Method for recovering staurosporine from staurosporine waste mother liquor

Abstract

The invention relates to a method for recovering staurosporine from staurosporine waste mother liquor, which comprises the following steps: concentrating the staurosporine waste mother liquor, and collecting a first solid; mixing the first solid with inorganic strong acid, low-carbon alcohol with volatility and water, heating to 60-80 DEG C, and dissolving; adding an adsorbent for heat adsorption; collecting liquid through first solid-liquid separation; cooling and crystallizing the liquid for 2-8 hours, and collecting a second solid in the crystallized liquid through second solid-liquid separation; washing the second solid with low-carbon alcohol and water, and collecting a third solid; the third solid and alkali are mixed in low-carbon alcohol, and acid in the third solid is removed through an acid-base neutralization reaction; adding water, mixing and crystallizing; and collecting solid containing staurosporine through third solid-liquid separation. The method can be used for recovering the staurosporine from the staurosporine waste mother liquor with high recovery rate and high purity, and is simple in process, short in time consumption and low in cost.

Description

Technical Field

[0001] The present application relates to the technical field of compound separation and purification, and in particular to a method for recovering staurosporine from staurosporine waste mother liquor. Background Art

[0002] Staurosporine is an indolecarbazole alkaloid isolated from the culture medium of Streptomyces. The first staurosporine compound (AM-2282) was isolated from the fermentation product of Streptomyces staurosporeus in 1977. It is widely used as a protein kinase C (PKC) inhibitor, with a broad spectrum of biological activities from antifungal to antihypertensive, and can induce G2 / M phase block of cancer cells, adjust G1 phase block, and induce cell apoptosis. In addition, staurosporine is a semi-synthetic raw material used in the clinical treatment of acute myeloid leukemia (AML) and is widely used in biological experiments. Staurosporine, especially high-purity reagent-grade staurosporine, has a high value.

[0003] In the process of producing staurosporine, a large amount of waste mother liquor containing staurosporine will be generated. Direct discharge will seriously pollute the environment and cause serious waste of valuable resources.

[0004] In order to recover staurosporine from the waste mother liquor, one method is to extract it with a water-immiscible organic solvent and then purify it with a chromatographic column. However, the effective extraction of staurosporine requires a large amount of water-immiscible organic solvent, the chromatographic purification is time-consuming and uses a large amount of eluent, the process is complicated, time-consuming and labor-intensive, resulting in a large amount of waste solvent, increasing costs, and bringing about solvent emission problems.

[0005] Therefore, it is very necessary to develop a method for recovering staurosporine from staurosporine waste mother liquor, which can not only increase the yield of staurosporine, but also reduce environmental pollution and create greater economic benefits. Summary of the invention

[0006] Based on this, the purpose of the present application is to provide a method for recovering high-purity staurosporine from staurosporine waste mother liquor without the need for chromatographic separation, which is low-cost, simple and efficient.

[0007] The technical solution is as follows:

[0008] Concentrating the staurosporine waste mother liquor to collect a first solid;

[0009] The first solid is mixed with a first solvent, and the mixture is heated to 60° C. to 80° C. to dissolve, thereby obtaining a first mixed solution, wherein the first solvent is composed of an inorganic strong acid, a volatile low-carbon alcohol, and water;

[0010] Using an adsorbent to perform thermal adsorption treatment on the first mixed liquid to prepare a second mixed liquid;

[0011] collecting liquid in the second mixed liquid through a first solid-liquid separation process;

[0012] The liquid in the second mixed liquid is subjected to a cooling and crystallization treatment for 2 hours to 8 hours, and a second solid in the crystallization liquid is collected through a second solid-liquid separation, wherein the second solid contains acid water compound crystals of staurosporine;

[0013] The second solid is washed with a second solvent to collect a third solid, wherein the second solvent consists of the low-carbon alcohol and water;

[0014] Mixing the third solid and a base in the low-carbon alcohol, removing the acid in the third solid by an acid-base neutralization reaction, and preparing a third mixed solution;

[0015] Mixing the third mixed solution with water to perform crystallization treatment to prepare a fourth mixed solution;

[0016] The solid containing staurosporine in the fourth mixed liquid is collected by a third solid-liquid separation process.

[0017] In one embodiment, the inorganic strong acid includes one or more of hydrochloric acid, phosphoric acid, sulfuric acid and nitric acid.

[0018] In one embodiment, the low-carbon alcohol includes one or more of ethanol, methanol, ethylene glycol, propanol and isopropanol.

[0019] In one embodiment, the volume ratio of the inorganic strong acid, the low-carbon alcohol and the water is 1:(15-25):(3-10).

[0020] In one embodiment, the volume mass ratio of the first solvent to the first solid is (10-15):1.

[0021] In one embodiment, the adsorbent includes one or more of activated carbon, molecular sieve, alumina, diatomaceous earth and silica gel.

[0022] In one embodiment, the mass ratio of the adsorbent to the first solid is (1-10):100.

[0023] In one embodiment, the temperature of the thermal adsorption treatment is 60° C. to 80° C., and the time is 1 h to 5 h.

[0024] In one embodiment, the temperature of the first solid-liquid separation treatment is 60°C to 80°C.

[0025] In one embodiment, the first solid-liquid separation treatment is performed by one or more of filtration, suction filtration and centrifugation.

[0026] In one embodiment, the second solid-liquid separation process is performed at room temperature.

[0027] In one embodiment, the second solid-liquid separation treatment is performed by one or more of filtration, suction filtration and centrifugation.

[0028] In one embodiment, in the second solvent, the volume ratio of the low-carbon alcohol to water is (15-25):(3-10).

[0029] In one embodiment, the base includes one or more of ammonia water, sodium carbonate, triethylamine, sodium hydroxide and potassium hydroxide.

[0030] In one embodiment, during the acid-base neutralization reaction, the pH of the system is 8 to 10 and the time is 2 h to 5 h.

[0031] In one embodiment, in the step of mixing the third mixed liquid with water for crystallization treatment, the volume ratio of the water to the third mixed liquid is (1-3):1.

[0032] In one embodiment, the third solid-liquid separation process is performed at room temperature.

[0033] In one embodiment, the third solid-liquid separation treatment method is one or more of filtration, suction filtration and centrifugation.

[0034] In one embodiment, the concentration process is carried out by vacuum distillation.

[0035] In one embodiment, the concentration treatment temperature is 40°C to 60°C.

[0036] In one embodiment, the vacuum degree of the concentration process is 0.06 MPa to 0.09 MPa.

[0037] In one embodiment, after the third solid-liquid separation step, the method further comprises washing and drying the solid containing staurosporine in sequence to prepare a pure staurosporine product.

[0038] In one embodiment, the washing treatment is water washing.

[0039] In one embodiment, the drying temperature is 80° C. to 110° C., and the vacuum degree is 0.08 MPa to 0.1 MPa.

[0040] This application has at least the following beneficial effects:

[0041] The method for recovering staurosporine from the waste mother liquor of staurosporine provided in the present application mainly comprises the steps of concentrating the mother liquor, adding an acid alcohol solvent to convert the intermediate containing staurosporine crystals into crystals containing acidified salts, and removing the pigment and some impurities by adsorption through the action of an adsorbent, improving the color purity of the crystals, and adding an alkali to remove the acid, so that the staurosporine crystals are free and then precipitated. The method can utilize the waste of the waste mother liquor of staurosporine, extract the staurosporine in the mother liquor, and the purity and recovery rate of staurosporine are both high, thereby improving the resource utilization rate of staurosporine fermentation, improving its economic benefits and reducing environmental pollution. In addition, the method of the present application also has the advantages of simple process, short time consumption, low energy consumption, simple equipment requirements, low production cost, and can realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A flow chart of a method for recovering staurosporine from staurosporine waste mother liquor as shown in one embodiment of the present application;

[0043] Figure 2 HPLC chart of the staurosporine waste mother liquor used in the examples of the present application;

[0044] Figure 3 HPLC chart of pure staurosporine recovered by the method for recovering staurosporine from staurosporine waste mother liquor as shown in Example 1 of the present application. DETAILED DESCRIPTION

[0045] The present application will be further described in detail below in conjunction with specific embodiments and accompanying drawings. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present application disclosure more thorough and comprehensive.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0047] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0048] Where “including,” “having,” and “comprising” are used herein, it is intended to cover a non-exclusive inclusion, and another component may also be added unless explicit limiting terms such as “only,” “consisting of,” etc. are used.

[0049] The words "optionally", "preferably", "more preferably", "preferably", "better", "better" and the like in this application refer to embodiments of the present application that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or in other circumstances. In addition, the statement of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of this application. That is, in this application, "optionally", "preferably", "more preferably", "preferably", "better", and the like are merely descriptions of implementation methods or examples with better effects, but do not constitute a limitation on the scope of protection of this application. For example, optionally, A is one or both of a and b, indicating that A can be selected from a, b, and a combination of a and b, and can also be selected from others.

[0050] In the present application, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0051] In this application, "above" or "below" includes the number itself. For example, "1 below" includes 1.

[0052] In the present application, "at least one" means more than one, such as one, two and more than two. "Multiple" or "several" means at least two, such as two, three, etc., and "multilayer" means at least two layers, such as two layers, three layers, etc., unless otherwise clearly and specifically defined. In the description of the present application, "several" means at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0053] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0054] Unless mentioned to the contrary, terms in the singular may include plural forms and should not be construed as being one in number.

[0055] The present application aims to solve the problem of environmental pollution and resource waste caused by direct discharge of a large amount of waste mother liquor containing staurosporine in the production process of staurosporine, and explores a method for recovering staurosporine from the waste mother liquor of staurosporine using a recrystallization method.

[0056] Staurosporine is usually prepared by separation from the fermentation broth of Streptomyces staurosporium. Since staurosporine is a fermentation product, fermentation impurities and pigments in the waste mother liquor are enriched in the mother liquor. If it is purified by crystallization, it will increase the difficulty of crystallization, the recovery rate is low, and the impurities are further enriched due to the introduction of new impurities. This is the difficulty of the present application.

[0057] Based on this, the purpose of the present application is to provide a method for recovering staurosporine from staurosporine waste mother liquor, which method has the advantages of low cost, simplicity and high efficiency, time and labor saving, and easy industrial application.

[0058] The technical solution is as follows:

[0059] Reference Figure 1 A method for recovering staurosporine from staurosporine waste mother liquor as shown in the examples of the present application comprises the following steps:

[0060] Concentrating the staurosporine waste mother liquor to collect a first solid;

[0061] The first solid is mixed with a first solvent, and the mixture is heated to 60° C. to 80° C. to dissolve, thereby obtaining a first mixed solution, wherein the first solvent is composed of an inorganic strong acid, a volatile low-carbon alcohol, and water;

[0062] Using an adsorbent to perform thermal adsorption treatment on the first mixed liquid to prepare a second mixed liquid;

[0063] collecting liquid in the second mixed liquid through a first solid-liquid separation process;

[0064] The liquid in the second mixed liquid is subjected to a cooling and crystallization treatment for 2 hours to 8 hours, and a second solid in the crystallization liquid is collected through a second solid-liquid separation, wherein the second solid contains acid water compound crystals of staurosporine;

[0065] The second solid is washed with a second solvent to collect a third solid, wherein the second solvent consists of the low-carbon alcohol and water;

[0066] Mixing the third solid and a base in the low-carbon alcohol, removing the acid in the third solid by an acid-base neutralization reaction, and preparing a third mixed solution;

[0067] Mixing the third mixed solution with water to perform crystallization treatment to prepare a fourth mixed solution;

[0068] The solid containing staurosporine in the fourth mixed liquid is collected by a third solid-liquid separation process.

[0069] In the present application, by concentrating the mother liquor, adding acid to convert staurosporine into staurosporine salt, and removing the pigment and some impurities by adsorption through the action of the adsorbent, the crystals of staurosporine salt are precipitated by utilizing the difference in solubility between staurosporine salt and impurities, and then adding alkali to remove the acid, so that the staurosporine crystals are free and precipitated again, the waste mother liquor of staurosporine can be used as waste, and the staurosporine in the mother liquor can be extracted, and the recovery rate of staurosporine is as high as 65% or more, and the recovery rate in some embodiments can reach more than 80%, and the purity of staurosporine is as high as 96.3% or more, and the purity in some embodiments can reach more than 97%, so as to achieve high recovery rate and high purity recovery of staurosporine, and significantly improve the resource utilization rate of staurosporine fermentation. In addition, in the present application, since the introduced acid and alkali undergo a neutralization reaction and enter the solution, only the same volatile low-carbon alcohol is introduced during the reaction process, and the low-carbon alcohol can be recovered after distillation treatment, and does not need to be discharged with the waste liquid, thereby improving its economic benefits and reducing environmental pollution. In addition, the method of the present application also has the advantages of simple process, short time consumption, low energy consumption, simple equipment requirements, low production cost, and can realize industrial mass production.

[0070] The method for recovering staurosporine from staurosporine waste mother liquor of the present application will be further described in detail below in a step-by-step manner.

[0071] S100: Concentrating the staurosporine waste mother liquor and collecting the first solid.

[0072] In one embodiment, in the concentration step, the concentration is carried out by vacuum distillation.

[0073] In one embodiment, the temperature of the concentration treatment is 40°C to 60°C, including but not limited to 40°C, 45°C, 50°C, 55°C or 60°C.

[0074] In one embodiment, the vacuum degree of the concentration treatment is 0.06 MPa to 0.09 MPa, including but not limited to 0.06 MPa, 0.07 MPa, 0.08 MPa or 0.09 MPa.

[0075] S200: mixing the first solid with a first solvent, heating to 60-80° C. to dissolve the solid, and obtaining a first mixed solution, wherein the first solvent is composed of a strong inorganic acid, a volatile low-carbon alcohol, and water.

[0076] In the present invention, the first solvent is composed of an inorganic strong acid, a volatile low-carbon alcohol and water. Wherein, the inorganic strong acid refers to an inorganic substance with strong acidity, such as one or more of hydrochloric acid, phosphoric acid, sulfuric acid and nitric acid. In the present application, for example, hydrochloric acid is further concentrated hydrochloric acid with a mass concentration of 36%. The low-carbon alcohol refers to an alcohol containing one to four carbons, such as one or more of ethanol, methanol, ethylene glycol, propanol and isopropanol, which can be miscible with water in any ratio. Further, in the first solvent, the volume ratio of the inorganic strong acid, the low-carbon alcohol and the water is 1: (15-25): (3-10), including but not limited to 1: 15: 3, 1: 15: 4, 1: 15: 5, 1: 15: 6, 1: 15: 7, 1: 15: 8, 1: 15: 9, 1: 15: 10, 1: 18: 3, 1: 18: 4, 1: 18: 5, 1: 18: 6 , 1:18:7, 1:18:8, 1:18:9, 1:18:10, 1:20:3, 1:20:4, 1:20:5, 1:20:6, 1:20:7, 1:20:8, 1:20:9, 1:20:10, 1:25:3, 1:25:4, 1:25:5, 1:25:6, 1:25:7, 1:25:8, 1:25:9 or 1:25:10.

[0077] In one embodiment, the volume mass ratio of the first solid to the first solvent is 1:(10-15), including but not limited to 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0078] In one embodiment, the first solid is added to the first solvent at a mass volume ratio of 1:(10-15), and the temperature is raised to 60° C. to 80° C. to dissolve, thereby obtaining a first mixed solution.

[0079] S300: using an adsorbent to perform thermal adsorption treatment on the first mixed liquid to prepare a second mixed liquid.

[0080] The adsorbent is an adsorbent that can effectively adsorb pigments and some impurities from the liquid, and the adsorbent can be selected from one or more of molecular sieves, alumina, diatomaceous earth, activated carbon and silica gel, preferably activated carbon. Further, the mass ratio of the adsorbent to the first solid in the first mixed liquid is (1-10):100, including but not limited to 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100 or 10:100.

[0081] In one embodiment, the temperature of the thermal adsorption treatment is 60°C to 80°C, including but not limited to 60°C, 65°C, 70°C, 75°C or 80°C.

[0082] In one embodiment, the thermal adsorption treatment time is 1 h to 5 h, including but not limited to 1 h, 2 h, 3 h, 4 h or 5 h.

[0083] S400: collecting liquid in the second mixed liquid through a first solid-liquid separation process.

[0084] In one embodiment, in the first solid-liquid separation step, the temperature of the first solid-liquid separation is 60° C. to 80° C., including but not limited to 60° C., 65° C., 70° C., 75° C. or 80° C. The specific method of the first solid-liquid separation is one or more of filtration, suction filtration and centrifugation.

[0085] S500: performing a cooling and crystallization treatment on the liquid in the second mixed liquid for 2 hours to 8 hours, and collecting a second solid in the crystallization liquid through a second solid-liquid separation, wherein the second solid contains acid water compound crystals of staurosporine.

[0086] It can be understood that the crystallization treatment method is natural cooling crystallization, and the crystallization time is 2h to 8h, including but not limited to 2h, 3h, 4h, 5h, 6h, 7h or 8h. Preferably, the crystallization time is 4h to 7h.

[0087] In one embodiment, the second solid-liquid separation process is performed at room temperature.

[0088] In one embodiment, the second solid-liquid separation treatment is performed by one or more of filtration, suction filtration and centrifugation.

[0089] S600: washing the second solid with a second solvent to collect a third solid, wherein the second solvent is composed of the low-carbon alcohol and water.

[0090] In one embodiment, the volume mass ratio of the second solid to the second solvent is 1:(10-15), including but not limited to 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0091] In one embodiment, the second solvent is composed of the low-carbon alcohol and water, and the volume ratio of the low-carbon alcohol to water is (15-25): (3-10). Preferably, the low-carbon alcohol is of the same type as the low-carbon alcohol in the first solvent. The third solid acid water compound crystal containing staurosporine is obtained after washing with the second solvent.

[0092] In one embodiment, the second solid is added to a second solvent at a mass volume ratio of 1:(10-15) for washing to obtain a third solid.

[0093] S700: mixing the third solid and a base in the low-carbon alcohol, removing the acid in the third solid through an acid-base neutralization reaction, and preparing a third mixed solution.

[0094] In one embodiment, the base is intended to neutralize the acid and includes one or more of ammonia, sodium carbonate, triethylamine, sodium hydroxide and potassium hydroxide, which compounds in water will produce hydroxide ions, thereby reacting with the hydrogen ions of the acid water compound containing staurosporine.

[0095] In one embodiment, during the acid-base neutralization reaction, the pH of the system is 8 to 10 and the time is 2 h to 5 h.

[0096] S800: Mix the third mixed solution with water to perform crystallization treatment to prepare a fourth mixed solution.

[0097] The third mixed solution is mixed with water, and the water acts as a precipitant, and staurosporine is gradually precipitated to obtain a fourth mixed solution of solid and liquid.

[0098] In one embodiment, in the step of mixing the third mixed liquid with water for crystallization treatment, the volume ratio of the water to the third mixed liquid is (1-3):1, including but not limited to 1:1, 2:1 or 3:1.

[0099] S900: collecting the solid containing staurosporine in the fourth mixed liquid through a third solid-liquid separation process.

[0100] In one embodiment, the temperature of the third solid-liquid separation is room temperature. Further, the specific method is one or more of filtration, suction filtration and centrifugation.

[0101] In one embodiment, after the third solid-liquid separation step, the method further comprises washing and drying the solid containing staurosporine in sequence to prepare a pure staurosporine product.

[0102] In one embodiment, the washing treatment is water washing until the washing liquid is neutral.

[0103] In one embodiment, after the water washing step, the collected solid is dried. Further, the drying temperature is 80°C to 110°C, including but not limited to 80°C, 85°C, 90°C, 95°C, 100°C, 105°C or 110°C. The vacuum degree of the drying is 0.08MPa to 0.1MPa, including but not limited to 0.06MPa, 0.07MPa, 0.08MPa, 0.09MPa or 0.1MPa.

[0104] The present application is further described below in conjunction with specific embodiments.

[0105] (1) The solvents used in the present application are not particularly limited and can be obtained from commercial sources.

[0106] (2) The specific preparation method of the staurosporine waste mother liquor used in this application can be referred to CN107446011A.

[0107] (3) The HPLC analysis method of staurosporine involved in the present application is: using octadecyl bonded silica gel as a filler, acetonitrile-0.1% triethylamine solution 500:500 as a mobile phase; the column temperature is 35°C, the flow rate is 1.0 ml / min, the isocratic elution is 60 min, and the detection wavelength is 292 nm.

[0108] Embodiment 1:

[0109] This embodiment provides a method for recovering staurosporine from staurosporine waste mother liquor, which is as follows:

[0110] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate at 50℃ and 0.08MPa to obtain 26.1g of brown solid. Add 300ml of hydrochloric acid ethanol solution (volume ratio is 36wt% concentrated hydrochloric acid: ethanol: water = 1:20:5). At this time, the solution is a suspension. Stir on the stirrer to release heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The solid begins to dissolve. At 65℃, slowly add 1.3g of activated carbon, and continue to heat up to 70℃. Filter while hot to remove insoluble matter. The filtrate is clear, and the temperature is naturally lowered and stirred, and a yellow solid precipitates. After 2 hours, the temperature was cooled to room temperature and stirring was continued for 4 hours. The turbid solution was filtered and washed with ethanol aqueous solution (volume ratio of ethanol: water = 20: 5) in small amounts for multiple times. The product was drained to obtain a total of 17.2 g of yellow solid. The product was poured into 180 ml of ethanol solution, and 22 wt% concentrated ammonia water was poured into it under stirring until the pH was ≥ 8. After stirring for 4 hours, 360 ml of water was poured into it to precipitate staurosporine. The solution was stirred until the temperature dropped to room temperature, filtered, and the solid was dried in a vacuum oven at 100°C and 0.09 MPa to obtain 15.5 g of yellow solid.

[0111] Example 2

[0112] This embodiment provides a method for recovering staurosporine from staurosporine waste mother liquor, which is as follows:

[0113] Take 5L of the mixed waste mother liquor (containing about 100g of staurosporine) and concentrate at 60℃ and 0.08MPa to obtain 130.1g of brown solid. Add 2L of hydrochloric acid ethanol solution (volume ratio is 36wt% concentrated hydrochloric acid: ethanol: water = 1:18:6), turn on the stirrer, and the solution will dissolve and heat up as it stirs, and it will turn black. When the temperature stops rising, turn on the heating and gradually increase the temperature. The solid begins to dissolve. At 60℃, slowly add 6.5g of activated carbon, continue to heat up to 75℃, and filter while hot. The filtrate is naturally cooled and stirred, and a yellow solid gradually precipitates. After 3 hours, the mixture was cooled to room temperature and stirred for 4 hours. The turbid solution was filtered and washed several times with ethanol-water solution (volume ratio of ethanol: water = 18:9), and the product was drained to obtain 90.8 g of yellow solid. The product was poured into 0.9 L of ethanol solution, and 22 wt% concentrated ammonia water was poured into the solution under stirring until the pH was ≥ 8. After stirring for 4 hours, 2 L of water was poured into the solution to precipitate staurosporine. The solution was stirred until the temperature dropped to room temperature, filtered, and the solid was dried in a vacuum oven at 100 ° C and 0.09 MPa to obtain 82.4 g of yellow solid.

[0114] Example 3

[0115] The difference between this embodiment and embodiment 1 is that ethanol is replaced with isopropanol, as follows:

[0116] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate at 50℃ and 0.08MPa to obtain 26.3g of brown solid. Add 300ml of hydrochloric acid isopropanol solution (volume ratio is 36wt% concentrated hydrochloric acid: isopropanol: water = 1:20:5). At this time, the solution is a suspension. Stir on the stirrer to release heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The solid begins to dissolve. At 65℃, slowly add 1.5g of activated carbon, and continue to heat up to 80℃. Filter while hot to remove insoluble matter. The filtrate is clear, and the temperature is naturally lowered and stirred, and a yellow solid precipitates. After 2 hours, cool to room temperature and continue stirring for 4 hours, filter, wash with isopropanol aqueous solution (volume ratio isopropanol: water = 20:5) in small amounts for multiple times, and drain the product to obtain a total of 16.1g of yellow solid. Pour the product into 200ml of isopropanol solution, pour 22wt% concentrated ammonia water under stirring to pH ≥ 8, stir for 4 hours, then pour 400ml of water to precipitate staurosporine, stir until the solution temperature drops to room temperature, filter, and dry the solid in a vacuum oven at 105°C, 0.09MPa to obtain 13.4g of yellow solid.

[0117] Example 4

[0118] The difference between this embodiment and embodiment 1 is that concentrated ammonia water is replaced with sodium hydroxide, as follows:

[0119] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate at 50℃ and 0.08MPa to obtain 26.0g of brown solid. Add 300ml of hydrochloric acid ethanol solution (volume ratio is 36wt% concentrated hydrochloric acid: ethanol: water = 1:20:5). At this time, the solution is a suspension. Stir on the stirrer to release heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The solid begins to dissolve. At 65℃, slowly add 1.3g of activated carbon, and continue to heat up to 70℃. Filter while hot to remove insoluble matter. The filtrate is clear, and the temperature is naturally lowered and stirred, and a yellow solid precipitates. After 2 hours, the temperature was cooled to room temperature and stirring was continued for 4 hours. The turbid solution was filtered and washed with ethanol aqueous solution (volume ratio of ethanol: water = 20: 5) in small amounts for multiple times. The product was drained to obtain 16.8 g of yellow solid. The product was poured into 180 ml of ethanol solution, and 10 wt% sodium hydroxide was poured into it under stirring until the pH was ≥ 9. After stirring for 4 hours, 360 ml of water was poured into it to precipitate staurosporine. The solution was stirred until the temperature dropped to room temperature, filtered, and the solid was dried in a vacuum oven at 100°C and 0.09 MPa to obtain 14.9 g of yellow solid.

[0120] Example 5

[0121] The difference between this embodiment and embodiment 1 is that hydrochloric acid is replaced with sulfuric acid, as follows:

[0122] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate at 50℃ and 0.08MPa to obtain 26.3g of brown solid. Add 300ml of sulfuric acid ethanol solution (volume ratio is 98wt% concentrated sulfuric acid: ethanol: water = 1:25:10). At this time, the solution is a suspension. Stir on the stirrer to release heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The color of the solution becomes darker and the solid begins to dissolve. At 65℃, slowly add 1.3g of activated carbon, and continue to heat up to 70℃. Filter while hot to remove insoluble matter. The filtrate is clear, and the temperature is naturally lowered and stirred, and a brown solid precipitates. After 2 hours, the temperature was cooled to room temperature and stirring was continued for 4 hours. The turbid solution was filtered and washed several times with ethanol aqueous solution (volume ratio of ethanol: water = 25:10) in small amounts, and the product was drained to obtain a total of 15.1 g of brown solid. The product was poured into 180 ml of ethanol solution, and 22 wt% concentrated ammonia water was poured into it under stirring until the pH was ≥ 8. After stirring for 4 hours, 360 ml of water was poured into it to precipitate staurosporine. The solution was stirred until the temperature dropped to room temperature, filtered, and the solid was dried in a vacuum oven at 100°C and 0.09 MPa to obtain 12.9 g of brown solid.

[0123] Comparative Example 1

[0124] The difference between Comparative Example 1 and Example 1 is that 1.3 g of activated carbon was not added for adsorption.

[0125] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate at 50℃ and 0.08MPa to obtain 26.1g of brown solid. Add 300ml of hydrochloric acid ethanol solution (volume ratio is 36wt% concentrated hydrochloric acid: ethanol: water = 1:20:5). At this time, the solution is a suspension. Stir on the stirrer to release heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The solid begins to dissolve, and continue to heat to 70℃. Filter while hot to remove insoluble matter. The filtrate is clear, and the temperature is naturally lowered and stirred, and a gray-yellow solid is precipitated. After 2 hours, the mixture was cooled to room temperature and stirred for 4 hours. The turbid solution was filtered and washed with ethanol aqueous solution (volume ratio of ethanol: water = 20:5) in small amounts for several times. The product was drained to obtain 17.8 g of gray-yellow solid. The product was poured into 180 ml of ethanol solution, and 22 wt% concentrated ammonia water was poured into the solution under stirring until the pH was ≥ 8. After stirring for 4 hours, 360 ml of water was poured into the solution to precipitate staurosporine. The solution was stirred until the temperature dropped to room temperature, filtered, and the solid was dried in a vacuum oven at 100 ° C and 0.09 MPa to obtain 15.6 g of gray-yellow solid.

[0126] Comparative Example 2

[0127] The difference between this comparative example 2 and example 1 is that after adding activated carbon to adsorb impurities, the temperature is directly lowered to room temperature to remove insoluble matter, as follows:

[0128] Take 1L of staurosporine waste mother liquor (containing about 20g of staurosporine) and concentrate it at 50℃ and 0.08MPa to obtain 26.2g of brown solid. Add 300ml of hydrochloric acid ethanol solution (volume ratio is 36wt% concentrated hydrochloric acid: ethanol: water = 1:20:5). At this time, the solution is a suspension. Stirring on the stirrer releases heat. When the temperature no longer rises, turn on the heating and gradually increase the temperature. The solid begins to dissolve. At 65℃, slowly add 1.3g of activated carbon, cool naturally and stir, and black solid precipitates. After 2 hours, cool to room temperature and continue stirring for 4 hours. Filter the turbid solution, wash it with ethanol aqueous solution (volume ratio is ethanol: water = 20:5) in small amounts and multiple times, and drain the product to obtain a total of 19.2g of black solid. Pour the filtrate into 22 wt% concentrated ammonia water while stirring until the pH is ≥ 8. After stirring for 4 hours, pour into 500 ml of water to precipitate a small amount of solid. Stir until the solution temperature drops to room temperature, filter, and dry the solid in a vacuum oven at 100°C and 0.09 MPa to obtain 2.5 g of brown solid.

[0129] Comparative Example 3

[0130] This comparative example 3 provides a method for recovering staurosporine from staurosporine waste mother liquor, which is as follows:

[0131] Take 1L of the mixed waste mother liquor (containing about 20g of staurosporine), add 300ml of hydrochloric acid ethanol solution (volume ratio of 36wt% concentrated hydrochloric acid: ethanol: water = 1:20:5), stir and heat on a stirrer and gradually raise the temperature to 80°C, pour in 22wt% concentrated ammonia water while stirring until the pH is ≥8, pour in 1L of water after stirring for 4 hours to precipitate staurosporine, stir until the solution temperature drops to room temperature, filter, and dry the solid in a vacuum oven at 100°C and 0.09MPa to obtain 3.6g of brown solid.

[0132] The color, purity and recovery rate data of staurosporine recovered in the above examples and comparative examples are shown in Table 1 below.

[0133] Table 1

[0134]

[0135] As can be seen from Table 1, according to the method for recovering staurosporine from staurosporine waste mother liquor shown in the embodiment of the present invention, the staurosporine waste mother liquor can be utilized to extract staurosporine in the mother liquor, and the staurosporine recovery rate is as high as 65% or more, and the recovery rate in some embodiments can reach more than 80%. The purity of staurosporine is as high as 96.3% or more, and the purity in some embodiments can reach more than 97%, achieving high recovery rate and high purity recovery of staurosporine, and significantly improving the resource utilization rate of staurosporine fermentation.

[0136] The difference between Comparative Example 1 and Example 1 is that after adding the adsorbent, although the data of Comparative Example 1 is better than that of other examples, since the staurosporine collected in Comparative Example 1 contains more unadsorbed impurity pigments, these impurities cannot be detected in HPLC, so the HPLC purity is higher, and its weight is counted into the total weight of staurosporine, so that the recovery rate is improved. In fact, the staurosporine obtained in Comparative Example 1 contains more impurities and is not truly pure staurosporine.

[0137] The difference between Comparative Example 2 and Example 1 is that after adding an adsorbent to adsorb impurities, the solution was not filtered while hot but directly cooled, so that the staurosporine originally dissolved in the solution was precipitated and mixed with the adsorbent and other impurities, resulting in lower purity and recovery rate of staurosporine.

[0138] The difference between Comparative Example 3 and Example 1 is that the mother liquor is not concentrated, and an alcohol aqueous solution of an inorganic acid is directly added to the mother liquor for acidification, and then an alkali is added to neutralize the acid. Moreover, before adding the alkali, no adsorbent is added to adsorb impurities and no impurities are removed by multiple solid-liquid separations. The purity and recovery rate of staurosporine are relatively low.

[0139] Figure 2 This is the HPLC chart of the staurosporine mother solution used in the examples of this application. Figure 3HPLC chart of pure staurosporine recovered from the waste mother liquor of staurosporine according to the method of recovering staurosporine from the waste mother liquor of staurosporine as shown in Example 1 of the present application, compared with Figure 2 and Figure 3 It can be seen that according to the method for recovering staurosporine from staurosporine waste mother liquor shown in Example 1 of the present invention, the staurosporine waste mother liquor can be utilized, the staurosporine in the mother liquor can be extracted, and the staurosporine pure product can be obtained.

[0140] In summary, the method for recovering staurosporine from staurosporine waste mother liquor provided in the present application can utilize the waste of staurosporine waste mother liquor, extract staurosporine in the mother liquor, improve the resource utilization rate of staurosporine fermentation, improve its economic benefits and reduce environmental pollution. In addition, the method of the present application also has the advantages of simple process, short time consumption, low energy consumption, simple equipment requirements, low production cost, and can realize industrial mass production.

[0141] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for recovering staurosporine from staurosporine waste mother liquor, characterized in that: The steps include: Concentrating the staurosporine waste mother liquor to collect a first solid; The first solid is mixed with a first solvent, and the mixture is heated to 60° C. to 80° C. to dissolve, thereby obtaining a first mixed solution, wherein the first solvent is composed of a strong inorganic acid, a volatile low-carbon alcohol, and water; Using an adsorbent to perform thermal adsorption treatment on the first mixed liquid to prepare a second mixed liquid; collecting liquid in the second mixed liquid through a first solid-liquid separation process; The liquid in the second mixed liquid is subjected to a cooling and crystallization treatment for 2 hours to 8 hours, and a second solid in the crystallization liquid is collected through a second solid-liquid separation, wherein the second solid contains acid water compound crystals of staurosporine; The second solid is washed with a second solvent to collect a third solid, wherein the second solvent consists of the low-carbon alcohol and water; Mixing the third solid and a base in the low-carbon alcohol, removing the acid in the third solid by an acid-base neutralization reaction, and preparing a third mixed solution; Mixing the third mixed solution with water to perform crystallization treatment to prepare a fourth mixed solution; The solid containing staurosporine in the fourth mixed liquid is collected by a third solid-liquid separation process.

2. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: The first solvent satisfies one or more of the following characteristics: The inorganic strong acid includes one or more of hydrochloric acid, phosphoric acid, sulfuric acid and nitric acid; The low-carbon alcohol includes one or more of ethanol, methanol, ethylene glycol, propanol and isopropanol; The volume ratio of the inorganic strong acid, the low carbon alcohol and the water is 1:(15-25):(3-10); The volume mass ratio of the first solvent to the first solid is (10-15):

1.

3. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: The thermal adsorption treatment step satisfies one or more of the following characteristics: The adsorbent includes one or more of activated carbon, molecular sieve, alumina, diatomaceous earth and silica gel; The mass ratio of the adsorbent to the first solid is (1-10):100; The temperature of the thermal adsorption treatment is 60°C to 80°C, and the time is 1h to 5h.

4. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: The first solid-liquid separation step satisfies one or more of the following characteristics: The temperature of the first solid-liquid separation treatment is 60°C to 80°C; The first solid-liquid separation treatment method is one or more of filtration, suction filtration and centrifugation.

5. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: The second solid-liquid separation step satisfies one or more of the following characteristics: The second solid-liquid separation treatment is carried out at room temperature; The second solid-liquid separation treatment method is one or more of filtration, suction filtration and centrifugation.

6. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: Meet one or more of the following characteristics: In the second solvent, the volume ratio of the low-carbon alcohol to water is (15-25):(3-10); The base includes one or more of ammonia water, sodium carbonate, triethylamine, sodium hydroxide and potassium hydroxide; During the acid-base neutralization reaction, the pH of the system is 8 to 10 and the time is 2 h to 5 h.

7. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: In the step of mixing the third mixed liquid with water for crystallization treatment, the volume ratio of the water to the third mixed liquid is (1-3):

1.

8. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: The third solid-liquid separation step satisfies one or more of the following characteristics: The third solid-liquid separation process is carried out at room temperature; The third solid-liquid separation treatment method is one or more of filtration, suction filtration and centrifugation.

9. The method for recovering staurosporine from staurosporine waste mother liquor according to any one of claims 1 to 8, characterized in that: The concentration step satisfies one or more of the following characteristics: The concentration treatment method is vacuum distillation; Optionally, the temperature of the concentration treatment is 40°C to 60°C, and the vacuum degree is 0.06MPa to 0.09Mpa.

10. The method for recovering staurosporine from staurosporine waste mother liquor according to claim 1, characterized in that: After the third solid-liquid separation step, the step of sequentially washing and drying the solid containing staurosporine to prepare a pure staurosporine product is also included; Optionally, the washing treatment is water washing; Optionally, the conditions for the drying treatment include: a drying temperature of 80° C. to 110° C. and a vacuum degree of 0.08 MPa to 0.1 MPa.

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