Research On Chemical Constituents Of Crude Polysaccharide From Nervilia Fordii And The Screening Of Anti-complement And Anti-inflammatory Activity

Objective:Nervilia fordii(Hance)Schltr.has significant heat clearing and detoxifying effect.To clarify its bioactive components of heat clearing and detoxifying,the active crude polysaccharide with anti-component activity have been studied.Isolation,purification,characterization,component analysis and structural elucidation were performed on the polysaccharides from Nervilia Fordii.Methods:1.The crude polysaccharides were isolated by water-extraction and alcohol-precipitation method,removed proteins by Sevage method,further purified by DEAE-cellulose and sephdex G-100 column chromatography;molecular weight was measured by HPGPC;monosaccharide composition of the polysaccharide was analyzed by precolumn derivatization HPLC;structure of the polysaccharide was analyzed by UV,IR,1D,2D-NMR,GC-MS.2.The optimal dilutions of sera(resources of complements)that gave submaximal lysis in the absence of complement inhibitors were determined to perfect the hemolysis assay to determine five homogeneous polysaccharides.3.The cell viability was determined by MTT assay.The model of inflammation was established by stimulating RAW264.7 with lipopolysaccharide(LPS)leading to NO production,and treated with DT2-1 DT2-2,DT4-1,DT4-2,and DT6-1,respectively.Results:1.Five homogeneous polysaccharides,DT2-1,DT2-2,DT4-1,DT4-2 and DT6-1,were isolated and purified.Their total sugar content were 78.64%,82.45%,86.13%,83.51,84.72%,protein content were 3.95%,6.38%,5.63%,4.65%,5.44%,glucuronic acid content were 8.44%,16.65%,4.01%,2.42%,7.17%,respectively.Their relative molecular weight are 1260,950,1070,810,930 KDa,respectively.DT2-1 mainly composed of Gal,Ara and Man in the ratio of 3.4:1.4:1,along with trace of Rha,Xyl and glucuronic acid;DT2-2 mainly composed of Ara,Gal and Rha in the ratio of 2.1:2:1,along with trace of Man,Rha and glucuronic acid;DT4-1 mainly composed of Glc,Gal and Ara Rha in the ratio of 2:1.6:1,along with trace of Man,Rha,Xyl and glucuronic acid;DT4-2 mainly composed of Glc and Gal in the ratio of 6.4:1,along with trace of Man,Rha,Ara,Xyl and glucuronic acid;DT6-1 mainly composed of Glc,and Gal in the ratio of 3:1,along with trace of Man,Xyl,Rha and glucuronic acid.Five homogeneous polysaccharides,with different MW and structures,were obtained from Nervilia Fordii for the first time.2.Functional analysis showed that DT4-1,DT4-2 and DT6-1 inhibited complement activation on both the classic and alternative pathways with CH50 value of 54.21±2.62,77.5412.12 and 51.46±2.38 ?g/ml,AP50 value of 76.68±5.14?92.58±6.42 and 91.35±6.42 ?g/ml,respectively.DT4-1,DT4-2 and DT6-1 were a little weaker than heparin through the classical and alternative pathway.However,DT2-1 and DT2-1 did not showed anti-complement activity on both the classic and alternative pathways.3.The inhibition abilities of five polysaccharides against the NO releasing from the LPS-induced RAW264.7 macrophages had been exmamined.DT2-2,DT4-1,DT4-2 and DT6-1(10?300 ?g/ml)showed significantly inhibited NO production activities in LPS-stimulated RAW264.7 cell.But DT2-1 did not show.Conclusion:Five polysaccharides were isolated from the crude polysaccharide,among them four polysaccharides showed the inhibition on the NO releasing and anti-complement with different levels.Thus,polysaccharides are presumed the active components with anti-inflammotary and immunological activities.