Isolation, Purification, Structural Elucidatuion And Biological Activity Of Polysaccharides From The Fruting Bodies Of Pleurotus Geesteranus

Pleurotus geesteranus, also known as white mushroom, with a scientific mane called yellow ear, is one of the most widely used edible mushroom genus in the world, and it gains its popularity because of its pleasant flavor, richness of nutritions, dietary fibers and high quality of proteins. It also contains eight amino acids enssetial to the dietary needs of man bodies, as well as a variety of valuable medicinal compounds.Here, we demonstrated the extractin and untrafiltration of polyaccharides from fruiting bodies of P. geesteranus, screening of their antioxidant activities, purification and physic-chemical characterizations of the functional group with high antioxidant activities, and structural analysis and anti-tumor study of the polysaccharides.In this study, the fruiting bodies of P. geesteranus were first degreased by95%ethanol, and then extracted with hot water to get the polysaccharide fractions, whose Antioxidant activities were further screened and graded by ultrafiltration into four crude polysaccharide groups. We found the PGPB (<100KDa) group had a relatively higher content than other groups, as well as the highest reducing ability, and the second highest hydroxyl radical and DPPH scavenging ability. Based on this finding, PGPB was selected for further separation and purification.PGPB crude was purified using anion-exchange chromtography (DEAE Sepharose Fast Flow) and gel-filtration chromatography (Sephacryl S-300and Sephacryl S-400), after which two water-soluble homogeneous polysaccharides were obtained:the water phase PGP1and salt phase PGP2, with molecular weights of1.3×104Da and>2.00×106Da, repectively. Then, the PGP1product was further characterized using UV absorption at wavelengths of280nm and260nm, indicating no evidence of protein and nucleic acid presence in the PGP1.The structure of PGP1after acetylation and methylation was investigated by both GC and GC-MS analyses, also combined with NMR spectroscopic methods (1H NMR,13C NMR;1H-1H COSY, HMQC, TOCSY, NOESY and HMBC spectra). In this chapter, we focus on the compositional and structural studies of the monosaccharide PGP1. The results showed that the main components of the polysaccharide were four monosaccharides, three of which were D-mannose, D-glucose and D-galactose, and the other one was possibly3(4)-O-methyl-hexose as we speculated based on GC-MS analysis. The methylated GC-MS analysis revealed the substituted positions were the end groups of the mannose,6-substituted galactose and glucose, and2,6-substituted3-O-methyl galactose with a molar ratio of1:0.25:1.14:0.94. Using NMR spectra analysis, the structure of PGP1is figured as followd: β-D-Manp (?)â†'6)-α-D-O-Me-D-Galp-(1â†'with a small amount of6-D-Glcp.Finally, the in vitro studies of anti-tumor activity were carried out using four groups of compounds obtained by ultrafiltration (i.e. PGPA, PGPB, PGPC, PGPD) and the other two purified groups (i.e. PGP1, PGP2). All the groups were founded to have inhibitory effects on the S180, in which the inhibition rate of tumor cell incubated with PGPD was significantly higher than the other three groups obtained by ultrafiltration, and presented a certain dose-dependent relationship. Also, the two components (PGP1, PGP2) purified from crude PGPB exihibited significantly higher inhibitory effect compared with the unpurified PGPB crude. Their anti-tumor effects were further comfirmed to be no toxic on normal cell CHL.