CN102796793A - Preparation method of ant toxin - Google Patents
Preparation method of ant toxin Download PDFInfo
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- CN102796793A CN102796793A CN2012103155912A CN201210315591A CN102796793A CN 102796793 A CN102796793 A CN 102796793A CN 2012103155912 A CN2012103155912 A CN 2012103155912A CN 201210315591 A CN201210315591 A CN 201210315591A CN 102796793 A CN102796793 A CN 102796793A
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Abstract
The invention discloses a preparation method of an ant toxin, and relates to a preparation method of a medicine raw material. Considering that ant has toxic and side effects when used as a medicinal material, the preparation method of the ant toxin is realized through the technical scheme consisting of the following steps of: 1, performing crushing treatment and enzymolysis on the ant 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 ant toxin prepared by the invention is a toxin which is prepared from traditional Chinese medicinal material ant or dried ant; 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 ant 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 ant.
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.
Ant is a kind of insect that social life habit is arranged, and belongs to Arthropoda, Insecta, Hymenoptera, Formicidae.
Ant effect: tonifying kidney and benefiting sperm, clearing and activating the channels and collaterals, removing toxic substances and promoting subsidence of swelling, physique enhancing and bone strengthening, strengthening vital QI to eliminate pathogenic factors.
The ant body contains 28 seed amino acids, and wherein Threonine, phenylalanine(Phe), Xie Ansuan, Isoleucine, leucine, Methionin, methionine(Met) are the human body essential amino acid, and contains various trace elements and multivitamin.The formic acid that contains has corrodibility, and formaldehyde is bigger to people's harm.
Modern pharmacological research shows: ant has anti-ageing, anti-inflammatory, two-ways regulation immunity, protects liver, influences internal secretion, antibiotic, anticancer, relieving asthma, the effect of spasmolysis.
Existing multiple ant goods, for example Ants Wine, ant king oral liquor, ant tea etc. on the market.
CN101810644A discloses the working method and the ant oil soft capsule of ant essential oil.The present invention be with ant with after the fried mistake of plant, remove ant, treat behind the oil cooling ant essential oil.
CN101928745A discloses the Preparation method and use of ant polypeptide, and this is invented ant cleaning, drying, pulverizing, supercritical CO
2Preparation ant protein is processed ant polypeptide with enzymolysis, the enzyme that goes out, sterilization, filtration, concentrated, drying again.
CN101991841A discloses a kind of pharmaceutical composition of treating cancer and preparation method thereof, and said composition is made up of Formica fusca, glossy ganoderma, selenium, protein powder.
CN102145127A discloses a kind of medicine of treating rheumatoid arthritis and preparation method thereof, and medicine is made up of Yunnan Dog Button, Formica fusca, scorpio, centipede, trypterygine, monkshood, the root of Chinese wild ginger, Radix Angelicae Sinensis, genseng, pseudo-ginseng, Ligusticum wallichii, rhizoma Gastrodiae, Wooly Datchmanspipe Herb, honey.
CN101954034A discloses a kind of Chinese medicine preparation of treating AIDS and preparation method thereof, forms reagent combination by Radix Codonopsis, the bighead atractylodes rhizome, Formica fusca, the root of large-flowered skullcap, Herb Gynostemmae Pentaphylli, Poria cocos, fructus amomi, Herba Andrographis, glossy ganoderma etc.
CN101919997A discloses a kind of prescription of treating neurodermatitis, is made up of the Paederus densipennis Bernh. of living, old tea, Chen Aiye, old ginger, garlic.
Yang Gui equality people studies the chemical ingredients of ant, and the Polyhachis vicina Roger protein contnt reaches 40-67%.
Ant can be used for the compatibility of multiple medicine as Chinese medicinal materials in principle, but not only contains protein in the chemical ingredients of ant, amino acid, VITAMINs, trace element, formaldehyde, formic acid, and it is definite to also have many compositions to still need.Therefore, comprehensive exploitation, thing to the greatest extent its material, can examine when producing, controlled, consistency of product is that Chinese medicinal materials is used for medicament preparation and needs the problem that solves.
Summary of the invention
The purpose of this invention is to provide the anatoxic preparation method of a kind of ant, this method has been eliminated allergen and toxic side effect that the ant body has, has made full use of the ant resource.
The objective of the invention is to realize: be raw material with the ant 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 ant, 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 ant toxoid bullion liquid phase;
(4) ant 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 ant toxoid, under 20-30 ℃ of condition, adsorb 10-24h, become stable ant toxoid.
The ant toxoid of the present invention preparation be dried with Chinese medicinal materials ant or ant be a kind of toxoid of feedstock production, its relative molecular weight is 35 * 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).Polyhachis vicina Roger in the ant is the specified unique a kind of dietotherapeutic ant of health ministry, and ant contains protein 45%, and contains fat and compositions such as lipid acid and aldehyde compound, and after chemical detoxification was handled, the allergen of ant was eliminated.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; Technological process is easy to control, the ant toxoid definite ingredients of preparation, good product consistency; 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 ant to put into mincer and rub the ant 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 6h-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 ant toxoid bullion liquid phase;
(4) 1L ant toxoid bullion liquid phase places microwave oven, and 1-10min carries out deactivation in power 0.5-2kw heating;
(5) 100ml ant 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 10h-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 ant toxoid 1000L adding 200-1000L content is 5-20g/L Al (OH)
3In the solution, under 20-30 ℃ of condition, adsorb 10h-24h, become stable ant toxoid.
Embodiment 2:
(1) 1000g ant 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 ant 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 ant toxoid bullion;
(5) 1000ml ant toxoid bullion is handled 2-5min with 10000-15000 Gauss magnetic induction, handles 3-5 deactivation;
(6) 1Kg ant 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 ant 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 ant toxoid.
Claims (5)
1. the anatoxic preparation method of ant is characterized in that said preparation method is following:
(1) is raw material with the ant, 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 ant toxoid bullion liquid phase, control pH=5.8-8.0, temperature 25-45 ℃;
(4) ant 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 ant toxoid, under 20-30 ℃ of condition, adsorb 10-24h, become stable ant toxoid.
2. the anatoxic preparation method of ant according to claim 1 is characterized in that said proteolytic enzyme is stomach en-or trypsinase.
3. the anatoxic preparation method of ant 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 ant 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 ant according to claim 1 is characterized in that said stablizer is Al (OH)
3Solution or Ca
3(PO
4)
2Solution.
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|---|---|---|---|
| CN2012103155912A CN102796793A (en) | 2012-08-31 | 2012-08-31 | Preparation method of ant toxin |
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|---|---|---|---|
| CN2012103155912A CN102796793A (en) | 2012-08-31 | 2012-08-31 | Preparation method of ant toxin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349329A (en) * | 2016-08-31 | 2017-01-25 | 惠州学院 | Method for extracting solenopsis invicta venom proteins |
-
2012
- 2012-08-31 CN CN2012103155912A patent/CN102796793A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349329A (en) * | 2016-08-31 | 2017-01-25 | 惠州学院 | Method for extracting solenopsis invicta venom proteins |
| CN106349329B (en) * | 2016-08-31 | 2019-07-26 | 惠州学院 | A kind of extracting method of red fire ant venom protein matter |
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Application publication date: 20121128 |