Studies On Extraction, Isolation, Structure And Activity Of Polysaccharides From Bellamya Purificata

Bellamya purificata belongs to phylum Mollusca, class Gastropoda, subclass Prosobranchia and family Vivipariidae, which is a species of mud snail found in fresh water in China. It has a flattened muscular foot for locomotion. It is widely distributed in the Yangtze River drainage area and most of the provinces in east-China. With the development of glycol-biology, polysaccharides isolated from various shellfish have attracted a great deal of attention because of their anti-tumor activity, immunoregulating activity and relatively low toxicity. In this paper, extraction, structure and activity of polysaccharides from bellamya purificata were investigated, that provide reference for utilization and exploration of bellamya purificata.According to the single factor experiment and reponse surface methodology, the optimal extraction parameters were established as follow:ratio of material to solvent was 1:28 (w/v), extraction temperature was 75℃and extraction time was 5.2h. The optimal extraction yeild of polysaccharide from bellamya purificata was 2.52% which was determined by phenol-sulfuric acid method. The extracted crude polysaccharide was named as CBPS.Durning the deprotein study, TCA method, sevag method, papain emzymolysis combined with sevag method and trypsin emzymolysis combined with sevag method have been compared. The optimum method of deprotein was a combination of trypsin and sevag method according to the rate of deproteinization, the rate of polysaccharide losing and reagent waste. The further purify methods such as dialysis, ion-exchange chromatography and gel-filtration chromatography was explored in this paper. Deproteined polysaccharide was dialyzed against distilled water, and then purified with DEAE-sephacel ion-exchange column (1.6×40cm) and Sepharose 6B gel column (1.6×120cm), collected two fractions which were named as BPS-1 and BPS-2, respectively. The purity of BPS-1 and BPS-2 were identified by HP GPC and CAME, the results revealed that both of them were homogeneous polysaccharides.Chemical methods were used to analysis the constituents of BPS-1 and BPS-2. it showed that the total sugar content of BPS-1 and BPS-2 are 99.14% and 97.06%, respectively. The glycuronic acid are 18.58% and 17.87%, glucosamine are 12.91% and 18.98%, sulfate group are 2.13% and 2.47%, respectively. The neutral monosaccharide composition of BPS-1 is 96.14% glucose,1.71% fucose,0.97% arabinose and 1.18% xylose, BPS-2 is 94.71% glucose,1.98% fucose,1.43% arabinose,0.58% xylose,0.44% mannose and 0.86% galactose in terms of the molar proportions according to the GC data. HP GPC analysis showes that the molecular weight of BPS-1 and BPS-2 are 7.2×106Da and 8.3×106Da, respectively.IR spectrum indicates that the sugar units of BPS-1 and BPS-2 are a-configuration of pyranose. Based on the data obtained from partial acid hydrolysis, periodate oxidation, smith degradation, methylation analysis, GLC-MS and NMR (1H,13C), the repeating unit of BPS-1 was established: the repeating unit of BPS-2 was:Anti-inflammatory activity of BPS-1 and BPS-2 were determined using xylene induced ear edema in mice. It indicated that the mouse ear edema inhibition rate of BPS-1 and BPS-2 are 14.02～57.56% and 11.07～56.46% in the concentration range of 0.05～1.0 mg/ml, respectivity. The antibacterial activity of BPS-1 and BPS-2 were determined by disk diffusion method, the results showed that BPS-1 and BPS-2 only have antibacterial activity against gram-negative bacterial, and the antibacterial activity of BPS-1 is slightly higher than BPS-2. The antioxidant activity of the BPS-1 and BPS-2 were invested in terms of scavening DPPH free radical. The results showed that BPS-1 and BPS-2 have no significant antioxidate capability by scavening DPPH free radical. The anti-tumor activity was measured by MTT method which suggesting that the polysacchardies BPS-1 and BPS-2 have no significant inhibition to HepG2 cell proliferation in vitro.