Preparation And Immunomodulatory Activities Of Polysaccharide From Tuber Sinense K. Tao Et Liu

Truffle, known as one of the three most famous delicious foods, is mainly distributed in Southwestern regions such as Sichuan, Yunnan and northwestern regions such as Xinjiang and Tibet in china. At present, little has been known for the biological activities of truffle polysaccharids. To develop the truffle mushroom resource, the research the optimal extraction process, isolation and purification of the water-soluble polysaccharide from dried fruiting bodies of Tuber sinense were studied. And the antioxidant activity of the crude polysaccharide from Tuber sinense and immuno-modulating activity of the puridied polysaccharide were researched. Specifically, the thesis mainly includes the following sections:1.The research on optimal extraction process of the crude polysaccharide from dried fruiting bodies of china truffle.Response surface methodology (R.SM) was employed to optimize the extraction conditions (extraction temperature, ratio of water to raw material and extraction time) of polysaccharides from the fruiting bodies of Chinese Truffle Tuber Sinense. A Box-Behnken Design was used for experimental design and analysis of the results to obtain the optimal processing parameters. The optimal conditions were extraction temperature 95?, ratio of water to raw material 16.4:1, extraction time 2.7h and the yield of polysaccharides was 10.23%. The crude polysaccharide obtained by the optimal extraction program was a pale brown powder, and the content of polysaccharides, proteins, uronic acid and sulfuric acid group of the powder was 42.79%,6.19%,1.43%,3.10%, respectively.2.The research on antioxidant activities of the CTP.In this study, the antioxidant activities of the CTP in vivo and vitro were eva-luated. As the results, CTP showed strong scavenging activity of superoxide radical and hydroxyl adical in a dose-dependent manner in vitro. But its reducing power is weak. CTP increased the activity of SOD and GSH-Px and decreased the level of MDA in serum of immunosuppressed mice. Meanwhile, CTP significantly incr eased the activity of SOD in liver and the content of MDA, but has no influence on activity of GSH-Px.3.The research on immuno-modulating activity of the crude polysaccharideCTP significantly promoted lymphocyte proliferation either alone or in combination with ConA(p<0.05),but did not promoted lymphocyte proliferation either alone or in combination with LPS(p>0.05). And the phagocytosis of mouse peritoneal macrophages was tested by neutral red uptake.The results showed that CTP significantly promoted neutral red phagocytosis of peritoneal macrophages and promote NO synthesis.CTP increased thymus index, and had no significant influence on spleen index in immunosuppressed mice. CTP enhanced Phagocytosis function of peritoneal macrophages by chicken red blood cell phagocytosis assay in immunosuppressed mice. CTP treatment significantly increased the number of red blood cells and the hemoglobin concentration. Meanwhile there is a trend toward increasing the number ofthe white blood cells in the peripheral blood, but the difference is not signifcant.4. Purification of truffle polysaccharideThe optimum bleaching temperature, processing time, resin weight and polysacc haride concentration is 40?,4h, 1g/mL and 4mg/mL, respectively. Under this con-ditions, the bleaching rate, protein removal rate and polysaccharides retention rate is 61.12%,46.87% and 70.58%, respectively.Water-soluble neutral polysaccharide was obtained by further separation and purification from bleached polysaccharide with DEAE-sepharose FF anion exchange resin. The protein in WSTP has been completely removed, and the polysaccharide content is 91.63%. The result of HPLC analysis showed WSTP contained two components, and the molecular weight ofthe two components are 1020620 Da and 30080Da respectively.5.Effect of WSTP on immuno-modulating activity in RAW264.7 cells.To study the effect of the WSTP on function of RAW264.7 cells, WSTP was added to the culture medium.24h later, WSTP significantly changed the morphology of RAW264.7 cells. Like LPS, WSTP significantly increased the cell volume, and reduced the number of macrophage. WSTP increased the synthesis of the NO in RAW.264.7 cells in a dose-dependent manner. There was a significant time effect on NO production. RAW.264.7 cells produced a time-dependent increase in the release of NO. WSTP increased the secretion of IL-1??IL-1??IL-6?and TNF-a in RAW.264.7 cells in a dose-dependent manner.6. The effects of WSTP on expression of IL-1??IL-1??IL-6 and TNF-a mRNA in RAW264.7 cells were studied by real-time PCR.The gene expression of IL-1??IL-1? IL-6?TNF-a mRNA were tested by RT-PCR. In comparison to the control group, there was a marked up-regulation in mRNA expression for the cytokines IL-l? IL-l?\IL-6 TNF-? of RAW264.7 cells in a dose-dependent manner from 10 to 200?g/mL after WSTP stimulation for 24h. Then IL-l??IL-1??IL-6 mRNA expression gradually decreased as the stimulating time prolonging, but TNF-a mRNA expression level was still high.