Separation,Structural Analysis And Immunomodulatory Activity Of Acidic Degradation Products Of Polysaccharides From Saccharina Japonica

The sulfated polysaccharides derived from Saccharina japonica exhibit complex and inhomogeneous characteristics both in structure and composition.Degradation of polysaccharide by inorganic acid is a commonly used depolymerization method for polysaccharide.In addition,alcohol precipitation is a traditional method of purifying water-soluble polysaccharide.Different extraction processes,separation and purification methods and degradation processes lead to differences in the molecular weight,structure,and composition of the polysaccharides,which in turn lead to differences in bioactivity.This article systematically studied the acidic degradetion products of polysaccharide from Saccharina japonica.Through the study of different preparation processes of oligosaccharides and polysaccharides,different fractions with variety of structures and compositions were obtained,and the activities of the corresponding components were investigated.A series of sulfated fucooligosaccharides were prepared from low molecular weight components,and the structure of the main oligosaccharides were characterized.The mechanism of the immunomodulatory activity of the sulfated hetropolysaccharide was investigated.Specifically,that are:1 By setting degradation time,precipitation concentration and choosing different settling agents,the chemical composition and monosaccharide composition of the products obtained by acid hydrolysis were analyzed.Through comparison,the influence of different conditions on the products was summarized.Based on this,different processes were set up to prepare seven components,including both pieces of oligosaccharides and components difficult to hydrolyze,named OF1,OF2,OF3,OF4,OF5,PF1 and SHP,respectively.OF1-OF5 are oligosaccharides mainly composed of fucose,while PF1 and SHP are heteropolysaccharides with a molecular weight no lower than 20 kD.The activity screening results showed that low molecular weight fractions could not stimulate the secretion of nitric oxide from RAW 264.7 macrophages,while the large molecular weight fraction showed significant stimulatory effect.The damage of NRK-52E cells induced by aristolochic acid was constructed.In the cell model,only OF3 and OF5 at 0.5 mg/m L and PF1 0.5 mg/mL and 1 mg/mL(p<0.05)showed a statistically significant protective effect on renal cell injury,and none of the other components showed activity.2 To elucidate the structure of OF1 and PF1,several separation methods combined with electrospray ionization mass spectrometry were employed,including preparative thin layer chromatography,strong anion exchange chromatography.OF1 mainly composed of sulfated fucose oligosaccharide fragments,and other heterosaccharides.The previous results showed that OF1 could not activate macrophages,but its fractionation components separated by ion chromatography had a significant immune induction.While a glucofucogalactan(F2)was obtained from PF1 by membrane filtration,which had a significant renal protective effect.3 The OF2 was separated by gel filtration chromatography.Six components of sulfated fucose oligosaccharides(named F1-F6)were prepared.Tandem mass spectrometry method for the 7 kinds of monosulfated and disulfated fucosaccharides has carried on the structure characterization,including monosulfated fucose(FucS),monosulfated fucobiose(Fuc₂S),monosulfated fucotriose(Fuc₃S),monosulfated fucoterose(Fuc₄S),disulfated fucotriose(Fuc₃S₂),disulfated fucoterose(Fuc₄S₂),disulfated fucopentaose(Fuc₅S₂).The anti-inflammatory effects of the six components were examined and the results showed that only F1,F2,and F3 could inhibit the release of nitric oxide from LPS-induced macrophages,and the other three components had no activity.4 SHP is a low-sulfated heteropolysaccharide.After degradation,it was found that its structural units contained glucuronan oligosaccharides(degree of polymerization 1-11)and mannoglucuronan oligosaccharides,and a small amount of sulfated fucosaccharide,heterozygous fucose-mannose-glucuronan oligosaccharides.Studies have been conducted on the immunomodulatory mechanism of SHP in vitro,and it was found that after SHP acted on RAW 264.7 macrophages,it can be recognized by the cell surface TLR-4 and generated the corresponding signal,which activated protein kinase B and mitogen-activated protein kinase,then transmited signals to the nucleus,activated the binding of nuclear factor kappa B and activator proteins to inflammation-related genes,thereby triggering the transcription and expression of inflammatory factors.