Study On Preparation And Antioxidant Activity Of Polysaccharide From Pleurotus Geesteranus SPMP-2a

Polysaccharides from edible fungus are important organic compounds which the human and animal bodies can easily absorb and utilize.They have many physiological functions such us significant anti-tumor,anti-oxidation,anti-aging and immune regulation.Polysaccharide from Pleurotus geesteranus which is delicious and popular edible fungus has certain antioxidant and antitumor activity.However,studies on polysaccharide from Pleurotus geesteranus is not deeper enough than other fungus such us Ganoderma lucidum,Cordyceps.Studies on selenium polysaccharide from Pleurotus geesteranus have not been reported.Therefore,polysaccharides from mycelium of Pleurotus geesteranus were studied in this paper.Selenium mycelium of Pleurotus geesteranus is the main raw material for preparing polysaccharide compounds from Pleurotus geesteranus.It is necessary to carry out the optimization of selenium enriched fermentation medium.As taking selenium mycelium as material,polysaccharide of Pleurotus geesteranus was extracted by ultrasonic assisted cellulase.The polysaccharide SPMP-2a which has high antioxidant activity was prepared through separation and purification.Its structure and its properties were determined by means of instrumental analysis and other means.Its antioxidant activity was comprehensive researched.The main results are as follows:1.The growth rate of mycelia as index,we found that the optimum selenium concentration was 20 μg/mL when Pleurotus geesteranus strains were cultured with selenium liquid fermentation.Under the concentration of selenium 20 μg/mL,the mycelium yield as index,single factor test was used to screen the best carbon source,nitrogen source and their concentration,KH2PO4 concentration,on the basis of single factor test,the Box-Behnken design and response surface analysis.The liquid culture medium of Pleurotus geesteranus for selenium enriched were that sodium selenite 0.002%,glucose 4.06%,soybean flour 1.51%,KH2PO4 0.3%,MgSO4·7H2O 0.15%.Pleurotus geesteranus fermented for 7 days with rotation speed 160 rpm,at 25 ℃,and the mycelium biomass was 1.9862 g/100 mL.Selenium content of the mycelium was 385.5μg/g.2.The extraction of polysaccharide from selenium mycelium of Pleurotus geesteranus were studied by ultrasonic assisted cellulase.On the basis of the single factor experiment,the optimum extraction conditions were obtained by orthogonal test.The amount of enzyme added was 2.0%,the ratio of material to liquid was 1:50,the ultrasonic time was 50 min,the ultrasonic power was 220 w.Under this condition,the extraction rate of polysaccharide from Pleurotus geesteranus was 19.77%,the selenium content of polysaccharide was 6.550 μg/mg.3.Free protein in Pleurotus geesteranus polysaccharide solution were removed with Sevag method,and pigment in them were removed with activated carbon.SPMP-1 was precipitated with 40%ethanol concentration.SPMP-2 was precipitated with 70%ethanol concentration.SPMP-3 was precipitated with 85%ethanol concentration.The total antioxidant capacity(TAC)in vitro were determined among SPMP-1,SPMP-2 and SPMP-3.The antioxidant ability of SPMP-2 was the highest.SPMP-2 was further separated by the molecular weight of 5 kDa and 50 kDa ultrafiltration membrane,SPMP-2a,SPMP-2b and SPMP-2c were obtained.The total antioxidant capacity(TAC)in vitro were determined among SPMP-2a,SPMP-2b and SPMP-2c,The strongest antioxidant capacity of three polysaccharides was SPMP-2a.SPMP-2a was studied by IR,UV,HPLC and colorimetric method.SPMP-2a is a selenium glycoprotein with the molecular weight of 3.18×104 Da.The polysaccharide content of SPMP-2a was 75.68%.The protein content of SPMP-2a was 18.95%.The uronic acid content of SPMP-2a was 21.64%.The selenium content of SPMP-2a was 0.688%.SPMP-2a was consisted of Man:Rha:Glc:Xyl:Ara = 1.87:1.00:17.86:3.28:3.12 and contains 17 kinds of amino acids.Glycopeptide linkage between peptide the sugar chain was O-glycoprotein.SPMP-2a is a kind of acid selenium compound polysaccharide with a-D glycopyranoside bond and Triple-helix.4.The antioxidant activities of polysaccharide SPMP-2a in vitro were investigated through the chemical test method.The results showed that SPMP-2a has certain DPPH free radicals,hydroxyl radicals and superoxide anion free radical scavenging ability.The scavenging ability of SPMP-2a and its concentration were an dose-dependent relationship.SPMP-2a also has a certain reduction capacity of metal ions and chelating ability,and showed a dose-response relationship.Therefore,SPMP-2a has some antioxidant activity in vitro.5.Hoechst33342 specific staining showed that HaCaT cell nuclei appeared dense stain light.Apoptotic cells in model group(with H2O2 treatment)was more than SPMP-2a group.Flow cytometry showed that the apoptosis rate of cells in SPMP-2a group were significantly decreased,the cell morphology gradually restored and the apoptosis rate decreased than H2O2 group.Western blot showed that the expression of Bcl-2 protein decreased in the model group.After SPMP-2a treatment,the expression of Bcl-2 protein increased.The results showed that SPMP-2a could promote Bcl-2 gene expression and regulation of H2O2 induced apoptosis in HaCaT cells.SPMP-2a pretreatment can improve the activity of SOD and CAT in damaged HaCaT cells,decrease the content of reactive oxygen species,avoid the oxidative stress damage,and improve the survival rate of damaged cells.Therefore,SPMP-2a can promote the activity of antioxidant enzymes in HaCaT cells,such as SOD and CAT,decreased intracellular ROS content,and inhibit the occurrence of apoptosis.With the increase of SPMP-2a concentration,the protective effect on the oxidative damage was more significant.The results indicated that SPMP-2a could inhibit the apoptosis of HaCaT cells induced by H2O2 and increase the antioxidant capacity of the cells.6.The mice were injected intraperitoneally with D-Gal to establish the aging model,and intragastric administration of different doses of SPMP-2a.The CAT,SOD,GSH-Px activity and MDA content in mice liver and brain tissue and AST,ALP,ALT in mice serum and other indicators were measured.SPMP-2a can significantly improve the aging mice induced by D-Gal liver index and spleen index,enhanced liver and brain tissue in SOD,GSH-Px and CAT activity,decreased the content of MDA in brain and liver tissues.The results indicated that SPMP-2a could significantly inhibit the degradation of organism of aging mice,improve the activity of antioxidant enzymes,enhance the ability of scavenging superoxide anion free radical and hydroxyl radical of aging mice.Therefore,SPMP-2a has anti-aging effect.