Method for purifying dehydroepiandrosterone

Abstract

The invention belongs to the technical field of purification, and particularly relates to a method for purifying dehydroepiandrosterone. According to the method, the good solvent and the large-scale solvent are sequentially used for purifying the dehydroepiandrosterone, the problems that a traditional purification method is complex in operation process, poor in purification effect on excessive reduction products and 4AD and the like are solved, and the purification method is easy to operate, mild in condition, low in equipment requirement, short in period, low in cost, easy for industrial production and suitable for industrial production. The purity of the product purified by the method disclosed by the invention is up to 99%, and the single impurity is less than 0.1%.

Description

Technical Field

[0001] The invention belongs to the technical field of purification, and in particular relates to a method for purifying dehydroepiandrosterone. Background Art

[0002] Dehydroepiandrosterone (DHEA) is an important steroidal API for humans and a precursor for the synthesis of a variety of steroidal hormones in humans. It has the ability to inhibit excessive mitochondrial production and reduce excessive 5-phosphoribosyl lipid production, ultimately inhibiting and preventing tumor growth, and has very significant anticancer drug activity. DHEA and its sulfate esters also have a variety of potential biological effects, such as anti-aging activity, anti-inflammatory activity, antiviral activity and antidepressant activity.

[0003] The preparation of high-purity dehydroepiandrosterone has an important position. Biotransformation has become an important method for the current industrial production of steroid hormones and dehydroepiandrosterone because of its extremely high stereoselectivity and efficient conversion ability. Anna Fryszkowska (Org. Process Res. Dev. 2016, 20, 1520-1528) et al. reported a method for preparing DHEA using carbonyl reductase, using 4AD to prepare 5AD by chemical method, and then reducing the carbonyl group at position 3 to obtain DHEA enzyme conversion liquid, extracting by ethyl acetate, drying with magnesium sulfate, and concentrating under reduced pressure to obtain DHEA crude product, followed by adding acetic acid for acidification, precipitating and filtering in methanol aqueous solution, washing the filter residue with hot methanol, and then washing it twice with methanol aqueous solution. After cumbersome steps, the final yield is 74%. It can be seen that the existing method is not only cumbersome to operate, but also needs to take into account the many wastes generated in industrial production. Therefore, there is an urgent need for a method that is simple to operate, environmentally friendly and can effectively purify dehydroepiandrosterone. Summary of the invention

[0004] In order to solve at least one of the above problems, the present invention provides a method for purifying dehydroepiandrosterone in a first aspect, comprising the following steps:

[0005] A. distilling the enzyme conversion liquid obtained by the bioconversion reaction of 3-ethoxy-androsta-3,5-diene-17-one under reduced pressure, filtering, adding good solvent 1, heating and reflux until the solution is clear, filtering, and concentrating the mother liquor under reduced pressure to obtain a crude dehydroepiandrosterone;

[0006] B. mixing the crude dehydroepiandrosterone with good solvent 2, heating and refluxing until the solution becomes clear, adding water, cooling and crystallizing, and filtering to obtain a first purified product;

[0007] C. The first purified product is mixed with a polar solvent, heated to reflux until the solution becomes clear, cooled to crystallize, and filtered to obtain the second purified dehydroepiandrosterone.

[0008] The dehydroepiandrosterone purification method provided by the present invention solves the problems of complicated operation of purifying dehydroepiandrosterone by enzyme conversion liquid obtained by bioconversion reaction of 3-ethoxy-androsta-3,5-diene-17-, poor purification effect on over-reduction products and 4AD, etc. The purification method of the present invention is simple to operate, mild conditions, low equipment requirements, short cycle, low cost, easy to industrialize, and the purity of the product is as high as 99%, and the single impurity is less than 0.1%.

[0009] According to a specific embodiment of the present invention, in step A, the temperature during the reduced pressure distillation is 38 degrees Celsius and the vacuum degree is -0.08 MPa. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0010] According to a specific embodiment of the present invention, the good solvent 1 is a ketone solvent.

[0011] According to a specific embodiment of the present invention, the ketone solvent includes any one of acetone and methyl ethyl ketone.

[0012] According to a specific embodiment of the present invention, the ketone solvent is acetone, and the mass percentage concentration of the acetone is 98%-100%. Under this condition, the dehydroepiandrosterone acquisition rate and purity are both high.

[0013] According to a specific embodiment of the present invention, the temperature during heating and reflux is 58° C., and the reflux time is 30 min. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0014] According to a specific embodiment of the present invention, the temperature during the reduced pressure concentration is 38 degrees Celsius and the vacuum degree is -0.08 MPa. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0015] According to a specific embodiment of the present invention, in step B, the good solvent 2 is a ketone solvent.

[0016] According to a specific embodiment of the present invention, the ketone solvent includes any one of acetone and methyl ethyl ketone.

[0017] According to a specific embodiment of the present invention, the ketone solvent is acetone, and the mass percentage concentration of the acetone is 98%-100%. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0018] According to a specific embodiment of the present invention, in step B, the temperature during heating and reflux is 60-70 degrees Celsius, and the reflux time is 30 minutes. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0019] The temperature-lowering crystallization condition is zero-degree crystallization, under which the dehydroepiandrosterone acquisition rate is relatively high.

[0020] According to a specific embodiment of the present invention, in step C, the polar solvent is an alcohol solvent.

[0021] According to a specific embodiment of the present invention, the alcohol solvent includes any one of methanol, ethanol and isopropanol.

[0022] According to a specific embodiment of the present invention, the alcohol solvent is methanol, and the mass percentage concentration of the methanol is 80%-100%. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0023] According to a specific embodiment of the present invention, in step C, the temperature during heating and reflux is 60-70 degrees Celsius, and the reflux time is 30 minutes. Under this condition, the dehydroepiandrosterone acquisition rate is relatively high.

[0024] According to a specific embodiment of the present invention, the temperature reduction crystallization condition is zero degree crystallization, under which the dehydroepiandrosterone acquisition rate is higher.

[0025] According to a specific embodiment of the present invention, the mass ratio of the good solvent 1 to the 3-ethoxy-androsta-3,5-diene-17-one is (7-9):1, under which the dehydroepiandrosterone acquisition rate is relatively high.

[0026] According to a specific embodiment of the present invention, the mass ratio of the good solvent 2 to the crude dehydroepiandrosterone is (3-5):1, under which the dehydroepiandrosterone acquisition rate is high.

[0027] According to a specific embodiment of the present invention, the mass ratio of the polar solvent to the first purified product is (5-7):1, under which the dehydroepiandrosterone acquisition rate is high.

[0028] According to a specific embodiment of the present invention, in step B, the mass ratio of the added water to the good solvent 2 is 1:(1.5-3), under which condition the dehydroepiandrosterone acquisition rate is high.

[0029] Compared with the prior art, the present invention has the following beneficial effects: the dehydroepiandrosterone purification method provided by the present invention solves the problems of the complicated operation process of the traditional purification method and the poor purification effect on the over-reduction product and 4AD. The purification method of the present invention is simple to operate, mild in conditions, low in equipment requirements, short in cycle, low in cost, easy to industrialize, and the purity of the product is as high as 99%, and the single impurity is less than 0.1%. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described in detail below, and are shown in the examples of the embodiments. The following embodiments are exemplary and intended to be used to explain the present invention, but cannot be understood as limiting the present invention.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0032] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0033] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0034] In this article, the term "reduced pressure distillation" (also known as vacuum distillation) is an important method for separating and purifying compounds, especially for the separation and purification of high-boiling point substances and those compounds that have been thermally decomposed, oxidized or polymerized before reaching the boiling point during atmospheric distillation.

[0035] In this article, the term "good solvent" refers to a solvent that has a strong solubility for polymer solutes and an interaction parameter χ with the polymer solute that is less than 0.5.

[0036] The term "biotransformation" refers to the metabolic process in which the chemical structure of a poison changes after being catalyzed by an enzyme, that is, the poison undergoes a qualitative change. Biotransformation is an important event that occurs before a poison is eliminated in an organism, and its typical outcome is the production of non-toxic or low-toxic metabolites. Therefore, biotransformation was once equated with detoxification. Specifically, in the present invention, the conditions for the biotransformation reaction are 3-ethoxy-androsta-3,5-diene-17-one in PBS buffer and DCM cosolvent, lipase (Novozym-51032) and reductase (EW-KRED-kit) are added, and the reaction is carried out at 30 degrees Celsius for 8 hours.

[0037] The present invention provides a method for extracting dehydroepiandrosterone from an enzyme conversion solution. The dehydroepiandrosterone obtained by the method of the present application has high purity and yield.

[0038] The crude dehydroepiandrosterone used in the present invention is obtained by biotransformation of 100 g of 3-ethoxy-androsta-3,5-diene-17-one by hydrolase and reductase. After the transformation is completed, the water-insoluble organic phase is removed by reduced pressure distillation, and the water is removed by suction filtration, and then 90 g of the desired purified crude product is dissolved with a good solvent, and the purity is 80%.

[0039] The technical solution adopted by the present invention to solve the technical problem is:

[0040] A method for extracting and purifying dehydroepiandrosterone from an enzyme conversion solution comprises the following specific steps: adding acetone and water to a crude dehydroepiandrosterone product, heating and refluxing to dissolve the product, cooling and crystallizing the product, filtering the product by suction, introducing the filter residue into methanol, heating and refluxing to dissolve the product by suction, cooling and crystallizing the product, filtering the product by suction, and drying the product to obtain a pure dehydroepiandrosterone product.

[0041] Preferably, a good solvent is used to extract the crude dehydroepiandrosterone, and the amount of the good solvent is 7-9 times the original amount of 3-ethoxy-androsta-3,5-dien-17-one.

[0042] Preferably, a good solvent is used in the first purification of dehydroepiandrosterone, and the amount of the good solvent is 3-5 times the quality of the crude product.

[0043] Preferably, a highly polar solvent is used in the second purification of dehydroepiandrosterone, and the amount of the highly polar solvent used is 5-7 times the quality of the crude product after the first purification.

[0044] Preferably, the good solvent is a ketone reagent, the ketone reagent is acetone, and the concentration of the acetone is 99.5%.

[0045] Preferably, the highly polar solvent is an alcohol reagent, the alcohol reagent is methanol, and the concentration of the methanol is 80%-100%.

[0046] The beneficial effects of the present invention are as follows: the dehydroepiandrosterone purification method provided by the present invention solves the problems that the existing dehydroepiandrosterone purification by enzyme conversion liquid obtained by bioconversion reaction of 3-ethoxy-androsta-3,5-diene-17- is complicated, and the purification effect of over-reduction products and 4AD is poor. The purification method of the present invention is simple to operate, mild in conditions, low in equipment requirements, short in cycle, low in cost, and easy to industrialize. The purity of the product is as high as 99%, and the single impurity is less than 0.1%.

[0047] The reagents used in the present invention can all be purchased from the market by conventional means, wherein the purchase sources and product numbers of the main reagents are shown in Table 1.

[0048] Table 1

[0049]

[0050] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Example 1

[0052] The extraction and purification method of dehydroepiandrosterone is as follows:

[0053] Step 1: Preparation of crude DHEA

[0054] 100 g of 3-ethoxy-androst-3,5-diene-17-one was subjected to a bioconversion reaction by hydrolase and reductase to obtain an enzyme conversion liquid, and the enzyme conversion liquid was distilled under reduced pressure at 38 degrees Celsius and -0.08 MPa to remove the water-insoluble organic phase, and the water layer was removed by suction filtration, and then 800 g of acetone was added to reflux and dissolve, and the insoluble matter was removed, and finally concentrated under reduced pressure to obtain 90 g of crude dehydroepiandrosterone (purity of 76.7%, containing 0.5% 5-androstenediol and 4% 4AD).

[0055] Step 2: First purification

[0056] 90 g of crude dehydroepiandrosterone was placed in a 2L bottle, 360 g of acetone was added, the temperature was raised to 65 degrees, refluxed to dissolve, 180 g of water was added, the temperature was lowered to 0 degrees for crystallization, and 77 g of white solid was obtained by suction filtration.

[0057] Step 3: Second purification

[0058] Transfer the white solid obtained in step 2 to a 1L bottle, add 540g of methanol, heat to 65 degrees, reflux to dissolve, cool to 0 degrees to crystallize, filter, and dry to obtain 65.4g of white solid.

[0059] Example 2

[0060] The preparation and purification method of dehydroepiandrosterone is as follows:

[0061] Step 1: Preparation of crude DHEA

[0062] 100 g of 3-ethoxy-androst-3,5-diene-17-one was subjected to a bioconversion reaction by hydrolase and reductase to obtain an enzyme conversion liquid, and the enzyme conversion liquid was distilled under reduced pressure at 38 degrees Celsius and -0.08 MPa to remove the water-insoluble organic phase, and the water layer was removed by suction filtration, and then 800 g of acetone was added to reflux and dissolve, and the insoluble matter was removed, and finally concentrated under reduced pressure to obtain 90 g of crude dehydroepiandrosterone (purity of 79.1%, containing 0.18% 5-androstenediol and 10.2% 4AD).

[0063] Step 2: First purification

[0064] Place 90 g of crude dehydroepiandrosterone in a 2L bottle, add 360 g of acetone, heat to 65 degrees, reflux to dissolve, add 180 g of water, cool to 0 degrees for crystallization, and filter to obtain 75 g of white solid.

[0065] Step 3: Second purification

[0066] Transfer the white solid obtained in step 2 to a 1L bottle, add 540g of methanol, heat to 65 degrees, reflux to dissolve, cool to 0 degrees to crystallize, filter, and dry to obtain 67.5g of white solid.

[0067] Example 3

[0068] The preparation and purification method of dehydroepiandrosterone is as follows:

[0069] Step 1: Preparation of crude DHEA

[0070] 10 g of 3-ethoxy-androst-3,5-diene-17-one was subjected to bioconversion reaction by hydrolase and reductase to obtain an enzyme conversion liquid, and the enzyme conversion liquid was distilled under reduced pressure at 38 degrees Celsius and -0.08 MPa to remove the water-insoluble organic phase, and the water layer was removed by suction filtration, and then 80 g of acetone was added to reflux and dissolve, and the insoluble matter was removed, and finally concentrated under reduced pressure to obtain 9 g of crude dehydroepiandrosterone (purity of 80%, containing 0.52% 5-androstenediol and 8% 4AD).

[0071] Step 2: First purification

[0072] 9 g of crude dehydroepiandrosterone was placed in a 250 ml bottle, 36 g of acetone was added, the temperature was raised to 65 degrees, refluxed to dissolve, 18 g of water was added, the temperature was lowered to 0 degrees for crystallization, and 7.0 g of white solid was obtained by suction filtration.

[0073] Step 3: Second purification

[0074] Transfer the white solid obtained in step 2 into a 100 ml bottle, add 54 g of methanol, heat to 65 degrees, reflux to dissolve, cool to 0 degrees to crystallize, filter, and dry to obtain 6.7 g of white solid.

[0075] Comparative Example 1

[0076] The difference from Example 1 is that in the first purification process of step 2, acetone is replaced by ethyl acetate, and the remaining steps are the same.

[0077] Comparative Example 2

[0078] The difference from Example 1 is that in the first purification process of step 2, acetone is replaced by a mixed solution of ethyl acetate and water, and the remaining steps are the same.

[0079] Comparative Example 3

[0080] The difference from Example 1 is that in the second purification process in step 3, methanol is replaced by dichloromethane, and the remaining steps are the same.

[0081] Comparative Example 4

[0082] The difference from Example 1 is that in the second purification process in step 3, methanol is replaced by isopropanol, and the remaining steps are the same.

[0083] Comparative Example 5

[0084] The difference from Example 1 is that in the second purification process in step 3, methanol is replaced by ethanol, and the remaining steps are the same.

[0085] Comparative Example 6

[0086] The difference from Example 1 is that the order of the solvents used in the first purification in step 2 and the second purification in step 3 is interchanged, and the remaining steps are the same.

[0087] Comparative Example 7

[0088] Take 10g of 3-ethoxy-androstane-3,5-diene-17-one and carry out bioconversion reaction with hydrolase and reductase to obtain enzyme conversion liquid, extract the enzyme conversion liquid three times with 400ml ethyl acetate, and then backwash the ethyl acetate three times with 400ml saturated brine. Then dry the ethyl acetate extract with anhydrous sodium sulfate and filter it, and distill the ethyl acetate under reduced pressure at 38 degrees and -0.08MPa to obtain 9.2g of crude dehydroepiandrosterone (purity is 70%, containing 0.52% over-reduction and 8% 4AD). After adding 65ml acetone and 0.5g activated carbon, reflux to the solution and stir for 1h, filter it while hot and cool it to -20 degrees in the refrigerator overnight to obtain 4.1g of white solid. After filtering, distill half of the solvent, continue to cool and crystallize, and finally obtain 2.7g. Add 6.8 g of the refined product to 34 ml of methanol, heat to 65 degrees, reflux to dissolve, cool to 0 degrees to crystallize, filter, and dry to obtain 5.3 g of a white solid.

[0089] The inventors used HPLC to analyze the components of the white solids obtained in Examples 1-3 and Comparative Examples 1-7. The results are shown in Table 2:

[0090] Table 2

[0091] Experimental Group Purity of dehydroepiandrosterone (%) Dehydroepiandrosterone yield (%) Impurity content (%) Example 1 99.41 72 Less than 0.1 Example 2 99.76 75 Less than 0.1 Example 3 99.6 77 Less than 0.1 Comparative Example 1 92.6 91 6.89(5-androstenediol) Comparative Example 2 95.34 85 1.30(4AD) Comparative Example 3 97.37 90 1.59(5-androstenediol) Comparative Example 4 99.26 69 0.31(5-androstenediol) Comparative Example 5 99.7 63 0.11(5-androstenediol) Comparative Example 6 99.38 44 0.14(5-androstenediol) Comparative Example 7 98.7 58 0.25(4AD)

[0092] It can be seen from Examples 1-3 and Comparative Examples 1-7 that the crude dehydroepiandrosterone with improved extraction method has higher purity, better properties and is easy to purify. At the same time, the removal rate of impurities can be well improved by adjusting the acetone ratio, and the purification yield is finally increased by 20%. The method in the embodiment has a more obvious advantage. The results of Comparative Example 2 and Comparative Example 1 show that when water is added to the purified solution, 5-androstenedione can be removed to a certain extent to increase the purity, but the overall yield is reduced.

[0093] The inventors further explored the solvent and feed amount in each reaction process, and each experimental group was set up as follows:

[0094] Experimental group 1: The difference from Example 1 is that in step 1, the mass of acetone is 225 g;

[0095] Experimental group 2: The difference from Example 1 is that in step 1, the mass of acetone is 630 g;

[0096] Experimental group 3: The difference from Example 1 is that in step 2, 180 g of water is not added;

[0097] Experimental group 4: The difference from Example 1 is that in step 3, the mass of methanol is 360 g;

[0098] Experimental group 5: The difference from Example 1 is that in step 3, the mass of methanol is 720 g;

[0099] The components of the white solids finally obtained from experimental groups 1-12 were analyzed by HPLC. The results of the first purification of the first three groups are shown in Table 3, and the results of the second purification of the last two groups are shown in Table 4:

[0100] Table 3

[0101] Experimental Group Purity of dehydroepiandrosterone (%) One fine recovery rate (%) Impurity content (%) Example 1 96.58 87 0.62(5-androstenediol) Experimental Group 1 92.87 90 6.14 (5-androstenediol) Experimental Group 2 97.30 79 0.53(5-androstenediol) Experimental Group 3 94.29 81 1.71(4AD)

[0102] According to the results of Example 1, Experimental Group 1 and Experimental Group 2 in Table 3, when the acetone content in step 1 is too low, although the yield of the crude dehydroepiandrosterone obtained by the first purification can be increased by 3%, the purity of the crude dehydroepiandrosterone obtained is greatly reduced (reduced by about 4%), which greatly increases the difficulty of subsequent purification; and when the acetone content in step 1 is too high, although the purity of the crude dehydroepiandrosterone obtained by the first purification can be slightly increased (0.88%), the yield of the crude dehydroepiandrosterone is greatly reduced (reduced by about 8%), which greatly reduces the yield of the dehydroepiandrosterone obtained by subsequent purification. Therefore, when the acetone feed amount in step 1 is about 8 times the original amount of 3-ethoxy-androsta-3,5-diene-17-one, the finally purified dehydroepiandrosterone has a higher purity and a higher yield.

[0103] According to the results of Example 1 and Experimental Group 3 in Table 3, when no water is added during the first purification in step 2, direct extraction with acetone not only reduces the purity of the finally purified dehydroepiandrosterone but also reduces the yield, which is contrary to the results of Comparative Example 2. This further illustrates that in the purification process, the selection of different reagents and the selection of different experimental conditions are crucial to the entire purification result.

[0104] Table 4

[0105] Experimental Group Purity of dehydroepiandrosterone (%) Secondary refinement recovery rate (%) Impurity content (%) Example 1 99.41 72 Less than 0.1 Experimental Group 4 98.78 81 0.53(4AD) Experimental Group 5 99.60 65 Less than 0.1

[0106] According to the results of Example 1 and Experimental Group 4 in Table 4, when the amount of methanol used in step 4 is too low during the second purification, the impurity content of the dehydroepiandrosterone obtained is relatively high, and when the impurity is higher than 0.1%, it is judged as a substandard product; according to the results of Example 4 and Experimental Group 5, when the amount of methanol used is too high, the final yield of the dehydroepiandrosterone obtained is relatively low, which will cause a lot of waste. It can be seen that when the amount of methanol added is about 6 times that of the crude product after the first purification, the dehydroepiandrosterone finally purified has a higher purity and a higher yield.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for purifying dehydroepiandrosterone, characterized in that: The following steps are involved: A. distilling the enzyme conversion liquid obtained by the bioconversion reaction of 3-ethoxy-androsta-3,5-diene-17-one under reduced pressure, filtering, adding good solvent 1, heating and reflux until the solution is clear, filtering, and concentrating the mother liquor under reduced pressure to obtain a crude dehydroepiandrosterone; B. mixing the crude dehydroepiandrosterone with good solvent 2, heating and refluxing until the solution becomes clear, adding water, cooling and crystallizing, and filtering to obtain a first purified product; C. The first purified product is mixed with a polar solvent, heated to reflux until the solution becomes clear, cooled to crystallize, and filtered to obtain the second purified dehydroepiandrosterone.

2. The method according to claim 1, characterized in that In step A, the temperature during the reduced pressure distillation is 38 degrees Celsius and the vacuum degree is -0.08 MPa; The good solvent 1 is a ketone solvent; The ketone solvent includes any one of acetone and methyl ethyl ketone; The ketone solvent is acetone, and the mass percentage concentration of the acetone is 98%-100%; The temperature during the heating reflux is 58°C and the reflux time is 30min; The temperature during the reduced pressure concentration is 38 degrees Celsius and the vacuum degree is -0.08 MPa.

3. The method according to claim 1, characterized in that In step B, the good solvent 2 is a ketone solvent; The ketone solvent includes any one of acetone and methyl ethyl ketone; The ketone solvent is acetone, and the mass percentage concentration of the acetone is 98%-100%.

4. The method according to claim 1, characterized in that: In step B, the temperature during heating and reflux is 60-70 degrees Celsius, and the reflux time is 30 minutes; The temperature reduction crystallization condition is zero degree crystallization.

5. The method according to claim 1, characterized in that: In step C, the polar solvent is an alcohol solvent; The alcohol solvent includes any one of methanol, ethanol and isopropanol; The alcohol solvent is methanol, and the mass percentage concentration of the methanol is 80%-100%.

6. The method according to claim 1, characterized in that In step C, the temperature during heating and reflux is 60-70 degrees Celsius, and the reflux time is 30 minutes; The temperature reduction crystallization condition is zero degree crystallization.

7. The method according to claim 1, characterized in that The mass ratio of the good solvent 1 to the 3-ethoxy-androsta-3,5-diene-17-one is (7-9):

1.

8. The method according to claim 1, characterized in that: The mass ratio of the good solvent 2 to the crude dehydroepiandrosterone is (3-5):

1.

9. The method according to claim 1, characterized in that: The mass ratio of the polar solvent to the first purified product is (5-7):

1.

10. The method according to claim 1, characterized in that In step B, the mass ratio of the added water to the good solvent 2 is 1:(1.5-3).

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