A method for extracting lincomycin hydrochloride

Abstract

The application relates to an extraction method of lincomycin hydrochloride, which comprises the following steps: S1, solid-liquid separation of lincomycin fermentation liquor after alkalization to obtain an alkalized liquor; S2, extraction of the alkalized liquor with a low-carbon alcohol to obtain an extraction liquor; S3, impurity removal and solid-liquid separation of the extraction liquor through a polyamide-ODS reverse-phase silica gel column to obtain a lincomycin separation liquor; and S4, decolorization of the lincomycin separation liquor with activated carbon, acidification and nanofiltration concentration in sequence. Through the low-carbon alcohol extraction, the polyamide-ODS reverse-phase silica gel column separation and the flocculation impurity removal technology, the technical problem that the content of group A is high and the content of group B is low in the lincomycin hydrochloride extraction and separation process is effectively solved, the lincomycin hydrochloride purification process is optimized, and the product quality of the lincomycin hydrochloride is improved.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic production technology, specifically relating to a method for extracting lincomycin hydrochloride. Background Technology

[0002] Lincomycin, also known as lincomycin or lincomycin hydrochloride, is an antibiotic isolated by Mason from Streptomyces cultures in 1962. Its main components are lincomycin A hydrochloride and lincomycin B hydrochloride, with lincomycin A hydrochloride being the predominant component. Its molecular formula is C. 18 H 34 N₂O₆S has a relative molecular weight of 406.56. The structural difference between the two components is that component A has a n-propyl group at position 4, while component B has an ethyl group. Therefore, components A and B have significant pharmacological differences; the effective activity of component B is only 25% of that of component A, and component B is more toxic. Pharmacopoes of various countries have corresponding requirements for the content of lincomycin hydrochloride B in finished lincomycin hydrochloride products; the proportion of component B must not exceed 5% of the sum of components A and B.

[0003] Numerous studies have addressed the production of lincomycin, including its biosynthetic pathway and extraction and purification processes. These studies primarily focus on improving lincomycin potency and reducing the potency of lincomycin B component during fermentation. Traditional processes using high-carbon alcohols, such as 2-octanol, suffer from strong odors, are environmentally unfriendly, have high boiling points and recovery temperatures leading to high impurity content, and offer limited removal of impurities through dissolution and crystallization, resulting in minimal optimization of the product's effective components. Traditional resin adsorption separation processes suffer from long production cycles and large amounts of resin regeneration wastewater, necessitating further improvements in resin performance. While the extraction process increases the content of lincomycin A component, the high content of other impurities, such as lincomycin B component, negatively impacts lincomycin potency. Given these shortcomings in existing lincomycin purification processes, a solution is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting lincomycin hydrochloride. By using low-carbon alcohol extraction pretreatment followed by separation and impurity removal using polyamide-ODS reversed-phase silica gel column chromatography, the technical problem of high content of component A and low content of component B in the extraction and separation process of lincomycin hydrochloride is effectively solved, thus optimizing the purification process of lincomycin hydrochloride and improving the product quality of lincomycin hydrochloride.

[0005] The technical solution adopted to achieve the above-mentioned objectives is as follows:

[0006] A method for extracting lincomycin hydrochloride, comprising the following steps:

[0007] S1: After alkalizing the lincomycin fermentation broth, the solid and liquid phases are separated to obtain the alkalized solution;

[0008] S2: Extract the alkalinized solution with a low-carbon alcohol to obtain an extract;

[0009] S3: The extract is subjected to solid-liquid separation by polyamide-ODS reversed-phase silica gel column chromatography to obtain lincomycin separation solution;

[0010] S4: The lincomycin separation solution is sequentially decolorized with activated carbon, acidified, and concentrated by nanofiltration to obtain the lincomycin hydrochloride product.

[0011] In step S1, the alkalization is performed by adjusting the pH value to 10-13 with sodium hydroxide solution.

[0012] Preferably, the concentration of the sodium hydroxide solution is 20%-30%.

[0013] In step S2, the lower alcohol is n-pentanol.

[0014] Preferably, the volume ratio of n-pentanol to alkalizing liquid is 1:1.

[0015] In step S3, the polyamide-ODS reverse silica gel column combination involves placing the polyamide in the upper layer and the ODS reverse silica gel in the lower layer.

[0016] Preferably, the polyamide-ODS reverse silica gel column filling volume ratio is 1:1.

[0017] Preferably, the silica gel column is filled with ODS reversed-phase silica gel in a 1:1 ratio in the lower layer and polyamide in the upper layer to form a polyamide-ODS reversed-phase silica gel column. The extract is then passed through the polyamide-ODS reversed-phase silica gel column for flocculation and impurity removal at a flow rate of 0.5 L / min, a temperature of 30 °C, and a pH of 10–13.

[0018] In step S4, acidification is performed by adjusting the pH value to 2-4 with hydrochloric acid.

[0019] Preferably, the concentration of the hydrochloric acid solution is 30%-40%.

[0020] The activated carbon decolorization process in step S4 further includes a plate and frame filtration step.

[0021] The lincomycin fermentation broth is obtained by fermentation culture of Streptomyces Lincolnensis using conventional methods in the art.

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

[0023] 1) In the extraction step, the present invention uses low-carbon alcohol to replace high-carbon alcohol, which lowers the boiling point of the recovered solvent and effectively avoids the thermal degradation of lincomycin. Then, the polyamide-ODS reversed-phase silica gel column is used for flocculation separation to remove impurities, which increases the content of lincomycin component A and reduces the content of component B. In addition, compared with the resin adsorption separation process, the production cycle is shortened by more than 20% compared with the traditional extraction process.

[0024] 2) The optimized lincomycin hydrochloride extraction method of this invention produces lincomycin hydrochloride products with a yield of over 78.9%, an A content of over 98.2%, and a B content of less than 1%. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] In the embodiments of the present invention, the lincomycin-containing fermentation broth is a fermentation broth of microorganisms conventionally used in the art for the preparation of lincomycin, generally obtained by fermentation culture of *Streptomyces lincolnensis* using conventional methods in the art. There is no particular limitation on the specific *Streptomyces lincolnensis* species; any species conventionally capable of producing lincomycin can be used, such as *Streptomyces lincolnensis* with accession number CCTCCM208064.

[0027] In this embodiment of the invention, the lincomycin content was determined using a high-performance liquid chromatography (USP standard) method. The chromatographic conditions were as follows: C8 packing material (4.6 mm * 25 cm * 5 μm); mobile phase: phosphoric acid solution: acetonitrile: methanol = 785:155:155; detection wavelength: 210 nm; flow rate: 1 ml / min; column oven temperature: 45 °C; injection volume: 10 μL. After injection and detection, the relative retention times of lincomycin B and lincomycin A components were recorded, and the content was calculated by peak area using the external standard method.

[0028] Example 1

[0029] Take 50 m3 of lincomycin fermentation broth with a potency of 9500 μg / ml (475 U / b), add 20% sodium hydroxide solution for alkalization, add n-pentanol to the alkalized solution, stir and mix evenly, maintain the temperature at 50-60℃ and the pH at 10-13, and allow to stand for extraction and separation for 1 hour to obtain the extract. In a silica gel column, pack the lower layer with ODS reversed-phase silica gel and the upper layer with polyamide to form a polyamide-ODS reversed-phase silica gel column. Filter the extract through the polyamide-ODS reversed-phase silica gel column for flocculation and impurity removal at a flow rate of 0.5 L / min, a temperature of 30℃, and a pH of 10-13. Then, decolorize by physical adsorption with activated carbon, remove the activated carbon by plate and frame filtration to obtain a decolorized solution. Acidify the decolorized solution with 30% hydrochloric acid solution to prepare lincomycin hydrochloride. Concentrate the acidified solution through nanofiltration membrane to obtain the final lincomycin hydrochloride product. The obtained product was analyzed by HPLC, and the yield was 79.3%, the content of lincomycin A hydrochloride was 98.7%, and the content of lincomycin B hydrochloride was 0.6%.

[0030] Example 2

[0031] Take 50 m3 of lincomycin fermentation broth with a potency of 9600 μg / ml (480 U / b), add 25% sodium hydroxide solution for alkalization, then add 60% n-pentanol to the alkalized solution, stir and mix thoroughly, maintain the temperature at 50-60℃ and the pH at 10-13, and allow to stand for extraction and separation for 1 hour to obtain the extract. In a silica gel column, pack the lower layer with ODS reversed-phase silica gel and the upper layer with polyamide to form a polyamide-ODS reversed-phase silica gel column. Filter the extract through the polyamide-ODS reversed-phase silica gel column for flocculation and impurity removal at a flow rate of 0.5 L / min, a temperature of 30℃, and a pH of 10-13. Then, decolorize by physical adsorption with activated carbon, remove the activated carbon by plate and frame filtration to obtain a decolorized solution. Acidify the decolorized solution with 35% hydrochloric acid solution to prepare lincomycin hydrochloride. Concentrate the acidified solution through nanofiltration membrane to obtain the final lincomycin hydrochloride product. The obtained product was analyzed by HPLC, and the yield was 79.4%, the content of lincomycin A hydrochloride was 98.2%, and the content of lincomycin B hydrochloride was 0.9%.

[0032] Example 3

[0033] Take 50 m3 of lincomycin fermentation broth with a potency of 9700 μg / ml (485 U / b), add 30% sodium hydroxide solution for alkalization, then add 60% n-pentanol to the alkalized solution, stir and mix thoroughly, maintain the temperature at 50-60℃ and the pH at 10-13, allow to stand for extraction and separation for 1 hour to obtain the extract. In a silica gel column, pack the lower layer with ODS reversed-phase silica gel and the upper layer with polyamide to form a polyamide-ODS reversed-phase silica gel column. Filter the extract through the polyamide-ODS reversed-phase silica gel column for flocculation and impurity removal at a flow rate of 0.5 L / min, a temperature of 30℃, and a pH of 10-13. Then, decolorize by physical adsorption with activated carbon, remove the activated carbon by plate and frame filtration to obtain a decolorized solution. Acidify the decolorized solution with 40% hydrochloric acid solution to prepare lincomycin hydrochloride. Concentrate the acidified solution through nanofiltration membrane to obtain the final lincomycin hydrochloride product. The obtained product was analyzed by HPLC, and the yield was 78.9%, the content of lincomycin A hydrochloride was 98.5%, and the content of lincomycin B hydrochloride was 0.7%.

[0034] Comparative Example 1

[0035] After the fermentation broth was placed in tanks, it was alkalized with sodium hydroxide, extracted with n-pentanol, filtered through a ceramic membrane, and then decolorized with activated carbon. The activated carbon was removed by filtration to obtain a decolorized solution. Hydrochloric acid solution was added to the decolorized solution for acidification to generate lincomycin hydrochloride solution. Finally, acetone was added for evaporation and crystallization to obtain lincomycin hydrochloride. The obtained product was analyzed by HPLC, with a yield of 76.9%, a lincomycin A hydrochloride content of 97.3%, and a lincomycin B hydrochloride content of 1.7%.

[0036] Comparative Example 2

[0037] After the fermentation broth was placed in a tank, it was alkalized with sodium hydroxide and extracted with 2-octanol. The mixture was stirred and stirred until homogeneous, and the temperature was maintained at 50-60℃ and the pH at 10-13. After standing for 1 hour to separate the layers, the extract was obtained. A silica gel column was formed by packing ODS reversed-phase silica gel in a 1:1 ratio in the lower layer and polyamide in the upper layer, creating a polyamide-ODS reversed-phase silica gel column. The extract was then subjected to flocculation separation and impurity removal on the polyamide-ODS reversed-phase silica gel column at a flow rate of 0.5 L / min, a temperature of 30℃, and a pH of 10-13. Activated carbon was added for decolorization, and the activated carbon was removed by filtration to obtain a decolorized solution. Hydrochloric acid solution was added to the decolorized solution for acidification to generate lincomycin hydrochloride solution. Finally, acetone was added for evaporation and crystallization to obtain lincomycin hydrochloride. The obtained product was analyzed by HPLC, with a yield of 77.4%, a lincomycin A content of 97.7%, and a lincomycin B content of 1.3%.

[0038] Comparative Example 3

[0039] After fermentation, the broth was alkalized with sodium hydroxide, extracted with 2-octanol, filtered through a ceramic membrane, and then decolorized with activated carbon. The activated carbon was removed by filtration to obtain a decolorized solution. Hydrochloric acid solution was added to the decolorized solution for acidification to generate lincomycin hydrochloride solution. Finally, nanofiltration was used for crystallization to obtain lincomycin hydrochloride. The obtained product was analyzed by HPLC, showing a yield of 77.2%, a lincomycin A hydrochloride content of 97.4%, and a lincomycin B hydrochloride content of 1.4%.

[0040] Comparative Example 4

[0041] After the fermentation broth was placed in tanks, it was alkalized with sodium hydroxide, extracted with 2-octanol, filtered through a ceramic membrane, and then decolorized with activated carbon. The activated carbon was removed by filtration to obtain a decolorized solution. Hydrochloric acid solution was added to the decolorized solution for acidification to generate lincomycin hydrochloride solution. Finally, acetone was added for evaporation and crystallization to obtain lincomycin hydrochloride. The obtained product was analyzed by HPLC, with a yield of 76.1%, a lincomycin A hydrochloride content of 96.5%, and a lincomycin B hydrochloride content of 2.1%.

Claims

1. A method for extracting lincomycin hydrochloride, comprising the following steps: S1: After alkalizing the lincomycin fermentation broth, the solid and liquid phases are separated to obtain the alkalized solution; S2: The alkalized solution is extracted with a low-carbon alcohol to obtain an extract, wherein... The lower alcohol is n-pentanol; S3: The extract is subjected to solid-liquid separation by polyamide-ODS reversed-phase silica column chromatography to obtain lincomycin separation solution, wherein the polyamide-ODS reversed-phase silica column chromatography is performed with polyamide in the upper layer and ODS reversed-phase silica in the lower layer. S4: The lincomycin separation solution is sequentially decolorized with activated carbon, acidified, and concentrated by nanofiltration to obtain the lincomycin hydrochloride product.

2. The extraction method of lincomycin hydrochloride according to claim 1, characterized in that... In step S1, the alkalization is performed by adjusting the pH value to 10-13 with sodium hydroxide solution.

3. The extraction method of lincomycin hydrochloride according to claim 1, characterized in that... The volume ratio of n-pentanol to alkalizing liquid is 1:

1.

4. The extraction method of lincomycin hydrochloride according to claim 1, characterized in that... The polyamide-ODS reverse silica gel column has a filling volume ratio of 1:

1.

5. The extraction method of lincomycin hydrochloride according to claim 1, characterized in that... Step S4 involves acidifying the solution with hydrochloric acid to adjust the pH value to 2-4.

6. The extraction method of lincomycin hydrochloride according to claim 1, characterized in that... Step S4, after the activated carbon decolorization, also includes a plate and frame filtration step.

7. The extraction method of lincomycin hydrochloride according to any one of claims 1-6, characterized in that: The lincomycin fermentation broth was obtained by fermenting and culturing *Streptomyces lincomycin* using conventional methods in the art.