Preparation method of prednisolone

Abstract

The invention discloses a preparation method of prednisolone, which comprises the following steps: inoculating arthrobacter simplex to a slant culture medium for preliminary culture, and then transferring the slant culture to a seed solution culture medium for enlarged culture to obtain a seed solution; transferring the seed solution into a fermentation culture medium, controlling the culture temperature to be 29-31 DEG C, culturing for 10-16 hours, adding hydrocortisone and absolute ethyl alcohol, controlling the conversion temperature to be 32-34 DEG C, and converting for 20-25 hours. The prednisolone preparation method provided by the invention has the characteristics of high fermentation conversion rate, short period and low production cost, and is very suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, and particularly relates to a method for preparing prednisolone. Background Technology

[0002] Prednisolone, also known as hydroprednisolone or dehydroprednisolone, has the chemical name 11β,17α,21-trihydroxypregn-1,4-diene-3,20-dione. It is a white or off-white crystalline powder; odorless, with a slightly bitter taste; and hygroscopic. It is soluble in methanol or ethanol, slightly soluble in acetone or dioxane, sparingly soluble in chloroform, and very slightly soluble in water. Its molecular formula is C1. 21 H 28 O5, with a molecular weight of 360.45, a melting point of 240℃, a boiling point of 570.6℃, [α]D +96°~104°, CAS: 50-24-8, has the following structural formula: .

[0003] Prednisolone is a corticosteroid drug mainly used for the following diseases: (1) Autoimmune and inflammatory diseases: rheumatoid arthritis, systemic lupus erythematosus, exfoliative dermatitis, pemphigus, etc.; (2) Allergic diseases: severe bronchial asthma, allergic dermatitis, eczema; (3) Hematologic diseases: thrombocytopenic purpura, granulocytopenia, acute lymphoblastic leukemia; (4) Others: nephrotic syndrome, adrenal insufficiency, neurodermatitis, etc. Currently, prednisolone is mainly prepared by two methods: fermentation and chemical synthesis. It has been reported that the fermentation method has a low conversion rate, is difficult to extract and purify in the later stage, and has a long production cycle, which is affected by a variety of factors. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing prednisolone, which aims to solve the technical problems of low fermentation conversion rate and long production cycle in the existing prednisolone fermentation preparation process.

[0005] The objective of this invention is achieved through the following technical solution: A method for preparing prednisolone includes the following steps: Arthrobacterium simplex was inoculated onto slant culture medium for initial culture, and then the slant culture was transferred to seed culture medium for expansion culture to obtain seed culture; The seed culture was transferred to the fermentation medium and the culture temperature was controlled at 29℃~31℃. After culturing for 10~16h, hydrocortisone and anhydrous ethanol were added and the conversion temperature was controlled at 32℃~34℃. After conversion for 20h~25h, prednisolone was obtained. The fermentation medium comprises the following components in weight percentage: glucose 0.5%–1.0%, yeast extract 0.5%–1.0%, peptone 0.2%–0.8%, potassium dihydrogen phosphate 0.2%–0.4%, inducing agent 0.01%–0.03%, and the remainder being water; the inducing agent is at least one selected from hydrocortisone, isopropyl-β-D-thiogalactoside, methyl-β-D-thiogalactoside, and salicylic acid. More preferably, the inducing agent is hydrocortisone.

[0006] In this invention, common *Arthrobacter simplex* is used as the starting strain. First, the strain is inoculated onto an agar slant for initial culture, then transferred to a seed culture medium for expansion culture to obtain a seed culture. Finally, the seed culture is inoculated into a fermentation medium for fermentation culture. Notably, the fermentation medium of this invention contains an inducer—hydrocortisone. This substance not only serves as a reaction substrate but also as an inducer, activating the synthesis of key enzymes in microbial metabolic pathways, thereby significantly improving substrate conversion efficiency.

[0007] Preferably, the culture temperature of both the slant culture medium and the seed culture medium is 29℃~31℃, and the culture time is 42h~48h.

[0008] Preferably, the slant culture medium contains the following components in weight percentage: glucose 0.8%~1.2%, yeast extract 0.6%~1.0%, agar 2.0%~2.5%, and the remainder is water.

[0009] Preferably, the slant culture medium contains the following components in weight percentage: 1.0% glucose, 0.8% yeast extract, 2.5% agar, and the remainder is water.

[0010] Preferably, the seed culture medium contains the following components in weight percentage: glucose 1.0%~1.5%, yeast extract 1.0%~1.5%, peptone 0.2%~0.8%, potassium dihydrogen phosphate 0.1%~0.5%, and the remainder is water.

[0011] Preferably, the seed culture medium comprises the following components in weight percentage: 1.0% glucose, 1.2% yeast extract, 0.5% peptone, 0.3% potassium dihydrogen phosphate, and the remainder being water.

[0012] Preferably, the fermentation medium comprises the following components in weight percentage: 0.75% glucose, 0.75% yeast extract, 0.5% peptone, 0.3% potassium dihydrogen phosphate, 0.01%~0.03% inducer, and the remainder being water.

[0013] Preferably, in the step of adding hydrocortisone and anhydrous ethanol after culturing for 10h to 16h, the amount of hydrocortisone added is 30g / L to 50g / L, and the amount of anhydrous ethanol added accounts for 4 to 6% of the fermentation broth volume.

[0014] Preferably, the amount of hydrocortisone added is 40 g / L, and the amount of anhydrous ethanol added accounts for 5% of the fermentation broth.

[0015] Preferably, the pH of the slant culture medium, seed culture medium and fermentation culture medium are all 6.8 to 7.0.

[0016] In this invention, the composition ratios of the slant culture medium, seed culture medium, and fermentation medium have been optimized to better promote the growth and metabolism of the microorganisms. For example, this invention selects yeast extract as a key nutrient component, which is rich in nitrogen sources, vitamins, and coenzymes, effectively meeting the diverse nutritional needs of the microorganisms. It is a comprehensive natural nutritional supplement that can significantly promote the growth and activity of microorganisms.

[0017] Compared with the prior art, the beneficial effects of the present invention include: This invention significantly improves the conversion rate of hydrocortisone, with a conversion rate exceeding 90% at the end of fermentation and a conversion time of only 20-30 hours. It has the advantages of high conversion efficiency, short cycle, and low production cost, and is suitable for industrial production. Attached Figure Description

[0018] Figure 1 The HPLC chromatogram of prednisolone prepared in Example 1 is shown. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] Example 1 A method for preparing prednisolone, the specific steps of which are as follows: (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL slant culture medium (by mass percentage, it consists of the following components: glucose 1.0%, yeast extract 0.8%, agar 2.5%, the remainder is water; pH is 6.86) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 48 h.

[0021] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.95), and the culture temperature was controlled at 29℃~31℃ for 42h.

[0022] (3) Fermentation culture: After 42 hours of culture, the seed culture was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: glucose 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, hydrocortisone 0.02%, the remainder being water; pH 6.91), and the culture temperature was 29℃~31℃.

[0023] (4) Fermentation conversion: After culturing the fermentation broth for 12 hours, add anhydrous ethanol (5% of the fermentation broth volume) and 40 g / L hydrocortisone. Control the temperature at 32℃~34℃ and convert for 24 hours before removing from the tank.

[0024] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 91.45%.

[0025] Example 2 A method for preparing prednisolone, the specific steps of which are as follows: (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL of slant culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 0.8%, agar 2.5%, the remainder is water; pH 6.92) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 48 h.

[0026] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.88), and the culture temperature was controlled at 29℃~31℃ for 48h.

[0027] (3) Fermentation culture: After 48 hours of culture, the seed culture was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: glucose 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, hydrocortisone 0.02%, the remainder being water; pH 6.98), and the culture temperature was 29℃~31℃.

[0028] (4) Fermentation conversion: After culturing the fermentation broth for 16 hours, add 5% anhydrous ethanol and 50 g / L hydrocortisone, control the temperature at 32℃~34℃, and convert for 28 hours before removing from the tank.

[0029] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 90.89%.

[0030] Example 3 A method for preparing prednisolone, the specific steps of which are as follows: (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL of slant culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 0.8%, agar 2.5%, the rest is water; pH is 6.81) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 40 h.

[0031] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.93), and the culture temperature was controlled at 29℃~31℃ for 44h.

[0032] (3) Fermentation culture: After 44 hours of culture, the seed culture was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: glucose 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, hydrocortisone 0.03%, the remainder being water; pH 6.90), and the culture temperature was 29℃~31℃.

[0033] (4) Fermentation conversion: After culturing the fermentation broth for 16 hours, add 5% anhydrous ethanol and 40 g / L hydrocortisone, control the temperature at 32℃~34℃, and convert for 28 hours before removing from the tank.

[0034] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 90.48%.

[0035] Comparative Example 1 A method for preparing prednisolone, the specific steps of which are as follows: (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL of slant culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 0.8%, agar 2.5%, the rest is water; pH is 6.84) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 48 h.

[0036] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.90), and the culture temperature was controlled at 29℃~31℃ for 42h.

[0037] (3) Fermentation culture: After 42 hours of culture, the seed culture was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: glucose 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.97), and the culture temperature was 29℃~31℃.

[0038] (4) Fermentation conversion: After culturing the fermentation broth for 12 hours, add 5% anhydrous ethanol and 40 g / L hydrocortisone, control the temperature at 32℃~34℃, and convert for 24 hours before removing from the tank.

[0039] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 84.90%.

[0040] Comparative Example 2 A method for preparing prednisolone, the specific steps of which are as follows: (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL of slant culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 0.8%, agar 2.5%, the remainder is water; pH is 6.94) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 48 h.

[0041] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: glucose 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.90), and the culture temperature was controlled at 29℃~31℃ for 48h.

[0042] (3) Fermentation culture: After 48 hours of culture, the seed culture was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: glucose 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.96), and the culture temperature was 29℃~31℃.

[0043] (4) Fermentation conversion: After culturing the fermentation broth for 16 hours, add 5% anhydrous ethanol and 50 g / L hydrocortisone, control the temperature at 32℃~34℃, and convert for 28 hours before removing from the tank.

[0044] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 81.45%.

[0045] By comparing Examples 1-2 with Comparative Examples 1-2, we found that when a certain concentration of the inducer hydrocortisone was added to the fermentation medium, the fermentation conversion rate of the present invention was improved to a certain extent compared with the case in the comparative examples where no inducer was added, and the conversion rate could reach more than 90%.

[0046] Comparative Example 3 (1) Slant culture: Take simple arthrobacterium and inoculate it into 50 mL slant culture medium (by mass percentage, it consists of the following components: corn starch 1.0%, yeast extract 0.8%, agar 2.5%, the remainder is water; pH is 6.82) for preliminary culture. The culture temperature is 29℃~31℃ and the culture period is 48 h.

[0047] (2) Seed culture: All the cultured slant bacteria were inoculated into 10L seed culture medium (composed of the following components by mass percentage: corn starch 1.0%, yeast extract 1.2%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.88), and the culture temperature was controlled at 29℃~31℃ for 42h.

[0048] (3) Fermentation culture: After 42 hours of culture, the seed liquid was transferred at a ratio of 10% to 100L of fermentation medium (composed of the following components by mass percentage: corn starch 0.75%, yeast extract 0.75%, peptone 0.5%, potassium dihydrogen phosphate 0.3%, the remainder being water; pH 6.95), and the culture temperature was 29℃~31℃.

[0049] (4) Fermentation conversion: After culturing the fermentation broth for 12 hours, add 5% anhydrous ethanol and 40 g / L hydrocortisone, control the temperature at 32℃~34℃, and convert for 24 hours before removing from the tank.

[0050] Detection and analysis: 1 mL of fermentation broth was added to 9 mL of ethyl acetate, sonicated for 10 min, and the supernatant was collected by centrifugation. Octadecylsilane-bonded silica gel was used as the packing material, and methanol / water (65:35) was used as the mobile phase. The detection wavelength was 240 nm and the injection volume was 10 μL. HPLC detection was performed, and the fermentation conversion rate was found to be 75.05%.

[0051] Comparing Example 1 and Comparative Example 3, it was found that in Comparative Example 3, not only was no inducer added to the fermentation medium, but the carbon source in its slant culture medium, seed culture medium, and fermentation medium was replaced with corn starch instead of glucose. This series of changes led to a significant decrease in fermentation conversion rate, to only 75.05%. This result indicates that the type of carbon source and the presence or absence of an inducer have a significant impact on fermentation efficiency, and specific carbon source selection and induction conditions are key factors in achieving high conversion rates.

[0052] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing prednisolone, characterized in that, Includes the following steps: Arthrobacterium simplex was inoculated onto slant culture medium for initial culture, and then the slant culture was transferred to seed culture medium for expansion culture to obtain seed culture; The seed culture was transferred to the fermentation medium and the culture temperature was controlled at 29℃~31℃. After culturing for 10~16h, hydrocortisone and anhydrous ethanol were added and the conversion temperature was controlled at 32℃~34℃. After conversion for 20h~25h, prednisolone was obtained. The fermentation medium comprises the following components in weight percentage: glucose 0.5%~1.0%, yeast extract 0.5%~1.0%, peptone 0.2%~0.8%, potassium dihydrogen phosphate 0.2%~0.4%, inducer 0.01%~0.03%, and the remainder being water; the inducer is at least one selected from hydrocortisone, isopropyl-β-D-thiogalactoside, methyl-β-D-thiogalactoside, and salicylic acid.

2. The method for preparing prednisolone according to claim 1, characterized in that, The slant culture medium and seed culture medium were cultured at temperatures ranging from 29°C to 31°C for 42 to 48 hours.

3. The method for preparing prednisolone according to claim 1, characterized in that, The slant culture medium contains the following components in weight percentage: glucose 0.8%~1.2%, yeast extract 0.6%~1.0%, agar 2.0%~2.5%, and the remainder is water.

4. The method for preparing prednisolone according to claim 3, characterized in that, The slant culture medium contains the following components in weight percentage: glucose 1.0%, yeast extract 0.8%, agar 2.5%, and the remainder is water.

5. The method for preparing prednisolone according to claim 1, characterized in that, The seed culture medium contains the following components in weight percentage: glucose 1.0%~1.5%, yeast extract 1.0%~1.5%, peptone 0.2%~0.8%, potassium dihydrogen phosphate 0.1%~0.5%, and the remainder is water.

6. The method for preparing prednisolone according to claim 1, characterized in that, The seed culture medium contains the following components in weight percentage: 1.0% glucose, 1.2% yeast extract, 0.5% peptone, 0.3% potassium dihydrogen phosphate, and the remainder is water.

7. The method for preparing prednisolone according to claim 6, characterized in that, The fermentation medium contains the following components in weight percentage: 0.75% glucose, 0.75% yeast extract, 0.5% peptone, 0.3% potassium dihydrogen phosphate, 0.02% inducer, and the remainder is water.

8. The method for preparing prednisolone according to claim 1, characterized in that, In the step of adding hydrocortisone and anhydrous ethanol after culturing for 10-16 hours, the amount of hydrocortisone added is 30 g / L to 50 g / L, and the amount of anhydrous ethanol added accounts for 4-6% of the fermentation broth volume.

9. The method for preparing prednisolone according to claim 8, characterized in that, The amount of hydrocortisone added is 40 g / L, and the amount of anhydrous ethanol added accounts for 5% of the fermentation broth.

10. The method for preparing prednisolone according to claim 1, characterized in that, The pH of the slant culture medium, seed culture medium, and fermentation medium is 6.8–7.0.

Images