CN102796794A - Preparation method of cantharis toxin - Google Patents
Preparation method of cantharis toxin Download PDFInfo
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- CN102796794A CN102796794A CN2012103158639A CN201210315863A CN102796794A CN 102796794 A CN102796794 A CN 102796794A CN 2012103158639 A CN2012103158639 A CN 2012103158639A CN 201210315863 A CN201210315863 A CN 201210315863A CN 102796794 A CN102796794 A CN 102796794A
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- blister beetle
- chinese blister
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Abstract
The invention discloses a preparation method of a cantharis toxin, and relates to a preparation method of a medicine raw material. Considering that cantharis has toxic and side effects when used as a medicinal material, the preparation method of the cantharis toxin is realized through the technical scheme consisting of the following steps of: 1, performing crushing treatment and enzymolysis on the cantharis serving as a raw material; 2, performing solid-liquid separation by a separator; 3, detoxifying; 4, deactivating by a chemical method or a physical method; 5, filtering to remove impurities through microfiltration and concentrating through ultrafiltration; 6, performing ultrasonic standing wave liquid phase preparation and chromatographic purification; and 7, performing stabilizing treatment. The cantharis toxin prepared by the invention is a toxin which is prepared from traditional Chinese medicinal material cantharis or dried cantharis; screening of a strain and a culture medium for producing the conventional toxin is not involved in a preparation process; the preparation method has the advantages of simple production process, energy conservation, no environmental pollution and easiness in control over process; and the prepared cantharis toxin has the advantages of clear components, low cost and suitability for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of pharmaceutical raw material, the anatoxic preparation method of especially a kind of Chinese blister beetle.
Background technology
Specificity toxicant-bacteriotoxin that a kind of pathogenic bacteria produced keeps immunogenicity through detoxification treatment, is referred to as toxoid.Toxoid is a kind of active immunity preparation, is used for the prevention and the treatment of bacterial poison disposition disease.
The anatoxic method of existing production commonly used is to select substratum and bacterial classification, and bacterial classification produces toxin after artificial culture, and detoxicated back forms toxoid.Very sophisticated at present toxoid has products such as Toxoid,tetanus, DT, cholera toxoid.
CN101869704A discloses single dose immunization against tetanus toxin cation dextran microspheres and preparation method thereof, it is characterized in that: be loaded on the positively charged ion Rocryl 400 dextran microspheres after through electrostatic interaction by Toxoid,tetanus and make; CN87104279 discloses the toxoid novel preparation method, comprise with a certain amount of oxidizer treatment at least the isolating toxin of part but keep its immunity simultaneously so that said toxin is carried out chemical ablation, after this therefrom obtain the toxin of deactivation; CN1798548 discloses based on containing adsorbed toxoid or contain the immunogenic composition of polysaccharide antigen microsphere, and this invention is toxoid antigen or the antigen that contains polysaccharide to be adsorbed on form immunogenic composition on the microsome.
Chinese blister beetle is the dry body of Meloidae insect south mylabris phalerata or yellow black mylabris cichorii; Hot, heat, very toxic; Return liver, stomach kidney channel, have broken knot and attack poison, dehematize is long-pending; The function of dredging water passages, southern mylabris phalerata contain Cantharidin 1-1.2%, wax 12% and resin, formic acid, yellow black mylabris cichorii contains Cantharidin 0.97-1.3%.
Human history with the existing two thousand years of Chinese blister beetle treatment disease, because Chinese blister beetle toxicity is bigger, so the synthetic various Chinese blister beetle verivates of people reach kind more than 100.
CN1966508 discloses a kind of method of extracting Cantharidin, comprises cold soaking, concentrates, technological process such as purifying.
CN1023367565A discloses the method for from Chinese blister beetle, extracting Cantharidin, uses acetone extraction, liquid concentrator crystallisation by cooling, secondary dissolving cooling crystallization.
CN101948528A discloses a kind of fibrinolytic protein from Chinese blister beetle, from Chinese blister beetle, extracts a kind of one-component albumen with fibrinolytic.
CN101633661 discloses the technology of preparation disodium cantharidinate, extracts Cantharidin and synthetic disodium cantharidinate by the Chinese blister beetle polypide.
CN1868471 discloses a kind of N-methylcantharidimide injection formulations and preparation method thereof, in N-methylcantharidimide, adds auxiliary agents such as phosphatide, Schardinger dextrins, Vinylpyrrolidone polymer, urea and increases the water-soluble of N-methylcantharidimide.
The purpose that tradition is concocted is in order to alleviate its toxicity, and wet rice commonly used pastes the pot stir-baked MYLABRIS with rice or fries two kinds of concocting methods heating and makes that the loss of Chinese blister beetle effective ingredient is more, and how making full use of this a kind of herb resource of Chinese blister beetle is the problem that domestic and international medicine circle studies for a long period of time.
Summary of the invention
In view of Chinese blister beetle has toxic side effect during as medicinal material, the present invention provides a kind of Chinese blister beetle anatoxic preparation method.
The objective of the invention is to realize: be raw material with the Chinese blister beetle 1,, after pulverization process, add proteolytic enzyme and carry out enzyme digestion reaction through following technological case; 2, separating machine solid-liquid separation; 3, detoxification; 4, chemical method or the deactivation of physics method; 5, micro-filtration filtering and impurity removing and ultrafiltration process concentrate; 6, ultrasonic standing wave liquid phantom preparing chromatogram purifying; 7, stabilization treatment.Concrete steps are following:
(1) is raw material with the Chinese blister beetle, after pulverization process, adds proteolytic enzyme and carry out enzyme digestion reaction;
(2) separate above-mentioned enzymolysis solution with separating machine with 3000-15000r/min;
(3) regulate collection liquid pH=5.8-8.0, controlled temperature 25-45 ℃, add H
2O
2Solution or formaldehyde carry out detoxification treatment, become Chinese blister beetle toxoid bullion liquid phase;
(4) Chinese blister beetle toxoid bullion liquid phase adopts chemical method or physics method to carry out inactivation treatment;
(5) filter with membrane pore size 0.2-5 μ m microporous filter, and, collect ultrafiltrated with membrane pore size 10-100nm ultrafilter uf processing;
(6) ultrafiltrated is dissolved in the solvent system, pumps into separation and purification continuously in the ultrasonic standing wave liquid phantom preparing chromatogram, ultrasonic standing wave frequency 2450-5000kHz, sound intensity 0.2-1.5kw, flow 0.2-1.0L/min, uv-spectrophotometric detector wavelength 260nm;
(7) stabilization treatment: add stablizer in the separation and purification Chinese blister beetle toxoid, under 20-30 ℃ of condition, adsorb 10-24h, become stable Chinese blister beetle toxoid.
The Cantharidin chemical name is outer-1,2-is suitable-and dimethyl--3,6-oxo bridge-hexahydro phthalic anhydride has severe toxicity, and the chmice acute toxicity test shows that the LD50 of Cantharidin is 1.25mg/kg, and LD50 is 82mg/kg after detoxification is handled.The Chinese blister beetle toxoid of the present invention preparation be dried with Chinese medicinal materials Chinese blister beetle or Chinese blister beetle be a kind of toxoid of feedstock production, its relative molecular weight is 37 * 10
4Da is the milk yellow unit for uniform suspension after the goods jolting, pH=6.6-7.4, and white lake content is less than 2.5mg/mL, and free formaldehyde content is less than 0.02% (g/mL).Preparation process of the present invention does not relate to bacterial classification and the screening of medium that traditional toxoid is produced, and production technique is simple, energy-conservation, non-environmental-pollution, and technological process is easy to control, the Chinese blister beetle toxoid definite ingredients of preparation, cost is lower, is fit to suitability for industrialized production.
Embodiment
Below in conjunction with embodiment the present invention is done further description.
Embodiment 1:
(1) selects for use fresh Chinese medicinal materials Chinese blister beetle to put into mincer and rub the Chinese blister beetle body,, regulate pressure 100000kpa with the further broken body of high-pressure homogenization crush method; Flow velocity is to 70cm/ml; Repeat broken 2-5 time,, collect liquid phase with whizzer 1250-3000r/min solid-liquid separation;
(2) collect liquid phase 1000ml adding 2000-10000ml pure water and mix back adding 0.5-10g stomach en-, keep 20-50 ℃ of temperature, enzymolysis 6-48h;
(3) separate above-mentioned enzymolysis solution with separating machine with 3000-15000r/min, collect liquid and use 0.2mol/LNa
2HPO
4-0.2mol/LNaH
2PO
4The damping fluid of forming is regulated pH=5.8-8.0, controlled temperature 25-45 ℃, adds 10-100gH
2O
2Solution detoxification treatment 15-45d becomes Chinese blister beetle toxoid bullion liquid phase;
(4) 1L Chinese blister beetle toxoid bullion liquid phase places microwave oven, and 1-10min carries out deactivation in power 0.5-2kw heating;
(5) 100ml Chinese blister beetle toxoid bullion liquid phase is added 0.2mol/LNa
2CO
3-0.2mol/LNaHCO
3Buffered soln is transferred pH=8-12, adds 50-100gNa again
2SO
4And the 20-200g atlapulgite, under 10-30 ℃ of condition, generate deposition and place 10-24h;
(6) above-mentioned material filters with membrane pore size 0.2-5 μ m microporous filter, and with membrane pore size 10-100nm ultrafilter uf processing, collects ultrafiltrated;
(7) the 1L ultrafiltrated of collecting is dissolved in the solvent system that 2-10L is made up of according to the volume ratio of 2:2:1:6 MTBE-propyl carbinol-acetonitrile-water, wherein adds the trifluoroacetic acid of 10-30mmol/L in the organic phase, aqueous phase adding 1-5mmol/L ammoniacal liquor; Pump in the ultrasonic standing wave liquid phantom preparing chromatogram; Ultrasonic standing wave frequency 2450-5000kHz, sound intensity 0.2-1.5kw, flow 0.2-1.0L/min; Uv-spectrophotometric detector wavelength 260nm, separation and purification continuously;
(8) separation and purification Chinese blister beetle toxoid 1000L adding 200-1000L content is 5-20g/L Al (OH)
3In the solution, under 20-30 ℃ of condition, adsorb 10-24h, become stable Chinese blister beetle toxoid.
Embodiment 2:
(1) 1000g Chinese blister beetle dry product with the 275-500r/min continuous pulverizing, and with 100-200 mesh screening, is collected powder with kibbler;
(2) the 1000g powder is put into microwave oven with power 0.5-2kw, heating 10-20min germicidal treatment naturally cools to 25-30 ℃ then;
(3) after Chinese blister beetle dry product powder adding 1-10L pure water mixes after the above-mentioned germicidal treatment, add 1.5-50g trypsinase, keep 25-50 ℃ of temperature, enzymolysis 5-48h;
(4) separate above-mentioned zymolyte with separating machine with 3000-15000r/min, the liquid of collection is used 0.2mol/L Na
2CO
3-0.2 mol/LNaHCO
3The buffered soln of forming is transferred pH=5.8-8, and controlled temperature 25-45 ℃, add 10-100g formaldehyde detoxification treatment 30-45d, become Chinese blister beetle toxoid bullion;
(5) 1000ml Chinese blister beetle toxoid bullion is handled 2-5min with 10000-15000 Gauss magnetic induction, handles 3-5 deactivation;
(6) 1Kg Chinese blister beetle quasi-mycin bullion is added ammoniacal liquor adjustment pH=7-9, add 100-300g (NH
4)
2SO
4And the 150-500g zeolite, normal temperature condition generates deposition down and inhales 10-24h;
(7) above-mentioned detoxification liquid filters with membrane pore size 0.2-10 μ m Microfilter and uses membrane pore size 10-100nm ultrafilter ultrafiltration and concentration again, collects liquid concentrator;
(8) the 1L liquid concentrator is dissolved in the solvent system that 2-10L is made up of according to the volume ratio of 2:2:1:6 MTBE-propyl carbinol-acetonitrile-water, wherein adds the 10-30mmol/L trifluoroacetic acid in the organic phase, and aqueous phase adds 1-5mmol/L ammoniacal liquor; Pump in the ultrasonic standing wave liquid phantom preparing chromatogram; Ultrasonic standing wave frequency 2450-5000kHz, sound intensity 0.2-1.5kw, flow 0.5-1.0L/min; Uv-spectrophotometric detector wavelength 260nm, separation and purification continuously;
(9) the 1L Chinese blister beetle toxoid of separation and purification adding 20.0-50.0L content is 10-20g/LCa
3(PO
4)
2In the solution, under 20-30 ℃ of condition, adsorb 10-24h, become stable Chinese blister beetle toxoid.
Claims (5)
1. the anatoxic preparation method of Chinese blister beetle is characterized in that said preparation method is following:
(1) is raw material with the Chinese blister beetle, after pulverization process, adds proteolytic enzyme and carry out enzyme digestion reaction;
(2) separate above-mentioned enzymolysis solution with separating machine with 3000-15000r/min;
(3) collect liquid and carry out detoxification treatment, become Chinese blister beetle toxoid bullion liquid phase, control pH=5.8-8.0, temperature 25-45 ℃;
(4) Chinese blister beetle toxoid bullion liquid phase adopts chemical method or physics method to carry out inactivation treatment;
(5) filter with membrane pore size 0.2-5 μ m microporous filter, and, collect ultrafiltrated with membrane pore size 10-100nm ultrafilter uf processing;
(6) ultrafiltrated is dissolved in the solvent system, pumps into separation and purification continuously in the ultrasonic standing wave liquid phantom preparing chromatogram, ultrasonic standing wave frequency 2450-5000kHz, sound intensity 0.2-1.5kw, flow 0.2-1.0L/min, uv-spectrophotometric detector wavelength 260nm;
(7) stabilization treatment: add stablizer in the separation and purification Chinese blister beetle toxoid, under 20-30 ℃ of condition, adsorb 10-24h, become stable Chinese blister beetle toxoid.
2. the anatoxic preparation method of Chinese blister beetle according to claim 1 is characterized in that said proteolytic enzyme is stomach en-or trypsinase.
3. the anatoxic preparation method of Chinese blister beetle according to claim 1 is characterized in that said solvent system is made up of according to the volume ratio of 2:2:1:6 MTBE-propyl carbinol-acetonitrile-water.
4. the anatoxic preparation method of Chinese blister beetle according to claim 3 is characterized in that adding the 10-30mmol/L trifluoroacetic acid in the organic phase of said solvent system, and aqueous phase adds 1-5mmol/L ammoniacal liquor.
5. the anatoxic preparation method of Chinese blister beetle according to claim 1 is characterized in that said stablizer is Al (OH)
3Solution or Ca
3(PO
4)
2Solution.
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| CN2012103158639A CN102796794A (en) | 2012-08-31 | 2012-08-31 | Preparation method of cantharis toxin |
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|---|---|---|---|
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059338A (en) * | 1990-08-29 | 1992-03-11 | 王日卫 | The preparation of cantharidia liposoluble pharmaceutics |
| CN1374310A (en) * | 2002-03-20 | 2002-10-16 | 贵州君之堂制药有限公司 | Prepn process of sodium cantharidinate |
| CN1634152A (en) * | 2004-10-28 | 2005-07-06 | 俞锋 | Raw material medicine of methylcantharidinimide and its preparation process |
| CN1686109A (en) * | 2005-04-01 | 2005-10-26 | 中山大学 | Application of cantharidin derivative as anti-tumour medicine |
| CN102268006A (en) * | 2011-06-15 | 2011-12-07 | 北京联合大学生物化学工程学院 | Method for extracting cantharidin from compound enzyme |
| CN102336765A (en) * | 2011-10-11 | 2012-02-01 | 江苏四环生物股份有限公司 | Method for extracting cantharidin from cantharides |
-
2012
- 2012-08-31 CN CN2012103158639A patent/CN102796794A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059338A (en) * | 1990-08-29 | 1992-03-11 | 王日卫 | The preparation of cantharidia liposoluble pharmaceutics |
| CN1374310A (en) * | 2002-03-20 | 2002-10-16 | 贵州君之堂制药有限公司 | Prepn process of sodium cantharidinate |
| CN1634152A (en) * | 2004-10-28 | 2005-07-06 | 俞锋 | Raw material medicine of methylcantharidinimide and its preparation process |
| CN1686109A (en) * | 2005-04-01 | 2005-10-26 | 中山大学 | Application of cantharidin derivative as anti-tumour medicine |
| CN102268006A (en) * | 2011-06-15 | 2011-12-07 | 北京联合大学生物化学工程学院 | Method for extracting cantharidin from compound enzyme |
| CN102336765A (en) * | 2011-10-11 | 2012-02-01 | 江苏四环生物股份有限公司 | Method for extracting cantharidin from cantharides |
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Application publication date: 20121128 |