The Studies Of Toxin Production Characteristic Of Protoceratium Reticulatum And Extraction And Purification Of Yessotoxin

Yessotoxins(YTXs) is a kind of polycyclic polyether marine biological toxins. Beside Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum was confirmed as the source of YTXs. YTXs have been detected in many countries, which was considered that has potential harm for human and ecosystem. it don’t cause diarrhea as classical diarrhetic shellfish toxins, but injure some tissue and organs, such as liver, heart, and nervous system. In 2007, YTX and analogue were found in China firstly. YTX was gradually known in our country, and caused take seriously, the demand for high purity YTX becomes more and more big. Because of the high purity YTX expensive, limits its application in various fields in China. Therefore,now it is very necessary to developed a method of purification YTX in our country. In this paper, mass culture of P. reticulatum in laboratory, using liquid chromatography- mass spectrometry(LC- MS/MS) detect toxin-producing conditions for qualitative quantitative of the growth curve of various periods each swarmer. And the extraction condition of YTX was optimized. And through the low pressure column chromatography purification YTX, in order to get high purity YTX samples.By LC-MS/MS toxin analysis, the strains cell can produce YTX and Homo-YTX, the toxin which YTX as main components, and in a growth cycle it can produce YTX 3.9~71 pg cell-1,at late stable growth of toxin-producing is highest.Based on the the single factor experiment, It was researehed that optimization and comparion of concentration of organic solvent and conditions, three independent variables such as ratio of solid to liquid, ultrasonic extraction time(min) and the extraction times were investigated. The results showed that extracting conditions were optimum when ratio of material to solution was 1:120; ultrasonic extraction time was 10 min; extraction times was twice,under the condition, the extraction rate was 97.7%.In order to get a further pure sample of YTX, Crude YTX extraets were purified by Low-Pressure colunin chromatography(LPCC) with three kinds of properties of alumina, C18 and RP-18 as carrier. With YTX peak time, recovery and variation coefficient as evaluation indicators, compared with the results of three kinds of properties of alumina, basic alumina was the best that purified YTX. The testing result shows it has the character of quick peak flowing out, the highest recycling rate and the least variation coefficient of all kinds of alumina. C18 and RP-18 have the character of quick peak flowing out, recovery(%) was 88.4±4.0 and 28.7±3.0, variation coefficient was 5% and 10%, respectively.In conclusion, the purity of the sample, which was after LPCC is 99% by LC-MS/MS analysis. The present study was first focus on separation and purification of YTX in China. The analytical investigation which supply the theory foundation for the extraction procedure of YTX.