Structural Characterization And Metabonomics Study Of Fucosylated Chondroitin Sulfate(FCS)in Holothurian Leucospilota

Fucosylated Chondroitin Sulfate(FCS)is a kind of holothurian Glycosaminoglycan(hGAG)extracted from the body wall of sea cucumber with many bioactivities,such as Antitumor metastasis,Antithrombotic,Anticoagulation,Antiviral,Antiinflammation and so on,which expected to exploit as a new kind of marine drug.FCS is composed of Chondroitin Sulfate(CS)backbone and sulfated Fucose branches,however the structure of FCS extracted from different holothurians and sea area and different extraction process will lead to the differences of structural characterization.Structural characterization is very important to the clarify and study of structure-activity relationship.In this page,bottom-up strategy,monosaccharide composition,sulfate content,disaccharide composition in backbone and Fucosylated branch analyses,were applied to elucidate the structure of FCS extracted from Holothurian leucospilota.1.The FCS was degraded by 2 M Trifluoroacetic acid(TFA)into monosaccharides and sulfate ions and analyzed by ion chromatography coupled with pulsed amperometric detector(PAD)and electrical conductivity(CD)detector respectively.The monosaccharides composition is Fucose,N-acetyl galactosamine Glucuronic Acid.The free and linked sulfate content are 0.07%and 30.7%respectively.2.In order to elucidate the disaccharides composition in CS backbone,the FCS was hydrolyzed with mild acid.The remained part was digested with CS lyase for disaccharide analysis.The products from each step were analyzed and the results revealed that mild sulfuric acid mainly released sulfated fucose branches,but also affected some residues and sulfo-groups on the backbone.The proposed backbone of the FCS is mainly composed of GlcA→GalNAc4S6S and GlcA→GalNAc6S,3.Over 140 oligosaccharide fragments were generated by catalytic oxidation and identified by high-performance size-exclusion chromatography combined with mass spectrograph(HPSEC-MS),including sulfated fucose oligosaccharides exclusively from branches,sulfated backbone fragments,and junctional fragments.Based on the results provided by the method,the branch is mainly located at N-Acetyl galactosamine(GalNAc).The longest branch observed is nonasaccharide,and most of the fucose on the branches are mono and/or di-sulfated.4.What’s more,we adopted the NMR technology to verify the results of disaccharides assay and oligosaccharide fragments characterization and support the conclusion.Besides,(Nuclear Magnetic Resonance)NMR analysis also completed the substitutional position of sulfated residues and the type of glycosidic bond between glycosyl.5.It is difficult to detect the polysaccharide and study its disposal process of FCS in vivo after oral or intragastric administration.In this study,according to two kinds of native FCS with different molecular weights,FCS derivates were obtained by mild acid hydrolysis.Corresponding metabolomics study was carried out with native FCS and FCS derivates.The healthy male mice were choosing for the metabonomics study after providing normal saline and FCS by intragastric administration,respectively.At the same time,venous blood was collected to analyze the metabolites.The results showed that it could significantly upregulate the metabolic function of intestinal flora and the metabolic level of the body in mice.Continuous intragastric administration of different characteristic polysaccharide compounds of FCS(A,dfA,B and dfB)significantly altered the concentrations of some metabolites in plasma of mice,and the concentrations of some metabolites in plasma remained significantly altered after drug withdrawal.In addition,compared with the vehicle group,intragastric administration of A,dfA,B,and dfB significantly affected the body weight of mice.In addition,in the recovery period,compared with the low branched chain and low molecular weight FCS derivatives dfA and dfB groups,the increase in the proportion of weight gain in groups A and B was more statistically significant,reflecting the differences in metabolism and body weight of mice caused by FCS with different structural characterization,such as,branched chain and molecular weight.The structural composition of FCS in Holothurian leucospilota was characterizedsystematically and entirely for the first time.With the analyzation of the metabolites after intragastric administration to study the potential bioactivity mechanism of FCS and the FCS could regulate the intestinal flora.