CN114539336B - Purifying method of avilamycin A - Google Patents
Purifying method of avilamycin A Download PDFInfo
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- CN114539336B CN114539336B CN202210079519.8A CN202210079519A CN114539336B CN 114539336 B CN114539336 B CN 114539336B CN 202210079519 A CN202210079519 A CN 202210079519A CN 114539336 B CN114539336 B CN 114539336B
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- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
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Abstract
The invention provides a purification method of avilamycin A, which comprises the following steps: A. adding ethyl acetate into mycelium containing avilamycin for leaching; B. concentrating the leaching solution under reduced pressure to obtain concentrated solution; C. adding chloro-n-butane and methylimidazole into a round-bottom flask, and performing rotary evaporation to obtain chlorinated 1-butyl-3-methylimidazole ionic liquid; D. preparing ionic liquid and water into a solution according to the molar ratio, placing the solution and concentrated solution into a constant-temperature oscillator, oscillating and standing; E. and D, layering the two-phase mixed liquid obtained in the step D in a liquid separating device, and dissolving out and crystallizing the upper ethyl acetate phase to obtain the avilamycin A. The invention has simple operation and can realize the separation of single components without using a liquid chromatographic column. Compared with the traditional solvent, the ionic liquid has the characteristics of low volatility, environmental protection, good chemical stability and thermal stability. The content of the avilamycin A can reach more than 95 percent.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a purification method of avilamycin A.
Background
The avilamycin belongs to oligosaccharide antibiotics of the family of the normal saccharomycin, is produced by fermenting Streptomyces viridochromogenes Tu 57, is a novel metabolism regulator and digestion promoter, mainly inhibits gram-positive bacteria, and has poor inhibition effect on gram-negative bacteria. Now, avilamycin of A-N14 different components is found, wherein the activity of the A component is the highest. The avilamycin has the advantages of small toxic and side effects, high antibacterial property, small residue, environmental safety, remarkable growth promoting effect and the like. As a new somatostatin feed additive, avilamycin has been widely used in livestock feeds.
The production technology of the avilamycin is monopolized by American Gift company, and the separation and purification of the avilamycin are less in related research in China and the technology is not mature. Most of domestic enterprises use premix with known content as a reference substance to control production, so that larger deviation of analysis results is generated. Therefore, the preparation of the single-component pure avilamycin can provide effective guarantee for the detection of the sample containing avilamycin, and has great practical significance for the research and production of avilamycin.
The efficient separation of the compounds with similar structures is one of the most challenging problems in the chemical industry, and the traditional separation method has the defects of low selectivity, high solvent consumption, high energy consumption and the like. The ionic liquid is a novel ionic compound which is completely composed of anions and cations and is in a liquid state near room temperature, has the characteristics of almost no volatilization, good stability, environmental protection and easiness in forming liquid-liquid two phases, provides a novel platform for the development of liquid-liquid extraction technology, and is widely studied in the separation of various substances such as biomacromolecules. The research of the ionic liquid-molecular solvent composite extractant provides a feasible way for solving the problems of high viscosity, high cost and the like of the ionic liquid.
CN 103012519A is prepared by leaching mycelium containing avilamycin with organic solvent, then crystallizing, silica gel column chromatography, eluting with ODS column, separating, recrystallizing, suction filtering, rinsing the crystal with pure water, oven drying to obtain pure product of avilamycin a and B, and purifying with complicated steps and twice column chromatography.
CN106146579 a extracts mycelium containing avilamycin by methanol solvent, then obtains pure product of avilamycin a and B by crystallization, sodium methoxide reaction, basic alumina column chromatography, recrystallization, etc., and the operation steps are complex.
Therefore, there is a need for a purification method of avilamycin a that is environmentally friendly, simple to operate and can reach higher purity.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a green, environment-friendly and efficient purifying method of avilamycin A.
The invention adopts the technical scheme that:
a purifying method of avilamycin A comprises the following steps:
A. adding ethyl acetate into mycelium containing avilamycin for leaching, stirring at room temperature, and filtering to obtain leaching solution;
B. concentrating the leaching solution obtained in the step A under reduced pressure to obtain concentrated solution;
C. adding n-butyl chloride and methylimidazole into a round-bottom flask, and refluxing for 8 hours at 100 ℃ under magnetic stirring; rotary evaporating to remove excessive chloro-n-butane to obtain chloridized 1-butyl-3-methylimidazole ionic liquid;
D. c, preparing a solution by using the 1-butyl-3-methylimidazole chloride ionic liquid and the water in a molar ratio, and adding the solution and the concentrated solution obtained in the step B into a triangular flask according to the volume ratio; then putting the triangular flask into a constant temperature oscillator, oscillating at the speed of 200r/min, and standing;
E. the two-phase mixed liquid obtained in the step D is placed in a liquid separating device for layering, an upper ethyl acetate phase is taken for dissolving out crystallization, and a lower ionic liquid phase is recovered;
F. e, placing the upper ethyl acetate phase in the step E into a crystallizer, stirring at normal temperature, and adding n-hexane into the liquid while stirring for crystallization;
G. and F, carrying out solid-liquid separation on the crystal slurry obtained in the step F, and drying the separated crystals under reduced pressure to obtain the avilamycin A.
Further, the volume ratio of mycelium to ethyl acetate in the step A is 1:8.
Further, the leaching solution in the step B is concentrated under reduced pressure by 6-9 times.
Further, the mole fraction of water in the aqueous solution of 1-butyl-3-methylimidazole chloride in the step D is 0.4-0.6; the volume ratio of the concentrated solution to the 1-butyl-3-methylimidazole chloride aqueous solution is 3:1-1:1.
Further, in the step D, the temperature is 30-35 ℃ when the device is oscillated at constant temperature and kept still.
Further, in the step F, the volume ratio of the ethyl acetate to the n-hexane is 1:2.
Further, the temperature at the time of drying in the step G is 50 ℃.
The beneficial effects obtained by the invention are as follows: the invention has simple operation and can realize the separation of single components without using a liquid chromatographic column. Compared with the traditional solvent, the ionic liquid has the characteristics of low volatility, environmental protection, good chemical stability and thermal stability. The content of the avilamycin A can reach more than 95 percent.
Drawings
FIG. 1 shows the content of each component in the mycelium of avilamycin;
FIG. 2 shows the content of the A component of the avilamycin after purification.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
A purifying method of avilamycin A comprises the following steps:
A. adding ethyl acetate into mycelium containing avilamycin for leaching, wherein the volume ratio of the mycelium to the ethyl acetate is 1:8, stirring at room temperature, and filtering to obtain leaching solution;
B. concentrating the leaching solution obtained in the step A under reduced pressure, wherein the multiple is 6-9 times, so as to obtain concentrated solution;
C. adding n-butyl chloride and methylimidazole into a round-bottom flask, and refluxing for 8 hours at 100 ℃ under magnetic stirring; rotary evaporating to remove excessive chloro-n-butane to obtain chloridized 1-butyl-3-methylimidazole ionic liquid;
D. c, preparing a solution by using the 1-butyl-3-methylimidazole chloride ionic liquid and the water in a molar ratio, and adding the solution and the concentrated solution obtained in the step B into a triangular flask according to the volume ratio; then placing the triangular flask into a constant temperature oscillator, oscillating at a speed of 200r/min at a temperature of 30-35 ℃, and then standing at a temperature of 30-35 ℃;
E. the two-phase mixed liquid obtained in the step D is placed in a liquid separating device for layering, an upper ethyl acetate phase is taken for dissolving out crystallization, and a lower ionic liquid phase is recovered;
F. e, placing the upper ethyl acetate phase in the step E into a crystallizer, stirring at normal temperature, and adding n-hexane into the liquid while stirring for crystallization;
G. and F, carrying out solid-liquid separation on the crystal slurry obtained in the step F, and drying the separated crystals under reduced pressure at 50 ℃ to obtain the avilamycin A.
The mole fraction of water in the chloridized 1-butyl-3-methylimidazole aqueous solution in the step D is 0.4-0.6; the volume ratio of the concentrated solution to the 1-butyl-3-methylimidazole chloride aqueous solution is 3:1-1:1. And in the step F, the volume ratio of the ethyl acetate phase to the n-hexane is 1:2.
Example 1
A. 50g of mycelium containing avilamycin, wherein the content of each component in the mycelium is shown in figure 1, is taken, 400mL of acetone is added for leaching, and the mixture is stirred and filtered at room temperature to obtain 390mL of leaching solution.
B. And (c) concentrating 390mL of the leaching solution obtained in the step A under reduced pressure to obtain 45mL of concentrated solution.
C. N-butane chloride and methylimidazole were added to the round bottom flask and refluxed for 8 hours at 100deg.C under magnetic stirring. Excess chloro-n-butane was removed by rotary evaporation to give chlorinated 1-butyl-3-methylimidazole ionic liquid as pale yellow transparent liquid, which was a white solid after cooling.
D. 45mL of the concentrate and 15mL of 1-butyl-3-methylimidazole chloride aqueous solution (water mole fraction: 0.4) were added to a triangular flask, followed by putting into a constant temperature shaker, shaking at a speed of 200r/min at 30℃and then standing at the same temperature.
E. And D, placing the two-phase mixed liquid obtained in the step D in a liquid separating device, layering, taking an upper ethyl acetate phase for next-step elution crystallization, and recovering a lower ionic liquid phase.
F. And E, placing 40mL of the upper ethyl acetate phase in the step E in a crystallizer, stirring at normal temperature, and adding 80mL of n-hexane into the liquid while stirring for crystallization.
G. And F, carrying out solid-liquid separation on the crystal slurry in the step F, and drying under reduced pressure after crystallization after separation, thereby obtaining the avilamycin A with the HPLC content of 97%.
Example two
A. 30g of mycelium containing avilamycin was extracted by adding 240mL of methanol, stirred at room temperature for 1 hour and filtered to obtain 240mL of extract.
B. And (c) concentrating 240mL of the leaching solution obtained in the step A under reduced pressure to obtain 30mL of concentrated solution.
C. N-butane chloride and methylimidazole were added to the round bottom flask and refluxed for 8 hours at 100deg.C under magnetic stirring. Excess chloro-n-butane was removed by rotary evaporation to give chlorinated 1-butyl-3-methylimidazole ionic liquid as pale yellow transparent liquid, which was a white solid after cooling.
D. 30mL of the concentrate and 30mL of 1-butyl-3-methylimidazole chloride aqueous solution (water mole fraction: 0.6) were added to a triangular flask, followed by putting into a constant temperature shaker, shaking at a speed of 200r/min at 35℃and then standing at the same temperature.
E. And D, placing the two-phase mixed liquid obtained in the step D in a liquid separating device, layering, taking an upper ethyl acetate phase for next-step elution crystallization, and recovering a lower ionic liquid phase.
F. And E, placing 30mL of the upper ethyl acetate phase in the step E in a crystallizer, stirring at normal temperature, and adding 60mL of n-hexane into the liquid while stirring for crystallization.
G. And f, carrying out solid-liquid separation on the crystal slurry in the step f, and drying under reduced pressure after crystallization after separation, so as to obtain the avilamycin A with the HPLC content of 98%, as shown in figure 2.
Example III
A. 80g of mycelium containing avilamycin is extracted by adding 640mL of methanol, stirred for 1h at room temperature and filtered to obtain 620mL of extract.
B. And (3) concentrating 620mL of the leaching solution obtained in the step A under reduced pressure to obtain 80mL of concentrated solution.
C. N-butane chloride and methylimidazole were added to the round bottom flask and refluxed for 8 hours at 100deg.C under magnetic stirring. Excess chloro-n-butane was removed by rotary evaporation to give chlorinated 1-butyl-3-methylimidazole ionic liquid as pale yellow transparent liquid, which was a white solid after cooling.
D. 80mL of the concentrate and 40mL of 1-butyl-3-methylimidazole chloride aqueous solution (water fraction 0.6) were added to a triangular flask, followed by putting in a constant temperature shaker, shaking at a speed of 200r/min at 35℃and then standing at the same temperature.
E. And D, placing the two-phase mixed liquid obtained in the step D in a liquid separating device, layering, taking an upper ethyl acetate phase for next-step elution crystallization, and recovering a lower ionic liquid phase.
F. And E, placing 80mL of the upper ethyl acetate phase in the step E in a crystallizer, stirring at normal temperature, and adding 160mL of n-hexane into the liquid while stirring for crystallization.
G. And F, carrying out solid-liquid separation on the crystal slurry in the step F, and drying under reduced pressure after crystallization after separation, thereby obtaining the avilamycin A with the HPLC content of 96%.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (7)
1. A purifying method of avilamycin A is characterized in that: the method comprises the following steps:
A. adding ethyl acetate into mycelium containing avilamycin for leaching, stirring at room temperature, and filtering to obtain leaching solution;
B. concentrating the leaching solution obtained in the step A under reduced pressure to obtain concentrated solution;
C. adding n-butyl chloride and methylimidazole into a round-bottom flask, and refluxing for 8 hours at 100 ℃ under magnetic stirring; rotary evaporating to remove excessive chloro-n-butane to obtain chloridized 1-butyl-3-methylimidazole ionic liquid;
D. c, preparing a solution by using the 1-butyl-3-methylimidazole chloride ionic liquid and the water in a molar ratio, and adding the solution and the concentrated solution obtained in the step B into a triangular flask according to the volume ratio; then putting the triangular flask into a constant temperature oscillator, oscillating at the speed of 200r/min, and standing;
E. the two-phase mixed liquid obtained in the step D is placed in a liquid separating device for layering, an upper ethyl acetate phase is taken for dissolving out crystallization, and a lower ionic liquid phase is recovered;
F. e, placing the upper ethyl acetate phase in the step E into a crystallizer, stirring at normal temperature, and adding n-hexane into the liquid while stirring for crystallization;
G. and F, carrying out solid-liquid separation on the crystal slurry obtained in the step F, and drying the separated crystals under reduced pressure to obtain the avilamycin A.
2. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: and in the step A, the volume ratio of the mycelium to the ethyl acetate is 1:8.
3. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: the leaching solution in the step B is decompressed and concentrated to be 6-9 times.
4. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: the mole fraction of water in the chloridized 1-butyl-3-methylimidazole aqueous solution in the step D is 0.4-0.6; the volume ratio of the concentrated solution to the 1-butyl-3-methylimidazole chloride aqueous solution is 3:1-1:1.
5. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: and D, oscillating at constant temperature and standing at the temperature of 30-35 ℃.
6. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: and in the step F, the volume ratio of the ethyl acetate phase to the n-hexane is 1:2.
7. The method for purifying avilamycin a according to claim 1, wherein the method comprises the following steps: the temperature at the time of drying in the step G is 50 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB948638A (en) * | 1959-07-23 | 1964-02-05 | Ciba Ltd | New antibiotic and process for its manufacture |
| CN101326275A (en) * | 2005-10-07 | 2008-12-17 | 阿拉巴马大学 | Multi-functional ionic liquid compositions |
| CN102977168A (en) * | 2012-12-17 | 2013-03-20 | 石家庄市兴柏生物工程有限公司 | Extraction and preparation method of abamectin B2a |
| CN106008624A (en) * | 2015-08-06 | 2016-10-12 | 河北圣雪大成制药有限责任公司 | Crystallization method for increasing content of avilamycin effective components A and B |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9278134B2 (en) * | 2008-12-29 | 2016-03-08 | The Board Of Trustees Of The University Of Alabama | Dual functioning ionic liquids and salts thereof |
| US20130252945A1 (en) * | 2012-03-23 | 2013-09-26 | Los Alamos National Security, Llc | Amorphous bioinorganic ionic liquid compositions comprising pharmaceuticals |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB948638A (en) * | 1959-07-23 | 1964-02-05 | Ciba Ltd | New antibiotic and process for its manufacture |
| CN101326275A (en) * | 2005-10-07 | 2008-12-17 | 阿拉巴马大学 | Multi-functional ionic liquid compositions |
| CN102977168A (en) * | 2012-12-17 | 2013-03-20 | 石家庄市兴柏生物工程有限公司 | Extraction and preparation method of abamectin B2a |
| WO2014094405A1 (en) * | 2012-12-17 | 2014-06-26 | 石家庄市兴柏生物工程有限公司 | Method for extraction and preparation of avermectin b2a |
| CN106008624A (en) * | 2015-08-06 | 2016-10-12 | 河北圣雪大成制药有限责任公司 | Crystallization method for increasing content of avilamycin effective components A and B |
Non-Patent Citations (2)
| Title |
|---|
| Kinetic study of Michael addition catalyzed by N-methylimidazole in ionic liquids: Residual N-methylimidazole in ionic liquids as a strong base;Meciarova, M等;Eur. J. Org. Chem.(第26期);4408-4411 * |
| 离子液体微波辅助萃取天然产物的研究进展;吴晟,张旭;甘肃科技;第37卷(第13期);36-39 * |
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