Efficient purification method of aureomycin A in streptomyces fermentation product

Abstract

The invention discloses a method for efficiently purifying aureomycin A in a streptomyces fermentation product, which only needs to perform drying, ultrasonic extraction, concentration and crystallization on the fermentation product, then dissolve the fermentation product with an organic solvent and then crystallize the fermentation product, and is simple to operate, low in equipment requirement, low in consumable cost, convenient in organic solvent recovery and low in cost. The problems of complex separation and purification steps, high equipment requirements, long time consumption and the like required by liquid-phase preparative chromatography in the existing method are solved. According to the invention, after the first crystallization and washing are completed, the purity of the aureomycin A in the obtained aureomycin A crude product is 87-92%. After recrystallization and secondary washing of the aureomycin A crude product, the purity of the aureomycin A crude product reaches 95% or above, the total yield reaches 47% or above, the total consumed time is 90-144 h, and the aureomycin A crude product is suitable for large-scale preparation, low in cost and high in benefit.

Description

(1) Technical Field

[0001] The present invention belongs to the field of chemical separation and purification, and particularly relates to a method for efficiently purifying chrysomycin A from streptomyces fermentation products. (2) Background Art

[0002] Chrysomycin A is a fermentation product derived from streptomyces, which has significant anti-tumor and anti-Gram-positive bacterial activities and great potential for new drug development. However, in the streptomyces fermentation products producing chrysomycin A, there are often two by-products, chrysomycin B and C. The structural formulas of chrysomycin A, B, and C are respectively:

[0003]

[0004] Since the structures of chrysomycin A and chrysomycin B, C are extremely similar, it is extremely difficult to prepare high-purity chrysomycin A by using traditional separation and purification means. At present, the large-scale separation and purification of high-purity chrysomycin A at home and abroad mainly rely on preparative chromatography separation. It is necessary to concentrate the extract to dryness and then redissolve it for a series of pretreatment processes such as extraction and decolorization. It has the disadvantages of high technical equipment requirements, high consumable costs, complex operation, and long time consumption. (3) Summary of the Invention

[0005] The purpose of the present invention is to provide a method for efficiently purifying chrysomycin A from streptomyces fermentation products, which avoids the use of liquid chromatography and reagents in the separation and purification process of chrysomycin A, solves the problems of the existing method with cumbersome steps, high cost, and long time consumption, and opens up a new method for separating and purifying chrysomycin A.

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

[0007] The present invention provides a method for efficiently purifying chrysomycin A from streptomyces fermentation products, and the method comprises the following steps:

[0008] (1) Filter the streptomyces fermentation product containing chrysomycin A, collect the wet bacterial residue, dry it in vacuum, and crush it (preferably pass through an 80-mesh sieve) to obtain a dry bacterial residue;

[0009] (2) Ultrasonically extract the dry bacterial residue with an organic solvent, and filter to obtain an extract;

[0010] (3) Concentrate the extract to supersaturation, and then let it stand for crystallization at room temperature; filter, and wash the obtained crystals with a solvent to remove impurities to obtain a crude product of chrysomycin A;

[0011] (4) Redissolve the crude product of chrysomycin A with an organic solvent, filter to remove insoluble impurities, then concentrate to supersaturation, let it stand for recrystallization at room temperature, filter, and wash the obtained crystals with a solvent to remove impurities to obtain a pure product of chrysomycin A.

[0012] Preferably, in step (1), the Streptomyces fermentation product containing chlortetracycline A is obtained by fermenting Streptomyces sp. 891-B6 CGMCC No. 21775.

[0013] Preferably, the Streptomyces fermentation product in step (1) is prepared as follows: Streptomyces sp. 891-B6 CGMCC No. 21775 is inoculated into ISP2 agar medium for activation for 6 - 7 days, and then inoculated into ISP2 liquid medium. After seed culture at 28 °C and 200 rpm for 48 - 72 h, a seed solution is obtained; the seed solution is inoculated into the fermentation medium at a volume concentration of 5%, and fermentation culture is carried out at 28 °C and 200 rpm for 168 - 192 h to obtain a fermentation broth.

[0014] The composition of ISP2 agar medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, 20.0 g / L agar powder, pH 7, sterilized at 121 °C for 20 min;

[0015] The composition of ISP2 liquid medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, pH 7.0, sterilized at 121 °C for 20 min;

[0016] The composition of the fermentation medium is: 5.0 g / L starch, 20.0 g / L glucose, 10.0 g / L soybean powder, 2.0 g / L calcium carbonate, pH 7, sterilized at 121 °C for 20 min.

[0017] Preferably, the vacuum drying in step (1) is carried out at 50 - 70 °C for 48 - 72 h, more preferably at 60 °C for 48 h.

[0018] Preferably, the organic solvent in step (2) is one or a mixture of several of methanol, ethanol, propanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume of the organic solvent used is 0.1 - 0.5 L / g based on the weight of the dry bacterial residue (preferably 0.3 L / g).

[0019] Preferably, the organic solvent in step (4) is one or a mixture of several of methanol, ethanol, propanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume of the organic solvent used is 1 - 5 L / g based on the weight of the crude chlortetracycline A (preferably 1.2 - 3.8 L / g).

[0020] Preferably, in step (2), the conditions for ultrasonic extraction are ultrasonic extraction at 100 - 200 W for 10 - 30 min (preferably ultrasonic extraction at 150 W for 25 min), and the extraction is repeated 1 - 3 times.

[0021] Preferably, in steps (1) to (4), the filtration method is one of plate and frame pressure filtration, vacuum filtration, and centrifugal filtration.

[0022] Preferably, in step (3), the extract is concentrated until the concentration of chlortetracycline A is 5 - 10 g / L. Concentrations lower than 5 g / L or higher than 10 g / L will affect the purity and mass recovery rate of chlortetracycline A.

[0023] Preferably, in step (3) or (4), the solvent is one or a mixture of several solvents selected from water, methanol, ethanol, propanol, butanol, ethyl acetate, acetone, acetonitrile, n - hexane, petroleum ether, dichloromethane, and chloroform.

[0024] Preferably, in step (3) or (4), the washing method is rinsing or slurrying.

[0025] Preferably, in step (4), it is concentrated until the concentration of chlortetracycline A is 4 - 12 g / L.

[0026] Preferably, when the organic solvent in step (1) is ethanol, the solvent in step (2) is pure water, the organic solvent in step (3) is ethyl acetate, and the solvent in step (4) is ethyl acetate; when the organic solvent in step (1) is methanol, the solvent in step (2) is ethanol, the organic solvent in step (3) is methanol, and the solvent in step (4) is acetonitrile; when the organic solvent in step (1) is ethanol, the solvent in step (2) is ethanol, the organic solvent in step (3) is ethanol, and the solvent in step (4) is ethanol.

[0027] The concentration in the present invention refers to a method of increasing the concentration of chlortetracycline A by solvent evaporation, including technical means such as vacuum evaporation and heating evaporation.

[0028] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: The method for separating and purifying chlortetracycline A from the fermentation products of Streptomyces producing chlortetracycline A only requires drying the fermentation products, ultrasonic extraction, concentrating and crystallizing, dissolving in an organic solvent, and then recrystallizing. The operation is simple, the equipment requirements are low, the consumable costs are low, and the recovery of organic solvents is convenient. It overcomes the problems of the existing methods, such as the cumbersome steps of liquid - phase preparative chromatography separation and purification, high equipment requirements, and long time consumption. After the first crystallization and washing are completed in the present invention, the purity of chlortetracycline A in the obtained crude chlortetracycline A is 87% - 92%. After the crude chlortetracycline A is recrystallized and washed for the second time, its purity reaches over 95%, and the total recovery rate reaches over 47%. It is suitable for large - scale preparation, with low cost and high efficiency, and the total time consumption is 90 - 144 h. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a flow chart for the purification of chlortetracycline A.

[0030] Figure 2Liquid chromatogram of crude chlortetracycline A in Example 2.

[0031] Figure 3 Liquid chromatogram of pure chlortetracycline A in Example 2.

[0032] Figure 4 Liquid chromatogram of crude chlortetracycline A in Example 3.

[0033] Figure 5 Liquid chromatogram of pure chlortetracycline A in Example 3.

[0034] Figure 6 Liquid chromatogram of crude chlortetracycline A in Example 4.

[0035] Figure 7 Liquid chromatogram of pure chlortetracycline A in Example 4.

[0036] Figure 8 Liquid chromatogram of crude chlortetracycline A in Comparative Example 1.

[0037] Figure 9 Liquid chromatogram of crude chlortetracycline A in Comparative Example 2.

[0038] Figure 10 Liquid chromatogram of pure chlortetracycline A in Comparative Example 2.

[0039] Figure 11 Liquid chromatogram of the extract in Comparative Example 4. (V) Specific Embodiments

[0040] 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:

[0041] Room temperature in the embodiments of the present invention refers to 25 - 30 °C.

[0042] The content, purity and recovery rate of chlortetracycline A are detected by high performance liquid chromatography as follows:

[0043] The chromatograph used is Shimadzu LC-20, the chromatographic column is Luna-C18, the particle size is 5 μm, the diameter of the chromatographic column is 4.6 mm, and the height is 250 mm. The injection volume is 5 μL, the mobile phase is acetonitrile: water = 50:50 (volume ratio), the detection wavelength is 254 nm, the retention time is 20 min, and the pump flow rate is 1 mL / min. The elution order is chlortetracycline B, chlortetracycline A, and chlortetracycline C.

[0044] The content, purity and mass recovery rate of chlortetracycline A are calculated according to the standard curve of chlortetracycline A prepared from the concentration of chlortetracycline A and the peak area.

[0045] The purity of chrysomycin A is characterized by the peak area ratio given by liquid chromatography.

[0046] Example 1. Preparation of chrysomycin fermentation broth

[0047] Streptomyces sp. 891 - B6 (CGMCC No. 21775, which has been disclosed in patent application CN113278545A) was inoculated into ISP2 agar medium for activation for 6 - 7 days, and then inoculated into ISP2 liquid medium. After seed culture at 28°C and 200 rpm for 48 - 72 h, a seed solution was obtained; the seed solution was inoculated into the fermentation medium at a volume concentration of 5%, and fermentation culture was carried out at 28°C and 200 rpm for 168 - 192 h to obtain the fermentation broth.

[0048] The composition of ISP2 agar medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, 20.0 g / L agar powder, pH 7, sterilized at 121°C for 20 min;

[0049] The composition of ISP2 liquid medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, pH 7.0, sterilized at 121°C for 20 min.

[0050] The composition of the fermentation medium is: 5.0 g / L starch, 20.0 g / L glucose, 10.0 g / L soybean powder, 2.0 g / L calcium carbonate, pH 7, sterilized at 121°C for 20 min.

[0051] Example 2. Isolation and purification of chrysomycin A

[0052] (1) 37 L of the fermentation broth prepared in Example 1 was centrifuged at 25°C and 3500 rpm for 20 min, the supernatant was removed to obtain wet bacterial residue, which was vacuum dried at 60°C for 48 h, pulverized and passed through an 80 - mesh sieve to obtain 505 g of dry bacterial residue.

[0053] (2) 1 g of the dry bacterial residue was added with 300 mL of ethanol, ultrasonic extraction was carried out at 150 W for 25 minutes, filtered, and the filter cake was repeatedly extracted with the same amount of ethanol 3 times, filtered, to obtain 850 mL of ethanol extract containing 0.11 g of chrysomycin A.

[0054] (3) 850 mL of the ethanol extract in step (2) was concentrated by reduced pressure evaporation to 20 mL, the concentration of chrysomycin A was about 5.5 g / L, allowed to stand for crystallization at room temperature, filtered, and the precipitated wet crystals were washed with a small amount of pure water to obtain 77.6 mg of crude chrysomycin A. 1 mg of the crude chrysomycin A was redissolved with ethanol, detected by liquid chromatography, the purity was 90.1%, and the mass recovery rate was 70.5%. The results are shown in Figure 2 .

[0055] (4) Dissolve 76.6 mg of crude chrysomycin A in 300 mL of ethyl acetate, filter to remove insoluble impurities, concentrate the ethyl acetate solution to 15 mL (chrysomycin A concentration 5.1 g / L), allow to crystallize at room temperature, filter, and wash the wet crystals with a small amount of ethyl acetate to obtain 57.7 mg of pure chrysomycin A. The total time taken is 92 h. Take 1 mg of pure chrysomycin A, redissolve it in ethanol, and detect it by liquid chromatography. The purity is 95.0%, and the total recovery rate is 52.5%. The results are shown in Figure 3 .

[0056] Example 3: Isolation and purification of chrysomycin A

[0057] (1) Centrifuge 37 L of the fermentation broth prepared in Example 1 at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial residue, dry it in vacuo at 60 °C for 48 h, pulverize it, and sieve it through a 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0058] (2) Take 10 g of the dry bacterial residue, add 3 L of methanol, extract it by ultrasonic wave at 150 W for 25 minutes, filter, and repeat the extraction of the filter cake with the same amount of methanol 3 times, filter to obtain 8.8 L of methanol extract, containing 1.12 g of chrysomycin A.

[0059] (3) Concentrate 8.8 L of the methanol extract in step (2) to 120 mL, chrysomycin A concentration 9.3 g / L, allow to crystallize at room temperature, filter, and wash the wet crystals with a small amount of ethanol to obtain 0.85 g of crude chrysomycin A. Take 1 mg of crude chrysomycin A, redissolve it in ethanol, and detect it by liquid chromatography. The purity is 87.3%, and the mass recovery rate is 75.9%. The results are shown in Figure 4 .

[0060] (4) Dissolve 0.85 g of crude chrysomycin A in 1 L of methanol, filter to remove insoluble impurities, concentrate the methanol solution to 75 mL (chrysomycin A concentration 11.3 g / L), allow to crystallize at room temperature, filter, and wash the wet crystals with a small amount of acetonitrile to obtain 0.68 g of pure chrysomycin A. The total time taken is 107 h. Take 1 mg of pure chrysomycin A, redissolve it in ethanol, and detect it by liquid chromatography. The purity is 95.0%, and the total recovery rate is 60.7%. The results are shown in Figure 5 .

[0061] Example 4: Isolation and purification of chrysomycin A

[0062] (1) Centrifuge 37 L of the fermentation broth prepared in Example 1 at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial cells, dry them in vacuo at 60 °C for 48 h, pulverize them, and sieve them through a 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0063] (2) Take 100 g of dry bacterial residue, add 30 L of ethanol, perform ultrasonic extraction at 150 W for 25 minutes, filter, repeat the extraction of the filter cake with the same amount of ethanol 3 times, filter to obtain 89 L of ethanol extract, containing 11.03 g of chrysomycin A.

[0064] (3) The 89 L of ethanol extract in step (2) was concentrated to 1.5 L (chrysomycin A concentration 7.3 g / L). Crystallization was carried out by standing at room temperature, filtered, and the precipitated wet crystals were washed with a small amount of ethanol to obtain 8.05 g of crude chrysomycin A. Take 1 mg of the crude chrysomycin A and redissolve it with ethanol, and perform detection by liquid chromatography. The purity was 89.1% and the recovery rate was 73.0%. The results are shown in Figure 6 .

[0065] (4) Dissolve 8.05 g of crude chrysomycin A in 10 L of ethanol, concentrate the ethanol solution to 1.2 L (chrysomycin A concentration 5.9 g / L), carry out crystallization by standing at room temperature, filter, and wash the precipitated wet crystals with a small amount of ethanol to obtain 5.27 g of pure chrysomycin A, with a total time consumption of 144 h. Take 1 mg of the pure chrysomycin A and redissolve it with ethanol, and perform detection by liquid chromatography. The purity was 95.2% and the total recovery rate was 47.8%. The results are shown in Figure 7 .

[0066] Comparative Example 1, Influence of Concentration Degree on the Separation and Purification of Chrysomycin A

[0067] (1) Centrifuge 37 L of the fermentation broth prepared in Example 1 at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial residue, dry it in a vacuum at 60 °C for 48 h, pulverize it, and pass it through an 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0068] (2) Take 10 g of dry bacterial residue, add 3 L of methanol, perform ultrasonic extraction at 150 W for 25 minutes, filter, repeat the extraction of the filter cake with the same amount of methanol 3 times, filter to obtain 8.9 L of methanol extract, containing 1.13 g of chrysomycin A.

[0069] (3) The 8.9 L of methanol extract in step (2) was concentrated to 80 mL, and the chrysomycin A concentration was 14.1 g / L. Since the chrysomycin A concentration was too high, a large amount of wet crystals precipitated during the concentration process. Filter and wash the precipitated wet crystals with a small amount of ethanol to obtain 0.62 g of crude chrysomycin A. Take 1 mg of the crude chrysomycin A and redissolve it with ethanol, and perform detection by liquid chromatography. It was found that the purity of the crude chrysomycin A was only 83.7% and the mass recovery rate was only 54.9%. The results are shown in Figure 8 .

[0070] Comparative Example 2, Influence of Washing on the Separation and Purification of Chrysomycin A

[0071] (1) Centrifuge the 37 L of fermentation broth prepared in Example 1 at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial residue, dry it in vacuo at 60 °C for 48 h, pulverize it, and sieve it through an 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0072] (2) Take 1 g of dry bacterial residue, add 300 mL of ethanol, perform ultrasonic extraction at 150 W for 25 minutes, filter, repeat the extraction of the filter cake with the same amount of ethanol 3 times, filter, and obtain 850 mL of ethanol extract containing 0.11 g of chlortetracycline A.

[0073] (3) Concentrate 850 mL of the ethanol extract in step (2) under reduced pressure to 20 mL, with the chlortetracycline A concentration of 5.5 g / L. Let it stand for crystallization at room temperature, directly filter the wet crystals without washing, and obtain 56.5 mg of crude chlortetracycline A. Take 1 mg of the crude chlortetracycline A and redissolve it with ethanol, and perform detection by liquid chromatography. The purity is 86.8%, and the mass recovery rate is 51.4%. The results are shown in Figure 9 . The purity is lower than that of Example 2.

[0074] (4) Dissolve 55.5 mg of crude chlortetracycline A in 300 mL of ethanol, concentrate the ethanol solution to 11 mL (chlortetracycline A concentration of 5.1 g / L), let it stand for crystallization at room temperature, directly filter the wet crystals without washing, and obtain 38.4 mg of pure chlortetracycline A. Take 1 mg of the pure chlortetracycline A and redissolve it with ethanol, and perform detection by liquid chromatography. The purity is 94.6%, the total recovery rate is 34.9%, and the total time-consuming is 92 h. The results are shown in Figure 10 . The purity does not reach 95.0%.

[0075] Comparative Example 3: Influence of the concentration of the extract on the separation and purification of chlortetracycline A

[0076] (1) Centrifuge the 37 L of fermentation broth prepared in Example 1 at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial residue, dry it in vacuo at 60 °C for 48 h, pulverize it, and sieve it through an 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0077] (2) Take 10 g of dry bacterial residue, add 3 L of methanol, perform ultrasonic extraction at 150 W for 25 minutes, filter, repeat the extraction of the filter cake with the same amount of methanol 3 times, filter, and obtain 8.9 L of methanol extract containing 1.13 g of chlortetracycline A.

[0078] (3) Concentrate 8.9 L of the methanol extract in step (2) to 300 mL, with the chlortetracycline A concentration of 3.8 g / L. The concentration is too low, and no crystals are found to precipitate after standing at room temperature.

[0079] Comparative Example 4: Influence of the extractant on the separation and purification of chlortetracycline A

[0080] (1) Centrifuge the fermentation broth prepared in Example 1 of 37 L at 25 °C and 3500 rpm for 20 min, remove the supernatant to obtain wet bacterial residue, dry it in vacuo at 60 °C for 48 h, pulverize it and sieve it through a 80-mesh sieve to obtain 505 g of dry bacterial residue.

[0081] (2) Take 1 g of dry bacterial residue, add 300 mL of pure water, perform ultrasonic extraction at 150 W for 25 minutes, filter, repeat the extraction of the filter cake with the same amount of pure water 3 times, filter to obtain 890 mL of extract, and detect it by liquid chromatography. The extract does not contain chlortetracycline A. The results are shown in Figure 11 .

Claims

1. A method for efficiently purifying chlortetracycline A from a Streptomyces fermentation product, characterized in that: The method comprises the following steps: (1) filtering the fermentation product of Streptomyces containing chrysomycin A, collecting the wet bacterial residue, vacuum drying, and crushing to obtain dry bacterial residue; (2) extracting the dried fungus residue with an organic solvent using ultrasonic wave, filtering, and obtaining an extract; (3) After the extract is concentrated to supersaturation, it is allowed to stand at room temperature for crystallization; filtered, and the obtained crystals are washed with a solvent to remove impurities, thereby obtaining a crude product of chrysomycin A; (4) The crude product of chrysotoxomycin A is redissolved in an organic solvent, insoluble impurities are removed by filtration, and then concentrated to supersaturation, allowed to stand at room temperature for recrystallization, filtered, and the obtained crystals are washed with a solvent to remove impurities, thereby obtaining pure chrysotoxomycin A.

2. The method according to claim 1, characterized in that In step (1), the Streptomyces fermentation product containing chrysomycin A is obtained by fermenting Streptomyces sp. CGMCC No.21775.

3. The method according to claim 1, characterized in that Step (1) The Streptomyces fermentation product is prepared according to the following steps: inoculating Streptomyces sp. CGMCC No. 21775 into ISP2 agar medium for activation for 6-7 days, then inoculating into ISP2 liquid medium, and culturing at 28° C. and 200 rpm for 48-72 hours to obtain seed liquid; inoculating the seed liquid into a fermentation medium at a volume concentration of 5%, and fermenting and culturing at 28° C. and 200 rpm for 168-192 hours to obtain a fermentation liquid; The composition of ISP2 agar medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, 20.0 g / L agar powder, pH 7, sterilized at 121°C for 20 min; The composition of ISP2 liquid medium is: 4.0 g / L glucose, 4.0 g / L yeast extract, 10.0 g / L malt extract, pH 7.0, sterilized at 121°C for 20 min; The composition of the fermentation medium is: 5.0 g / L starch, 20.0 g / L glucose, 10.0 g / L soybean powder, 2.0 g / L calcium carbonate, pH is 7, and sterilized at 121°C for 20 min.

4. The method according to claim 1, characterized in that The organic solvent in step (2) is one or a mixed solvent of methanol, ethanol, propanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume amount of the organic solvent is 0.1-0.5 L / g based on the weight of the dry mushroom residue.

5. The method according to claim 1, characterized in that The organic solvent in step (4) is one or a mixed solvent of methanol, ethanol, propanol, butanol, ethyl acetate, acetone, and acetonitrile; the volume of the organic solvent used is 1-5 L / g based on the weight of the crude chrysomecin A.

6. The method according to claim 1, characterized in that In step (2), the ultrasonic extraction conditions are 100-200W ultrasonic extraction for 10-30 minutes, and the extraction is repeated 1-3 times.

7. The method according to claim 1, characterized in that In step (3), the chloramphenicol A is concentrated to a concentration of 5-10 g / L.

8. The method according to claim 1, characterized in that In step (4), the chloramphenicol A is concentrated to a concentration of 4-12 g / L.

9. The method according to claim 1, characterized in that In step (3) or (4), the solvent is one or a mixed solvent of water, methanol, ethanol, propanol, butanol, ethyl acetate, acetone, acetonitrile, n-hexane, petroleum ether, dichloromethane, and chloroform.

10. The method according to claim 1, characterized in that In step (3) or (4), the washing method is rinsing or pulping.

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