Method for extracting and primarily purifying tetrodotoxin

A tetrodotoxin, a preliminary technology, applied in the field of chromatographic columns, to achieve high recovery, continuous process, and semi-automated effects

Inactive Publication Date: 2015-01-28
WUXI CHROM MATRIX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These characteristics make the purification and purification of tetrodotoxin full of technical challenges

Method used

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  • Method for extracting and primarily purifying tetrodotoxin
  • Method for extracting and primarily purifying tetrodotoxin
  • Method for extracting and primarily purifying tetrodotoxin

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0052] Embodiment 1: the preparation method of the weak cation exchange chromatographic column of bonded glutamic acid and / or polyglutamic acid

[0053] The preparation method of the weak cation exchange chromatographic column of the present invention can use the preparation method disclosed in the field, wherein a kind of preparation method is:

[0054] (1) According to the document J.Amer.Chem.Soc., 80, 3361etssq., 1958, polyglutamic acid with a degree of polymerization of 8 was synthesized;

[0055] (2) Spherical B-type diol silica gel was selected as the chromatographic silica gel skeleton, and the skeleton was treated with sodium periodate to prepare 20 μm aldehyde-based chromatographic silica gel. The parameter of described B type diol silica gel is shown in table 1;

[0056] (3) The polyglutamic acid with a degree of polymerization of 8 is bonded to the above-mentioned aldehyde chromatographic skeleton through Schiff base reaction to prepare the weak cation exchange ch...

Embodiment 2

[0060] Embodiment 2: the method for extracting tetrodotoxin from the visceral tissue of 10g puffer fish

[0061] (1) Homogenization: Accurately weigh 10 g of puffer fish ovary as raw material, use Polytron, and homogenize at high speed;

[0062] (2) Dialysis: Use 1% acetic acid to soak the obtained homogenate, put it into a dialysis bag, stir and dialyze with 4 times 1% acetic acid in a 4°C freezer, replace the new 1% acetic acid dialysate every 2 hours, and repeat the acetic acid dialysis 20 times At this time, LC-MS / MS was used to detect that the concentration of tetrodotoxin in the final acetic acid dialysate was lower than 0.1 μg / L (analysis);

[0063] (3) Reverse C18+weak anion liquid chromatography separation: use C18+WAX mixed bed C18+weak anion exchange double-layer chromatography column to adsorb and remove impurities, and collect the first eluate containing all tetrodotoxin and its analogues;

[0064] (4) Weak cation chromatographic column extraction: pass the first...

Embodiment 3

[0067] Embodiment 3: the method for extracting tetrodotoxin from 1kg puffer fish visceral tissue

[0068] 1kg of wild puffer fish ovary was mixed with dry ice and homogenized using Robot Coupe. After sublimation of dry ice in a freezer at -20°C, part or all of it was soaked in 10 liters of 1% acetic acid, and repeated dialysis and extraction with ceramic membrane equipment required 16 times. The tetrodotoxin acetic acid aqueous solution obtained by dialysis flows through a C18+WAX mixed bed, and then flows through the weak cation exchange chromatographic column of the present invention. Tetrodotoxin and its analogues were retained on the second column and eluted with 500 mL of 2M acetic acid. Then freeze-dry to obtain 1.54 g of tetrodotoxin dry powder with a purity of 87%.

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Abstract

The invention relates to a method for extracting tetrodotoxin from globefish internal organ tissues and then primarily purifying the tetrodotoxin. The globefish internal organ tissues are subjected to homogenate, dialysis, and reversed-phase weak anionic chromatography to remove the impurities, then tetrodotoxin is condensed and purified by a weak cationic exchange technology, and finally the tetrodotoxin is subjected to freeze drying so as to obtain the tetrodotoxin dry powder with a purity of 80%. In the provided method and technology, LC-MS or LC-MS / MS is used to detect the tetrodotoxin concentration in each step so as to guarantee that no tetrodotoxin is lost or discarded. The tetrodotoxin yield of the provided method is 10 to 100 times more than that of conventional methods recorded in literature and current patents. The provided technology especially the continuous fluid and special weak cationic exchange materials can process 10 kilograms of globefish ovary at one time, and is capable of obtaining 10 to 20 grams of tetrodotoxin dry powder with a purity of 80% to 90%.

Description

technical field [0001] The invention relates to the technical field of chromatographic columns, in particular to a method for extracting and preliminarily purifying tetrodotoxin, in particular to a method for extracting and preliminarily purifying tetrodotoxin using a weak cation exchange chromatography column Background technique [0002] Tetrodotoxin (TTX) is an alkaloid contained in puffer fish (commonly known as puffer fish) and other organisms. Its molecular formula is C 11 h 17 o 8 N 3 , with a molecular weight of 319, is an aminoperhydroquinazoline compound, one of the most toxic neurotoxins found in nature, and can block sodium ion channels on nerve excitatory membranes with high selectivity and high affinity . Tetrodotoxin is a small molecular weight, non-protein neurotoxin, its toxicity is more than 1250 times higher than that of highly toxic sodium cyanide, and 0.5mg can be fatal. Tetrodotoxin binds to the SS1 / SS2 subunits of sodium channels with high specif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D491/22
CPCC07D491/22
Inventor 杨育晖
Owner WUXI CHROM MATRIX BIOTECH
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