Chemical Modification And Biological Activity In Vitro Of Pleurotus Eryngii Polysaccharides

As biological macromolecular, polysaccharide has important biological activities, such as antitumor, antiviral, antibacterial, enhance immunity and anti-oxidation effect, the activity and its structure has a certain relationship, so the research of the structure and activity of polysaccharide has great important practical significance for us. Pleurotus eryngii is one of the treasures of edible fungi and has the most effective components-polysaccharides. In this work, crude polysaccharides WPP obtained by hot water extraction, then separated through fiber column and gel column, it was further fractionated to give two new polysaccharide fraction named washing polysaccharide WPP1and brine washing polysaccharide WPP2. Two kinds of polysaccharide purity were detected by HPLC. At the same time, three kinds of polysaccharide were modified and analysed its structure by using infrared absorption spectroscopy and atomic force microscopy, then compared its antitumor function and antioxidant activity with the undecorated polysaccharide. Research conclusions are as follows:(1) The optimum extraction conditions of Pleurotus eryngii polysaccharide:solid-liquid ratio1:30, extraction time3h, extraction temperature90℃, the extraction yield4.52%; Two kinds of polysaccharide purity WPP1and WPP2were detected by HPLC, the results show that they were uniform polysaccharide.(2) Four kinds of chemical modification, namely sulfate, phosphorylation, carboxy methylation and acetylation, were used on the polysaccharide, especially the sulfated modification was acted on washing polysaccharide and salt washing polysaccharide, to obtain the sulfating Pleurotus eryngii polysaccharide S-WPP, phosphorylation Pleurotus eryngii polysaccharide P-WPP, carboxymethylated Pleurotus eryngii polysaccharide C-WPP, acetylated Pleurotus eryngii polysaccharide Ac-WPP1, sulfate washing Pleurotus eryngii polysaccharide S-WPP1and sulfate salt washing the Pleurotus eryngii polysaccharide S-WPP2, Through infrared spectrometer and measurement on substitution degree to judge whether the various chemical modification is successful, and through the atomic force microscope imaging analysis polysaccharide structure changes. The experimental results showed that S-WPP, S-WPP1, S-WPP2, P-WPP, C-WPP and Ac-WPP’s substitution degree were0.116,0.164,0.223,0.139,0.858, and0.632. There were respectively S=O and C-O-S stretching vibration absorption peak at1240cm-1and810cm-1on the S-WPP1, S-WPP2and S-WPP’s infrared spectrogram. P-O-C stretching vibration absorption peak become stronger at1062.72cm-1on the P-WPP’s infrared spectra. The symmetric and asymmetric absorption peak of C=O in-COO occurred at at1593.11cm-1and1431.10cm-1on C-WPP’s infrared spectra. The C-O and C=O absorption peak in-COCR appeared at1745.48cm-1and1253.66cm-1on Ac-WPP’s infrared spectra. All of these shows that polysaccharide of four kinds of chemical modification are successful, consistent with substitution degree test results. Atomic force microscope imaging results as follows:WPP mainly composed of two or more polysaccharides that have different physical and chemical properties, of which one is long-chain and multibranched polysaccharide structure. Four kinds of chemical modification were acted on polysaccharides, the result showed that the molecular and advanced structure of polysaccharides were changed varying degrees.(3) Through MTT colorimetric method to study nine kinds of Pleurotus eryngii polysaccharides, namely the S-WPP, P-WPP, C-WPP, Ac-WPP, WPP, WPP1and WPP2, S-WPP1and S-WPP2which have inhibition effect on K562cells in vitro proliferation. According to the results of nine kinds of polysaccharides at different concentration effect after24h,48h and72h, all of those have certain anti-tumor effect, among them, three kinds of polysaccharide, sulfate modified S-WPP, S-WPP1and S-WPP2have the highest activity in experiment. P-WPP, C-WPP and Ac-WPP are higher than that of WPP’s antitumor activity in vitro. Research shows that the effect of growth inhibition after chemical modification on the Pleurotus eryngii polysaccharide were raised on K562cells in vitro.(4) Comparison before and after the chemicial modification of Pleurotus eryngii polysaccharide in vitro antioxidant effect, we can learn from the test of total reducing power about polysaccharides:five kinds of polysaccharide reduction ability is weak, after four kinds of chemical modifications acted on polysaccharides have reduces the total reducing ability. Polysaccharide on the scavenging of the O2-, Results show that the five kinds of polysaccharides have weaker work on eliminating O2-, but chemical modified polysaccharide have enhance this effect. Polysaccharide on the scavenging of the·OH, Results show that the five kinds of polysaccharides have stronger work on eliminating·OH, and chemical modified polysaccharide have enhance this effect. Polysaccharide to scavenge DPPH· results show that the five kinds of polysaccharides on clear away the DPPH· have certain ability, Compared with the Pleurotus eryngii crude polysaccharide WPP, S-WPP, C-WPP and Ac-WPP’s ability to scavenge DPPH· is weaker, P-WPP’s ability to scavenge DPPH· is strong. The result of Polysaccharide’s effect on lipid peroxidation induced by Fe+show that the five kinds of polysaccharide have the certain role on inhibition, the chemical modification to further strengthen this effect.