Detection And Analysis Of Uronic Acid-Containing Polysaccharides From Mollusks By HPLC-MS

Uronic acid-containing polysaccharides(UACPs)are important nutriceuticals in mollusks,but systematic analysis and regular understanding about UACPs from shellfishes still lacks at present.In the present study,UACPs from 29 shellfishes of Gastropod and Bivalvia were analyzed by HPLC-MS.At the same time,the effects of origins and sampling time on UACPs in shellfish were analyzed,and the distribution of UACPs in different tissues was quantitatified.First of all,the extraction process of shellfish polysaccharides was optimized.According to the optimum process,the samples were incubated with trypsin and papain for 4 h and 3 h successively,and then the supernatant was collected and precipitated with 3 times volume of ethanol to afford crude polysaccharides.The content of sulfated polysaccharides in shellfishes were detected by 1,9-dimethylmethylene blue(DMB).The results revealed that the contents of sulfated polysaccharides were concentrated in the range of 1.5%to 2.0%,and there was a significant difference between the sulfated polysaccharide contents of the same tissues from Mya arenaria Linnaeus(BMA)and Coelom.actra antiquate(BCAX).Second,the shellfish UACPs were qualitatively and quantitatively detected.Crude polysaccharides isolated from mollusks were hydrolyzed with 1.3 M trifluoroacetic acid(TFA)at 105℃ for 3 h to offer disaccharides,and then the hydrolysates were analyzed by HPLC-MS/MS upon 1-phenyl-3-methyl-5-pyrazolone(PMP)derivatization.The results showed that the prevalence of glycosaminoglycans(GAGs)UACPs,including chondroitin sulfate(CS),heparin(HP),U8 and U11 as well as 2 non-GAGs UACPs,including U6 and U10 in mollusks.Among all the detected shellfishes,unlike Bivalve,Gastropoda contained little or no HP,and the species of UACPs from shellfishes living in the intertidal zone were less than those of living benthic shellfishes.As indicated by the dendrogram of hierarchical clustering,the distribution of UACPs in shellfishes was related with the genetic relationship at a certain degree.Habitats and sampling times may also influence the populations and contents of UACPs from shellfishes by comparing Meretrilx pethechialis and Tegillarca granosa bought from Zhejiang and Fujian provinces,and Cyclina sinensis collected in March and October 2016.Furthermore,the distributions of UACPs in different tissues from BMA and BCAX were analyzed.Notably,UACP compositions in different tissues of the same shellfish differed,while those in the same tissue(mantle,siphon or gill lamella)from two different shellfishes were also obviously distinct.To learn more structural information about UACPs in 8 shellfishes,crude polysaccharides were dissolved in 2 M TFA(AGSP)or 0.2 M TFA(CS)individually and heated at 100℃ for 1 h to obtain diversity of oligosaccharides for HPLC-MSn analysis.The finding of the tetrasaccharide,PMP labled β-GlcA(1→2)-a-Man(1→4)-β-GlcA(1→2)-a-Man,proved the structure of AGSP.And the structure of CS was identified as PMP labeled GlcA-(1→3)-GalNAc and β-GlcA(1→3)-P-GalN6S were detected.Finally,the data of UACPs in 48 shellfishes obtained by the pesent study as well as previous studies of our team were systematically collected to construct the data management platform of nutrients in marine products.The data obtained from this study not only enriched knowledge of UACPs from mollusks,but also provided reference data for nutrition assessment of these edible shellfishes and the scientific basis for the explorment of UACPs from mollusks.