Characterization Of Polysaccharide From Astragalus Radix As A Macrophage Stimulator

Astragalus membrabaceus is used as a chinese herb medicine, containing polysaccharide,protein,alkaloid,amino,flavonids,glycoside,trace element etc.Astragalus polysaccharide(APS) is main biological active conponent.APS have morpharmacol effect,includingimmunomodulation,lowerbloodsugar,antistress,antitumor,antivirus,antio xidant etc. Extraction of APS have been the focus in the traditional chinede medicine and the modern researchof chinese herbal medicine owing to the multibioactive and well clinical therapeutic effect.Macrophage played a significant role in the host defense,and it is the key participants in the innate immune response.when acticated they inhibit the growth of a wide variety of tumor cells and microorgnisms.in addition,macrophage can be functioned as antigen-presenting cells and interact with T lymophocytes to modulate the adaptive immune reponse.furthermore,macrophage was involved in tissue remodeling during embrygenesis,wound repair,clearance of apoptotic cells and hematopoiesis.Concretely this study included two parts In the first part, five fractions water soluble polysaccharide named Astragalus polysaccharide(APS) was obtained by hot water extraction,alcohol precipitation, gel-permeation chromatography and ultra filtration,with the weight-average molecular weight(MW) of about2.4×i05,1.0x105, 6.9x104,3.2×104,1.6×104Da detected by Gel Permeation Chromatog-Size Exclusion Chromatography(GPC-SEC).The 6.9x104 Da fraction was selected to the further research owing to its component homogenicity,high yield and purity.In the second part,macrophages-like RAW264.7 cells were cultured.Fluorescence material 2-aminoacridone (2-AMAC) labeled APS(2-AMAC-APS) was applied to investigate the binding of RAW264.7 macrophage to APS, the observation was carried out using laser-scanning microscope. Griess and ELISA method was applied to investigate the production including NO, TNF-a and GM-CSF by RAW264.7 macrophage.PMB the specificity inhibitor of LPS was used to exclude the contamination of APS.The extraction and purification of APS illustrated:rude APS was obtained by hot water extraction,alcohol precipitation,then the crude APS applied to gel chromatography. water soluble polysaccharide with the average MW of 6.9×104Da was obtained,weighted 0.92g,suger containing 92%The binding of RAW264.7 macrophage to APS demonstrated that,2-AMAC labled APS can specificity bined to macrophage in a time-dependent manner,and the binding effect can be blocked by unlabled APS,it is concerned that the inhibition can be possibily owing to the special receptor saturationBioactive investigation of APS shown:RAW264.7 macrophage treated with various concentration of APS(0,12.5,25,50,100(μ/ml) for 12h,the50, 100μg/ml group could remarkably increase the production of NO(P<0.05),and the 25,50,100μg /ml group also siginificantly increase the level of TNF-a, GM-CSF secreted by macrophage,LPS treated was used as positive control..To test for the possilble contamination of bacterial LPS in APS,we examine the effect of PMB on APS-induced production of NO.The result demonstrated that the activation of macrophages by APS was not due to the contamination of LPS in APS.Conclusively, hot water extraction,alcohol precipitation combined with GPC-SEC method can both easily and effectively applied to extract and charicterize APS from Astragalus radix,the production with the yield of 0.91%,the content of polysaccharide were 92%,MW 6.9×104Da.APS can activate RAW264.7 macrophage in a dose-dependent manner,increase the level of NO, TNF-a, GM-CSF.The results suggested that APS possess potent immunomodulatory activity by stimulating macrophage and could be used as an immunotherapeutic adjuvant.