Extraction And Purification Of Polysaccharide From Grifola Frondosa And Research Of Antioxidant Activity

The Grifola frondosa is a medium-temperature,oxygen fungus.The rich Grifola frondosa polysaccharide has significant biological activity in antiviral,immunomodulation and anti-tumor.In this paper,the dry matter of Grifola frondosa was used as raw material,after the crude polysaccharide(S-GFP)obtained by Microwave water extraction of cellulase-papain complex enzymes and the crude polysaccharide(J-GFP)obtained by low-temperature alkali extraction were deproteinize by the sevage method and decolorize by the 001*7 macroporous resins.four purified polysaccharides were separated using DEAE-52 cellulose chromatography column.To the four purified polysaccharides,Purity identification,chemical structure characterization and the hydrolyzate composition were studied,and the antioxidant activity was studied.Combined with the single factor test results,The results of response surface showed that the optimal extraction conditions of Microwave water extraction of cellulase-papain complex enzymes extracting Grifola frondosa polysaccharide were as follows:under the conditions of microwave extraction time of 7 min,solid-water ratio of 1:27,complex enzymes ratio of 1:1,and enzyme-solid ratio of 2%,the theoretical yield of Grifola frondosa polysaccharide was 75.58 mg/g,the actual yield was 73.25 mg/g,and the relative deviation was 3.17%.The optimal extraction conditions of low-temperature alkali extracting Grifola frondosa polysaccharide were as follows:under the conditions of solid-water ratio of 1:55,Na OH concentration was 0.46 mol/L,and extraction time was2.15 h,the theoretical yield of Grifola frondosa polysaccharide was 128.29 mg/g,the actual yield was 123.29 mg/g,and the relative deviation was 4.33%.The optimal extraction conditions of the two methods were applied to the extraction of polysaccharides from Grifola frondosa,and the obtained crude polysaccharides were deproteinize by the sevage method,decolorize by the 001*7 macroporous resins and separated using DEAE-52cellulose chromatography column.Four purified polysaccharides S-GFP1,S-GFP2,J-GFP1and J-GFP2 were obtained.The samples'purity and chemical structure characterization were studied of four extracted polysaccharides from Grifola frondosa.The UPLC-MS-MS spectrometry were used to determined of the hydrolyzate product.The purity identification results showed that sugar content of the four extracted polysaccharides was 96.23%,95.37%,97.95%,89.29%.UV scanning showed that they didn't contain nucleic acids and proteins.The HPLC analysis result showed S-GFP1,S-GFP2 and J-GFP1 have high purity.The chemical structure characterization result showed relative molecular weight of S-GFP1,S-GFP2 and J-GFP1 were 1845.48 k Da,29.69 k Da and 4.99 k Da respectively.IR Scaning indicated S-GFP1 and J-GFP1 was?-pyranose,S-GFP2 was?-pyranose.Among them,S-GFP2 and J-GFP1 containing carboxyl group,S-GFP2 and S-GFP1 contain phosphate group.The congo red experiment showed S-GFP2 and J-GFP1 had triple-helix structure.S-GFP1,S-GFP2 and J-GFP1 using 3mol/L Hydrochloric acid was hydrolyzed for 1h,separated on an Agilent Polaris 3 NH₂column(2.0 mm×150 mm)using 10 mmol/L ammonium formate/water solution-acetonitrile as the mobile phase gradient elution.The electrospray ionization tandem quadrupole mass spectrometric analysis was carried out in the negative ion mode using multiple reaction monitoring (MRM).The method was applied to analyze the samples of glucose(30.80%-35.43%),galactose(33.19%-39.41%),arabinose(0.10%-2.22%),rhamnose(5.05%-14.72%),sucrose(11.02%-13.00%)were detected in most of the samples.However,S-GFP2 contains glucuronic acid(5.21%),raffinose(19.31%)were detected in J-GFP1.The antioxidant in vitro experiments of S-GFP,J-GFP,S-GFP1,S-GFP2 and J-GFP1showed that the maximum reducing power reached 0.43,0.49,0.82,1.01 and 0.67respectively.The maximum DPPH radical scavenging rate is 25.84%,13.39%,23.54%,50.38%and 23.59%respectively.The maximum superoxide anion scavenging rate is54.88%,62.36%,79.23%,87.23%and 78.34%respectively.The maximum hydroxyl radical scavenging rate is 77.88%,68.28%,81.59%,90.21%and 85.02%respectively.In general,the reducing power,superoxide anion and hydroxyl radical scavenging effect of Grifola frondosa polysaccharides were stronger,and DPPH radical scavenging ability was weaker.The overall trend is that the antioxidant effect of purified polysaccharides from Grifola frondosa is better than crude polysaccharides.The antioxidant effect of Microwave water extraction of cellulase-papain complex enzymes polysaccharides is better than low-temperature alkali extraction polysaccharides.