| Lentinus edodes is a medicinal and food homologous fungus,rich in protein,polysaccharides,amino acids and other nutrients and trace elements.The polysaccharide in Shiitake mushroom is an important active ingredient,with antioxidant,antitumor,immunomodulatory and other biological activities.In this project,taking Lentinus edodes as the research object,the optimal process conditions for deep eutectic solvent-enhanced subcritical water extraction of Lentinus edodes polysaccharide were first explored through univariate experiments and response surface experiments.Then,DEAE-52 cellulose column and Sephadex G-100 gel column were used to separate and purify polysaccharides.And the structure of purified components was analyzed.Finally,the antioxidant and immunomodulatory activities of purified fractions of lentinus edodes polysaccharide were preliminarily studied in vitro by cell experiments.Main research contents and results:(1)Optimization of the process of deep eutectic solvent enhancement for extraction of Lentinus edodes polysaccharide from subcritical water.The optimization process was achieved by using single-factor experiments and response surface experiments,and the optimal process conditions for extracting polysaccharides from subcritical water were obtained:extraction temperature 147.23℃,extraction time 17.58 min,liquid-solid ratio 40:1 mL/g,the water content of DES 39.76%.Under the optimal extraction conditions,the extraction rate of polysaccharides was 6.26±0.08%,which was 19.24%and 17.01%higher than that through subcritical water extraction(5.25±0.05%)and hot water extraction(5.35±0.11%),respectively.(2)Isolation,purification and structural analysis of Lentinus edodesThe DEAE-52 cellulose column and the Sephadex G-100 gel column were used to separate and purify the Lentinus edodes polysaccharide to obtain two purification components,LEP1 and LEP2.Both components have high purity.The structural characteristics of purified Lentinus edodes polysaccharides were clarified by using monosaccharide composition analysis,molecular weight analysis,SEM,AFM,periodic acid oxidation and Smith degradation,ultraviolet spectroscopy,FT-IR,methylation and NMR.The results showed that LEP1 and LEP2 were composed of Man,Glc and Gal,and their monosaccharides had molar ratios of 1:12.97:7.48 and 1:51.15:5.29,respectively.The molecular weights of polysaccharides are 9.878×104 Da and 1.976×104 Da,respectively.The results of periodic acid oxidation and Smith degradation showed that LEP1 and LEP2 contained 1→3 or 1→3,6,1→or 1→6,1→2 or 1→4 glycosidic bonds,but the proportions were different.The methylation and NMR results further showed that the sugar residue composition of the two purified components was similar.Both of components were sugar residue structures with→4)-β-D-Glcp-(1→as the main chain,and the main chain and side chain and terminal were mainly linked by 1→4 and 1→6 glycosidic bonds.The structural differences of the two polysaccharides mainly lay in the connection type of the side chain and the proportion of sugar residues of different types.SEM and AFM results showed that LEP2 has a looser structure and lower particle height than LEP1.In aqueous solution,the polysaccharides exhibit chain aggregation and chain conformation in which the branched chain structure was intertwined.(3)Study on antioxidant activity and immunomodulatory activity of Lentinus edodes polysaccharide.A model of H2O2-induced oxidative damage in HepG2 cells was established,and the antioxidant activity of polysaccharide components was studied by detecting MDA,SOD,ROS and CAT content,as well as apoptosis and mitochondrial membrane potential changes.The immunomodulatory activity of Lentinus edodes polysaccharide was evaluated by studying the NO release ability,phagocytosis and secretion levels of inflammatory factors IL-6,IL-1β and TNF-α of RAW 264.7 cells.The results of antioxidant experiments showed that LEP1 and LEP2 had the strongest antioxidant activity when added at a concentration of 100 μg/mL.At this added concentration,the apoptosis rates LEP1(13.4%)and LEP2(9.5%)of HepG2 cells decreased significantly compared to the model group(26.5%).The contents of ROS and MDA in LEP1 decreased by 21.0%and 48.4%,respectively,and the contents of CAT and SOD increased by 53.0%and 39.7%,respectively.The contents of ROS and MDA in LEP2 incubation decreased by 32.7%and 65.6%,respectively,and the contents of CAT and SOD increased by 69.0%and 62.1%,respectively.The results of immunoactivity experiments showed that the cells had the strongest immunoactivity when the polysaccharide was added at a concentration of 200 μg/mL.At this added concentration,the phagocytic capacity and NO content of RAW 264.7 incubated by LEP1 increased by 34.5%and 188.0%,respectively,and the contents of IL-6,IL-1β and TNF-α in cells increased by 83.0%,271.8%and 194.5%,respectively.The phagocytic capacity and NO content of LEP2-incubated macrophages increased by 49.6%and 221.9%,respectively,and the contents of IL-6,IL-1β and TNF-α in macrophages increased by 103.4%,300.0%and 225.8%,respectively.The results of activity experiments showed that the antioxidant activity and immunomodulatory activity of LEP2 were stronger at the same added concentration. |
PDF Full Text Request