Preparation,Physicochemical Properties And Immunomodulatory Effects On CD4~+ T Cells Of Polysaccharides From Maca

Objective:Four maca polysaccharide components with different molecular weights were extracted from maca by using a graded alcohol precipitation method.Comparatively study the differences in physical and chemical properties of maca polysaccharide components,as well as the effects on the proliferation of CD4~+T cells and secretion of cytokine,to promote the structure-activity relationship of maca polysaccharides.Based on the results of privious experiments,the most active maca polysaccharide component was determined.Fluorescent labeling of this component and the ways to act on CD4~+T cells were explored.Methods:(1)Four grades of maca polysaccharides(MCP1,MCP2,MCP3,MCP4)were extracted from maca powder using graded alcohol precipitation method,and the extraction rates were calculated,the total sugar contents,uronic acid contents and protein contents were determined separately.The molecular weights and monosaccharide compositions of each component of maca polysaccharides were determined by size exclusion chromatography and high-performance liquid chromatography,the ultraviolet spectrum and Fourier transform infrared spectrum of maca polysaccharides were measured by UV spectrophotometer and infrared spectrometer,respectively,and their chemical structures were analyzed.(2)Immunomagnetic beads were used to isolate and purify CD4~+T cells derived from mouse spleen.After treatment with maca polysaccharides,the proliferation of CD4~+T cells was measured by CFSE labeling method,the secretion of IFN-?and IL-4 was detected by ELISA.Based on the above experiments,the most active component(MCP2)was selected for fluorescent labeling.(3)After reacted with tyramine and sodium boron cyanide,then MCP2 reacted with FITC to bind FITC on MCP2.The MCP2 labeled with FITC was purified with a Sephadex G-200 chromatography column,and the eluent with the highest content of polysaccharide and FITC was collected.The eluate was freeze-dried to obtain MCP2-Tyr-FITC and the efficiency of fluorescent labeling was measured.MCP2 and MCP2-Tyr-FITC were added to the culture system of CD4~+T cells,the proliferation of cells was measured by the CCK-8 method,and laser confocal microscopy and flow cytometry were used to detect the mode of action between MCP2-Tyr-FITC and CD4~+T cells.Results:(1)MCP1 and MCP2 had similar extraction rates,total sugar contents,uronic acid contents and protein contents,while the extraction rate,uronic acid content and protein content of MCP2 were significantly different from those of MCP3 and MCP4.The molecular weights of maca polysaccharide components(MCPs)from MCP1 to MCP4 showed a clear trend from high to low.In addition,the purity of MCP2 was higher than that of MCP1,MCP3 and MCP4.MCPs were composed of monosaccharides such as galacturonic acid(GalUA),galactosamine(GalN),glucose(Glc),galactose(Gal)and arabinose(Ara),but the proportion of monosaccharide composition was not exactly the same.The content of GalN in MCP2 was significantly higher than the other three polysaccharides.There were only trace amounts of protein in MCPs and there was no significant difference in chemical structures.(2)MCPs all had the function of promoting CD4~+T cells proliferation and IFN-?secretion,and the optimal concentration was 50?g/mL.In addition,the activities of promoting CD4~+T cells proliferation and IFN-?secretion of MCP2 were higher than MCP1,MCP3 and MCP4.However,MCPs have no significant effect on IL-4secretion.(3)The fluorescence labeling efficiency of MCP2-Tyr-FITC was 4.9%.Compared with MCP2,the effect of MCP2-Tyr-FITC on the proliferation of CD4~+T cells was not significantly different at 50?g/mL.Besides,the results showed that MCP2-Tyr-FITC could bind to the membrane of CD4~+T cells.Conclusion:(1)Four components of maca polysaccharides(MCPs)were successfully obtained by using a graded alcohol precipitation method.The molecular weights and monosaccharide compositions of MCPs were different.Among them,MCP2 had the highest purity,and its GalN content was significantly higher than the other three components.(2)MCPs all had the function of promoting CD4~+T cells proliferation and IFN-?secretion(p<0.01),and the optimal concentration was 50?g/mL.Among them,the function of promoting CD4~+T cells proliferation and IFN-?secretion of MCP2 were significantly stronger than other three components.(3)MCP2-Tyr-FITC could bind to the membrane of CD4~+T cells.