Method for preparing high-purity dehydrated tetrodotoxin monomer through silica gel column chromatography separation

Abstract

The invention relates to a method for preparing a high-purity 4, 9-dehydrated tetrodotoxin monomer through separation. According to the method, an extracted and prepared tetrodotoxin crude product is used as a raw material, silica gel column chromatography separation is performed twice, and elution and separation are performed by using acidic alcohol aqueous solutions with different concentrations to prepare the high-purity 4, 9-dehydrated tetrodotoxin. Through high performance liquid chromatography detection, the purity of the prepared 4, 9-dehydrated tetrodotoxin reaches 98% or above. The method for preparing the high-purity 4, 9-dehydrated tetrodotoxin monomer through large-scale separation is easy and convenient to operate, high in yield and low in cost, and a foundation is provided for research of 4, 9-dehydrated tetrodotoxin and development of application products.

Description

Technical Field

[0001] The present technology relates to a natural heterocyclic compound and a method for separating, purifying and preparing a high-purity dehydrated tetrodotoxin monomer. Background Art

[0002] 4,9-anhydrotetrodotoxin is an amino-perhydroquinazoline-type compound with a structure extremely similar to that of tetrodotoxin. It is a very important derivative of tetrodotoxin, coexists with tetrodotoxin in nature, and there is a certain mutual conversion relationship between the two. It mainly exists in the ovaries, livers, skins and intestines of pufferfish. Like tetrodotoxin, 4,9-anhydrotetrodotoxin can block the Na + channels of nerve cell membranes, but it specifically acts on different sodium ion channel subtypes, and thus has different biological and medicinal functions. At the same time, the biological activity and toxicity of 4,9-anhydrotetrodotoxin are much lower than those of tetrodotoxin, and it has better biological safety.

[0003] The content of 4,9-anhydrotetrodotoxin in organisms is extremely low, and there are isomers in organisms that are very similar to 4,9-anhydrotetrodotoxin in molecular structure, molecular weight and physical and chemical properties. This has caused great difficulties in obtaining high-purity and mass-producing 4,9-anhydrotetrodotoxin. According to literature reports, in 1984, Munetomo Nakamura et al. used 39 liters of 0.1% acetic acid to extract 1.5 kg of pufferfish liver, and through a series of chromatographic column separations and purifications, and through steps such as concentration, precipitation and drying, 13.4 mg of 4,9-anhydrotetrodotoxin was obtained. In 1987, Takeshi Yasumoto et al. also obtained 20 mg of 4,9-anhydrotetrodotoxin from 3.5 kg of newts by the same method. The above-mentioned separation and preparation processes are not only very complex, but also have very low yields.

[0004] According to the invention specification (publication number CN1058717C), Huang Zhiqiang et al. used the purified tetrodotoxin mother liquor, separated it by high-pressure liquid chromatography, and then desalted it through ion exchange resin and biogel to obtain dehydrated tetrodotoxin crystals. By this method, 60 mg of 4,9-anhydrotetrodotoxin with a purity of more than 97% was obtained from 2 g of tetrodotoxin crude product.

[0005] According to the invention specification (publication number CN1361107A), Lu Yingzhu et al. separated 4,9-anhydrotetrodotoxin by high-performance liquid chromatography, then precipitated the sample by adjusting the pH of the concentrated solution, and finally dried it to obtain 4,9-anhydrotetrodotoxin. By this method, 65 mg of 4,9-anhydrotetrodotoxin with a purity of more than 97% can be obtained from 1 g of crude product.

[0006] According to the invention specification (publication number CN101391999A), Yi Ruizao et al. used crude tetrodotoxin as the raw material, and used a preparative high performance liquid chromatography-mass spectrometry instrument system to purify 4,9-anhydrotetrodotoxin in the raw material, and then obtained 4,9-anhydrotetrodotoxin crystals through vacuum concentration and low-temperature crystallization. Summary of the Invention

[0007] In view of the disadvantages of using high-value instrument equipment such as high performance liquid chromatography and low separation and purification yield in the current process for preparing high-purity 4,9-anhydrotetrodotoxin, the method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography of the present invention provides a new method for preparing high-purity 4,9-anhydrotetrodotoxin with low cost, high yield and suitable for large-scale production.

[0008] The technical solution of the present invention is to obtain high-purity 4,9-anhydrotetrodotoxin by purifying crude tetrodotoxin through silica gel column chromatography.

[0009] The present invention includes the following steps: Using crude tetrodotoxin extracted from puffer fish viscera or the waste liquid recovery after separating and preparing tetrodotoxin as the raw material, dissolving it with high-concentration acidic fatty alcohol, and loading it onto a silica gel chromatography column; For the silica gel chromatography column loaded as described in 1) above, elute it with acidic alcohol of different concentrations in a gradient manner and control the flow rate. Collect the eluent in portions with each column volume as one portion, and detect the content and purity of 4,9-anhydrotetrodotoxin in each portion of the eluent by HPLC.

[0010] 3) Combine the elution phases with high content of 4,9-anhydrotetrodotoxin separated in 2) above, and concentrate and evaporate to dryness.

[0011] 4) Perform second silica gel column chromatography separation and purification on the dried product obtained in 3) above in the same method as in 1) and 2) above. Analyze and detect each phase of the separation liquid by HPLC, and collect and combine the elution phases with the content of 4,9-anhydrotetrodotoxin greater than 98%.

[0012] 5) If the purity of the 4,9-anhydrotetrodotoxin obtained in 4) above does not reach more than 98%, it can be separated and purified again by silica gel column chromatography according to the methods in 1), 2) and 3) above.

[0013] 6) Vacuum-concentrate the 4,9-anhydrotetrodotoxin eluent with a content greater than 98% obtained in 4) or 5) above, and perform acid dissolution and alkali precipitation twice to obtain 4,9-anhydrotetrodotoxin powder with a content greater than 98%.

[0014] In step 1), the content of 4,9-anhydrotetrodotoxin in the crude tetrodotoxin used or the waste liquid recovery after separating and preparing tetrodotoxin is about 30%. The sample is dissolved and loaded onto the column with a solution of lower fatty alcohols such as 98% methanol or ethanol containing 1% weak acid (such as phosphoric acid or acetic acid). The ratio of the sample loading amount to the column volume during the first silica gel column separation is 0.1 - 1.0 mg / cm 3 (the optimal is 0.3mg / cm 3 ).

[0015] In step 2), the eluent used is a solution of lower fatty alcohols (such as methanol or ethanol) with different concentrations (99% - 95%) containing 1% weak acid (such as phosphoric acid or acetic acid). During the elution process, the flow rate is controlled at 0.5 - 1.5 BV / h. The eluent is collected in portions of each column volume, and the content and purity of 4,9-anhydrotetrodotoxin in each portion of the eluent are detected by HPLC.

[0016] In step 3), the elution phases with high 4,9-anhydrotetrodotoxin content are combined and evaporated to dryness under vacuum. The temperature for vacuum concentration is 50 - 60°C.

[0017] In step 4), the dry matter obtained in the above step 3) is purified by second silica gel column chromatography in the same method as in steps 1) and 2). The purity of 4,9-anhydrotetrodotoxin in each portion of the eluent is detected by HPLC, and the elution phases with 4,9-anhydrotetrodotoxin content greater than 98% are collected and combined.

[0018] In step 5), if the purity of the 4,9-anhydrotetrodotoxin obtained in step 4) does not reach more than 98%, it can be purified by third silica gel column chromatography.

[0019] In step 6), the eluent with 4,9-anhydrotetrodotoxin purity greater than 98% obtained is evaporated to dryness under vacuum. The dried product is purified by acid dissolution and alkali precipitation. That is, the sample is dissolved with an aqueous solution of about 1% weak acid (such as acetic acid or phosphoric acid), and then the solution is adjusted to alkaline (about pH 9.0) with a weak base (such as ammonia water), and left to stand for precipitation. The precipitate is collected by centrifugation. The acid dissolution and alkali precipitation are repeated once. The precipitate is washed with a small amount of organic solvents such as acetone or absolute ethanol and dried under vacuum to obtain white crystals of 4,9-anhydrotetrodotoxin with a purity greater than 98%. Description of the Drawings

[0020] Figure 1 Sample before silica gel column chromatography separation Figure 2 Sample after the first silica gel column chromatography separation Figure 3 Sample after the second silica gel column chromatography separation Specific Implementation Method

[0021] 1.1) Raw materials: crude tetrodotoxin or the waste liquid recovery after separating and preparing tetrodotoxin. The content of 4,9-anhydrotetrodotoxin in the used raw materials is 32.121% (see attachment Figure 1 ).

[0022] 1.2) Chromatographic column for the first separation: a silica gel column chromatography with a height of 84 cm, a diameter of 6 cm, and a mesh size of 60 - 100; 1.3) Sample dissolution and loading: Take 800 mg of the crude product of the raw materials described in 1.1), dissolve it with a 98% methanol solution containing 1% acetic acid, and load it onto the silica gel chromatography column described in 1.2).

[0023] 1.4) Gradient solvent elution: Elute with a gradient methanol solution containing 1% acetic acid from 99% to 95%. First, elute with 99% methanol for 2 column volumes, then change to 97% methanol to elute for 4 column volumes, and finally elute with 95% methanol for 7 column volumes. Analyze each elution phase by HPLC.

[0024] 1.5) After the first column chromatography, the elution phase with a 4,9-anhydrotetrodotoxin content greater than 98% is extremely small. Therefore, select the elution phases with a 4,9-anhydrotetrodotoxin content greater than 60% to collect and combine, and perform a second silica gel column chromatography separation.

[0025] 1.6) Vacuum concentrate and dry the collected phase with a 4,9-anhydrotetrodotoxin content greater than 60% described in 1.5) at 50 - 60 °C to obtain a 169 mg sample with a 4,9-anhydrotetrodotoxin content of 73.470% (see attachment Figure 2 ).

[0026] 2.1) Raw materials: 169 mg of 4,9-anhydrotetrodotoxin with a purity of 73.470% after one pass through the column; 2.2) Chromatographic column for the second separation: a silica gel chromatography column with a height of 92 cm, a diameter of 4.5 cm, and a mesh size of 60 - 100; 2.3) Sample dissolution and loading: Dissolve the sample with a 4,9-anhydrotetrodotoxin content of 73.470% obtained from the first column chromatography described in 2.1) with a 98% methanol solution containing 1% acetic acid, and load it onto the silica gel chromatography column described in 2.2).

[0027] 2.4) Solvent gradient elution: Elute with a gradient methanol solvent containing 1% acetic acid from 99% to 95%. First, elute with 99% methanol for 9 column volumes, then change to 95% methanol to elute for 4 column volumes. Analyze the 4,9-anhydrotetrodotoxin in each elution phase by HPLC. Combine the elution phases with a 4,9-anhydrotetrodotoxin content greater than 98%.

[0028] 2.5) Vacuum concentration and drying of 4,9-anhydrotetrodotoxin: The eluate with a 4,9-anhydrotetrodotoxin content greater than 98% described in 2.4) is vacuum concentrated and dried at 50 - 60 °C.

[0029] 2.6) After dissolving the vacuum concentration and drying product obtained in the above step 2.5) with 1% aqueous acetic acid solution, the solution is adjusted to alkaline (pH 9.0) with pure ammonia water, precipitated for more than two hours, and centrifuged. The precipitate is acid-dissolved and alkali-precipitated again in the same way. The precipitate is washed with a small amount of acetone or absolute ethanol and then vacuum dried to obtain 34.6 mg of white crystals. After sampling and HPLC detection, the content of 4,9-anhydrotetrodotoxin is 98.654% (see attachment Figure 3 )

Claims

1. A method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography, characterized in that it comprises the following steps: 1) Using the crude tetrodotoxin extracted from pufferfish viscera or the waste liquid recovery after separating and preparing tetrodotoxin as raw materials, dissolving it with high-concentration acidic alcohol, and loading it onto a silica gel chromatography column; 2) For the silica gel chromatography column loaded as described in 1) above, eluting it with acidic alcohol of different concentrations in a gradient manner and controlling the flow rate. Collect the eluent in portions with each column volume as one portion, and detect the content and purity of 4,9-dehydrated tetrodotoxin in each portion of the eluent by HPLC; 3) Combining the elution phases with high 4,9-dehydrated tetrodotoxin content separated in 2) above, and concentrating and evaporating it to dryness under vacuum; 4) Subjecting the dried product obtained in 3) above to a second silica gel column chromatography purification in the same manner as in 1) and 2) above. Analyzing and detecting each phase of the separation liquid by HPLC, and collecting and combining the elution phases with a 4,9-dehydrated tetrodotoxin content greater than 98%; 5) If the purity of the 4,9-dehydrated tetrodotoxin obtained in 4) above does not reach more than 98%, it can be purified again by silica gel column chromatography according to the methods in 1), 2) and 3) above; 6) Concentrating the 4,9-dehydrated tetrodotoxin eluent with a content greater than 98% obtained in 4) or 5) above to dryness under vacuum, and performing acid dissolution and alkali precipitation twice to obtain 4,9-dehydrated tetrodotoxin powder with a content greater than 98%.

2. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that in step 1), the content of dehydrated tetrodotoxin in the crude tetrodotoxin or the waste liquid recovery after separating and preparing tetrodotoxin used is about 30%.

3. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that in step 1), the high-concentration acidic alcohol is a solution of lower aliphatic alcohol such as 98% methanol or ethanol containing 1% weak acid (such as phosphoric acid or acetic acid, etc.).

4. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that In step 1), the ratio of the sample loading amount to the column volume during the first silica gel column separation is 0.1 - 1.0 mg / cm 3 (optimally 0.3 mg / cm 3 ).

5. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that in step 2), the eluent used in silica gel column chromatography is gradient elution with 99% - 95% lower aliphatic alcohol (such as methanol or ethanol, etc.) containing 1% weak acid (such as phosphoric acid or acetic acid, etc.).

6. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that in step 2), the flow rate during silica gel column chromatography elution should be controlled at 0.5 - 1.5 BV / h.

7. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that in step 3), the temperature used during vacuum concentration is 50 - 60 °C.

8. The method for separating and preparing high-purity dehydrated tetrodotoxin monomer by silica gel column chromatography according to claim 1, characterized in that In the acid dissolution and alkali precipitation described in step 6), after dissolving with an aqueous solution of about 1% weak acid (such as acetic acid or phosphoric acid, etc.), the solution is adjusted to alkaline (about pH 9.0) with a weak base (such as ammonia water), and then precipitated. The precipitate is collected by centrifugation, washed with a small amount of organic solvents such as acetone or absolute ethanol, and dried under vacuum to obtain high-purity 4,9-anhydrotetrodotoxin powder.

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