Structure And Anticoagulant Activity Of Fucan Sulfate From Holothuria Floridana

Fucan sulfate（FS）,one kind of sulfated polysaccharide rich in L-fucose,is widely distributed in echinoderms and algae.FS is an important acidic polysaccharide in the body wall of sea cucumber.The repeated structural units of FS vary in a species-specific manner.In addition,the structure of FS from sea cucumber has a more regular chemical structure than that of fucoidan form algae.FS has a wide range of pharmacological activities,such as anticoagulant,hypolipidemic,antiviral,anti-inflammatory and gastric mucosal protective effects.The molecular weight of natural FS is usually high,and its signals cannot be attributed directly by nuclear magnetic resonance spectroscopy,which makes it difficult to clarify its structure-activity relationship.In the previous work,the chemical structure and pharmacological activity of fucosylated glycosaminoglycan（HfFG）from Holothuria floridana have been studied.In this study,FS in the body wall of Holothuria floridana was studied.HfFS was purified and its physico-chemical properties were studied.Then a series of oligosaccharides were obtained from mild acid hydrolyzed HfFS and their structures were identified.The"bottom up"strategy was used to deduce the exact structure of native HfFS.Furthermore,the anticoagulant activities of HfFS and its low-molecular weight products were preliminarily analyzed,which provides a reference for the study of the chemical structure and biological activity of natural FS.The following are the methods and main results of this study:Methods（1）Isolation and physico-chemical properties of HfFS:The polysaccharide in the dried body wall of H.Florida was extracted by means of enzymatic and alkaline hydrolysis.The proteins were removed by isoelectric point method.After decolorization by H₂O₂,it was further purified by FPA98 strong anion chromatography to obtain purified HfFS.Its molecular weight was determined by HPGPC method.Its monosaccharide composition was analyzed by PMP derivatization after complete acid hydrolysis.The content of sulfate group was detected by ion chromatography.And its basic structural characteristics were analyzed by infrared spectrum analysis,optical rotation measurement and NMR spectrum.（2）Kinetics of mild acid hydrolysis of HfFS:10 m M of trifluoroacetic acid as the acid treatment agent was added to the solution of HfFS at certain temperature.A small reaction mixture was taken out and quenched with aqueous Na OH solution,and collected as one sample every half an hour.After 5 h,the reaction was stopped.The samples were collected and measured by HPGPC to study the hydrolysis kinetics of glycosidic bond.This measurement could be carried out at different temperatures.Meanwhile,the sulfate group contents of a series of derivatives were detected by ion chromatography to explore the effect of mild acid hydrolysis on the sulfate groups shedding on HfFS.（3）Structures of HfFS derivatives and oligosaccharides:HfFS was depolymerized by H₂O₂to obtain the low-molecular-weight derivatives,which were analyzed by NMR to characterize their basic structures.Meanwhile,a series of oligosaccharide products with different degrees of polymerization were obtained by mild acid hydrolysis with trifluoroacetic acid.The chemical structures of oligosaccharides were determined by 1D/2D NMR spectra and ESI-Q-TOF MS.Then,the exact chemical structure of HfFS was analyzed.（4）A series of low-molecular-weight HfFS derivatives were prepared by mild acid hydrolysis.The anticoagulant activities of HfFS and its derivatives were analyzed by plasma coagulation time method,including APTT,PT and TT activities,and their effects on coagulation function and structure-activity relationship were analyzed.Results（1）Extraction and physico-chemical properties analysis of HfFSHfFS with good homogeneity was obtained by extraction and purification.The yield was 0.96%,and the molecular weight of HfFS was 443.4 k Da.The results of monosaccharide composition showed that HfFS composed of fucose and its optical rotation is[α]_D^25℃=-232.68.The results of ion chromatography showed that the content of sulfate group is 30.44%.The results of infrared and¹H NMR spectrum showed that it has the basic structural characteristics of fucan sulfate.（2）Kinetics of mild acid hydrolysisThe hydrolysis kinetics in trifluoroacetic acid mild acid hydrolysis process of HfFS was studied at 50℃and 80℃,respectively.The molecular weight of the depolymerization product was measured and fitted.At 50℃,the reaction rate equation was Mw_50℃=48892.74+409501.31 e^-1.2530x（R²=0.95）,and the reaction rate constant was 0.0039 h^-1.The rate equation at 80℃is Mw_80℃=2183.01+441229.61e^{-8.5848 x}（R²=0.99）,and the reaction rate constant was 0.1548 h^-1.It showed that temperature has an important effect on the reaction rate of partial acid hydrolysis of HfFS.Meanwhile,the content of sulfate group of the derivatives was detected by ion chromatography.The results showed that the content of sulfate group was relatively stable at the reaction temperature of 50℃and 80℃.（3）Structural analysis of low-molecular-weight derivatives and oligosaccharides from HfFSdHfFS,the derivative of HfFS by hydrogen peroxide,has the regular pentasaccharide repeat structure unit:-{L-Fuc_2S4S-α（1,3）-L-Fuc-α（1,3）-L-Fuc-α（1,3）-L-Fuc_2S-α（1,3）-L-Fuc_2S}_n-,with novel chemical structure.Oligosaccharides from HfFS were prepared by mild acid hydrolysis.The final products were reduced with Na BH₄and then were purified by Bio-Gel columns chromatography and SAX-HPLC separation.Six homogeneous oligosaccharides were obtained:Hf-41,Hf-39,Hf-37-a,Hf-37-b,Hf-35 and Hf-34.The chemical structures are as follows:Hf-41:L-Fuc_2S4S-olL-Fuc_2S-α（1,3）-L-Fuc-ol L-Fuc_4S-α（1,3）-L-Fuc-ol L-Fuc_2S4S-α（1,3）-L-Fuc-olHf-39:L-Fuc_2S4S-α（1,3）-L-Fuc-olL-Fuc_2S-α（1,3）-L-Fuc_2S-olHf-37-a:L-Fuc-α（1,3）-L-Fuc_2S-α（1,3）-L-Fuc-olL-Fuc_4S-α（1,3）-L-Fuc-α（1,3）-L-Fuc-α（1,3）-L-Fuc-olHf-37-b:L-Fuc-α（1,3）-L-Fuc_2S4S-α（1,3）-L-Fuc-olHf-35:L-Fuc_2S4S-α（1,3）-L-Fuc-α（1,3）-L-Fuc_2S-α（1,3）-L-Fuc-olHf-34:L-Fuc_2S4S-α（1,3）-L-Fuc-α（1,3）-L-Fuc_2S-α（1,3）-L-Fuc_2S-olThe analysis of oligosaccharide structure showed that its structural sequence is basically consistent with the basic structural characteristics of HfFS.There may be a small amount of 2-position sulfate group hydrolysis in the process of mild acid hydrolysis.Meanwhile,there are also a small number of irregular structure areas in HfFS.HfFS is fucan sulfate with relatively regular pentasaccharide structural repeating units.（4）Anticoagulant activityAnticoagulation experiments showed that HfFS could significantly prolong the time activity of plasma activated partial thromboplastin.HfFS and its low-molecular-weight derivatives showed anticoagulant activity through inhibiting intrinsic coagulation pathway in a chain length dependent manner.With the decrease of polymerization degree,its anticoagulant activity gradually weakened.When the molecular weight was less than 11.5k Da,it showed no obvious anticoagulant activity.Meanwhile,HfFS and its derivatives did not significantly affect the anticoagulant activity of extrinsic and common coagulation pathways.Conclusion:In this study,a fucan sulfate（HfFS）was isolated from H.floridana by enzymatic alkaline hydrolysis and ion exchange chromatography.Its molecular weight was 443.4 k Da.The content of sulfate group was 30.44%.The"bottom up"strategy was used to analyze the structure of HfFS.The low-molecular-weight derivatives of HfFS were obtained by peroxidative depolymerization.A series of fucooligosaccharides were obtained by mild acid hydrolysis,and the structures were analyzed by 1D/2D NMR.Results indicated that HfFS was mainly composed of regular repeating pentasaccharide units:-{L-Fuc_2S4S-α（1,3）-L-Fuc-α（1,3）-L-Fuc-α（1,3）-L-Fuc_2S-α（1-3）-L-Fuc_2S}_n-.Anticoagulant activity experiments showed that HfFS A could significantly prolong the APTT,and the potencies decreased with the reduction in molecular weights.When the molecular weight was less than 11.5 kDa,it showed no significant anticoagulant activity.