Study On Isolation Purification Structural Characterization And Activity Of Polysaccharide From Sanghuangporus Vaninii

Objective: Based on the actual production and the existing problems,the optimal production process of prepared SHP was selected to provide the basis for the production process of prepared mulberry leaves,improve the production rate,reduce the production cost,and provide quality assurance for prepared SHP.The extraction method,purification and separation of crude polysaccharide,physical and chemical properties,structural characterization,anti-inflammatory and immune regulation of polysaccharide from SHP were studied.It provides a theoretical basis for the comprehensive development and utilization of the active components of Phellinus igniarius polysaccharides and the medicinal resources of SHP.Methods:1.Using the single factor experimental design method,taking the moistening temperature and time as the investigation factors,and taking the polysaccharides,total phenols,ergosterol and the color of the prepared pieces of SHP as the evaluation indexes,the optimal production process of the prepared pieces of SHP was selected through the comprehensive scoring method.2.The crude polysaccharide of Sanghuangporous vaninii(Ljub)L.W.Zhou& Y.C.Dai(SHP)obtained by water extraction,ethanol precipitation,protein removal and freeze drying was separated and purified by Cellulose DEAE-52 cellulose ion exchange column,and then separated and purified by Sephadex G-100 column of dextran gel chromatography to obtain a uniform polysaccharide.The absolute molecular weight was determined by GPC-RI-MALS;The monosaccharide composition was determined by ion chromatography;Methylation analysis;Nuclear magnetic resonance spectrum;The structure of the polysaccharide was analyzed by FT-IR and SEM.3.CCK-8 method was used to detect the effect of SHP on the proliferation of mouse monocyte macrophage RAW264.7;Using ELISA kit to determine the effect of Sanghuang homopolysaccharide on LPS-induced nitric oxide and inflammatory cytokine tumor necrosis factor in TNF-α,IL-6 and IL-1βImpact of release.Result:1.The best production process of SH decoction pieces: take the original medicinal materials,remove impurities,wash,soften at 50 ℃,cut thick pieces,dry at 50 ℃,and sift away debris.2.After water extraction and alcohol precipitation,deproteinization and freeze-drying,9.538 g crude polysaccharide(SHP)from Phellinus igniarius was obtained.Three polysaccharides,SHP-W,SHP-1 and SHP-2,were separated and purified by Cellulose-DE-52 celluloseion exchange column.Three homogeneous polysaccharide components SHP-W-1,SHP-1-1 and SHP-2-1 were further purified by Sephadex G-100 column chromatography with dextran gel.The structures of SHP-W-1,SHP-1-1and SHP-2-1 were preliminarily analyzed by gel chromatography differential multiangle laser light scattering GPC-RI-MALS,monosaccharide composition,methylation analysis,FT-IR,and scanning electron microscopy(SEM).The weight average molecular weight,number average molecular weight and average molecular weight of SHPW-1 are 16.075 k Da,15.506 k Da and 16.478 k Da respectively,and the polydispersity coefficient is 1.037.SHP-W-1 is composed of Fuc,Ara,Rha,Gal,Glu,Xyl,Man and Rib.SHP-W-1 has 17 glycosidic bonds.The weight average molecular weight,number average molecular weight and average molecular weight of SHP-1-1 are 333.599 k Da,43.427 k Da and3516.544 k Da,respectively,and the polydispersity coefficient is 7.682;SHP-1-1 is composed of Fuc,Ara,Rha,Gal,Glu,Xyl,Man and Man-UA;SHP-1-1 has 25 glycosidic bonds.The weight average molecular weight,number average molecular weight and average molecular weight of SHP-2-1 are 563.032 k Da,59.641 k Da and 4205.062 k Da respectively,and the polydispersity is 9.44;SHP-2-1 is composed of Fuc,Ara,Rha,galactose,Glu,Xyl,Man and Man-UA;SHP-2-1 has 25 glycosidic bonds.SHP-W-1,SHP-1-1 and SHP-2-1 are spherical molecules.3.The results of CCK-8 method showed that SHP-W-1 was dosedependent in the range of 10～150 μg/m L,and SHP-1-1 and SHP-2-1 were dose-dependent in the range of 10～100 μg/m L.With the increase of concentration,the effect on cell proliferation becomes more obvious.All three polysaccharides can reduce the content of NO in LPS-induced RAW264.7 macrophages and inhibit TNF-α,IL-6 and IL-1β It shows good anti-inflammatory activity.Conclusion:In this study,the optimal process of industrial production of Sanghuang decoction pieces was investigated,and the crude polysaccharide of Sanghuang was obtained by water extraction and alcohol precipitation,protein removal dialysis and other methods.On this basis,the crude polysaccharide was further isolated,purified and structurally characterized to explore its anti-inflammatory activity in vitro.This study provides a theoretical basis for the active components of Sanghuang polysaccharide and the comprehensive development and utilization of Sanghuang medicinal resources.