The Studies On Structure Identification,Biological Activity Of Polysaccharide(LDS-1) From Lyophyllum Decastes(Fr.:Fr.) Sing

The edible and medicinal Lyophyllum decastes?Fr.:Fr.?Sing is a fungus belonging to Lyophyllum,Tricholomataceae.It was found that the polysaccharides extracted from Lyophyllum decastes?Fr.:Fr.?Sing could participate in many biological activities.At present,studies on Lyophyllum decastes?Fr.:Fr.?Sing were mainly focused on extracting crude polysaccharides to stimulate cells to participate in the immune response in vitro,and there were no related reports on the fine structure of the polysaccharide from Lyophyllum decastes?Fr.:Fr.?Sing,and there was a lack of systematic study on the mechanism of in vivo and in vitro stimulating cells to participate in the immune response.In this study,polysaccharides with high purification degree were extracted from the Lyophyllum decastes?Fr.:Fr.?Sing,which were collected from xiaojin county,sichuan province.And the structure of polysaccharides from Lyophyllum decastes?Fr.:Fr.?Sing was studied.Meanwhile,the in vivo and in vitro biological activities of the extracted polysaccharide from Lyophyllum decastes?Fr.:Fr.?Sing were systematically analyzed.It is expected to further understand the fine structure and immune mechanism of the high biological activity of the polysaccharides from Lyophyllum decastes?Fr.:Fr.?Sing.The main results of this paper are as follows:?1?The taxonomic status of Lyophyllum decastes?Fr.:Fr.?Sing was identified by ITS sequence analysis of internal transcribed spacer and morphological alignment.This fungus was identified as Lyophyllum decastes?Fr.:Fr.?Sing.?2?The fruit body of Lyophyllum decastes?Fr.:Fr.?Sing was used as the experimental material,and the crude polysaccharides were exstracted and purified to obtained the polysaccharides?LDS-1?by hot water extraction,alcohol precipitation,DEAE cellulose-52 chromatography,DEAE-FF gel chromatography,and super-clean water dialysis methods.Finally,the average weight molecular?Mw?of the polysaccharides was determined by high performance gel osmosis chromatography?HPLC-RID?.The polysaccharides were a homogeneous polysaccharide of 8681 Da.?3?The exact structure of LDS-1 was analyzed by Fourier Transform Infrared Spectrum?FT-IR?,High Performance Liquid Chromatography,Differential Refractive Detector?HPLC-RID?,Nuclear magnetic resonance ¹H spectroscopy?¹H-NMR?,Nuclear magnetic resonance ¹³C spectroscopy(¹³C-NMR),Heteronuclear Multiple QuantumCorrelation?HMQC?,Heteronuclear Multiple Bond Correlation?HMBC?,H-H correlation spectroscopy?¹H-¹H COSY?and Gas ChromatograPhy-Mass Spectrum?GC-MS?.Based on the above results,it was speculated that the monosaccharides unit structure of LDS-1 was composed of Glcpcose monosaccharides residues and Galpactose monosaccharides residues with a ratio of2:1.The monosaccharides unit structure of LDS-1 was mainly composed of 1,4-?-Glcpcose monosaccharides residues,in which two?-Galpactose monosaccharides residues were connected by a branch chain at 6-O of one Glcpcose monosaccharides residues,and the branched chain was composed of two?-Galpactose monosaccharides residues,which were combined in the way of 1,6-linkage.The C1 position of the Galpactose monosaccharides residue was connected to the branched chain,and the6-O position was connected to the other Galpactose monosaccharides residue.?4?By detecting the effects of LDS-1 on the cytological activities of T cells,B cells and RAW264.7 cells within the concentration range,the results showed that LDS-1 significantly promoted the proliferation of immune cells.By detecting the effects of LDS-1 on the secretion of immune factors by immune cells within the concentration range,the results showed that LDS-1 promoted the secretion of IgA,IgD,IgE,IgG,IgM,TNF-?,TNF1-?and IL-2,and the highest secretion rate of IgE was 46.22%.The phagocytosis of RAW264.7 cells were tested by neutral red and fluorescent microspheres.The results showed that LDS-1 promoted RAW264.7phagocytosis,and the phagocytosis rate of neutral red phagocytosis was up to60.67%.The effects of LDS-1 on the proliferation of CT26.WT,MFC,L929 and S180 tumor cells were detected in the concentration range.And the results showed that LDS-1 could inhibit the proliferation of tumor cells,and the highest inhibition rates of MFC cell was 45.01%.The inhibition rate of S180 tumor in mice was 54.32%by LDS-1stimulating.?5?By analyzing the transcriptome data of mouse S180 tumor tissue,it was found that LDS-1 mainly down-regulated creb,cd14,traf6,ikk,nf?b and other genes to reduce the expression of DNA,inhibit the proliferation and differentiation,and anti-aging of tumor cells.In the Ras signaling pathway,LDS-1 down-regulated rassf1,nore1,mst1,pak,rac,ikk,nf?b and other genes,negatively regulating the cell cycle and inhibit tumor cells to infect and metastasize.In the PI3K-Akt signaling pathway,egf,ras,raf1,pkcs,creb,ikk and nf?b were down-regulated to mediate DNA repair of tumor cells,reduce Glcpcose uptake and indirectly regulate tumor cell survival.LDS-1 negatively regulated the cell cycle of tumor cells and inhibited the ability of tumor cells to escape apoptosis and differentiation by up-regulation of p15INK4b,Wnt,FZD,C/EBP,PDGF,IkB?and other genes in cell circulation,Wnt signaling pathway and nuclear regions.Based on the above results,it can be known that the polysaccharide of Lyophyllum decastes?Fr.:Fr.?Sing?LDS-1?is a novelty polysaccharide.LDS-1 had the effects of promoting immunity and anti-tumor activity,and the research of LDS-1provided a theoretical basis for LDS-1's application in food and medicine.