Method for efficiently extracting and separating high-purity tetrodotoxin from pufferfish viscera

A method for efficiently extracting, separating and preparing tetrodotoxin from pufferfish viscera, comprising steps of:

• (1) cutting fresh or thawed pufferfish visceral tissues into small pieces, adding acidic alcohol, and homogenizing a resultant in a homogenizer, wherein the acidic alcohol is a mixture of acetic acid and ethanol;
• (2) adding a flow aid or a filter aid into a homogenate obtained in the (1), stirring and mixing a resultant uniformly, and then loading the resultant into a chromatographic column according to a column packing method of column chromatography;
• after standing, completely eluting tetrodotoxin (TTX) in materials by a column chromatography eluting method using the same acidic alcohol, measuring a TTX content in the eluent fractions by HPLC until no TTX exists in the eluent, and collecting the TTX-containing eluent fractions as an extract solution;
• (3) concentrating the TTX extract solution in the (2) to a water phase under reduced pressure in a vacuum concentrator, recovering ethanol for repeated use, and performing extraction on the water phase with an organic solvent to remove fat-soluble substances, wherein a water phase obtained is a TTX extract concentrated solution;
• (4) separating the TTX extract concentrated solution obtained in the (3) by pre-treated ion exchange resin column chromatography, after sample loading, first eluting impurities with neutral ammonium salt, pure water and low acidic solution successively, then eluting TTX with a higher acidic aqueous solution, collecting eluent phases having high TTX contents, and concentrating the TTX eluent phases by nanofiltration to dryness, to obtain a TTX crude product, wherein the nanofiltration comprises concentrating the TTX eluent separated by a cation exchange resin column chromatograph by a nanofiltration membrane, and the nanofiltration concentration membrane is a nanofiltration member with molecular weight cut-off of 100-200 Dalton;
• and
• (5) subjecting the TTX crude product to repeated acid-dissolution and alkali-precipitation or crystallisation multiple times in an aqueous solution or organic solvent, to obtain high-purity TTX, wherein a purity of the TTX is 98% or higher upon HPLC analysis.