Studies On Extraction And Purification Technology Of Tetrodotoxin

Tetrodotoxin (TTX) is a marine biotoxin with very strong toxicity and the micro-agent has good local anesthesia and analgesia effect. TTX is also one kind of rare marine pharmaceuticals resources, which is widespread in the puffer fish. Puffer fish resources are abundant in the South China Sea, and their toxicity is very complicated. So far, besides few low toxic parts of specific species are processed for export, many high toxic globefish species and body parts are discarded in the producing process, which has resulted in the new waste of resource and pollution of exterior circumstance.In this paper, toxicity distributing situation of common puffer fish in the South China Sea was investigated. Meanwhile, ultrasonic-assisted extraction and column chromatography purification technology of TTX were studied systematically, in order to find an effective, cheap and easily operational method for TTX production. The research results were as follows:1. Species identification was identified by Morphological character and toxicity distributing situation was investigated using biological test in mice and enzyme linked immunosorbant assay (ELISA) to132puffer fish collected along the coast of Fujian and Guangdong Province. The results showed that these fishes could be divided into12species, and Gastrophysus and Takifugu were the main species. Gastrophysus was the dominant species accounted for75.8%of the total sample size. The TTX content in different species and different parts of globefish body was significantly different. Gonad and liver were rich in TTX, but most skin and muscle were non-toxic or low toxic. Gastrophysus lunaris was considered as the best raw materials to extract TTX, due to the whole fish body was toxic and even the TTX content in the gonad could be up to251.4MU.2. The extraction conditions of TTX using traditional water bath method and ultrasound-assisted method were optimized by L9(34) orthogonal test and response surface methodology (RSM) respectively, and then the obtained optimum conditions were compared. The results were showed as follows:the optimum extraction conditions of traditional water bath method were:0.1%acetic acid as the solvent, liquid-solid ratio4:1, water bath temperature80℃, and extraction times2(10min per time). Under such conditions, TTX extract amount from the gonad of Gastrophysus lunaris was50.23μg/g. The optimum ultrasound-assisted extraction conditions were liquid-solid ratio3:1, ultrasonic frequency40kHz, ultrasonic power40W, ultrasonic temperature40℃and ultrasonic time1(22min per time). Under such conditions, the TTX extract amount was63.57ug/g, which increased by26.6%than the traditional water bath method. 3. The TTX samples extracted by ultrasound-assisted method as the raw materials were purified by D152ion exchange resin, granular activated carbon and Bio-Gel P2column chromatography in turn. The results were showed as flows:the optimum technological conditions of D152resin column chromatography were:crude TTX extracts of pH5.0, sample flow rate of6mL/min, acetic acid eluant concentration of2%, elution flow rate of1.8BV/h and eluant dosage of7BV. The recovery rate and purity with this method were98.4%and0.33%respectively. The optimum conditions of granular activated carbon column chromatography were: resin-purified TTX sample of pH7.0, sample volume of50mL, adsorption velocity of2mL/min, acetic acid gradient elution concentration of1%～5%, elution flow rate of6.0BV/h and eluant dosage of8BV. The recovery rate and purity with this method were92.5%and4.93%respectively. The technological conditions of the gel column chromatography were:activated carbon-purified TTX sample of pH7.0, sample volume of4.0mL, sample concentration of450μg/mL, elution flow rate of6.0BV/h, elution with1BV pure water and2BV1%acetic acid in turn. The recovery rate and purity with this method were89.4%and46.6%respectively.After three-step column chromatography purification, the total recovery rate of the TTX was81.4%and the purity of the TTX sample increased from0.01%to46.6%. The removal rate of protein impurities and total sugar were99.7%and99.8%respectively. The fat and the pigment hade been removed completely.