| Object:1.To optimize the decolorization process of Brassica rapa L polysaccharides on macroporous adsorption resin.2.To isolate and identify the structures of the purified polysaccharides from Brassica rapa L.3.To preliminary evaluate the immunological activity of Brassic rapa L.polysaccharides based on the function of RAW264.7 cells.Mehtods:1.The decolorization process of Brassica rapa L polysaccharides macroporous adsorption resin was optimized by using Response surface methodology.On the basis of the comprehensive score?M?including the rate of polysaccharide retention,decolorization rate and protein removal rate.The sample concentration,flow rate and sample volume on the decolorization process of macroporous adsorption resin were optimized.2.The purified polysaccharides from Brassica rapa L.were separated and isolated by the combination of anion-exchange chromatography and gel permeation chromatography,and strctures were characterized by UV,HPGPC,FT-IR,GC-MS and NMR technology.3.To evaluated the immunological activity of Brassica rapa L.polysaccharides on RAW264.7 macrophages,CCK-8 assay was used to test the proliferation activity,and ELISA was chosed to determine in the secretory activity NO,TNF-?and IL-6 cytokines from macrophages RAW264.7.Results:1.The decolorization process of Brassica rapa L.polysaccharides macroporous adsorption resin was optimized,as follows:the concentration of the sample 1%,the flow rate 3.6 BV·h-1,and the sample loading amount 0.75BV.2.Two neutral polysaccharides?BRNP-1 240mg,BRNP-2250mg?and two acidic polysaccharides?BRAP-1 300mg,BRAP-2 1.26g?were separated and purified from Xinjiang Brassica rapa L.by the combination of DEAE-650M ion-exchange chromatography and gel permeation on Sepharose 6B and Sephacryl S-300 column.The apparent molecular weight of two neutral polysaccharides BRNP-1 and BRNP-2 were 6873Da and 4751Da,two acidic polysaccharides were6080Da?BRAP-1?,7590Da?BRAP-2?.Sugar composition analyses showed that BRNP-1and BRNP-2 were all composed only glucose?Glc?,while BRAP-1 contains arabinose?Ara?,Glc,galactose?Gal?and galacturonic acid?GalA?in a molar ratio of2.07:4.53:2.20:1,and BRAP-2 contains rhanose?Rha?,Ara,Glc,Gal and GalA in a molar ratio of 1.06:1:5.03:2.22:1.50.Methylation analysis showed that both BRNP-1 and BRNP-2 mainly consists of?1?4?-linked glucopyrannosyl?Glcp?,?1?4,6?-linked Glcp,termianally linked Glcp residues.Combined the methylation analyses and 1D-and2D-NMR analyses,both BRNP-1 and BRNP-2 contain the backbone composed of?-D-?1?4?-linked Glcp residues.In terms of BRNP-1,there is t-linked-?-D-Glcp branched at O-6 position of?-D-?1?4?-linked Glcp every eight backbone-glycosyl residues.And degree of branch?DB?is calculated as 0.243.As for BRNP-2,there is t-linked?-D-Glcp branched at O-6 position of?-D-?1?4?-linked Glcp every nine backbone-glycosyl residues.And DB is calculated as 0.238.3.Bioassay results showed both BRNP-1 and BRNP-2 could stimulate the maximum proliferation of macrophage RAW264.7 at the concentration 400?g·mL-1,and stimulate the release of NO,and secretion of IL-6 and TNF-?.Conclusion:1.The results of optimized the decolorization process of Brassica rapa L polysaccharides on macroporous adsorption resin were stable and reliable,simple and feasible.It will be benefit to the isolation and purification of polysaccharides from Xinjiang Brassica rapa L..2.Two neutral polysaccharides and two acidic polysaccharides were obtained from Xinjiang Brassica rapa L..Among them,two neutral polysaccharides BRNP-1 and BRNP-2 were first obtained from Xinjiang Brassica rapa L..Furthermore,combination of FT-IR,HPGPC,GC-MS,and NMR,BRNP-1 and BRNP-2 were structurally characterized firstly.It provides the material basis for biological activity research of polysaccharides from Xinjiang Brassica rapa L..3.Results of bioassay exhibited that both BRNP-1 and BRNP-2 showed significantly the immunostimulating activity based on the proliferation of macrophages,the release of NO,and secretion of IL-6 and TNF-?.It provided us preliminary information for the coming investigation on the anti-tumor effects of Brassica rapa L.polysaccharides. |
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