The Study Of The Structure And Bioactive Of The Polysaccharides From Phellinus Nigricans

The submerged culture medium of Phellinus nigricans and the extraction, purification, structure analysis and bioactivities of mycelium polysaccharide and medium polysaccharide were studied in this paper. The relationship between the structure and bioactivities of polysaccharides was also discussed.The effects of four kinds of carbon sources and nine kinds of nitrogen sources on the fungus Phellinus nigricans mycelia were studied. The results showed that the optimal carbon and nitrogen sources were starch and corn meal. The optimal combination was sucrose and corn meal. The optimal extraction conditions of polysaccharide from the mycelia of Phellinus nigricans were studied by orthogonal experiments. The optimal extracted conditions for hot water extraction were confirmed: the temperature 100℃, four times, two hours per times and the diffusion ratio 1:30. The effects of these influential factors on extracted efficiency were displayed as follows: extracted times > the diffusion ratio > extracted time.The two crude water-soluble polysaccharides, PNW and PNM, were isolated from the mycelia and its culture media of Phellinus nigricans, respectively. The results of the pharmaceutical experiments showed that oral administration of PNW and PNM inhibited the growth of transplant tumor of mice-transplanted Sarcoma 180 in vivo. Moreover, a higher inhibition ratio of PNW was obtained when compared with PNM. GC analysis indicated that PNW was composed of mannose and glucose with the molar ratio of 2.68:1.00, and PNM was composed of mannose, galactose and glucose with the molar ratio of 2.02:1.00:3.21.PNW1 and PNM1 were purified from PNW and PNM respectively using a combination of techniques such as frozen-thawed, deproteinization by Sevag method, anion-exchange chromatography on a column of DEAE-cellulose and gel filtration chromatography on a column of Sepharose CL-6B. PNW1 and PNM1 appeared as single symmetrical peak on HPLC, and the average molecular weights were 33 kDa and 29 kDa which were calculated through calculation using dextran standard. The results of monosaccharide composed analysis showed that glucose, galactose and mannose were the dominant monosaccharide units in the two polysaccharides and a small amount of arabinose and fucose were also existed; however, there was a difference in the corresponding molar ratios between PNW1 (3.26:8.77:6.44:1.00:1.35) and PNM1 (20.06:8.72:6.94:1.00:0.76).The major structural features of PNW1 and PNM1 were elucidated using partial acid hydrolysis, periodate oxidation, Smith degradation, 13C NMR, methylation and GC-MS. On the base of these results, the repeated units of PNW1 and PNM1 were established. On the base of the results obtained above, it was possible to conclude that the repeated unit of PNW1 contained a backbone composed of 1→6-linked-galactose and glucose, and 1→6-linkage-mannose branches attached to O-2 of galactose and glucose. PNM1 was composed of a backbone of 1→6-linked-galactose and glucose with 1→6- linkage- mannose branches attached to O-2 of glucose. The terminal monosaccharides were glucose, arabinose, fucose and mannose.At the dose of 100, 200, and 400mg/kg, both PNW1 and PNM1 exhibited anti-tumor activities against mice-transplanted Sarcoma 180 in vivo. PNW1 had better activity than PNM1 and the maximal tumor inhibition rate was 74.7% at the does of 400 mg/kg. While, no direct cytotoxic activity against Sarcoma 180 was observed in vitro. The significant increase in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-α) in serum was observed, that coincided with decreasing the tumor weight significantly. PNW1 and PNM1 could stimulate lymphocytes proliferation and increase production of nitric oxide (NO) and TNF-αin macrophages. The results indicate that both lymphocyte and macrophages were activated by polysaccharide from mycelium and culture medium of Phellinus nigricans. The anti-tumor effect of the polysaccharide was not directly tumoricidal but rather immunostimulating. PNW1 and PNM1 had the similar molecular weight of about 30 kDa and similar monosaccharide composition, but their molar ratios of monosaccharide were different. Their structures had common feature such as 1→6 linked backbone attached 1→6 mannose branches at O-2. All these results supposed that the fungus heteropolysaccharide with 1→6 linked backbone, O-2 branch and molecular weight of about 30 kDa had anti-tumor and immunomodulating activities. The differences between the two polysaccharides indicated that the type of sugar residue effects the anti-tumor activation of polysaccharide.