Study On Purification And Anticancer Activity Of Polysaccharides From Pleurotus Nebrodensis By Alkali Extraction

Most fungi polysaccharides have functions of anti-tumor, involved in immune regulation, Research and development of fungus polysaccharide has become one of the hot researches in fungi fields. P. nebrodensis was taken as the object in this paper, after a discussion of the extraction, purification of P. nebrodensis polysaccharide on the basis of previous studies, this paper discussed its basic structure and physicchemical properties and focused on the study of effects of P. nebrodensis polysaccharide on survival and proliferation of HepG-2 hepatoma cells.More studies of its function mechanism were conducted too.Crude P. nebrodensis polysaccharide were extracted by alkali extraction with the extact rate of 5.14% and purified by agarose gel column chromatography with 4 fractions obtained:PNA-1, PNA-2, PNA-3 and PNA-4. The total polysaccharide content was measured to be 84.17%, which contained 3.70% of protein.The solubility, viscosity, thermal properties, crystallinity of P. nebrodensis polysaccharide were studied, The molecular mass of PNA-2 was determined to be 37 kDa by HPLC.Infrared spectrum analysis of PNA-2 indicated a typical peak of β-polysaccharide, and also contained peaks of pyran keys and furan keys; PNA-2 monosaccharide composition were analysized by gas chromatography, which suggested the composition of a large amounts of glucose, a small amount of mannose, galactose, trace of ribose.This study took HepG-2 cells as reaserch objectand to investigate its inhibiton, DNA damage and apoptosis induced by PNA-2. Inhibitory effect of P. nebrodensis polysaccharide on the proliferation HepG-2 cells firstly studied by MTT method to determine the test groups were the 12.5 μg/mL,62.5 μg/mL and 125μg/mL of PNA-2;Cell scratch assay showed a notable inhibition of HepG-2 cells PNA-2 treated. Typical apoptotic morphological feature and apoptotic bodies in HepG-2 cells induced by PNA-2 at the above concentrations were observed by scanning electron micrographs and AO/EB staining.A statistically significant (p< 0.05) induction in the DNA damage was observed by the comet assay in cells exposed to 12.5 to 125 μg/mL PNA-2 for 48 h.A preliminary discussion is made on the mechanism of apoptosis of HepG-2 cells induced by PNA-2 in the test.PNA-2 also disrupted the mitochondrial membrane potential evaluated by Rhodamine-123 staining. Further analysis by qRT-PCR indicated that the expression of CYT, caspase-3 and caspase-9 mRNA was increased significantly at the concentration of 62.5 and 125 μg/mL PNA-2 for 48 h. However, the mRNA expression of Bcl-2 was decreased significantly. Taken together, these findings suggest that PNA-2 can inhibit HepG-2 cell proliferation, cause DNA damage and induce apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway.