CN107641128B - Method for efficiently extracting tetrodotoxin - Google Patents
Method for efficiently extracting tetrodotoxin Download PDFInfo
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Abstract
The invention relates to a method for extracting and producing tetrodotoxin with higher purity in a large scale and high efficiency by taking puffer fish viscera as raw materials, and the product can be used for drug rehabilitation, pain relief and blood pressure reduction pharmaceutical preparations. The method solves the problems that the prior methods for extracting and purifying the tetrodotoxin have a plurality of defects, such as high-efficiency liquid phase method with high output purity has expensive equipment (high-pressure liquid phase instrument), and for example, the immersion fractionation method has the problems of low efficiency, low product purity and the like, and provides the method for extracting and preparing the tetrodotoxin on a large scale, which has simple and high-efficiency process, high yield and excellent quality, has the product purity as high as 97-99 percent, and directly meets the requirements of biological research and clinical application. Another object of the present invention is to provide various preparations containing the tetrodotoxin product prepared by the method.
Description
Technical Field
The invention relates to the field of extraction of chemical substances, in particular to a method for extracting and producing tetrodotoxin in a large-scale and high-efficiency manner by taking blowfish viscera as raw materials. The product can be used for treating drug addiction, relieving pain and lowering blood pressure.
Background
Tetrodotoxin (TTX) is a low molecular weight alkaloid substance extracted from the viscera of puffer fish. Due to the unique chemical structure, the compound is a specific blocker of a cell sodium ion channel. Therefore, it has an irreplaceable position as a biological reagent in the field of biological research. The pharmacological effects of tetrodotoxin in clinical medicine have been reported for clinical intervention of alkaloid addiction in the early 30 s. The analgesic therapy is also being reported. Therefore, it shows a very important position in both the field of biological research and in the clinical application of medicine.
TTX is a typical sodium channel blocker, which selectively binds to sodium channel receptors on the cell membrane surface of muscles and nerve cells, blocks voltage-dependent sodium channels, thereby blocking animal potentials, inhibiting conduction of excitation between nerve and muscle, resulting in disorders of physiological functions associated therewith, mainly causing paralysis of muscles and nerves. The structure-activity relationship shows that the active groups of TTX are guanidine amino at 1, 2 and 3 positions and hydroxyl at nearby C-4, C-9 and C-10 positions, and guanidine is protonated at physiological pH value to form a positive electricity active region to interact with electronegative carbonyl of a sodium ion channel receptor protein, so that ions are blocked from entering a channel. The sodium ion receptor has at least 6 specific target molecule binding sites, and TTX binds to sodium channel receptor site I. TTX receptors are located on the outer side of excitable cell membranes and near the outer mouths of sodium channels, and TTX binds to the receptor sites and blocks sodium ions from accessing the outer mouths of the channels. Research shows that TTX acts specifically on sodium channel and has no direct effect on potassium and calcium channels, synapses of neuromuscular and cholinergic finger enzymes. In addition, the toxin can enter the center through the blood brain barrier, and has obvious inhibition effect on the center. Overall, respiratory and cardiovascular suppression of TTX is a result of both central and peripheral co-action.
TTX is a very useful tool for neurophysiological studies because it blocks sodium ion channels in cells in very small amounts, has no effect on potassium ion channels, and is highly metastatic.
Clinical medicine proves that TTX is a good sedative, spasmolytic and analgesic, has a good analgesic effect on malignant pain such as cancer, and has an analgesic effect of 12-20 hours and no addiction compared with traditional analgesics such as dolantin and morphine, although the response time is slightly longer. TTX also shows good effect in the aspect of drug rehabilitation recently, and a common drug rehabilitation person only needs to inject 10 mu g of TTX once a day, 5-7 days are a treatment course, drug addiction can be controlled, and the frequently-occurring drug rehabilitation syndrome in common drug rehabilitation can be greatly reduced.
However, tetrodotoxin injection (aqueous solution) is sensitive to temperature and is easily degraded by the influence of temperature, and the degradation is faster at higher temperature. Once the content of tetrodotoxin as effective active substance is less than 90% of its labeled amount or the relative content of related substances exceeds the limit specified by medical standards (greater than the main peak area of control solution), it is not satisfactory for clinical application. Experiments prove that the tetrodotoxin injection (water injection) is unstable at room temperature, and in order to ensure the quality of the tetrodotoxin injection and prevent the content of the tetrodotoxin from decreasing and the content of related substances from increasing, the tetrodotoxin injection must be stored in a refrigerator at the temperature of 4-8 ℃. Therefore, the clinical application of tetrodotoxin is difficult and inconvenient, the low temperature of 4-8 ℃ is kept in all links of storage, transportation, loading and unloading, wholesale, retail, hospitals and application correspondingly, otherwise, the high temperature can influence the clinical effect, and therefore, the problem needs to be solved as soon as possible, and a reliable product which is safe and stable and can be stored for a long time at room temperature is developed. In order to solve the above problems, it is a simple method to freeze-dry tetrodotoxin. The storage period of the unstable bioactive substances in the water solution can be prolonged after freeze-drying and dehydration, and the unstable bioactive substances can be regenerated by adding sterilized water for injection when the bioactive substances are clinically applied. However, when tetrodotoxin is used in medicine, the dosage of each dose is only 0.5-60 μ g, so that the trace tetrodotoxin in solution can not form required solid residue after freeze-drying and dehydration, and a pharmaceutically acceptable excipient is required to be added to provide a scaffold to which the trace tetrodotoxin can be attached, and the solution can form a solid after freeze-drying and dehydration.
Although the prior methods for extracting and purifying tetrodotoxin are various, the methods have certain defects, such as the high-performance liquid phase method with high output purity also has the problem of expensive equipment (high-pressure liquid phase instrument), and for example, the immersion fractionation method has the problems of low efficiency, low product purity and the like.
The Chinese patent office discloses a tetrodotoxin preparation method in 2008, 6.18.A patent with an authorization publication number of CN101891751B, which adopts amino acid as a cosolvent, not only can play a role of the cosolvent to increase the solubility of tetrodotoxin in water, but also can form a high-water-solubility salt solution with tetrodotoxin, and provides a method for producing tetrodotoxin with higher purity and less impurities.
Disclosure of Invention
In order to solve the problems that although the existing methods for extracting and purifying the tetrodotoxin are various, the methods have certain defects, such as the high-efficiency liquid phase method with high output purity also has the problem of expensive equipment (high-pressure liquid phase instrument), and such as the immersion fractionation method has the problems of low efficiency, low product purity and the like, the method for extracting and preparing the tetrodotoxin on a large scale is provided, and has the advantages of simple and high-efficiency process, high yield, excellent quality and high product purity of 97-99 percent, and directly meets the requirements of biological research and clinical application.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for efficiently extracting tetrodotoxin, which comprises the following steps:
1) mashing blowfish viscera, homogenizing, soaking in alcohol-water mixed solution containing acetic acid under stirring for 1-2 hr, centrifuging at 5800-; removing fat from the filtrate with fat removing solution to obtain yellowish initial venom;
2) decolorizing and purifying the yellowish primary venom with activated carbon at low temperature, filtering to obtain activated carbon, further adsorbing and purifying with macroporous adsorbent, and filtering to obtain primary venom clear liquid;
3) freeze-drying the primary venom clear liquid and concentrating under reduced pressure to reduce the volume to one third to one half of the original volume to obtain concentrated tetrodotoxin venom, performing microfiltration and ultrafiltration, collecting filtrate, performing microfiltration and ultrafiltration for 2-3 times, and mixing the filtrates to obtain low-impurity tetrodotoxin venom;
4) carrying out nanofiltration treatment on the low-impurity tetrodotoxin venom to collect filtrate, eluting the nanofiltration membrane to collect eluent, repeating the operation again, and combining the eluents obtained by two-time collection to obtain a liquid tetrodotoxin crude product;
5) performing water bath distillation treatment on the liquid tetrodotoxin crude product, collecting residual liquid, adjusting the pH value of the residual liquid subjected to the water bath distillation treatment to 8.5-9.5 by using ammonia water, and standing at a low temperature to obtain tetrodotoxin crystals;
6) dissolving tetrodotoxin crystal in acetic acid-methanol-water system solution, adjusting pH to 8.5-9.5 with ammonia water, drying at low temperature under reduced pressure, crystallizing and purifying the product again, and drying the crystallized product to obtain the desired tetrodotoxin.
Preferably, both step 5) and step 6) are preferably adjusted to a pH of 9 to 9.2.
Preferably, the alcohol in the alcohol-water mixed solution containing acetic acid in the step 1) is methanol, ethanol or a mixed alcohol of the methanol and the ethanol, the content of the acetic acid is 0.8-1%, the content of the alcohol is 10-12%, and the balance is water.
Preferably, the degreasing solution in the step 1) is any one of chloroform, diethyl ether, benzene and petroleum ether.
Preferably, the macroporous adsorbent in the step 2) is any one of a D101 macroporous adsorbent or a D201 macroporous adsorbent.
Preferably, the lyophilization in step 3) is divided into three stages: (1) pre-freezing for 2-3 hours at the temperature of-20 to-35 ℃; (2) drying for 10-12 hours at low pressure and at the temperature of-20 to-25 ℃; (3) and (3) heating and drying for 3-4 hours at the temperature of-5 ℃.
Preferably, the microfiltration pressure in the step 3) is 0.1-0.3MPa, preferably 0.1-0.15 MPa; the ultrafiltration pressure is 0.1-0.3MPa, preferably 0.2-0.25 MPa; the pressure applied by nanofiltration in the step 4) is 0.1-0.3MPa, preferably 0.1-0.15 MPa.
Preferably, the concentration under reduced pressure in the step 3) is carried out under the condition that the vacuum degree under reduced pressure is 1.2-2.0 Pa; and 5) keeping the low-temperature standing temperature at 10-35 ℃.
Preferably, in the acetic acid-methanol-water system in the step 6), the content of acetic acid is 0.5-0.8%, the content of methanol is 8-10%, and the balance is water; the low-temperature reduced pressure drying is carried out under the conditions that the pressure is 0.01-0.02MPa and the temperature is 5-10 ℃.
The method is characterized in that the extraction is carried out under the condition of water bath, the temperature of the water bath is preferably 90-100 ℃, because Tetrodotoxin (TTX) has good thermal stability, can not be decomposed by common heating and cooking, and the TTX is slightly soluble in water, is insoluble in partial organic matters but has good solubility for an acetic acid solution with certain concentration, the TTX can be promoted to be dissolved and extracted by heating the water bath under an acetic acid-alcohol-water solution system, the alcohol is preferably methanol, and the stirring ensures that the TTX in the solvent is uniformly distributed and can promote the dissolution process; in addition, because the globefish viscera contains a large amount of protein, most of the dissolved protein can be denatured to generate precipitate in the water bath heating process, so that the filtration is convenient, and most of the protein contained in the solution system can be removed; the obtaining rate of TTX can be improved by stirring and water-bathing for many times; because the raw material is globefish viscera which contains a large amount of animal fat, the interference of animal fat on TTX extraction in the subsequent process can be reduced by adding the degreasing liquid, the degreasing liquid is methanol or ethanol solution of any one of chloroform, ether, benzene and petroleum ether, preferably methanol solution of chloroform, the weight percentage of chloroform is 10-20%, preferably 10-12%, the animal fat in the filtrate is removed by an extraction mode, and the components in the degreasing liquid do not have adverse effect on the TTX dissolved in the original filtrate.
The activated carbon is preferably one of wood activated carbon, animal bone/blood activated carbon or peat activated carbon, and is granular activated carbon activated by steam, the activated carbon has rich original sources and low price, is simple to prepare and activate, is favorable for the condition that a large amount of activated carbon is needed for large-scale production of TTX, and has stable properties, does not pollute the yellowish primary venom in the purification and decoloration processes, introduces impurities, and is easy to filter and remove by a filtration mode; the macroporous adsorbent is one of a D101 macroporous adsorbent or a D201 macroporous adsorbent, the D201 macroporous adsorbent is optimally selected, the D201 is an anion exchange resin which has excellent affinity to weakly acidic substances, and the TTX is used as an alkaloid, so that the D201 macroporous adsorbent has no influence on the TTX when used for adsorption and impurity removal, and the yield of the TTX is not reduced.
The microfiltration and ultrafiltration are both in a physical impurity filtering mode, the microfiltration and ultrafiltration can be used for more thoroughly removing the high molecular weight impurities in the solution, and the microfiltration and ultrafiltration can improve the filtering effect of the ultrafiltration so as to prevent the high molecular weight substances from blocking the ultrafiltration membrane or damaging the ultrafiltration membrane; ultrafiltration is preferably carried out by an ultrafiltration membrane with the pore diameter of 500-700A, the molecular weight of TTX is 319, the ultrafiltration membrane with the pore diameter has good permeability to TTX, impurities with the molecular weight larger than TTX are almost completely filtered out in the ultrafiltration process after microfiltration, only a few components with the molecular weight very close to that of TTX and components with the molecular weight smaller than that of TTX exist in filtrate, nanofiltration is preferably carried out by a nanofiltration membrane with the pore diameter of 1-2nm in the subsequent nanofiltration process, most of component substances with the molecular weight smaller than that of TTX can permeate, and only substances with the molecular weight very close to that of TTX are remained in eluent obtained by eluting from the nanofiltration membrane, wherein TTX is mainly used as the main component, and impurities are basically removed in the microfiltration-ultrafiltration-nanofiltration triple membrane filtration.
The eluent for eluting the nanofiltration membrane is acetic acid-methanol-water solution, the concentration of acetic acid is 0.5-1.5%, the concentration of methanol is 5-12%, and the balance is water, preferably the concentration of acetic acid is 0.8-0.9%, the concentration of methanol is 9-10%, and the balance is water.
The steps of freeze drying and decompression concentration are all used for reducing the content of acetic acid, alcohol and water in a solution system where the TTX is located and improving the concentration of the TTX, so that the TTX is close to a saturated state in the solution system where the TTX is located, and a good bedding effect is achieved on subsequent precipitated crystals.
Adding ammonia water into the solution system of TTX to neutralize acetic acid in the solution system, adjusting pH value, reducing the solubility of TTX to make it in a supersaturated state, separating out TTX, and standing to separate out TTX to improve the purity of TTX crystal; the alkali liquor can neutralize acetic acid, ammonia water is selected as the alkali liquor, firstly, the ammonia water is suitable for fine adjustment of pH by being used as the alkali liquor due to weak alkalinity and strong controllability of the ammonia water, secondly, the TTX is extremely unstable to alkali, reacts with most of alkali substances and generates impurities with stable chemical properties, and the ammonia water is a weak alkali substance, so that the occurrence of the condition is reduced or even avoided; the purity of TTX can be further improved by dissolving the crystals after the primary crystallization, drying the crystals at a low temperature under reduced pressure, and then recrystallizing the crystals.
On the other hand, the invention also provides various preparations containing the tetrodotoxin product prepared by the method, including oral capsules, oral tablets, buccal tablets, intramuscular injection, intravenous drip and other dosage forms. And various types of transdermal absorption preparations including, but not limited to, transdermal patches, sprays, smears, and the like. The tetrodotoxin product content in each dosage form is as follows: 10-50ng per tablet or capsule of oral dosage form; 1-5ng per ml of injection; the transdermal absorption preparation contains 5-50ng of each monomer.
The invention has the beneficial effects that: tetrodotoxin has good thermal stability, can not be decomposed by common heating and cooking, and TTX is slightly soluble in water, is insoluble in partial organic matters but has good solubility for an acetic acid solution with a certain concentration, so that the TTX can be promoted to be dissolved and extracted by heating in a water bath under an acetic acid-alcohol-water solution system; common filtration, activated carbon and macroporous adsorbent adsorption can quickly remove precipitates and high molecular weight impurities, and then multiple membrane filtration combining microfiltration, ultrafiltration and nanofiltration is matched, so that most of impurities in a solution system where TTX is located can be quickly and thoroughly removed, and the solution system with high TTX purity can be efficiently prepared; the method for efficiently extracting tetrodotoxin provided by the invention has the advantages of strong practicability, high extraction efficiency and high TTX purity, and each process can be completed by mechanical automation or mechanical manual operation, so that each working step can reduce the direct contact between workers and venom as much as possible, on one hand, the danger of production operation is reduced, the safety of the workers is ensured, and on the other hand, the method is more favorable for realizing large-scale production, reducing pollution and protecting environment.
The improvement of the preparation of the invention compared with the similar medicament containing tetrodotoxin of the prior art lies in the selection of a specific dosage range, for example, the patent document with the patent number of CN1227102A published by the Chinese patent office, the dosage range of the medicament provided by the proposal is too large, 0.001-0.45 mu g, and the lowest dosage of the medicament containing tetrodotoxin is too low, thereby the therapeutic effect can not be achieved at all, and the medicament can cause the drug resistance and the immunological reaction of the organism while not playing the therapeutic effect, therefore, the dosage selected by the invention is a safe dosage with good using effect on the basis of the pharmacokinetic research. In the dosage range, the better dose-effect relationship can be ensured, the times of medication of patients are reduced, and unnecessary pain and burden of the patients are avoided.
The tetrodotoxin product can be used as a medicine for clinically giving up alkaloid addiction dependent drug syndromes, namely commonly called drug addiction stopping medicine, can also replace analgesics such as morphine, dolantin and the like, and clinical medicines for relieving spasm, calming, local anesthesia, resisting pruritus, tachycardia, reducing blood pressure and the like.
Drawings
FIG. 1 is a standard curve of tetrodotoxin assay prepared by the present invention.
FIG. 2 is an ion flow diagram of HPLC-MS/MS detection mass spectrum of tetrodotoxin prepared by the present invention.
Detailed Description
Example 1: extraction of tetrodotoxin
1) Mashing 50kg blowfish viscera, homogenizing, soaking in alcohol-water mixed solution containing acetic acid under stirring for 1 hr, centrifuging at 6200r/m speed, stirring in water bath, filtering precipitate, repeating the above steps for more than 3 times, mixing filtrates, and concentrating; removing fat from the filtrate with fat removing solution to obtain yellowish initial venom; wherein the content of acetic acid in the mixed solution is 1 percent, the content of alcohol is 12 percent, and the balance is water; the degreasing solution is methanol solution of chloroform, and the chloroform is 10%;
2) decolorizing and purifying the yellowish primary venom with wood activated carbon at low temperature, filtering to obtain activated carbon, further adsorbing and purifying with D201 macroporous adsorbent, and filtering to obtain primary venom clear liquid;
3) freeze drying and concentrating the clear liquid of the initial toxin solution under reduced pressure to reduce the volume to one third of the original volume to obtain concentrated tetrodotoxin toxin solution, performing microfiltration and ultrafiltration, collecting filtrate, performing microfiltration and ultrafiltration on the filtrate for 2 times, and combining the obtained filtrate to obtain low-impurity tetrodotoxin toxin solution; freeze-drying is divided into three stages: (1) pre-freezing for 2 hours at-35 ℃; (2) drying at-25 deg.C for 10 hr under low pressure; (3) heating and drying for 3 hours at the temperature of 5 ℃; the pressure of the reduced pressure concentration is 1.2 Pa; the external pressure applied by microfiltration is 0.1MPa, the external pressure applied by ultrafiltration is 0.25MPa, and the aperture of an ultrafiltration membrane is 500A;
4) carrying out nanofiltration treatment on the low-impurity tetrodotoxin venom to collect filtrate, eluting a nanofiltration membrane to collect eluent, carrying out nanofiltration treatment on the filtrate, eluting the nanofiltration membrane to collect the eluent, and combining the eluents obtained by two times of collection to obtain a liquid tetrodotoxin crude product; the nanofiltration membrane is selected from nanofiltration membrane with the aperture of 1 nm; the eluent is acetic acid-methanol-water solution, the concentration of acetic acid is 0.8%, the concentration of methanol is 10%, and the balance is water;
5) performing water bath distillation treatment on the liquid tetrodotoxin crude product, collecting residual liquid, adjusting the pH value of the residual liquid subjected to the water bath distillation treatment to 9.2 by using ammonia water, and standing at a low temperature to obtain tetrodotoxin crystals; the low temperature is 10 ℃;
6) dissolving tetrodotoxin crystal in acetic acid-methanol-water system solution, adjusting pH value of the solution to 9.2 with ammonia water, drying at low temperature under reduced pressure, crystallizing and purifying the product again, and drying the crystallized product to obtain the desired tetrodotoxin; in the acetic acid-methanol-water system, the content of acetic acid is 0.8 percent, the content of methanol is 10 percent, and the balance is water; the low-temperature reduced-pressure drying is carried out under the conditions that the pressure is 0.01MPa and the temperature is 5 ℃.
Example 2: extraction of tetrodotoxin
1) Mashing 50kg blowfish viscera, homogenizing, soaking in alcohol-water mixed solution containing acetic acid under stirring for 2 hr, centrifuging at 5800r/m speed, stirring in water bath, filtering precipitate, repeating the above steps for more than 2 times, mixing filtrates, and concentrating; removing fat from the filtrate with fat removing solution to obtain yellowish initial venom; wherein the content of acetic acid in the mixed solution is 0.8 percent, the content of alcohol is 10 percent, and the balance is water; the degreasing solution is methanol solution of chloroform, and the chloroform is 20%;
2) decolorizing and purifying the yellowish primary venom with wood activated carbon at low temperature, filtering to obtain activated carbon, further adsorbing and purifying with D101 macroporous adsorbent, and filtering to obtain D101 macroporous adsorbent to obtain primary venom clear liquid;
3) freeze drying and concentrating the clear liquid of the initial toxin solution under reduced pressure to reduce the volume to one half of the original volume to obtain concentrated tetrodotoxin toxin solution, performing microfiltration and ultrafiltration, collecting filtrate, performing microfiltration and ultrafiltration on the filtrate for 3 times, and combining the obtained filtrate to obtain low-impurity tetrodotoxin toxin solution; freeze-drying is divided into three stages: (1) pre-freezing for 3 hours at-20 ℃; (2) drying at-20 deg.C for 12 hr under low pressure; (3) heating and drying for 4 hours at the temperature of-5 ℃; the pressure of the reduced pressure concentration is 1.2 Pa; the external pressure applied by microfiltration is 0.1MPa, the external pressure applied by ultrafiltration is 0.25MPa, and the aperture of an ultrafiltration membrane is 500A;
4) carrying out nanofiltration treatment on the low-impurity tetrodotoxin venom to collect filtrate, eluting a nanofiltration membrane to collect eluent, carrying out nanofiltration treatment on the filtrate, eluting the nanofiltration membrane to collect the eluent, and combining the eluents obtained by two times of collection to obtain a liquid tetrodotoxin crude product; the nanofiltration membrane is a nanofiltration membrane with the aperture of 2 nm; the eluent is acetic acid-methanol-water solution, the concentration of acetic acid is 0.9 percent, the concentration of methanol is 9 percent, and the rest is water;
5) performing water bath distillation treatment on the liquid tetrodotoxin crude product, collecting residual liquid, adjusting the pH value of the residual liquid subjected to the water bath distillation treatment to 9 by using ammonia water, and standing at a low temperature to obtain tetrodotoxin crystals; the low temperature is 35 ℃;
6) dissolving tetrodotoxin crystal in acetic acid-methanol-water system solution, adjusting pH to 9 with ammonia water, drying at low temperature under reduced pressure, crystallizing and purifying the product again, and drying the crystallized product to obtain the desired tetrodotoxin; the acetic acid-methanol-water system contains 0.5% of acetic acid, 8% of methanol and the balance of water; the low-temperature reduced-pressure drying is carried out under the conditions that the pressure is 0.02MPa and the temperature is 10 ℃.
Example 3: extraction of tetrodotoxin
1) Mashing 50kg blowfish viscera, homogenizing, soaking in alcohol-water mixed solution containing acetic acid under stirring for 2 hr, centrifuging at 6200r/m speed, stirring in water bath, filtering precipitate, repeating the above steps for more than 3 times, mixing filtrates, and concentrating; removing fat from the filtrate with fat removing solution to obtain yellowish initial venom; wherein the content of acetic acid in the mixed solution is 1 percent, the content of alcohol is 10 percent, and the balance is water; the degreasing solution is methanol solution of chloroform, and the chloroform is 15%;
2) decolorizing and purifying the yellowish primary venom with wood activated carbon at low temperature, filtering to obtain activated carbon, further adsorbing and purifying with D201 macroporous adsorbent, and filtering to obtain primary venom clear liquid;
3) freeze drying and concentrating the clear liquid of the initial toxin solution under reduced pressure to reduce the volume to one half of the original volume to obtain concentrated tetrodotoxin toxin solution, performing microfiltration and ultrafiltration, collecting filtrate, performing microfiltration and ultrafiltration on the filtrate for 3 times, and combining the obtained filtrate to obtain low-impurity tetrodotoxin toxin solution; freeze-drying is divided into three stages: (1) pre-freezing for 3 hours at-35 ℃; (2) drying at-25 deg.C for 12 hr under low pressure; (3) heating and drying for 3 hours at the temperature of-5 ℃; the pressure of the reduced pressure concentration is 2.0 Pa; the external pressure applied by microfiltration is 0.15MPa, the external pressure applied by ultrafiltration is 0.20MPa, and the aperture of an ultrafiltration membrane is 700A;
4) carrying out nanofiltration treatment on the low-impurity tetrodotoxin venom to collect filtrate, eluting a nanofiltration membrane to collect eluent, carrying out nanofiltration treatment on the filtrate, eluting the nanofiltration membrane to collect the eluent, and combining the eluents obtained by two times of collection to obtain a liquid tetrodotoxin crude product; the nanofiltration membrane is selected from nanofiltration membrane with the aperture of 1 nm; the eluent is acetic acid-methanol-water solution, the concentration of acetic acid is 0.9 percent, the concentration of methanol is 10 percent, and the rest is water;
5) performing water bath distillation treatment on the liquid tetrodotoxin crude product, collecting residual liquid, adjusting the pH value of the residual liquid subjected to the water bath distillation treatment to 9.2 by using ammonia water, and standing at a low temperature to obtain tetrodotoxin crystals; the low temperature is 10 ℃;
6) dissolving tetrodotoxin crystal in acetic acid-methanol-water system solution, adjusting pH value of the solution to 9.2 with ammonia water, drying at low temperature under reduced pressure, crystallizing and purifying the product again, and drying the crystallized product to obtain the desired tetrodotoxin; in the acetic acid-methanol-water system, the content of acetic acid is 0.8 percent, the content of methanol is 8 percent, and the balance is water; the low-temperature reduced-pressure drying is carried out under the conditions that the pressure is 0.02MPa and the temperature is 5 ℃.
The tetrodotoxin product prepared by the method has the quality and purity detected and determined by the indexes listed in the following table:
high pressure liquid phase analysis using 20RBAXC18And the column detects the wavelength of 200-210nm, and calculates by a peak area normalization method through retention time and peak value comparison according to the scanning drawing result.
LD50 acute toxicity test was determined using a simplified probability method.
Formulation example 1
Accurately weighing 20.0mg of tetrodotoxin, adding 50ml of citrate solution with the citric acid content of 60mg/100ml, fully dissolving, adding conventional tabletting excipient which is 100g of starch, fully mixing uniformly, drying, evaporating and tabletting. Each tablet has a net weight of 100mg and contains 20ng tetrodotoxin.
Formulation example 2
Accurately weighing tetrodotoxin 15.0mg, adding citrate 100.0mg, adding injectable water 2000ml, stirring for dissolving completely, and packaging under aseptic condition to obtain 1.0 ml/branch containing tetrodotoxin 5 ng.
Formulation example 3
Accurately weighing 20.0mg, adding citrate 20.0mg, adding water for injection 100ml, adding azone 1000.0mg and sodium alginate 3000.0mg, stirring for dissolving completely, using calcium chloride as shaping agent, and making into 1000 pieces each containing tetrodotoxin 10 ng.
Claims (9)
1. A method for efficiently extracting tetrodotoxin, which is characterized by comprising the following steps:
1) mashing blowfish viscera, homogenizing, soaking in alcohol-water mixed solution containing acetic acid under stirring for 1-2 hr, centrifuging at 5800-; removing fat from the filtrate with fat removing solution to obtain yellowish initial venom;
2) decolorizing and purifying the yellowish primary venom with activated carbon at low temperature, filtering to obtain activated carbon, further adsorbing and purifying with macroporous adsorbent, and filtering to obtain primary venom clear liquid;
3) freeze-drying the primary venom clear liquid and concentrating under reduced pressure to reduce the volume to one third to one half of the original volume to obtain concentrated tetrodotoxin venom, performing microfiltration and ultrafiltration, collecting filtrate, performing microfiltration and ultrafiltration for 2-3 times, and mixing the filtrates to obtain low-impurity tetrodotoxin venom;
the ultrafiltration is carried out by using an ultrafiltration membrane with the aperture of 500-700A;
4) carrying out nanofiltration treatment on the low-impurity tetrodotoxin venom to collect filtrate, eluting the nanofiltration membrane to collect eluent, repeating the operation again, and combining the eluents obtained by two-time collection to obtain a liquid tetrodotoxin crude product;
5) performing water bath distillation treatment on the liquid tetrodotoxin crude product, collecting residual liquid, adjusting the pH value of the residual liquid subjected to the water bath distillation treatment to 8.5-9.5 by using ammonia water, and standing at a low temperature to obtain tetrodotoxin crystals;
6) dissolving tetrodotoxin crystal in acetic acid-methanol-water system solution, adjusting pH to 8.5-9.5 with ammonia water, drying at low temperature under reduced pressure, crystallizing and purifying the product again, and drying the crystallized product to obtain the desired tetrodotoxin;
wherein, the pressure applied by the microfiltration in the step 3) is 0.1-0.3 MPa; the ultrafiltration pressure is 0.1-0.3 MPa;
the pressure applied by nanofiltration in the step 4) is 0.1-0.3MPa, and the nanofiltration membrane in the step 4) is a nanofiltration membrane with the aperture of 1-2 nm.
2. The method for extracting tetrodotoxin with high efficiency as claimed in claim 1, wherein the pH value of step 5) and the pH value of step 6) are both adjusted to 9-9.2.
3. The method for extracting tetrodotoxin as claimed in claim 1, wherein the alcohol in the alcohol-water mixture containing acetic acid in step 1) is methanol, ethanol or a mixture of methanol and ethanol, the acetic acid content is 0.8-1%, the alcohol content is 10-12%, and the balance is water.
4. The method for efficiently extracting tetrodotoxin according to claim 1, wherein the lipid removing solution in step 1) is any one of chloroform, diethyl ether, benzene and petroleum ether.
5. The method for efficiently extracting tetrodotoxin according to claim 1, wherein the macroporous adsorbent in step 2) is any one of a D101 macroporous adsorbent and a D201 macroporous adsorbent.
6. The method for extracting tetrodotoxin with high efficiency as claimed in claim 1, wherein the lyophilization in step 3) is divided into three stages: (1) pre-freezing for 2-3 hours at the temperature of-20 to-35 ℃; (2) drying for 10-12 hours at low pressure and at the temperature of-20 to-25 ℃; (3) and (3) heating and drying for 3-4 hours at the temperature of-5 ℃.
7. The method for extracting tetrodotoxin with high efficiency as claimed in claim 1, wherein the microfiltration applied pressure in step 3) is 0.1-0.15 MPa; the pressure applied by ultrafiltration is 0.2-0.25 MPa; the pressure applied by nanofiltration in the step 4) is 0.1-0.15 MPa.
8. The method for extracting tetrodotoxin with high efficiency as claimed in claim 1, wherein the concentration under reduced pressure in step 3) is performed under a reduced vacuum degree of 1.2-2.0 Pa; and 5) keeping the low-temperature standing temperature at 10-35 ℃.
9. The method for extracting tetrodotoxin with high efficiency as claimed in claim 1, wherein the acetic acid-methanol-water system in step 6) has an acetic acid content of 0.5-0.8%, a methanol content of 8-10%, and the balance being water; the low-temperature reduced pressure drying is carried out under the conditions that the pressure is 0.01-0.02MPa and the temperature is 5-10 ℃.
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| EP4166559A4 (en) * | 2021-05-04 | 2024-01-31 | Zhongyang Biotechnology Shanghai Co Ltd | Method for efficiently extracting and separating high-purity tetrodotoxin from pufferfish viscera |
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| CN111036078B (en) * | 2018-10-14 | 2022-04-26 | 深圳市健元医药科技有限公司 | Post-treatment method of GnRH antagonist |
| CN111122752B (en) * | 2020-02-26 | 2022-06-10 | 浙江清华长三角研究院 | Preparation method of tetrodotoxin component analysis standard substance |
| CN112920192A (en) * | 2021-02-03 | 2021-06-08 | 宋江 | Tetrodotoxin extraction and purification method |
| WO2024007068A1 (en) * | 2022-07-06 | 2024-01-11 | Wex Pharmaceuticals Inc. | Process for the extraction and purification of tetrodotoxin |
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| CN1245406C (en) * | 2003-06-24 | 2006-03-15 | 中国海洋大学 | Method for extracting tetrodotoxin from globefish livers |
| CN101891751B (en) * | 2008-06-18 | 2011-12-21 | 上海亿年生物科技有限公司 | Method for preparing tetrodotoxin |
| CN102584843A (en) * | 2011-01-18 | 2012-07-18 | 王秀菊 | Extraction method for high-purity tetrodotoxin |
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