Isolation, Purification, Biological Activity And Structure Analysis Of Polysaccharides From

Volvariella volvacea (straw or paddy straw mushroom), belonged to the Agaricales of Basidiomycetes, is the third largest cultivated edible mushroom worldwide. Carbohydrates are the main nutritional components of V. volvacea and polysaccharides from V. Volvacea are important for possessing immunomodulatory and antioxidant activity. In this paper, optimization of extraction, purification of polysaccharides from V. volvacea was investigated. Moreover, we preliminarily studied immunomodulatory and antioxidant activity of the polysaccharides through in vitro models, and characterized the structures of the polysaccharides and discussed the structure-activity relationship of the polysaccharides. The main results are as follows:1. Studying the polysaccharides extraction process from V. volvacea, focused on the optimization of aqueous extraction, microwave-assisted extraction and papain extraction, the preferable extraction conditions of aqueous extraction by the single factor experiment and response surface methodology were 79℃ for 4.4h with the ratio of material to water 1:65(g/mL) repeatly, the maximum yield of the polysaccharides was 17.14%. The optimal extraction condition of microwave-assisted extraction by the single factor experiment and Orthogonal Design were 400W and 70℃ for 20min followed by 80℃ water bath for 4h, the yield of the polysaccharides was 27.96%, the results of papain extraction by the single factor experiment and Uniform Design were pH 6.5 and 62℃ for 2h with 0.2% papain dosage, the maximum yield of the polysaccharides was 23.51%. Under the optimal conditions of three extraction processes, the maximum yield of the polysaccharides decreased in the order of the Microwave-assisted extraction> the papain extraction> the aqueous extraction.2. Optimizition of the separation and purification of the V. volvacea polysaccharides, researching the impact of the method of Sevag and neutral protease-Sevag on deproteinization and the effects of activated carbon and H2O2 on decoloration.Theresults revealed that the most effective method for deproteinization was neutral protease-Sevage, the conditions were 40% neutral protease dosage,45℃ for 2h, followed by deproteinizing with Sevag reagent twice, the removal rate of protein was 92.21%, and the retention of the polysaccharides was 78.32%. Moreover, the more impactful method for decoloration was H2O2 oxidation, the optimal separation conditions were 20% H2O2 dosage,60℃ and pH 9 for 3h, the removal rate of color was 99.45% and retention of the polysaccharides was 69.12%. The polysaccharide was purified subsequently by DEAE-52 ion exchange column chromatography and Sephadex G-100 gel chromatography column, and the three kinds of polysaccharides were obtained, named as VVP-1,VVP-2 and VVP-3, respectively. Protein characteristic absorption peaks were shown in the UV-visible spectras of VVP-1, VVP-2 and VVP-3.3. The bioactivities of the V. volvacea polysaccharides were preliminarily investigated by studying the antioxidant activities of the polysaccharides and the impacts of the polysaccharides on the proliferation of immunological cells and liver cancer HepG2 cells in vitro.In antioxidant assays in vitro, the polysaccharides have feeblish DPPH-radical scavenging activity, O2-·radical scavenging activity, reducing power and strong-OH radical scavenging acetivity. The DPPH·radical scavenging activity and ·OH radical scavenging acetivity of the polysaccharides samples decreased in the order of VVP>VVP-1>VVP-2>VVP-3, O2-· radical scavenging activities of the polysaccharides samples depends on their concentrations. When the concentrations of the samples were 6mg/mL, the activity decreased in the order of VVP-2>VVP-1>VVP-3. In the cellular experiment in vitro,the results showed that the V. Volvacea polysaccharides exhibited the activities to promote the proliferation of macrophage and splenic lymphocyte,decreased in the order of VVP>VVP-1>VVP-2>VVP-3;furthermore, they inhibited the proliferation of liver cancer HepG2 cells and the VVP-2 had the stronger activity than the VVP-1 and VVP-3.4. The structural characterizations of the V. volvacea polysaccharides were investigated in a variety of methods, such as scanning electron microscope (SEM),Thin-layer chromatography(TCL), Gas Chromatography-Mass Spectrometer(GC-MS), Infrared Spectroscopy(IR), periodic acid oxidation and Nuclear Magnetic Resonance(NMR). The SEM spectra showed that VVP was sponginess, VVP-1 was smooth schistose, and VVP-2 was dendritic. The FT-IR specetra of VVP-1 and VVP-2 were similar, they both were polysaccharides with the characteristic absorption peaks, the three bands in the region of 1010 and 1200cm-1 were assigned to the deformation vibration of the pyranose. Monosaccharide compositional analysis showed that both VVP-1 and VVP-2 consisted of Arabinose, Xylose, Fructose, Galactose, Glucose and Mannose. In the periodic acid oxidation assays, the results indicated that VVP-1 and VVP-2 both contained 1â†'6 and 1â†'4 or 1â†'2 linked glycosidic bonds.13CNMR specetra revealed that VVP-1 was the pyranoses with α-and β-configurations, and VVP-2 mainly consisted of the pyranoses with β-configurations.