| Phellinus igniarius (L.: Fr) Quel, is rare medicinal fungi contained polysaccharides besides many other kind of compounds. Polysaccharides of Phellinus igniarius has been reported to possess antitumor activities and inhibit tumor recrudescence and metastasis. The chemical composition and biological activities of crude polysaccharides of Phellinus sp. have been investigated and there is no reports relating to the chemical structure about the pure polysaccharides isolated from cultivated P. igniarius. In order to use this natural antitumor resource, the extraction, isolation, purification, structure characterization and pharmacological tests of the polysaccharides from the cultivated fruiting bodies of P. igniarius were carried out. It will be of scientific significance in researching and developing rare medicial fungi and be useful for searching new natural antitumor medicine.The crude polysaccharides of P. igniarius PIP30,PIP60,PIP80 and PIWE1-10,PIWE10-100,PIWE100 were obtained by ethanol precipitation and ultrafiltrate respectively after hot water extracting. P30U,P30W,P30S1 and P30S2 were isolated from PIP30, and P60U,P60W,P60S1 and P60S2 were isolated from PIP60, and P1W,P1S1 and P1S2 were isolated from PIWE1-10, and P10W,P10S1 and P10S2 were isolated from PIWE10-100, and P100U,P100W,P100S1 and P100S2 were isolated from PIWE100 using DEAE Sepharose F. F. The proliferation rates of lymphocyte in vitro of each polysaccharide fractions obtained during the isolation and purification were detected in order to observe their immune activities. The results showed that crude polysaccharides both obtained from ethanol precipitation and ultrafiltrate had the promoting effects on lymphocyte proliferation in various degrees. P30U,P30W,P30S1,P30S2,P60U,P60W,P60S2,P1S1,P1S2,P10S1,P10S2,P100U,P100W,P100S1 and P100S2 had the promoting effects on lymphocyte proliferation. P60W,P60S1,P1S1,P10S1 and P100S1 were further purified by gel-filtration chromatography (Sephacryl S-100-500 ) repeatedly. Five water-soluble homogeneous polysaccharides (P60w1,P60s1,P1SP1,P10SP1and P100SP1) were obtained. Lack of absorption at 280 nm and 260 nm by UV scanning indicated that contained no protein and nucleic acid. IR spectra showed that the sugar characteristic groups were existed. HPLC produced a single symmetrical peak, indicated homogeneity and their relatively molecular weigh were 1.71×10~4 Da,2.07×10~4 Da,1.48×10~4 Da,2.20×10~4 Da and 2.56×10~4 Da respectively. P60w1,P1SP1,P10SP1 and P100SP1 had the promoting effects on lymphocyte proliferation.A special monosaccharide 3-O-methylgalactose was identified by high performance anion exchange chromatography (HPAEC) and GC/MC. This monosaccharide existed both in fruiting body and mycelia polysaccharides of P. igniarius further indicate that it was possibly characteristic sugar in P. igniarius polysaccharides.The structures of five homogeneous polysaccharides were investigated using chemical (Sugar analysis, Methylation analysis, etc.) and spectroscopic methods (1D NMR: 1H NMR, 13C NMR,DEPT spectra; 2D NMR: 1H-1H COSY, TOCSY, HMQC, NOESY and HMBC spectra; etc.) to confirm the sugar composition, the number of sugar residues, the form of glycosyl linkage, the configuration of anomeric center, the sequence of sugar residues. The structure of P60w1 with high immune activity in vitro was elucidated in detail. Sugar analysis showed this polysaccharide was composed of fucose, galactose, glucose, mannose and 3-O-Me-galactose in the ratio of 1.12:2.26:1.00:1.03:1.11. Its main chain was composed ofα-D-1,6 linked galactosyl,α-D-1,2,6 linked 3-O-Me-galactosyl,β-D-1,2,4 linked 3-O-Me-galactosyl andα-D-1,2,6 linked mannose, side chain was composed of terminalα-L-fucose andα-D-glucose. The proposal structure is as follows:Sugar analysis showed other four polysaccharides were composed of fucose, 3-O-Me-galactose, galactose, xylose, mannose, glucose and a minor of arabinose, and the ratios of galactose and 3-O-Me-galactose were high, and the ratio of each sugar differed from each polysaccharide. Methylation analysis showed galactose and 3-O-Me-galactose were mainly 1,6 and 1,2,6 terminal linkage, fucose was terminal linkage, mannose and glucose had the different linkage in these polysaccharides.Antitumor experiments in mouse model with ethanol extract PIEE, crude polysaccharides PIP60, isolated polysaccharide P60S2 and pure polysaccharide P60w1 showed that PIEE, PIP60, P60S2 and P60w1 all had the inhibit effects on mouse liver cancer H22, and the effect of P60w1 was better than crude polysaccharides and positive control. P60w1and P60S2 had the inhibit effects on mouse Lewis lung cancer at the dosage 100 mg/(kg·d). Pure polysaccharides inhibiting tumor effect may have no relation to inhibiting tumor cell proliferation and potentially inhibited tumor by regulating immune function of body.
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