The Preparation, Structure Analysis And Biological Activity Of Morel Polysaccharide And Its Sulfated Derivatives

Many biological activities of polysaccharides from Morchella have been generally recognized,and the biological activities of polysaccharides are closely related to their structure,so the study on the structure of polysaccharides has laid a foundation for the exploration of biological activity.In recent years,it is also a hot topic that the bioactivity of polysaccharides can be improved by structural modification.Therefore,in this paper,Morchella polysaccharides were extracted by hot water and alcohol precipitation,and the structure,immunomodulatory activity and antitumor activity of the polysaccharides modified by sulfation were studied,so as to develop natural drugs that can enhance immune regulation.The main research results are as follows:（1）The molecular identification of Morchella was carried out by using ITS sequence analysis technology,and the identification results showed that the strain was Morchella sextelata,which was a new strain fungi.Crude polysaccharides were extracted from Morchella mycelium by ultrasonic crushing,alcohol precipitation,protein removal and degreasing.Then,using the full-automatic protein purification system,the MSP-II polysaccharide was obtained after the separation and purification by DEAE-cellulose column chromatography and Sephadex G-25column chromatography.The polysaccharide of MSP-II content was about 91.56%.The purity and molecular weight of MSP-II were identified by GPC.It was found that MSP-II was a homogeneous polysaccharide component with a molecular weight of 2.34×10⁴.（2）The monosaccharide components of Morchella polysaccharide MSP-II were analyzed by PMP pre-column derivation.The results showed that MSP-II mainly consisted of arabinose,glucose,galactose and galacturonic acid,and contained a small amount of mannose,rhamnose,fucose and glucuronic acid.The molar ratio of monosaccharide is:Glu:Ara:Gal:Man:Fuc:Gal:Glu=34.95:8.7:9.55:4.55:5.0:1.45:12.7:7.65.The UV full spectrum scanning of Morchella polysaccharide MSP-II showed that the maximum UV absorption value of MSP-II was about200 nm.There was no absorption peak at 260 nm and 230 nm,indicating that MSP-II contained almost no protein and nucleic acid.FT-IR spectra were used to analyze the structure of MSP-II,and the results showed that MSP-II had typical absorption peaks of polysaccharides.The presence ofα-glucosidic bonds in the structure of MSP-II has been shown to be closely related to the immunomodulatory activity of polysaccharides.In addition,there is a small amount of alpha-glycosidic bond configuration in MSP-II.The polysaccharide msp-ii of morchella was analyzed by NMR,and the ¹H and ¹³C NMR spectra of the polysaccharide were determined.The¹H NMR and ¹³C NMR data showed that the repeat unit of MSP-II contained three glycosidic bond connections,and the sugar residue wasα-andβ-glucosidic configuration.（3）MTT assay was used to detect the effect of Morchella polysaccharides on the growth of mouse macrophages RAW264.7.The results showed that Morchella polysaccharides could significantly improve the growth of mouse macrophages RAW264.7,indicating that Morchella polysaccharides could promote the growth of mouse macrophages.The neutral red phagocytic assay was used to detect the phagocytic activity of Morchella polysaccharides on mouse macrophages RAW264.7,and the results showed that MSP-II could significantly improve the phagocytic activity of macrophages.The NO release amount of Morchella polysaccharide on mouse macrophages RAW264.7 was detected with NO kit.The experimental results showed that MSP-II could promote the release of NO,indicating that Morchella polysaccharide could enhance the immunity of mouse macrophages.Flow cytometry was used to detect the effect of MSP-II on the ROS release of mouse macrophage RAW264.7.The results showed that the ROS release showed a gradually increasing trend with the increase of MSP-II concentration,which proved that Morchella polysaccharide could promote the ROS release.（4）UV spectrum scanning was performed on the polysaccharides of morchis after sulfation,and the results showed that the polysaccharides after sulfation still did not have the presence of nucleic acids and proteins,and there was no new absorption peak.Compared with the original polysaccharides,the FT-IR spectra of the sulfated polysaccharides showed that the sulfated polysaccharides not only retained the characteristic absorption peak of the polysaccharides,but also had S=O absorption peak,indicating that the sulfated polysaccharides were successful.MTT assay was used to detect the inhibitory effect of sulfated polysaccharides on tumor cells Hela.The experimental results showed that sulfated polysaccharides were not toxic to normal cells,but could inhibit Hela cell proliferation.Flow cytometry was used to detect the antitumor activity of sulfated polysaccharides,and cell apoptosis,cell cycle and mitochondrial membrane potential were tested.The results further indicated that sulfated polysaccharides could promote the apoptosis of Hela cells.