Isolation,Purification,Structural Elucidation And Bioactivity Of Polysaccharides(PBP60)from Philomycus Bilineatus

In this paper,the extraction,purification,sugar structure elucidation and bioactivities for polysaccharides(PBP60)from Philomycus bilineatus were systematic studied.The crude polysaccharides(PBP60)were isolated from Philomycus bilineatus by shear processing,enzymolysis extraction,ethanol precipitation.AAP60-A,AAP60-B,AAP60-C and AAP60-D were obtained after DEAE-Sepharose Fast Flow anion-exchange chromatography.PBP60-A was mainly composed of carbohydrate,with little protein.PBP60-B,PBP60-C and PBP60-D contained carbohydrate,protein,uronic acid and hexosamine.PBP60-A2 was purified by Sephacryl S300 chromatography after DEAE Sepharose Fast Flow anion-exchange chromatography.HPLC producing a single symmertrical peak indicated it was homogeneous and its molecular weight was 39 kDa.Lack of absorption at 260 nm and 280 nm by UV scanning indicated that it contained no protein and nuleic.AAP60-A,AAP60-B,AAP60-C and AAP60-D were analysised by FT-IR spectroscopy,Acetylation and PMP deriviative analysis.Results demonstrated that AAP60-A was mainly composed of Man,Rha,Glc,Gal,Xyl and Fuc in the mole ratio of 1.17:0.59:1.71:1.00:0.47:0.22.AAP60-B was made up of Man,GlcN,Rha,GalN,GlcU,Glc,Gal,Xyl and Fuc in the mole ratio of 0.90:0.31:1.15:0.37:0.24:1.02:3.84:0.93:1.99.AAP60-C and AAP60-D were composed of Man,GlcN,Rha,GalN,GlcU,Glc,Gal,Xyl,Fuc and 3(4)-O-methyl-hexose.The structure of PBP60-A2 was investigated by FT-IR spectroscopy,GC-MS analysis and NMR spectroscopic methods(1D NMR and 2D NMR).AAP60-A2 was comprosed of Man,Glc and Gal in the mole ratio of 0.16:1.00:0.12.Methlation revealed the primary structure of PBP60-A2 mainly consised of 1-linked Glcp,1,6-linked Galp,1,2-linked Manp,1,6-linked Glcp,1,4-linked Glcp and1,4,6-linked Glcp.NMR analysis futher showed the structure of PBP60-A2 is consisted of α-D-(1→4)-glucopyranan,α-D-(1→4,6)-glucopyranan,β-D-(1→4)-glucopyranan backbone with a terminal α-D-glucosyl unit.it also contained α-D-(1→6)-glucopyranan branch chain and little 1,2-α-D-Mannopyranan residues.The bioactivitiesof PBP60-A,PBP60-B,PBP60-C and PBP60-D focus on antioxidant activity and antitumor activity.The antioxidant activities were studied via different methods,including ABTS assay,DPPH assay,hydroxyl radical assay,superoxide radical assay and reducing power assay,The four fractions exhibited significant antioxidant activities in a dose-dependent manner,AAP60-D presented relative stronger antioxidant activity among the four polysaccharides.The inhibition effects of the four fractions on the growths of A549 cells,MCF-7cells,S180 cells and CHL cells were evaluated in vitro by MTT assay.AAP60-C showed high antitumor activity against A549,MCF-7,S180 cells in vitro,and only at highest concentration exhibited inhibition rate against he proliferation of CHL cells in vitro.