Isolation,Structural Characterization And IEC-6 Cell Migration Activities Of Polysaccharides From Atractylodes Macrocephala Koidz

BackgroundsAtractylodes macrocephala koidz is a chinese traditional medicine with the function of strengthening spleen and replenishing Qi,also it is the composition of famous prescriptions such as Sijunzi soup,Shenlingbaizhu powder and Buzhongyiqi soup etc.Chinese traditional medicines of strengthening spleen and replenishing Qi can promote renovation of the damage of gastric mucosa,gastric mucosa protection is the important targets.Cell migration play a key role during the gastrointestinal tract to restoring the continuity of the surface epithelium after extensive destruction.Our previous research found that polysaccharide of Atractylodes macrocephala koidz was able to promote IEC-6 migration,and could completely reverse DFMO-induced inhibition of IEC-6 migration,which came into contact with polyamine mediated potassium channel activation of signaling pathways.But the polysaccharide is complicated,the chemical structure is unknown.In this paper,we attempt to screen and isolate the polysaccharide,and validate whether these polysaccharide fractions exhibited promoting migration activities.ObjectiveIn the present study,we attempt to screen and isolate the bioactive polysaccharide fractions from the roots of Atractylodes macrocephala koidz using bioassay-guided fractionation,their chemical stuctures were then characterized on basis of spectroscopic data and physiochemical properties.Methods1.Bioassay-guided fractionation was performed to screen and isolate bioactive polysaccharide fractions from the roots of Atractylodes macrocephala koidz by aqueous extraction,ethanol precipitation and purification followed by column chromatography on DEAE-cellulose and Sephadex LH-20.2.The chemical structures of bioactive polysaccharide fractions were then elucidated on the basis of acidic hydrolysis,gel permeation chromatography-multi-angle laser light scattering(GPC-MALLS),environmental scanning electron microscope and spectroscopic data,including FT-IR,1D-and 2D-NMR analysis.3.The in vitro IEC-6 wound healing model was carried out to validate whether these polysaccharide fractions exhibited promoting migration activities.4.The Real-Time Cell Analysis(RTCA)assays were performed to evaluate effects of these isolated polysaccharides on IEC-6 cell proliferation.Results1.Bioassay-guided fractionation led to isolation of two new polysaccharides fractions YY13008 and YY21714 from the roots of Atractylodes macrocephala koidz by aqueous extraction,ethanol precipitation and column chromatography purification on DEAE-cellulose and Sephadex LH-20.2.The chemical structures of polysaccharides YY13008 and YY21714 were then elucidated on the basis of acidic hydrolysis,environmental scanning electron microscope and spectroscopic data,including IR,1D-and 2D-NMR analysis.The polysaccharide fractions YY13008 and YY21714 were determined as homogeneous,with molecular weight of 6546 and 2898,respectively,as determined by a gel permeation chromatography-multi-angle laser light scattering(GPC-MALLS)method.The polysaccharide fractions YY13008 and YY21714 were found to be fructans containing almost exclusively a(21)-linked ?-D-fructofuranosyl repeating units with terminal a-glucopyranosyl unit.3.The in vitro wound healing assays revealed that the treatment of the polysaccharide fractions YY13008 and YY21714 with difluoromethylornithine(DFMO),a specific inhibitor of ornithine decarboxylase,was able to completely reverse DFMO-induced inhibition of IEC-6 migration,however,the treatment of YY13008 or YY21714 alone did not affect the basal rate of IEC-6 migration.4.The Real-Time Cell Analysis(RTCA)assays showed that the treatment of the polysaccharide fractions YY13008 and YY21714 stimulated cell proliferation of IEC-6 at the concentrations of 300-400 ug/mL.ConclusionBioassay-guided fractionation led to isolation of two homogeneous polysaccharide fractions YY13008 and YY21714 from the roots of of Atractylodes macrocephala koidz.Both of them were found to be fructans containing almost exclusively(2?1)-linked ?-D-fructofuranosyl repeating units with terminal a-glucopyranosyl unit.Although the treatment of YY13008 or YY21714 alone did not affect the basal rate of IEC-6 migration,the polysaccharide fractions YY13008 and YY21714 were able to completely reverse DFMO-induced inhibition of IEC-6 migration and promote IEC-6 cell proliferation.These findings differ from the previous reports.