Efficient purification method of N-methylensansalvamide in fusarium fermentation product

Abstract

The invention discloses an efficient purification method of N-methylensansalvamide in a fusarium fermentation product, the method only needs organic solvent ultrasonic extraction, then organic solvent extraction and drying, the operation is simple, the equipment requirement is low, the consumable cost is low, and the organic solvent is convenient to recycle. After purification is completed, the purity of the obtained N-methylsansalvamide product reaches 95% or above, the recovery rate is at least 65%, and the method is suitable for large-scale fermentation preparation.

Description

(1) Technical Field

[0001] The present invention belongs to the field of chemical separation and purification, and particularly relates to a method for large-scale purification of high-purity N-methylsansalvamide. (2) Background Art

[0002] N-methylsansalvamide (abbreviated as SA) is a secondary metabolite derived from Fusarium sp., belonging to the class of cyclic depsipeptides, and has significant activity in inhibiting tumor cells (bladder cancer, colorectal cancer, breast cancer, etc.), showing great potential for new drug development. The problem of drug source is one of the important factors affecting new drug development. Efficient preparation of sufficient high-purity N-methylsansalvamide is an important guarantee for new drug development. The structural formula of N-methylsansalvamide is:

[0003]

[0004] Due to the complex components in the fermentation products of Fusarium sp., the preparation of high-purity N-methylsansalvamide is challenging. Currently, liquid chromatography technology is used for the separation and purification of N-methylsansalvamide both at home and abroad, which requires high equipment, complex operation, long time consumption, and low efficiency. There is no report on the large-scale preparation of high-purity N-methylsansalvamide. (3) Summary of the Invention

[0005] The object of the present invention is to provide an efficient purification method for N-methylsansalvamide in the fermentation products of Fusarium sp., which avoids the use of liquid chromatography instruments and chromatographic reagents in the process of its large-scale preparation, and opens up an efficient method for large-scale purification of high-purity N-methylsansalvamide.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides an efficient purification method for N-methylsansalvamide in the fermentation products of Fusarium sp., and the method comprises the following steps:

[0008] (1) Filter the fermentation products of Fusarium sp. containing N-methylsansalvamide, collect the wet mycelium, dry it to a mass water content of 5 - 9%, and crush it to obtain dry mycelium powder;

[0009] (2) After dissolving the dry mycelium powder with an organic solvent, perform ultrasonic extraction, filter it to obtain an extract, concentrate it to 15% of the original volume, and dry it to a mass water content of 7% to obtain a crude extract;

[0010] (3) After dissolving the crude extract in an organic solvent, an extractant and water are added, and the mixture is allowed to stand for extraction. The extractant layer is taken and dried to a moisture content of 3% by mass to obtain pure N-methylsansalvamide.

[0011] Preferably, the Fusarium fermentation product containing N-methylsansalvamide in step (1) is obtained by fermenting and culturing Fusarium sp. CCTCC NO: M 20241573.

[0012] Preferably, the fermentation product in step (1) is prepared according to the following steps:

[0013] 1) Inoculate Fusarium CCTCC NO: M 20241573 into a PDA medium and activate it at 30 °C for 3 d; the composition of the PDA medium is: 200.0 g / L potatoes, 20.0 g / L glucose, 15.0 - 20.0 g / L agar, natural pH, water as the solvent, sterilized at 121 °C for 20 min;

[0014] 2) Inoculate the strain obtained in step 1) into a PDB medium and culture it at 35 °C and 180 rpm for 72 h to obtain a seed solution; the composition of the PDB medium is: 200.0 g / L potatoes, 20.0 g / L glucose, natural pH, water as the solvent, sterilized at 121 °C for 20 min;

[0015] 3) Inoculate the seed solution obtained in step 2) into the fermentation medium at an inoculation amount of 5% by volume concentration and ferment it statically at 25 °C for 13 d to obtain a fermentation broth containing N-methylsansalvamide;

[0016] The composition of the fermentation medium is: 22.5 g / L sucrose, 16.5 g / L tryptone, 0.024 g / L yeast extract, 20.0 g / L sea salt, pH 6.5, water as the solvent, the liquid loading amount of the fermentation broth is 50% of the container volume, and sterilized at 121 °C for 20 min.

[0017] Preferably, in steps (1) and (2), filtration includes atmospheric pressure filtration, vacuum filtration, plate and frame pressure filtration, and centrifugation.

[0018] Preferably, in steps (1), (2) and (3), the drying method is low-temperature drying at 40 - 60 °C, vacuum drying at 35 - 45 °C, and freeze drying at -50 °C to -30 °C.

[0019] Preferably, in step (2), the organic solvent is one or a mixture of several of methanol, ethanol, propanol, isopropanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume dosage of the organic solvent is 10 - 50 mL / g (preferably 30 mL / g) based on the mass of the dry bacterial powder.

[0020] Preferably, the conditions for ultrasonic treatment in step (2) are ultrasonic treatment at 150 - 200 W for 35 - 45 min.

[0021] Preferably, in step (3), the organic solvent is one or a mixture of several of methanol, ethanol, acetone, isopropanol, and acetonitrile; the volume dosage of the organic solvent is 10 - 30 mL / g (preferably 18 - 22 mL / g) based on the mass of the crude extract.

[0022] Preferably, in step (3), the extractant is one or a mixture of several of petroleum ether, chloroform, n - hexane, cyclohexane, heptane, and toluene.

[0023] Preferably, in step (3), the volume ratio of the extractant to the organic solvent and water is 10:7 - 9:6 - 8.

[0024] Preferably, when the organic solvent in step (3) is methanol, the extractant is n - hexane, and the volume ratio of the extractant to methanol and water is 10:8:7; when the organic solvent is ethanol, the extractant is petroleum ether, and the volume ratio of the extractant to ethanol and water is 10:9:6; when the organic solvent is isopropanol, the extractant is n - hexane, and the volume ratio of the extractant to isopropanol and water is 10:7:8.

[0025] Preferably, in step (3), the number of extraction times is at least 1 time.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: The method for separating and purifying N - methylsansalvamide from the fermentation product of Fusarium only requires ultrasonic extraction with an organic solvent, followed by extraction with an organic solvent and drying. The operation is simple, the equipment requirements are low, the consumable cost is low, and the recovery of the organic solvent is convenient. After purification, the purity of the obtained N - methylsansalvamide product reaches over 95%, and the recovery rate is at least 65%. It is suitable for large - scale preparation, with low cost and high efficiency. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a flow chart for large - scale purification of high - purity N - methylsansalvamide.

[0028] Figure 2 It is a liquid chromatogram of the fermentation broth containing N - methylsansalvamide in Example 1.

[0029] Figure 3 It is a liquid chromatogram of the pure product of N - methylsansalvamide in Example 2.

[0030] Figure 4 It is a liquid chromatogram of the pure product of N - methylsansalvamide in Example 3.

[0031] Figure 5 It is the liquid chromatogram of pure N-methylsansalvamide in Example 4. (V) Specific implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Fusarium sp. R1-UV-4-3a was deposited in the China Center for Type Culture Collection on July 15, 2024, with the deposit number CCTCC NO: M 20241573, and has been disclosed in the patent application CN 119020173A.

[0034] In the embodiments of the present invention, the peak area of SA is detected by high performance liquid chromatography to calculate the content, purity and recovery rate. The specific steps are as follows:

[0035] High performance liquid chromatography detection is carried out using a Shimadzu LC-20AT high performance liquid chromatography system, equipped with a Phenomenex C18 chromatographic column (5μm, 4.6mm×250mm) and an ultraviolet detector (detection wavelength 210nm), isocratic elution, and a flow rate of 1.0 mL / min. The mobile phase is an aqueous solution containing 0.1% formic acid - acetonitrile (volume ratio 30:70).

[0036] SA standard curve equation: y = 16624x - 16725 (R 2 = 0.9999).

[0037] The purity is the ratio of the peak area of N-methylsansalvamide to the sum of all peak areas.

[0038] Example 1. Fermentation broth containing N-methylsansalvamide

[0039] (1) Inoculate Fusarium R1-UV-4-3a into a PDA medium and activate it at 30°C for 3 days. The composition of the PDA medium is: 200.0 g / L potatoes, 20.0 g / L glucose, 15.0 - 20.0 g / L agar, natural pH, the solvent is water, and sterilize at 121°C for 20 min;

[0040] (2) Inoculate the strain in step (1) into a PDB medium and culture it at 35°C and 180 rpm for 72 h to obtain a seed solution. The composition of the PDB medium is: 200.0 g / L potatoes, 20.0 g / L glucose, natural pH, the solvent is water, and sterilize at 121°C for 20 min.

[0041] (3) Inoculate the seed liquid obtained in step (2) into the fermentation medium at an inoculum volume concentration of 5%, and ferment statically at 25 °C for 13 d to obtain a fermentation broth containing N-methylsansalvamide. Collect the mycelia in the fermentation broth, extract the mycelia with an organic solvent (such as methanol), and concentrate the extract by rotary evaporation to obtain a crude extract. Dissolve the crude extract in chromatographic-grade methanol, filter it through a 0.22-μm organic membrane, and detect it by high-performance liquid chromatography. The results are shown in Figure 2 as follows.

[0042] The fermentation medium consists of: sucrose 22.5 g / L, tryptone 16.5 g / L, yeast extract 0.024 g / L, sea salt 20.0 g / L, pH 6.5, the solvent is water, the loading volume of the fermentation broth is 50% of the container volume, and sterilize at 121 °C for 20 min.

[0043] Example 2. Isolation and purification of N-methylsansalvamide

[0044] (1) Filter 500 mL of the fermentation broth prepared by the method of Example 1 with gauze, remove the filtrate to obtain wet bacterial cells, dry them in vacuo at 60 °C until the moisture content by mass is 7%, and pulverize them to obtain 2.4 g of dry bacterial powder.

[0045] (2) Take 1 g of the dry bacterial powder, add 30 mL of acetone, ultrasonically extract it for 40 minutes under the condition of 175 W, filter it to obtain an acetone extract; concentrate it to 15% of the original volume, and dry it at 60 °C until the moisture content by mass is 7% to obtain 0.13 g of a crude extract, containing 0.054 g of N-methylsansalvamide.

[0046] (3) Dissolve 0.13 g of the crude extract obtained in step (2) in methanol, with a solid-liquid ratio of dissolution of 22 mg / mL, then add water and n-hexane to form an extraction system. Finally, the volume ratio of n-hexane, methanol, and water is 10:8:7. Let it stand and layer at room temperature, take the upper n-hexane layer (the target compound N-methylsansalvamide is enriched in the n-hexane layer), dry it with a nitrogen blower, and then freeze-dry it at -40 °C until the moisture content by mass is 3% to obtain 0.041 g of pure N-methylsansalvamide. Detect it by high-performance liquid chromatography ( Figure 3 ), with a purity of 95.3% and a mass recovery rate of 75.6% (relative to the content of N-methylsansalvamide in the crude extract).

[0047] Control Example 1. Isolation and purification of N-methylsansalvamide

[0048] (1) Filter 500 mL of the fermentation broth prepared by the method of Example 1 through gauze, remove the filtrate to obtain wet mycelium, dry it in vacuo at 60 °C until the mass water content is 7%, and pulverize it to obtain 2.4 g of dry mycelium powder.

[0049] (2) Take 1 g of the dry mycelium powder, add 30 mL of acetone, ultrasonically extract it for 40 minutes under the condition of 175 W, filter it to obtain an acetone extract; concentrate it to 15% of the original volume, and dry it at 60 °C until the mass water content is 7% to obtain 0.13 g of a crude extract, containing 0.054 g of N-methylsansalvamide.

[0050] (3) Dissolve 0.13 g of the crude extract in step (2) with methanol, and the solid-liquid ratio of dissolution is 22 mg / mL. Then add water and n-hexane to form an extraction system. Finally, the volume ratio of n-hexane, methanol and water is 10:7:20. Let it stand and layer at room temperature, take the upper n-hexane layer (the target compound N-methylsansalvamide is enriched in the n-hexane layer), dry it with a nitrogen blower and then freeze-dry it at -40 °C until the mass water content is 3% to obtain 0.044 g of pure N-methylsansalvamide. Detect it by high performance liquid chromatography, the purity is 91.3%, and the mass recovery rate is 78.6% (relative to the content of N-methylsansalvamide in the crude extract).

[0051] Example 3, Separation and Purification of N-methylsansalvamide

[0052] (1) Centrifuge 3000 mL of the fermentation broth prepared by the method of Example 1 at 4000 rpm for 20 min, remove the supernatant, collect the wet mycelium, dry it in vacuo at 55 °C until the mass water content is 8%, and pulverize it to obtain 14.1 g of dry mycelium powder.

[0053] (2) Take 10 g of the dry mycelium powder, add 300 mL of ethanol, ultrasonically extract it for 35 minutes under the condition of 200 W, filter it to obtain an ethanol extract; concentrate it to 15% of the original volume, and dry it at 50 °C until the mass water content is 7% to obtain 1.41 g of a crude extract, containing 0.52 g of N-methylsansalvamide.

[0054] (3) Dissolve 1.41 g of the crude extract in step (2) with ethanol, and the solid-liquid ratio of dissolution is 18 mg / mL. Then add water and petroleum ether to form an extraction system. Finally, the volume ratio of petroleum ether, ethanol and water is 10:9:6. Let it stand and layer at room temperature, take the upper petroleum ether layer, dry it at 40 °C until the mass water content is 3% to obtain 0.38 g of pure N-methylsansalvamide. Detect it by high performance liquid chromatography ( Figure 4 ) with a purity of 95.8% and a recovery rate of 73%.

[0055] Control Example 2, Isolation and Purification of N-methylsansalvamide

[0056] (1) 3000 mL of the fermentation broth prepared by the method of Example 1 was centrifuged at 4000 rpm for 20 min, the supernatant was removed, the wet bacterial cells were collected, vacuum dried at 55 °C until the mass water content was 8%, and then pulverized to obtain 14.1 g of dry bacterial powder.

[0057] (2) 10 g of the dry bacterial powder was taken, added with 300 mL of ethanol, and ultrasonically extracted under 200 W for 35 minutes, then filtered to obtain an ethanol extract; it was concentrated to 15% of the original volume and dried at 50 °C until the mass water content was 7% to obtain 1.41 g of a crude extract, containing 0.52 g of N-methylsansalvamide.

[0058] (3) 1.41 g of the crude extract from step (2) was dissolved in ethanol with a solid-liquid ratio of 18 mg / mL, and then water and petroleum ether were added to form an extraction system. Finally, the volume ratio of chloroform, ethanol, and water was 10:9:6. It was allowed to stand and layer at room temperature, and the lower chloroform layer was taken, and dried at 40 °C under low temperature until the mass water content was 3% to obtain 0.38 g of pure N-methylsansalvamide with a purity of 90.6% and a recovery rate of 67%.

[0059] Example 4, Isolation and Purification of N-methylsansalvamide

[0060] (1) 30 L of the fermentation broth prepared by the method of Example 1 was vacuum filtered to remove the filtrate, and the wet bacterial cells were obtained, vacuum dried at 40 °C until the mass water content was 8%, and then pulverized to obtain 136 g of dry bacterial powder.

[0061] (2) 100 g of the dry bacterial powder was taken, added with 3000 mL of methanol, and ultrasonically extracted under 150 W for 45 minutes, then filtered to obtain a methanol extract; it was concentrated to 15% of the original volume and dried at 50 °C until the mass water content was 7% to obtain 12.17 g of a crude extract, containing 5.1 g of N-methylsansalvamide.

[0062] (3) 12.17 g of the crude extract from step (2) was dissolved in isopropanol with a solid-liquid ratio of 20 mg / mL, and then water and n-hexane were added to form an extraction system. Finally, the volume ratio of n-hexane, isopropanol, and water was 10:7:8. It was allowed to stand and layer at room temperature, and the upper n-hexane layer was taken, and dried under reduced pressure at 45 °C until the mass water content was 3% to obtain 3.315 g of pure N-methylsansalvamide, detected by high performance liquid chromatography ( Figure 5 ) with a purity of 95.1% and a recovery rate of 65%.

[0063] Control Example 3, Isolation and Purification of N-methylsansalvamide

[0064] (1) 30 L of the fermentation broth prepared by the method of Example 1 was filtered under reduced pressure to remove the filtrate, obtaining wet bacterial cells, which were dried in vacuo at 40 °C until the moisture content by mass was 8%, and then pulverized to obtain 136 g of dry bacterial powder.

[0065] (2) 100 g of the dry bacterial powder was taken, added with 3000 mL of methanol, and ultrasonically extracted for 45 minutes under the condition of 150 W, and then filtered to obtain a methanol extract; it was concentrated to 15% of the original volume and dried at 50 °C until the moisture content by mass was 7% to obtain 12.17 g of a crude extract, containing 5.1 g of N-methylsansalvamide.

[0066] (3) 12.17 g of the crude extract from step (2) was dissolved in isopropanol, and the solid-liquid ratio of the dissolution was 20 mg / mL. Then, water and n-hexane were added to form an extraction system. Finally, the volume ratio of n-hexane, isopropanol, and water was 10:7:8. It was allowed to stand and separate layers at room temperature, and the upper n-hexane layer was taken and dried under reduced pressure at 70 °C until the moisture content by mass was 3% to obtain 3.1 g of pure N-methylsansalvamide, with a purity of 92.1% and a recovery rate of 61.7%.

Claims

1. A method for the efficient purification of N-methylsansalvamide from the fermentation product of Fusarium, characterized in that, The method includes the following steps: (1) Filter the Fusarium fermentation product containing N-methylsansalvamide, collect the wet mycelium, dry it to a mass water content of 5-9%, and pulverize it to obtain dry mycelium powder. (2) After dissolving the dry mycelium powder with an organic solvent, perform ultrasonic extraction, filter to obtain an extract, concentrate it to 15% of the original volume, and dry it to a mass water content of 7% to obtain a crude extract. (3) After dissolving the crude extract with an organic solvent, add an extractant and water, let it stand for extraction, take the extractant layer, and dry it to a mass water content of 3% to obtain pure N-methylsansalvamide.

2. The method according to claim 1, wherein The Fusarium fermentation product containing N-methylsansalvamide in step (1) is obtained by fermenting and culturing Fusarium sp. CCTCC NO: M 20241573.

3. The method according to claim 1, characterized in that, The fermentation product in step (1) is prepared according to the following steps: 1) Inoculate Fusarium CCTCC NO: M 20241573 into PDA medium and activate it at 30 °C for 3 days; the composition of the PDA medium is: 200.0 g / L potatoes, 20.0 g / L glucose, 15.0-20.0 g / L agar, natural pH, and the solvent is water. 2) Inoculate the strain in step 1) into PDB medium, culture it at 35 °C and 180 rpm for 72 h to obtain a seed solution; the composition of the PDB medium is: 200.0 g / L potatoes, 20.0 g / L glucose, natural pH, and the solvent is water. 3) Inoculate the seed solution in step 2) into the fermentation medium at an inoculation amount of 5% (v / v), and ferment it statically at 25 °C for 13 days to obtain a fermentation broth containing N-methylsansalvamide; the composition of the fermentation medium is: 22.5 g / L sucrose, 16.5 g / L tryptone, 0.024 g / L yeast extract, 20.0 g / L sea salt, pH 6.5, and the solvent is water.

4. The method according to claim 1, characterized in that, The drying methods in steps (1), (2), and (3) are low-temperature drying at 40-60 °C, vacuum drying at 35-45 °C, or freeze-drying at -50 °C to -30 °C.

5. The method according to claim 1, characterized in that The organic solvent in step (2) is one or a mixture of several of methanol, ethanol, propanol, isopropanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume of the organic solvent used is 10-50 mL / g based on the mass of the dry mycelium powder.

6. The method according to claim 1, wherein The conditions for ultrasonic treatment in step (2) are ultrasonic treatment at 150-200 W for 35-45 min.

7. The method according to claim 1, characterized in that The organic solvent in step (3) is one or a mixture of several of methanol, ethanol, acetone, isopropanol, and acetonitrile; the volume of the organic solvent used is 10-30 mL / g based on the mass of the crude extract.

8. The method according to claim 1, characterized in that, The extractant in step (3) is one or a mixture of several of petroleum ether, chloroform, n-hexane, cyclohexane, heptane, and toluene.

9. The method according to claim 1, wherein In step (3), the volume ratio of the extractant to the organic solvent and water is 10:7-9:6-8.

10. The method according to claim 1, characterized in that, When the organic solvent in step (3) is methanol, the extractant is n-hexane, and the volume ratio of the extractant to methanol and water is 10:8:7; when the organic solvent is ethanol, the extractant is petroleum ether, and the volume ratio of the extractant to ethanol and water is 10:9:6; when the organic solvent is isopropanol, the extractant is n-hexane, and the volume ratio of the extractant to isopropanol and water is 10:7:8.

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