| China has a vast sea area,and rich marine biological resources.Marine organisms contain many active compounds with unique structure,which have great potential for drug development and application,and also make more and more domestic and foreign scholars turn their attention to the exploration of marine active compounds.In this study,polysaccharides extracted from eggs of Glyptocidaris crenularis,Strongylocentrotus intermedius and Strongylocentrotus nudus from the Yellow Sea and Bohai Sea,as well as the extracellular polysaccharides of deep-sea bacteria in the Western Pacific Ocean were isolated and purified.And their physical and chemical properties were analyzed.The immunoregulatory activity,antiviral activity of the polysaccharides and the mechanism were explored.The structure of active polysaccharides was characterized.This study is expected to provide a theoretical basis for elucidating the structure-function relationship of sea urchin polysaccharides and marine bacterial polysaccharides,as well as the development of marine functional products and drugs.Main study results are as follows:(1)Sea urchin polysaccharides were extracted by hot water and alkali solution in turn.The water extraction rates of polysaccharides from S.nudus,G.crenularis and S.intermedius were 11.1%,9.4%and 24.9%,respectively,and the alkali extraction rates were 6.2%,2.7%and 3.9%,respectively.The crude polysaccharides were enzymatically hydrolyzed using alkaline protease and papain,and total 24 fractions were obtained after separation by ion exchange column.Five purified polysaccharides(ZS-1B,MS-1B,HS-1B,MSGA and HJ2A)were obtained after separation and purification by Sephadex G gel filtration column.The physicochemical properties of purified polysaccharides at all levels(35 fractions in total)were determined,and the fractions with higher total sugar content included ZS-1(78.90%),HS-1(76.50%),HJ-1(53.40%),HJ-2(49.60%)and MS-1(66.00%)etc.The protein content of ZS-1B,HS-1B,MS-1B,HJ2A and MSGA were 8.60%,7.00%,13.20%,0.10%and 0.30%.All polysaccharides contained no sulfate groups.The average molecular weights(Mw)of polysaccharides were quite different(4 k Da～600 k D),and the Mw of ZS-1B,HS-1B and MS-1B that purified by gel column chromatography were about 4 k Da.The Mw of MSG-A and HJ-2A were 2.65×10~4 k Da and 8.06×10~3 k Da,respectively.Sea urchin polysaccharides was mainly composed of Man,Glc N and Glc,and some fractions also contained small amounts of Rha,Glc A,Gal N,Gal and Fuc.Among them,ZS-1B,MS-1B,and HS-1B were composed of Man,Glc N,and Glc with molar ratios of 5.18:1.00:4.13,7.78:1.91:1.00,and 5.06:1.00:8.01,respectively,and MSGA and HJ2A were only composed of Glc.In vitro activity study indicated that MSGA and HJ2A had potential immunoenhancing activity.(2)98 strains of bacteria from the cold seeps of the South China Sea and one strain of Bacillus sp.BS11 from the Yapu trench were used to ferment and extract extracellular polysaccharides.More than 70 extracellular polysaccharides were prepared.The study of activity screening in vitro showed that exopolysaccharides from Halomonas sp.2E1and 2C11(EPS2E1 and EPS2C11)have potential immunoenhancing activity.EPS11showed strong immunosuppressive activity.EPS11 and EPS2E1 were purified,and their physicochemical properties were analyzed.The total sugar content of EPS11 and EPS2E1 were 49.5%and 83.1%,the protein content was 30.2%and 7.9%,the Mw of EPS11 and EPS2E1 were 306.0 k Da and 47.0 k Da,respectively.EPS2E1 mainly contains Man and Glc with a molar ratio of 3.76:1.EPS11 included Man,Glc N,Gal N,Glc,Gal,Rha,and Glc A with a molar ratio of 13.08:7.84:8.23:4.73:7.03:1.00:1.03.EPS11 is a glycoprotein containing 20 amino acids with a total amino acid content of30.5%,of which Glu,Asp,Ser and Gly are more abundant.The protein in EPS11 has homology with eight known proteins,and the two proteins with the highest match are alkaline bacillopeptidase F and actin.(3)The structures of active polysaccharides MSGA,HJ2A,EPS2E1,ZS-1B,MS-1B and HS-1B were characterized by FT-IR,methylation analysis,NMR and MS.MSGA and HJ2A are glycogens in sea urchins,and contained Glc-(1→,→4)-Glc-(1→,and→4,6)-Glc-(1→residues,with molar ratios of 1.01:2.75:1.00 and 2.19:3.18:1.00,respectively.They all has an average of one branch per five residues;EPS2E1 is a highly branched polysaccharide with the backbone mainly containing→2)-Man-(α-1→and→2,6)-Man-(α-1→with molar ratios of 2.45:1.00.Other residues,such as→4)-Glc-(α-1→,→6)-Man-(α-1→and→3)-Glc-(β-1→,also contained in the chain.HS-1B,MS-1B,and ZS-1B had diverse linkage patterns but similarities.They all contained(1→2),(1→4),(1→6),(1→3,6)and(1→4,6)linkage patterns.HS-1B mainly contained(1→4),MS-1B mainly contains(1→2),while the proportions of(1→2),(1→4),(1→6)and(1→4,6)in ZS-1B are close,all of which have branches at positions 3,4 or 6 of the backbone residues.(4)RAW264.7 macrophages were used as an in vitro model to study the immunoregulatory activity of two sea urchin glycogens and three microbial exopolysaccharides.MSGA and HJ2A was not cytotoxic in the concentration range of0～800μg/m L,and EPS2E1 and EPS2C11 were not cytotoxic in the concentration range of 0～200μg/m L.They both had effects of Inflammatory stimulation,and up-regulated the transcription of inflammation-related genes by promoting the phosphorylation of IκB and P65 in the NF-κB signaling pathway,as well as ERK1/2,JNK,or P38 in the MAPKs pathway.They could induce the differentiation of RAW264.7 macrophages into M1 type,and significantly promoted the production of inflammatory mediators and key enzymes(NO,COX-2,IL-1,IL-6,and TNF-α).EPS11 could significantly inhibit the production of NO,COX-2,IL-1,IL-6,and TNF-αin M1 macrophages by inhibiting the phosphorylation of P38,ERK1/2,JNK,IκB,and P65 proteins in MAPKs and NF-κB signaling pathways at concentrations ranging from 0 to 200μg/m L.It might as well promot NF-κB ubiquitination.So,EPS11 inhibited the transcription of inflammation-related genes,reducing the intracellular transduction of inflammatory signals,and without cytotoxicity.(5)The activity of sea urchin polysaccharides(MS-1B,HS-1B,ZS-1B,MSGA and HJ2A)against SARS-Co V-2 virus was investigated by surface plasmon resonance(SPR)technique and pseudo virus model.The results of SPR showed that MS-1B,HS-1B,ZS-1B,MSGA and HJ2A had strong affinity for S-protein,and they interacted with each other in a dose-dependent manner.The model of pseudo virus binding to HEK293was used to demonstrate that MSGA,HJ2A,HS-1B and ZS-1B could competitively inhibit the binding of S-protein to ACE2 and prevent SARS-Co V-2 virus from entering cells,which might be one of the mechanisms of the antiviral activity of sea urchin polysaccharides,while MS-1B had no obvious binding ability to S-protein.At present,researches on sea urchins and deep-sea bacteria mainly focus on metabolism,evolution or aquaculture,etc.Although some scholars have paid more and more attention to polysaccharides in recent years,there are still great difficulties in the analysis of their fine structure.And the research on the mechanism of biological activity is still not in-depth enough.This study provided a theoretical basis for elucidation of the structure-activity relationship of sea urchin and deep-sea bacterial polysaccharides,as well as the development of marine functional products and drugs. |
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