| Objectives:As a traditional seafood treasure,sea cucumbers are rich in nutrients and biologically active ingredients,including acidic polysaccharides with special chemical structure and significant biological activity.Fucan sulfate(FS)is one of the acidic polysaccharides contained in the body wall of sea cucumber.It is generally believed that compared with fucoidan sulfate(Fucoidan)derived from seaweed,the chemical structure of sea cucumber FS is relatively regular,and FS from different species has diverse structural characteristics.It is known that sea cucumber FS has biological activities such as immune regulation,anticoagulation,and anti-inflammation.Our group reported earlier the chemical structure of a FS derived from the body wall of Pattalus mollis.Subsequent research showed that in addition to the reported FS,the body wall of P.mollis also contains several FS with significant differences in physiochemical properties.Based on this,this thesis systematically analyzes the type,chemical structure and anticoagulant activity of FS contained in the body wall of P.mollis,so as to analyze the diverse structural characteristics of FS contained in a single species of sea cucumber,and analyze its biological activity through the comparison of anticoagulant activity.The structure-activity relationship provides abundant and reliable basic research data for the discovery and application of sea cucumber polysaccharide active ingredients.Methods:(1)P.mollis body wall polysaccharide was extracted by combined enzymatic hydrolysis-alkaline hydrolysis,and the obtained polysaccharide extract was separated by fractionation and ion exchange column chromatography to obtain uniform FS(five kinds,FS-Ⅰ,FS-Ⅱ,FS-Ⅲ,FS-Ⅳ and FS-Ⅴ,where FS-Ⅴ is the same as the PmFS previously reported by the research group).(2)The physiochemical properties of uniform PmFS(FS-Ⅰ,FS-Ⅱ,FS-Ⅲ,and FS-Ⅳ)were analyzed,including molecular weight and distribution,monosaccharide composition,sulfate content and optical rotation,etc.(3)The basic structure of FS-Ⅰ,FS-Ⅱ,FS-Ⅲ,FS-Ⅳ and FS-Ⅴ were characterized by spectroscopic method.Among them,due to the large molecular weight of FS-Ⅰ and FS-Ⅲ,the peroxide depolymerization method was used to depolymerize them into dFS-Ⅰ and dFS-Ⅲ.The basic structure of the depolymerized product was analyzed by 1D/2D NMR spectra(4)Among the five kinds of PmFS,the structure of FS-V is more complicated,and there may be a branched chain structure.Due to the serious overlap of 1D/2D NMR signals of the natural sugar,it is still difficult to determine the exact structure of its branches(side chain length,side chain arrangement,etc.)characteristics.For this reason,in this thesis,we used mild acid hydrolysis to depolymerize FS-V,and purified a series of oligosaccharide compounds from the depolymerized product by gel chromatography and strong anion chromatography,and tried to deduce the exact structure of FSV from the chemical structure of the obtained series of oligosaccharides.("bottom-up" strategy).(5)The correlation between their chemical structure and activity potency was analyzed based on the analysis of the characteristics of anticoagulant activity of FS-Ⅰ,FS-Ⅱ,FS-Ⅲ and FS-Ⅳ.Results:(1)Separation and purification and analysis of its physiochemical properties of PmFSExtraction by combined enzymatic hydrolysis-alkaline hydrolysis,fractionation,ion exchange chromatography and molecular exclusion chromatography purificationLead to Five FS components from the body wall of sea cucumber P.mollis:FS-Ⅰ,FS-Ⅱ,FS-Ⅲ,FS-Ⅳ and FS-Ⅴ.Analysis of monosaccharide composition showed that FS-Ⅰ,-Ⅱ,-Ⅲ,-Ⅳ and-Ⅵ were all composed of L-fucose(Fuc).Conductivity analysis showed that its sulfate group(-SO3-)content was 32.2%,29.6%,28.6%,35.9%and 30.4%,respectively;analysis by HPGPC method showed that its weight average molecular weight(Mw)were 238.3 kDa,81.0 kDa,82.0 kDa,23.2 kDa and 6.12 kDa,respectively.(2)Chemical structure analysis of FS-Ⅰ,FS-Ⅱ,FS-Ⅲ and FS-ⅣThe 1D/2D NMR spectrum analysis of the prototype or depolymerized FS-Ⅰ,-Ⅱ,-Ⅲ and-Ⅳ shows:the chemical structure of FS-Ⅰ is {-3)-L-FucS-α(1-}n,and in the formula,FucS is Fuc2S(44%),Fuc4S(36%),Fuc2S4S(10%)or Fuc0S(10%),n is about 960;FS-Ⅱ has a chemical structure similar to FS-Ⅰ,that is,FS-Ⅱ is also a linear polysaccharide formed by FucS connected by a 1,3 glycosidic bonds,and its degree of polymerization is low(n is about 330);the chemical structure of FS-Ⅲ is {-4)-L-Fuc2S-α(1,4)-L-Fuc2S-α(1,4)-L-FucS-(α1-}n,and in the formula,FucS is Fuc3S(～50%)or Fucos(50%),n is about 118;the chemical structure of FS-Ⅳ is {-(4-L-Fuc2S3S-α1)2-(4-L-Fuc2S-α1)4-}n,and in the formula,n is approximately 14.(3)Mild acid hydrolysis of FS-V and purification and structure analysis of a series of oligosaccharide compounds:Previous studies have shown that the chemical structure of FS-V may be {-4-L-Fuc2S-α1,4-[L-Fuc4s/3s-α1,3]-L-Fuc2S-α1,4-L-Fuc2S-α1-}n.There may be relatively regular arrangement of monosaccharide side chains.Since the spectral signal showing the side chain connection position and the length of the side chain overlap seriously,the exact structure of the side chain is still difficult to confirm.In this study,a series of oligosaccharide components F1-F6 were separated from dFS-V by gel column chromatography after depolymerization to prepare the depolymerized product dFS-V by a mild acid hydrolysis method.The structure of the oligosaccharide component F1-F6 was analyzed by spectroscopy.F1:Fuc0S-ol;F2:Fuc4S-ol(70%),Fuc2S-ol(17%)&Fuc3S-ol(13%);F3:Fuc4S-α1,3-Fuc0S-ol(66%),Fuc2S-α1,4-Fuc0S-ol(15%),Fuc2S-α1,3-Fuc0S-ol(13%),Fuc3S-α1,4-Fuc0S-ol(6%);F4:Fuc2S4S-α1,3-Fuc0S-ol;F5:Fuc2S-α1,4-Fuc2S-α1,4-Fuc0S-ol(67%),Fuc4S-α1,4-Fuc4S-α1,3-Fuc0S-ol(17%),Fuc2S4S-α1,4-Fuc0S-α1,3-Fuc0S-ol(10%),Fuc3S-α1,4-Fuc3S-α1,4-Fuc0S-ol(6%);F6:Fuc2S-α1,4-Fuc2S-α1,4-Fuc2S-ol(60%),Fuc2S-α1,4-Fuc2S-α1,4-Fuc3S-ol(40%).Combined with the analysis structure of the original sugar structure and the oligosaccharide component F1-F6,revision of the chemical structure of FS-V should be:{-3-R-L-Fuc2S-α1,4-(L-Fuc2S/3S-ol,4-)x-}n In the formula,R is Fuc(2S)4S-α1,3/4-Fuc4S(0S)-α1-or Fuc(2S)4S-;x is 1 or 2.(4)Anticoagulant activity analysis of FS-Ⅰ,FS-Ⅱ,FS-Ⅲ and FS-ⅣFS-Ⅰ,FS-Ⅱ,FS-Ⅲ and FS-Ⅳ can significantly prolong the APTT of human quality control plasma,indicating that they all have a certain anticoagulant activity that inhibits the intrinsic coagulation pathway.The concentrations of FS-Ⅰ,FS-Ⅱ,FS-Ⅲ and FS-Ⅳ required to double plasma APTT are 11.81,20.22,23.34,and 23.59μg/mL,respectively.It can be seen that the activity potency of FS-Ⅱ,FS-Ⅲ and FS-Ⅳ is similar,while that of FS-Ⅰ is relatively strong.Inhibition of iXase activity analysis shows that FS-Ⅰ and FS-Ⅱ have similar and strong iXase inhibitory activities(IC50 are about 64.86 and 75.57 ng/mL,respectively).Because FS-Ⅰ has stronger anticoagulant activity,it suggests that the anticoagulant activity mechanism of FS-Ⅰ may not be limited to the inhibition of iXase;FS-Ⅲ and FS-Ⅳ also have relatively weak iXase inhibitory activity(ICso is about 109.7,161.2 ng/mL,respectively),because FS-Ⅲ and FS-Ⅳ have similar activity intensity with FS-Ⅱ,indicating that its anticoagulant activity mechanism is also not limited to the inhibition of iXase.Conclusion:(1)The body wall of P.mollis contains at least five kinds of FS,this is the first reported sea cucumber with the largest number of FS types so far.Generally speaking,the types of the same kind of polysaccharides contained in animals are limited.For example,glucans are basically glycogen components.P.mollis containing such a variety of FS is extremely rare in the animal kingdom.(2)Among the FS contained in P.mollis,there are linear polysaccharides withα1,3 glycosidic bonds,linear polysaccharides with α1,4 glycosidic bonds,and polysaccharides that consisted of the mainchain with α1,4 glycosidic bonds and the branches(monosaccharides or disaccharides)linking to the mainchain via α1,3 glycosidic bonds.The Fuc in the FS contained in P.mollis has various types of sulfation.There are not only Fuc without sulfate group substitution,but also all types of Fuc substituted with monosulfate groups such as C2,C3 and C4,and the 2,4-,3,4-di-O-sulfate substitution.This demonstrates the structural diversity of the FS from P.mollis.The physiological significance of the diverse FS contained in P.mollis remains to be elucidated.(3)The FS contained in the body wall of P.mollis has certain anticoagulant and iXase inhibitory activities.According to the correlation analysis of iXase inhibitory activity and its anticoagulant activity,the anticoagulant activity mechanism of the FS should not be limited to inhibit the activity of iXase.The structure-activity relationship analysis shows that the strength of its anticoagulant activity is related to its Mw and the degree of sulfation;and the way of glycosidic bond connection and the presence of side chains may be the key factors affecting the strength of its iXase inhibitory activity.The results of this study suggest that the presence of a special form of side chain may help improve the activity of FS to inhibit iXase and the selectivity of its pharmacological effects. |
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