Chemical Structure And Antioxidant Activity Of Polysaccharide FSI From Holothuria Fuscopunctata

Sea cucumber has been regarded as a valuable nourishing food and medicinal material since ancient times,and its body wall is the main edible and medicinal part.Chinese medicine believes that"its nature is warming and nourishing,and the effect is comparable to Panax ginseng C.A.Meyer,so it is called sea cucumber".A Supplement to Compendium of Materia Medica recorded:sea cucumber can tonify the kidney,benefit the essence,urinate and strengthen the Yang.In recent years,with the deepening of the research on sea cucumber,it is found that polysaccharides such as fucoidan of its body wall have a wide range of pharmacological activities such as anticoagulation,antioxidation,anti-tumor and anti-inflammatory.Unlike algae-derived fucoidan（fucoidan）,most fucan sulfate（FS）from sea cucumbers usually possess regular linear structures,and a few of them were branched with side chains.The linear structure is mostly composed of repeating units of oligosaccharides.The structures of FS from different sea cucumbers are usually in a species-specific manner,and main differences are the number of residues in the repeating unit,sequence of these residues,glycosidic bonds,and degree of sulfation.The biological activities are highly dependent on the structure.For example,it has been reported that the presence of sulfate group is a necessary condition for low-molecular-weight FS to exhibit anticoagulation.Therefore,clarifying the structure of polysaccharides will facilitate the structure-activity relationship study and further promote its application.Our previous study found that the basic structure of FSI from Holothuria fuscopunctata is a tetrasaccharide repeating unit linked by novelα1,3 andα1,4 glycosidic bonds alternately.The structure was stated as:{-3-L-Fuc_2S4S-α1,4-L-Fuc-α1,3-L-Fuc_2S-α1,4-L-Fuc-α1-}_n.However,the previous structural analysis of FSI was based on the monosaccharide composition and 1D/2D NMR spectra of the low-molecular-weight product depolymerized by the free-radical cleavage method.In addition,the signals in NMR spectra were overlapped and complex due to the various sulfation types,especially,the H-3,H-4 signals of Fuc_0S were usually overlapped and difficult to distinguish.Consequently,using the glycosidic bond cleavage method to obtain pure oligosaccharide,analyzing accurately oligosaccharide structure is an effective way to elucidate the structure of native FSI.Given that the anticoagulant activity of FSI has been reported in our previous paper,along with abundant literatures showed that FS possessed antioxidant activity,here,the antioxidant activity of FSI and dFSI-2 were evaluated in vitro by free radical generation system.Methods:（1）Enzymatic hydrolysis-alkaline hydrolysis method was used to extract acidic polysaccharide from the body wall of Holothuria fuscopunctata,and strong anion exchange chromatography was used to purify the crude polysaccharide FSI after graded salting out and alcohol precipitation.The purity and molecular weight of native FSI was analyzed by HPGPC;the sulfate content was determined by conductivity method and ion chromatography method;The low molecular weight products dFSI-1 and dFSI-2 of FSI were prepared by peroxidative depolymerization and the molecular weight was determined;At the same time,the hydrolyzate dFSI was prepared by optimizing the conditions of mild acid hydrolysis.（2）1D/2D NMR was used to analyze the structure of dFSI-1 and determine the basic structure characteristics of FSI.GPC（gel permeation chromatography）method was used to separate and purify dFSI,a series of oligosaccharides with single peak were obtained.Their chemical structures were comprehensively analyzed by NMR and MS（mass spectrometry）technique.Based on the information of oligosaccharide structure and the basic structure characteristics of FSI,the"bottom up"strategy was used to confirm the glycosidic bonds type and structural sequence of native FSI.（3）The antioxidant activity of FSI and its peroxidative depolymerized product dFSI-2were evaluated using free radical generating system in vitro.Results:（1）FSI purification,physicochemical properties and depolymerization treatment（1）Enzymatic hydrolysis-alkali hydrolysis extraction,salting-out alcohol precipitation separation,strong anion exchange column chromatography to obtain purified fucan sulfate FSI.（2）HPGPC analysis showed that the purity of FSI was about 95%（area normalization）.Its weight-average Molecular Weight（Mw）was 470.6 k Da by GPC method.The content of sulfate group by conductivity method and ion chromatography was about 23%and 25.7%,respectively.（3）The depolymerization products dFSI-1 and dFSI-2 were obtained by peroxidative depolymerization,and their molecular weights were about 10 k Da and 4.6 k Da by GPC analysis.（4）Through comparative study,the depolymerization product dFSI was obtained by mild acid hydrolysis in 10 m M trifluoroacetic acid（TFA）at 80℃.（2）FSI basic structure and definite structure analysis（1）By analyzing the 1D/2D NMR spectrum of dFSI-1,it is inferred that the basic structural unit of FSI is:{-3-L-Fuc_2S4S-α1,4-L-Fuc-α1,3-L-Fuc_2S-α1,4-L-Fuc-α1-}_n（2）Six oligosaccharide fractions（FI～FVI）were separated from dFSI by repeated GPC chromatography.1D/2D NMR and QTOF-MS spectral analysis confirmed the chemical structures of the obtained oligosaccharides（components）FI～FVI,respectively:FI-a:L-Fuc_2S-α1,3-L-Fuc-ol;FI-b:L-Fuc_4S-α1,3-L-Fuc-ol;FII:L-Fuc_2S4S-α1,3-L-Fuc-ol;FIII:L-Fuc_2S4S-α1,3-L-Fuc-α1,3-L-Fuc-ol;FIV-a:L-Fuc_4S-α1,3-L-Fuc_2S4S-α1,3-L-Fuc-ol;FIV-b:L-Fuc_2S4S-α1,3-L-Fuc_2S-α1,3-L-Fuc-ol;FV-a:L-Fuc_2S4S-α1,3-L-Fuc-α1,3-L-Fuc_2S-α1,3-L-Fuc-ol;FV-b:L-Fuc_4S-α1,3-L-Fuc_2S4S-α1,3-L-Fuc-α1,3-L-Fuc-ol;FV-c:L-Fuc_2S-α1,3-L-Fuc-α1,3-L-Fuc_2S4S-α1,3-L-Fuc-ol;FV-d:L-Fuc_4S-α1,3-L-Fuc_4S-α1,3-L-Fuc_2S-α1,3-L-Fuc-ol;FVI:L-Fuc_2S4S-α1,3-L-Fuc_2S-α1,3-L-Fuc_2S-α1,3-L-Fuc-ol。（3）Comparing the structural characteristics of the obtained oligosaccharides（component）FI～FVI,it can be seen that the reducing ends of all oligosaccharides are Fuc-ol,which is consistent with the post-treatment of end group reduction.In ¹H-¹H ROSEY（rotating frame overhauser effect specctrocopy）spectrogram of dFSI-1,it can be clearly observed that Fuc_0Sis directly connected with Fuc_2S4S,and the non-reducing terminal of trisaccharide and tetrasaccharide is mainly Fuc_2S4S,the reduction end is Fuc-ol indicate that theα1,3 glycoside bond between Fuc_0S and Fuc_2S4S is easily cleaved by mild acid hydrolysis.（4）The analysis of the structure of oligosaccharides showed that all the fucose oligosaccharides were linked byα1,3 glycosides,but there was noα1,4 glycosides,which was different from the previous conclusion that the basic structural units of FSI were alternately linked byα1,3,α1,4 glycosides.The analysis shows that in previous studies,the NMR signals of native FSI and its depolymerized product dFSI-1 overlap seriously and are difficult to distinguish.In particular,the H-3 signal（4.07ppm）and H-4 signal（3.97ppm）of the residue Fuc_0S show almost equal chemical shift,leading to misjudgment of the type of glycoside bond.（5）Based on described above the basic structure analysis of FSI,the chemical structure analysis of the obtained series of oligosaccharides,and the"bottom up"macromolecular compound structure analysis strategy,the chemical structure of FSI can be stated as:{-3-L-Fu_2S4S-α1,3-L-Fuc_（2S）-α1,3-L-Fuc_2S-α1,3-L-Fuc-α1-}_n（3）Antioxidant activity analysis in vitro（1）The results of antioxidant activity in vitro showed that FSI and dFSI-2 showed strong scavenging ability on superoxide anion radical（·O₂^-）.In the concentration range of 0.025-0.2mg/m L,FSI（Mw 470.6 k Da）and dFSI-2（Mw 4.6 k Da）showed a concentration-dependent increase in scavenging activity on·O₂^-.The IC₅₀ were 65.71 and 83.72μg/m L,respectively.In comparison,the antioxidant activity of FSI was stronger than dFSI-2,and the activity of FSI was similar to the positive control Vc.（2）FSI and dFSI-2 had no significant scavenging activity on DPPH radical（DPPH·）,hydroxyl radical（·OH）and ABTS radical.Conclusion:（1）Through the structural analysis of a series of oligosaccharide fragments,the chemical structure of FSI derived from Holothuria fuscopunctata was confirmed as:{-3-L-Fu_2S4S-α1,3-L-Fuc_（2S）-α1,3-L-Fuc_2S-α1,3-L-Fuc-α1-}_n.（"bottom-up"strategy）In other words,FSI is a macromolecular fucoidan with relatively regular tetrasaccharide structure units connected in sequence.（2）The Fuc residues contained in FSI are connected byα1,3 glycosidic bonds,and there is noα1,4 type glycosidic bond,which is inconsistent with the previous report,the reason is that the previous study was mainly based on the ¹H-¹H ROESY spectrum of low molecular weight products analysis,in fact,the terminal hydrogen on fucose and H-4 on the adjacent fucose are close due to the spatial distance,and there is a correlation signal,so a structural misjudgment occurs.（3）Tetrasaccharide FV represents the basic structural units contained in native FSI.Native FSI also contains a small amount of irregular structure,which is mainly manifested by the substitution of a sulfate group on the second Fuc residue in the tetrasaccharide structural unit.（4）Theα1,3 glycosidic bond between Fuc and Fuc_2S4S in FSI is easily cleaved by mild acid hydrolysis.（5）FSI and dFSI-2 have strong scavenging ability on superoxide anion free radicals in in vitro antioxidant experiments.