Structural Analysis And Antiviral Activity Of Polysaccharides From Sargassum Henslowianum C. Ag.

Objective Sargassum henslowianum is the most common algal species along the coast of China.Phaeophyta sargassum is an important natural source of sulfuric acid polysaccharides.A large number of studies have confirmed the anti-HSV activity of polysaccharides,especially the fucoidan.However,due to the complexity of polysaccharide structure and the limitations of analytical methods,the research on polysaccharides from Sargassum henslowianum was limited to the extraction process and monosaccharide composition,and its pharmacological activity was therefore limited to the study on crude polysaccharide,and its pharmacodynamic material basis and mechanism were still unclear.Therefore,we plan to study the structure and antiviral mechanism of polysaccharide from Sargassum henslowianum,so as to lay a scientific foundation for elucidation of its pharmacodynamic material basis and mechanism.Methods 1.The crude polysaccharides?SH?from Sargassum henslowianum were obtained by hot water extraction and alcohol precipitation,and then were separated and purified by anion exchange column DEAE-650M,agarose gel column Sephrose 6B and dextran gel column Sephacryl S-500 to obtain polysaccharides with homogeneous charge and molecular weight.2.High performance gel permeation chromatography?HPGPC?was used to determine the homogeneity and molecular weight of polysaccharide from Sargassum henslowianum.The total sugar content,uronic acid content and sulfate content of the homogenous polysaccharides were determined by phenol-sulfuric acid method,m-hydroxybiphenyl method and elemental analysis.The monosaccharide compositions of neutral polysaccharide SHNP and acid polysaccharide SHAP-1 and SHAP-2 were analyzed by chemical derivatization combined with GC and PMP derivatization combined with HPLC.Fourier transform infrared spectroscopy?FT-IR?was used to detect the desulfurization of polysaccharides.GC-MS analysis was performed on SHNP and SHAP-1 and SHAP-2 after desulfurization by methylation analysis to determine the connection mode of polysaccharides.The spatial configuration of monosaccharides in polysaccharides was studied by ¹H and ¹³C NMR.HSQC was used to classify the hydrocarbons of each monosaccharide in detail.Finally,HMBC is used to determine the connection position and sequence of each monosaccharide.The chemical structure of polysaccharides was investigated by the above methods.3.Three purified polysaccharides were tested for cytotoxicity and antiviral activity by CCK-8,trypan blue and flow cytometry.The antivirus activity of the active polysaccharides was preliminarily screened by using the selective index SI of antivirus.Then,through virus adsorption experiment,virus invasion experiment and direct killing virus experiment,the antivirus target of polysaccharide samples was preliminarily explored.Results 1.The yield of SH was 6%.After the separation and purification of SH by DEAE-650M,Sephrose 6B and Sephacryl S-500,SHNP,SHAP-1 and SHAP-2 were obtained,and the yield was 3.5%,0.017%and 0.011%,respectively.2.The results of structural analysis showed that the relative molecular weights of SHNP,SHAP-1 and SHAP-2 were 8.4×10³,6.0×10⁵ and 4.2×10⁵,respectively.The total sugar content was 100%,94.61%and 84.78%,respectively.The content of uronic acid was 0%,1.9%and1.7%.The content of sulfate was 31.90%and 31.80%respectively.The monosaccharide composition analysis of SHNP showed that only glucose?Glc?and SHAP-1 and SHAP-2showed that both of them were composed of a large amount of fucose?Fuc?and a small amount of galactose?Gal?,with a ratio of about 3:1?Fuc:Gal?.Three polysaccharides did not contain uronic acid,which was basically consistent with the results of the determination of uronic acid by m-hydroxybiphenyl method.After desulfurization,DS-SHAP-1 and DS-SHAP-2 were completely desulfurized by FT-IR.The results of methylation analysis and NMR analysis on SHNP,DS-SHAP-1 and DS-SHAP-2 showed that the main chain of SHNP was composed of 1,3-linked?-D-Glucan.The main chain of SHNP consists of 1,3-linked?-D-Glucan,which were linked by O-3 position of 1,3,6-linked-?-D-Glucan.The main chains of DS-SHAP-1 and DS-SHAP-2 polysaccharides are composed of 1,3-linked?-L-Fucp residues and 1,2-linked Galp residues,with a ratio of about 4:1.The results of methylation analysis showed that the two polysaccharides were connected in similar ways,with the difference in molecular weight and degree of sulfuric acid.3.Cytotoxic assay results showed that the three polysaccharide samples of SHNP,SHAP-1and SHAP-2 showed no obvious cytotoxic effect on Vero cells,which ensured the safety of polysaccharides as potential drugs.Next,the antiviral activity of the three polysaccharide samples was screened,and the results showed that the neutral polysaccharide SHNP had certain antiviral effect,but not obvious.Two sulfuric acid polysaccharides SHAP-1 and SHAP-2 almost completely inhibited herpes I virus infection in Vero cells at 10 g/mL,respectively.Both sulfuric acid polysaccharides completely inhibited herpes II virus?HSV-2?infection of Vero cells at the dose of 1 g/mL.The antivirus effect of sulfated polysaccharides on HSV-2 was more obvious.In the study of antiviral mechanism,SHAP-1 and SHAP-2 completely inhibit the adsorption of herpes type II virus to host cells at the dose of 100 g/mL,and can play an antiviral role by effectively inhibiting the adsorption of herpes virus to the host cell surface.However,when interfering with the invasion of virus into host cells,the results showed that only about 1000g/mL of the sample could inhibit the invasion of half of the virus into host cells,indicating that the sample had a certain inhibitory effect on the invasion of virus into host cells,but it was not obvious.Conclusions Three polysaccharides isolated from Sargassum henslowianum have a definite structure.The sulfuric acid polysaccharide has definite anti-HSV-2 effect and can provide scientific basis for the development and utilization of the rich resources of Sargassum henslowianum.