An extraction method to improve the bioavailability of tylosin tartrate

Abstract

This invention relates to an extraction method for improving the bioavailability of tartrate tylosin. The method includes subjecting tartrate fermentation broth to a single acid-dissolution and alkali-precipitation treatment, followed by static crystallization to obtain crude tartrate. This crude tartrate is then subjected to extraction and back-extraction steps, followed by decolorization and ultrafiltration to obtain refined tartrate. Finally, it is spray-dried to obtain tartrate tylosin. This invention optimizes the extraction process of tartrate tylosin by combining a single acid-dissolution and alkali-precipitation crystallization purification process with extraction and back-extraction processes, effectively improving the bioavailability of tartrate tylosin.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic extraction technology, and in particular relates to an extraction method for improving the bioavailability of tylosin tartrate. Background Technology

[0002] Tylvaline, also known as 3-acetyl-4"-isovalery tylosin (AIV) or super tylosin, is a macrolide antibiotic primarily obtained through fermentation of *Streptomyces thermotolerans*. It was initially discovered by Eco Animal Health Products Ltd. in the UK. Tylvaline is alkaline and typically forms stable salts with tartaric acid. The structural formula of tylvaline tartrate is shown in formula (I).

[0003]

[0004] Currently, research on tylvaporin mainly involves its biosynthetic pathway and fermentation process. The biosynthetic pathway shows that tylvaporin synthesis produces numerous intermediates, such as 2'-O-acetyl-tylosin, 2',4"'-O-diacetyl-tylosin, and 2',4",4"'-O-triacetyl-tylosin. Isovalerate is then added to synthesize 2',4"'-O-diacetyl-4"-O-isovalerate tylosin and 3,2',4"'-O-triacetyl-4"-O-isovalerate tylosin, ultimately yielding 3-O-acetyl-4"-O-isovalerate tylosin (acetylisovalerate tylosin). However, the biopotency of the product obtained through normal production using the publicly available extraction process for tylvaporin tartrate is low, affecting product sales. Summary of the Invention

[0005] The purpose of this invention is to provide an extraction method for improving the bioavailability of tartrate tylosin. The method mainly involves a one-step acid-dissolution, alkaline precipitation, and crystallization purification process for tylosin fermentation broth, combined with extraction and back-extraction processes. This significantly optimizes the extraction process of tartrate tylosin and effectively enhances its bioavailability.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An extraction method for improving the bioavailability of tylosin tartrate includes the following steps:

[0008] (1) Tiamulin fermentation broth was subjected to acid dissolution and alkali precipitation once, and then allowed to stand and crystallize to obtain crude tiamulin.

[0009] (2) Dissolve the crude tylosin obtained in step (1) in water and then extract it with an extractant. Cool down and separate the solid and liquid to obtain tylosin extract.

[0010] (3) The extract obtained in step (2) is back-extracted with tartaric acid solution to obtain tylosin back-extract;

[0011] (4) The back-extraction solution obtained in step (3) is decolorized and then filtered through an ultrafiltration membrane to obtain tylosin refined solution.

[0012] (5) After spray drying the purified liquid obtained in step (4), the finished product of tartrate tylosin is obtained.

[0013] In step (1), the acid dissolution is carried out with dilute sulfuric acid, the pH of the acid dissolution is 3.8-4.0, and the acid dissolution time is 65-80 min.

[0014] In step (1), the alkaline precipitation is precipitated with an 8% liquid alkali solution, and the pH of the alkaline precipitation is adjusted to 7.1-7.5.

[0015] In step (1), the crystal growth temperature is 35-40℃ and the crystal growth time is 25-30min.

[0016] In step (2), the water dissolution temperature is 50-60℃.

[0017] In step (2), the extractant is butyl acetate.

[0018] The volume ratio of butyl acetate to filtrate is 10-12:1.

[0019] In step (3), the back-extraction solution is a 5% tartaric acid solution, the back-extraction pH is 3.6-3.8, and the back-extraction temperature is 2-5℃.

[0020] In step (4), the ultrafiltration is performed using a 0.1 μm ultrafiltration membrane.

[0021] In step (5), the inlet air temperature of the spray drying process is 150°C and the outlet air temperature is 60°C.

[0022] The technical solution of the present invention has at least the following beneficial technical effects:

[0023] This invention significantly optimizes the extraction process of tartrate tartrate tartrate by combining a single acid-dissolution, alkaline precipitation, and crystallization purification process with the synergistic effect of extraction and back-extraction. This effectively enhances the bioavailability of tartrate tartrate tartrate. The extraction method of this invention increases the bioavailability of tartrate tartrate tartrate to over 840 μg / ml. Detailed Implementation

[0024] The detection method of this invention's embodiments and comparative examples: Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile-0.15 mol / L ammonium acetate solution-acetic acid (45:45:10) was used as the mobile phase; the flow rate was 1.5 ml / min, and the detection wavelength was 280 nm. Calculations were performed using the peak area normalization method.

[0025] Example 1

[0026] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3 The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 3.8, and the mixture was stirred and acidified for 65 min. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.1 with 8% liquid alkali, and the temperature was maintained at 35℃. The mixture was allowed to stand for crystal growth for 25 min, and then filtered through a plate and frame filter to obtain crude tylosin. Next, hot water was added and stirred to dissolve the crude product. The product was then extracted with 10 times its volume of butyl acetate. After the temperature was lowered to room temperature, the extract was separated and back-extracted with 5% tartaric acid solution. The pH was adjusted to 3.6, and the temperature was maintained at 2℃. Activated carbon was then added and stirred for decolorization for 30 min. The activated carbon was removed by plate and frame filtration. The decolorized solution was filtered through a 0.1μm ultrafiltration membrane to obtain a purified solution. Finally, the purified solution was spray-dried to obtain tylosin tartrate. The biopotency of the obtained tylosin tartrate was 845 μg / ml as determined by HPLC.

[0027] Example 2

[0028] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3 The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 3.9, and the mixture was stirred and acidified for 70 min. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.3 with 8% liquid alkali, and the temperature was maintained at 37℃. The mixture was allowed to stand for crystal growth for 28 min, and then filtered through a plate and frame filter to obtain crude tylosin. Next, hot water was added and stirred to dissolve the tylosin, and then it was extracted with 11 times its volume of butyl acetate. After the temperature was lowered to room temperature, the extract was separated and back-extracted with 5% tartaric acid solution. The pH was adjusted to 3.7, and the temperature was maintained at 4℃. Activated carbon was then added and stirred for decolorization for 35 min. The activated carbon was removed by plate and frame filtration, and the decolorized solution was filtered through a 0.1μm ultrafiltration membrane to obtain a purified solution. Finally, the purified solution was spray-dried to obtain tylosin tartrate. The biopotency of the obtained tylosin tartrate was 842 μg / ml as determined by HPLC.

[0029] Example 3

[0030] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 4.0, and the mixture was stirred and acidified for 80 min. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.5 with 8% liquid alkali, and the temperature was maintained at 40℃. The mixture was allowed to stand for crystal growth for 30 min, and then filtered through a plate and frame filter to obtain crude tylosin. Next, hot water was added and stirred to dissolve the crude product. The product was then extracted with 12 times its volume of butyl acetate. After the temperature was lowered to room temperature, the extract was separated and back-extracted with 5% tartaric acid solution. The pH was adjusted to 3.8, and the temperature was maintained at 5℃. Activated carbon was then added and stirred for decolorization for 40 min. The activated carbon was removed by plate and frame filtration. The decolorized solution was filtered through a 0.1μm ultrafiltration membrane to obtain a purified solution. Finally, the purified solution was spray-dried to obtain tylosin tartrate. The biopotency of the obtained tylosin tartrate was 847 μg / ml as determined by HPLC.

[0031] Comparative Example 1

[0032] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3 The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 4.0, and the mixture was stirred and acidified for 80 minutes. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.5 with 8% liquid alkali, and the temperature was maintained at 40℃. The mixture was allowed to stand for crystal growth for 30 minutes, and then filtered through a plate and frame filter to obtain crude tylosin. Next, hot water was added and stirred to dissolve the crude product. The product was then extracted with 12 times its volume of butyl acetate. After the temperature was lowered to room temperature, the extract was separated and back-extracted with 5% tartaric acid solution. The pH was adjusted to 3.8, and the temperature was maintained at 5℃. Activated carbon was then added and stirred for decolorization for 40 minutes. The activated carbon was removed by plate and frame filtration. Finally, the purified solution was spray-dried to obtain tylosin tartrate. The biopotency of the obtained tylosin tartrate was 830 μg / ml as determined by HPLC.

[0033] Comparative Example 2

[0034] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3 The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 4.0, and the mixture was stirred and acidified for 80 minutes. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.5 with 8% liquid alkali, and the temperature was maintained at 40℃. The mixture was allowed to stand for crystal growth for 30 minutes. After plate and frame filtration, crude tivalvicin was obtained. The above acid-dissolution and alkali-precipitation steps were repeated twice to obtain tivalvicin. After dissolving in hot water, the pH was adjusted to 4.0 with tartaric acid. Activated carbon was added and stirred for decolorization for 40 minutes. The activated carbon was removed by plate and frame filtration. The decolorized solution was filtered through a 0.1μm ultrafiltration membrane to obtain a purified solution. Finally, the purified solution was spray-dried to obtain tivalvicin tartrate. The biopotency of the obtained tivalvicin tartrate was determined to be 826μg / ml by HPLC.

[0035] Comparative Example 3

[0036] Take 50 ml of tylosin fermentation broth with a potency of 28000 U / ml. 3 The total amount was 1400U. First, dilute sulfuric acid was added to adjust the pH to 4.0, and the mixture was stirred and acidified for 80 minutes. The acidified solution was obtained by plate and frame filtration. The pH was then adjusted to 7.5 with 8% liquid alkali, and the temperature was maintained at 40℃. The mixture was allowed to stand for crystal growth for 30 minutes. After plate and frame filtration, crude tivalvicin was obtained. The above acid-dissolution and alkali-precipitation steps were repeated twice to obtain tivalvicin. After dissolving in hot water, the pH was adjusted to 4.0 with tartaric acid. Activated carbon was added and stirred for decolorization for 40 minutes. The activated carbon was removed by plate and frame filtration. Finally, the purified solution was spray-dried to obtain tivalvicin tartrate. The biopotency of the obtained tivalvicin tartrate was 814 μg / ml as determined by HPLC.

Claims

1. An extraction method for improving the bioavailability of tylosin tartrate, comprising the following steps: (1) Tiamulin fermentation broth was subjected to acid dissolution and alkali precipitation once, and then allowed to stand and crystallize to obtain crude tiamulin. (2) Dissolve the crude tylosin obtained in step (1) in water and then extract it with an extractant. Cool down and separate the solid and liquid to obtain tylosin extract. (3) The extract obtained in step (2) is back-extracted with tartaric acid solution to obtain tylosin back-extract; (4) The back-extraction solution obtained in step (3) is decolorized and then filtered through an ultrafiltration membrane to obtain tylosin refined solution. (5) After spray drying the purified liquid obtained in step (4), the finished product of tartrate tylosin is obtained.

2. The extraction method according to claim 1, characterized in that... The acid dissolution in step (1) is carried out with dilute sulfuric acid, the pH of the acid dissolution is 3.8-4.0, and the acid dissolution time is 65-80 min.

3. The extraction method according to claim 1, characterized in that... The alkaline precipitation in step (1) is precipitated with an 8% liquid alkali solution, and the pH of the alkaline precipitation is adjusted to 7.1-7.

5.

4. The extraction method according to claim 1, characterized in that... The crystal growth temperature in step (1) is 35-40℃ and the crystal growth time is 25-30min.

5. The extraction method according to claim 1, characterized in that... The water dissolution temperature in step (2) is 50-60℃.

6. The extraction method according to claim 1, characterized in that... The extractant in step (2) is butyl acetate.

7. The extraction method according to claim 6, characterized in that... The volume ratio of butyl acetate to filtrate is 10-12:

1.

8. The extraction method according to claim 1, characterized in that... The back-extraction solution in step (3) is a 5% tartaric acid solution, the back-extraction pH is 3.6-3.8, and the back-extraction temperature is 2-5℃.

9. The extraction method according to claim 1, characterized in that... The ultrafiltration in step (4) is performed using a 0.1 μm ultrafiltration membrane.

10. The extraction method according to claim 1, characterized in that... The inlet air temperature of the spray dryer in step (5) is 150°C and the outlet air temperature is 60°C.

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