阅读说明：本技术 一种氢化可的松的纯化提取方法 (Purification and extraction method of hydrocortisone ) 是由 冯蕾 付林 廖俊 曾建华 傅文利 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种氢化可的松的纯化提取方法,将发酵液pH值调整至6.5至7.5后过滤,滤液经浓缩除去杂质,浓缩液经稀释后采用大孔吸附树脂AB-8进行吸附,经乙酸丁酯洗脱后,洗脱液经水洗析晶后获得氢化可的松。本发明所述提取方法通过多级协同操作,逐步提升氢化可的松的提取纯度,提取过程不会采用大量的萃取剂,可减少废水排放,生产成本低,适于工业化应用。(The invention discloses a purification and extraction method of hydrocortisone, which comprises the steps of adjusting the pH value of fermentation liquor to 6.5-7.5, filtering, concentrating filtrate to remove impurities, diluting the concentrated solution, adsorbing by using macroporous adsorption resin AB-8, eluting by butyl acetate, and washing eluent by water for crystallization to obtain the hydrocortisone. The extraction method provided by the invention has the advantages that the extraction purity of the hydrocortisone is gradually improved through multi-stage cooperative operation, a large amount of extractant is not adopted in the extraction process, the wastewater discharge can be reduced, the production cost is low, and the extraction method is suitable for industrial application.)

1. A purification and extraction method of hydrocortisone is characterized by comprising the following steps: filtering the fermentation liquor, concentrating, diluting the concentrated solution, adsorbing by using macroporous adsorption resin, eluting, concentrating, washing and crystallizing the eluent to obtain the hydrocortisone.

2. The purification and extraction method according to claim 1, wherein the macroporous adsorption resin is AB-8.

3. The purification and extraction method of claim 1, wherein the volume ratio of the macroporous adsorbent resin to the concentrated solution is 0.1-0.2; the diameter-height ratio of the macroporous adsorption resin is 1: (1-8), wherein the flow rate of the concentrated solution on the column is 0.5-1.0 BV/h.

4. The purification and extraction method of claim 1, wherein the elution solvent is butyl acetate, the amount of the butyl acetate is 1-1.5 times of the volume of the concentrated fermentation broth, and the elution speed is 0.5-1.5 BV/h.

5. The purification and extraction method according to claim 1, wherein the fermentation broth is adjusted to 6.5-7.5 before filtration.

6. The purification and extraction method of claim 1, wherein the concentration is performed by a wiped film evaporator to collect the lower heavy component.

7. The purification and extraction method according to claim 6, wherein the concentration step is performed to concentrate the fermentation broth to 1/3-1/4 of the original volume.

8. The purification and extraction method as claimed in claim 1, wherein the elution solution is washed with water, the solvent phase is concentrated until the hydrocortisone wet product is separated out, then the solution is frozen at 0-5 ℃, after complete separation, the solution is centrifuged, and then washed with frozen butyl acetate, dried, and dried to obtain the hydrocortisone.

9. The purification and extraction method according to claim 8, wherein the concentration process is carried out under vacuum at 80 ± 10 ℃ to distill off butyl acetate.

Technical Field

The invention belongs to the field of chemical pharmacy, and relates to a purification and extraction method of chemical hydrocortisone.

Background

Hydrocortisone is a commonly used corticoid drug, has wide clinical application, is mainly used for treating adrenal insufficiency, autoimmune diseases, allergic diseases, acute leukemia, ophthalmia and Hodgkin's disease, is also used for comprehensive treatment of hyperpyrexia caused by some serious infections and the like, and has the following structural formula:

the existing production process comprises synthesis and fermentation, Chinese patent publication No. CN110804640A proposes a production fermentation process of hydrocortisone, and fermentation conversion liquid obtained after the fermentation and conversion operation process contains a certain content of epi-hydrocortisone, unconverted RSA and other impurities. Epi-hydrocortisone as an isomer of hydrocortisone has substantially no pharmaceutical effect, is difficult to separate from hydrocortisone as a major by-product from a fermentation broth, and has the following structural formula:

due to the existence of isomer body surface hydrocortisone, how to improve the extraction purity of hydrocortisone always becomes a bottleneck restricting the industrial extraction of a fermentation method.

The traditional fermentation process mostly adopts a solvent extraction method to extract hydrocortisone generated after fermentation, the used extractant is butyl acetate or isobutyl acetate, a strategy of repeated extraction is generally adopted, although the method can recover more target products, a large amount of extractant is consumed in the process, according to six-stage cross flow reported in the literature, "research on continuous countercurrent extraction of hydrocortisone by a centrifugal extractor [ J ]" (fine chemical, 2001, volume 18, No. 1), the volume ratio of butyl acetate is 0.9: 1, the yield is 68.1%, the total usage of butyl acetate is in direct proportion according to the amount of the fermentation liquor, the loss is huge, although the extractant can be recycled, the recycling process is time-consuming and labor-consuming, and simultaneously generates a large amount of industrial wastewater, which increases the burden of sewage treatment and brings great difficulty to laboratory research and industrial production of hydrocortisone.

Therefore, the development of a new method for purifying and extracting the fermentation broth hydrocortisone has very important practical significance.

Disclosure of Invention

The invention provides a method for extracting hydrocortisone, which is used for at least solving one of the problems in the prior art.

In view of this, the scheme of the invention is as follows:

a purification and extraction method of hydrocortisone comprises the following steps: filtering the fermentation liquor, concentrating, diluting the concentrated solution, adsorbing by using macroporous adsorption resin, eluting, concentrating, washing and crystallizing the eluent to obtain the hydrocortisone.

According to an embodiment of the invention, the macroporous adsorbent resin is AB-8.

According to the embodiment of the invention, the volume ratio of the macroporous adsorption resin to the concentrated solution is 0.1-0.2; the diameter-height ratio of the macroporous adsorption resin is 1: (1-8), wherein the flow rate of the concentrated solution on the column is 0.5-1.0 BV/h.

According to the embodiment of the invention, butyl acetate is used as the elution solvent, the dosage of the butyl acetate is 1-1.5 times of the volume of the concentrated fermentation liquor, and the elution speed is 0.5-1.5 BV/h.

According to an embodiment of the invention, the fermentation broth is adjusted to 6.5-7.5 before filtration.

According to the embodiment of the invention, the concentration adopts a scraper film evaporator, and the lower heavy component is collected; further, the concentration step is used for concentrating the fermentation liquor to 1/3-1/4 of the original volume.

According to the embodiment of the invention, after the eluent is washed by water, the solvent phase is concentrated until a hydrocortisone wet product is separated out, then the hydrocortisone wet product is frozen at 0-5 ℃, after the hydrocortisone wet product is completely separated out, the hydrocortisone wet product is centrifuged, and the hydrocortisone wet product is washed by using frozen butyl acetate, dried and dried to obtain the hydrocortisone. Preferably, the concentration process is maintained at 80 ± 10 ℃ under vacuum to distill off butyl acetate.

Compared with the prior art, the invention has the beneficial effects that:

1. the purification and extraction method provided by the invention has the advantages that the extraction purity of the hydrocortisone is gradually improved through multi-stage cooperative operation, a large amount of extractant is not adopted in the extraction process, the wastewater discharge can be reduced, the production cost is low, and the method is suitable for industrial application.

2. The purification and extraction method can obviously reduce the content of epi-hydrocortisone and has obvious significance for improving the product quality.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention is further described in detail with reference to the following detailed description. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The invention provides an extraction method of hydrocortisone, which comprises the following steps:

1. adjusting pH of the fermentation liquid to 6.5-7.5 with 10% sulfuric acid solution, and filtering to remove degerming thread to obtain filtrate.

2. And concentrating by using a scraper film evaporator to obtain heavy components. The specific operation steps are as follows: putting the filtrate into a feeding tank, pumping the extracting solution into a scraper film evaporator by using a feeding pump, setting and controlling the heating temperature of the scraper film evaporator, turning on a stirring motor of the scraper film evaporator, vacuumizing the scraper film evaporator by using a water ring vacuum pump, introducing a cooling medium into a condenser, concentrating the extracting solution by using the scraper film evaporator until the extracting solution reaches 1/3-1/4 of the original volume, paying attention to real-time observation to prevent excessive concentration and causing the degradation of nutrient components in the culture medium due to excessive concentration so as to influence subsequent extraction, and after the concentration is finished, feeding the concentrated solution into an extraction tank to be extracted.

3. And (2) adsorbing the concentrated solution by using macroporous adsorption resin, wherein the macroporous adsorption resin can be common macromolecular adsorption resin which does not contain exchange groups and has a macroporous structure, such as AB-8, and the diameter-height ratio of a macroporous adsorption resin column is 1: 1-8; the macroporous adsorption resin is mainly used for adsorbing the effective component hydrocortisone in the fermentation liquor. The dosage of the resin is 0.1-0.2 times of the volume of the fermentation liquor, and the flow rate of the resin on the column is 0.5-1.0 BV/h. If the dosage of the macroporous absorption resin is too small, the flow rate of the macroporous absorption resin on the column is too fast or the height-diameter ratio is too small, the effective components can not be fully absorbed; if the dosage of the macroporous absorption resin is too much, the flow rate of the macroporous absorption resin on the column is too slow or the height-diameter ratio is too large, the production period is prolonged and the production cost is increased.

4. And (4) eluting the resin after adsorption by using butyl acetate, and combining the eluates. The dosage of the butyl acetate is 1-1.5 times of the volume of the fermentation liquor, and the elution speed is 0.5-1.5 BV/h.

5. Washing the eluate with pure water, concentrating the solvent phase in a concentration tank until a large amount of hydrocortisone wet product is separated out, freezing at 0-5 deg.C, keeping the temperature for about 1 hr, centrifuging, washing with frozen butyl acetate, spin-drying, and drying to obtain hydrocortisone extract.

The principle of the extraction method provided by the invention is as follows:

1. the purpose of adjusting the pH value of the fermentation liquor is to avoid the phenomenon that the compound is unstable under the alkaline condition and create a stable acid-base environment for the later extraction process; on the other hand, a small amount of impurities can be separated out, so that the subsequent entering of the impurities into the macroporous adsorption resin is avoided to influence the adsorption performance;

2. before extraction, a scraper film evaporator is used for concentration, so that on one hand, a small amount of light components in the fermentation liquor can be removed, the influence of impurities in the subsequent adsorption process is further reduced, on the other hand, the volume of the filtrate is reduced to 1/3-1/4 of the original volume, the volume of the fermentation liquor is reduced, the discharge amount of wastewater is reduced, the industrial production is facilitated, on the other hand, the concentration of the fermentation liquor is properly improved, and the subsequent macroporous resin adsorption efficiency is facilitated;

3. according to the steroid stereochemistry, the 11 β hydroxyl group of hydrocortisone is the axial substituent, whereas the 11 α hydroxyl group of epi-hydrocortisone is the equatorial substituent, and compared to hydrocortisone, the ring two and ring three steric structures of epi-hydrocortisone deviate from the boat conformation, epi-hydrocortisone exhibits greater steric hindrance, and thus the 11 α hydroxyl group undergoes less hydrogen bonding than the 11 β hydroxyl group, resulting in that the hydrocortisone can be more easily adsorbed. The reason for using macroporous adsorbent resin materials is that, by selecting a weakly polar resin material, the fermentation liquid components are more easily adsorbed by a weakly polar adsorbent than polar media (such as ethanol or water), and the whole molecule is weakly polar compared with a large non-polar steroid nucleus despite the fact that hydrocortisone molecules have several polar groups. Therefore, in an alcohol-water system, the effect of adopting the nonpolar or low-polarity adsorption resin is better. In addition, the molecular weight of hydrocortisone is relatively large, and the steroid nucleus is of a planar structure, so that the requirement on the pore diameter is met, in addition, the larger the internal surface area of the adsorbent is, the stronger the adsorption capacity is, for example, the macroporous adsorption resin AB-8 has the performances of high crosslinking degree, high specific surface area, non-polarity without functional groups and the like, the continuous pore structure of the macroporous adsorption resin AB-8 endows the macroporous adsorption resin with excellent adsorption performance, and in addition, the polymer structure of the AB-8 ensures that the macroporous adsorption resin AB-8 has excellent physical and chemical properties and thermal stability, has strong regeneration capacity and is suitable for industrial production.

4. The reason for using butyl acetate as the resolving solvent is that butyl acetate has a significantly different partition coefficient than hydrocortisone and epi-hydrocortisone. According to steroid stereochemistry, the 11 α hydroxyl group in epi-hydrocortisone acts less strongly with solvents than the 11 β hydroxyl group of hydrocortisone, resulting in a lower partition coefficient for epi-hydrocortisone. The butyl acetate has a higher separation coefficient, and the analytic amounts of hydrocortisone and epi-hydrocortisone are different, so that the content of epi-hydrocortisone in the extraction process can be further reduced.

In the invention, the AB-8 macroporous adsorption resin is Tulsimer, a Dusheng. Before column filling, the resin exchange column is cleaned by clear water, part of attached impurities are cleaned, water 1/3 is injected into the column, a small amount of resin is taken, and the resin is filled into the column from a manhole at the top of the exchange column to observe whether resin leaks. And after the inspection is finished, adding the resin into the column for pretreatment, soaking and cleaning for 3h by adopting 1mol/LNaOH solution, soaking by adopting pure water, cleaning to be neutral, and removing residual organic molecules, pore-forming agents and the like in resin pore channels until the cleaning outlet liquid or the soaking liquid is not turbid and tasteless for later use.

In the invention, the detection content of hydrocortisone in the fermentation stock solution is 1.8 g/L.

Example 1

Adjusting the pH value of the fermentation conversion solution to 7.5 by using a 10% sulfuric acid solution, filtering to remove hyphae to obtain 45L of filtrate, putting the extract into a feed tank, pumping the extract into a scraper film evaporator by using a feed pump, setting and controlling the heating temperature of the scraper film evaporator to be more than 75 ℃, turning on a stirring motor of the scraper film evaporator, setting the linear speed of the scraper to be 1m/s, vacuumizing the scraper film evaporator by using a water ring vacuum pump, introducing a cooling medium into a condenser, and concentrating the extract by using the scraper film evaporator to obtain 12L of concentrated solution.

The concentrated solution is adsorbed by macroporous adsorption resin AB-8 after pretreatment by stages, and the diameter-height ratio of an adsorption column is 1: 1; the amount of the resin used was 1.2L, and the flow rate of the resin on the column was 0.5 BV/h. And (3) eluting the resin after adsorption by using butyl acetate, wherein the using amount of the butyl acetate is 12L, and the elution speed is 0.5 BV/h.

Transferring the eluate to a water washing tank, injecting about 10L of purified water into the water washing tank, washing and layering, transferring the solvent layer to a 50L concentration tank for concentration, and collecting butyl acetate at the temperature of 80 +/-10 ℃ under the control of vacuum degree; when the volume is about 2L, a large amount of hydrocortisone wet product is separated out, the concentration is stopped, the wet product is transferred to a refrigerator at 0-5 ℃ to be cooled for 1 hour, the centrifugation is carried out, butyl acetate is used for washing and spin-drying, the drying is carried out at 50-70 ℃ for 8-10 hours, 40.8g of hydrocortisone is obtained, and the content of the hydrocortisone obtained in the embodiment is 99.3% through the determination of a High Performance Liquid Chromatography (HPLC) method.

Example 2

Adjusting the pH value of the fermentation conversion solution to 6.9 by using a sulfuric acid solution with the concentration of about 10%, filtering to remove hyphae to obtain filtrate with the concentration of about 45L, putting the extracting solution into a feeding tank, pumping the extracting solution into a scraper film evaporator by using a feeding pump, setting and controlling the heating temperature of the scraper film evaporator to be more than 75 ℃, turning on a stirring motor of the scraper film evaporator, setting the linear speed of the scraper to be 0.5m/s, vacuumizing the scraper film evaporator by using a water ring vacuum pump, introducing a cooling medium into a condenser, and concentrating the extracting solution by using the scraper film evaporator to obtain concentrated solution with the concentration of about 15L.

The concentrated solution is adsorbed by macroporous adsorption resin AB-8 after pretreatment by stages, and the diameter-height ratio of an adsorption column is 1: 2; the amount of the resin is 2L, and the flow rate of the resin on the column is 0.8 BV/h. And (3) eluting the resin after adsorption by using butyl acetate, wherein the using amount of the butyl acetate is 18L, and the elution speed is 1.0 BV/h.

Transferring the eluate to a water washing tank, injecting about 12L of purified water into the water washing tank, washing and layering, transferring the solvent layer to a 50L concentration tank for concentration, and collecting butyl acetate at the temperature of 80 +/-10 ℃ under the control of vacuum degree; when the volume is about 2L, a large amount of hydrocortisone wet product is separated out, the concentration is stopped, the wet product is transferred to a refrigerator at 0-5 ℃ to be cooled for 1 hour, the centrifugation is carried out, butyl acetate is used for washing and spin-drying, the drying is carried out at 50-70 ℃ for 8-10 hours, 41.1g of hydrocortisone is obtained, and the content of the hydrocortisone obtained in the embodiment is 99.7 percent through the determination of a High Performance Liquid Chromatography (HPLC).

Example 3

Adjusting the pH value of the fermentation conversion solution to 6.5 by using a sulfuric acid solution with the concentration of about 10%, filtering to remove hyphae to obtain filtrate with the concentration of about 45L, putting the extracting solution into a feeding tank, pumping the extracting solution into a scraper film evaporator by using a feeding pump, setting and controlling the heating temperature of the scraper film evaporator to be more than 75 ℃, turning on a stirring motor of the scraper film evaporator, setting the linear speed of the scraper to be 0.8m/s, vacuumizing the scraper film evaporator by using a water ring vacuum pump, introducing a cooling medium into a condenser, and concentrating the extracting solution by using the scraper film evaporator to obtain concentrated solution with the concentration of about 12L.

The concentrated solution is adsorbed by macroporous adsorption resin AB-8 after pretreatment by stages, and the diameter-height ratio of an adsorption column is 1: 8; the amount of the resin used was 2.4L, and the flow rate of the resin on the column was 1.0 BV/h. And (3) eluting the resin after adsorption by using butyl acetate, wherein the using amount of the butyl acetate is 18L, and the elution speed is 1.5 BV/h.

Transferring the eluate to a water washing tank, injecting about 12L of purified water into the water washing tank, washing and layering, transferring the solvent layer to a 50L concentration tank for concentration, and collecting butyl acetate at the temperature of 80 +/-10 ℃ under the control of vacuum degree; when the volume is about 2L, a large amount of hydrocortisone wet product is separated out, the concentration is stopped, the wet product is transferred to a refrigerator at 0-5 ℃ to be cooled for 1 hour, the centrifugation is carried out, butyl acetate is used for washing and spin-drying, the drying is carried out at 50-70 ℃ for 8-10 hours, 40.2g of hydrocortisone is obtained, and the content of the hydrocortisone obtained in the embodiment is 99.4% through the determination of a High Performance Liquid Chromatography (HPLC).

Comparative example 1

Hydrocortisone in the fermentation broth was extracted in the same manner as in example 3 except that the amount of the macroporous adsorbent resin AB-8 was 3L. The method gives 40.4g of hydrocortisone, and the content of hydrocortisone obtained in this example is 99.4% as determined by High Performance Liquid Chromatography (HPLC).

Comparative example 2

Hydrocortisone in the fermentation broth was extracted in the same manner as in example 3 except that the amount of the macroporous adsorbent resin AB-8 was 1L. This procedure gave 35.8g of hydrocortisone, which was found to have a cortisol content of 96.3% by High Performance Liquid Chromatography (HPLC).

Comparative example 3

Hydrocortisone in the fermentation broth was extracted in the same manner as in example 3 except that the flow rate on the column was 1.5BV/h and the elution rate was 1.8 BV/h. The method gives 40.1g of hydrocortisone, and the content of hydrocortisone obtained in this example is 96.3% as determined by High Performance Liquid Chromatography (HPLC).

Comparative example 4

Hydrocortisone in the fermentation broth was extracted in the same manner as in example 1 except that the flow rate of the upper column was 0.3BV/h and the elution rate was 0.3 BV/h. 41.3g of hydrocortisone is obtained by the method, the purification yield is 93.0%, and the content of the hydrocortisone obtained in the example is 99.6% by High Performance Liquid Chromatography (HPLC) method.

Comparative example 5

Hydrocortisone in the broth was extracted in the same manner as in example 1, except that the pH of the broth was not adjusted and was found to be 7.9. The method obtains 34.8g of hydrocortisone, and the content of the hydrocortisone obtained in the example is 95.8% determined by a High Performance Liquid Chromatography (HPLC) method.

As can be readily seen from the above examples 1-3, the purity of the purified hydrocortisone provided by the present invention can be 99.7. In comparative example 1, the increase of the amount of the macroporous adsorbent resin had no effect on the increase of the purity, whereas when the amount of the macroporous adsorbent resin was too small (comparative example 2), the purity was remarkably decreased. Comparative example 3 the higher flow velocity of upper column can influence the impurity adsorption rate, and the higher analytic flow velocity can destroy impurity adsorption balance and then influence final extract purity. Comparative example 4 the flow rate and the resolution rate of the upper column were both relatively slow, but the purity was not greatly improved, and the efficiency was low, and thus it was not suitable for use in production. In the comparative example 5, the fermentation liquor is weakly alkaline without pH value adjustment, part of impurities cannot be removed by filtration or subsequent macroporous resin adsorption, and in addition, the adsorption force of the target component hydrocortisone on the macroporous adsorption resin material is weakened under the weakly alkaline condition, and both factors can influence the final purity of the hydrocortisone.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, and it is not intended to be limited to the specific details, representative experimental examples and examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.