CN110790805A - Method for extracting chenodeoxycholic acid from pig bile paste - Google Patents
Method for extracting chenodeoxycholic acid from pig bile paste Download PDFInfo
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- CN110790805A CN110790805A CN201911111097.2A CN201911111097A CN110790805A CN 110790805 A CN110790805 A CN 110790805A CN 201911111097 A CN201911111097 A CN 201911111097A CN 110790805 A CN110790805 A CN 110790805A
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- 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
Abstract
The application discloses a method for extracting chenodeoxycholic acid from pig bile paste, which comprises the steps of saponification, complex crystallization, extraction esterification, acetylation and the like, and achieves the purpose of separating the hyodeoxycholic acid, the hyocholic acid and the chenodeoxycholic acid from the pig bile paste at one time. The three products obtained by the method have high purity, the reagents are all cheap and easy to obtain, most of solvents can be recycled, and the generated wastewater is less.
Description
Technical Field
The invention belongs to the field of separation and purification, and particularly relates to a method for extracting chenodeoxycholic acid from porcine bile paste.
Background
Chenodeoxycholic Acid (CDCA) with the chemical name of 3 α,7 α -dihydroxy-5 β -cholanic acid, CAS number: 474-25-9, molecular formula of C24H40O4, molecular weight of 392.57 and structural formula shown as follows:
chenodeoxycholic acid is a substance mainly extracted from bile, is mainly used for reducing the saturation of cholesterol in the bile, is mainly used for treating gallstones clinically, is also a main raw material for synthesizing ursodeoxycholic acid (UDCA) at present, is formally developed for treating cholesterol stones in 1972 by the UK, and is applied as a first medicament for correcting saturated bile and dissolving gallstones.
Chenodeoxycholic acid is the main component of fowl bile such as chicken, duck and goose, and can be directly extracted from fowl bile. However, the chenodeoxycholic acid products in the current market are basically extracted from chicken bile due to technical limitation, but the chicken bile raw material is limited in supply and high in price, so that the chenodeoxycholic acid extraction by taking chicken bile paste as the raw material is low in profit and limited in yield; the goose is fed in a small quantity, and the bile is difficult to be used as a raw material for extracting the chenodeoxycholic acid. The feeding amount of the duck is huge, but compared with the composition of the chicken bile, the composition of the duck bile is complex, and the chenodeoxycholic acid cannot be extracted from the duck bile by applying a method for extracting the chenodeoxycholic acid from the chicken bile. The pig gall has rich supply in China and low price, and the chenodeoxycholic acid content in the waste pig gall paste after the bilirubin is extracted can reach more than 20%.
Chinese patent CN201710153404.8 discloses a method for preparing chenodeoxycholic acid. The method comprises the following steps in sequence: refining hyodeoxycholic acid, preparing hyodeoxycholic acid methyl ester, eliminating 6-hydroxy and preparing chenodeoxycholic acid. The preparation method of chenodeoxycholic acid has simple steps and wide and sufficient sources of used raw materials. The chenodeoxycholic acid prepared by the method has high yield. However, the invention is only aiming at further refining and synthesizing the existing hyodeoxycholic acid and is completely different from the technical scheme of the inventor.
Chinese patent CN201710753687.X discloses a method for combined extraction of hyodeoxycholic acid and chenodeoxycholic acid, which relates to a method for producing hyodeoxycholic acid and chenodeoxycholic acid by taking hyocholic extract after bilirubin extraction as a raw material. Extracting hyodeoxycholic acid from the pig bile paste with acetone, crystallizing, concentrating the crystallized mother liquor, preparing calcium salt, acidifying to obtain crude product of chenodeoxycholic acid, and purifying with recrystallization method to obtain high-purity chenodeoxycholic acid product.
The pig bile paste is waste of pig product processing enterprises, is large in quantity, is easy to obtain raw materials, and mainly comprises hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid as organic components. The main difficulty for the technology of extracting chenodeoxycholic acid from the porcine bile paste is the separation of chenodeoxycholic acid (CDCA) from the hyocholic acid (HCA) and hyodeoxycholic acid (HDCA), the structures of which are shown as follows:
as shown by structural comparison, the structural difference between hyodeoxycholic acid (HDCA) and a target extract chenodeoxycholic acid (CDCA) is that only one hydroxyl group is substituted, and hyocholic acid (HCA) has one more hydroxyl group than chenodeoxycholic acid (CDCA), so that the structural similarity makes the separation of the three substances difficult.
The existing extraction methods mainly comprise a barium salt method, a calcium salt precipitation method, a crystallization extraction method and an ion exchange resin method, the barium salt can bring serious environmental problems, when the calcium salt is used, a large amount of water is used, water resources are greatly wasted, the crystallization extraction method needs a large amount of organic solvent, mother liquor needs to be repeatedly separated and crystallized, the production period is long, large-scale production cannot be realized, the extraction yield is low, the purity of the obtained product is generally lower than 90%, and the process cannot be used for separating a plurality of components in the pig bile at one time.
Therefore, under the condition that the prior art has the problems, it is very significant to develop an extraction technology which can effectively extract hyodeoxycholic acid, hyocholic acid and chenodeoxycholic acid from the porcine bile paste at one time and has the advantages of good effect, high efficiency, low cost, high purity and the like.
Disclosure of Invention
The application mainly aims to provide a method for extracting hyodeoxycholic acid, hyocholic acid and chenodeoxycholic acid from porcine bile paste at one time.
The technical scheme of the method is based on the purpose of extracting chenodeoxycholic acid from the pig bile paste, and is formed by further optimizing and improving the prior art. In order to achieve the above objects and solve the problems in the prior art, the present application adopts the following technical solutions:
a method for extracting chenodeoxycholic acid from pig bile paste can be summarized as steps of saponification, complex crystallization, extraction esterification, acetylation, silica gel filtration, reflux hydrolysis and the like, wherein the filtration mode of the silica gel filtration step is that silica gel is paved in a filter funnel for filtration.
Preferably, the thickness of the silica gel is 2-5 cm or 8-12 cm, and the silica gel is applied to a pilot plant test and a pilot plant test respectively.
Preferably, the method also comprises a mother liquor recovery step, wherein the mother liquor recovery step comprises the following steps: the crystallized mother liquor is concentrated to remove the alkane solvent and then is reused in the step of acid regulation and extraction before the next batch of methyl esterification.
Through multiple experiments, the experimenter finds out a better experimental scheme for extracting hyodeoxycholic acid, hyocholic acid and chenodeoxycholic acid from the porcine bile paste at one time, and the experimental scheme is as follows:
a method for extracting chenodeoxycholic acid from porcine bile paste comprises the following steps:
(1) saponification: adding sodium hydroxide and water into the pig bile paste, heating and refluxing for saponification, wherein the original pig bile paste contains a large amount of amino acids, and the saponification is carried out to separate hyodeoxycholic acid, hyocholic acid and chenodeoxycholic acid from the pig bile paste.
(2) Complexing and crystallizing: cooling the saponified reaction liquid to 30-50 ℃, adding acid to adjust the pH value, adding inorganic magnesium salt to form salt and crystallize, stirring for 4 hours, filtering to obtain a filter cake and a filtrate, and adding water to the hyodeoxycholic acid magnesium salt and adding acid to dissociate to obtain a hyodeoxycholic acid crude product. The method utilizes the solubility difference of the chenodeoxycholic acid magnesium salt and the hyodeoxycholic acid magnesium salt in water to separate the chenodeoxycholic acid magnesium salt and the hyodeoxycholic acid magnesium salt, wherein the hyodeoxycholic acid magnesium salt is in a solid state, the other two magnesium salts are in liquid states, the separation can be realized by simple filtration, and the added acid is one or a mixture of more of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid, preferably hydrochloric acid.
(3) Extraction and esterification: and (3) dropwise adding hydrochloric acid into the filtrate obtained in the step (2) to adjust the pH value, adding ethyl acetate, adjusting the acidity for extraction, concentrating the extract until the reaction system becomes oily, adding methanol and concentrated sulfuric acid for reflux stirring, carrying out esterification, adding solid sodium bicarbonate to adjust the pH value after the esterification is finished, filtering to obtain a bile acid methyl ester methanol solution, concentrating to obtain oily matter, extracting chenodeoxycholic acid and hyocholic acid from an aqueous phase to an organic phase through extraction, further removing impurities, and changing carboxyl groups in the hyocholic acid and the hyodeoxycholic acid into methyl ester groups through esterification so as to change the polarities of the hyodeoxycholic acid and the hyodeoxycholic acid.
(4) Acetylation: adding an acetylation reagent into the oily substance obtained in the step (3) for heating reflux reaction, cooling to room temperature after the reaction is finished, filtering, and concentrating the filtrate to obtain an oily substance, wherein hydroxyl groups in the hyocholic acid and the chenodeoxycholic acid are acetylated in the step to further change the polarity of the hyocholic acid and the chenodeoxycholic acid.
(5) Silica gel filtration: adding an alkane solvent into the oily matter obtained in the step (4), heating to completely dissolve the oily matter obtained in the step (4), adding silica gel powder, cooling, spreading a layer of silica gel in a filter funnel for filtering, washing with the alkane solvent to obtain a filtrate, wherein a filter cake is triacetyl hyocholic acid methyl ester, hydrolyzing the triacetyl hyocholic acid methyl ester recovered from the filter cake to obtain hyocholic acid, wherein the polarity difference between the triacetyl hyocholic acid methyl ester and diacetyl chenodeoxycholic acid methyl ester is utilized, the triacetyl hyocholic acid methyl ester with larger polarity is adsorbed by utilizing the adsorption difference of the silica gel, and the diacetyl chenodeoxycholic acid is eluted.
(6) Refluxing and hydrolyzing: concentrating the filtrate obtained in the step (5) to recover the solvent, adding a sodium hydroxide aqueous solution for heating reflux hydrolysis, cooling to adjust acidity, adding ethyl acetate for extraction, continuously adding acid to adjust pH, heating, stirring for standing for layering, removing a water layer, cooling again for crystallization, and dropwise adding an alkane solvent to expel crystals to obtain chenodeoxycholic acid.
(7) Mother liquor recovery: and concentrating the crystallization mother liquor to remove the alkane solvent, and then, mechanically applying to the next batch of magnesium salt filtrate to perform acid regulation extraction.
Preferably, the temperature for heating reflux to carry out saponification in the step (1) is 95-105 ℃, and the saponification duration is 8-15 hours.
Preferably, the inorganic magnesium salt in step (2) is selected from one or more of magnesium chloride and magnesium sulfate.
Preferably, the stirring temperature in the step (2) is 35-50 ℃.
Preferably, the pH of the filter cake in the step (2) is adjusted to 2-3 by adding acid.
Preferably, acid is added in the step (2) to adjust the pH value to 9-11, hydrochloric acid is added dropwise in the step (3) to adjust the pH value to 6-7, and solid sodium bicarbonate is added to adjust the pH value to 7-8.
Preferably, the acetylation reagent in the step (4) is one selected from acetic anhydride, sodium acetate combination or acetyl chloride and triethylamine combination, the temperature is raised to 120-140 ℃, and the reflux time is 3-4 hours.
Preferably, the specification of the silica gel in the step (5) is selected from one or more of 100-200 meshes, 200-300 meshes, 300-400 meshes and 400-500 meshes. The thickness of the silica gel depends on the weight of the material, and generally, the silica gel is 2 to 5cm thick in a laboratory pilot scale and 8 to 12cm thick in a factory pilot scale.
Preferably, the temperature for dissolving the oily substance by heating in the step (5) is 60-70 ℃.
Preferably, the temperature of the silica gel powder added in the step (5) is 20-40 ℃.
Preferably, the mass of the silica gel powder added in the step (5) is 0.1-1 time of that of the pig gall cream.
Preferably, the heating temperature for dissolving the oily substance by adding the alkane solvent in the step (5) is 40-70 ℃.
Preferably, the temperature of the heating reflux in the step (6) is 80-120 ℃, and the reflux time is 8-15 pilot plant test.
Preferably, the temperature for cooling in the step (6) is 40-50 ℃, the pH value of acidity is adjusted to 7-8, the pH value is continuously adjusted to 2-4 by adding acid, the temperature for heating and stirring is 40-60 ℃, and the temperature for cooling and crystallizing is-15-5 ℃.
Preferably, the alkane solvent in step (5) and step (6) is one or more of hexane, heptane, dichloromethane, cyclohexane and petroleum ether.
The application also comprises hyodeoxycholic acid, hyocholic acid and chenodeoxycholic acid prepared by the method.
The application also includes the use of the method in the purification of chenodeoxycholic acid.
The main difficulty of the technology for extracting chenodeoxycholic acid from the porcine bile paste is the separation of the three substances of the hyodeoxycholic acid, the hyocholic acid and the chenodeoxycholic acid. The hyodeoxycholic acid is an isomer of a target product chenodeoxycholic acid, has similar structure and chemical properties, and is successfully separated from the hyodeoxycholic acid magnesium salt by adding an inorganic magnesium salt and utilizing the solubility difference of the chenodeoxycholic acid magnesium salt and the hyodeoxycholic acid magnesium salt in water, so that the problem is solved. The structure of the hyocholic acid is very similar to that of the chenodeoxycholic acid, and the experiments of the inventor show that the methyl ester acetylation product triacetyl hyocholic acid methyl ester of the hyocholic acid and the methyl ester acetylation product diacetyl chenodeoxycholic acid methyl ester of the chenodeoxycholic acid can be well separated when the methyl ester acetylation product triacetyl hyocholic acid methyl ester and the methyl ester acetylation product diacetyl chenodeoxycholic acid methyl ester of the chenodeoxycholic acid are filtered through silica gel, the triacetyl hyocholic acid methyl ester with larger polarity can be adsorbed by the silica gel, and the diacetyl. And hydrolyzing the triacetyl hyocholic acid methyl ester adsorbed by the silica gel to obtain the hyocholic acid. The rest filtrate is diacetyl chenodeoxycholic acid methyl ester, and chenodeoxycholic acid can be obtained by reflux hydrolysis, extraction, cooling and crystallization. Therefore, the whole scheme can separate and extract the hyodeoxycholic acid, the hyocholic acid and the chenodeoxycholic acid at one time, not only can effectively obtain a target product of the hyodeoxycholic acid, but also can use the separated hyocholic acid and the hyodeoxycholic acid for other purposes, does not cause material waste, and has excellent separation effect through silica gel.
The extraction method of our prescription also solves a plurality of technical problems of the chenodeoxycholic acid in the past production process. For example, in the step of extraction and esterification, the carboxyl groups of chenodeoxycholic acid and hyocholic acid are modified into methyl groups, so that the problem that the chenodeoxycholic acid and the hyocholic acid are difficult to dissolve is solved. For example, in the actual production process of chenodeoxycholic acid, the separation of the oily substance in the step (5) is a problem influencing the experimental result, and in the past production process, if the oily substance in the step (5) is directly dissolved and heated by the alkane solvent and then is directly cooled, the oily substance is very easily separated out again in the process, so that the material of the chenodeoxycholic acid is lost and the subsequent silica gel filtration is influenced. Thereby my side has improved technical scheme, has added the silica gel powder for oily thing can not appear at the in-process of cooling, has solved the problem that oily thing appears easily among the cooling process. The extraction esterification and acetylation in the steps of the method are all group modification on products to be separated, and related polarity and physicochemical properties are changed, so that the final silica gel filtration step is facilitated.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
1. the separation of the two components is realized by utilizing the difference of the adsorption degrees of triacetyl hyocholic acid methyl ester and diacetyl chenodeoxycholic acid methyl ester in silica gel, the separation effect is very good, the content of hyocholic acid in the obtained chenodeoxycholic acid crude product is lower than 5%, and the content of hyocholic acid in the chenodeoxycholic acid crude product is lower than 1% after the chenodeoxycholic acid crude product is refined.
2. The method separates the chenodeoxycholic acid magnesium salt and the hyodesoxycholic acid magnesium salt by utilizing the solubility difference of the chenodeoxycholic acid magnesium salt and the hyodesoxycholic acid magnesium salt in water, reduces the content of hyodesoxycholic acid to be below 3% after separation, and is lower than 1% after refining.
3. The purity of hyodeoxycholic acid and hyocholic acid separated during the extraction of chenodeoxycholic acid is higher, and the hyodeoxycholic acid and hyocholic acid can be used for other purposes.
4. The reagent used in the process of extracting chenodeoxycholic acid is cheap and easy to obtain, most of the solvent can be recycled, and the generated wastewater is less.
5. The application of the method can improve the extraction yield of the chenodeoxycholic acid by extracting and recycling the mother liquor in the extraction process of the chenodeoxycholic acid.
Drawings
FIG. 1 is a flow chart of my party solution;
FIG. 2 is a detection spectrum of a finished product prepared in example 1 of my party;
FIG. 3 is a detection profile of the filtrate after saponification of my example 1;
FIG. 4 is a detection profile of the filtrate after removal of hyodeoxycholic acid according to my example 1;
FIG. 5 is a detection spectrum of the filtrate after silica gel filtration of our example 1 after concentration and saponification;
FIG. 6 is a detection spectrum of the finished product prepared in example 2 of my party;
FIG. 7 is the detection spectrum of the finished product prepared in my example 3.
Detailed Description
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example 1
A method for extracting chenodeoxycholic acid from porcine bile paste is an example of a laboratory lab bench and comprises the following steps:
(1) saponification: 3kg of pig gall cream (HPLC content 22.1%) is taken, and the pig gall cream is dark brown and is a massive solid. Putting the mixture into a 30L reaction kettle, adding 9kg of water and 1.2 kg of sodium hydroxide, heating the mixture to 100 ℃, refluxing the mixture for saponification, and keeping the saponification for 12 hours.
(2) Complexing and crystallizing: and (3) refluxing and preserving the heat of the saponified reaction solution for 8 hours, cooling to 48 ℃, adding hydrochloric acid to adjust the pH value to 10, adding a magnesium chloride aqueous solution (1.5 kg of magnesium chloride hexahydrate is dissolved in 3.3 kg of water), stirring for 4 hours at 50 ℃, filtering, and leaching with 0.6 kg of water to obtain a filtrate and a filter cake. The filter cake is hyodeoxycholic acid magnesium salt, and the hyodeoxycholic acid magnesium salt obtained by filtering is added with water and is dissociated by adding acid to obtain a hyodeoxycholic acid crude product.
(3) Extraction and esterification: and (3) dropwise adding hydrochloric acid into the filtrate obtained in the previous step to adjust the pH value to 7, adding 12.5L of ethyl acetate, continuously dropwise adding hydrochloric acid to adjust the pH value to 3, heating to 50 ℃, fully stirring for 1 hour, standing for layering, and removing a water layer to obtain an ethyl acetate solution. Concentrating the ethyl acetate solution to be oily, adding 6L of methanol, dripping 0.1kg of concentrated sulfuric acid, heating to 68 ℃, refluxing for 3h, esterifying, detecting by using TLC (thin layer chromatography), cooling to room temperature after complete reaction, adding sodium bicarbonate solid to adjust the pH to 8, filtering, washing a filter cake by 0.6L of methanol, and concentrating the filtrate to be oily.
(4) Acetylation: and (3) adding 3L of acetic anhydride and 0.15kg of sodium acetate into the oily substance obtained in the step (3), heating to 120 ℃ again until refluxing for 3 hours, detecting by TLC that the reaction is complete, cooling to room temperature, filtering, and concentrating the filtrate to obtain the oily substance.
(5) Silica gel filtration: and (3) adding 12L of heptane into the oily matter in the step (4), heating to 70 ℃ to completely dissolve the oily matter, adding 500g of silica gel powder to prevent the oily matter from being separated out again in the cooling process, cooling to 20 ℃, filtering through silica gel (silica gel with the thickness of about 3cm is paved in a filter funnel), leaching with 6L of heptane to obtain filtrate and filter cake, and adding water into triacetyl methyl hyocholate recovered from the filter cake to hydrolyze to obtain the hyocholic acid.
(6) Refluxing and hydrolyzing: concentrating the filtrate obtained in the step (5) to an oily substance, adding 0.3kg of sodium hydroxide and 3kg of water, heating to 103 ℃, refluxing for 12 hours, detecting by TLC to react completely, cooling to 50 ℃, adding hydrochloric acid to adjust the pH to 7, adding 3L of ethyl acetate, continuously dropwise adding hydrochloric acid to adjust the pH to 3, heating to 60 ℃, fully stirring for 1 hour, standing for layering, discarding the water layer, cooling to 3 ℃ to precipitate a solid, dropwise adding 6L of heptane, stirring for 2 hours, filtering to obtain 0.9 kg of a wet chenodeoxycholic acid product, and drying to obtain 0.52 kg of a finished chenodeoxycholic acid product.
(7) Mother liquor recovery: and concentrating the crystallization mother liquor to remove the alkane solvent, and then, mechanically applying to the next batch of magnesium salt filtrate to perform acid regulation extraction.
The finished product was submitted for inspection and the results are shown in the following table:
| variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 97.824% | 0.319% | 0.831% |
The detection map is shown in FIG. 2, and the specific parameters are shown in the following table:
| peak number | Retention time | Height | Area of | Area% | Degree of separation (USP) | Theoretical plate number (USP) | Tailing factor |
| 1 | 14.880 | 421 | 9196 | 0.219 | -- | 10949 | 0.924 |
| 2 | 18.760 | 498 | 13389 | 0.319 | 6.051 | 11056 | -- |
| 3 | 19.468 | 574 | 14821 | 0.353 | 1.009 | 12703 | -- |
| 4 | 24.206 | 1082 | 34923 | 0.831 | 6.269 | 13923 | 0.813 |
| 5 | 31.440 | 474 | 19051 | 0.454 | 7.812 | 14867 | 0.861 |
| 6 | 55.942 | 25614 | 4108632 | 97.824 | 8.271 | 2091 | 2.469 |
| Total of | 28663 | 4200012 | 100.000 |
Therefore, the purity of the chenodeoxycholic acid prepared by the method is very high and reaches a level of more than 97%. The content of the hyocholic acid and the hyodeoxycholic acid is also very low, is below 1 percent, and the content of impurities is well controlled.
The method also takes key nodes in the preparation process for detection, and the detection results are shown in the following graphs in fig. 3-5 respectively after saponification, hyodeoxycholic acid removal and silica gel filtration hydrolysis are finished:
after saponification
| Variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 43.864% | 18.739% | 28.526% |
After removing hyodeoxycholic acid
| Variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 73.939% | 21.379% | 1.800% |
After silica gel filtration and hydrolysis
| Variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 91.080% | 4.280% | 1.448% |
As can be seen from the table, the removal effect of hyodeoxycholic acid and hyocholic acid is remarkable.
Example 2
A method for extracting chenodeoxycholic acid from porcine bile paste, which is a second embodiment of lab bench, comprising the following steps:
(1) saponification: 3kg of pig gall cream (HPLC content 22.1%) is taken, and the pig gall cream is dark brown and is a massive solid. Putting the mixture into a 30L reaction kettle, adding 9kg of water and 1.2 kg of sodium hydroxide, heating the mixture to 100 ℃, refluxing the mixture for saponification, and keeping the saponification for 12 hours.
(2) Complexing and crystallizing: and (3) refluxing and preserving the heat of the saponified reaction solution for 8 hours, cooling to 40 ℃, adding hydrochloric acid to adjust the pH value to 11, adding a magnesium chloride aqueous solution (1.8 kg of magnesium sulfate heptahydrate is dissolved in 3.3 kg of water), stirring for 4 hours at 35 ℃, filtering, and leaching with 0.6 kg of water to obtain a filtrate and a filter cake. The filter cake is hyodeoxycholic acid magnesium salt, and the hyodeoxycholic acid magnesium salt obtained by filtering is added with water and is dissociated by adding acid to obtain a hyodeoxycholic acid crude product.
(3) Extraction and esterification: and (3) dropwise adding hydrochloric acid into the filtrate obtained in the previous step to adjust the pH value to 7, adding 12.5L of ethyl acetate, continuously dropwise adding hydrochloric acid to adjust the pH value to 2, heating to 50 ℃, fully stirring for 1 hour, standing for layering, and removing a water layer to obtain an ethyl acetate solution. Concentrating the ethyl acetate solution to be oily, adding 6L of methanol, dripping 0.1kg of concentrated sulfuric acid, heating to 68 ℃, refluxing for 3h, esterifying, detecting by using TLC (thin layer chromatography), cooling to room temperature after complete reaction, adding sodium bicarbonate solid to adjust the pH to 8, filtering, washing a filter cake by 0.6L of methanol, and concentrating the filtrate to be oily.
(4) Acetylation: and (3) adding 3L of acetic anhydride and 0.15kg of sodium acetate into the oily substance obtained in the step (3), heating to 140 ℃ again until refluxing for 3 hours, detecting by TLC that the reaction is complete, cooling to room temperature, filtering, and concentrating the filtrate to obtain the oily substance.
(5) Silica gel filtration: and (3) adding 12L of heptane into the oily matter in the step (4), heating to 70 ℃ to completely dissolve the oily matter, adding 600g of silica gel powder to prevent the oily matter from being separated out again in the cooling process, cooling to 20 ℃, filtering through silica gel (silica gel with the thickness of about 3cm is paved in a filter funnel), leaching with 6L of heptane to obtain filtrate and filter cake, and adding water into triacetyl methyl hyocholate recovered from the filter cake to hydrolyze to obtain the hyocholic acid.
(6) Refluxing and hydrolyzing: concentrating the filtrate obtained in the step (5) to an oily substance, adding 0.3kg of sodium hydroxide and 3kg of water, heating to 100 ℃, refluxing for 12 hours, detecting by TLC to react completely, cooling to 50 ℃, adding hydrochloric acid to adjust the pH to 7, adding 3L of ethyl acetate, continuously dropwise adding hydrochloric acid to adjust the pH to 3, heating to 60 ℃, fully stirring for 1 hour, standing for layering, discarding the water layer, cooling to 3 ℃ to precipitate a solid, dropwise adding 6L of heptane, stirring for 2 hours, filtering to obtain 1.0 kg of a wet chenodeoxycholic acid product, and drying to obtain 0.55 kg of a finished chenodeoxycholic acid product.
(7) Mother liquor recovery: and concentrating the crystallization mother liquor to remove the alkane solvent, and then, mechanically applying to the next batch of magnesium salt filtrate to perform acid regulation extraction.
The finished product was submitted for inspection and the results are shown in the following table:
| variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 97.919% | 0.833% | 0.827% |
The detection map is shown in fig. 6, and the specific parameters are shown in the following table:
| peak number | Retention time | Height | Area of | Area% | Degree of separation (USP) | Theoretical plate number (USP) | Tailing factor |
| 1 | 14.943 | 154 | 3696 | 0.081 | -- | 9959 | -- |
| 2 | 19.550 | 1104 | 37892 | 0.833 | 6.616 | 9711 | 0.727 |
| 3 | 24.320 | 1021 | 37631 | 0.827 | 5.487 | 10593 | 0.810 |
| 4 | 25.849 | 39 | 1017 | 0.022 | 1.821 | 19839 | 1.103 |
| 5 | 28.038 | 57 | 1757 | 0.039 | 2.802 | 18320 | 0.825 |
| 6 | 29.025 | 54 | 1684 | 0.037 | 1.184 | 19177 | 1.161 |
| 7 | 31.603 | 240 | 11020 | 0.242 | 2.518 | 10922 | 0.867 |
| 8 | 56.026 | 26726 | 4455678 | 97.919 | 7.714 | 1914 | 2.446 |
| Total of | 29396 | 100.000 |
Therefore, the purity of the chenodeoxycholic acid prepared by the method is very high and reaches a level of more than 97%. The content of the hyocholic acid and the hyodeoxycholic acid is also very low, is below 1 percent, and the content of impurities is well controlled.
Example 3
A method for extracting chenodeoxycholic acid from porcine bile paste is an example of pilot plant test in a factory, and comprises the following steps:
(1) saponification: 90kg of pig gall cream (HPLC content 22.1%) is taken, and the pig gall cream is dark brown and is a massive solid. Putting the mixture into a 1000L reaction kettle, adding 270 kg of water and 36 kg of sodium hydroxide, heating the mixture to 98 ℃, refluxing the mixture for saponification, and keeping the saponification time for 12 hours.
(2) Complexing and crystallizing: and (3) refluxing and preserving the heat of the saponified reaction solution for 8 hours, cooling to 44 ℃, adding hydrochloric acid to adjust the pH to 9, adding a magnesium chloride aqueous solution (45 kilograms of magnesium chloride hexahydrate is dissolved in 100 kilograms of water), stirring for 4 hours at 42 ℃, filtering, and leaching with 18 kilograms of water to obtain a filtrate and a filter cake. The filter cake is hyodeoxycholic acid magnesium salt, and the hyodeoxycholic acid magnesium salt obtained by filtering is added with water and is dissociated by adding acid to obtain a hyodeoxycholic acid crude product.
(3) Extraction and esterification: and (3) putting the filtrate obtained in the previous step into an enamel kettle, adding hydrochloric acid to adjust the pH value to 7, adding ethyl acetate 375L, continuously dropwise adding hydrochloric acid to adjust the pH value to 3, heating to 48 ℃, fully stirring for 1 hour, standing for layering, and removing a water layer to obtain an ethyl acetate solution. Concentrating the ethyl acetate solution to be oily, adding 180L of methanol, dripping 3kg of concentrated sulfuric acid, heating to 69 ℃, refluxing for 3h, esterifying, adding sodium bicarbonate solid after TLC detection reaction is completed, adjusting the pH to 8, filtering, washing a filter cake by 18L of methanol, and concentrating the filtrate to be oily.
(4) Acetylation: and (4) adding 90L of acetic anhydride and 4.5kg of sodium acetate into the oily substance obtained in the step (3), heating to 130 ℃ again until refluxing for 3 hours, detecting by TLC that the reaction is complete, cooling to room temperature, filtering, and concentrating the filtrate to obtain the oily substance.
(5) Silica gel filtration: and (3) adding 360L of heptane into the oily matter in the step (4), heating to 70 ℃ to completely dissolve the oily matter, adding 54kg of silica gel powder to prevent the oily matter from being separated out again in the cooling process, cooling to 20 ℃, filtering through the silica gel (the silica gel with the thickness of about 8cm is paved in a filter), leaching with 180L of heptane to obtain filtrate and a filter cake, and adding water into the triacetyl methyl hyocholate recovered from the filter cake to hydrolyze to obtain the hyocholic acid.
(6) Refluxing and hydrolyzing: concentrating the filtrate obtained in the step (5) to an oily substance, adding 9kg of sodium hydroxide and 90kg of water, heating to 96 ℃, refluxing for 12 hours, detecting by TLC (thin layer chromatography) to complete the reaction, cooling to 50 ℃, adding hydrochloric acid to adjust the pH to 7, adding 90L of ethyl acetate, continuously dropwise adding hydrochloric acid to adjust the pH to 3, heating to 58 ℃, fully stirring for 1 hour, standing for layering, removing a water layer, cooling to 0 ℃ to separate out a solid, adding 180L of heptane, stirring for 2 hours, centrifuging to obtain 22 kg of a wet chenodeoxycholic acid product, and drying to obtain 17 kg of a chenodeoxycholic acid finished product.
(7) Mother liquor recovery: and concentrating the crystallization mother liquor to remove the alkane solvent, and then, mechanically applying to the next batch of magnesium salt filtrate to perform acid regulation extraction.
The finished product was submitted for inspection and the results are shown in the following table:
| variety of (IV) C | Chenodeoxycholic acid | Hyocholic acid | Hyodeoxycholic acid |
| Purity of | 97.423% | 0.759% | 0.684% |
The detection map is shown in FIG. 7, and the specific parameters are shown in the following table:
| peak number | Retention time | Height | Area of | Area% | Degree of separation (USP) | Theoretical plate number (USP) | Tailing factor |
| 1 | 14.121 | 103 | 2344 | 0.207 | -- | 9681 | 0.900 |
| 2 | 17.692 | 184 | 4780 | 0.423 | 5.191 | 7795 | -- |
| 3 | 18.290 | 345 | 8581 | 0.759 | 0.810 | 11776 | -- |
| 4 | 22.573 | 250 | 7742 | 0.684 | 5.857 | 13119 | 0.798 |
| 5 | 29.054 | 152 | 5710 | 0.505 | 7.348 | 14185 | -- |
| 6 | 52.665 | 12302 | 1102069 | 97.423 | 13.543 | 7038 | 1.451 |
| Total of | 13335 | 1131225 | 100.000 |
Therefore, the purity of the chenodeoxycholic acid prepared by the method is very high and reaches a level of more than 97%. The content of the hyocholic acid and the hyodeoxycholic acid is also very low, is below 1 percent, and the content of impurities is well controlled.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.
Claims (10)
1. A method for extracting chenodeoxycholic acid from porcine bile paste comprises the steps of saponification, complex crystallization, extraction esterification, acetylation and reflux hydrolysis of the porcine bile paste, and is characterized by further comprising the step of silica gel filtration, wherein the silica gel filtration mode is that silica gel is paved in a filter funnel for filtration.
2. The method of claim 1, wherein the silica gel has a thickness of 2 to 5cm or 8 to 12 cm.
3. A method for extracting chenodeoxycholic acid from porcine bile paste comprises the following steps:
(1) saponification: adding sodium hydroxide and water into the pig gall paste, heating and refluxing for saponification;
(2) complexing and crystallizing: cooling the saponified reaction liquid to 30-50 ℃, adjusting the pH to be alkaline, adding an inorganic magnesium salt to form salt and crystallize, filtering to obtain a filtrate and a filter cake, adding an acid to the filter cake to adjust the pH, stirring, and filtering to obtain hyodeoxycholic acid;
(3) extraction and esterification: adding ethyl acetate into the filtrate obtained in the step (2), adjusting acid property for extraction, concentrating the extract until the reaction system becomes oily, adding methanol and concentrated sulfuric acid for reflux stirring, carrying out esterification, adding solid sodium bicarbonate to adjust the pH value after the esterification is finished, filtering to obtain a bile acid methyl ester methanol solution, and concentrating to obtain oily matter;
(4) acetylation: adding an acetylation reagent into the oily substance obtained in the step (3) for reflux reaction, cooling to room temperature after the reaction is finished, filtering, and concentrating the filtrate to obtain an oily substance;
(5) silica gel filtration: adding an alkane solvent into the oily matter obtained in the step (4), heating to completely dissolve the oily matter obtained in the step (4), adding silica gel powder, cooling, spreading a layer of silica gel in a filter funnel for filtering, washing with the alkane solvent to obtain a filtrate, wherein a filter cake is triacetyl hyocholic acid methyl ester, and hydrolyzing the triacetyl hyocholic acid methyl ester recovered from the filter cake to obtain hyocholic acid;
(6) refluxing and hydrolyzing: concentrating and recovering the filtrate obtained in the step (5), adding a sodium hydroxide aqueous solution for reflux hydrolysis, cooling to adjust acidity, adding ethyl acetate for extraction, cooling to crystallize, and dropwise adding an alkane solvent to expel crystals to obtain chenodeoxycholic acid.
4. The method of claim 3, wherein the inorganic magnesium salt in step (2) is selected from one or more of magnesium chloride and magnesium sulfate.
5. The method of claim 3, wherein the pH is adjusted to an alkaline pH of 9 to 11 in step (2), and solid sodium bicarbonate is added to adjust the pH to 7 to 8 in step (3).
6. The method of claim 3, wherein the acetylating reagent in step (4) is selected from one of acetic anhydride, a combination of sodium acetate or a combination of acetyl chloride and triethylamine.
7. The method according to claim 3, wherein the mass of the silica gel powder added in the step (5) is 0.1-1 times of that of the pig bile paste.
8. The method of claim 3, wherein the alkane solvent in step (5) and step (6) is one or more selected from hexane, heptane, dichloromethane, cyclohexane and petroleum ether.
9. Hyodeoxycholic acid, hyocholic acid, chenodeoxycholic acid, prepared by the process according to any one of claims 1 to 8.
10. Use of a process according to any one of claims 1 to 8 for the purification of chenodeoxycholic acid.
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