Studies On The Structure Identification And Bioactivities Of Polysaccharide (BSF-X) And The Immune Activities Of Polysaccharide (BSF-1) From Boletus Speciosus Frost

Boletus speciosus Frost that belongs to Boletaceae of Boletus,is a kind of edible large fungus.B.speciosus has strong immune activity and anti-tumor function,and is ideal for natural edible fungi.At present,most of the studies of B.speciosus Frost were concentrated on the cultivation-domestication and cultivation techniques,the nutrient characteristics of the fruiting bodies and the structure of polysaccharides,while the biological activity of polysaccharides from B.speciosus Frost were seldom reported.In this study,the polysaccharides of B.speciosus Frost were extracted and purified by hot water extraction and column chromatography,and its structure was identified by chemical methods and spectroscopy methods.The bioactivities of anti-tumor and immunoregulatory activities were studied using modern biotechnology.Raw264.7 cell was used as a modle to study the mechanism of immunoregulatory activity.These studies on the extraction and purification,structural identification and biological activity of polysaccharides from B.speciosus Frost provided a theoretical basis for the study and development of the economic value and medicinal value of B.specious Frost,and for the further large-scale cultivation of domesticated B.specious Frost and the development and utilization of polysaccharide.Also,it provided a prerequisite for the regulation of immune regulation activity,anti-tumor and its mechanism.In this paper,there were four parts,which contained the extraction,isolation and purification of polysaccharide from B.speciosus Frost;the study of structural analysis and identification;the study of immunomodulatory activities and antitumor activities;and the preliminary study on the mechanism of immunoregulatory activity.The crude polysaccharide of B.speciosus Frost was obtained by hot water extraction and ethanol precipitation from the fruting badies,and the acquisition rate was 15.12%.The crude polysaccharide of B.speciosus Frost was purified by DEAE-cellulose column chromatography to obtain pure polysaccharide,and named BSF-X.We found that there were not protein and nucleic acid by UV-free scan,and detected polysaccharide content by sulfuric acid phenol method.High-performance gel permeation chromatography detected that the Mw of BSF-X was 141309 Da.The structure of polysaccharide was studied by infrared spectrum,GC-MS and NMR.The results showed that the molecular weight of BSF-X was 141309 Da,and it was composed of β-D-glucosepyranose and α-D-galactosepyranose,which ratio of these two kinds of monosaccharides was 2: 1.The polysaccharide of BSF-X had a skeleton that the main chain was(1 → 4)-β-D-glcp,and a side chain had a →1,6)-α-D-galp on 6-O,and 2-O of side chain on galactosepyranose was attached to → 4)-β-D-glcp.The proliferation of three kinds of immune cells(B lymphocytes,T lymphocytes and macrophages)were detected by CCK-8 kit.The cell cycle of the three cells were detected by the cycle kit,and the ability of phagocytosis of macrophages by the neutral red method,and the abilities of releasing IL-6,IL-23,TNF-α on macrophages by ELISA kit.These results found that BSF-X in a certain concentration range could promote the proliferation of three immune cells,and it significantly improved the cell numbers in the S phase.It also evidently promoted the ability of swallowing the neutral red and the abilities of releasing immune factors(IL-6,IL-23 and TNF-α)in macrophages.The effect of BSF-X on the growth of L929 cells in vitro was studied by CCK-8.The results showed that BSF-X could inhibit the growth of L929 cells with a significantly dose dependent on the concentration of BSF-X in a certain range.Through the experimental analysis of S180 tumor model,BSF-X could inhibit the growth of S180 tumor in vivo,and the tumor inhibition rate was 61.35%.These experiments indicated that BSF-X had a strong immunomodulatory activity and anti-tumor activity.The results suggested that polysaccharide from B.speciosus Frost(BSF-1)could promote the proliferation of B cell,T cell and macrophages,and achieved the proliferation of macrophage cells by shorting cell cycle of G0/G1 phase and promoting cell cycle of S phase and G2/M phase,which might induce cell division.In the case of macrophages of Raw264.7 cells,the total RNA was extracted,and these studies about the differential gene analysis of the transcriptome,and GO annotation and KEGG/GO pathway analysis found that 22.22% DEG belongs to the MAPK signal pathway.In the MAPK signaling pathway,BSF-1 could play a role by stimulating EGF to bind to epidermal growth factor receptor(EGFR)on the cell surface,which could lead to cell proliferation,differentiation and survival.The process also increased intracellular calcium levels,glycolysis and protein synthesis,which ultimately lead to cell proliferation and DNA synthesis.This process could fully explain the mechanism of BSF-1 on macrophage proliferation.