induced iNOS expression levels in a dose-dependent manner(p < 0.05). It inhibits the production of 5-LOX in cells, but not in dose-dependence. Further, in dextran sulfate sodium (DSS)-induced colitis model, the results showed that exopolysaccharide attenuated body weight loss, diarrhea, fecal blood, and the shortening of colon and improved histological changes. Furthermore, exopolysaccharide treatment would reduce NO production and some cytokines' secretion such as IL-4 and IL-17A. These results indicate that exopolysaccharide might be exploited as an effective anti-inflammatory agent for application in IBD. Secondly, ultrasound technology was applied to modify the physicochemical properties (MW and viscosity) of this fungal exopolysaccharide, and fractions of different MWs were obtained through ultrasonic degradation method. Effect of the MW degradation, viscosity and anti-inflammatory property of exopolysaccharide under ultrasonic treatment were optimized with response surface methodology. The best ultrasonic treatment parameters were obtained with a three-variable-three-level Box-Behnken design. The optimized conditions for efficient anti-inflammatory activity include: Initial concentration - 0.4%; ultrasonic power - 600 W; and duration of ultrasonic treatment - 9 min. Under these conditions, the NO inhibition rate is 95 ± 0.03% which agreed closely with the predicted value (96%). Average MW of exopolysaccharide decreased after ultrasonic treatments, but no significant change in the preliminary structure by infrared spectroscopy analysis. The viscosity of degraded exopolysaccharide dropped compared with native exopolysaccharide. The results suggest that ultrasound technology is an effective approach to reduce the MW of exopolysaccharide. Our results also showed that exopolysaccharide from S. commune was degraded into three fractions (low, medium, and high MW) by ultrasonic treatment. The changes of MW, atomic force microscope morphology, X-ray diffraction, particle size distribution and viscosity analysis indicate the triple helical structure of exopolysaccharide was dissociated into single helical structure and random coiled structure by breaking of inter- and intramolecular hydrogen bonds. The medium and high MW exopolysaccharide had the mixture of triple helix and single helix conformation. Moreover, the low MW exopolysaccharide exhibit random coiled conformation. As for their anti-inflammatory effect in DSS-induced colitis mice model, the results showed that medium and high MW exopolysaccharide significantly recovered DSS-induced colitis in body weight loss, shortening of colon lengths, colon weight loss, diarrhea and rectal bleeding, histological score, myeloperoxidase (MPO) activity, NO and cytokines (IFN-γ, IL-10 and IL-17) production in inflamed tissues. Moreover, exopolysaccharide with medium and high MW reduced DSS-induced infiltration of macrophages. These results showed that medium and high MW exopolysaccharide had intestinal anti-inflammatory activity. The degraded exopolysaccharide with medium and high MW had a triple and single-helical structure. These results suggested that the intestinal anti-inflammatory activity of exopolysaccharide from S. commune is related to both helical structure and MW.

Amanita ponderosa é uma espécie de cogumelos silvestres, comestível, característica de alguns microclimas mediterrânicos, existente na Península Ibérica. Neste estudo, avaliaram-se as propriedades biológicas e toxicológicas destes cogumelos e culturas, com vista a valorizar o seu potencial biotecnológico. Desenvolveu-se também uma metodologia de monitorização, utilizando técnicas de microanálise e imunológicas para screening e análise da especificidade de produção dos compostos bioativos. Os corpos de frutificação apresentaram um relevante conteúdo mineral e um perfil molecular correlacionável com o local de colheita. Tanto os cogumelos como as culturas e seus extratos revelaram baixa toxicidade, in vitro e in vivo, apresentaram propriedades antioxidantes, capacidade hepatoprotetora e efeito antiproliferativo em células MDA-MB-231. Estes resultados sugerem que A. ponderosa e/ou os seus extratos podem constituir uma importante fonte de compostos bioativos, com potencial valor nutracêutico e medicinal, podendo ser utilizados como suplementos alimentares, coadjuvantes no tratamento de doenças hepáticas e/ou tumorais; Characterisation of Amanita ponderosa wild mushrooms: production of metabolites with biological activity Abstract: Amanita ponderosa is a species of wild edible mushrooms that grows in some Mediterranean microclimates in the Iberian Peninsula. In this study, we evaluated the biological and toxicological properties of these mushrooms and cultures, in order to enhance their biotechnological potential. A monitoring methodology was also developed using microanalysis and immunological techniques, for screening and specificity evaluation of bioactive compounds production. The fruiting bodies presented a relevant mineral content and a characteristic molecular profile correlated with the geographical location. Either mushrooms or cultures and extracts have shown low toxicity in vitro and in vivo, and presented antioxidant properties, hepatoprotective effect and MDA-MB-231 antiproliferative activity. These results suggest that A. ponderosa and their extracts may constitute an important source of bioactive compounds with antioxidant benefits, nutraceutical potential and medicinal value, that can be used as dietetic supplements and as co-adjuvant of liver and cancer disease treatments.

碩士
國立臺灣大學
漁業科學研究所
101
Cytokine and growth factor secreted by tumor cells can actively recruit blood monocytes at the tumor microenvironment, where the recruiting monocytes differentiated into tumor-associated macrophages which promote tumor progression, thereby resulting a favorable tumor microenvironment for tumor growth. The tumor microenvironment subverted the immune system and enhanced tumor growth through strong immune suppressive mechanisms within the tumor microenvironment. The current study is to investigate whether oral mushroom polysaccharides have any effect on molecular profiles within the serum, lung and tumor microenvironment and tumor-associated macrophage phenotypes. The current study showed that oral Schizophyllan treatment significantly increased IFN-γ mRNA expression, but significantly reduced COX-2 mRNA expression within the lung. For LLC1 tumor model, oral Schizophyllan or oral Ganoderma lucidum polysaccharides treatments significantly reduced TGF-β production in serum. In addition, IL-12 and IFN-γ mRNA expression were significantly increased, but IL-6, IL-10, COX-2 and TGF-β mRNA expression were substantially declined within the tumor microenvironment after oral Schizophyllan or oral Ganoderma lucidum polysaccharides treatments. Moreover, oral Ganoderma lucidum polysaccharides treatment significantly increased M1 phenotype of tumor-associated macrophages, but significantly reduced M2 phenotype of tumor-associated macrophages. Taken together, our study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides through altering the tumor microenvironment.

Athymic nude mice were employed as the in vivo model to study the detailed mechanism of how the three sclerotial polysaccharides act to inhibit the growth of human xenografted tumors in vivo. Using immunohistochemical staining, it was found that the presence of F4/80 + macrophages was related to the reduction of tumor size of the HL-60 xenograft. mRNA extracted from the spleens were reverse-transcribed to cDNA and detected by real-time PCR so that a variety of genes related to the toll-like receptors being up-regulated or down-regulated due to the injection of mushroom sclerotial polysaccharides were determined. Combining the results from dectin-1 regulation, it was concluded that both hot water-soluble sclerotial polysaccharides, PTRW and PRW, having a structure of polysaccharide-protein complexes were responsible for activating and thus binding to CR3 or toll-like receptors while PRSon with structure of pure beta-glucan was responsible for activating the expression of dectin-1 receptor, which led to the subsequent activation of host immune system in immunopotentiation and antitumor activities.
In the future, further investigation of the detailed structure of mushroom sclerotial polysaccharides is required to explain the immunomodulatory mechanism so that the effective dosage for immunomodulation as well as antitumor effects can be determined. Furthermore, phage display can be applied to find out any novel glucan receptors specific to the mushroom sclerotial polysaccharides.
In vitro antitumor study indicated that PTRW had a significant (p<0.05) inhibitory effect (>40%) on the human monocytic leukemic cells (THP-1) in addition to HL-60 and K562 cells. In vitro immunomodulatory study showed that both PRW and PRSon had significant proliferative effects (p<0.05) on human normal spleen monocyte/macrophage cell, MD. Moreover, PRSon was shown to have a significant increase (p<0.05) in the growth of human natural killer cells, NK-92M1; however, PTRW showed a significant inhibition (p<0.05) on this cell line.
Mushroom sclerotia have a rich source of polysaccharides when compared with fruit bodies. It was previously reported that the polysaccharides from novel mushroom sclerotia, namely, Pleurotus tuber-regium and Polyporus rhinocerus, had potent in vitro and in vivo antitumor effects. In this project, hot water-soluble sclerotial polysaccharides of Pleurotus tuber-regium (PTRW), hot water-soluble and sonication-assisted cold alkali-soluble sclerotial polysaccharides of Polyporus rhinocerus (PRW and PRSon, respectively) were chosen for investigation of their in vivo and in vitro immunomodulatory effects.
Polysaccharides have long been proposed to exert their antitumor and thus immunomodulating functions through glucan receptors and among the four being discovered, Dectin-1 has drawn most attention recently. In the in vivo study, PRSon showed an increase in the expression of Dectin-1 on mice spleen MNCs while PTRW showed an increase in the expression of the previously widely-reported complement receptor (CR3). There was also an increase of Dectin-1 expression on PEC in the mice injected with PRSon. In the in vitro study, the three mushroom sclerotial polysaccharides were incubated with NK-92M1, MD and THP-1 cells. There was a significant increase (p<0.05) of Dectin-1 expression on NK-92MI cells incubated with PTRW. On the other hand, PTRW caused a significant decrease ( p<0.05) of Dectin-1 expression while PRSon showed a significant increase (p<0.05) on THP-1 cells. The cytokine profile of extra-cellular media indicated that the inhibition of THP-1 cells by PTRW should be related to the innate immunity. In the in vitro study, human primary immune cells, CD56+ NK cells were used to incubate with sclerotial polysaccharides and there was a significant stimulation (p<0.05) of their growth when compared with the control.
The in vivo immunomodulatory study was carried out by injecting the abovementioned sclerotial polysaccharides intraperitoneal to 7-8 weeks old healthy male BALB/c mice. The spleens excised from groups injected with PTRW and PRW were found to have significant increase of weight ( p<0.001). Flow cytometric analysis revealed that the NK cell population in spleen mononuclear cells (MNCs) of mice injected with PRW and PRSon was increased when compared with the control. In addition, ail three sclerotial polysaccharides showed a large increase of T helper cell population as well as CD4+/CD8+ ratio in spleen MNCs. On the other hand, the macrophage population in peritoneal exudates cells (PEC) was found to be increased in the groups of mice injected with PTRW and PRW.
Lai, Kin Ming Connie.
Adviser: Cheung Chi Keung.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3412.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 120-137).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

碩士
國立臺灣大學
漁業科學研究所
101
Mushroom polysaccharides are distinguished as important immunostimulant in animal body. Administrate glucan to animal inducing different isotype immunoglobulin secretion. Immunoglobulin A is the major antibody in the intestinal mucus, and transcytosis of IgA across epithelia is mediated by the poly-Ig receptor. Neutralization is important protection mechanism against antigen by IgA in gut. In the present study simulate acting intestinal mucosal immune system. Further to study the effects of mushroom polysaccharide on immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM) concentration in serum and small intestine washing fluid (SIWF) and intestine tissue poly-immunoglobulin receptor (poly-Ig receptor) mRNA expression. Feeding mice polysaccharide IgA and IgG in SIWF are increase significantly, and IgG and IgM in serum are also increase significantly. The results suggest that the effect of polysaccharides was induced at intestinal mucosa firstly, and induced body circulation immune response further. Poly-Ig receptor mRNA expression increase significantly, too. Our study highlights the efficacious effect of mushroom polysaccharides increasing immunoglobulin concentration in intestinal tract and serum immunoglobulin concentration, and increase poly-Ig receptor mRNA expression in intestine tissue. Mushroom polysaccharide may stimulate intestinal mucosal immune system to protect the intestinal tract from being damaged by the bacterial over-population.

碩士
中州科技大學
保健食品系
104
The mushroom or related health care products were variety on the market, including of the extract was from the single mushroom such as Ganoderma lucidum, Antrodia camphorata or others or, the compound productions were made from various mushrooms extract. By this productions, we mainly investigate to compare the effect of one type extract from Ganoderma lucidum or Agaricus blazei Murill and the compound extracts mixture from Ganoderma lucidum and Agaricus blazei Murill (1:1 mixture) on the immunomodulation function, so as to assess the proliferation of T cells and B cells in the spleen, Nature killer cell (NK cell) activity and the phagocytic cells capacity. Experimental results shown that the proliferation of T cell and B cell were significantly enhanced in the concentration of the 160 mg / kg / day by the oral feeding unilateral mushroom polysaccharides for six weeks but, the significantly enhancement activity was presented from the compound mushrooms polysaccharides for six weeks in the concentration of 120 mg / kg / day. This presented data indicated that oral feeding with compound mushrooms extract was higher effective promotion in the T cell and B cell proliferation than the oral with unilateral mushroom polysaccharides. In the nature killer cell activity experiment and the phagocytic cell activity assay, the experimental results shown that since the spleen nature killer cell was exhibited significantly activity than the control group and, the similar experimental situation was also presented in the spleen phagocytic capacity assay. Experimental results shown that the cytotoxic activity in the nature killer cell and the phagocytotic capacity in the phagocytic cells, it was significantly enhanced in the concentration of the 40 mg / kg / day by the oral feeding unilateral mushroom polysaccharides for six weeks but, the significantly enhancement activity was presented from the compound mushrooms polysaccharides for six weeks in the concentration of 20 mg / kg / day. This presented data indicated that oral feeding with compound mushrooms extract was higher effective promotion in cytotoxic and phagocytotic capacity than the oral with unilateral mushroom polysaccharides. Therefore, oral with compound mushrooms extracts was better than the unilateral mushroom polysaccharides n the proliferation of T cells and B cells, cytotoxic activity from NK cell and the phagocytotic capacity of phagocytic cells which were observed in the various dose concentration experiment.

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

Poon Shuk-ching.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 113-125).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
Abstract (Chinese Version) --- p.iv
Table of Contents --- p.vi
List of Tables --- p.x
List of Figures --- p.xi
List of Abbreviations --- p.xiii
Chapter Chapterl 1 --- ntroduction --- p.1
Chapter Chapter 2 --- Literature Review --- p.5
Chapter 2.1 --- Mushroom Polysaccharides From Basidiomycetes --- p.5
Chapter 2.1.1 --- Antitumor and Immunomodulatory Activity --- p.6
Chapter 2.1.2 --- Antiviral Activity --- p.9
Chapter 2.1.3 --- Hypoglycermic Activity --- p.11
Chapter 2.1.4 --- Free Radical Scavenging Activity --- p.11
Chapter 2.2 --- Mushroom Dictyophora indusiata --- p.13
Chapter 2.2.1 --- Nutritional Value --- p.13
Chapter 2.2.2 --- Structural Characteristic of Dictyophora indusiata Polysaccharides --- p.14
Chapter 2.2.3 --- Biological Activity --- p.16
Chapter 2.3 --- In vivo Antitumor Study --- p.19
Chapter 2.4 --- Induction of Cytokines Production in Immune System --- p.21
Chapter 2.5 --- In vitro Antitumor Study --- p.25
Chapter 2.6 --- Cell Cycle Regulation --- p.28
Chapter Chapter 3 --- Materials & Methods --- p.34
Chapter 3.1 --- Extraction --- p.34
Chapter 3.1.1 --- Extraction of Dictyophora indusiata Polysaccharides --- p.34
Chapter 3.1.2 --- Purification of Dictyophora indusiata Polysaccharides --- p.35
Chapter 3.1.2.1 --- Preparation of DEAE-cellulose Ion Exchanger --- p.35
Chapter 3.1.2.2 --- Fractionation --- p.35
Chapter 3.2. --- Characterization of Dictyophora indusiata Polysaccharides --- p.39
Chapter 3.2.1 --- Polysaccharide Content Determination --- p.39
Chapter 3.2.2 --- Protein Content Determination --- p.39
Chapter 3.2.3 --- Gas Chromatography (GC) --- p.40
Chapter 3.2.4 --- Uronic Acid Content Determination --- p.42
Chapter 3.2.5 --- High Performance Liquid Chromatography (HPLC) --- p.43
Chapter 3.3 --- In vivo Studies --- p.44
Chapter 3.3.1 --- Animals --- p.44
Chapter 3.3.2 --- Maintenance of Sarcoma 180 Cell Line --- p.44
Chapter 3.3.3 --- Effect of DI3 Fraction on Sarcoma 180 Solid Tumor --- p.45
Chapter 3.3.4 --- Effect of DI3c Fraction on Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin 2 (IL-2) Production --- p.47
Chapter 3.3.4.1 --- Treatment of Mice --- p.47
Chapter 3.3.4.2 --- Preparation of Mouse Serum --- p.47
Chapter 3.3.4.3 --- Enzyme-linked Immunosorbent Assay (ELISA) for TNF-α Production --- p.48
Chapter 3.3.4.4 --- Enzyme-linked Immunosorbent Assay (ELISA) for IL-2 Production --- p.49
Chapter 3.4 --- In vitro Studies --- p.51
Chapter 3.4.1 --- Maintenance of Cell Lines --- p.51
Chapter 3.4.2 --- Effect on Cancer Cell Lines --- p.52
Chapter 3.4.3 --- Cytotoxicity on Normal Cell Line --- p.52
Chapter 3.4.4 --- Trypan Blue Exclusion Method --- p.53
Chapter 3.4.5 --- MTT Assay --- p.54
Chapter 3.4.6 --- BrdU Incorporation --- p.55
Chapter 3.5 --- Statistical Analysis --- p.56
Chapter Chapter 4 --- Results --- p.57
Chapter 4.1 --- Extraction and Fractionation of Polysaccharides from Dictyophora indusiata --- p.57
Chapter 4.1.1 --- Percentage Yield of Crude DI Polysaccharides --- p.57
Chapter 4.1.2 --- Percentage Yield of DI3 Fractions --- p.57
Chapter 4.2 --- Characterization of DI3 Fractions --- p.62
Chapter 4.2.1 --- Polysaccharide and Protein Contents of DI3 Fractions --- p.62
Chapter 4.2.2 --- Relative Monosaccharide and Uronic Acid Content in Different DI3 Fractions --- p.62
Chapter 4.2.3 --- Estimated Molecular Weight of DI3 Fractions --- p.65
Chapter 4.3 --- Antitumor Effect of Dictyophora indusiata Polysaccharides In vivo --- p.70
Chapter 4.3.1 --- In vivo Antitumor Effect of Crude DI Polysaccharides --- p.70
Chapter 4.3.2 --- In vivo Antitumor Effect of Various Fractions of DI3 --- p.70
Chapter 4.3.3 --- Effect of DI3c on TNP-α and IL-2 Production in Mice --- p.78
Chapter 4.4 --- In vitro Effects of DI3 Fractions on Cell Density and Viability on Normal and Cancer Cell Lines --- p.86
Chapter 4.4.1 --- Effects of DI3 Fractions on Cell Density and Viability of Human Leukemic HL-60 and K-562 and Mouse Sarcoma 180 Cells --- p.86
Chapter 4.4.2 --- Effects of DI3 Fractions on the Growth of Human Liver Cancer HepG2 and Normal Monkey Kidney Vero Cells --- p.86
Chapter 4.4.3 --- Effect of DI3b Fraction on Proliferation of HL-60 Cells Determined by BrdU Incorporation --- p.94
Chapter Chapter 5 --- Discussions --- p.96
Chapter 5.1 --- Extraction and Characterization of DI3 Fractions --- p.96
Chapter 5.2 --- Antitumor Effects of Dictyophora indusiata Polysaccharides --- p.101
Chapter 5.3 --- Further Studies --- p.109
Chapter Chapter 6 --- Conclusion --- p.111
References --- p.113

Choi, Man Wing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 126-139).
Abstracts in English and Chinese.
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Antigen presenting cells (APC) in Immune systems --- p.1
Chapter 1.1.1 --- Dendritic cells --- p.2
Chapter 1.1.1.1 --- Differentiation of dendritic cells in mice --- p.2
Chapter 1.1.1.2 --- Maturation of dendritic cells --- p.3
Chapter 1.1.1.3 --- Stimulation and polarization of T cells stimulated by dendritic cells --- p.6
Chapter 1.1.2 --- Monocyte and macrophage --- p.7
Chapter 1.1.2.1 --- Differentiation of monocyte and macrophage in humans --- p.7
Chapter 1.1.2.2 --- Changes involved in differentiation of monocytes into macrophages --- p.9
Chapter 1.2 --- "Isolation, structure and activity of mushroom polysaccharides" --- p.13
Chapter 1.2.1 --- Sources of mushroom polysaccharides --- p.13
Chapter 1.2.2 --- Extraction methods --- p.14
Chapter 1.2.3 --- Structure-Activity Relationship (SAR) of mushroom polysaccharides --- p.15
Chapter 1.2.4 --- Previous studies on immunomodulatory effects of mushroom sclerotial polysaccharides --- p.18
Chapter 1.3 --- Recognition of β-glucan by specific receptors --- p.20
Chapter 1.3.1 --- Complement Receptor 3 (CR3) --- p.22
Chapter 1.3.1.1 --- Introduction of CR3 --- p.22
Chapter 1.3.1.2 --- Expressions of CR3 to recognize fungi --- p.22
Chapter 1.3.2 --- Dectin-1 --- p.24
Chapter 1.3.2.1 --- Introduction of Dectin-1 --- p.24
Chapter 1.3.2.2 --- Structure of Full-length Dectin-1 --- p.26
Chapter 1.3.2.2.1 --- Isoforms of Dectin-1 in Mice --- p.28
Chapter 1.3.2.2.2 --- Isoforms of Dectin-1 in Humans --- p.28
Chapter 1.3.2.3 --- Immune responses triggered by of Dectin-1 --- p.29
Chapter 1.3.3 --- Toll-like 2 receptor (TLR2) --- p.31
Chapter 1.3.3.1 --- Introduction of TLR2 --- p.31
Chapter 1.3.3.2 --- Structure of TLR2 --- p.33
Chapter 1.3.3.3 --- Immune responses triggered by TLR2 --- p.34
Chapter 1.4 --- Research Objectives --- p.35
Chapter Chapter 2 --- Materials and Methods --- p.38
Chapter 2.1 --- Materials --- p.38
Chapter 2.1.1 --- Mushroom sclerotia --- p.38
Chapter 2.1.1.1 --- Polysaccharide extraction from mushroom sclerotia --- p.38
Chapter 2.1.2 --- Antibodies and reagents --- p.41
Chapter 2.1.3 --- Human acute leukocyte monocytic cell line and culture medium --- p.42
Chapter 2.1.4 --- Preparation of murine bone marrow-derived immature dendritic primary cells (immature BMDCs) --- p.43
Chapter 2.2 --- Methods --- p.45
Chapter 2.2.1 --- Chemical Analysis --- p.45
Chapter 2.2.1.1 --- Measurement of monosaccharide profile --- p.45
Chapter 2.2.1.1.1 --- Acid depolymerisation --- p.45
Chapter 2.2.1.1.2 --- Neutral sugar derivatization --- p.45
Chapter 2.2.1.1.3 --- Gas chromatography (GC) --- p.46
Chapter 2.2.1.2 --- Determination of total sugar by phenol-sulfuric acid method --- p.47
Chapter 2.2.1.3 --- Determination of protein content by Lowry-Folin Method --- p.48
Chapter 2.2.1.4 --- Size exclusion chromatography by high pressure liquid chromatography (HPLC) --- p.49
Chapter 2.2.1.5 --- Endotoxin detection --- p.50
Chapter 2.2.2 --- Measurement of Bioactivities --- p.51
Chapter 2.2.2.1 --- Trypan blue exclusion assay --- p.51
Chapter 2.2.2.2 --- MTT cell proliferation assay --- p.51
Chapter 2.2.2.3 --- BrdU cell proliferation assay --- p.53
Chapter 2.2.2.4 --- Expression of cell surface markers --- p.54
Chapter 2.2.2.5 --- Phagocytosis / Endocytosis of FITC-labeled dextrans --- p.55
Chapter 2.2.2.6 --- Nitric oxide production assay --- p.55
Chapter 2.2.2.7 --- Reactive oxygen species production --- p.57
Chapter 2.2.2.8 --- Determination of cytokine profile using cytokine antibody array --- p.58
Chapter 2.2.2.9 --- Cell cycle analysis --- p.59
Chapter 2.2.2.10 --- Expression of surface receptors --- p.60
Chapter 2.2.2.11 --- Statistical analysis --- p.61
Chapter Chapter 3 --- Results and Discussion --- p.61
Chapter 3.1 --- Chemical characteristics of sclerotial polysaccharides --- p.61
Chapter 3.1.1. --- The yield of sclerotial polysaccharides --- p.61
Chapter 3.1.2 --- Total carbohydrate content of sclerotial polysaccharides --- p.65
Chapter 3.1.3 --- Protein content of sclerotial polysaccharides --- p.66
Chapter 3.1.4 --- Monosaccharide profiles of sclerotial polysaccharides from PR by gas chromatography (GC) --- p.66
Chapter 3.1.5 --- Molecular weight of sclerotial polysaccharides from PR by size exclusion chromatography (SEC) --- p.69
Chapter 3.1.6 --- Endotoxin test --- p.73
Chapter 3.2 --- Immune responses for human monocytic cell line THP-1 --- p.74
Chapter 3.2.1 --- MTT cell viability assay --- p.74
Chapter 3.2.2 --- BrdU cell proliferation assay --- p.75
Chapter 3.2.3 --- Change in cell morphology of THP-1 --- p.79
Chapter 3.2.4 --- Phenotypic maturation of THP-1 --- p.81
Chapter 3.2.5 --- Up-regulated phagocytic ability of THP-1 --- p.84
Chapter 3.2.6 --- Increased nitrite production in THP-1 --- p.86
Chapter 3.2.7 --- Production of reactive oxygen species --- p.88
Chapter 3.2.8 --- Human cytokines profile array --- p.90
Chapter 3.2.9 --- Cell cycle analysis --- p.93
Chapter 3.2.10 --- Surface receptors expression --- p.95
Chapter 3.2.11 --- Summary --- p.98
Chapter 3.3 --- Immune responses for murine immature BMDCs --- p.102
Chapter 3.3.1 --- Inhibition effects on murine immature BMDCs --- p.102
Chapter 3.3.2 --- Change in cell morphology of murine immature BMDCs --- p.103
Chapter 3.3.3 --- Phenotypic maturation of murine immature BMDCs --- p.105
Chapter 3.3.4 --- Down-regulation of endocytosis in murine immature BMDCs --- p.106
Chapter 3.3.5 --- Increased nitrite production --- p.109
Chapter 3.3.6 --- Decreased expression of CD 11c in PRW-treated immature BMDCs --- p.109
Chapter 3.3.7 --- Cytokine profile detection --- p.112
Chapter 3.3.8 --- Surface receptors expression --- p.116
Chapter 3.3.9 --- Summary --- p.119
Chapter Chapter 4 --- Conclusion and future works --- p.123
Appendix --- p.125
References --- p.126

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2012
Hoje em dia, devido ao prolongamento da esperança média de vida, bem como o aumento das agressões sofridas pelo meio ambiente envolvente, o cancro é uma das doenças mais proeminentes no seculo XXI. Neoplasmas, conjuntos anormais de células cromossomicamente mutadas, levam ao crescimento relativamente autónomo de tecido danificado. As causas destas mutações variam entre internas - condições imunitárias, hormonais, heritabilidade - ou externas - como tabagismo, exposição a radiação ou agentes químicos infecciosos (Zaidman et al., 2005). As células neoplásicas caracterizam-se pela perda das suas funções especializadas e ganham por sua vez características biológicas tais como a produção de sinais de crescimento, inibição da apoptose, elevado potencial replicativo e a capacidade de invadir tecidos circundantes (Zaidman et al., 2005; Zong et al.,, 2012). A esta metastização dos tecidos está associada 90% da responsabilidade da morte por cancro (Zong et al.,, 2012). Apesar da eficiência dos fármacos gerados até hoje no combate ao cancro, o seu efeito tóxico causa na maioria dos casos um decréscimo acentuado na qualidade de vida dos doentes. Assim, torna-se importante descobrir novos agentes anti cancerigenos com efeitos tóxicos mais baixos (Cao et al., 2010) que possam ser utilizados em terapias complementares. Virámo-nos então de novo para as tradições mais antigas da medicina Chinesa e Mediterrânea. Polissacáridos são biopolímeros constituídos por várias unidades de monossacáridos ligados através de ligações glicosidícas, que podem apresentar diferentes conformações, não só nas suas unidades constituintes, mas também na conformação das suas ligações, atribuindo-lhes assim diversas funções biológicas. Estes estão presentes desde o armazenamento de energia (amido), formação de componentes estruturais (celulose), participantes na comunicação célula a célula, possuindo ainda papéis importantes no sistema imunitário, na fertilização, desenvolvimento, agregação plaquetária e prevenção de patogéneses (Zong et al., 2012). Desde a medicina ancestral chinesa que os cogumelos têm sido reportados como tendo propriedades imunomoduladoras - actuando como modificadores da resposta biológica - e possuindo propriedades antivirais, antimicrobiais, circulatórias e nas ultimas décadas anti cancerigenas. (Wasser, 2010). A descoberta do Lentinian, um polissacárido com propriedades anti cancerígenas, despoletou nos anos 60 a pesquisa que tem sido dedicada ate hoje ao campo dos polissacáridos (Cao et al., 2010). Ao longo do tempo, diversos polissacáridos produzidos por cogumelos, têm demonstrado largos efeitos inibidores em vários tipos de tumores como Sarcoma 180, carcinoma de Lewis e Yoshida Sarcoma (Zhang et al., 2007). Existem três mecanismos propostos de actuação destes polissacáridos: (1) actividade preventiva, (2) aumento da resposta imunitária do hospedeiro e (3) inibição directa quando em contacto com células cancerígenas (Zhang et al., 2007). Na sua maioria os cogumelos que possuem polissacáridos biologicamente activos pertencem ao grupo dos basidiomicetes contendo cerca de 700 espécies descritas (Wasser, 2002). O seu poder antioxidante também desempenha um papel fundamental contra o desenvolvimento cancerígeno. Anti oxidantes naturais encontrados em fruta, vegetais, cogumelos e outros recursos alimentares têm sido investigados para prevenção de doenças coronárias e outras como o cancro (Kozarski et al.,2012). Assim, na procura de compostos não tóxicos que possuíssem propriedades anticancerígenas investigou-se também um pouco dos efeitos que as folhas de oliveira possam ter no combate ao cancro. A oliveira (Olea europaea) é uma árvore da família das Oleaceae, (Zaid et al., 2012) com folhas alongadas, que se encontra ao longo de toda região Mediterrânea desde há mais de 7000 anos (Lalas et al., 2011). Ao longo da história as folhas de Olea europaea têm sido exploradas para a prevenção de hipertensão, carcinogénese, diabetes, arteroesclerose bem como outras doenças do foro mais comum (Bouallagui et al., 2011; Zaid et al., 2012). A folha de oliveira foi ainda utilizada ao longo da história como um meio de combate á malária (Lee et al., 2009) A incidência de cancro ao longo do mediterrâneo tem sido das mais baixas quando comparada com outras partes do mundo, principalmente quando nos referimos ao cancro da mama, do endométrio, próstata e leucemia. (Bouallagui et al., 2011; Zaid et al., 2012) Assim reunimos neste trabalho, o estudo dos efeitos de algumas espécies conhecidas (e outras menos conhecidas) de cogumelos, com o estudo dos efeitos de algumas variedades de oliveiras nacionais, na inibição do crescimento de células tumorais. Para isso, foram efectuados vários doseamentos de polissacáridos, proteínas, açucares e compostos fenólicos e foram aplicadas diversas amostras das espécies amostradas a quatro linhas de carcinoma humano: HeLa, carcinoma cervical, A549 carcinoma pulmonar; A431 carcinoma epidermóide e OE21 carcinoma esófagofaringeal. De entre os variados resultados obtidos, pudemos concluir que a aplicação de extra polissacáridos da estirpe Ganoderma carnosum revelou um alto teor inibitório, e que os intra polissacáridos de todas as estirpes amostradas mostraram uma capacidade antioxidativa acima de 50%. As folhas de oliveira por sua vez, foram também capazes de mostrar um potencial anti proliferativo das células tumorais bastante satisfatório. Acreditamos que nestes compostos podem estar presentes as condições para a sua aplicação como terapia complementar às terapias anticancerígenas existentes.
Cancer is widely known nowadays as the disease of the century. Not only by the number of infected people worldwide but also by the amount of research dedicated to this field and its increasing success rate. Neoplasms, which are abnormal masses or colonies of cells produced by a relatively autonomous new growth of tissue, arise normally by the clonal expansion of a single cell that has undergone mutation in the chromosomal DNA, caused by a chemical, physical or biological agent (Zaidman et al., 2005). One of the most characterizing features of cancers, besides it’s ability to resist treatment, it’s his ability to metastasize and invade surrounding tissues, causing 90% of cancer deaths ( Zong et al., 2012). Although the efficiency of chemotherapy for the majority of cancers has improved over the last three decades, the drugs used in this treatment, contain high toxic effects that cause severe reduction in quality of life. Therefore, it is important to develop novel potent, but low toxic anti-cancer reagents, including natural products (Cao et al., 2010). The desire to find this new chemical drug as lead us to look back in time and to report to times of ancient Chinese and Mediterranean medicine, in order to encounter natural products that can in the future be developed as efficient co-chemotherapeutical agents. During this work we will test mushroom polysaccharides as well as olive leaves phenolic compounds, in human carcinoma cell lines, in order to discover more about their anti-tumor effects. Amongst our findings, extrapolysaccharides from Ganoderma lucidum were the most promising and all intrapolysaccharides were found to have a 50% rate of antioxidative power. Olive leaves were also very promising in the inibithion of human carcinoma cells in vitro.

碩士
國立臺灣大學
食品科技研究所
100
Edible mushroom polysaccharides are regarded as potential immune-stimulating substances, e.g. such as β-(1, 3)-D-glucan, (1, 6) branched (1, 3)-β-D-glucans, α-glucans, α-(1, 6)-galactans, mannans, and glycogen-like polysaccharides. The immunomodulatory activities of vary with sugar composition, glycosidic linkage pattern and molecular weight of the polysaccharide. This study intends to use the principal component statistical analysis for 21 species of edible mushrooms cultivated in Taiwan to establish the correlation of dietary fiber content and the sugar profile of indigestible water-soluble polysaccharide (ND-WSP), including the content, sugar composition, glycosidic linkage pattern, molecular weight, the content and degree of branch of β-(1, 3)-D-glucan, as parameters with the relationship of their taxonomic classification. The total dietary fiber, insoluble dietary fiber, and soluble dietary fiber content of these mushrooms were 24.78~83.76%, 19.62~76.5% and 0.56~5.09%, respectively. ND-WSP, the major composition of soluble dietary fiber were 1~4.16% in mushrooms dry weight, glucose, galactose and mannose are majority in their sugar composition, Agriucus. blazei, Auricularia auricula-juda and Tremella fuciformis contained higher uronic acid, and glucuronic acid were the major component. Xylose was the major monosaccharide in Agrocybe cylindracea, A. auricula-juda and T. fuciformis, and Lentinula edodes contained a high amount of fucose. The major glycosdic linkage pattern in ND-WSP were 1, 6-Galp; 1, 3-Glcp; 1, 6-Glcp; 1, 2-Manp; 1, 3-Manp and 1, 6-Manp, etc, and with several branching points, such as 1, 2, 6-Galp; 1, 2, 3-Galp; 1, 3, 6-Glcp; 1, 4, 6-Glcp; 1, 3, 4, 6-Glcp and 1, 3, 6-Manp, etc. There were two molecular weight distribution for most of ND-WSP in the range of 790-2370 and 8.5-20.3 kDa. The content of β-(1, 3)-D-glucan in ND-WSP were in the range of 0.69-41.66% and their degree of branch were 0.20-0.40. The mushrooms ND-WSP stimulated RAW 264.7 macrophages cell line releasing TNF-α, NO. ND-WSP of A. blazei and A. auricula-juda showed the highest activities among selected mushrooms. For selected 21 mushrooms, PCA analysis group them into four categories according to their sugar profile of ND-WSP and immune-stimulating activities on RAW 264.7 which was correlated to their taxonomic classification.

Preliminary results of the influence of Schizophyllum commune polysaccharides on the inhibition of mycotoxin synthesis in Aspergillus flavus and Aspergillus carbonarius Jelena Loncar1,3, Slaven Zjalić1 , Paola Cescutti2 Alice Motola 3, Massimo Reverberi3 1) Department of Ecology, Aquaculture and Agriculture, University of Zadar, Mihovila Pavlinovića 1, 23 000 Zadar, Croatia 2) Department of Life Sciences, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy 3) Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Abstract There has been interest in the use of mushroom polysaccharides to control the synthesis of key mycotoxins in staple commodities. Tramesan(c), an exo-polysaccharide produced by Trametes versicolor, is among the most efficient and studied ones. The objective of the present study were to analyse the effects of secreted polysaccharides of Schizophyllum commune on production of aflatoxin B1 (AFB1) and ochratoxin A (OTA) produced by Aspergillus flavus and Aspergillus carbonarius respectively. Comparison of the effects of commercial schizophyllan and partialy purified S. commune polysaccharides indicated that the inhibition of AFB1 and OTA synthesis may be due to other types of polysaccharide(s) rather than schizophyllan. Preliminary results suggest that the mechanism of efficacy of the S. commune polysaccharides could be analogous to that found previously for Tramesan®, i.e., by enhancing the antioxidant response in the target fungal cells.
Oligosaccharides derived from Tramesan: their structure and activity on mycotoxin inhibition in Aspergillus flavus and Aspergillus carbonarius Jelena Loncar1,3, Barbara Bellich2, Paola Cescutti2, Roberto Rizzo2, Slaven Zjalić1, Alessia Parroni3, Massimo Reverberi3 1) Department of Ecology, Aquaculture and Agriculture, University of Zadar, Mihovila Pavlinovića 1, 23 000 Zadar, Croatia 2) Deptartment of Life Sciences, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy 3) Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Abstract Food and feed safety, due to its direct influence on human health, has acquired a paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture to control pests, could present both environmental problems and health hazards, has led to a large limitation of their use. Since 2014, the European Community (EC) has banned about 50% of crop protection chemicals used in agriculture. In plant cultivation, chemicals were also the main tool used against fungal pathogens and against the contamination of food/feed stuff with toxic secondary metabolites, mycotoxins. There is thus a drive to develop more environmentally friendly “green” approaches and eco-compatible tools to control mycotoxigenic fungi and toxin contamination of staple foodstuffs. Different mushroom metabolites showed the potential to act as control agents against fungal growth and mycotoxin production. This study has examined the use of a polysaccharide Tramesan, extracted from the basidiomycete Trametes versicolor for controlling mycotoxin biosynthesis by Aspergillus flavus (aflatoxin B1) and Aspergillus carbonarius (ochratoxin A). This study evaluated component of the repeating unit of the polysaccharide produced by T. versicolor which was active in inhibition of aflatoxin B1 synthesis. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by column chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Although fragments smaller than seven units showed no significant effect on mycotoxin inhibition, heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these compounds could be used as natural alternatives to crop protection chemicals for control of these two mycotoxins in economically important commodities.
Effect of mushroom culture filtrates on inhibition of mycotoxins produced by Aspergillus flavus and Aspergillus carbonarius Jelena Loncar1,3, Barbara Bellich2, Paola Cescutti2, Alice Motola3, Slaven Zjalić1, Massimo Reverberi3 1) Department of Ecology, Aquaculture and Agriculture, University of Zadar, Mihovila Pavlinovića 1, 23 000 Zadar, Croatia 2) Deptartment of Life Sciences, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy 3) Department of Environmental Biology, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Abstract Mycotoxins are secondary metabolites produced by certain fungi that have hazardous effects on humans and animals, which in turn result in disease and economic losses. Two of the mycotoxins of greatest public health and agroeconomic significance are aflatoxin B1 (AFB1), and ochratoxin A (OTA). There is a need for the development of natural compounds for the control of these mycotoxins in food and feed. There has been interest in the use of mushroom polysaccharides for controlling mycotoxins. In this study 42 isolates of different ligninolytic mushrooms were assayed for their ability to inhibit the synthesis of OTA and AFB1. The results showed that 23 strains produce metabolites able to inhibit the synthesis of OTA by >20%, and 32 strains that produced metabolites that inhibited AFB1. Only two strains, Trametes versicolor strain TV117 and Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. It is known that T. versicolor produces a polysaccharide Tramesan, able to inhibit the synthesis of AFB1 and was probably responsible for mycotoxin inhibition. The results suggest that S. commune metabolites were able to inhibit both OTA and AFB1 synthesis may be based on polysaccharides.

博士
國立臺灣大學
生化科學研究所
101
Ganoderma lucidum (Reishi), a mushroom commonly used as a Chinese herb medicine, contains complex polysaccharides that have been used as an antitumor supplement with a rarely understood mechanism of immune response. Here we demonstrated that the mice immunized with a fucose-enriched Reishi polysaccharide fraction (designated as FMS) were able to induce antibodies against murine Lewis lung carcinoma (LLC1) cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular monocyte chemoattractant protein-1, MCP-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycan antigens, with the antigenic structure located in the non-reducing termini, i.e., Fuca1-2Galb1-3GalNAc-R(where R represents reducing end), typically found in Globo H and related tumor antigens. Although the composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-a-galactan, a fucose-dependent nanoLC-tandem MS analysis uncovered the Fuca1-2Gal, Fuca1-3/4Man, Fuca1-4Xyl and Fuca1-2Fuc linkages in the non-reducing termini of the FMS glycans, underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycan antigens. In summary, our development of carbohydrate immunomodulation-based therapy, which successfully correlated the high-throughput glycan microarray analysis with detailed structural analyses of glycan antigens, should be applicable to other medicinal polysaccharides.

by Liu Fang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 159-178).
ACKNOWLEGEMENTS --- p.i
ABSTRACT --- p.ii
TABLE OF CONTENTS --- p.v
LIST OF TABLES --- p.ix
LIST OF FIGURES --- p.xi
ABBREVIATIONS --- p.xiv
Chapter Chapter 1. --- General Introduction --- p.1
Chapter Chapter 2. --- Literature Review --- p.7
Chapter 2.1. --- Biologically active polysaccharides --- p.7
Chapter 2.2. --- Antitumor activities of polysaccharides --- p.11
Chapter 2.2.1. --- In vivo studies --- p.11
Chapter 2.2.2. --- In vitro studies --- p.15
Chapter 2.3. --- Antitumor mechanisms of polysaccharides --- p.17
Chapter 2.4. --- Structure and antitumor activities of polysaccharides --- p.25
Chapter 2.4.1. --- The effect of molecular mass --- p.26
Chapter 2.4.2. --- The impact of branching configuration --- p.21
Chapter 2.4.3. --- The relationship between antitumor activity and conformation --- p.28
Chapter 2.4.4. --- Improvement of antitumor activity by chemical modification --- p.29
Chapter 2.5. --- Other biological activities --- p.30
Chapter 2.5.1. --- Antiviral activity --- p.30
Chapter 2.5.2. --- Antimicrobial activity --- p.31
Chapter 2.5.3. --- Free radical scavenging activity --- p.32
Chapter 2.5.4. --- Hepatic protective effect --- p.32
Chapter Chapter 3. --- Isolation and Characterization of a Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.34
Chapter 3.1. --- Introduction --- p.34
Chapter 3.2. --- Materials and methods --- p.36
Chapter 3.2.1. --- Strain --- p.36
Chapter 3.2.2. --- Culture conditions --- p.36
Chapter 3.2.3. --- Extraction of T. lobayense --- p.39
Chapter 3.2.4. --- Purification of polysaccharide-protein complex --- p.40
Chapter 3.2.5. --- Molecular mass determination --- p.43
Chapter 3.2.6. --- High performance liquid chromatography --- p.43
Chapter 3.2.7. --- SDS-polyacrylamide gel electrophoresis --- p.44
Chapter 3.2.8. --- Ultraviolet scanning --- p.44
Chapter 3.2.9. --- Chemical analysis --- p.45
Chapter 3.2.10. --- Experimental animals --- p.47
Chapter 3.2.11. --- In vivo antitumor assay --- p.48
Chapter 3.2.12. --- Safety tests --- p.49
Chapter 3.2.13. --- Statistical analysis --- p.51
Chapter 3.3. --- Results --- p.51
Chapter 3.3.1. --- Extraction and purification --- p.51
Chapter 3.3.2. --- Biochemical analysis --- p.52
Chapter 3.3.3. --- Chemical analysis --- p.60
Chapter 3.3.4. --- In vivo antitumor activity --- p.68
Chapter 3.3.5. --- Safety evaluation --- p.68
Chapter 3.4. --- Discussion --- p.75
Chapter 3.5. --- Summary --- p.84
Chapter Chapter 4. --- "Immunomodulating, Antitumor and other Biological Activities of Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense" --- p.85
Chapter 4.1. --- Introduction --- p.85
Chapter 4.2. --- Materials and methods --- p.87
Chapter 4.2.1. --- Experimental animals --- p.87
Chapter 4.2.2. --- Cultivation of tumor cells --- p.87
Chapter 4.2.3. --- Preparation of peritoneal exudate cells and splenocytes --- p.87
Chapter 4.2.4. --- Mitogenic response of T cells --- p.89
Chapter 4.2.5. --- Responses of peritoneal exudate cells --- p.89
Chapter 4.2.6. --- In vitro antitumor assay --- p.92
Chapter 4.2.7. --- Transmission electron microscope --- p.93
Chapter 4.2.8. --- Evaluation of other biological activities --- p.94
Chapter 4.2.9. --- Statistical analysis --- p.99
Chapter 4.3. --- Results --- p.99
Chapter 4.3.1. --- Immunomodulating activity --- p.99
Chapter 4.3.2. --- In vitro antitumor action --- p.107
Chapter 4.3.3. --- Observation on tumor regression induced by PSPC --- p.107
Chapter 4.3.4. --- Other biological actions --- p.112
Chapter 4.4. --- Discussion --- p.121
Chapter 4.4.1. --- Immunomodulating activity --- p.121
Chapter 4.4.2. --- Antitumor activity --- p.125
Chapter 4.4.3. --- Other biological activities --- p.127
Chapter 4.5. --- Summary --- p.130
Chapter Chapter 5. --- Induction of Gene Expression of Immunomodulating Cytokines by Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.132
Chapter 5.1. --- Introduction --- p.132
Chapter 5.2. --- Materials and methods --- p.135
Chapter 5.2.1. --- Experimental animals --- p.135
Chapter 5.2.2. --- Preparation of peritoneal exudate cells and splenocytes --- p.136
Chapter 5.2.3. --- RNA extraction --- p.137
Chapter 5.2.4. --- Reverse transcription- polymerase chain reaction --- p.137
Chapter 5.2.5. --- Dot blot --- p.138
Chapter 5.2.6. --- Hybridization --- p.141
Chapter 5.3. --- Results --- p.142
Chapter 5.3.1. --- mRNA phenotyping of cytokines and cytokine receptors in normal mice --- p.142
Chapter 5.3.2. --- mRNA phenotyping of cytokines and cytokine receptors in tumor-bearing mice --- p.142
Chapter 5.4. --- Discussion --- p.150
Chapter 5.5. --- Summary --- p.153
Chapter Chapter 6. --- General Discussion and Conclusion --- p.155
REFERENCES --- p.159

Lam Wai Yee.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 124-148).
Abstracts in English and Chinese.
Chapter Chapter 1: --- Introduction --- p.1
Chapter 1.1 --- Lipoproteins --- p.1
Chapter 1.1.1 --- General structure --- p.1
Chapter 1.1.2 --- Chylomicrons --- p.2
Chapter 1.1.3 --- Very-low-density lipoprotein (VLDL) --- p.3
Chapter 1.1.4 --- Low-density lipoprotein (LDL) --- p.4
Chapter 1.1.5 --- High-density lipoprotein (HDL) --- p.4
Chapter 1.1.6 --- Lipoprotein metabolism --- p.5
Chapter 1.1.6.1 --- Exogenous pathway --- p.5
Chapter 1.1.6.2 --- LDL receptor pathway --- p.6
Chapter 1.1.6.3 --- Reverse cholesterol transport --- p.6
Chapter 1.2 --- Cholesterol homeostasis --- p.8
Chapter 1.2.1 --- Role of Acyl-CoA: Cholesterol Acyltransferase (ACAT) in intracellular cholesterol regulation --- p.8
Chapter 1.2.2 --- Cholesterol biosynthesis --- p.9
Chapter 1.2.3 --- Bile acid metabolism --- p.10
Chapter 1.3 --- Coronary heart disease (CHD) --- p.14
Chapter 1.3.1 --- Risk factors of CHD --- p.16
Chapter 1.3.2 --- Lipoprotein cholesterol and CHD --- p.18
Chapter 1.4 --- Animal models for hypercholesterolemic study --- p.20
Chapter 1.5 --- Physico-chemical properties of water-soluble dietary fiber (SDF) --- p.22
Chapter 1.5.1 --- Water-holding capacity --- p.23
Chapter 1.5.2 --- Viscosity --- p.24
Chapter 1.5.3 --- Adsorption or entrapment of organic molecules --- p.25
Chapter 1.5.4 --- Fermentability --- p.25
Chapter 1.6 --- Hypocholesterolemic effect of SDF and proposed mechanisms --- p.26
Chapter 1.7 --- Medicinal properties of edible mushrooms --- p.28
Chapter 1.7.1 --- Background information --- p.28
Chapter 1.7.2 --- Hypocholesterolemic effect of edible mushrooms --- p.29
Chapter 1.7.3 --- Previous studies on edible jelly mushrooms --- p.31
Chapter 1.8 --- Objectives
Chapter Chapter 2: --- Materials and Methods --- p.34
Chapter 2.1 --- Materials --- p.34
Chapter 2.1.1 --- Sample preparation --- p.34
Chapter 2.1.2 --- Animal model --- p.35
Chapter 2.2 --- Methods --- p.35
Chapter 2.2.1 --- Extraction scheme of mushroom water-soluble non-starch polysaccharides (SNSPs) --- p.35
Chapter 2.2.2 --- Proximate analyses of samples --- p.36
Chapter 2.2.2.1 --- Crude protein --- p.36
Chapter 2.2.2.2 --- Fat --- p.37
Chapter 2.2.2.3 --- Total dietary fiber --- p.38
Chapter 2.2.2.4 --- Soluble and insoluble dietary fiber --- p.39
Chapter 2.2.2.5 --- Ash --- p.40
Chapter 2.2.2.6 --- Moisture --- p.41
Chapter 2.2.3 --- Chemical characterization of mushroom SNSPs --- p.41
Chapter 2.2.3.1 --- Monosaccharide composition by gas chromatography --- p.41
Chapter 2.2.3.2 --- Total carbohydrate content --- p.44
Chapter 2.2.3.3 --- Uronic acid content --- p.44
Chapter 2.2.3.4 --- Soluble protein content --- p.45
Chapter 2.2.4 --- Rheological study of mushroom SNSPs --- p.46
Chapter 2.2.4.1 --- Determination of intrinsic viscosity [ η] of mushroom SNSPs --- p.46
Chapter 2.2.4.2 --- Determination of apparent viscosity [ηap] of mushroom SNSPs --- p.48
Chapter 2.2.5 --- In vivo study --- p.50
Chapter 2.2.5.1 --- Animal diets --- p.50
Chapter 2.2.5.1.1 --- Study for hypocholesterolemic potential of mushroom SNSPs --- p.50
Chapter 2.2.5.1.2 --- Study for dose-dependent effect on hypocholesteolemic potential of Auricularia polytricha (AP) SNSP --- p.50
Chapter 2.2.5.2 --- Feeding experiments --- p.51
Chapter 2.2.5.2.1 --- Screening for hypocholesterolemic potential of mushroom SNSPs --- p.51
Chapter 2.2.5.2.2 --- Dose-dependent effect on hypocholesterolemic potential of AP SNSP --- p.52
Chapter 2.2.5.3 --- Blood samples collection --- p.52
Chapter 2.2.5.4 --- Plasma preparation --- p.53
Chapter 2.2.5.5 --- Liver samples collection and preparation --- p.53
Chapter 2.2.5.6 --- Fecal samples collection and preparation --- p.53
Chapter 2.2.5.7 --- Determination of plasma lipid profiles --- p.54
Chapter 2.2.5.7.1 --- Plasma total cholesterol (TC) analysis --- p.54
Chapter 2.2.5.7.2 --- Plasma high-density lipoprotein cholesterol (HDL-C) analysis --- p.54
Chapter 2.2.5.7.3 --- Plasma triglycerides (TG) analysis --- p.55
Chapter 2.2.5.8 --- Determination of hepatic cholesterol profile by gas chromatography --- p.56
Chapter 2.2.5.9 --- Determination of hepatic enzymes activity --- p.58
Chapter 2.2.5.9.1 --- Preparation of hepatic microsomes --- p.58
Chapter 2.2.5.9.2 --- Determination of 3-hydroxy-3-methyl- glutaryl-Coenzyme A reductase (HMG-CoA reductase) activity --- p.58
Chapter 2.2.5.10 --- Determination of fecal lipid profiles by gas chromatography --- p.61
Chapter 2.2.5.10.1 --- Separation of fecal neutral and acidic sterols --- p.61
Chapter 2.2.5.10.2 --- Fecal neutral sterol analysis --- p.61
Chapter 2.2.5.10.3 --- Fecal acidic sterol analysis --- p.62
Chapter 2.2.6 --- Statistical analysis --- p.63
Chapter Chapter 3: --- Results and Discussion --- p.65
Chapter 3.1 --- Proximate analysis of edible jelly mushrooms --- p.65
Chapter 3.2 --- Yield of mushroom SNSP crude extracts --- p.67
Chapter 3.3 --- Chemical characterization of mushroom SNSPs --- p.68
Chapter 3.3.1 --- Total carbohydrate content --- p.68
Chapter 3.3.2 --- Uronic acid content --- p.68
Chapter 3.3.3 --- Soluble protein content --- p.68
Chapter 3.3.4 --- Monosaccharide composition --- p.69
Chapter 3.4 --- Rheological behavior of mushroom SNSPs --- p.71
Chapter 3.4.1 --- Intrinsic viscosity [η] --- p.71
Chapter 3.4.2 --- Apparent viscosity [ηap] --- p.75
Chapter 3.5 --- In vivo hypocholesterolemic potential of mushroom SNSPs --- p.78
Chapter 3.5.1 --- Effect on body weight and diet intake --- p.79
Chapter 3.5.2 --- Effect on plasma TC concentration --- p.81
Chapter 3.5.3 --- Effect on plasma HDL-C concentration --- p.84
Chapter 3.5.4 --- Effect on plasma TG concentration --- p.86
Chapter 3.5.5 --- Effect on hepatic cholesterol profile --- p.89
Chapter 3.5.6 --- Effect on HMG-CoA reductase activity by AA and AP SNSPs --- p.92
Chapter 3.5.7 --- Effect on neutral and acidic sterols excretion by AA and AP SNSPs --- p.93
Chapter 3.5.8 --- Correlation between hypocholesterolemic potential and viscosity of mushroom SNSPs --- p.97
Chapter 3.6 --- In vivo dose-dependent effect on hypocholesterolemic potential of AP SNSP --- p.99
Chapter 3.6.1 --- Effect on body weight and diet intake --- p.100
Chapter 3.6.2 --- Effect on plasma TC concentration --- p.102
Chapter 3.6.3 --- Effect on plasma HDL-C concentration --- p.105
Chapter 3.6.4 --- Effect on plasma TG concentration --- p.107
Chapter 3.6.5 --- Effect on hepatic cholesterol profile --- p.110
Chapter 3.6.6 --- Effect on HMG-CoA reductase activity --- p.113
Chapter 3.6.7 --- Effect on neutral and acidic sterols excretion --- p.114
Chapter 3.6.8 --- Correlation between dosage and hypocholesterolemic effect of AP SNSP --- p.119
Chapter Chapter 4: --- Conclusions and Future works --- p.121
List of References --- p.124
Related Publications --- p.149

Wong Ka-kei.
Thesis submitted in: December 2004.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 123-139).
Abstracts in English and Chinese.
THESIS COMMITTEE --- p.i
ACKNOWNLEDGEMENT --- p.ii
ABSTRACT (ENGLISH VERSION) --- p.iii
ABSTRACT (CHINESE VERSION) --- p.v
LIST OF TABLES --- p.ix
ABBREVIATIONS --- p.xiii
Chapter CHAPTER 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Edible mushrooms --- p.1
Chapter 1.1.1 --- Classification and terminology --- p.1
Chapter 1.1.2 --- Mode of nutrition --- p.3
Chapter 1.1.3 --- World consumption --- p.3
Chapter 1.1.4 --- Nutritional values of edible mushroom --- p.6
Chapter 1.1.5 --- Medicinal values of mushrooms --- p.7
Chapter 1.2 --- Mushroom mycelium --- p.11
Chapter 1.2.1 --- Uses and applications --- p.11
Chapter 1.2.2 --- Submerged fermentation (SmF) --- p.12
Chapter 1.2.3 --- Factors affecting the growth of mycelium in submerged fermentation --- p.14
Chapter 1.2.3.1 --- Nutritional requirements - Carbon sources --- p.14
Chapter 1.2.3.2 --- Nutritional requirements ´ؤ Nitrogen sources --- p.16
Chapter 1.2.3.3 --- Nutritional requirements ´ؤ Minerals --- p.16
Chapter 1.2.3.4 --- Environmental factors ´ؤ Temperature --- p.17
Chapter 1.2.3.5 --- Environmental factors - Aeration --- p.17
Chapter 1.2.3.6 --- Environmental factors - Agitation --- p.18
Chapter 1.2.4 --- Optimization of growth of mycelium and production of EPS --- p.18
Chapter 1.3 --- Mushroom polysaccharides --- p.21
Chapter 1.3.1 --- Biologically active mushroom polysaccharides --- p.21
Chapter 1.3.2 --- Chemical structures of mushroom polysaccharides --- p.21
Chapter 1.3.2.1 --- β-glucans --- p.23
Chapter 1.3.2.2 --- α-glucans --- p.25
Chapter 1.3.2.3 --- Mannans --- p.26
Chapter 1.3.2.4 --- Protein-bound polysaccharides --- p.26
Chapter 1.3.2.5 --- Other heteroglycans --- p.28
Chapter 1.4 --- Mushrooms under investigation --- p.28
Chapter 1.4.1 --- Pleurotus tuber-regium (Fr.) Sing. (PTR) --- p.28
Chapter 1.4.2 --- Agrocybe cylindracea (AC) --- p.30
Chapter 1.4.3 --- Grifola frondosa (GF) --- p.31
Chapter 1.5 --- Objectives and experimental design --- p.32
Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.35
Chapter 2.1 --- Source of mushroom mycelium --- p.35
Chapter 2.2 --- Effect of different carbon sources on submerged fermentation --- p.37
Chapter 2.2.1 --- Production of mycelium by submerged fermentation using 250 mL and 1L shake-flasks --- p.37
Chapter 2.2.2 --- Scale-up production of mycelium of PTR using fermentor --- p.39
Chapter 2.2.3 --- Concentration of dissolved oxygen in 250 mL and 1L shake-flasks. --- p.39
Chapter 2.3 --- Isolation and fractionation of mushroom polysaccharides --- p.40
Chapter 2.3.1 --- Isolation of exo-polysaccharides (EPS) from culture medium by ethanol precipitation --- p.40
Chapter 2.3.2 --- Isolation of EPS from culture medium by ultra-filtration --- p.40
Chapter 2.3.3 --- Hot water extraction of PTR mycelium --- p.41
Chapter 2.3.4 --- Fractionation of HWE by fractional ethanol precipitation --- p.41
Chapter 2.4 --- Chemical composition of HWE and EPS --- p.42
Chapter 2.4.1 --- Phenol-sulphuric acid method --- p.42
Chapter 2.4.2 --- Modified Lowry method --- p.43
Chapter 2.4.3 --- Monosaccharide composition analysis of HWE and EPS --- p.43
Chapter 2.4.3.1 --- Acid depolymerization --- p.43
Chapter 2.4.3.2 --- Neutral sugar derivatization --- p.44
Chapter 2.4.3.3 --- Determination of neutral sugar composition by gas chromatography (GC) --- p.45
Chapter 2.4.3.4 --- Uronic acid content --- p.46
Chapter 2.5 --- Structural studies of HWE and EPS --- p.47
Chapter 2.5.1 --- High Pressure Liquid Chromatography (HPLC) --- p.47
Chapter 2.5.2 --- Methylation study and gas chromatography- mass spectrometry (GC-MS) --- p.48
Chapter 2.5.2.1 --- Preparation of dry dimethyl sulfoxide (DMSO) --- p.48
Chapter 2.5.2.2 --- Preparation of methylsulfinyl methyl sodium (CH3SOCH2-Na+) --- p.48
Chapter 2.5.2.3 --- Methylation --- p.49
Chapter 2.5.2.4 --- Extraction of methylated polysaccharide --- p.49
Chapter 2.5.2.5 --- Acid depolymerization and preparation of aditol acetate derivatives --- p.50
Chapter 2.5.2.6 --- Determination of partially methylated alditol acetates (PMAAs) by gas chromatography-mass spectrometry (GC-MS) --- p.50
Chapter CHAPTER 3 --- RESULTS AND DISCUSSION --- p.51
Chapter 3.1 --- "Production of mycelium and EPS of PTR, AC and GF by submerged fermentation in 250 mL shake-flask with liquid medium containing different carbon sources" --- p.51
Chapter 3.1.1 --- "Mycelial biomass production of PTR, AC and GF" --- p.51
Chapter 3.1.2 --- "Production of EPS of PTR, AC and GF" --- p.57
Chapter 3.1.3 --- "Characterization of EPS of PTR, AC and GF" --- p.62
Chapter 3.1.3.1 --- Carbohydrate and protein content --- p.62
Chapter 3.1.3.2 --- Monosaccharide composition --- p.67
Chapter 3.1.4 --- Summary --- p.72
Chapter 3.2 --- "Production of mycelium, EPS of PTR by submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources" --- p.75
Chapter 3.2.1 --- Mycelial production of PTR --- p.75
Chapter 3.2.2 --- EPS Production of PTR --- p.80
Chapter 3.2.3 --- Chemical characteristics of EPS of PTR --- p.83
Chapter 3.2.3.1 --- Carbohydrate and protein content --- p.83
Chapter 3.2.3.2 --- Monosaccharide composition --- p.85
Chapter 3.2.4 --- Structural characteristics of EPS of PTR --- p.87
Chapter 3.2.4.1 --- Molecular weight of EPS of PTR by HPLC --- p.87
Chapter 3.2.4.2 --- Glycosyl linkages of EPS of PTR by GC-MS of PMAA --- p.90
Chapter 3.2.5 --- Summary --- p.93
Chapter 3.3 --- Hot water extraction of mycelium of PTR from the scale-up submerged fermentation in 1L shake-flask and 8L fermentor with liquid medium containing different carbon sources --- p.95
Chapter 3.3.1 --- Yield of hot water extract (HWE) of mycelium of PTR --- p.95
Chapter 3.3.2 --- Chemical characteristics of HWE of PTR --- p.101
Chapter 3.3.2.1 --- Carbohydrate and protein content --- p.101
Chapter 3.3.2.2 --- Monosaccharide composition --- p.104
Chapter 3.3.3 --- Structural characteristics of HWE of PTR --- p.112
Chapter 3.3.3.1 --- Molecular weight of HWE of PTR by HPLC --- p.112
Chapter 3.3.3.2 --- Glycosyl linkages of HWE of PTR by GC-MS ofPMAA --- p.116
Chapter 3.3.4 --- Summary --- p.119
Chapter CHAPTER 4 --- CONCLUSIONS AND FUTURE WORKS --- p.120
Chapter 4.1 --- Conclusions --- p.120
Chapter 4.2 --- Future works --- p.121
REFERENCES --- p.123

by Wang He-Xiang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1996.
Includes bibliographical references (leaves 161-179).
ACKNOWLEDGEMENTS --- p.i
ABSTRACT --- p.ii
LIST OF CONTENTS --- p.v
LIST OF TABLES --- p.xi
LIST OF FIGURES --- p.xii
LIST OF ABBREVIATIONS --- p.xvi
Chapter CHAPTER 1. --- General Introduction --- p.1
Chapter CHAPTER 2. --- Literature Review --- p.5
Chapter 2.1. --- Lectins --- p.5
Chapter 2.1.1. --- Aspects of lectins --- p.5
Chapter 2.1.2. --- Isolation and purification of lectins --- p.8
Chapter 2.1.3. --- Characteristics of lectins --- p.9
Chapter 2.1.4. --- Effects of lectins on biological activities --- p.10
Chapter 2.1.4.1. --- The role of lectins in plant defence --- p.11
Chapter 2.1.4.2. --- The specificity of some legume lectins --- p.13
Chapter 2.1.4.3. --- Some properties of animal lectins --- p.14
Chapter 2.1.4.4. --- Hypotensive activity of the lectins --- p.18
Chapter 2.1.4.5. --- Lectins in immunology --- p.20
Chapter 2.2. --- Mushroom Lectins and Polysaccharides --- p.24
Chapter 2.2.1. --- General aspects of mushroom lectins and polysaccharides --- p.24
Chapter 2.2.2. --- Mushroom lectins --- p.25
Chapter 2.2.2.1. --- Hericium erinaceum lectin --- p.26
Chapter 2.2.2.2. --- Lactarius deterrimus lectin --- p.26
Chapter 2.2.2.3. --- Laetiporus sulfureus lectin --- p.27
Chapter 2.2.2.4. --- Grifola frondosa lectin --- p.28
Chapter 2.2.2.5. --- Volvariella volvacea lectin --- p.28
Chapter 2.2.2.6. --- Flammulina veltipes lectin --- p.29
Chapter 2.2.2.7. --- Ischnoderma resinosum agglutinin --- p.31
Chapter 2.2.2.8. --- Lectins from Agaricus spp --- p.31
Chapter 2.2.3. --- Mushroom polysaccharides --- p.34
Chapter 2.2.3.1. --- Lentinan --- p.35
Chapter 2.2.3.2. --- "PSK (trade name, Krestin)" --- p.35
Chapter 2.2.3.3. --- PSP (Polysaccharopeptide) --- p.37
Chapter 2.2.3.4. --- PSPC (polysaccharide-peptide complex) --- p.38
Chapter CHAPTER 3. --- Isolation and Characterization of Two Distinct Lectins from the Cultured Mycelium of the Edible Mushroom Tricholoma mongolicum --- p.44
Chapter 3.1. --- Introduction --- p.44
Chapter 3.2. --- Materials and Methods --- p.45
Chapter 3.2.1. --- Strain and culture condition --- p.45
Chapter 3.2.2. --- Extraction --- p.46
Chapter 3.2.3. --- Purification --- p.46
Chapter 3.2.4. --- Hemagglutination activity --- p.47
Chapter 3.2.5. --- Test of hemagglutination inhibition by various carbohydrates --- p.47
Chapter 3.2.6. --- MW estimation by gel filtration and SDS- PAGE --- p.48
Chapter 3.2.7. --- Glycoprotein staining with PAS reagent --- p.49
Chapter 3.2.8. --- Carbohydrate content --- p.49
Chapter 3.2.9. --- Thermal stability --- p.49
Chapter 3.2.10. --- pH stability --- p.49
Chapter 3.2.11. --- Effect of cations --- p.50
Chapter 3.2.12. --- Amino acid analysis --- p.50
Chapter 3.2.13. --- Antiproliferative activity of lectins --- p.50
Chapter 3.2.14. --- Statistics --- p.51
Chapter 3.3. --- Results --- p.51
Chapter 3.3.1. --- Extraction and purification --- p.51
Chapter 3.3.2. --- General characteristics of lectins --- p.52
Chapter 3.3.3. --- Antiproliferative activity of lectins --- p.54
Chapter 3.4. --- Discussion --- p.55
Chapter 3.5. --- Summary --- p.58
Chapter CHAPTER 4. --- The Immunomodulatory and Antitumor Activities of Lectins from the Mushroom Tricholoma mongolicum --- p.79
Chapter 4.1. --- Introduction --- p.79
Chapter 4.2. --- Materials and Methods --- p.81
Chapter 4.2.1. --- Lectins --- p.81
Chapter 4.2.2. --- Animals --- p.81
Chapter 4.2.3. --- Assay for antitumor activity --- p.81
Chapter 4.2.4. --- Assessment of tumor growth and host survival after lectin treatment --- p.82
Chapter 4.2.5. --- Mitogenic activity of lectins --- p.82
Chapter 4.2.6. --- Production of nitrite ions in response to lectin treatment --- p.83
Chapter 4.2.7. --- Preparation of concanavalin A-stimulated lymphokines --- p.84
Chapter 4.2.8. --- Assay for macrophage activating factor --- p.85
Chapter 4.2.9. --- Production of tumor necrosis factor (TNF) --- p.86
Chapter 4.2.10. --- Bioassay for tumor necrosis factor --- p.86
Chapter 4.2.11. --- Statistics --- p.87
Chapter 4.3. --- Results --- p.87
Chapter 4.3.1. --- Antitumor activity --- p.87
Chapter 4.3.2. --- Assessment of tumor growth and host survival --- p.87
Chapter 4.3.3. --- Mitogenic activity --- p.88
Chapter 4.3.4. --- Production of nitrite ions --- p.89
Chapter 4.3.5. --- Production of macrophage activating factor --- p.89
Chapter 4.3.6. --- Tumor necrosis factor assay --- p.90
Chapter 4.4. --- Discussion --- p.90
Chapter 4.5. --- Summary --- p.94
Chapter CHAPTER 5. --- Hypotensive and Vasorelaxing Activities of a Lectin (TML-1) from the Edible Mushroom Tricholoma mongolicum --- p.109
Chapter 5.1. --- Introduction --- p.109
Chapter 5.2. --- Materials and Methods --- p.111
Chapter 5.2.1. --- Animals --- p.111
Chapter 5.2.2. --- In vivo blood pressure measurement in rats --- p.112
Chapter 5.2.3. --- Study employing blockade of autonomic ganglion transmission --- p.113
Chapter 5.2.4. --- Study employing alpha-adrenergic blockade --- p.113
Chapter 5.2.5. --- Study employing beta-adrenergic blockade --- p.114
Chapter 5.2.6. --- Study employing cholinergic blockade --- p.114
Chapter 5.2.7. --- Study employing histaminergic blockade --- p.114
Chapter 5.2.8. --- Study employing inhibitor of the renin- angiotensin system --- p.115
Chapter 5.2.9. --- Preparation of right atrium for in vitro studies --- p.115
Chapter 5.2.10. --- Preparation of aorta for in vitro studies --- p.116
Chapter 5.2.11. --- Adenosine receptor binding assays --- p.116
Chapter 5.2.12. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.118
Chapter 5.2.13. --- Statistics --- p.118
Chapter 5.3. --- Results --- p.118
Chapter 5.3.1. --- Blood pressure changes in vivo --- p.118
Chapter 5.3.2. --- Pharmacological studies using receptor antagonists --- p.119
Chapter 5.3.3. --- Adenosine receptor binding assay --- p.119
Chapter 5.3.4. --- Effects on the right atrium in vitro --- p.120
Chapter 5.3.5. --- Effect of TML-1 on vascular relaxation --- p.120
Chapter 5.3.6. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.120
Chapter 5.4. --- Discussion --- p.120
Chapter 5.5. --- Summary --- p.123
Chapter CHAPTER 6. --- A Polysaccharide-Peptide Complex with Immunoenhancing and Antitumor Activities from Cultured Mycelia of the Mushroom Tricholoma mongolicum --- p.134
Chapter 6.1. --- Introduction --- p.134
Chapter 6.2. --- Materials and Methods --- p.135
Chapter 6.2.1. --- Extraction --- p.135
Chapter 6.2.2. --- Purification --- p.135
Chapter 6.2.3. --- PSP for purpose of comparison --- p.136
Chapter 6.2.4. --- Polysaccharide and protein contents --- p.136
Chapter 6.2.5. --- MW determination of F1 using gel filtration --- p.136
Chapter 6.2.6. --- Animals --- p.136
Chapter 6.2.7. --- Antiproliferative activity assay --- p.137
Chapter 6.2.8. --- Mitogenic activity --- p.137
Chapter 6.2.9. --- Production of nitrite ions --- p.138
Chapter 6.2.10. --- Macrophage activating factor assay --- p.138
Chapter 6.2.11. --- Antitumor activity assay --- p.139
Chapter 6.2.12. --- Statistics --- p.139
Chapter 6.3. --- Results --- p.140
Chapter 6.3.1. --- Purification of polysaccharide-peptide complex --- p.140
Chapter 6.3.2. --- Antiproliferative activity --- p.140
Chapter 6.3.3. --- Mitogenic activity in vitro --- p.140
Chapter 6.3.4. --- Molecular weight of Fl --- p.141
Chapter 6.3.5. --- Mitogenic activity in vivo --- p.141
Chapter 6.3.6. --- Production of nitrite ions --- p.141
Chapter 6.3.7. --- Production of macrophage activating factor --- p.141
Chapter 6.3.8. --- Antitumor activity in vivo --- p.142
Chapter 6.4. --- Discussion --- p.142
Chapter 6.5. --- Summary --- p.144
GENERAL DISCUSSION --- p.155
CONCLUSIONS --- p.158
REFERENCES

碩士
慈濟科技大學
放射醫學科學研究所
106
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.

Dissertação de Mestrado Integrado em Engenharia Química apresentada à Faculdade de Ciências e Tecnologia
Presently, the rapid rate of growth of the affected population with premature aging and numerous diseases is evident being cancer and neurodegenerative diseases, such as Alzheimer's, Parkinson's and amyotrophic lateral sclerosis, the most prevalent and requiring more attention. One of the main causes for the onset of these diseases is the uncontrolled production of reactive oxygen species (ROS), derived from an excessive production of free radicals. In order to reduce oxidative damage and considering the restrictions on the use of synthetic antioxidants, there is an increasing demand for natural products that contain a high antioxidant potential.Among the existing natural products, mushrooms have been the subject of countless studies, due to their beneficial effects on health in addition to their nutritional properties. These are fungi quite rich in bioactive compounds, such as polysaccharides, phenolic compounds, vitamins and secondary metabolites. Some of the referred compounds have numerous antioxidant properties that are very beneficial to the organism. This master's thesis project is focused on the study of the Tricholoma equestre mushroom species, which has not been much studied until now. The scope of the work was to investigate the effect of polysaccharides and of phenolic compounds, extracted from Tricholoma equestre species, on the production of ROS in neuronal cells. The experiments were performed in brain slices from Wistar rats, at the mossy fiber synapses from CA3 hippocampal area. Polysaccharides and phenolic compounds extracts obtained from the Tricholoma mushroom, were used to prepare different solutions with concentrations of 0.1, 0.5 and 1 g.L-1, by adding the extracts to the artificial cerebrospinal fluid (ACSF), which has the composition of the neuronal extracellular medium. The aim of this part of the work was to study the effect of those compounds on neuronal ROS production using the fluorescent ROS indicator H2DCFDA.The results show that the lower polysaccharides concentrations (0.1 and 0.5 g.L-1) did not cause significant ROS changes while the higher dose (1 g.L-1) led to a decrease of the ROS signals of about 2.7 % with respect to the baseline. The experiments carried out with the phenolic compounds solutions gave more accentuated changes that were opposite to the previous one. The larger increases in ROS production were observed for the concentrations of 0.1 and 1 g.L-1, having the fluorescence signals amplitudes of about 18 % and 14 %, respectively, while for 0.5 g.L-1 the enhancement was approximately 6 %. These facts indicate that the phenolic compounds have a peculiar concentration dependent effect, occurring the larger ROS changes for the lower concentration, 0.1 g. L-1, whose signals have a similar profile to that of the higher concentration, 1 g. L-1.The last group of experiments was made with the synthetic compound, 4-hydroxybenzoic acid (1 g.L-1), which is the only identified phenolic compound of the studied mushroom species. The results indicate that this acid causes similar changes to those observed for the same concentration of the phenolics extract, with a maximum value of about 14 % upon the removal of the acid.In conclusion, and due to the obtained results, it is undeniable that mushrooms have antioxidant properties that affect the performance of cellular metabolism, acting on ROS signals and triggering different responses of the organism to these stimuli.
Atualmente, é evidente a rápida taxa de crescimento da população afetada com envelhecimento prematuro e por numerosas doenças como o cancro e doenças neurodegenerativas, tais como as doenças de Alzheimer, de Parkinson e a esclerose lateral amiotrópica, que são as mais frequentes e que requerem mais atenção. Uma das causas principais para o início destas doenças é a produção descontrolada de espécies reativas de oxigénio (ROS), derivadas da produção excessiva de radicais livres. De forma a reduzir danos oxidativos e considerando a restrição no uso de antioxidantes sintéticos, verifica-se uma procura crescente de produtos naturais com um elevado potencial antioxidante. Entre os produtos naturais existentes os cogumelos têm sido objeto de inúmeros estudos devido aos seus efeitos benéficos na saúde para além das suas propriedades nutricionais. Estes são fungos muito ricos em compostos bioativos, tais como polissacarídeos, compostos fenólicos, vitaminas e metabolitos secundários. Alguns dos compostos referidos tem muitas propriedades antioxidantes que são muito benéficas para o organismo. Este projeto de tese de mestrado foca-se no estudo da espécie de cogumelos Tricholoma equestre que, até à data, não tem sido muito estudado. O objetivo do estudo era investigar o efeito de polissacarídeos e de compostos fenólicos, extraídos da espécie Tricholoma equestre, na produção de ROS em células neuronais. As experiências foram realizadas em fatias cerebrais de ratos Wistar nas sinapses das fibras musgosas da área CA3 do hipocampo. Extratos de polisacarídeos e de compostos fenólicos, obtidos do cogumelo Tricholoma, foram usados para preparar diferentes concentrações de 0.1, 0.5 e 1 g.L-1, adicionando os extratos ao fluído cerebroespinal artificial (ACSF), que tem a composição do meio neuronal extracelular. O objetivo desta parte do trabalho era estudar o efeito daqueles compostos na produção de ROS neuronal usando o indicador de ROS fluorescente H2DCFDA.Os resultados mostram que as concentrações mais baixa de polissacarídeos (0.1 and 0.5 g.L-1) não causaram variações significativas de ROS enquanto que a dose mais alta (1 g.L-1) levou a uma diminuição dos sinais de ROS de cerca de 2.7 % em relação à linha base. As experiências realizadas com as soluções de compostos fenólicos originaram variações mais acentuadas que eram opostas à anterior. Os maiores aumentos na produção de ROS foram observados para as concentrações de 0.1 e 1 g.L-1, tendo os sinais de fluorescência respetivamente amplitudes de cerca de 18 % e 14 %, enquanto que para 0.5 g.L-1 o aumento era aproximadamente 6 %. Estes factos indicam que os compostos fenólicos têm um efeito dependente da concentração que é peculiar, ocorrendo as maiores variações de ROS para a menor concentração, 0.1 g.L-1, cujos sinais têm um perfil semelhante aos da concentração mais elevada, 1 g. L-1. O último grupo de experiências foi feito com o composto sintético 4-hydroxybenzoic acid (1 g.L-1), que é o único composto fenólico identificado na espécie de cogumelos estudada. Os resultados indicam que este ácido causa variações de ROS semelhantes às que foram observadas para a mesma concentração do extrato de fenólicos, com um valor máximo de cerca de 14 % após a retirada do ácido.Em conclusão, e pelos resultados obtidos, é inegável que os cogumelos possuem propriedades antioxidantes que afetam o desempenho de metabolismo celular, atuando nos sinais de ROS e desencadeando diferentes respostas do organismo a estes estímulos.
Universidade de Coimbra - Compra de materiais, reagentes e equipamentos referentes a realização experimental da dissertação de mestrado.

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

源自菌菇的具有生物活性的多糖(含β-葡聚糖)能有效調節先天性免疫,早在二十世紀末就被應用于臨床。現有關於菌菇類β-葡聚糖的免疫調節活性以及其結構-活性之間的關係的研究相對匱乏，因此限制了臨床應用。巨噬細胞遍佈宿主的全身，是先天性免疫中主要的細胞。本研究將從藥用菌核虎乳靈芝(PR)中提取純化出具有新穎結構的多糖，并研究其對巨噬細胞的免疫活性。本研究亦試圖比較不同來源的β-葡聚糖在結構和活性方面的差異以更好地闡述其結構-活性的關係。
通過用熱水提取和冷鹼(1M NaOH)依次提取虎乳靈芝的多糖組分，得到水溶性組分(PRW) 和三種鹼溶性組分(PRA-1, PRA-2A和PRA-2C)。從這些組分中，PRW經過超濾純化得到新穎的糖蛋白複合物PRW1，顆粒化PRA-1(PRA-1p)也成功製備出。
PRW的多糖部分是由葡萄糖和甘露糖構成的雜多糖。PRW1分子量小於50 kDa，由45.7% 的雜多糖和44.2% 蛋白质構成的糖蛋白複合物。根據單糖組成，糖鏈結構，和紅外光譜分析的結果，PRW1的多糖部分b-D-mannoglucan由→1)-D-Glcp-(4→, →1)-D-Glcp-(6→ and →1)-D-Manp-(2→主鏈 (摩爾比例為5:4:6) 和末端D-Glcp支鏈構成 (分支度是0.62)。
PRA-1, PRA-2A和PRA-2C具有類似的化學結構，均為超多分枝 (1,3)-β-D-葡聚糖, 分枝位點在葡聚糖的O-6位，分支度分別是 0.89，0.87， 0.89。根據凝膠滲透色譜的結果，PRA-1, PRA-2A和PRA-2C 平均分子量為 24190, 28350 和 15950 kDa。掃描電鏡和透射電鏡的結果顯示，PRA-1p呈現均勻的球狀結構 (半徑範圍 0.5-1 微米)，而PRA-1呈現棒狀結構 (長度範圍0.1-1 微米)。
非虎乳靈芝來源的β-葡聚糖中，大麥β-葡聚糖是通過b-(1,3)-(1,4)連接。羧甲基化curdlan (CM-curdlan)， curdlan 和 pachyman 均是線性b-(1,3)-葡聚糖，laminarin和酵母葡聚糖均是由b-(1,3)主鏈和b-(1,6)支鏈構成，分支度分別是 0.3 和0.16。CM-curdlan, curdlan, 大麥β-葡聚糖, laminarin，pachyman和酵母葡聚糖的分子量依次是4378.6 kDa，3270.6 kDa，444.2 kDa，9.5 kDa，2.2 kDa，2.3 kDa。
體外免疫調節研究表示，PRA-1p， PRW and PRW1，CM-curdlan, curdlan and酵母葡聚糖能顯著激活鼠源性RAW264.7 巨噬細胞。
PRW1顯著增強RAW264.7中一氧化氮的產生，并引起RAW264.7形態改變。PRW1亦能刺激RAW264.7分泌細胞因子(包括G-CSF, GM-CSF, IL-6, IL12p40/70，MCP-1，MCP-5，MIP-1-α，MIP-2，RANTES，sTNFRI and TNF-α)。機制上來說，PRW1的處理在15分鐘內通過激活ERK來激活巨噬細胞，處理6小時后細胞內誘導性氧化氮合酶 (iNOS) 的表達顯著上調。
PRA-1p 能顯著增強RAW264.7中一氧化氮和活性氧的產生，引起RAW264.7形態改變，使細胞吞噬更多FITC標記的葡聚糖，表示了顯著提高巨噬細胞的吞噬能力。PRA-1p刺激RAW264.7顯著分泌更多細胞因子(包括CTACK，G-CSF，MCP-1，MIP-1α，MIP-2，RANTES，sTNFRI TIMP-1)。PRA-1p可能通過引起包括iNOS，NF-κB，ERK 和 AKT的活化，從而活化巨噬細胞。
CM-curdlan, curdlan 和 酵母葡聚糖能誘導人外周血單個核細胞分泌細胞因子(包括GM-CSF, GRO 和IL-6) 從而發揮免疫調節作用。
通過對比虎乳靈芝菌核多糖和非虎乳靈芝來源的葡聚糖在結構以及對小鼠巨噬細胞的免疫活性方面的差異，總結出b-(1,3)連接的葡聚糖較b-(1,3;1,4)連接的葡聚糖具有更強的免疫活性。對於b-(1,3)連接的葡聚糖，其線性結構和分枝結構以及分子量大小和水溶性都能影響葡聚糖對小鼠巨噬細胞的免疫活性。本研究亦揭示了顆粒結構對於增強b葡聚糖免疫活性的重要性。本研究為葡聚糖的結構免疫活性構效關係提供了重要的思路，並且為設計用於臨床的具免疫治療活性的b葡聚糖提供了方向。該研究中從虎乳靈芝菌核里提取的的新型糖蛋
Bioactive polysaccharides derived from mushrooms and fungi with b-glucans in particular are potent stimulants of innate immunity and have been used in clinical therapies since the late 20th century. The lack of detailed knowledge in the immunomodulating effects of b-glucans and their structure-activity relationship have limited their clinical applications. Macrophages, which are distributed throughout the body of the host, are the major effector cells of innate immunity. In this project, the sclerotia of a medicinal mushroom Polyporus rhinocerus (PR) was chosen from which novel structurally polysaccharides were isolated and purified and were used to study the immunomodulatory effect on macrophages. In order to delineate the structure-activity relationship, the macrophage activation by the PR mushroom polysaccharides with structural differences and b-glucans from other sources was compared in this project.
By differential solvent fractionation, a crude water-soluble fraction (PRW) and three alkali-soluble fractions (PRA-1, PRA-2A and PRA-2C) were obtained from the sclerotia of PR. Two specific samples from these fractions including a novel water-soluble polysaccharide-protein complex (PRW1) purified by membrane ultrafiltration from PRW using membrane ultrafiltration and a particulate form of PRA-1 (PRA-1p) were also prepared.
The polysaccharide moiety of PRW was a hyper-branched heteropolysaccharide which was composed of glucose and mannose. PRW1 had a molecular weight of less than 50 kDa and was found to be a highly branched heteropolysaccharide-protein complex with 45.7% polysaccharide and 44.2% protein. Based on the monosaccharide analysis, methylation study and Fourier transform infrared spectroscopy, the carbohydrate moiety of PRW1 was found to be a b-D-mannoglucan with its backbone containing →1)-D-Glcp-(4→, →1)-D-Glcp-(6→ and →1)-D-Manp-(2→ residues with a molar ratio of 5:4:6 and having terminal D-Glcp as side chain with a degree of branching of 0.62. PRA1 and PRA-2A and PRA-2C had similar structure which were hyper-branched β-glucans with a backbone composed of →1)-D-Glcp-(3→ linkages and side chains having the major branching points at the O-6 positions with a degree of branching of 0.89, 0.87 and 0.89, respectively. By gel permeation chromatograph (GPC), PRA-1, PRA-2A, PRA-2C were found to have an average molecular weight of 24190, 28350 and 15950 kDa,respectively. Results from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that PRA-1 and PRA-1p had a spherical conformation with diameter ranging from 0.5-1 μm and a rod-like structure with length ranging from 0.1-1 μm, respectively.
The chemical structure of the b-glucans derived from non-PR sources was determined by methylation analysis. It was found that barley b-glucan was a linear b-(1,3)-(1,4)-glucan; carboxymethyl (CM)-curdlan, curdlan and pachyman were linear b-(1,3)-glucan while laminarin and yeast b-glucan were b-(1,6)-branched b-(1,3)-glucan with a DB of 0.3 and 0.16, respectively. The molecular weight of these non-PR b-glucans determined by GPC was as follows: 4378.6 kDa for CM-curdlan, 3270.6 kDa for curdlan, 444.2 kDa for barley b-glucan, 9.5 kDa for laminarin, 2.2 kDa for yeast b-glucan and 2.3 kDa for pachyman.
In vitro immunomodulatory studies showed that PRA-1p, PRW and PRW1, CM-curdlan, curdlan and yeast b-glucan significantly activated murine macrophage RAW264.7.
Specifically, PRW1 significantly induced nitric oxide (NO) production and stimulated morphological changes in RAW264.7 cells. PRW1 treatment enhanced the release of a variety of cytokines including G-CSF, GM-CSF, IL-6, IL12p40/70, MCP-1, MCP-5, MIP-1-
In addition, CM-curdlan, curdlan and yeast b-glucan exhibited immunomodulatory effects on human peritoneal blood mononuclear cells (PBMCs) by inducing cytokine production including GM-CSF, GRO, IL-6.
By comparing the structure and the in vitro immumomodulatory effect onmurine macrophges of PR polysaccharides and the non-PR β-glucans, it was confirmed that the β-(1,3)-glucans showed macrophage-activating effect while mixed linkage β-(1,3;1,4)-gluca
Liu, Chaoran.
Thesis Ph.D. Chinese University of Hong Kong 2016.
Includes bibliographical references (leaves ).
Abstracts also in Chinese.
Title from PDF title page (viewed on …).
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.

碩士
中興大學
食品暨應用生物科技學系所
99
Some plant polysaccharides had been considered as having prebiotic effect which can selectively stimulate the growth of probiotics. The mushroom polysaccharides are well known for their immunomodulatory, anti-tumor and anti-cancer activity, however, litter information is available showing if they could be used as prebiotics. In this study, stipes of Lentinula edodes, bases of Pleurotus eryngii and Flammulina velutipes were used as raw materials for polysaccharides extraction. The effect of mushroom polysaccharides on viability of Lactobacillus acidophilus BCRC 10695, Lactobacillus casei BCRC14080 and Bifidobacterium longum BCRC 14664 under refrigerated storage and on their gastrointestinal tolerance were investigated. The viability of probiotics, proteolysis and angiotensin-converting enzyme (ACE) inhibitory activity of the fermented milk, which supplemented with mushroom polysaccharides were also investigated during cold storage. Results showed that mushroom polysaccharides enhanced survival of probiotics in MRS broth under refrigerated storage. Supplemented mushroom polysaccharides in lower concentrations (0.1-0.5% (w/w)) had better effect to enhance the probiotics viability than in higher concentration (1.0% (w/w)). The survival rate of probiotic bacteria in simulated gastric acid (pH 2.0) and bile juice (4.5%. pH 8.0) was higher compared to control group. Probiotic bacteria with 0.5% (w/w) mushroom polysaccharides supplementation improved their tolerance to such conditions. Supplementation with mushroom polysaccharides could keep the higher microbial counts of L. acidophilus BCRC 10695, improve viability of L. casei BCRC 14080 and retard the death of B. longum BCRC 14664 in fermented milk during cold storage. The presence of high functionality of proteolysis in fermented milk containing B. longum with 0.1 % (w/w) FVP (F. velutipes polysaccharide) at 28 days in cold storage was observed; 20% ACE inhibition was observed in the fermented milk In addition, the presence of prebiotics or storage period did not influence the ACE inhibitory activity. This study indicated that mushroom polysaccharides enhanced probiotics survival in MRS during cold storage and improve their tolerance in simulated gastric and bile juice. Supplementation with mushroom polysaccharides also increased the population of probiotics and functionality of the fermented milk, which suggest that mushroom polysaccharides are potential prebiotics.

碩士
國立屏東科技大學
動物科學與畜產系所
105
Abstract Student ID: M10226005 Title of Thesis: Effects of dietary supplementation of fermented byproduct and nano Zn enhanced mushroom polysaccharides complex on growth performances, carcass traits, and intestinal properties of chicks. Total Page: 137 Name of Institute: Department of Animal Science, National Pingtung University of Science and Technology Graduate Date: June, 2017 Degree Conferred: Master Name of Student: Pei-Chia Adviser: How-Hong Hsieh, Ph. D. The Contents of Abstract in This Thesis: The objectives of this study were to evaluate the effects of dietary supplementation of fermented byproduct and nano Zn enhanced mushroom polysaccharides complex on growth performance, carcass traits, and intestinal properties of broiler chicks. In Exp. 1, three hundred and fifty two day-old Avian broilers were randomly allotted into 4 treatments × 4 replications experimental design with 22 birds in each pen. In Exp. 1, the 4 treatment groups were: BD (basal diet), UP 2.5 (contain Upro 2.5%), UP 5.0 (contain Upro 5.0%) and FM 2.5 (contain fish meal 2.5%). In Exp. 2, three hundred and sixty day-old male red native chickens were randomly allotted into 3 diets (fish meal, upro, upro plus) × 2 nano Zn enhanced mushroom polysaccharides complex (add or not add) × 2 replications experimental design (FM2.5, FMW2.5, UP2.5, UPW2.5, UPP2.5, UPPW2.5). All experimental diets were equal in energy and crude protein content. Feed and water were provided ad libitum. In Exp. 1, birds were weighed individually and feed consumption were recorded at 14, 28 and 35 days of age. Four birds from each treatment were randomly selected at the end of growth trial for gut microflora test, carcass characteristics, jejunum and ileum mucosa morphology and serum biochemistry determination. In Exp. 2, birds were weighed individually and feed consumption were recorded at 28, 56 and 80 days of age. Six birds from each treatment were randomly selected at the end of growth trial for microflora test, carcass characteristics, jejunum and ileum mucosa morphology, and serum biochemistry determination. In Exp. 2, at 73 days of age six birds from each treatment were placed into individual cages for metabolic test. The result of Exp. 1 showed that the UP2.5% and FM2.5% groups were significantly increased in body weight, average daily feed intake, average daily gain, and improved in feed conversion ratio. The result of intestinal properties showed that the number of coliforms in the jejunum were significantly reduced in UP2.5% and UP5.0% groups, and lactobacilli counts were significantly increased in UP2.5% group. Moreover, the UP2.5%, UP5.0% and FM2.5% groups were significantly increased in villus height of jejunum and ileum, also significantly increased in ileum villus height/crypt depth ratio as compared to the BD group. The result of serum biochemistry showed that the UP2.5% and FM2.5% groups were significantly increased calcium concentration. The result of carcass characteristics were not significantly different among all groups. The results of Exp. 2 showed that the UPP2.5% and GZn water groups were significantly increased in body weight, average daily gain, and improved in feed conversion ratio, the protein retention rate of UPP2.5% and GZn water group were higher than other groups, however the nutrient utilization of ash were significantly increased in UPP2.5% and UP2.5% groups. The results of intestinal properties test showed that the UPP2.5% and GZn water groups were significantly increased in villus height of jejunum and ileum, also significantly increased in ileum villus height/crypt depth ratio. Moreover, the UPP2.5% and GZn water groups were significantly increased in numbers of lactobacilli in the jejunum, and coliforms counts were significantly reduced in UPP2.5% groups. The results of serum biochemistry test showed that the UP2.5%, UPP2.5% and GZn water groups were significantly reduced on serum total cholesterol, however, the alkaline phosphatase concentration were significantly reduced in GZn water groups. Moreover, the UPP2.5% groups were significantly increased on serum Newcastle disease antibody titers. The results of carcass characteristics showed that the GZn water groups were significantly increased on carcass yield, also, the relative spleen weight were significantly increased in UPP2.5% groups. The overall results indicated that the replacement of fish meal with Upro and nano Zn enhanced mushroom polysaccharides complex in chicken diets and water can save feed cost and improved growth performances, carcass traits, and intestinal properties. Key words: Chicken, Growth performance, Intestinal properties, Mushroom polysaccharides, Nano Zn

by Lee Man Yi.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (leaves 125-137).
Abstract also in Chinese.
THESIS COMMITTEE --- p.i
ACKNOWLEDGEMENTS --- p.ii
ABSTRACT --- p.iii
ABSTRACT (Chinese version) --- p.v
TABLE OF CONTENTS --- p.vi
LIST OF TABLES --- p.xi
LIST OF FIGURES --- p.xv
LIST OF ABBREVIATIONS --- p.xvii
Chapter CHAPTER ONE: --- INTRODUCTION --- p.1
Chapter 1.1 --- Background of mushrooms --- p.1
Chapter 1.1.1 --- Cultivated mushrooms --- p.1
Chapter 1.1.1.1 --- Volvariella volvacea --- p.3
Chapter 1.1.1.2 --- Pleurotus sajor-caju --- p.4
Chapter 1.1.1.3 --- Pleurotus tuber-regium --- p.5
Chapter 1.1.2 --- Chemical composition and nutritional value --- p.6
Chapter 1.1.3 --- Composition and structure of fungal cell wall --- p.9
Chapter 1.1.4 --- Medicinal attributes of β-glucan in mushrooms --- p.10
Chapter 1.1.5 --- Structure and antitumor activity β-glucan --- p.11
Chapter 1.2 --- Dietary fiber --- p.14
Chapter 1.2.1 --- Composition of dietary fiber --- p.14
Chapter 1.2.2 --- Preparation of dietary fiber --- p.18
Chapter 1.3 --- Structural analysis of polysaccharides --- p.19
Chapter 1.3.1 --- Isolation of polysaccharides --- p.19
Chapter 1.3.2 --- Methylation analysis --- p.20
Chapter CHAPTER TWO : --- MATERIALS AND METHODS --- p.22
Chapter 2.1 --- Sources and preparation of mushroom samples --- p.22
Chapter 2.1.1 --- V. volvacea --- p.22
Chapter 2.1.2 --- P. sajor-caju --- p.22
Chapter 2.1.2.1 --- Fungal strain --- p.22
Chapter 2.1.2.2 --- Production of spawn --- p.22
Chapter 2.1.2.3 --- Production of fruiting bodies --- p.23
Chapter 2.1.3 --- p. tuber-regium --- p.23
Chapter 2.2 --- Analysis of mushroom composition --- p.24
Chapter 2.2.1 --- Moisture content --- p.24
Chapter 2.2.2 --- Starch content --- p.24
Chapter 2.2.2.1 --- Total glucose --- p.24
Chapter 2.2.2.2 --- Free glucose --- p.25
Chapter 2.2.2.3 --- Measurement of glucose content --- p.25
Chapter 2.2.2.4 --- Total starch content --- p.25
Chapter 2.2.3 --- Crude protein content --- p.26
Chapter 2.2.4 --- Amino acid analysis --- p.27
Chapter 2.3 --- Preparation of mushroom fiber material --- p.28
Chapter 2.3.1 --- Enzymatic method --- p.28
Chapter 2.3.1.1 --- Total dietary fiber (TDF) --- p.28
Chapter 2.3.1.2 --- Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF) --- p.29
Chapter 2.3.2 --- Chemical method --- p.29
Chapter 2.3.2.1 --- Cell wall material --- p.29
Chapter 2.4 --- Chemical composition of mushroom fiber material --- p.31
Chapter 2.4.1 --- Monosaccharide composition of non-starch polysaccharides (NSP) --- p.31
Chapter 2.4.1.1 --- Acid depolymerisation --- p.31
Chapter 2.4.1.2 --- Neutral sugar derivatization --- p.31
Chapter 2.4.1.3 --- Determination of neutral sugars by gas chromatography (GC) --- p.32
Chapter 2.4.1.4 --- Uronic acid content --- p.32
Chapter 2.4.2 --- Resistant starch content --- p.33
Chapter 2.4.3 --- Residual protein content --- p.34
Chapter 2.5 --- Fractionation of mushroom fiber material --- p.34
Chapter 2.5.1 --- Solvent extraction --- p.34
Chapter 2.5.2 --- Anion-exchange chromatography --- p.35
Chapter 2.5.3 --- Gel permeation chromatography --- p.36
Chapter 2.6 --- Structural analysis of mushroom fiber material --- p.37
Chapter 2.6.1 --- Linkage analysis by methylation --- p.37
Chapter 2.6.1.1 --- Preparation of methylsufinyl carbanion (Dimsyl) --- p.37
Chapter 2.6.1.2 --- Preparation and dissolution of sample --- p.37
Chapter 2.6.1.3 --- Methylation --- p.38
Chapter 2.6.1.4 --- Hydrolysis --- p.38
Chapter 2.6.1.5 --- Reduction and acetylation --- p.39
Chapter 2.6.1.6 --- Determination of partially methylated alditol acetate (PMAA) by gas chromatograph-mass spectrometry (GC-MS) --- p.39
Chapter 2.6.2 --- Fourier-transform infrared (FTIR) spectroscopy --- p.40
Chapter CHAPTER THREE : --- RESULTS AND DISCUSSION --- p.41
Chapter 3.1 --- Chemical composition of mushrooms --- p.41
Chapter 3.1.1 --- Moisture content --- p.41
Chapter 3.1.2 --- Carbohydrate content --- p.41
Chapter 3.1.3 --- Protein content --- p.44
Chapter 3.1.4 --- Amino acid profile --- p.44
Chapter 3.1.5 --- Dietary fiber content --- p.48
Chapter 3.1.6 --- Cell wall material --- p.53
Chapter 3.1.7 --- Comparison of the yield and composition of TDF and CWM --- p.55
Chapter 3.1.8 --- "Monosaccharide composition of the dietary fiber (TDF, IDF and SDF) and cell wall material (CWM)" --- p.57
Chapter 3.2 --- Fractionation of TDF and CWM --- p.69
Chapter 3.2.1 --- Solvent extraction --- p.69
Chapter 3.2.2 --- Monosaccharide composition of solvent fractionated TDF and CWM --- p.71
Chapter 3.2.3 --- Anion-exchange chromatography --- p.78
Chapter 3.2.4 --- Gel permeation chromatography --- p.82
Chapter 3.2.5 --- Monosaccharide composition of fractionated fiber material by anion-exchange chromatography --- p.84
Chapter 3.3 --- Structural analysis --- p.86
Chapter 3.3.1 --- Partially methylated alditol acetate (PMAA) --- p.86
Chapter 3.3.1.1 --- Alkali-extracted water-soluble fractions of V. volvacea fiber material --- p.95
Chapter 3.3.1.2 --- Alkali-extracted water-soluble fractions of P. sajor-caju fiber material --- p.99
Chapter 3.3.1.3 --- Alkali-extracted water-soluble fractions of P. tuber-regium fiber material --- p.102
Chapter 3.3.1.4 --- Alkali-extracted water-insoluble fractions of the mushroom fiber material --- p.106
Chapter 3.3.1.5 --- Alkali- and acid- resistant fractions of the mushroom fiber material --- p.109
Chapter 3.3.2 --- Infrared spectroscopy --- p.112
Chapter 3.4 --- "β (l→3), (→4) glucan" --- p.119
Chapter CHAPTER FOUR : --- CONCLUSION --- p.121
REFERENCES --- p.125
RELATED PUBLICATIONS --- p.137

碩士
國立中興大學
食品科學系
89
Wood ears are common edible fungi in China. Five kinds of wood ears are already commercially available in Taiwan, including black, red, jin, snow and silver ears. Mushroom fruit bodies and mycelia can be used as foods and food flavoring, to produce specifically chemical and medical substances, such as water soluble polysaccharides, vitamins, organic acids, nucleotides and enzymes, as well as to pharmaceuticals such as antibiotics, anticancer and antidisease drugs. In addition, mushrooms can supply antioxidant substances. However, so far no complete information is available for the antioxidant properties of wood ears in Taiwan. However, studies on the physiologically effects of wood ears have not been conducted. This research used wood ears as samples to study the proximate composition, antioxidant properties of methanolic extracts, the monosaccharide constituents and the structural analysis of polysaccharides. Their structures were elucidated by spectroscopic analysis and comparison with known compounds. For all methanolic extracts from ear mushrooms, the antioxidant activity in the 1,3-diethyl-2-thiobarbituric acid method were moderate (38.6~74.6%) at 1.0 to 5.0 mg/mL. Methanolic extracts for red, jin and snow ears showed excellent antioxidant activity in the conjugated diene method at 5.0 mg/mL. At 5.0 mg/mL, reducing powers in the descending order of sonw＞black ≈ red ≈ jin＞silver ears. The scavenging effect of methanolic extracts from ear mushrooms on 1,1-diphenyl-2-picryl hydrazyl radicals (DPPH·) was excellent except for that from silver ears. Ear mushrooms were not good scavengers for hydroxyl free radicals but good chelators for ferrous ions. Naturally occurring antioxidants, including ascorbic acid, tocopherols and total polyphenols, were found in methanolic extracts. However, b-carotene was not detected. Total antioxidant components were 15.69, 30.09, 27.83, 49.17, and 31.70 mg/g for black, red, jin, snow, and silver ears, respectively. In ear mushrooms, the total sugar contents of water extract and alkaline extract polysaccharides were 74~92%. The compositions of neutral monosaccharide were the major have xylose, mannose, glucuronic acid and some ears have glucose. Ear mushrooms of water extract and alkaline extract polysaccharides was separated using gel filtration, and showed a molecular weight of about 2×106 Da. Their structure were elucidated by 1H-NMR spectra of water extract and alkaline extract polysaccharides showed that they contained RCH3, RCH2, CH3-CO- and CH2O-. That can be have a b-D-glucan chain. IR spectroscopy showed that water extract polysaccharides contained OH, C-H, C=O, C=C, C-O, CH-O-CH-, but that alkaline extract polysaccharides not have C=O, C-O. The thermal behaviors of water extract and alkaline extract polysaccharides were examined by DSC, except for silver ear showed one endotherm but the anther not have. It could be study their physiological activities, advance analysis. The research and X-ray can be have a b-D-glucan chain. For the application in healthy food, further research on the mechanism of polysaccharide properties is in progress.

Medicinal mushrooms are known for their traditional use for the treatment of various diseases including diabetes, cancer and cardiovascular diseases. Mushrooms have been traditionally used for curing numerous life-threatening diseases such as cancer, Tuberculosis, Parkinson’s and Alzheimer. Cancer is one of the leading causes of death worldwide and finding better therapeutics to treat this disease is an urgent need. Literature demonstrates the potential of mushroom polysaccharides for their therapeutic value with significant immunostimulatory and anticancer activities with least side effects. A few polysaccharides from medicinal mushrooms (e.g. Polysaccharide Krestin, Polysaccharopeptide) are already in clinical use to treat cancer. This research therefore aims to isolate polysaccharides with immunomodulatory, prebiotic and anticancer properties from traditionally known medicinal mushrooms Ganoderma lucidum, Cordyceps sinensis and Trametes versicolor. The objectives of this research are to study the biological activities relevant to anticancer therapy and to evaluate the structure- function relationship of mushroom polysaccharides. It is therefore expected that these mushrooms have tremendous potential for the discovery of polysaccharides with immune enhancing, prebiotic and anti-cancer properties and hence chosen for this study. A total of six polysaccharides have been identified in this thesis from two medicinal mushrooms (G. lucidum and C. sinensis). Four of these fractions (GLP-1, GLP-2, GLP-3, CSP-1) have displayed significant radical scavenging activities. These activities correlated well with molecular masses and structural elements. GLP-1 and GLP-3 displayed highly significant immunostimulatory activities with respect to both cytokines and are extremely important candidate for immunotherapeutic applications. CSPs and GLPs have also displayed significant prebiotic activities that are highly significant. Overall, the results presented in this thesis strongly suggest immune enhancing, prebiotic and direct anticancer activities of polysaccharides from G. lucidum and C. sinensis (GLPs and CSPs). It is therefore concluded that these polysaccharides form potential candidates for combination “immuno-chemotherapy”. It is therefore suggested that, pre-treatment of cancer patients with immunostimulatory and prebiotic mushroom polysaccharides identified in this research to activate cancer microenvironment and to improve gut microbiota, followed by therapy with suitable immune checkpoint inhibitors is expected to produce enhanced outcomes for cancer patients. It is in this context that the immune enhancing and prebiotic mushroom polysaccharides discovered in this research have great potential in the treatment of cancer.

碩士
中臺科技大學
食品科技研究所
98
This research used Agrocybe cylindracea (DC: Fr.) Mre, Flammulina velutipes (Fr.) Sing, Pleurotus eryngii (DC: Fr.) Quel, Hypsizigus marmoreus (Peck.) Bigelow, white mutant of Hypsizigus marmoreus fruit bodies to study the proximate composition and structure, and spectral analysis and antioxidant properties of various hot-water-extracted polysaccharide-rich. With regard to the various hot-water extractions of polysaccharide-rich, the yields were higher and in the order of white mutant of H. marmoreus after boiling for 0 hour (6.58%) > H. marmoreus after boiling for 2 hour (6.01%) > H. marmoreus after boiling for 1 hours (5.70%). In the total sugar content, the P. eryngii after boiling for 2 hour was the highest (65.63%). In the protein content of hot-water extracted polysacchaides from various mushrooms, the A. cylindracea were higher and in the range of 19.97% ~ 21.97%. The hot-water extracted polysacchaides from A. cylindracea, F. velutipes, P. eryngii, H. marmoreus, white mutant of H. marmoreus mainly composed of glucose and xylose. Using gel filtration, the molecular weights of hot-water extracted polysacchaides after boiling for 0 hour were in the descending of H. marmoreus > P. eryngii > white mutant of H. marmoreus > A. cylindracea > F. velutipes and in the range of 8 × 103 Da ~ 3.5 × 104 Da. After boiling for 1 hour; the molecular weights of hot-water extracted polysacchaides were in the descending of A. cylindracea > H. marmoreus ~ P. eryngii > F. velutipes > white mutant of H. marmoreus and in the range of 1.2 × 104 Da ~ 2.6 × 104 Da. In addition, after boiling for 2 hour; the molecular weights of hot-water extracted polysacchaides were in the descending of P. eryngii > A. cylindracea > F. velutipes > H. marmoreus > white mutant of H. marmoreus and in the range of 1.1 × 104 Da ~ 4.3 × 104 Da. Using the conjugated diene method, the hot-water extracted polysaccharide-rich from H. marmoreus after boiling for 0 hour was the highest antioxidant activity (95.04%) at 10 mg/mL. In addition, at 10 mg/mL the antioxidant activity of hot-water extracted polysaccharide-rich from white mutant of H. marmoreus after boiling for 2hour was the highest (95.04%). With regard to reducing power, the hot-water extracted polysaccharide-rich from white mutant of H. marmoreus after boiling for 0 and 1 hours were higher at 10 mg/mL. In addition, the reducing power of the hot-water extracted polysaccharide-rich from A. cylindracea after boiling for 2 hour was the highest and 1.12. With regard to scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals, the hot-water extracted polysaccharide-rich from F. velutipes were higher than other mushroom and in the range of 46.84% ~ 59.08%. The hot-water extracted polysaccharide from white mutant of H. marmoreus after boiling for 0 hour was the highest chelating ability on ferrous ion as 92.60% at 10 mg/mL. The scavenging abilities of ABTS + free radicals of the hot-water extracted polysaccharide-rich from A. cylindracea at 10 mg/mL were higher as 98%. Overall, the polysaccharide-rich of edible mushroom of A. cylindracea, F. velutipes, H. marmoreus, white mutant of H. marmoreus and P. eryngii exhibited various levels of the antioxidant properties, and indicated that polysaccharide-rich components was one of the major antioxidant components. Keywords: Edible mushroom, antioxidant properties, polysaccharide-rich

博士
國立臺灣大學
食品科技研究所
102
The bioactive polysaccharides, (1,3)-β-D-glucans with (1,6)-β-D-glucosyl branches, are components of structural polysaccharides of fungal cell walls and have been classified as biological response modifiers (BRM). In this study, we used enzymatic-high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method to determine the amount and degree of branching (DB) of (1,3;1,6)-β-D-glucans in samples including the fruity bodies of edible mushrooms cultivated in Taiwan and the mycelium products of Ganoderma lucidum by submerged cultifation. Alkaline solution (0.5 M NaOH) extraction could increased the yield of (1,3;1,6)-β-D-glucans to 4.4 – 16.4 folds compared with the one from hot water extraction. Except the fruiting body of Ganoderma lucidum, which exhibits ligniform, the DB of edible mushrooms reveal no difference by various extraction methods. The (1,3)-β-D-glucans of G. lucidum comprised of water-soluble branching (DB 0.24) component and essential linear component that occurred in the alkaline solution extraction. Moreover, the content of insoluble dietary fiber (IDF)-(1,3;1,6)-β-D-glucans contents were 5.11 – 202.50 mg/g (dry basis), and soluble dietary fiber (SDF)-(1,3;1,6)-β-D-glucans were 0.18 – 15.36 mg/g (dry basis). The results indicated that the majority of (1,3;1,6)-β-D-glucans occurred in the insoluble dietary fiber of mushroom. We also investigated bioactivity of non-digestible polysaccharides (ND-PS) from various mushroom species by two reporter cell platforms (RAW 264.7 containing constructed plasmid, iNOS promoter-luciferase or COX-2 promoter-luciferase), and the TNF-alpha and NO concentrations in medium were determined as well. The results indicated that the immune-stimulation activities of (1,3;1,6)-β-D-glucans are mild, and some other bioactive polysaccharides may also contribute to the immune modulation activity. Moreover, we confirmed the strong positive correlations between iNOS or COX-2-directed luciferase reporter platform and the ELISA-based assay for medium TNF-alpha through this data set. This suggested that the promoter-luciferase assays successfully reflect the TNF-alpha concentration levels and the platform is applicable as a high throughput screening for the detection of mushroom polysaccharides with immune-modulatory activities. We further analyzed twelve cultivation products of Ganoderma lucidum mycelium samples. Although the results display that the amount of (1,3;1,6)-β-D-glucans significantly varied in different fermentation conditions, the DB and molecular weight of (1,3;1,6)-β-D-glucans restrict to a narrow range. For the high aggregating tendency of (1,3;1,6)-β-D-glucans, we successfully purified (1,3;1,6)-β-D-glucans by 35% ethanol precipitation method. We further confirmed the purity of (1,3;1,6)-β-D-glucans and demonstrated its bioactivity by TNF-alpha releasing assay in RAW 264.7 cells.

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

碩士
國立中興大學
食品科學系
89
Effect of heating (70-100℃, 1-10min) on cultured winter mushroom ( Flammulina velutipes ) was investigated to decide processing condition for preparation of a seasoned retort pouch product. Changes in quality of the products resulted from sterilization and storage was determined. On the other hand, water and weak base soluble polysaccharides were individually extracted from cap, stem and foot parts of mushroom body to evaluate the availability of disused feet during processing. The results obtained were as follows: 1. Hardness, fracturability and adhesiveness of winter mushroom’s stem decreased with increasing heating temperature and time, but no change was observed for springiness. For cap, hardness, adhesiveness, chewiness and gumminess also decreased with increasing heating temperature and time, but both springiness and cohesiveness were the reverse. 2. SEM showed that heating resulted in the extraction of soluble polysaccharides from mushroom body. The degree of heating damage can be determined according to hyphae soften, surface pilei and interior pilei collapsed. 3. Color, total plate counts, pH, fracturability, adhesiveness and springiness of the prepared products were not significantly different after sterilization at 85, 90 and 100℃. But POV, TBA and hardness increased with increasing sterilization temperature, except hardness of stem. 4. Considering all possible items relating to safety, quality and cost of product, heating at 90℃was appropriate as the sterilization condition for preparing the seasoned retort pouch product. 5. Rapid degradation in color, oil oxidation, pH and texture was observed for the products stored at 37℃, but slow degradation for that of stored at 25 and 4℃. No matter storage temperatures, no microorganisms were found in all products within 12 weeks. Storage limits were 2, 8, and 12 weeks for the products stored at 37, 25, and 4℃, respectively. 6. Winter mushroom was a good source of polysaccharides for containing 49.23% of carbohydrate (dry matter). The highest extraction rate 8.78% was obtained from stems with hot water, while 5.51% was obtained from feet with weak base solution. 7. Gel filtration showed the molecular weight of water soluble polysaccharides were 89 kD, while 50~60 kD and lower 13.7 kD for that of alkaline-extractable polysaccharides. The molecular weight for proteins extracted by both solutions was lower 13.7kD. 8. IR spectrums showed both polysaccharides extracted by hot water and weak base solution had similar functional groups in their molecules. The polysaccharides extracted from feet showed a specific absorption band.

碩士
國立中興大學
食品暨應用生物科技學系所
106
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.