Isolation And Identification Of Monosialogangliosides In Sea Urchins And Sea Cucumbers

Sea urchins and sea cucumbers are species which belong to echinodermata. A variety ofbiologically active substances have been isolated from the echinoderm species, such as: saponins,glycolipids, carotenoids, naphthoquinones. Gangliosides of glycosphingolipids containing sialicacid, are one of the most important bioactive compositions of echinodermata. Gangliosides havebeen proved to be involved in numerous biological processes and play essential roles in severepathologies, with predilection in those of the central nervous system. It is known that thegangliosides present in sea urchins and sea cucumbers possess unique structures and thereforethey are expected to be promising medicinal resources.In this paper, two types of monosialogangliosides of Hemicentrotus pulcherrimus, nonsulfomonosialoganglioside (NMG) and sulfo monosialoganglioside (SMG), are isolated andidentified. The fragmentation pattern of these two monosialogangliosides in ESI-MS areinvestigated. Then the structures of gangliosides from Anthocidaris crassispina, Glyptocidariscrenularis and Apostichopus japonicus are identified using ESI-MS and the molecular species ofthese ganglisodes are conducted with an RPLC-MS/MS equipment. In the final part, a method todetermine the content of NMG and SMG in echinoderms is established based on HPLC-MStechnology. With this method, the content of the gangliosides in10different sea cucumbers and3sea urchins is assayed. The study is conducted as follow:1. Two ganglisides, HPG-A and HPG-B, are isolated from the germen ofHemicentrotus pulcherrimus using silica gel column chromatography and DEAE-Sephadex A25chromatography. Three monomers designated as HPG-A1, HPG-A2and HPG-A3are separatedfrom HPG-A and one monomer designated as HPG-B1is acquired from HPG-B withsemi-preparative HPLC. HPG-A is identified to be a mixture of Neu5Gcα2-6Glcβ1-1Cer and asmall amount of Neu5Acα2-6Glcβ1-1Cer by GC-MS, NMR and ESI-MS technology. Hp-B isidentified to be8-SO4-Neu5Gcα2-6Glcβ1-1Cer. The main fatty acids in the two above are C22:1 and C22:1h, and the long-chain bases are t16:0and t18:0. The four monomers were identified tobe Neu5Gcα2-6Glcβ1-1-t16:0-C22:1(Mass1078.7), Neu5Gcα2-6Glcβ1-1-t18:0-C22:1(Mass1106.7), Neu5Gcα2-6Glcβ1-1-t18:0-C22:1h (Mass1122.7) and8-SO4-Neu5Gcα2-6Glcβ1-1-t18:0-C22:1h (Mass1202.7). The fragmentation pattern of the twomonosialogangliosides is investigated in negative ion mode by ESI-MS/MS. Glycosidic bond ofsialic acid in NMG is easily undergo cleavage in CID-MS/MS, and fragment of sialic acid,m/z=306.1, will be produced as well as two ring cleavage fragments0,4A2-CO2and0,2X1-CO2,which is verdicted as the evidence of α2-6linked sialic acid. HPG-B1produces no ring cleavagefragment, but the sialic acid fragment produced by glycosidic bond will continue to undergocleavage. Therefore the attachment position of sulfuric acid ester can be identified.2. Gangliosides in Anthocidaris crassispina, Glyptocidaris crenularis and Apostichopusjaponicus are isolated and the structures are verified by using ESI-MS equipment. Themolecular species of monoganglioside without sulfate group is assayed using HPLC coupledwith tandom mass spectrometer. NMGs and SMGs were identified to exist in both sea urchinsamples while only NMG was identified in sea cucumber. A new type ganglioside molecularwith structure KDNα2-6Glc1-1Cer was identified in sea urchin Glyptocidaris crenularis.Molecular species of NMGs in each sample were determinate through HPLC coupled with MSspectrometer. It can be deduced the major long chain bases are mainly t16:0and t18:0in seaurchin NMGs and d18:1in sea cucumber NMGs. There were no obvious difference in fattyacid compositions.3. The sulfo and nonsulfo monosialogangliosides in ten different sea cucumbers and threesea urchins were extracted according to a Svennerholm method. The variation of compositionand content of sulfo-and nonsulfo-monosialogangliosides in the samples is compared by MRMmode of HPLC-MS. The molecular composition of sulfo and nonsulfo monosialogangliosides isassayed by ESI-MS Scan and then the content of the two gangliosides in these samples isdetermined via detecting sialic acid ion in MRM mode. Among sea urchins, Glyptocidariscrenularis containing the highest amount of NMG（382.4μg/100mg）while Anthocidaris crassispina having the highest amount of SMG(437.2μg/100mg). There was only NMG detectedin sea cucumbers and Parastichopus Califormicus having the highest content(13.1μg/100mg).