Method for improving solubility of acetylisovaleryltylosin tartrate in trichloromethane

Abstract

The invention relates to a method for improving the solubility of acetylisovaleryltylosin tartrate in trichloromethane, which comprises the following steps: performing acid dissolution impurity removal and alkalization crystal growing treatment on acetylisovaleryltylosin tartrate fermentation liquor, dissolving with trichloromethane to form turbid liquid, performing secondary filtration treatment, and finally performing spray drying to obtain the acetylisovaleryltylosin tartrate. The production process of the acetylisovaleryltylosin tartrate is optimized, the dissolvability of the acetylisovaleryltylosin tartrate in trichloromethane is effectively improved while the product quality is guaranteed, the dissolvability of the acetylisovaleryltylosin tartrate can reach 10% or above, the quality of a later preparation is guaranteed, and the method is more suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to a method for improving the solubility of tylvalosin tartrate in chloroform, belonging to the technical field of antibiotic extraction. Background Art

[0002] Tylvalosin is also known as acetylisovaleryl tylosin (3-acetyl-4"-isovalery tylosin, AIV), and at the same time, it is also called super tylosin. It is a macrolide antibiotic, mainly obtained by fermentation of Streptomyces thermotolerans, and was initially discovered by Elanco Animal Health Limited in the UK. Tylvalosin is alkaline and usually exists stably as salts with tartaric acid, phosphoric acid, etc. The structural formula of tylvalosin tartrate is as follows.

[0003]

[0004] Currently, the research on tylvalosin mainly involves the biosynthetic pathway of tylvalosin and the fermentation process of tylvalosin, etc. The biosynthetic pathway shows that many intermediate products will be produced during the synthesis of tylvalosin, such as 2'-O-acetyl tylosin, 2',4”'-O-diacetyl tylosin, 2',4”,4”'-O-triacetyl tylosin, and then isovaleric anhydride is added to synthesize 2',4”'-O-diacetyl-4”-O-isovaleryl tylosin, 3,2',4”'-O-triacetyl-4”-O-isovaleryl tylosin, and finally 3-O-acetyl-4”-O-isovaleryl tylosin (acetylisovaleryl tylosin) is obtained. The products obtained by normal production according to the extraction process of tylvalosin tartrate have poor solubility in chloroform or will form flocculent precipitates after dissolution, resulting in the inability to use veterinary drug preparations produced with tylvalosin tartrate as raw materials or not meeting the quality standards of veterinary drug production, that is, flocculent precipitates will appear when 1 g of tylvalosin tartrate is dissolved in 10 ml of chloroform. Therefore, there is an urgent need for a method to improve the solubility of tylvalosin tartrate in chloroform, which is of extremely important significance for its production and sales. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the solubility of tylvalosin tartrate in chloroform. By acid-dissolving and removing impurities and alkalizing and crystal-nurturing treatment of the tylvalosin fermentation broth, combined with secondary filtration treatment of the turbid liquid formed by dissolving in chloroform, while ensuring the product quality, the solubility of tylvalosin tartrate is effectively improved, the quality of the subsequent preparation is ensured, and it is more suitable for industrial production.

[0006] The technical solution adopted to achieve the above invention purpose is as follows:

[0007] A method for improving the solubility of tylvalosin tartrate in chloroform, comprising the following steps:

[0008] (1) Adjust the pH value of the tylvalosin fermentation broth to 3.5 - 4.0 with acid, and obtain the tylvalosin acidified solution after solid-liquid separation;

[0009] (2) Adjust the pH value of the tylvalosin acidified solution obtained in step (1) to 6.5 - 7.0 with alkali, and obtain the crude tylvalosin after crystallization and solid-liquid separation;

[0010] (3) Dissolve the crude tylvalosin obtained in step (2) in chloroform and let it stand to obtain a turbid tylvalosin chloroform solution;

[0011] (4) Filter the turbid solution obtained in step (3) once, and obtain the primary filtrate of tylvalosin after solid-liquid separation;

[0012] (5) Filter the primary filtrate obtained in step (4) through a liquid filter for a second time, and obtain the secondary filtrate of tylvalosin after solid-liquid separation;

[0013] (6) Obtain the refined tylvalosin after spray drying the secondary filtrate obtained in step (5);

[0014] (7) Dissolve the refined tylvalosin obtained in step (6) in water, adjust the pH to 3.5 - 4.0 with solid tartaric acid, and then spray dry to obtain the finished product of tylvalosin tartrate.

[0015] Among them, the acid in step (1) is an aqueous solution of sulfuric acid or hydrochloric acid with a concentration of 8.0 - 10.0 wt%.

[0016] Among them, the alkali in step (2) is an aqueous solution of sodium hydroxide or potassium hydroxide with a concentration of 8.0 wt%.

[0017] Among them, the crystallization temperature in step (2) is 50 - 70 °C.

[0018] Among them, the crystallization time in step (2) is 25 - 30 min.

[0019] Among them, the standing time in step (3) is 1.5 - 2.5 h.

[0020] Among them, the primary filtration in step (4) is filtration with diatomaceous earth as the base.

[0021] Among them, the liquid filter in step (5) is a 0.45 μm titanium rod filter.

[0022] Among them, the inlet air temperature of the spray drying in steps (6) and (7) is 150 °C, and the outlet air temperature is 60 °C.

[0023] Among them, the temperature for dissolving tylvalosin in step (7) in water is 0 to 10°C.

[0024] Among them, after the tylvalosin described in step (7) is dissolved in water at 0 to 10°C, the titer in the solution is 100,000 to 150,000 μg / ml, and then solid tartaric acid is added to adjust the pH end point to 3.5 to 4.0.

[0025] The technical solution of the present invention has at least the following beneficial technical effects:

[0026] 1. When 1 g of tylvalosin tartrate is dissolved in 10 ml of chloroform, flocculants appear and cannot meet the quality standards. After preparation manufacturers use such raw materials to produce preparations, flocculants will also appear and cannot be sold, resulting in waste of resources and economic losses. Through this invention, the solubility of tylvalosin can be effectively improved, and the obtained solution is clear and transparent; its solubility reaches more than 10%.

[0027] 2. Effectively ensure the product quality, yield and purity data. Specific embodiments

[0028] The following will clearly and completely describe the implementation scheme of the present invention in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The description of at least one exemplary embodiment below is actually only illustrative and in no way constitutes a limitation on the present invention and its application or use. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0029] The HPLC detection method for the following examples and comparative examples: Chromatographic conditions: Octadecylsilane-bonded silica gel is used as the filler; acetonitrile - 0.15 mol / L ammonium acetate solution - acetic acid (45:45:10) is used as the mobile phase; the flow rate is 1.5 ml / min, and the detection wavelength is 280 nm. Calculated by the peak area normalization method, and do parallel in the same way. The content of tylvalosin A should not be less than 80%.

[0030] Quality standard: According to the CVP quality standard requirements, tylvalosin tartrate is easily soluble in chloroform. Weigh 1 g of tylvalosin tartrate finished product and add it to 10 ml of chloroform, stir for 30 s until completely dissolved, and after standing for 5 min, the solution is clear and transparent without precipitation and flocculants.

[0031] Example 1

[0032] Take 50 m of tylvalosin fermentation broth with a tank discharge titer of 27,000 U / ml 3, with a total of 1350 U per billion, add 8% hydrochloric acid to adjust the pH of the fermentation broth to 3.5, and let it stand for acidification for 1 h; after removing the bacterial residue by plate and frame filtration, use 8% liquid caustic soda solution to adjust the pH of the acidified liquid back to 6.5, raise the temperature to 50 °C, carry out crystal cultivation for 25 min, and then filter through plate and frame to obtain crude tilmicosin; dissolve the obtained crude tilmicosin with chloroform, let it stand for 1.5 h, then filter with diatomaceous earth as a base to obtain the primary filtrate, and then filter with a 0.45 μm titanium rod filter to obtain the secondary filtrate; the filtrate is spray-dried to obtain tilmicosin; finally, dissolve it with water at 0 °C, add solid tartaric acid to adjust the pH to 3.5, and spray-dry to obtain tilmicosin tartrate. Weigh 1 g of the obtained tilmicosin tartrate, add it to 10 ml of chloroform solution, stir for 30 s until completely dissolved, let it stand for 5 min, the solution is clear and transparent, with a purity of 89% and a yield of 94.2%.

[0033] Example 2

[0034] Take 50 m of tilmicosin fermentation broth with a discharging tank titer of 27000 U / ml 3 , with a total of 1350 U per billion, add 9% sulfuric acid to adjust the pH of the fermentation broth to 3.7, and let it stand for acidification for 1.5 h; after removing the bacterial residue by plate and frame filtration, use 8% liquid caustic soda solution to adjust the pH of the acidified liquid back to 6.7, raise the temperature to 60 °C, carry out crystal cultivation for 27 min, and then filter through plate and frame to obtain crude tilmicosin; dissolve the obtained crude tilmicosin with chloroform, let it stand for 2.0 h, first filter with diatomaceous earth as a base to obtain the primary filtrate, and then filter with a 0.45 μm titanium rod filter to obtain the secondary filtrate; the filtrate is spray-dried to obtain tilmicosin; finally, dissolve it with water at 5 °C, add solid tartaric acid to adjust the pH to 3.7, and spray-dry to obtain tilmicosin tartrate. Weigh 1 g of the obtained tilmicosin tartrate, add it to 10 ml of chloroform solution, stir for 30 s until completely dissolved, let it stand for 5 min, the solution is clear and transparent, with a purity of 88.5% and a yield of 94.3%.

[0035] Example 3

[0036] Take 50 m of tilmicosin fermentation broth with a discharging tank titer of 27000 U / ml 3, with a total of 1.35 billion units being 1350U, 10% hydrochloric acid was added to adjust the pH of the fermentation broth to 4.0, and it was allowed to stand and acidify for 2 h. After removing the bacterial residue by plate-and-frame filtration, the pH of the acidified solution was adjusted back to 7.0 with 8% liquid caustic soda solution, the temperature was raised to 70 °C, and crystallization was carried out for 30 min, followed by plate-and-frame filtration to obtain crude tilmicosin. The obtained crude tilmicosin was dissolved in chloroform, allowed to stand for 2.5 h, first filtered with diatomaceous earth as a base to obtain a primary filtrate, and then filtered with a 0.45 μm titanium rod filter to obtain a secondary filtrate. The filtrate was spray-dried to obtain tilmicosin. Finally, it was dissolved in water at 10 °C, and solid tartaric acid was added to adjust the pH to 4.0, and then spray-dried to obtain tilmicosin tartrate. 1 g of the obtained tilmicosin tartrate was weighed and added to 10 ml of chloroform solution, stirred for 30 s until completely dissolved, allowed to stand for 5 min, and the solution was clear and transparent, with a purity of 88.2% and a yield of 94.5%.

[0037] Comparative Example 1

[0038] The tilmicosin fermentation broth was acidified to pH 3.8 with 8% sulfuric acid, acidified for 10 min until stable, and then filtered through a plate-and-frame filter to obtain the acidified solution. The pH was then adjusted to 6.7 with 8% sodium hydroxide solution, the temperature was slowly raised to 50 °C, crystallization was carried out for 30 min, and the crude tilmicosin was obtained by plate-and-frame filtration. It was then dissolved in citric acid and the pH was adjusted to 3.8, cooled to 5 °C to obtain a solution, activated carbon was added, stirred, and filtered to obtain a decolorized solution. The pH was adjusted to 6.7 with 8% liquid caustic soda solution, the temperature was slowly raised to 60 °C, crystallization was carried out for 30 min, and after plate-and-frame filtration and spray-drying, tilmicosin was obtained. Finally, it was dissolved in water at 5 °C, and solid tartaric acid was added to adjust the pH to 4.0, and then spray-dried to obtain tilmicosin tartrate. 1 g of the obtained tilmicosin tartrate was weighed and added to 10 ml of chloroform solution, stirred for 30 s until completely dissolved, the solution was light black, allowed to stand for 5 min, and there were no flocculants in the solution, with a purity of 84.6% and a yield of 94.7%.

[0039] Comparative Example 2

[0040] The tilmicosin fermentation broth was acidified to pH 3.8 with 8% sulfuric acid, acidified for 10 min until stable, and then filtered through a plate-and-frame filter to obtain the acidified solution. The pH was then adjusted to 6.7 with 8% sodium hydroxide solution, the temperature was slowly raised to 50 °C, crystallization was carried out for 30 min, and the crude tilmicosin was obtained by plate-and-frame filtration. It was then dissolved in citric acid and the pH was adjusted to 3.8, filtered with diatomaceous earth as a base, and after filtration, the pH was adjusted to 6.7 with 8% liquid caustic soda solution, the temperature was slowly raised to 60 °C, crystallization was carried out for 30 min, and after plate-and-frame filtration and spray-drying, tilmicosin was obtained. Finally, it was dissolved in water at 5 °C, and solid tartaric acid was added to adjust the pH to 4.0, and then spray-dried to obtain tilmicosin tartrate. 1 g of the obtained tilmicosin tartrate was weighed and added to 10 ml of chloroform solution, stirred for 30 s until completely dissolved, there were flocculants after standing for 5 min, with a purity of 85.1% and a yield of 94.4%.

[0041] Comparative Example 3

[0042] The tilmicosin fermentation broth was acidified to pH 3.8 with 8% sulfuric acid. After being stable for 10 min, the acidified solution was obtained by plate-and-frame filtration. Then, the pH was adjusted to 6.7 with 8% sodium hydroxide solution, and the temperature was slowly raised to 50 °C. After crystallization for 30 min, the crude tilmicosin was obtained by plate-and-frame filtration. Then, it was dissolved in chloroform, and activated carbon was added and stirred for decolorization. The decolorized solution was filtered through a 0.45-μm titanium rod filter, and then through plate-and-frame filtration and spray drying to obtain tilmicosin. Finally, it was dissolved in water at 5 °C, and solid tartaric acid was added to adjust the pH to 4.0, and then spray dried to obtain tilmicosin tartrate. 1 g of the obtained tilmicosin tartrate was weighed and added to 10 ml of chloroform solution. After stirring for 30 s until completely dissolved and standing for 5 min, the solution was clear and transparent, with a purity of 86.5% and a yield of 94.3%.

[0043] The tilmicosin tartrate prepared in the above Examples 1-3 and Comparative Examples 1-3 was tested for its solubility according to the test method specified in Article 16 of the General Provisions in the First Part of Chinese Veterinary Pharmacopoeia 2020 Edition. The results are shown in Table 1.

[0044] Table 1 Solubility data of Examples and Comparative Examples

[0045] Serial number Category Solubility 1 Example 1 12.5％ 2 Example 2 13.4％ 3 Example 3 11.9％ 4 Comparative Example 1 7.4％ 5 Comparative Example 2 8.6％ 6 Comparative Example 3 9.2％

Claims

1. A method for improving the solubility of tylvalosin tartrate in chloroform, characterized in that It includes the following steps: (1) Adjust the pH value of the tilmicosin fermentation broth to 3.5 - 4.0 with acid, and obtain the tilmicosin acidified solution after solid-liquid separation; (2) Adjust the pH value of the tilmicosin acidified solution obtained in step (1) to 6.5 - 7.0 with alkali, and obtain the crude tilmicosin after crystallization and solid-liquid separation; (3) Dissolve the crude tilmicosin obtained in step (2) in chloroform and let it stand to obtain a turbid tilmicosin chloroform solution; (4) Filter the turbid solution obtained in step (3) once, and obtain the primary tilmicosin filtrate after solid-liquid separation; (5) Filter the primary filtrate obtained in step (4) through a liquid filter for a second time, and obtain the secondary tilmicosin filtrate after solid-liquid separation; (6) Obtain the high-quality tilmicosin after spray drying the secondary filtrate obtained in step (5); (7) Dissolve the high-quality tilmicosin obtained in step (6) in water, add solid tartaric acid to adjust the pH to 3.5 - 4.0, and then spray dry to obtain the finished product of tilmicosin tartrate.

2. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, wherein The acid described in step (1) is an aqueous solution of sulfuric acid or hydrochloric acid with a concentration of 8.0 - 10.0 wt%.

3. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, wherein The alkali described in step (2) is an aqueous solution of sodium hydroxide or potassium hydroxide with a concentration of 8.0 wt%.

4. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, characterized in that The crystallization temperature described in step (2) is 50 - 70 °C.

5. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, characterized in that The crystallization time described in step (2) is 25 - 30 min.

6. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, characterized in that The standing time described in step (3) is 1.5 - 2.5 h.

7. The method for improving the solubility of tiamulin tartrate in chloroform according to claim 1, characterized in that The primary filtration described in step (4) is filtration with diatomaceous earth as the base layer.

8. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, wherein The liquid filter described in step (5) is a 0.45 μm titanium rod filter.

9. The method for improving the solubility of tiamulin tartrate in chloroform according to claim 1, characterized in that The inlet air temperature and outlet air temperature of the spray drying described in steps (6) and (7) are 150 °C and 60 °C respectively.

10. The method for improving the solubility of tylvalosin tartrate in chloroform according to claim 1, wherein The temperature for dissolving the tilmicosin in water described in step (7) is 0 - 10 °C.

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