Study On Extraction, Isolation And Purification Of Polysaccharide From Boletus Edulis And Its Antioxidant Activity

Boletus edulis (alias black porcini), belonging to Boletus with the "high protein, low fat, low calorie" and other features, mainly distributes in Sichuan, Guizhou, Yunnan, Europe and other places, which is a species of edible fungi with great nutritional and commercial value. In the present work, the extraction conditions of polysaccharides from Boletus edulis were studied through different extraction, and then optimized the extraction process by response surface methodology. The crude polysaccharide was purified, and the molecular weight and monosaccharides were measured. Moreover, the antioxidant of crude polysaccharide and purified polysaccharide were also evaluated. The main results obtained are as follows:1. Five extraction processes for extracting Boletus edulis polysaccharides were compared:namely, hot water extraction, alkali extraction, microwave-assisted extraction, ultrasound-assisted extraction and enzyme-assisted extraction method. The extraction rates of those five extraction processes were 14.73%,17.3%,12.55%, 14.37%and 18.16%, respectively. By comparing the results of those five extraction methods, enzyme-assisted extraction method had advantages in the extraction conditions and efficiency, so it was optimized by response surface methodology. The optimum conditions about the cellulose assisted extraction optimized by response surface methodology are listed as below:enzymatic quantities 834U·g-1, pH value 5.4, ezymohydrolysis temperature 51℃, ezymohydrolysis time 53min, water bath temperature 50℃, water bath time 2.5h, solid-liquid rate 1:40(g:mL). Under these conditions, the extraction rate of Boletus aereus polysaccharide was 18.46%.2. The protein and small molecules incrude polysaccharide of Boletus edulis were taken off through Sevage method and the dialysis bag, respectively to obtain refined polysaccharide. The crude polysaccharide was eluted with the distilled water and 0.3mol·L-1NaCl, respectively, through the DEAE-52 cellulose column chromatography and two kinds of polysaccharides were obtained:PB-1 and PB-2. Then, PB-1 and PB-2 were eluted with the distilled water with the Sephadex G-100 column chromatography to obtain the components PB-1A and PB-2A, respectively. PB-1A and PB-2A were proved to be homogeneous polysaccharide using gel detection, specific rotation of pure determination and UV spectroscopy examination.3. Experiments showed that PB-1A and PB-2A can be dissolved in water, dilute acid, dilute alkali, especially in hot water. However, they were undissolved in methanol, ethanol, acetone, ether, chloroform and other organic solvents. Moreover, PB-IA and PB-2A didn’t contain starch, amino acids, free monosaccharide, sugar, protein, and polyphenols. The molecular weight of PB-1A and PB-2A are 87.03kDa and 24.38kDa tested by gel filtration. PB-1A contains five kinds of monosaccharides tested by HPLC, namely, mannose, glucose, galactose, xylose, fucose, and the molar ratio is 0.80:0.20:1.36:0.21:1.00. PB-2A also contains these five monosaccharides, and the molar ratio is 1.61:0.45:1.00:0.43:1.16.4. The in vitro antioxidant activity of crude polysaccharide of Boletus edulis, PB-1A and PB-2A were evaluated by hydroxyl radical scavenging, DPPH radical scavenging ability and reducing power methods. The results showed that the antioxidant activity of crude and purified polysaccharides displayed a dose-dependent manner. Crude polysaccharide had good reducing power and DPPH scavenging activity, but possessed a weak hydroxyl radical scavenging capacity.The IC50 of crude polysaccharide of hydroxyl radical scavenging capacity, DPPH radical scavenging capacity and reducing power were 3.29mg·mL-1, 0.60mg·mL-1 and 1.13mg·mL-1, respectively. The IC50 of PB-IA of hydroxyl radical scavenging capacity, DPPH radical scavenging capacity and reducing power were 0.96mg-mL"1, 0.34mg·mL-1 and 0.65mg·mL-1, respectively. For PB-2A, the IC50 of hydroxyl radical scavenging capacity, DPPH radical scavenging capacity and reducing power are 0.67mg·mL-1, 0.14mg·mL-1 and 0.47mg·mL-1, respectively. From the above results, the antioxidant capacity of crude polysaccharide was significantly weaker than that of purified polysaccharide, and the antioxidant capacity of PB-2A was better than PB-IA.