Extraction,Purification,and Immunoregulation Activity Of Water Soluble Polysaccharide From Auricularia Auricular Judge

Auricularia auricular judae,a well-known edible and medicinal mushroom,belonging to the families of Basidiomycota and Agaricomycotina,which is rich in nutrients.Furthermore,Auricularia auricular polysaccharide is the most important component that have multiple pharmacological activities and bioactivities.Many studies have demonstrated that Auricularia auricular judae possess beneficial biological activities such as immunomodulatory,antitumor and antiinflammatory.However,there is little data for the immunomodulating actions of Auricularia auricular polysaccharides and the dada of the relationships between the immunostimulating activity and structural characteristics of the polysaccharide is even less.As such,the aim of this study was to investigate optimization of the hot-water extraction technology,isolation and purification,immunomodutatory activity and structrul characterization from Auricularia auricular judae.Many results are listed as follows:(1)In this research,the hot-water extraction technology of polysaccharide from Auricularia auricular judae was studied by a single-factor test and the orthogonal test respectively.The influence of factors were analyzed,including liquid-solid ratio,stirring speed,extraction time,extraction temperature and the particle size of Auricularia auricular.The optimal technology condition was obtained,based on the best yield rate of the polysaccharide.The optimal parameters for this method were selected 80 mesh as the particle size,with the solid-liquid ratio of 30:1(ml/g),the stirring speed of 200 r/min at 100 ? for 70 min.Under this condition,the yield rate of the polysaccharide could up to be 8%.(2)Auricularia auricular judae was extracted on the basis of the best technology parameter.The extracts were deproeined,then removed the low-molecular-weight matter.Two polysaccharides were precipitated from different concentrations of ethanol,then dialyzed and freeze-dried.The total sugar content was detected by phenol sulfuric acid method,and the results were 96.35 % and 98.36 % respectively;uronic acid content was determined by sulfuric carbazole method,and the results were 23.759 % and 30.14 % respectively.(3)Two polysaccharides were isolated and purified by ion exchange column chromatography and gel filtration column chromatography.Everyone was devided a mainly pure fraction,named AAP-10-Se and AAP-80-Se.(4)The immunomodulatory effects of AAP-10-Se and AAP-80-Se were evaluated in cyclophosphamide(Cy)-induced immunosuppressed mice.The results showned that two polysaccharide fractions accelerated recovery of spleen indice,promoted peritoneal macrophage phagocytosis,enhanced NK cell activity,as well as T cell proliferation response.These results indicate that AAP-10-Se and AAP-80-Se can enhance animal nonspecific and specific immunity,and they could be a potential immunomodulatory agent.In addition,the immunomodulatory effect of AAP-10-Se is better than AAP-80-Se.(5)The phenomenon result showed that AAP-10-Se and AAP-80-Se not contained nucleic acid and protein.In IR spectrum,they displayed the characteristic absorptions of carboxyl group and beta pyranoid glycosidic linkages in the two polysaccharide fractions.(6)The homogeneity and molecular weight of the polysaccharide fractions was determined by high-performance gel-permeation chromatography(HPGPC),and the results showed that they were homogenerous polysaccharides.The Mn and Mw of AAP-10-Se was 6.56×105 Da and 4.44×106 Da,while the Mn and Mw of AAP-80-Se was 3.75×105 Da and 1.49×106 Da.The molecular weight of AAP-10-Se was larger than AAP-80-Se.(7)Monosaccharide composition of AAP-10-Se and AAP-80-Se were determined using PMP pre-column derivatization method and sugar nitriles acylation Gas chromatography method.The result of the former showed AAP-10-Se was composed of mannose and glucose with the ratio of 14.34% : 85.66%,and AAP-80-Se was consisted of manmose,glucruonic acid,glucose and xylose with the ratio of 53.6% : 12.5% : 25.71% : 8.2%;and the result of the latter showed that AAP-10-Se was composed of mannose and glucose with the ratio of 20.4% : 79.6%,and AAP-80-Se was consisted of xylose,manmose and glucose with the ratio of 8.33% : 69.44% : 22.33%.There are some differences from the two polysaccharide fractions.AAP-10-Se mainly consists glucose,while AAP-80-Se mainly contains mannose with glucruonic acid,being an acidic heropolysaccharide.PMP pre-column derivatization method can reflect not only monosaccharide composition but also uronic acid content,which is more comprehensive than the sugar nitriles acylation Gas chromatography method.(8)The results of Scanning electron microscopy(SEM)and X-ray diffraction(XRD)showed that the polysaccharide fractions were both amorphous and non-crystalline structure.