Structure And Bioactivities Of Extracellular Polysaccharide From Culture Medium Of Phellinus Pini

Phellinus pini is a Basidiomycetous fungus belonging to Hymenochaetaceae.In this paper,Phellinus pipi fermentation broth was obtained in submerged flask fermentation.The crude extracellular polysaccharide (CPPE) was isolated from culture medium by ethanol precipitation. The protein and total sugar of CPPE content were 31.68% and 33.56% respectively determined by Kjeldahl nitrogen and phenol-sulfuric acid method.PPE was purified from CPPE using a combination of techniques,such as:frozen- thawed, deproteinated by enzyme/Sevag method and Sepharose CL-6B column chromatography.PPE was light yellow powders. Its specific rotation [α]D20 is +21.3°.The content of the carbohydrate was 92.84%.HPLC analysis manifested that PPE was uniform fraction and the molecular weight was about 39kDa.GC analysis indicated that the PPE was composed of Man,Glc and Gal with the molar ratio of 38.4:1.76:1.The major structural features of PPE was elucidated using partial acid hydrolysis, periodate oxidation,Smith degradation, methylation and IR,GC,GC-MS,13C-NMR.On the basis of these results,the repeating units of PPE was established.It was possible to conclude that a repeating unit of PPE contained a backbone composed ofα-(1→2)-Manp.every eight sugar residues of the backbone consist a repeating unit,four branches located at 0-6 ofα-(1→2)-Manp.The branched chain was consisted ofα-(1→3)-Manp andα-(1→6)-Manp with the ratio of 3.71:1.The terminal sugar was Man.The immunomodulatory effect of PPE was detected by Lymphocyte proliferation assay with Methyl Thiazolyl Tetrazolium(MTT).The bioassay showed that PPE could stimulate mouse splenic lymphocytes proliferation and increase the ConA or LPS-induced lymphocytes proliferation in vitro.