Isolation,Purification,Structural Characterization And Biological Activity In Vitro Of Polysaccharides From The Fruting Bodies Of Phellinus Pini

Phellinus pini was a rare medicinal fungi of the genus Phellinus in the family of Hymenochaetaceae.The polysaccharides from the genus Phellinus had been studied to have good biological activities,such as anti-tumor,antioxidant,antiviral and immunomodulatory activity,and so on.Compared with other mushroom polysaccharides of the same genus Phellinus,the studies of polysaccharides from Phellinus pini were relatively less at present.The fruiting bodies of Phellinus pini were used as the research object in the study.The isolation,purification,physical-chemical properties,structural characterization and biological activities in vitro were researched systematically in this paper.It would be some value in the area of research and applications of Phellinus pini for pharmaceuticals or functional products.The dissertation mainly was divided into three parts.Researches on extraction,isolation,purification,physic-chemical properties,structural characterization,in vitro antioxidant and ?-Glucosidase inhibitory activity were discussed in this paper.The results were as follows:(1)The fruiting bodies powder of Phellinus pini were degreased by 95%ethanol.After pretreatment,three polysaccharide fractions(PP30,PP60 and PP80)were obtained by boiling-water extraction and ethanol precipitation at corresponding concentrations.The antioxidant activities of three fractions of water-souble crude polysaccharides from Phellinus pini were evaluated by in vitro assay of DPPH radical,ABTS radical,hydroxyl radical and ferric-reducing antioxidant power.And in vitro a-Glucosidase inhibitory activity were also studied.All the antioxidant results were in good accordance with each other,the antioxidant power of crude polysaccharides decreased in the order of PP80>PP60>PP30 in the four assays.The results of in vitro ?-Glucosidase inhibitory activity of polysaccharides were also in the same order in the experiment concentration range.According to the results of the in vitro experiment,PP80 was selected for further separation and purification.(2)PP80 was selected for further study and purified with ion exchange chromatography on DEAE-Sepharose fast flow.And two major elution peaks:PP80W and PP80S,were detected.Then PP80W was further purified by High Resolution Sephacryl S-200 gel-permeation chromatography,and a major polysaccharide named as PPW-1 was isolated.PPW-1 appeared as a single symmetrical peak in the HPLC chromatograph,indicating that PPW-1 was a homogeneous polysaccharide,with an average molecular weight of 3.2×10-3 Da.The results of UV scanning indicated that PP80W-1 didn't contain protein but trace amount of nucleic acid.IR spectra showed PP80W-1 was a neutral polysaccharide.(3)The structure characterization of PP80W-1 was elucidated by GC analysis,GC-MS analysis and NMR spectrum analysis.PP80W-1 was hydrolyzed with TFA,reduced with NaBH4 and acetylated with acetic anhydride,GC analysis indicated that PP80W-1 was composed of D-mannose,D-glucose,D-galactose and a small mount of L-rhamose.At the same time we found and further verified that PP80W-1 contained 3-O-methyl-D-galactose.The IR,methylation analysis and NMR analysis showed that the structure of PP80W-1 mainly contained 1,6-linked Glcp,1,3-linked Glcp,1-linked Glcp,1-linked Galp,1,6-linked 3-O-Me Galp,1,3,6-linked Manp,1,4-linked Manp and 1-linked Rhap.?6)-?-D-Glcp-(1? and ?3)-?-D-Glep-(1?were main bond types and may constitute the main chain.?3,6)-?-D-Manp-(1? was the branch chain.Terminal group was ?-D-Glcp-(1? and a small amount of?-D-Galp-(1?.In addition,the backbone of PP80W-1 also contained?6)-?-3-O-Me-D-Galp-(1???4)-?-D-Manp-(1?and a small amount of?-L-Rhap-(1?.