Chemical Structures And Antioxidant Activities Of Polysaccharides From Phyllophorus Proteus

Free radicals are closely related to some diseases and health status of the human body.A considerable amount of studies have reported that polysaccharides from various sources have good antioxidant activity,while acidic polysaccharides exhibit better antioxidant activity than neutral polysaccharides.However,there are limited studies on the antioxidant activities of sea cucumber polysaccharides,and it is unclear whether there are significant differences in the antioxidant activities of sea cucumber polysaccharides with different structures.In this study,three kinds of polysaccharides（PPP-1a,PPP-1b,PPP-2）were extracted from the body wall of Phyllophorus proteus.The physicochemical properties,molecular structures and antioxidant activities of the polysaccharides were investigated to discover the different structural types of sea cucumber acid polysaccharides and to provide a material basis and reference for the study of structure-antioxidant activity relationships of sea cucumber polysaccharides.The main research contents are as follows:（1）Different sea cucumber polysaccharides were obtained from the dry body wall of Phyllophorus proteus by enzymatic/alkaline hydrolysis extraction,isoelectric point deproteinization,hydrogen peroxide decoloration,salting out method,alcohol precipitation,and column chromatography.The purity and molecular weight of the polysaccharides were analyzed by high performance gel chromatography（HPLC）,the sulfuric acid/carboxylic acid molar ratio was determined by the conductivity method,the sulfate content was determined by the barium chloride-gelatin method,the monosaccharide compositions were analyzed by the PMP derivatization-HPLC method,and the intrinsic viscosity and optical rotation were measured by viscometer and polarimeter.Finally,¹H NMR and IR spectroscopy were used to identify the basic structural characteristics of polysaccharides.（2）The depolymerization product of PPP-1b was prepared by depolymerization with hydrogen peroxide,and the basic chemical structural characteristics of depolymerized PPP-1b were analyzed by 1D and 2D NMR to speculate the basic structural features of the PPP-1b.（3）The depolymerized PPP-2 product was prepared by selective N-deacetylation and deaminative cleavage.The small-molecule impurities in the depolymerized PPP-2 product were removed through salting-out,alcohol precipitation and dialysis,followed by the size-exclusion chromatogrphy to obtain the homogenous oligosaccharides of PPP-2.The structural characteristics of the oligosaccharide compounds were analyzed by 1D and 2D NMR.（4）The abilities of the crude polysaccharide PPP,and the purified polysaccharides,PPP-1a,PPP-1b,and PPP-2 to scavenge DPPH free radicals,hydroxyl radicals,superoxide anion radicals,and ABTS radicals were detected to evaluate their antioxidant activities.Result:（1）Three homogeneous polysaccharides,PPP-1a,PPP-1b,and PPP-2,were obtained from the dried body wall of the Phyllophorus proteus.The purity of the three polysaccharides was more than 99%determined by HPLC.The molecular weights were 369.6,41.73,and57.76 kDa,respectively,the sulfate content was 20.6%,25.2%,and 28.0%,respectively,the intrinsic viscosities were 172.44,62.84,and 39.61 mL/g,respectively,and the specific rotations were-77°,-138.5°,and-42.5°,respectively,indicating that all three polysaccharides were L-optical substances.Analysis of monosaccharide composition showed that PPP-1a was composed of galactosamine（GalNAc）,galactose（Gal）and fucose（Fuc）in a molar ratio of 1.00:1.24:8.04,PPP-1b contained only Fuc,belonging to Fucans.PPP-2 was composed of glucuronic acid（GlcA）,GalNAc,and Fuc,with a molar ratio of 1.00:1.17:1.77and a molar ratio of sulfuric acid to carboxylic acid of 3.32,belonging to fucosylated glycosaminoglycans..（2）Depolymerised products of PPP-1b were obtained by hydrogen peroxide with the molecular weight about 5 kDa.The 1D and 2D NMR spectra showed that PPP-1b was composed of Fuc substituted by different kinds of sulfuric acid,mainly includin g Fuc_0S,Fuc_2S,Fuc_3S,and Fuc_4S,Fuc_2S4S types.The structure of PPP-1b was complex and should be further studied.（3）The depolymerization product of PPP-2 was obtained by the N-deacetylation a nd deaminative cleavage.The oligosaccharide compounds 1-4 was obtained by the gel chromatography.HPLC analysis revealed that compounds 1-4 were trisaccharide,pent aose,hexaose,and nonasaccharide,respectively.1D and 2D NMR spectra revealed that the chemical structure of compound 1 was:L-Fuc_2S4S-α1,3-D-GlcA-β1,3-D-anTal_4S6S-ol,compound 2 was a pentaose containing different sulfated fucose types,the main che mical structure of compound 3 was:L-Fuc_2S4S-α1,3-D-GlcA-β1,3-D-GalNAc_4S6S-β1,4-[L-F uc_2S4S-α1,3-]-D-GlcA-β1,3-D-anTal_4S6S-ol,which may also contain 17.5%L-Fuc4S-α1,3-D-GlcA-β1,3-D-GalNAc_4S6S-β1,4-[L-Fuc_4S-α1,3-]-D-GlcA-β1,3-D-anTal_4S6S-ol,and the ma in chemical structure of compound 4 was:L-Fuc_2S4S-1,3-D-GlcA-β1,3-{D-GalNAc_4S6S-β1,4-[L-Fuc_2S4S-α1,3-]-D-GlcA-β1,3-}2-D-anTal_4S6S-ol,still containing Fuc_0S,Fuc_3S,Fuc_4S su bstitution types of nonasaccharides.Based on these analysis,it can be speculated that PPP-2 was mainly a polysaccharide composed of a trisaccharide structural unit{D-Gal NAc_4S6S-β1,4-[L-Fuc_2S4S-α1,3-]-D-GlcA-β1,3-}n,containing a small amount of other sulf ated Fuc residues.（4）The in vitro antioxidant activity of crude polysaccharides and three polysaccharide components of Phyllophorus proteus showed that Phyllophorus proteus polysaccharides had good antioxidant capacity,which showed a dose-effect relationship,and these polysaccharide had the highest ability to scavenge superoxide anion radicals.When the concentration was 4mg/mL,the scavenge rates of PPP-1a,PPP-1b and PPP-2 to superoxide anion radicals were91.15%,92.47%and 99.66%,respectively,which was similar to vitamin C.Conclusion:In this study,three structurally different polysaccharides were extracted from Phyllophorus proteus.PPP-1a could be a heteropolysaccharide compound with complex structure.PPP-1b was composed of Fuc substituted by different kinds of sulfuric acid,mainly including Fuc_0S,Fuc_2S,Fuc_3S,and Fuc_4S,Fuc_2S4S types.The structure of PPP-1b was complex and should be further studied.The structure of depolymerized oligosaccharides of PPP-2 by N-deacetylation and deaminative cleavage shows that.PPP-2 was glycosylated glycosaminoglycan mainly a polysaccharide composed of a trisaccharide structural unit{D-GalNAc_4S6S-β1,4-[L-Fuc_2S4S-α1,3-]-D-GlcA-β1,3-}_n,containing a small amount of other sulfated Fuc residues.Studies have shown that all three polysaccharides have good antioxidant activity,and the ability to remove superoxide anion free radicals is comparable to that of vitamin C.The fucosylated glycosaminoglycan has slightly stronger antioxidant activity than fucoidan.Therefore,the antioxidant activity of sea cucumber polysaccharides deserves further study,and this study can provide some data for the development and utilization of sea cucumbers.