Structural Analysis Of Several Fucoidan From Sea Cucumber And Their Structure-Activity Relationship Study

Sea cucumber fucoidan (SC-FUC) is a kind of marine polysaccharide, extractedfrom the body wall of sea cucumber. Various bioactivities of SC-FUC were found andit shows a promising future in functional food industry. A prerequisite for developingnew functional food is structure-activity relationship study of bioactive compounds.However, due to the scarce structural analysis of SC-FUC, the structure-activityrelationship study and industrial application of SC-FUC were impeded. In order tosolve this problem, structures of fucoidan from three species of sea cucumber wereelucidated. Meanwhile, combined with bioactive study of these SC-FUCs, thestructure-activity relationship study of SC-FUC was investigated.Fucoidanase is an important tool for structural analysis of fucoidan, but there isno commercial fucoidanase available. The researchers in our lab have isolated andpurified a kind of fucoidanase solution named CZ1127FUCase from marine bacteriaFlavobacteriaceae sp.CZ1127. Based on these previous researches, the enzymaticmode was further studied by using fucoidan from Isostichopus badionotus (Ib-FUC)with determined structure as substrate. The results indicated that the fucoidanase wasan endo-acting enzyme. By elucidating structures of oligosaccharides in enzymaticproducts, it was confirmed that the fucoidanase possessed a novel linkage specificityto3-α-L-Fucp2(OSO3-)1â†'3-α-L-Fucp-1. Thus, CZ1127FUCase could be used astools for structural analysis of other SC-FUC with unknown structures.A kind of fucoidan was extracted from the body wall of sea cucumber Thelenotaananas, with the molecular weight1284KDa and sulfated content of28.2±3.5%. Itsmonosaccharide composition is only fucose. The fucoidan from sea cucumber T.ananas (Ta-FUC) was hydrolyzed by CZ1127FUCase to obtain oligosaccharides andlow molecular weight fucoidan. Through ESI-CID-MS/MS and NMR analysis, thestructure of these enzymatic products were elucidated, unified and proved to contain atetrafucose repeating unit: α-L-Fucp-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†' 3-α-L-Fucp2(OSO3-). For that reason, the structure of Ta-FUC was hypothesized asfollowing structure:[â†'3-α-L-Fucp-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp2(OSO3-)-1â†']n,which is a novel structure.The efficiency of preparing oligosaccharides could be very low if only CZ1127FUCase was used to hydrolyze fucoidan from sea cucumber Acaudina molpadioides(Am-FUC). Thus, the crude enzyme solution which contains fucoidanase, sulfataseand α-L-fucosidase was utilized to depolymerize Am-FUC for oligosaccharides. Theenzymatic products were analyzed by LC-MS for oligosaccharides composition andtandem SuperdexTMPeptide was used for target purification of the mainoligosaccharides in enzymatic products. Through ESI-CID-MS/MS and NMRanalysis, the structures of four oligosaccharides was elucidated as following：α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp,α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp,α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp andα-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp. CZ1127FUCase wasused to hydrolyze Am-FUC to obtain the low molecular weight fucoidan. Its structurewas investigated by NMR and indicated as [â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2(OSO3-)â†']n. Finally, based on all the informationabove, the structure of Am-FUC was deduced as [â†'3-α-L-Fucp-1â†'3-α-L-Fucp2,4(OSO3-)-1â†'3-α-L-Fucp-1â†'3-α-L-Fucp2(OSO3-)-1â†']n,which is also a novelstructure.Through infra-red spectrum, NMR and methylation analysis, the structure offucoidan from sea cucumber Apostichopus japonicus (Aj-FUC) was elucidated. Theresults showed that it mainly consisted of α-L-Fucp2(OSO3-) and α-L-Fucp withα1â†'3and α1â†'4linkage. The branch may occur at C-4of fucose residue. CZ1127FUCase was used to hydrolyze Aj-FUC to obtain the low molecular weight fucoidan.Its structure was deduced as [â†'3-α-L-Fucp2(OSO3-)-1â†'3(α-L-Fucp-1â†'4-α-L-Fucp-1â†'4)-α-L-Fucp2(OSO3-)-1â†'3-α-L-Fucp2(OSO3-)â†']nby NMR analysis. The structure of Aj-FUC might be complex and heterogeneous. The structure of lowmolecular weight fucoidan occupied80%of overall structure of Aj-FUC. Therefore，the repeating units in the structure of low molecular weight fucoidan could representthe main structural characteristics of Aj-FUC.Moreover, the structure-activity relationship study about antioxidant andantitumor of Ta-FUC, Ib-FUC, Am-FUC and Aj-FUC were conducted. The resultsindicated that the antioxidant and antitumor of SC-FUC is not only related to sulfatecontent but also to the delicate structure of SC-FUC such as the distribution of sulfateesters and branch.To sum up, in this paper, we used the fucoidanase as a unique tool to prepareoligosaccharides of SC-FUC for the first time. Through ESI-CID-MS/MS and NMR,the analytic method of delicate structure of SC-FUC was established. Furthermore,three novel structures of SC-FUC were discovered. Finally, the structure-activityrelationship study of SC-FUC was investigated and the results indicated that thebioactivities of SC-FUC have a close relationship with their delicate structure. Thisresearch laid a solid foundation on further inverstigtion and development of SC-FUC...