The Study Of Isolation And Purification Of Undaria Pinnatififida Polysaccharides And Mechanism Of Immunomodulation

Objective:In this study,extraction,separation,purification,and determination of basic structure of Undaria polysaccharide were studied.The antitumor and immunoregulatory activities of Undaria pinnatifida polysaccharide were studied,and in-depth explored the immunoregulatory mechanism of Undaria pinnatifida polysaccharide and lay a theoretical basis and scientific basis for the comprehensive development and the utilization of Undaria pinnatifida polysaccharide.Methods:The polysaccharides of Undaria pinnatifida were extracted by water extraction and alcohol precipitation,the protein was removed by sevag reagent,and the crude polysaccharide was obtained by freeze-drying.The crude polysaccharides were separated by DEAE Sepharose fast flow,the component polysaccharide UPIs 1-3 was obtained,and the purity of the UPIs 1-3 were identified by G-100.High performance gel permeation chromatography,ion chromatography,fourier infrared spectroscopy,ultraviolet spectroscopy,Congo red test,and scanning electron microscopy(SEM)were used to analyze the basic structural characteristics of UPIs 1-3.Two types of colon cancer tissues from different sources were cultured as colon cancer organs,and then UPIs 1-3 were used to intervene in the two types of colon cancer organs in the range of 0.01-10 mg/mL,and the IC50 value were measured via cell viability.The mouse macrophage RAW264.7 cells were used to study the toxic effects of UPIs 1-3 on RAW264.7 cells and the effects of phagocytosis.The immunoactive component polysaccharides UPIs 1-3 were screened by the secretion of NO and cytokines(TNF-α,IL-6 and IL-1β)determined by Griess and Elisa kit methods,respectively.UPI-3 with strong activity was obtained.The effect of UPI-3 on the expression of macrophage-related cytokine mRNA was studied,and the immunoregulatory mechanism of polysaccharide was analyzed from the transcriptome and metabolome.Results:UPIs 1-3 were obtained from the crude polysaccharides of Undaria pinnatifida.The three component polysaccharides were homogeneous,and their molecular weights were 7.212 kDa,13.924 kDa,and 55.875 kDa,respectively.The total sugar contents were 56 ±0.21%,41.4± 0.11%,and 79.4± 0.15%,respectively.All of them contained uronic acid and sulfuric acid group,and they were acidic polysaccharides.The monosaccharide composition of UPIs 1-3 was detected by ion chromatography.UPI-1 and UPI-2 were mainly composed of mannuronic acid,and the content of mannuronic acid was more than 99%,while UPI-3 was mainly composed of fucose and glucuronic acid with the percentage of 31.51%and 36.39%,respectively.Under alkaline conditions,there were no significant difference between the maximum absorption wavelengths of UPIs 1-3 and Congo red solution and pure Congo red solution,indicating that they do not contain a triple-helix structure.SEM observations showed that UPI-1 and UPI-2 were chip and weak intermolecular interactions,UPI-3 was fragmented and strong intermolecular interactions.UPIs 1-3 had toxic effects on the organoids of the two types of colon cancer.When the polysaccharide concentration of the three components was 10.0 mg/mL,the cell viability of the two types of organoids were 0.50%,2.00%,7.00%and 15.43%,1.10%and 1.68%,respectively.The IC50 values of UPIs 1-3 on two types of colon cancer organoids were 2.81[2.30,3.35],3.87[2.30,6.89],4.51[1.82,11.67]and 3.33[-0.027,17.42],4.02[2.29,7.30],3.42[0.22,14.70]mg/mL,indicating that all three polysaccharides have antitumor activity.In the concentration range of 1-1000 μg/mL,UPIs 1-3 acted on RAW264.7 macrophages,and all of them had a certain proliferation effect and enhanced phagocytosis.what’s more,UPI3 had a strong enhancement effect Within the concentration range of 12.5-100 mg/mL,the secretion of immune factors NO,TNF-α and IL-6 could be promoted.UPI-3 with the strongest immune activity was screened out.UPI-3 induced the expression of iNOS,TNF-α,IL-6 mRNA,which was consistent with the corresponding expression trend of cytokines,indicating that UPI-3 has significant immunoregulation function.Based on the analysis of transcriptome and metabolome,we found that immune related pathways include Toll-like receptor,NF kappa B,TNF,IL-17,MAPK and PI3K Akt signaling pathways,arachidonic acid metabolism pathway,glycerophosphate metabolism pathway,amino acid biosynthesis pathway,with 39 genes and 13 metabolites annotated.UPI-3 bound to membrane receptors on the surface of macrophages,and then activated Toll-like receptors,NF-kappa B,prostaglandin and other immune-related intermediates or metabolites signaling pathways to improve the transcriptional expression of related protein genes.Thereby exerting immunomodulatory activity.Conclusion:Three component polysaccharides UPIs 1-3 were isolated from the crude polysaccharides of Undaria pinnatifida,all of which were homogeneous acid heteropolysaccharides.The monosaccharides of UPI-1 and UPI-2 are mainly composed of mannuronic acid,while UPI-3 is mainly composed of fucose and glucuronic acid.UPIs 1-3 do not contain triple-stranded helical structures.UPIs 1-3 show antitumor activity against both colon tumor organoids.UPIs 1-3 shown immune activity against macrophages,and UPI-3 was the strongest.UPI-3 could significantly increase iNOS,TNF-α,IL-6 mRNA expression and cytokine(NO,TNF-α,IL-6)expression.UPI-3 exerted its role in immune regulation by activating Toll-like receptors,NF-kappa B,and prostaglandin,and other immune-related signaling pathways.