| Agaricus Blazei Murill is a precious edible-medicinal fungus. Its antitumor activity is on the forefront among the fungi with antitumor activity, and it has a great development value and good application prospect. Water-soluble polysaccharides were extracted from the fruiting body of Agaricus Blazei Murill, and were isolated and purified. Physicochemical properties, structural analysis, and immunological activity of polysaccharide franctions were also studied.A crude polysaccharide was extracted using hot water and precipated using alcohol from Agaricus Blazei Muril. The extraction rate and yield of polysaccharide were determinated by phenol-sulfuric acid method. The conditions of hot water reflux extraction of polysaccharide were: temperature: 100 oC, ratio of material to water: 1:20, time: 4 h. The extraction rate of polysaccharide was 8.17%, and the yield of polysaccharide was 12.2%. As a simple method with convenient, this method would not cause the degradation of polysaccharide, and the polysaccharide extraction rate was high.The effect of 5 kinds of resins and sevag method on the deproteinization and decolorization capacities were compared. And it’s found that the capacities of resins were better than sevag method. D101 and D315 gained the highest deproteinization, decolorization and polysaccharide retention capacities among five kinds of resins, macroporous adsorption resins D101, HPD-100 and LX-22, ion exchange resin D113 and D315. The resin D101 don’t have the selectivity for charge property and charge intensity. Conditions D101 static adsorption with were: temperature: 40 oC, time: 7 h. The deproteinization rate was 61.85%, the decolorization rate was 62.4%, and the polysaccharide retention rate was 80.65%. A crude polysaccharide was decolorized and deproteinated by D101, then ABM was obtained. ABM was brown powder. The polysaccharide content of ABM is 83%(by phenol-sulfuric acid method), and the protein content is 4.4%(by Kjeldahl method).ABM was purified using DEAE Sepharose Fast Flow and Sepharose CL-6B chromatography, and the yield and total polysaccharide content of each franction were determinated. 7 kinds of polysaccharide franctions were obtained, were ABM1-1, ABM1-2, ABM1-3, ABM2-1, ABM2-2, ABM2-3 and ABM2-4, respectively. The proportion of 7 kinds of polysaccharide franctions to polysaccharides(ABM1 and ABM2) were 6.5%, 18.26%, 25.84%, 8.7%, 7.7%, 5.7% and 25.13%, respectively. Results form phenol-sulfuric acid assay showed that 7 kinds of polysaccharide franctions contained 94.11%, 93.28%, 88.60%, 86.12%, 84.98%, 82.18% and 75.32% carbohydrate, respectively.High performance size row exclusion chromatography and differential refractive detector(HPSEC-RI), size exclusion chromatography and light scattering(SEC-LLS) were used to analyze the relative molecular weight(Mw), absolute molecular weight and advanced conformation of each franction. HPSEC-RI indicated that Mws of ABM1-1, ABM1-2, ABM1-3, ABM2-1, ABM2-2, ABM2-3 and ABM2-4 were 1.08×106 Da, 4.08×105 Da, 1.13×104 Da, 1.07×106 Da, 4.9×105 Da, 7.7×104 Da and 1.45×104 Da, respectively. The results of the molecular weight distribution of all the seven were all a single symmetrical peak. The absolute molecular weights of 7 kinds of polysaccharides measured by SEC-LLS were 2.450×107 g/mol, 4.727×106 g/mol, 1.486×104 g/mol, 8.595×106 g/mol, 5.503×106 g/mol, 5.302×104 g/mol and 2.322×104 g/mol(the results were slightly higher than that of HPSEC-RI), respectively. The results of the advanced conformation of 7 franctions measured by SEC-LLS have shown that the advanced conformation of ABM1-1 may be compliant chains; the advanced conformation of ABM1-2, ABM1-3, ABM2-1 and ABM 2-2 may be rigid chains; the advanced conformation of ABM2-3 and ABM2-4 may be coiled into a ball.The monosaccharides composition and glycosidic bond configuration of each franction were measured by RP-HPLC, IR and NMR. The results of RP-HPLC revealed that the mole percentage of glucose in ABM1-1, ABM1-2, ABM2-1 and ABM2-2 were 97.38%, 95.94%, 95.848% and 95.98%, respectively, beyond 95%, so the polysaccharide parts of ABM1-1, ABM1-2, ABM2-1 and ABM2-2 were dextrans. The mole percentage of glucose in ABM1-3 was 62.83%, and ABM1-3 contained some galactose(28.43%), fucose(5.54%) and mannose(1.46%), so the polysaccharide part of ABM1-3 was mainly composed of glucose, galactose, fucose and mannose. The mole percentage of glucose in ABM2-3 and ABM2-4 were respectively 93.128% and 91.048%, beyond 91%. The mole percentage of glucose acid in ABM2-3 and ABM2-4 were respectively 2.14% and 2.40 %, so the polysaccharide parts of ABM2-3 and ABM2-4 were the dextrans containing a small amount of glucose acid. By IR and NMR analyses, the primary structures of ABM1-1, ABM1-2 and ABM2-2 were likely composed of α-(1→4) and β-(1→6). ABM1-3 had β and α configurations, and fucose. The primary structure of ABM1-3 was likely composed of(1→2) and(1→6). The primary structure of ABM2-1 was likely composed of α-(1→4) and α-(1→6). The primary structures of ABM2-3 and ABM2-4 were likely composed of β-(1→6) and β-(1→3), in the ratio of 1:1.The effect of the polysaccharides from the fruiting body of Agaricus Blazei Muril in vitro on the promotion of the proliferation of the spleen cells and macrophages was detected by MTT method. The results have shown that the three dose concentration(10, 25 and 50 μg/ml) of polysaccharides from Agaricus Blazei Muril could improve the proliferation on normal mouse spleen cells. The proliferation rates of ABM, ABM1-1 and ABM1-2 were more than 30%, were obvious. The three dose concentration(10, 50 and 100 μg/ml) of polysaccharides from Agaricus Blazei Muril could improve the proliferation on normal mouse macrophages. The results of polysaccharides and LPS were better than that of polysaccharides, also better than that of LPS, which showed that polysaccharides could promote the proliferation of macrophages activated by LPS. The effect of ABM1-2 on the proliferation of macrophages activated by LPS was the most significant one. |
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