Method for purifying nemadectin in fermentation liquor

Abstract

The invention discloses a method for purifying nemadectin in fermentation liquor, and belongs to the technical field of biological pharmacy. The method comprises the following steps: enriching a nemadectin crude product by using macroporous resin; refining the nemadectin crude product through a silica gel chromatographic column; and separating out high-purity nemadectin fine powder through crystallization. According to the purification method, the yield reaches 50% or above, the nemadectin purity can reach 98% or above, various impurities are effectively controlled, and high-purity nemadectin can be produced on a large scale.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a method for purifying nimoxetine from fermentation broth. Background Technology

[0002] Nemadectin is a macrolide antibiotic produced by Streptomyces fermentation. It possesses strong antiparasitic activity, particularly effective against mites and nematodes. Its structure is similar to avermectin, but it exhibits superior biological activity in certain areas. Nemadectin can be used as a precursor in the synthesis of highly effective antiparasitic drugs such as moxidectin.

[0003] The chemical structural formula of nimocritin is as follows: .

[0004] Currently, the purification methods for nimocritin mainly include solvent extraction, column chromatography, and crystallization. However, due to the complex composition of its fermentation broth, the variety of impurities, and their similar structures, especially its homologues and oxidation products, which are difficult to separate, traditional methods result in low yields, low purity, large solvent consumption, and serious environmental pollution, making it difficult to achieve large-scale production.

[0005] Therefore, developing a nimocriptine purification method that is simple in process, has high yield, good purity, is environmentally friendly, and suitable for industrial production is of great practical significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for purifying nimocritin from fermentation broth. This method is simple, has a high yield, good purity, uses environmentally friendly solvents, and is suitable for large-scale production.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for purifying nimocritin from fermentation broth, comprising the following steps: (a) The nimocriptine fermentation broth was separated into solid and liquid phases. The bacterial residue was extracted with a first organic solvent. The extract was subjected to macroporous resin chromatography. The eluent was collected and concentrated until no liquid residue remained. An equal volume of extractant was added for extraction twice. The organic phases of the two extractions were combined and concentrated under reduced pressure at 35-50°C to a paste to obtain the first paste. (b) Dissolve the first paste in a second organic solvent with a mass-to-volume ratio of 0.6~0.8:100 g / mL to prepare a column loading solution, perform silica gel chromatography, collect the eluent of qualified nimoxine components, concentrate to a paste, and obtain the second paste. (c) Dissolve the second paste in a third organic solvent with a mass-to-volume ratio of 1.2~1.6:100 g / mL, add n-hexane dropwise until crystals precipitate, filter and dry using 30~50μm qualitative filter paper to obtain the finished product Nimoxetine.

[0008] In step (a), the specific method of performing macroporous resin chromatography on the extract includes: loading the extract onto a macroporous adsorption resin and eluting with an aqueous solution of a first organic solvent with a volume concentration of 50% to 70%, wherein the first organic solvent is selected from ethanol, acetone, and methanol, preferably ethanol.

[0009] Preferably, the macroporous adsorption resin is selected from polystyrene resins, and more preferably LX-T28, LX-30, LX-60, LX-T83, LX-98, PAD900, and SP-207.

[0010] More preferably, LX-30, PAD900, and SP-207.

[0011] In step (b), the specific steps of silica gel chromatography include: loading the obtained loading solution onto a silica gel chromatography column, using a hexane solution with a volume concentration of 55% as the mobile phase, and performing chromatographic detection, wherein the second organic solvent is selected from ethyl acetate and dichloromethane, preferably ethyl acetate.

[0012] Preferably, the packing material of the silica gel chromatography column is selected from MEG silica gel, BANGKAI silica gel, and marine silica gel.

[0013] Marine silicone is preferred.

[0014] Preferably, the silica gel chromatography has a pore size of 100 mesh to 200 mesh.

[0015] Preferably, in step (c), the third organic solvent is selected from ethyl acetate and dichloromethane, with dichloromethane being more preferred.

[0016] Preferably, the extractant is ethyl acetate.

[0017] The present invention has the following beneficial effects: This invention enriches crude nimoxetine with macroporous resin, then refines it by silica gel chromatography, and finally obtains high-purity nimoxetine by crystallization. This method is simple, uses environmentally friendly solvents, has a high yield (over 50%), and good purity (≥98%), making it suitable for industrial production. Attached Figure Description

[0018] Figure 1 The HPLC chromatogram of nimocriptine fermentation broth; Figure 2 The HPLC chromatogram of the nimocriptine product in Example 7 is shown. Detailed Implementation

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available products and can be purchased on the market; among them, Bangkai silicone was purchased from Bangkai Company, Meigao silicone was purchased from Meigao Technology Company, and Haiyang silicone was purchased from Qingdao Haiyang Silicone Company.

[0021] The fermentation strain used in this embodiment of the invention is a Gram-positive Streptomyces hydrophila obtained by our company. Streptomyces hygroscopicus The sample was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34582 and deposit date of May 19, 2025.

[0022] The Gram-positive Streptomyces hydrophila and its mutant strains used in this invention, such as mutant strains that produce nimocritin obtained through X-ray irradiation, ultraviolet irradiation, nitrogen mustard treatment, or genetic engineering, are also included within the scope of this invention.

[0023] The present invention will be further described below through embodiments, but these descriptions are not intended to further limit the scope of the invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the present invention still fall within the protection scope of the present invention.

[0024] Example 1 The fermentation broth obtained from the fermentation of nimoxetine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue. The residue was then extracted with ethanol to obtain an ethanol extract. 1 L of the ethanol extract containing nimoxetine (2.3 g of nimoxetine) was loaded onto an LX-30 resin column. After loading, the column was washed with water and then eluted with a 50% ethanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 40°C until no liquid residue remained. 800 mL of chloroform was added for extraction. After stirring and allowing to stand, the chloroform phase was separated and concentrated to a paste. 50 mL of ethyl acetate was added to dissolve the chloroform phase, yielding the preparative column buffer. The concentration was 36.8 mg / mL. This preparative column buffer was packed into a silica gel column. The mobile phase was a 50:50 (V:V) solution of ethyl acetate and n-hexane. The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. After filtration and drying, 1.33 g of the finished product meeting USP quality standards was obtained (total impurities = 1.69%; chromatographic purity of the nimoxetine main peak = 98.31%), with an overall yield of 57.83%.

[0025] Example 2 The fermentation broth obtained from the fermentation of nimoxetine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue, which was then extracted with methanol to obtain a methanol extract. 1 L of the methanol extract containing nimoxetine (2.3 g of nimoxetine) was loaded onto an SP-207 resin column. After loading, the column was washed with water, followed by elution with a 60% methanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 45°C until no liquid residue remained. 800 mL of dichloromethane was added for extraction, and after stirring and separation, a dichloromethane phase was obtained. This dichloromethane phase was then concentrated to a paste, dissolved in 50 mL of dichloromethane to obtain the preparative column buffer. The concentration was 36.8 mg / mL. The preparative column buffer was packed into a marine silica gel column. The mobile phase was ethyl acetate:n-hexane = 40:60 solution (V:V). The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. The product was filtered and dried to obtain 1.41 g of the finished product that met the USP quality standard (total impurities = 1.89%; chromatographic purity of the nimoxetine main peak = 98.11%), with an overall yield of 61.3%.

[0026] Example 3 The fermentation broth obtained from the fermentation of nimocriptine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue. The residue was then extracted with acetone to obtain an acetone extract. 1 L of the acetone extract containing nimocriptine (2.3 g of nimocriptine) was loaded onto an LX-60 resin column. After loading, the column was washed with water, followed by elution with 70% acetone-water (V:V) solution. The eluent was concentrated under reduced pressure at 35°C until no liquid residue remained. 800 mL of ethyl acetate was added for extraction. After stirring and allowing to stand, the ethyl acetate phase was separated and concentrated to a paste. 50 mL of ethyl acetate was added to dissolve the paste, yielding the preparative column buffer. The concentration was 36.8 mg / mL. This preparative column buffer was packed into a Bonker silica gel chromatography column. The mobile phase was ethyl acetate:n-hexane = 60:40 solution (V:V). The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. The product was filtered and dried to obtain 1.16 g of the finished product that met the USP quality standard (total impurities = 1.21%; chromatographic purity of the nimoxetine main peak = 98.79%), with an overall yield of 50.43%.

[0027] Example 4 The fermentation broth obtained from the fermentation of nimocriptine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue. The residue was then extracted with acetone to obtain an ethanol extract. 1 L of the ethanol extract containing nimocriptine (2.3 g of nimocriptine) was loaded onto a PAD900 resin column. After loading, the column was washed with water and then eluted with a 55% ethanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 50°C until no liquid residue remained. 800 mL of hexane was added for extraction. After stirring and allowing to stand, the liquid was separated to obtain the hexane phase. The hexane phase was then concentrated to a paste and dissolved in 50 mL of dichloromethane to obtain the preparative column buffer. The concentration was 36.8 mg / mL. This preparative column buffer was packed into a marine silica gel column. The mobile phase was ethyl acetate:n-hexane = 45:55 (V:V). The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. After filtration and drying, 1.26 g of the finished product meeting USP quality standards was obtained (total impurities = 1.51%; chromatographic purity of the nimoxetine main peak = 98.49%), with an overall yield of 54.78%.

[0028] Example 5 The fermentation broth obtained from the fermentation of nimoxetine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue. The residue was then extracted with ethanol to obtain an ethanol extract. 1 L of the ethanol extract containing nimoxetine (2.3 g of nimoxetine) was loaded onto an LX-98 resin column. After loading, the column was washed with water, followed by elution with a 65% ethanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 40°C until no liquid residue remained. 800 mL of ethyl acetate was added for extraction. After stirring and allowing to stand, the ethyl acetate phase was separated and concentrated to a paste. 50 mL of ethyl acetate was added to dissolve the paste, yielding the preparative column buffer. The concentration was 36.8 mg / mL. This preparative column buffer was packed into a silica gel column. The mobile phase was ethyl acetate:n-hexane = 55:45 (V:V). The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. The product was filtered and dried to obtain 1.23 g of the finished product that met the USP quality standard (total impurities = 1.56%; chromatographic purity of the nimoxetine main peak = 98.44%), with an overall yield of 53.48%.

[0029] Example 6 The fermentation broth obtained from the fermentation of nimocriptine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue, which was then extracted with ethanol to obtain an ethanol extract. 1 L of the ethanol extract containing nimocriptine (containing 2.3 g of nimocriptine) was loaded onto an LX-30 resin column. After loading, the column was washed with water, followed by elution with a 55% ethanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 50°C until no liquid residue remained. 800 mL of dichloromethane was added for extraction, and after stirring and separation, a dichloromethane phase was obtained. This dichloromethane phase was then concentrated to a paste, dissolved in 50 mL of dichloromethane to obtain the preparative column buffer. The concentration was 36.8 mg / mL. The preparative column buffer was packed into a Bonker silica gel chromatography column. The mobile phase was ethyl acetate:n-hexane = 50:50 solution (V:V). The effective components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified fraction eluted with silica gel was concentrated to a paste. 14 mL of ethyl acetate was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. The product was filtered and dried to obtain 1.3 g of the finished product that met the USP quality standard (total impurities = 1.34%; chromatographic purity of the nimoxetine main peak = 98.66%), with an overall yield of 56.52%.

[0030] Example 7 The fermentation broth obtained from the fermentation of nimocriptine-producing bacteria was separated into solid and liquid phases using a plate and frame filter press to obtain filter residue, which was then extracted with ethanol to obtain an ethanol extract. 1 L of the ethanol extract containing nimocriptine (2.3 g of nimocriptine) was loaded onto an LX-30 resin column. After loading, the column was washed with water, followed by elution with a 65% ethanol-water (V:V) solution. The eluent was concentrated under reduced pressure at 45°C until no liquid residue remained. 800 mL of ethyl acetate was added for extraction, and after stirring and settling, the ethyl acetate phase was obtained. This ethyl acetate phase was then concentrated to a paste, and 50 mL of ethyl acetate was added to dissolve it, yielding the preparative column buffer. The concentration was 36.8 mg / mL. This preparative column buffer was packed into a silica gel column. The mobile phase was an ethyl acetate:n-hexane solution (55:45, V:V). The active components were collected, and the total impurities were controlled to be ≤2.5%. The chromatographic purity of the nimoxetine main peak was ≥97.5%. The qualified components eluted with silica gel were concentrated to a paste. 14 mL of dichloromethane was added to the pre-crystallization solution at a concentration of 10:100 g / mL. Then, n-hexane was added dropwise until a solid precipitated. The product was filtered and dried to obtain 1.44 g of the finished product that met the USP quality standard (total impurities = 0.98%; chromatographic purity of the nimoxetine main peak = 99.02%), with an overall yield of 62.61%.

[0031] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made without creative effort should be included within the protection scope of the present invention.

Claims

1. A method for purifying nimocidin in a fermentation broth, characterized by, The method comprises the following steps: The fermentation liquor of nimorazole is subjected to solid-liquid separation, the residue is extracted with a first organic solvent, the extract is subjected to macroporous resin chromatography, the eluate is collected and concentrated to no liquid residue, extracted twice with an equal volume of extractant, and the organic phases of the two extractions are combined and concentrated to a paste at 35-50 DEG C under reduced pressure to obtain a first paste; The first paste is dissolved in a second organic solvent in a mass-volume ratio of 0.6-0.8:100 g / mL to prepare column loading liquid, subjected to silica gel chromatography, the eluate of the qualified nimorazole component is collected and concentrated to a paste to obtain a second paste; The second paste is dissolved in a third organic solvent in a mass-volume ratio of 1.2-1.6:100 g / mL, and n-hexane is added dropwise until crystals are precipitated, which are filtered and dried with 30-50 mu m qualitative filter paper to obtain finished nimorazole.

2. The purification method of claim 1, wherein, The macroporous resin chromatography of the extract is specifically as follows: the extract is loaded onto macroporous adsorption resin, and eluted with a 50%-70% volume concentration aqueous solution of the first organic solvent.

3. The purification method of claim 2, wherein, The first organic solvent is selected from ethanol, acetone and methanol, and is preferably ethanol.

4. The purification method of claim 1, wherein, The macroporous adsorption resin is selected from polystyrene resins, and is preferably LX-T28, LX-30, LX-60, LX-T83, LX-98, PAD900 and SP-207, and is more preferably LX-30, PAD900 and SP-207.

5. The purification method of claim 1, wherein, The silica gel chromatography specifically comprises the following steps: the obtained column loading liquid is loaded onto a silica gel chromatography column, and a 55% volume concentration n-hexane solution of the second organic solvent is used as the mobile phase for chromatographic detection.

6. The purification method of claim 5, wherein, The second organic solvent is selected from ethyl acetate and dichloromethane, and is preferably ethyl acetate.

7. The purification method of claim 5, wherein, The filler of the silica gel chromatography column is selected from Meigu silica gel, Bangke silica gel and Haoyan silica gel, and is preferably Haoyan silica gel.

8. The purification method of claim 7, wherein, The pore size of the silica gel chromatography is 100-200 mesh.

9. The purification method of claim 1, wherein, The third organic solvent is selected from ethyl acetate and dichloromethane, and is preferably dichloromethane.

10. The purification method of claim 1, wherein, The extractant is ethyl acetate.

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