CN111995652B - Method for separating chenodeoxycholic acid from duck bile - Google Patents
Method for separating chenodeoxycholic acid from duck bile Download PDFInfo
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- CN111995652B CN111995652B CN202010804258.2A CN202010804258A CN111995652B CN 111995652 B CN111995652 B CN 111995652B CN 202010804258 A CN202010804258 A CN 202010804258A CN 111995652 B CN111995652 B CN 111995652B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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Abstract
The invention discloses a preparation method for separating chenodeoxycholic acid from duck bile, which comprises the following steps: the method comprises the steps of forming ester from saponified and free duck gall paste with alcohol under acid catalysis, separating chenodeoxycholic acid ester through a solvent, then reacting with acetic anhydride to generate a 3, 7-diacetyl chenodeoxycholic acid ester crude product, refining the crude product through a single solvent to obtain a high-purity 3, 7-diacetyl chenodeoxycholic acid ester refined product, and then hydrolyzing with alkali to obtain chenodeoxycholic acid. The raw material duck bile used in the invention is cheap and easy to obtain, the process operation is simple, a large amount of solvent is avoided being repeatedly treated, and the obtained chenodeoxycholic acid has higher purity and is more suitable for industrial production.
Description
Technical Field
The invention relates to a preparation method of chenodeoxycholic acid, in particular to a method for separating chenodeoxycholic acid from duck bile.
Background
Chenodeoxycholic acid (Chenodeoxycholic Acid, abbreviated as CDCA)), which is a steroid compound, has a chemical name of 3 alpha, 7 alpha-dihydroxyl-5 beta-cholanic acid, and is a natural cholic acid which is extracted from goose bile for the first time in 1848. Chenodeoxycholic acid has cholesterol-correcting effect, and is used as the first medicine for correcting saturated bile and dissolving cholelithiasis, and is also the raw material for synthesizing ursodeoxycholic acid and other steroid compounds.
The bile of chicken, duck, goose and the like contains chenodeoxycholic acid which can be directly extracted from the bile of chicken, and the chenodeoxycholic acid in the market is mainly extracted from the chicken bile at present. Meanwhile, the duck breeding amount in China is huge, duck bile resources are rich, but the duck bile is not fully utilized, and due to the complex components of the duck bile, the chenodeoxycholic acid in the duck bile cannot be directly extracted by the method for extracting the chenodeoxycholic acid from the chicken bile.
The existing extraction method for extracting chenodeoxycholic acid from bile of chicken, duck, goose and the like mainly comprises an organic solvent extraction method, a barium salt method, a calcium salt method, a silica gel column and the like. However, the traditional extraction method has complex technological process, low purity of the obtained product, repeated treatment of the organic solvent for many times, more three wastes, low yield and difficult industrialization.
Disclosure of Invention
In order to solve the problem of effectively separating chenodeoxycholic acid with higher purity from duck bile, fully utilizing duck bile resources, increasing raw material sources of the chenodeoxycholic acid and improving utilization efficiency of poultry bile resources, the invention provides a method for separating the chenodeoxycholic acid from the duck bile.
The duck bile paste obtained after the saponification and the ionization of the duck bile mainly contains chenodeoxycholic acid and duck cholic acid, wherein the chenodeoxycholic acid and the duck cholic acid are structurally different from each other only by 23-site groups, and both the chenodeoxycholic acid and the duck cholic acid contain carboxylic acid groups, have larger polarity and are difficult to separate by directly refining with a solvent. The method comprises the steps of firstly forming esters with alcohol under acid catalysis, separating the esters with a solvent to obtain chenodeoxycholic acid ester, then acetylating the chenodeoxycholic acid ester with acetic anhydride, purifying the chenodeoxycholic acid ester with single solvent to obtain 3, 7-diacetyl chenodeoxycholic acid ester with higher purity, and finally hydrolyzing the chenodeoxycholic acid ester to obtain the chenodeoxycholic acid. The method has the advantages of mild reaction conditions, simple process, low-cost and easily-obtained reagents, high yield, lower cost and obvious purification effect, and is suitable for industrial production.
Specifically, it comprises the following steps:
(1) Dissolving duck gall paste in alcohol, dropwise adding concentrated sulfuric acid at room temperature, maintaining room temperature for stirring reaction, adjusting pH to 6-7 with inorganic base after reaction, filtering, concentrating, adding into organic solvent, heating, refluxing, cooling, and slowly crystallizing to obtain chenodeoxycholate;
(2) Dissolving chenodeoxycholic acid ester obtained in the step (1) in acetic anhydride, slowly dripping water to separate out solid after the reflux reaction is finished, and filtering to obtain a 3, 7-diacetyl chenodeoxycholic acid ester crude product;
(3) Dissolving the crude 3, 7-diacetyl chenodeoxycholic acid ester obtained in the step (2) in an organic solvent, heating, cooling and crystallizing, and filtering to obtain a fine product of 3, 7-diacetyl chenodeoxycholic acid ester;
(4) Adding the 3, 7-diacetyl chenodeoxycholic acid ester refined product obtained in the step (3) into alkaline water, refluxing for hydrolysis reaction, adjusting the pH of the reaction solution to 2-3 by hydrochloric acid after the reaction is finished, slowly separating out solid, and filtering to obtain chenodeoxycholic acid.
Preferably, the alcohol in step (1) of the process of the present invention is selected from one or more of methanol, ethanol, isopropanol, preferably methanol.
Further, the inorganic base in step (1) of the present invention is selected from one or more of potassium carbonate, sodium bicarbonate, preferably sodium bicarbonate.
Preferably, the organic solvent in step (1) of the process of the present invention is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetone, toluene, isopropyl ether, methyl tert-butyl ether, preferably isopropyl ether.
Preferably, the organic reagent in step (3) of the process of the present invention is selected from one or two of toluene, tetrahydrofuran, acetonitrile, acetone, ethyl acetate, dichloromethane, chloroform, preferably toluene.
Further, the base described in step (4) of the process of the present invention is selected from one or more of NaOH, KOH, liOH, preferably NaOH.
Detailed Description
The following examples are given to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
Adding fresh duck bile 25kg (25L in volume) into a reaction kettle, adding 1.5kg sodium hydroxide, heating and refluxing for 24 hr for saponification, adjusting pH to 2-3 with concentrated hydrochloric acid, slowly precipitating solid, filtering, washing to neutrality to obtain brown paste, and drying at 70deg.C for 10 hr. Dissolving the paste with 30kg of ethanol, adding 15kg of petroleum ether, washing for 2 times, and concentrating the ethanol solution under reduced pressure to obtain 3.5kg of duck gall paste.
Example 2
Adding 1.6kg of duck gall paste into 8L of methanol, adding 100ml of 98% concentrated sulfuric acid after dissolving, reacting for 12 hours at 25-35 ℃, adjusting the pH to 6-7 by using sodium bicarbonate after finishing, filtering, concentrating the filtrate under reduced pressure to obtain oily matter, adding 5L of isopropyl ether into the oily matter, heating and refluxing, then cooling to 25-35 ℃, stirring for 1-2 hours, filtering, leaching the filter cake with 1L of isopropyl ether once, collecting the filter cake, and drying at 45-55 ℃ to obtain 656g of chenodeoxycholic acid methyl ester.
Example 3
Adding 1.6kg of duck gall paste into 8L of methanol, adding 100ml of 98% concentrated sulfuric acid after dissolving, reacting for 12 hours at 25-35 ℃, adjusting the pH to 6-7 by using sodium bicarbonate after finishing, filtering, concentrating the filtrate under reduced pressure to obtain oily matter, adding 6L of chloroform into the obtained oily matter, heating and refluxing, then cooling to 25-35 ℃, stirring for 1-2 hours, filtering, leaching a filter cake with 1.5L of chloroform once, collecting the filter cake, and drying at 45-55 ℃ to obtain 620g of chenodeoxycholic acid methyl ester.
Example 4
Adding 1.6kg of duck gall paste into 8L of ethanol, adding 100ml of 98% concentrated sulfuric acid after dissolving, reacting for 12 hours at 25-35 ℃, adjusting the pH to 6-7 by using sodium bicarbonate after finishing, filtering, concentrating the filtrate under reduced pressure to obtain oily matter, adding 5L of isopropyl ether into the obtained oily matter, heating and refluxing, then cooling to 25-35 ℃, stirring for 1-2 hours, filtering, leaching the filter cake with 1L of isopropyl ether once, collecting the filter cake, and drying at 45-55 ℃ to obtain 570g of chenodeoxycholic acid ethyl ester.
Example 5
556g of chenodeoxycholic acid methyl ester obtained in example 2, 500mL of acetic anhydride and 100g of sodium acetate are added into a reaction bottle, heating reflux is carried out for 4-5 hours, the temperature is reduced to 25-35 ℃, 3L of water is slowly added dropwise, solids are slowly separated out, filtration is carried out, a filter cake is leached by water, the filter cake is collected, and then the filter cake is dried at 55-65 ℃ to obtain 313 g of 3, 7-diacetyl chenodeoxycholic acid methyl ester crude product, and the yield is 91.9%.
Example 6
100g of the crude 3, 7-diacetyl chenodeoxycholic acid methyl ester obtained in example 5 is dissolved in 400mL of toluene, heated and refluxed for 0.5h, cooled to 25-35 ℃, stirred for 1 hour, filtered, the filter cake is leached with 50mL of toluene once, the filter cake is collected, and the filter cake is dried at 45-55 ℃ to obtain 64.3g of refined 3, 7-diacetyl chenodeoxycholic acid methyl ester, and the yield is 64.3%.
Example 7
100g of the crude 3, 7-diacetyl chenodeoxycholic acid methyl ester obtained in example 5 is dissolved in 500mL of methyl tertiary butyl ether, heated and refluxed for 0.5h, cooled to 25-35 ℃, stirred for 1 hour, filtered, the filter cake is leached once with 60mL of methyl tertiary butyl ether, the filter cake is collected, and the filter cake is dried at 45-55 ℃ to obtain 65g of 3, 7-diacetyl chenodeoxycholic acid methyl ester refined product, and the yield is 65.0%.
Example 8
100g of the crude 3, 7-diacetyl chenodeoxycholic acid methyl ester obtained in example 5 is dissolved in 400mL of chloroform, heated and refluxed for 0.5h, cooled to 25-35 ℃, stirred for 1 hour, filtered, and the filter cake is once filtered with 50mL of chloroform, and the filter cake is collected and dried at 45-55 ℃ to obtain 63.5g of the 3, 7-diacetyl chenodeoxycholic acid methyl ester refined product, and the yield is 63.5%.
Example 9
50g of the chenodeoxycholic acid methyl ester obtained in example 2 is added into 10% sodium hydroxide solution (100 ml), stirred at room temperature for 5 hours, after the reaction is finished, the pH is adjusted to 2-3 by concentrated hydrochloric acid, stirred at room temperature for 1 hour, filtered, and the filter cake is washed to be neutral by water and dried to obtain 42.4g of chenodeoxycholic acid, wherein the yield is 87.9% and the purity is 73.5%.
Example 10
60g of the 3, 7-diacetyl chenodeoxycholic acid methyl ester refined product obtained in example 6 is added into 10% sodium hydroxide solution (200 mL), heated and refluxed for 12 hours, after the reaction is finished, the reaction solution is cooled to room temperature, the pH is regulated to 2-3 by concentrated hydrochloric acid, the reaction solution is stirred for 1 hour at room temperature, the filtration is carried out, the filter cake is washed to be neutral by water, and the reaction solution is dried to obtain 44.3g of chenodeoxycholic acid, the yield is 92.3%, and the purity is 97.1%.
Example 11
65g of the refined 3, 7-diacetyl chenodeoxycholic acid methyl ester obtained in example 7 is added into 10% sodium hydroxide solution (210 mL), heated and refluxed for 12 hours, after the reaction is completed, the reaction solution is cooled to room temperature, the pH is adjusted to 2-3 by concentrated hydrochloric acid, the reaction solution is stirred for 1 hour at room temperature, the filtration is carried out, the filter cake is washed to be neutral by water, and the reaction solution is dried, thus 49.4g of chenodeoxycholic acid is obtained, the yield is 94.6%, and the purity is 93.4%.
Example 12
63.5g of 3, 7-diacetyl chenodeoxycholic acid ester obtained in example 8 is added into 10% sodium hydroxide solution (210 mL), heated and refluxed for 12 hours, after the reaction is finished, the reaction solution is cooled to room temperature, the pH is adjusted to 2-3 by concentrated hydrochloric acid, the mixture is stirred for 1 hour at room temperature, the mixture is filtered, the filter cake is washed to be neutral by water and dried, and 47.4g of chenodeoxycholic acid is obtained, the yield is 93.2%, and the purity is 94.8%.
In conclusion, the chenodeoxycholic acid preparation method disclosed by the invention is low in cost, high in purity and simple to operate, and can be suitable for industrial application.
While the invention has been described above with respect to a general description and specific examples, the implementation of the invention is not limited to the examples described above, but many other modifications or improvements may be devised by those skilled in the art, which fall within the scope of the invention as claimed.
Claims (5)
1. A preparation method for separating chenodeoxycholic acid from duck bile is characterized by comprising the following steps:
(1) Dissolving duck gall paste in alcohol, dropwise adding concentrated sulfuric acid at room temperature, maintaining room temperature for stirring reaction, adjusting pH to 6-7 with inorganic base after reaction, filtering, concentrating filtrate, adding organic solvent, heating for reflux, cooling and crystallizing to obtain chenodeoxycholate;
(2) Dissolving chenodeoxycholic acid ester obtained in the step (1) in acetic anhydride, slowly dripping water to separate out solid after the reflux reaction is finished, and filtering to obtain a 3, 7-diacetyl chenodeoxycholic acid ester crude product;
(3) Dissolving the 3, 7-diacetyl chenodeoxycholic acid ester crude product obtained in the step (2) in an organic solvent, heating to dissolve, cooling for crystallization, and filtering to obtain a 3, 7-diacetyl chenodeoxycholic acid ester refined product; the organic solvent is toluene;
(4) Adding the 3, 7-diacetyl chenodeoxycholic acid ester obtained in the step (3) into alkaline water, refluxing for hydrolysis reaction, adjusting the pH of the reaction solution to 2-3 by hydrochloric acid after the reaction is finished, slowly separating out solid, and filtering to obtain chenodeoxycholic acid.
2. The method according to claim 1, characterized in that: the alcohol in the step (1) is selected from one or more of methanol, ethanol and isopropanol.
3. The method according to claim 1, characterized in that: the organic solvent in the step (1) is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, acetone, toluene, isopropyl ether and methyl tertiary butyl ether.
4. The method according to claim 1, characterized in that: the inorganic base in the step (1) is selected from one or more of potassium carbonate, sodium carbonate and sodium bicarbonate.
5. The method according to claim 1, characterized in that: the base in step (4) is selected from one or more of NaOH, KOH, liOH.
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| CN111995652B true CN111995652B (en) | 2023-06-27 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102827234A (en) * | 2012-08-30 | 2012-12-19 | 苏州天绿生物制药有限公司 | Method for separating and purifying chenodeoxycholic acid from duck gall |
| CN105418716A (en) * | 2015-12-25 | 2016-03-23 | 成都市新功生物科技有限公司 | Method for synthesizing chenodeoxycholic acid by using duck cholic acid extracted from duck bile |
| CN105481926A (en) * | 2015-12-25 | 2016-04-13 | 成都市新功生物科技有限公司 | Method for directly preparing chenodeoxycholic acid from duck bile |
| CN109422794A (en) * | 2017-08-28 | 2019-03-05 | 天津科技大学 | A method of extracting chenodeoxycholic acid from duck bile |
| CN110790805A (en) * | 2019-11-14 | 2020-02-14 | 湖南九典制药股份有限公司 | Method for extracting chenodeoxycholic acid from pig bile paste |
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- 2020-08-12 CN CN202010804258.2A patent/CN111995652B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827234A (en) * | 2012-08-30 | 2012-12-19 | 苏州天绿生物制药有限公司 | Method for separating and purifying chenodeoxycholic acid from duck gall |
| CN105418716A (en) * | 2015-12-25 | 2016-03-23 | 成都市新功生物科技有限公司 | Method for synthesizing chenodeoxycholic acid by using duck cholic acid extracted from duck bile |
| CN105481926A (en) * | 2015-12-25 | 2016-04-13 | 成都市新功生物科技有限公司 | Method for directly preparing chenodeoxycholic acid from duck bile |
| CN109422794A (en) * | 2017-08-28 | 2019-03-05 | 天津科技大学 | A method of extracting chenodeoxycholic acid from duck bile |
| CN110790805A (en) * | 2019-11-14 | 2020-02-14 | 湖南九典制药股份有限公司 | Method for extracting chenodeoxycholic acid from pig bile paste |
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