Isolation And Purification Of Polysaccharide From Pleurotus Eryngii And Its Antioxidation And Antitumor Function

Researches of mushroom polysaccharide have become a hot spot in China and abroad, they have shown that polysaccharides is one of the most valuable of the active ingredient in the edible and medicinal mushroom, they have many biological activities, such as anti-tumor, anti-cancer, lowering blood glucose and strengthening the immune system and other. Pleurotus (Pleurotus eryngii), is from Basidiomycetes Pleurotaceae (Pleurotaceae) Pleurotus, edible mushrooms belong to the class.This paper examined the anti-oxidation, anti-tumor activities of the polysaccharide of purified Pleurotus eryngii, clarified the Pleurotus eryngii polysaccharide radical scavenging and anti-tumor effect, supported for the development and application, provided a theoretical foundation and technical of Pleurotus eryngii polysaccharide.PEP was extracted from the P. eryngii used a multistep purification procedure including hot-water extraction and repeated ethanol precipitation. The extraction rate of the dried PEP reached approximately 5.4%(w/w). The total carbohydrate content of PEP was determined to be 61.3%. The compositional monosaccharides of PEP were identified by HPLC-UV technique. The results showed that PEP was composed of D-mannose, D-ribose, L-rhamnose, D-glucuronic acid, D-glucose, D-xylose, D-galactose and D-fucose with the corresponding mole percentage was 9.8%,1.6%,0.15%,0.8%, 62.8%,0.05%,24.4% and 0.4%, respectively. Which D-glucose, D-galactose and D-mannose content more. It was shown that PEP is a heteropolysaccharide composed of eight monosaccharide composition. Through a series of anti-oxidation experiments, the antioxidant capacity was evaluated in vitro, indicated that PEP has antioxidant activities in vitro, and exhibited dose-dependent effects.DEAE-52 cellulose column chromatography and Sephadex G-100 chromatography column used to purified the Pleurotus eryngii polysaccharides for further study. DEAE-52 cellulose column chromatography eluted by two kinds of polysaccharides which were homogeneous components, named PEP-1, PEP-2. UV scanning results showed that,260-280 nm had no absorption peak, and the PEP-1, PEP-2 does not contain nucleic acid and protein. The infrared spectra showed that pyranose ring structure were present in the PEP-1 and PEP-2. Through the monosaccharide composition of HPLC-UV for further testing, the results showed that, monosaccharide composition of PEP-1 contain mannose, glucose and galactose, monosaccharide composition of PEP-2 contain mannose, glucose and galactose. Analysis by HPLC gel permeation chromatography, the molecular weight of PEP-1 is 25362 Da, PEP-2 is 463396 Da, respectively.Through the MTT and LDH assay, PEP-2 had more obvious effect of growth inhibition on human hepatocellular carcinoma HepG2 cells compared with PEP-1, and the activities showed dose effect relationship with the concentration. DAPI staining showed that HepG2 cells appeared apoptosis, and the chromatin condensation, nuclear fragmentation and apoptotic body characteristics. The apoptosis assay used Annexin V-FITC/PI, PEP-2 treated cells in 6 h, then appeared 20.49% of early apoptosis, late apoptosis accounted for 4.27%; and after 24 h, early apoptosis reached 35.71%, late apoptosis also increases to 20.63%. Obtained by flow cytometry, PEP-2 arrested in the human hepatocellular carcinoma HepG2 cells in S phase.The used of flow cytometry in ROS levels of PEP-2-induced HepG2 cells showed that as time goes on, intracellular H2O2 levels were significantly increased, therefore we concluded that, when the PEP-2 exert an anti-tumor effect, ROS-mediated the HepG2 cells apoptosis.In this study, two kinds of polysaccharides separated and purificated from Pleurotus eryngii, and their structures was preliminary testing and observation, and screened to obtain a component inhibiting PEP-2 (molecular weight is 35 W Da) with significant proliferation of liver cancer cells. ROS plays a mediating role in cell apoptosis. The result of our study pointed out the direction thinking of the study of antitumor mechanism of polysaccharides in the future, and provided a theoretical basis for the development of functional food factors or drugs.