Extraction,Isolation,Purification,Structural Characterization And Immunomodulatory Activity In RAW264.7 Cells Of The Polysaccharides From Pueraria Lobate (willd.) Ohwi Root

The dry root of Pueraria Lobata(willd.)Ohwi had been recorded in the famous Chinese medical book "shen nong sutra" around thousands of years ago.Because it is rich in isoflavones,its medicinal value is very high.In this paper,two kinds of water-soluble active polysaccharide GE-1 and GE-2 were isolated and purified by using Pueraria Lobata(willd.)Ohwi root as raw materials.Both polysaccharide are compared on the structure and immune activity.The results showed that: Ethanol reflux method is used to remove the flavonoids in Pueraria Lobata(willd.)Ohwi root.Enzymatic hydrolysis is used to remove starch and cellulose.The Sevag method is adopted to remove protein and the method of "water extract-alcohol precipitation" is used to get water soluble crude polysaccharide from root of Pueraria Lobata(willd.)Ohwi.After separation and purification of DEAE-Sepharose Fast Flow and glucan gel column chromatography,the two components of GE-1 and GE-2 are obtained.GE-1 is neutral polysaccharide,GE-2 is acid polysaccharide,and the content of uronic acid was 1.5%.The molecular weight of GE-1 was 9.9kDa,and the molecular weight of GE-2 was 12.3kDa.By scanning electron microscopy,it is found that GE-1 has the reticular structure with small pores on the surface.GE-2 has the strip structure with a smooth surface.Ultraviolet full-band scan show that GE-1 and GE-2 contain no protein and nucleic acid substances,so is is not glycoprotein.The infrared spectra shows that GE-1 and GE-2 contain polysaccharide characteristic absorption peaks,and it only contain the alpha-type pyranose without the alpha furanose.The monosaccharide composition analysis show that GE-1 and GE-2 are composed of glucose,and without other kinds of monosaccharides.High iodate oxidation,Smith degradation and methylation showe that the GE-1 has the 1,4-glcp and 1,6-glcp glycosidic bond,and the molar ratio between them is 1.72:2.64.GE-2 contains 1,3-Glcp,1,4-Glcp,1,3,6-glcp and 1,6-glcp bonds.The molar ratio of each glucoside bond is 1.90:5.52:1.43:2.45.One-dimensional NMR analysis shows that GE-1 has the straight chain structure with →4)-α-D-Glc(1→ and →6)-α-D-Glc(1→ sugar residues.GE – 2 has the branched chain of →6)-α-D-Glc(1→,its straight chain structure is composed of→3)-α-D-Glc(1→,→4)-α-D-Glc(1→ and →3,6)-α-D-Glc(1→ sugar residues.Both GE-1 and GE-2 have immunoregulation activity,which can promote the secretion of cytokines NO,TNF-α and IL-6 in mouse macrophages RAW264.7.However,the promotion effect of GE-1 is very weak,and the promotion effect of GE-2 is stronger.GE-2 can specifically interact with the surface receptor SR,GR and TLR4 of mouse macrophage RAW264.7 to activate the immune response,thus affecting the secretion of cytokines NO,TNF-α and IL-6.