Method for recovering dehydroepiandrosterone mother liquor substance by using enzyme reaction technology
Through enzyme reaction technology and multi-stage purification steps, the problems of incomplete reaction and impurities in the production process of dehydroepiandrosterone were solved, and the preparation of high-purity dehydroepiandrosterone was achieved, reducing costs and environmental pollution.
Patent Information
- Application Number
- CN202411846647.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-16
- Publication Date
- 2025-03-18
AI Technical Summary
In the prior art, during the production process of dehydroepiandrosterone, the esterides are not thoroughly reacted after the enzyme reaction, resulting in impurity of the product and impurities such as 4-AD, and androsin glycol and isomers, which affects the preparation of high-purity DHEA.
The dehydroepiandrosterone mother liquor was recovered by enzyme reaction technology, and the purity of dehydroepiandrosterone was gradually improved through the steps of primary purification, esterification, dual-stage purification, enzyme reaction and tertiary purification, to achieve a purity of ≥99%.
High purity preparation of dehydroepiandrosterone is achieved, which improves recycling and utilization, simplifies operating procedures, reduces production costs, and reduces environmental pollution.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of steroid drug preparation, and in particular to a method for recovering dehydroepiandrosterone mother liquor by utilizing enzyme reaction technology. Background Art
[0002] Dehydroepiandrosterone, English name: Dehydroepiandrosterone, chemical name: 3β-hydroxy-5-androsten-17-one, molecular formula: C 19 H 28 O2. Dehydroepiandrosterone is an important intermediate and raw material for the manufacture of steroid drugs. It also has the functions of delaying aging, maintaining youthful vitality, enhancing physical fitness, improving mood and sleep, improving memory, regulating immune system function, and improving body immunity.
[0003] Domestic manufacturers of dehydroepiandrosterone generally use androstenedione (4-AD), a product of plant sterol fermentation, as a raw material to synthesize dehydroepiandrosterone (DHEA) through esterification, ketalization, reduction, and hydrolysis. The synthesis route is as follows:
[0004] In recent years, with the rapid development of biotechnology, more and more scholars have used androstenedione (4-AD) as a raw material to synthesize dehydroepiandrosterone through two steps of conversion and enzyme reduction. The route is as follows: Route 1 is:
[0005] Route 2 is:
[0006] The inventors took the mother liquor in route 2 as the research object. In the process of realizing the present invention, the inventors found that there are at least the following problems in the prior art: after the enzyme reaction of the second step esterification product of the route, about 0.5-3% of the substrate will not react thoroughly, and after the enzyme reaction, about 0.5-1.5% of 4-AD will be produced, and 0.5-1.5% of androstenediol and its isomers will be produced; to prepare high-purity DHEA, it is necessary to purify the crude dehydroepiandrosterone to obtain DHEA with higher purity. The refined mother liquor contains DHEA (60-70%), 4-AD (5%-10%), esterification (5-10%), androstenediol and its isomers (5-10%) and other main components, and the recycling of this part of the material is very important. Summary of the invention
[0007] In order to solve the problems existing in the prior art, the present invention proposes a method for recovering dehydroepiandrosterone mother liquor by using enzyme reaction technology. The method has simple and rapid process and high product purity, which is beneficial to reducing the production cost of dehydroepiandrosterone, reducing the cost of treating solid waste and reducing environmental pollution.
[0008] To this end, the technical solution adopted by the present invention is: a method for recovering dehydroepiandrosterone mother liquor using enzyme reaction technology, characterized in that: Using dehydroepiandrosterone mother liquor as raw material, after primary purification, esterification, secondary purification, enzyme reaction, and tertiary purification, dehydroepiandrosterone is obtained with a purity of ≥99%; The process is:
[0009] The specific processing method includes the following steps: Step A: Add dehydroepiandrosterone mother liquor to a reaction bottle, add solvent 1 for primary purification, stir evenly, heat to reflux to dissolve, slowly add drinking water dropwise, keep warm and reflux, then cool and crystallize, filter, and dry in an oven to obtain a purified dehydroepiandrosterone mother liquor; Step B: adding solvent 2 to the purified dehydroepiandrosterone mother liquor in step A, and then adding a catalyst to the reaction bottle, controlling the temperature and stirring the reaction, adding drinking water for water separation after the reaction is completed, filtering, washing with water until neutral, and draining the material; adding solvent 3 to the material, performing secondary purification, controlling the temperature and stirring, cooling, filtering, and draining the material to obtain dehydroepiandrosterone esterification; Step C: adding the dehydroepiandrosterone ester obtained in step B, water, solvent 4, auxiliary materials, buffer salt, enzyme into a reaction bottle, controlling the temperature, adjusting the pH with alkaline solution, and completing the reaction; and filtering to obtain a crude dehydroepiandrosterone product; Step D: The crude dehydroepiandrosterone in step C is subjected to three-stage purification by five solvent recrystallizations and dried to obtain a high-purity finished product.
[0010] Preferably, the solvent 1 in step A is one of methanol, ethanol, acetone or a mixed solvent, and the weight ratio of the solvent 1 to the dehydroepiandrosterone mother liquor is 2-10:1, the amount of drinking water added is 0.1-2:1, and the dropwise addition time is 1-4h; the temperature for cooling and crystallization is controlled at 0-20°C, and the drying temperature is: 60-70°C.
[0011] Preferably, the second solvent in step B is acetic anhydride, the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 2-5:1, and the cooling temperature is controlled at 0°C-20°C; the added catalyst is p-toluenesulfonic acid, the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 0.05-0.1:1, the temperature control reaction temperature range is 5-35°C, the stirring time is 1-10h, and the weight ratio of water for hydrolysis to the purified dehydroepiandrosterone mother liquor is 5-30:1; the third solvent added to the material is one of methanol, ethanol or acetone, the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 1-2:1, the stirring temperature is controlled at 15-50°C, the temperature is reduced to 0-20°C before filtration, and the dehydroepiandrosterone ester is obtained after drying.
[0012] Preferably, the solvent four in step C is one of tert-butyl alcohol, isopropanol, and ethyl acetate, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 3-10:1; the weight ratio of drinking water to the dehydroepiandrosterone ester is 5-12:1; the auxiliary material is glucose, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 0.5-1.5:1; the buffer salt is one of sodium dihydrogen phosphate and potassium dihydrogen phosphate, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 0.02-1.0:1; the reaction The temperature should be controlled at 10-40°C, and the reaction time is 5-20h; the enzyme solution is a mixed solution of hydrolase: reductase: glucose dehydrogenase: coenzyme = 10:10:2:1, and the weight ratio of the enzyme solution to the dehydroepiandrosterone ester is 0.5-1:1; the alkali solution is one of sodium carbonate or sodium hydroxide, and the concentration is 10-20%; the pH is controlled between 6.4-6.8, and the reaction time is 6-24h; after the reaction is completed, the crude dehydroepiandrosterone is obtained by suction filtration, and the purity is ≥92.5%.
[0013] Preferably, the solvent five used for the three-stage purification of recrystallization in step D is one of methanol, ethanol, acetone, and ethyl acetate or a mixture thereof, and the weight ratio of the solvent to the crude dehydroepiandrosterone is 5-20:1, the weight ratio of the residual volume of the crystallization solvent to the crude dehydroepiandrosterone is 1-2:1 (ml / g), the crystallization temperature is controlled at 0-35°C, the crystallization time is 0.5-3h, the drying temperature is controlled at 60-70°C, and the obtained dehydroepiandrosterone has a purity of ≥99%.
[0014] Beneficial effects: The method for recycling dehydroepiandrosterone mother liquor provided by the present invention comprises the following steps: step A is to remove androstenediol and its isomer impurities in the mother liquor by refining with a solvent; step B is to mainly convert 4-AD in the mother liquor into an esterified product by esterification, which is beneficial to subsequent enzyme reaction; step C is to convert the esterified product into the target product dehydroepiandrosterone by enzyme reaction; and step D is to improve the purity of dehydroepiandrosterone by purification.
[0015] Compared with the existing technology, this method is easy to operate and efficient, and has the following advantages: (1) high recovery rate, qualified products, and dehydroepiandrosterone purity ≥ 99%; (2) the treatment method is simple and easy to operate, with a large treatment volume; (3) cost savings and reduced environmental pollution. DETAILED DESCRIPTION
[0016] The technical solution of the present invention is clearly and completely described below through specific embodiments, but the embodiments should not be construed as limiting the present invention.
[0017] Embodiment 1: (1) Add 100 g of dehydroepiandrosterone mother liquor to a reaction bottle, add 200 g of methanol and stir evenly, then heat to 70°C, maintain for 1 hour, dropwise add 50 g of drinking water, and after the dropwise addition is complete, slowly cool to 5°C, filter, and dry in an oven at 60°C to constant weight, collecting 85 g of the purified dehydroepiandrosterone mother liquor.
[0018] (2) 85 g of the purified dehydroepiandrosterone mother liquor obtained in (1) was added to a reaction flask, and 170 g of acetic anhydride was added. The mixture was stirred and cooled to 10° C., and then 4.25 g of p-toluenesulfonic acid was added to the reaction flask. The mixture was stirred and reacted at 20° C. for 2 h. TLC was taken to confirm that the reaction was complete. The reaction solution was added to 850 g of water, stirred thoroughly, filtered, washed with water until neutral, and dried. The obtained material was put into a reaction flask, 85 g of methanol was added, stirred, heated to 40° C., then cooled to 20° C., filtered, and dried to obtain a dehydroepiandrosterone ester with a wet weight of 82.5 g.
[0019] (3) Add 82.5 g of the dehydroepiandrosterone ester in (2), 247.5 g of tert-butyl alcohol, 825 g of drinking water, 82.5 g of glucose, and 1.65 g of sodium dihydrogen phosphate into a reaction flask, control the temperature to 20°C, add 41.25 g of enzyme solution, control the pH value between 6.4 and 6.8, and keep the reaction temperature for 10 h. After the TLC reaction is completed, filter, wash with water until neutral, and drain to obtain 75.5 g of crude dehydroepiandrosterone with a wet weight of 93.75% purity.
[0020] (4) Add 75.5 g of the crude dehydroepiandrosterone obtained in (3) and 755 g of methanol to a reaction flask, stir and heat until completely dissolved, filter, evaporate 600 g of methanol from the filtrate, cool to 20°C for crystallization for 3 h, filter with suction, and dry at 60°C to obtain 68.2 g of refined dehydroepiandrosterone with a purity of 99.45%.
[0021] Embodiment 2: (1) Add 100 g of dehydroepiandrosterone mother liquor to a reaction bottle, add 200 g of ethanol and stir evenly, then heat to 85°C, maintain for 1 hour, and drop 100 g of drinking water. After the dropwise addition is complete, slowly cool to 5°C, filter to obtain a purified dehydroepiandrosterone mother liquor, and dry in an oven at 65°C until constant weight is obtained to obtain 87.5 g of the material.
[0022] (2) Add 87.5 g of the purified dehydroepiandrosterone mother liquor obtained in (1) to a reaction flask, add 262.5 g of acetic anhydride, stir and cool to 15° C., then add 8.75 g of p-toluenesulfonic acid to the reaction flask, keep at 25° C. and stir for 3 h, take a TLC sample, and after confirming that the reaction is complete, add the reaction solution to 1750 g of water, stir well, filter, wash with water until neutral, and drain the material; put the obtained material into a reaction flask, add 175 g of ethanol, stir and heat to 50° C., then cool to 30° C., filter, and drain to obtain a dehydroepiandrosterone ester with a wet weight of 85.5 g.
[0023] (3) Add 85.5 g of the dehydroepiandrosterone ester in (2), 427.5 g of isopropanol, 427.5 g of drinking water, 68.4 g of glucose, and 8.55 g of potassium dihydrogen phosphate into a reaction flask, control the temperature to 25° C., add 64.125 g of enzyme solution, control the pH value between 6.4 and 6.8, and keep the reaction temperature for 15 h. After the TLC reaction is completed, filter, wash with water until neutral, and drain to obtain 77.5 g of crude dehydroepiandrosterone with a wet weight of 93.56% purity.
[0024] (4) Add 77.5 g of the crude dehydroepiandrosterone obtained in (3) to a reaction flask, add 775 g of ethanol, stir and heat until completely dissolved, filter, evaporate 620 g of ethanol from the filtrate, cool to 15°C for crystallization for 1 h, filter with suction, and dry at 65°C to obtain 66.8 g of refined dehydroepiandrosterone with a purity of 99.35%.
[0025] Embodiment three: (1) Add 100 g of dehydroepiandrosterone mother liquor to a reaction bottle, add 200 g of acetone and stir evenly, then heat to 65°C, maintain for 2 h, dropwise add 200 g of drinking water, and after the dropwise addition is complete, slowly cool to 0°C, filter to obtain a purified dehydroepiandrosterone mother liquor, and dry in an oven at 70°C until constant weight is obtained to obtain 83 g of the material.
[0026] (2) 83 g of the purified dehydroepiandrosterone mother liquor obtained in (1) was added to a reaction flask, and 166 g of acetic anhydride was added. The mixture was stirred and cooled to 10° C., and then 6.64 g of p-toluenesulfonic acid was added to the reaction flask. The mixture was stirred and reacted at 20° C. for 2 h, and then a TLC sample was taken. After confirming that the reaction was complete, the reaction solution was added to 1660 g of water, stirred thoroughly, filtered, washed with water until neutral, and dried. The obtained material was put into a reaction flask, 83 g of acetone was added, stirred, heated to 40° C., and then cooled to 10° C., filtered, and dried to obtain a dehydroepiandrosterone ester with a wet weight of 81.2 g.
[0027] (3) Add 81.2 g of the dehydroepiandrosterone ester in (2), 324.8 g of Triton X-100, 812 g of drinking water, 81.2 g of glucose, and 16.24 g of sodium dihydrogen phosphate into a reaction flask, control the temperature to 28°C, add 81.2 g of enzyme solution, control the pH value between 6.4 and 6.8, and keep the reaction for 15 h. After the TLC reaction is completed, filter, wash with water until neutral, and drain to obtain 75.1 g of crude dehydroepiandrosterone with a wet weight of 94.35% purity.
[0028] (4) Add 75.1 g of crude dehydroepiandrosterone obtained in (3) and 901.2 g of acetone to a reaction flask, stir and heat until completely dissolved, filter, evaporate 751 g of acetone from the filtrate, cool to 0°C for crystallization for 1 h, filter with suction, and dry at 70°C to obtain 67.5 g of refined dehydroepiandrosterone with a purity of 99.5%.
[0029] Parts not described in detail in this specification are well-known technologies in the art.
[0030] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.
Claims
1. A method for recovering dehydroepiandrosterone mother liquor using enzyme reaction technology, characterized in that: Using dehydroepiandrosterone mother liquor as raw material, after primary purification, esterification, secondary purification, enzyme reaction, and tertiary purification, dehydroepiandrosterone is obtained with a purity of ≥99%; The specific processing method includes the following steps: Step A: Add dehydroepiandrosterone mother liquor to a reaction bottle, add solvent 1 for primary purification, stir evenly, heat to reflux to dissolve, slowly add drinking water dropwise, keep warm and reflux, then cool and crystallize, filter, and dry in an oven to obtain a purified dehydroepiandrosterone mother liquor; Step B: adding solvent 2 to the purified dehydroepiandrosterone mother liquor in step A, and then adding a catalyst to the reaction bottle, controlling the temperature and stirring the reaction, adding drinking water for water separation after the reaction is completed, filtering, washing with water until neutral, and draining the material; adding solvent 3 to the material, performing secondary purification, controlling the temperature and stirring, cooling, filtering, and draining the material to obtain dehydroepiandrosterone esterification; Step C: adding the dehydroepiandrosterone ester obtained in step B, water, solvent 4, auxiliary materials, buffer salt, enzyme into a reaction bottle, controlling the temperature, adjusting the pH with alkaline solution, and completing the reaction; and filtering to obtain a crude dehydroepiandrosterone product; Step D: The crude dehydroepiandrosterone in step C is subjected to three-stage purification by five solvent recrystallizations and dried to obtain a high-purity finished product.
2. The method for recovering dehydroepiandrosterone mother liquor by using enzyme reaction technology according to claim 1, characterized in that: The solvent 1 in step A is one of methanol, ethanol, acetone or a mixed solvent, and the weight ratio of the solvent 1 to the dehydroepiandrosterone mother liquor is 2-10:1, the amount of drinking water added is 0.1-2:1, and the dropwise addition time is 1-4h; the temperature for cooling and crystallization is controlled at 0-20°C, and the drying temperature is: 60-70°C.
3. A method for recovering dehydroepiandrosterone mother liquor using enzyme reaction technology according to claim 1 or 2, characterized in that: The second solvent in step B is acetic anhydride, and the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 2-5:1, and the cooling temperature is controlled at 0°C-20°C; the added catalyst is p-toluenesulfonic acid, and the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 0.05-0.1:1, the temperature control reaction temperature range is 5-35°C, the stirring time is 1-10h, and the weight ratio of water for hydrolysis to the purified dehydroepiandrosterone mother liquor is 5-30:1; the third solvent added to the material is one of methanol, ethanol or acetone, and the weight ratio of the solvent to the purified dehydroepiandrosterone mother liquor is 1-2:1, the stirring temperature is controlled at 15-50°C, the temperature is reduced to 0-20°C before filtration, and the dehydroepiandrosterone ester is obtained after drying.
4. The method for recovering dehydroepiandrosterone mother liquor by using enzyme reaction technology according to claim 3, characterized in that: The solvent four in step C is one of tert-butyl alcohol, isopropanol and ethyl acetate, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 3-10:1; the weight ratio of drinking water to the dehydroepiandrosterone ester is 5-12:1; the auxiliary material is glucose, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 0.5-1.5:1; the buffer salt is one of sodium dihydrogen phosphate and potassium dihydrogen phosphate, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 0.02-1.0:1; the reaction temperature is controlled at 10-40°C, and the reaction time is 5-20h; the enzyme solution is a mixed solution of hydrolase: reductase: glucose dehydrogenase: coenzyme = 10:10:2:1, and the weight ratio of the solvent to the dehydroepiandrosterone ester is 0.5-1:1; the alkali solution is one of sodium carbonate or sodium hydroxide, and the concentration is 10-20%; The pH value is controlled between 6.4 and 6.8, and the reaction time is 6 to 24 hours. After the reaction is completed, the crude dehydroepiandrosterone is obtained by filtration with a purity of ≥ 92.5%.
5. A method for recovering dehydroepiandrosterone mother liquor using enzyme reaction technology according to claim 1, 2 or 4, characterized in that: The solvent five used for the three-stage purification of recrystallization in step D is one of methanol, ethanol, acetone, and ethyl acetate or a mixture thereof, and the weight ratio of the solvent to the crude dehydroepiandrosterone is 5-20:1, the weight ratio of the residual volume of the crystallization solvent to the crude dehydroepiandrosterone is 1-2:1 (ml / g), the crystallization temperature is controlled at 0-35°C, the crystallization time is 0.5-3h, the drying temperature is controlled at 60-70°C, and the purity of the obtained dehydroepiandrosterone is ≥99%.