CN1680382A - Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof - Google Patents
Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof Download PDFInfo
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- CN1680382A CN1680382A CNA2005100131155A CN200510013115A CN1680382A CN 1680382 A CN1680382 A CN 1680382A CN A2005100131155 A CNA2005100131155 A CN A2005100131155A CN 200510013115 A CN200510013115 A CN 200510013115A CN 1680382 A CN1680382 A CN 1680382A
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- tetraodotoxin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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- Y02P20/582—Recycling of unreacted starting or intermediate materials
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Abstract
Production of tetradotoxin by two-step resin method and tetradotoxin preparation are disclosed. It is carried out by taking viscus of puffer fish as materials, extracting by extracting pot, purifying by two-step resin method, and secondary crystallizing. It can be used for clinical abstinence natural base addiction dependent medicine syndrome, analgesic and depressor medicine. Its advantages include safety, simple, high quality and purity of product, and high productivity.
Description
[technical field]: the present invention relates to a kind of is the preparation method of raw material large-scale production tetraodotoxin with the globe fish internal organ.Its product can be used as the clinical medicament that is used for drug rehabilitation and analgesia.Biological reagent as ion channel blocking effect research.
[background technology]: tetraodotoxin (being called for short TTX) is the lower molecular weight alkaloids substance that a kind of effluent filefish internal organ extract.Because its unique chemical structure makes it become the single-minded blocker of cellular sodium ionic channel.Therefore it has irreplaceable position in the biological study field as biological reagent.Tetraodotoxin shown pharmacotoxicological effect on clinical medicine just has been useful on the clinical report of giving up alkaloids habituation venereal disease disease as far back as the thirties in last century.Aspect analgesia therapy, also constantly there is report to release.Therefore, no matter it has all shown crucial status in the biological study field or in the clinical medicine application facet.
Though tetraodotoxin has important effect in biological study field and clinical medicine application, himself is one and has extremely strong toxic highly toxic substance.Simultaneously, because tetraodotoxin is extremely low in the unit content of globe fish internal organ, bring sizable difficulty for the extraction purifying.So select a kind of safe, easy and simple to handle, method that large-scale production that higher yields is arranged prepares tetraodotoxin, just demonstrate very important effect.
The method of extracting tetraodotoxin at present has many kinds, but these methods are all existing some shortcoming in varying degrees.As high-efficient liquid phase technique: use apparatus expensive (high-pressure liquid phase instrument), and output capacity of product is low, is difficult to satisfy large-scale production; Membrane filter method: well-known membrane filtration principle is in a certain molecular weight interval range, the material that molecular weight cut-off is roughly the same.Material to the roughly the same structure of molecular weight, different in kind then can't separate, and therefore is difficult to the tetraodotoxin purifying; Activated carbon method: gac is a kind of non-selective sorbent material, and it is to the material high adsorption capacity, and material is eluted weak effect, and the yield of product is had direct influence; These have all restricted the large-scale production of tetraodotoxin.
[summary of the invention]: one of purpose of the present invention is to solve the problem that method use apparatus expensive, output capacity of product of extracting tetraodotoxin are low, be difficult to large-scale production that has now; provide a kind of and can extract the method for preparing tetraodotoxin on a large scale; this method not only extraction process is simple; the productive rate height; and good quality of product; purity directly meets biological study and requirements for clinical application up to 96-99%.
The product that the inventive method is extracted can directly be prepared as various preparations, and therefore, another technical problem that the present invention will solve provides the preparation that a class is given up drug dependence.
For solving the problems of the technologies described above, the present invention at first provides a kind of method for preparing tetraodotoxin, and this method comprises the steps:
1) with globe fish internal organ (fish liver, fish ovaries etc.) fragmentation, add deionized water low temperature and stir extraction down, obtain extracting solution, with extracting solution heating, solidifying egg white; Hang elimination and remove solid substance; Obtain little yellow venom just;
2) under the first venom cold condition, after the absorption of nonpolar adsorption resin R1 resin stirring at low speed, leach resin; Obtain the inclusion-free crude venom;
3) crude venom is carried out ion-exchange type resin R2 resin upper prop exchange absorption; Crude venom is finished by ion-exchange type resin R2 resin absorption, after washing; Use elutriant acetic acid: water pH=3.5 carries out wash-out, collects dense fraction, with bioassay method spike monitoring, the dense fraction liquid of acquisition, i.e. tetraodotoxin crude product;
4) gained liquid tetraodotoxin crude product is put vacuum concentration equipment, carry out concentrate under reduced pressure at low temperature, be concentrated into small volume, get high density tetraodotoxin liquid; Concentrate under reduced pressure at low temperature wherein, be in temperature less than 50 ℃, carry out under the reduced vacuum degree 0.8-1.1Pa condition;
5) the high density tetraodotoxin liquid pH=7.5-9.0 of adjusting gained makes and reaches iso-electric point, puts to leave standstill under the 4-8 ℃ of condition to obtain the tetraodotoxin crystallization;
6) with the tetraodotoxin crystallization with inorganic or organic acidity agent dissolves, and transfer pH to reach iso-electric point with alkaline reagents again, make product secondary crystal purifying.The crystalline product that obtains both had been the tetraodotoxin product after drying.
The extraction of aforesaid method is in double-deck extractor, cools off extraction with water coolant.Once can extract raw material more than 500 kilograms (globe fish internal organ).Method of the present invention adopts extract at low temperature.Too high to reduce tetraodotoxin Yin Wendu, cause the product dehydrogenation and quality decline and productive rate reduction.Above-mentioned " low temperature " preferably is meant about 2-8 ℃.
" nonpolar adsorption resin " in the aforesaid method can be the import or the homemade nonpolar adsorption resin of all size.Preferred NKA-9 resin (production of Tianjin Chemical Plant of Nankai Univ.), but be not limited to import or the homemade nonpolar adsorption resin that other model resin of the same type is meant all size.
For example: X-5,3520 etc. (Tianjin products)
Above-mentioned " ion-exchange type resin " can be the import or the homemade ion-exchange type resin of all size.Preferred D
151Resin, but be not limited to other model resin of the same type, for example: D
152, D
111, (Tianjin product) Amberlyst XN1004 (U.S.'s product).
Nonpolar adsorption resin adopts and filters or centrifugation method after absorption impurity finishes in the aforesaid method, separates the inclusion-free toxin solution.Resin is reused after removing impurity with ethanol elution; The eluent of ion-exchange type resin equally also can be the eluent of any conventional this resinoid of wash-out, the preferred acetic acid that still is not limited to: aqueous systems, hydrochloric acid: aqueous systems etc.
Operable various organic acids of secondarily purified crystallization and mineral acid chemical reagent are as purified crystals reagent in the aforesaid method.These organic acids or mineral acid comprise and being not limited to: for example hydrochloric acid, acetic acid, picric acid etc.
On the other hand, the present invention also provides the various preparations of the tetraodotoxin product that contains this law preparation, comprises formulations such as oral capsule, oral tablet, lozenge, intramuscular injection, intravenous injection, intravenous drip.And all types of transdermal absorption formulations (including, but not limited to percutaneous plaster, sprays, be coated with and touch agent etc.).The tetraodotoxin product content is in each formulation: oral dosage form; Every (or each capsule) contains 10-50ng.Injection; Every milliliter contains 10-50ng, transdermal absorption formulation; Each uses monomer to contain 1-50ng.
Advantage of the present invention and positively effect: the present invention is by " two-step resin method ", promptly by selecting specific resin coupling for use; Remove whole non-tetraodotoxin impurity with nonpolar adsorption resin absorption.Carry out purifying and concentrated inclusion-free crude venom with the ion-exchange type resin.Make extraction process simplify, efficient improves, and the product purity height of gained can be realized scale operation.And this leaching process can be under semiclosed condition; Both hung filter pocket-sealing whip attachment jar-purifying resin column etc. to seal double-deck extractor-sealing heating kettle-sealing.Between each operation,, carry out liquid with liquor pump and carry, make each algorithm avoid operator to contact as far as possible with the direct of venom with pipe link.Help accomplishing scale production so on the one hand, reduce on the other hand and pollute, help environment protection.
Above-mentioned preparation of the present invention and prior art similarly contain the selection that improvements that the medicament of tetraodotoxin compares are specific dosage range.Cross the low 0.001-0.45ug that only is as its using dosage of patent (CN 1227102A), this dosage can't reach result of treatment at all.Cross low tetraodotoxin dosage simultaneously, can make body produce resistance and immunological response.As the coupled thing immune animal of the tetraodotoxin of using low dosage, can prepare monoclonal antibody.(patent CN 1465403A) so the selected dosage of the present invention are on the basis of pharmacokinetic, one of proposition both safely, the using dosage with good drug abstinence arranged.Under this dosage range, can guarantee dose-effect relationship preferably.The use of this dosage has simultaneously also reduced patient's medication number of times, has avoided bringing unnecessary painful and burden to patient.
This tetraodotoxin product can be used as and is used for clinical alkaloids habituation dependence medicine syndromes (drug rehabilitation) medication of giving up, and also can be used as the analgesia medication and substitutes morphine, pethidine etc.And spasmolysis, calmness, toponarcosis, antipruritic, and tachycardia and clinical application such as hypotensive.
[embodiment]:
Example 1: the extraction of tetraodotoxin
1, get 100 kilograms in globe fish internal organ (fish liver, fish ovaries), extractor is dropped in broken back, adds 2 times of deionized waters, and (2-8 ℃) stirs and extracted 30 hours under the cold condition.
2, extract finish after, from extractor lower liquid outlet connecting tube, will extract mixed solution and pump into the heating tank jar, be heated to boiling with mixed solution 10-15 minute after, be cooled to rapidly below 30 ℃ immediately.
3, from the end opening pipeline after with protein coagulation mixed solution hangs filter pocket and carries out (hanging filter cloth is 200 orders) and hang filter, obtain little yellow transparent clear liquid, be tetraodotoxin venom just.
4, above-mentioned clear liquid is pumped into high-order R1 resin (A-8) adsorption tanks by connecting tube, carry out adsorption-edulcoration matter, whip attachment 5 hours leaches resin, obtains the water white transparency clear liquid.
5, transparent clear liquid is connected with R2 exchange ion resin bed (post) by pipeline, carries out R2 resin (D
151) absorption.After (resin column is high 1.5 meters, 0.2 meter of diameter) treats that whole liquid adsorption finish, use the elutriant wash-out instead.(elutriant pH=4.0) collects and to contain the dense part elutriant (be about total extracted amount 1/60) that heats up in a steamer of tetraodotoxin and be crude product tetraodotoxin liquid.
6, crude product tetraodotoxin liquid is carried out concentrating under reduced pressure, the concentrate under reduced pressure at low temperature temperature is 45 ℃.The reduced vacuum degree is 1.0Pa.Crude product tetraodotoxin liquid is concentrated into original volume 1/50.
7, collect concentrated solution to centrifuge tube, behind the adjusting pH=7.5-9.5, under cold condition, left standstill 5-10 hour, obtain crystallization tetraodotoxin (0.7 gram/ten ten thousand that is about total extracted amount).
The crystallization tetraodotoxin is secondarily purified, with 1.5 times of 1N acetums, and complete dissolving crystallized tetraodotoxin.Regulate pH=8.5 with basic solution.Stand at low temperature 10 hours.Centrifugal collection crystallization tetraodotoxin, dry back obtain pure product tetraodotoxin (0.6 gram/ten ten thousand that is about total extracted amount).
The tetraodotoxin product of method for preparing, its quality, the listed index of purity following table detect, determine product purity:
Quality product, purity detecting
| Detect title | Conclusion |
| Outward appearance | Range estimation is for white crystalline powder, the microscopically hour hand shape crystalline powder that is white in color. |
| Melting point | Product does not have melting point, 220 ℃ of charings. |
| Ultimate analysis | C:40.24 ± 0.3 H:5.53 ± 0.4 N:12.83 ± 0.3 |
| Visible spectrum | Through the scanning of visible region 530-700nm all wave band, do not have any impurity peaks and occur. |
| UV spectrum | Through the scanning of ultraviolet region 190-300nm all wave band, confirm product outside there is a special absorption peak at the 195-210nm place, other no any impurity absorption peak in zone. |
| Infrared spectra | Measure through KCl pressed disc method infrared scan, confirming has special absorption peak at 3300,3230,1667,1611 places. |
| Nuclear magnetic spectrogram | Measure through nmr spectrometer, confirm product 2.34,3.94, there is special absorption peak at 4.03,4.25,5.46 places. |
| High pressure liquid phase analysis | Adopt 20RBAX C 18Post detects wavelength 200-210nm, according to the drawing result of scanning,, calculates and assert that product purity reaches 99% with the peak area normalization method by retention time and peakedness ratio. |
| LD 50Acute toxicity test | Adopt and simplify probability method mensuration, assert the LD of product 50Be 10.97ug/kg ± 0.72 |
| Viable cell diaphragm tongs technology detects | Use the viable cell of vitro culture, according to product pair cell Na +The minimum concentration of passage generation blocking action and shortest time result determine product purity and biological activity. |
Embodiment 2: the preparation of tetraodotoxin tablet
Accurately take by weighing tetraodotoxin 20mg, add the citrate solution 50ml that contains 30mg, fully after the dissolving.Add conventional film-making vehicle (as starch, aluminium hydroxide, glucose) 100g again, after fully being mixed, dry transpiring moisture film-making.The every heavy 100mg of self sheet contains tetraodotoxin 20ng.
Embodiment 3: the preparation of tetraodotoxin injection
Accurately take by weighing tetraodotoxin 30mg, add Citrate trianion 100mg, add injection water 1000ml, stir down fully dissolving, packing 1.0ml/ props up under the aseptic condition.Every contains tetraodotoxin 20ng.
Embodiment 4: the preparation of transdermal absorption formulation
Accurately take by weighing tetraodotoxin 10mg, add Citrate trianion 20mg, add injection water 100ml, add azone 1.0g, sodium alginate 3.0g stirs down fully dissolving.With calcium chloride is formagen, makes 1000 of pasters.Every contains tetraodotoxin 10ng.
Claims (8)
1, a kind of two-step resin legal system is equipped with the method for tetraodotoxin, it is characterized in that this method comprises the steps:
1) with the fragmentation of globe fish internal organ, add deionized water low temperature and stir extraction down, must extract mixed solution, be heated with solidifying egg white, hang elimination except that solid substance, obtain little yellow venom just;
2) just venom stirring velocity 10-30 rev/min, adsorbed 5 hours through nonpolar adsorption resin resin whip attachment; After absorption finishes, leach resin, obtain the inclusion-free crude venom;
3) with resin column on the crude venom, through ion-exchange type resin resin absorption, absorption finishes after washing, is elutriant with 4% acetum pH=3.5, carries out wash-out; Collect high dense fraction, the liquid of acquisition, i.e. tetraodotoxin crude product;
4) the liquid tetraodotoxin crude product with gained carries out concentrate under reduced pressure at low temperature, is concentrated into small volume, gets high density tetraodotoxin liquid;
5) regulate gained high density tetraodotoxin liquid pH=7.5-9.0, make to reach iso-electric point, put and leave standstill the crystallization of acquisition tetraodotoxin under the cold condition;
6) with the crystallization of gained tetraodotoxin with inorganic or organic acidity agent dissolves, and transfer pH=7.5-9.0 with alkaline reagents again, reach iso-electric point, with product crystallization purifying once more, the crystalline product of acquisition is required tetraodotoxin product after drying.
2,, it is characterized in that described inorganic or organic acid reagent is hydrochloric acid, acetic acid or picric acid according to the method for claim 1.
3, according to the method for claim 1, it is characterized in that described method 1) step in extraction carry out at 2-8 ℃.
4, according to the method for claim 1, it is characterized in that concentrate under reduced pressure at low temperature, temperature is carried out under the reduced vacuum degree 0.8-1.1Pa condition less than 50 ℃.
5, according to the method for claim 1, it is characterized in that extracting be at double-deck extractor interlayer with the water coolant cooling that circulates, temperature remains on carries out in 2-8 ℃.
6, a kind of oral tablet that contains the prepared tetraodotoxin of claim 1, the content that it is characterized in that every tetraodotoxin is 10-50ng.
7, a kind of injection that contains the prepared tetraodotoxin of claim 1, the tetraodotoxin content in it is characterized in that every is 10-50ng.
8, a kind of transdermal absorption formulation that contains the prepared tetraodotoxin of claim 1 is characterized in that each uses monomeric tetraodotoxin content to be 10-50ng.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100131155A CN1323081C (en) | 2005-01-19 | 2005-01-19 | Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100131155A CN1323081C (en) | 2005-01-19 | 2005-01-19 | Preparation of tetraodotoxin by two-step resin method and tetraodotoxin preparation thereof |
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| CN1680382A true CN1680382A (en) | 2005-10-12 |
| CN1323081C CN1323081C (en) | 2007-06-27 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891751B (en) * | 2008-06-18 | 2011-12-21 | 上海亿年生物科技有限公司 | Method for preparing tetrodotoxin |
| EP2638908A1 (en) * | 2012-03-16 | 2013-09-18 | Phytotox SpA | Paralytic Shellfish Poison |
| CN110776662A (en) * | 2019-11-19 | 2020-02-11 | 常州大学 | Preparation method and application of modified brominated butyl rubber porous material |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1187355C (en) * | 2000-11-22 | 2005-02-02 | 南宁枫叶药业有限公司 | Method for refining high-purity tetradoxin |
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2005
- 2005-01-19 CN CNB2005100131155A patent/CN1323081C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891751B (en) * | 2008-06-18 | 2011-12-21 | 上海亿年生物科技有限公司 | Method for preparing tetrodotoxin |
| EP2638908A1 (en) * | 2012-03-16 | 2013-09-18 | Phytotox SpA | Paralytic Shellfish Poison |
| WO2013135884A1 (en) | 2012-03-16 | 2013-09-19 | Phytotox Spa | Paralytic shellfish poison |
| US9593120B2 (en) | 2012-03-16 | 2017-03-14 | Algenis Spa | Paralytic shellfish poison |
| CN110776662A (en) * | 2019-11-19 | 2020-02-11 | 常州大学 | Preparation method and application of modified brominated butyl rubber porous material |
| CN110776662B (en) * | 2019-11-19 | 2022-02-11 | 常州大学 | Preparation method and application of modified brominated butyl rubber porous material |
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| Publication number | Publication date |
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| CN1323081C (en) | 2007-06-27 |
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