Studies On Molecular Species Analysis And Bioactivities Of Sphingolipids From Sea Cucumbers

Sea cucumber belongs to Echinodermata Holothuroidea. Due to its high edibleand medicinal value，sea cucumber is listed as the first of the "eight treasures" for along time. Cerebroside and ceramide are main sphingolipids in sea cucumber, havevariety of biological activity, such as anti-tumor, regulating cell growth, etc. But thecerebroside and ceramide from sea cucumbers have complex structures and difficultto identify the structure and analysis the molecular composition. So, a rapid andeffective identification method has been highly needed at present.In this paper, sphingolipids which extracted from a variety of sea cucumbershave carried on the qualitative and quantitative determination. Though highlyeffective liquid phase method, three kinds of long chain bases are prepared. A studyon anti-HepG2cell activity of three cerebrosides, ceramide and long-chainbase(d17:1,d18:2) from Cucumaria frondosa are carried out. The main contents in thisthesis are concluded as follows:1. By employing HPLC-ELSD method, uses TSKgel CN-80Ts as column,n-hexane-isopropanol-dichloromethane-methanol as mobile phase, achieve thequantitation of cerebroside and ceramide from20kinds sea cucumber simultaneously.Detection Limit of cerebroside and ceramide are0.01μg and0.001μg, respectively.Among the sixteen kinds of sea cucumbers, contain various cerebrosides andceramides. The contents ranges of cerebroside and ceramide is respectively from0mg/g to4.44mg/g and from0.24mg/g to2.21mg/g.In addition, we use LC-ESI-QQQ to analysis cerebrosides in Pearsonothuriagraeffei、Parastichopus japonicus、Bohadschia argus、Cucumaria frondosa、Acaudinamolpadioides、Parastichopus Californicus、Apostichopus japonicus, seven differentkinds of sea cucumber along with Apostichopus japonicus from three differentproducing area. Meanwhile we analysis ceramides in Cucumaria frondosa、 Apostichopus japonicus and Acaudina molpadioides by using LC-ESI-QQQ. Resultsshowed that there is a dramatic difference of cerebroside series among seven kinds ofsea cucumbers. Cerebrosides in Parastichopus Californicus is the most complicated,which possess58different kinds of cerebrosides. We reckoned d17:1as thecharacteristic structure of sea cucumber by analysis the constitution of cerebrosideseries，d17:1-C24:1h is the characteristic structure of cerebroside. Cluster analysis iscarried out on the sea cucumber cerebroside, results show that the molecular speciesclassification of cerebroside was significantly associated with sea cucumber varieties.Cerebrosides in Apostichopus japonicus from three different production areamanifested that the series of cerebrosides are based on the production area. Theceramide series of Cucumaria frondosa、Apostichopus japonicus and Acaudinamolpadioides showed that ceramides and cerebrosides share a similar constitution, butdiffer in ratio.By analyzing and quantifying cerebrosides and ceramides in sea cucumber, wepartly figure out the distinctive difference of cerebrosides have connections withproduction area and types of sea cucumber. And provide groundwork for furtherconstruction of the fingerprint of cerebrosides. Moreover, this research provide dataand foundation for the development of the sea cucumber, nutritional evaluation andquality control.2. By using acidolysis to get LCB from Cucumaria frondosa total lipids, andcolumn chromatography to get the purified LCB, the purity of total LCBs is94.7%.Using HPLC, TSKgel ODS-4as the separation column, methanol-PBS as the mobilephase, separated d17:1, d18:2and18:3, three LCB monomers, which providereference for the structure-function relationship of sea cucumber sphingolipids.3. Studied proliferation activity of ceramides, cerebrosides and LCBs inCucumaria frondosa towards HepG2cell. MTT method to detect the influence of thetest materials to HepG2proliferation during24h and48h. Results showed that amongsix different sphingolipids, only d17:1and d18:2have significant anti-proliferationactivity, and had dose depend relationship. IC50of d17:1and d18:2toward HepG2 were5.22、5.24μg/mL in24h,4.96、5.32μg/mL in48h. Cerebrosides and ceramidesshowed almost no inhibitive efficacy. Results showed that sea cucumber LCBs are notonly the skeleton of sphingolipids，but also the anti-tumor components. Theseresearches provided reference for the development of marine drug and functionalfood.