Isolation,Purification,Structure Characterization And Immunoregulatory Effect Of Polysaccharide From Cordyceps Militaris

Cordyceps militaris,a traditional edible and medicinal mushroom,which has been widely considered as a folk tonic food in China and other Asian countries due to similar pharmacological activities to the well-known Chinese traditional medicine Cordyceps sinensis.As the main active ingredient in C.militaris,polysaccharide has multiple pharmacological activities,such as immunomodulatory,anti-tumor,hypoglycemic,hypolipidemic and anti-obesity.Modern medical research has shown that the immunomodulatory activity of polysaccharides plays an important role in improving the body's immune defense mechanisms in the elderly and cancer patients.In order to identify the structure and immunomodulatory activity of C.militaris polysaccharides,a variety of chromatographic techniques were used to separate and purify three homogeneous polysaccharides from the fruiting bodies of C.militaris in this study.Finally,a novel component CMP-III with excellent immunomodulatory activity was successfully screened.Subsequently,the CMP-III was characterized by a variety of optical and chemical analysis methods.The RAW264.7 macrophage cell line was used as a cell model to investigate the phagocytosis of macrophages,NO and immune-related cytokines(IL-6,TNF-?)secretion and m RNA expression,as well as the regulation of MAPKs,NF-?B signaling pathway-related protein levels,and then explore the immune regulation of macrophages and its mechanism.The main findings of this paper were as follows:(1)Macroporous resins for simultaneous decoloration and deproteinization of C.militaris polysaccharidesThe resin NKA-9 with the best adsorption effect was selected from ten macroporous adsorption resins with different properties.The decoloration ratio,deproteinization ratio and polysaccharide recovery ratio were 80.69%,62.51%and 78.08%,respectively.The static adsorption results showed that the optimal adsorption conditions for NKA-9 resin were adsorption time 120 min,room temperature,natural p H,initial concentration 2.5mg/m L;Dynamic adsorption results showed that the optimum sample flow rate was 0.5BV/h,the optimal sample processing volume is 4 column volumes.(2)Screening for the excellent activity of the new polysaccharides of C.militarisThe crude polysaccharide of C.militaris was preliminarily purified by DEAE-52column.The three components were eluted with distilled water,0.1M and 0.3 M Na Cl solution,and further purified by Sephadex G-100 and G-200 column chromatography.Three components of CMP-I,CMP-II,and CMP-III were obtained,respectively.It was found by GPC that CMP-I and CMP-II were homogeneous components with molecular weights of 5.948×10³ Da and 1.529×10⁴ Da,respectively,but similar components have been reported in previous paper.Screening for CMP-III components as new components through the molecular weight,homogeneity detection and cell activity tracking,which has the value of further research.(3)Physicochemical properties and structural analysis of CMP-IIIThe content of carbohydrate,protein,uronic acid and sulfuric radical and total polyphenols in CMP-III were determined according to the reported methods of phenol-sulfuric acid,coomassie brilliant blue,m-hydroxybiphenyl,Ba Cl₂-gelatin and Folin phenol method,respectively.The results showed that the total sugar content of the CMP-III component was 89.47%,the protein content was 0.895%,the uronic acid content was2.628%,the sulfuric radical content was 2.550%,and the total polyphenol content was0.815%.Molecular weight determination,monosaccharide composition analysis,infrared spectroscopy,methylation reaction and NMR showed that the weight average molecular weight(Mw)of CMP-III was 4.796×10⁷ Da,exhibiting a tightly packed spherical conformation in water and DMSO when 1 mg/m L at 25°C.This component consists of glucose,mannose and galactose,accounting for 86.6%,10.7%and 2.7%,respectively.The preliminary determination of FT-IR spectroscopy is mainly?-pyran type polysaccharide.Combined with methylation and NMR results,it is speculated that CMP-III has a backbone of?4)-?-D-Glcp-(1?4)-?-D-Manp-(1?,?-D-Manp branched at C-6 by side chain of?4,6)-?-D-Manp-(1?.(4)Immunomodulatory activity of CMP-IIIUsing macrophage RAW264.7 model,the results showed that the safe concentration of CMP-III was below 200?g/m L,which was enhanced by stimulation with different concentrations of CMP-III(25,50,100,200?g/m L).CMP-III activates MAPKs signaling pathway by inducing phosphorylation of ERK1/2,JNK1/2 and p38 proteins in macrophages,and also stimulates phosphorylation of I?B?and NF-?B p65 via NF-?B signaling pathway.The NF-?B p65 is translocated from the cytoplasm to the nucleus,regulates the expression levels of i NOS,IL-6 and TNF-?m RNA are significantly increased.The phagocytic activity of RAW264.7 cells can also significantly promote the expression of secretion of NO and immune-related cytokines IL-6 and TNF-?by macrophages,ultimately exerting immunomodulatory activity.