Aqueous two-phase simultaneous extraction and purification of a polysaccharide from Grifola frondosa: Process optimization, structural characteristics and antioxidant activity

Traditional method for Grifola frondosa polysaccharide (CGFP) separation is water extraction or other method assistance, ethanol precipitation and further purification via column chromatography. Due to the strict control conditions, inefficient and not environmental-friendly, the method is hard to scale up and implement, and even limited the biological activity study and application of Grifola frondosa polysaccharide. Therefore, an ammonium sulfate/ethanol aqueous two phases (ATPS) was developed to extract and preliminary purify the polysaccharide from Grifola frondosa (GFPA). The optimized conditions were concluded as the ATPS composition of ammonium sulfate 17% and ethanol 27.9%, extraction pH 3.22 based on single-factor and response surface methodology experiments. Under this condition, the recovery yield and content of GFPA were 90.21% and 66.7%, respectively. The structural characteristics investigation revealed that GFPA exhibited a slight change in weight average molecular weight (Mw), molar ratio of monosaccharide and microstructure as compared to CGFP. Results indicated that the Mw of GFPA was 2.954 × 106 Da, which was lower than that of CGFP. Both of them are heteropolysaccharides with pyranose ring, and mainly composed of glucose. GFPA exhibited a rod shape and reticular structure with smooth surface, while CGFP showed a rough surface. Also, Congo red test indicating the presence of triple-helix structure in both of them, while AFM observation suggesting a spherical structure tended to aggregate occurred. The chemical antioxidant activities of GFPA were increased in a dose-dependent manner. Moreover, GFPA can effectively prevent RAW264.7 cells from H2O2-induced damage through reducing the release of MDA, LDH and ROS, and enhancing the levels of SOD, CAT and GSH-Px. Furthermore, GFPA showed more significant and effective than CGFP at all the tested dosages. Thus, it can be seen that the ATPS method established in this paper possess the merits of efficient, environmental-friendly and simple operation, which can successfully be applied for Grifola frondosa polysaccharide large scale separation, and make the application of GFPA possible. It has reference value for other large-scale polysaccharide separation. The resulting also revealed that GFPA has the potential value of developing as a natural antioxidant agent.