Structure Characterization And Antioxidant Activity Of Polysaccharide TLH-G From Tricholoma Lobayense

T.lobayense is a nutritious and precious edible fungus.In this paper,In order to screen the polysaccharide from T.lobayense with the high antioxidant activity,a novel polysaccharide TLH-G was screened from different kinds of fruiting body..And the physical and chemical properties and structure characteristics of TLH-G were studied in detail.Combining with the previous studies on the structure-activity relationship of polysaccharides from T.lobayense,the factors that influence the high antioxidant activity of polysaccharides were further analyzed.The results are as follows:1.Screening of different T.lobayense strainsThere was obvious antagonistic effect between the strains using the plate culture method in the macroscopic view.The phylogenetic relationships of the three species were analyzed by ITS sequencing.They were found to be more than 99%similar to Tricholoma giganteum(Registration number:KF360838.1,KF360837.1,AF042591.1).It is determined that they were different species T.lobayense,combined with their respective morphology of cultivated fruiting body.According to the extraction and HPLC analysis,the distribution of polysaccharides of three different kinds of fruiting bodie swas different,and had species diversity,which was closely related to the genetic factors of T.lobayense itself.The fruiting body polysaccharide obtained from the Gaoyou strain contains three fractions which are consistent with the earlier study,and the smaller molecular weight polysaccharide fraction from Gaoyou was proved to be most understanding antioxidant activity.The results showed that the polysaccharide of Gaoyou mushroom had excellent antioxidant ability.So we choose the fruiting body polysaccharide obtained from the Gaoyou strain for the next exprement.2.Antioxidant activity of T.lobayense polysaccharide TLH-G(1)The crude polysaccharide was obtained by flash extraction,and then the protein was removed with Savage methods.T.lobayense polysaccharides TLH-G was seperated by ultrafiltration.HPLC-ELSD proved that TLH-G was homogeneous,the molecular weights was 4.1×103 Da.The extraction yield of TLH-G was 2.8%.The total sugar content of TLH-G was up to 99%.The uronic acid content was 13.85%,which resulted to good water solubility.Ultraviolet spectroscopy showed that TLH-G had no protein and nucleic acid.The fourier transform infrared spectroscopy(FT-IR)showed that there was the typical absorption peak and only pyranoid ring conformation in TLH-G.(2)The results of antioxidant activities in vitro showed,TLH-G showed high antioxidant activity in the system of scavenging DPPH and total antioxidant.The IC50 of TLH-G was 430 ?g/mL in the free radical scavenging assay for DPPH.The IC50 of TLH-G in the determination of total antioxidant capacity was greater than 500 ?g/mL.The antioxidant activities of nine common fungi polysaccharides were compared and evaluated by the free radical scavenging assay for DPPH.And we found that TLH-G had the lowest IC50.The results demonstrated that the novel polysaccharide TLH-3 from T.lobayense was a natural and efficient antioxidant.2.Physical and chemical properties and structure of TLH-Q which was separated,was identificated to further analyse the structure-activity relationship,compared with its relationship between antioxidant activity and structure of TLH-3.(1)The molecular weight of TLH-G was 4.1 x 103 Da.It was further proved that the smaller the molecular weight,the stronger the antioxidant activity was.At the same time,the polysaccharide TLH-G and TLH-3 have strong water-solubility,which further indicated that the antioxidation activity of polysaccharide from T.lobayense was closely related to its water-solubility.(2)The monosaccharide composition showed that the monosaccharide molar ratios of TLH-G was mannose:rhamnose:arabinose:glucuronic acid:galacturonic acid:glucose:galactose = 1.00:0.56:0.14:0.33:<0.10:14.13:2.92.Compared with the monosaccharide composition of TLH-3,the absolute content of glucose and galactose and a certain amount of rhamnose were found to be an important factor in the high antioxidant activity of polysaccharides TLH-3 and TLH-G More importantly,galactose and galacturonic acid content was directly related to the activity of TLH-3 and TLH-Q which is an important factor affecting antioxidant activity of polysaccharides.(4)Methylated analysis demonstrated that TLH-G was maily composed by the mixture of 1,6-linked-Glcp.In addition,there is a certain proportion of 1,6-linked-Galp.The branching degree of TLH-G was 0.21,which belonged to small branch polysaccharide,while the branching degree of TLH-3 was 0.74,which was a highly branched polysaccharide.This indicated that the degree of branching is another factor affecting the antioxidant activity of polysaccharides TLH-G and TLH-3.The higher the branching degree is,the stronger the antioxidant activity is.(5)The structure of TLH-G was predicted by NMR.The structures of TLH-G was(1?6)-?-D-Glcp and(1?6)-?-D-Galp.There is a branch point:(1 ? 3,6)-a-D-Manp.The results were consistent with the results of methylation GC-MS.The multiple glycosidic bond types were the important reason for the high antioxidant activity of T.lobayense polysaccharides,compared with TLH-3.The important role of 1,6-a-D-lingked-Glcp,1,4-a-D-linked-Glcp and 1,3-?-D-linked-Galp in TLH-3 was further explained by the difference of the activitivity of T.lobayense polysaccharides.In addition 1,6-?-D-linked-Glcp and special structural features make TLH-G strong antioxidant activity.In conclusion,this study described a novel polysaccharides screened and isolated from the fruiting body of Gaoyou T.lobayense had outstanding antioxidant activities.Furthermore,combined with the structure-activity relationship of TLH-3,the results showed that the molecular weight,water-solubility and other factors,such as monosaccharide composition,branching degree and glycosidic bond type,had important influence on antioxidant activity of polysaccharides.This study provided theoretical support for the construction of the structure-activity relationship of polysaccharides from T.lobayense,and was of great significance for the development of high activity T.lobayense polysaccharides.