Preparation method of high content ammonium salt of chenodeoxycholic acid and chenodeoxycholic acid

Abstract

The application discloses a preparation method of high-content ammonium chenodeoxycholate and chenodeoxycholic acid, relates to the technical field of chenodeoxycholic acid extraction, and aims at solving the problem that the prior art cannot prepare chenodeoxycholic acid products with a content of about 98% to meet customer demands, and comprises the following steps: using water A to dissolve gall paste, extracting and separating the gall paste with an organic solvent, adding water B to the organic solvent layer, adjusting the pH of the organic solvent layer to 5.5-6.8 by using an alkali, adding an organic alkali to the organic solvent layer under stirring until the pH of the organic solvent layer is 8-12, adjusting the pH of the organic solvent layer to 5.5-8.0 by using an acid, and filtering and washing the organic solvent layer to obtain ammonium chenodeoxycholate and a filtrate; the content of chenodeoxycholic acid in the ammonium chenodeoxycholate reaches 98% or more, the impurities of cholic acid and other maximum single impurities are significantly reduced, the ammonium chenodeoxycholate can meet more extensive customer demands, and the product has higher added value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the field of chenodeoxycholic acid extraction technology. Background Technology

[0002] Chenodeoxycholic acid (CDCA) can reduce cholesterol saturation in bile, thus having a good therapeutic effect on gallstones. Chenodeoxycholic acid is also an important raw material for the chemical synthesis of ursodeoxycholic acid (3α,7β-dihydroxy-5-cholanoic acid, UDCA); ursodeoxycholic acid is the main active component of bear bile and has clear therapeutic effects on various diseases of the liver and gallbladder.

[0003] Chenodeoxycholic acid is mainly extracted or semi-synthesized from animal bile. The main sources of extraction are: 1. Chicken and goose bile; 2. Pig bile; 3. Duck bile.

[0004] Patent application number CN200610046855.3 describes an industrial-scale preparation method for extracting and purifying chenodeoxycholic acid from pig bile. The process includes: using the mother liquor after bilirubin extraction from pig bile as raw material to prepare total cholic acid; saponification and acidification to obtain crude chenodeoxycholic acid; decolorization and defatting to prepare barium chenodeoxycholic acid precipitate; potassium carbonate removal and acidification; and silica gel column purification. This patent involves toxic heavy metal barium salts and the silica gel column purification step, which is inconvenient for large-scale production.

[0005] Patent application CN201010288350.4 describes a process from fresh or frozen poultry bile to obtain crude bile acids or crude bile acid calcium salts via saponification. The crude bile acid ester solution is then heated with an aqueous solution of an organic nitrogenous compound A to remove most of the hydrophilic impurities from the crude bile acids. The resulting crude bile acid ester solution reacts with an organic nitrogenous compound B, precipitating a precipitate. After filtration, decolorization, and purification, chenodeoxycholic acid with a purity of approximately 95% can be obtained. However, this invention suffers from drawbacks such as cumbersome steps, a long purification cycle, the need for calcium salt formation, and the requirement to use gasoline for ester removal.

[0006]

[0007] Bile extracts contain numerous impurities; for example, chicken and goose bile extracts contain only about 26±6% chenodeoxycholic acid. The largest impurity in these extracts—cholic acid—accounts for approximately 7±2%, with other bile acid-related impurities making up about 1%. Additionally, there are large amounts of non-cholic acid impurities such as fats, fatty acids, pigments, and proteins, resulting in a complex composition. As shown in the diagram above, bile acids and chenodeoxycholic acid have similar structures. Selectively and cost-effectively removing bile acid impurities is one of the challenges in extracting chenodeoxycholic acid. The high proportion and complex composition of impurities in bile extracts make it difficult to prepare high-content chenodeoxycholic acid.

[0008] The patent application with application number CN201910502719.8 dissolves bile paste in an aqueous sodium hydroxide solution, adds butyl acetate and hydrogen peroxide, and stirs to obtain a decolorizing solution. By precisely controlling the pH value, the solution is separated and washed with alkaline water to remove some impurities. The chenodeoxycholic acid washing solution is purified by crystallization with sec-butylamine or tert-butylamine, and then desalted to obtain chenodeoxycholic acid. This invention removes a large amount of bile acid impurities, pigment impurities, and oil impurities by forming a salt with tert-butylamine or sec-butylamine, and the chenodeoxycholic acid content can reach 92%. However, since both chenodeoxycholic acid and bile acids contain carboxyl groups, these carboxyl groups will form salts with the organic amines (tert-butylamine, sec-butylamine) protected in the patent. The content of bile acid impurities is relatively high, and bile acid impurities cannot be removed by salt formation alone. Multiple alkaline water washes are required to remove bile acid impurities, and a high content of chenodeoxycholic acid is still not obtained. The invention produces a chenodeoxycholic acid product with a content of approximately 92%, which can meet the quality requirements of some customers, but cannot meet the needs of customers with high content (approximately 98%) chenodeoxycholic acid. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing high-content chenodeoxycholic acid ammonium salt and chenodeoxycholic acid, so as to meet the customer demand that the existing technology cannot prepare chenodeoxycholic acid products with a content of about 98%.

[0010] To achieve the above objectives, the present invention specifically adopts the following technical solution: a method for preparing high-content chenodeoxycholate ammonium salt, comprising the following steps: extracting bile paste with water A, separating the liquid with an organic solvent, adding water B to the organic solvent layer, adjusting the pH to 5.5-6.8 with alkali, adding an organic base under stirring to the pH to 8-12, adjusting the pH to 5.5-8.0 with acid, filtering and washing to obtain chenodeoxycholate ammonium salt and filtrate A, wherein the organic solvent is butyl acetate, ethyl acetate or isopropyl acetate.

[0011] In this technical solution, bile paste is dissolved in a two-part system of organic solvent and water, and the pH is adjusted to 5.5-6.8 with alkali. Within this pH range, a large amount of cholic acid reacts with sodium hydroxide to produce sodium cholate. Sodium cholate dissolves in the aqueous solution and will not be precipitated by forming a salt with tert-butylamine acid, thus removing the impurity—cholic acid. An organic alkali is then added with stirring to adjust the pH to the desired level. 8-12 (Terbutamine and other organic bases are alkaline; controlling the appropriate pH range allows for control of the appropriate amount of organic base added). Then, adjust the pH to 5.5-8.0 with acid. Within this pH range, chenodeoxycholic acid can form salts with organic bases more fully, resulting in more complete precipitation. This allows chenodeoxycholic acid to preferentially form salts with organic bases, precipitating as solid chenodeoxycholic acid ammonium salt for purification, while very little cholic acid forms salts with organic bases. A large amount of cholic acid is removed by dissolving in the solution. At the same time, under these conditions, impurities such as pigments and oils are also more easily removed from the solution. There is no need to use hydrogen peroxide for decolorization and impurity removal (hydrogen peroxide has certain dangers in industrial storage and use), and a white product with high content of chenodeoxycholic acid can be obtained.

[0012] Further, an organic base is added with stirring until the pH reaches 8.5-10. The organic base includes tert-butylamine, sec-butylamine, or α-phenylethylamine.

[0013] Furthermore, the pH is adjusted to 5.8-6.3 using an alkali, such as sodium hydroxide or potassium hydroxide.

[0014] Further, the pH is adjusted to 6.0-7.2 using sulfuric acid or hydrochloric acid.

[0015] Furthermore, the weight ratio of cholecystokinin, water A, organic solvent and water B is 1:0.5-3:2-5:0.5-2.

[0016] Further, the pH of filtrate A is adjusted to 5.7-6.9, and after standing, it is separated to obtain an organic layer A1 and an aqueous layer C1. Aqueous layer C1 is back-extracted with an organic solvent to obtain an organic layer A2 and an aqueous layer C2. Organic layers A1 and A2 are combined to obtain a recovered chenodeoxycholic acid organic solvent solution. Aqueous layer C2 is acidified to precipitate a solid, and cholic acid is recovered (the organic solvent is butyl acetate, ethyl acetate, or isopropyl acetate).

[0017] A method for preparing a high-content chenodeoxycholic acid ammonium salt, wherein the chenodeoxycholic acid ammonium salt prepared by this method is used to prepare a high-content chenodeoxycholic acid, includes the following steps:

[0018] S1. Ammonium chenodeoxycholate salt is mixed with an organic solvent, acid water is added until the salt is completely dissolved, water is added until the solution is clear, and the mixture is allowed to stand and separate. Organic layer C is taken, alkaline water is added and stirred, and the mixture is allowed to stand and separate to obtain aqueous layer A (aqueous layer A can be used for water B) and organic layer B. The organic solvent is butyl acetate, ethyl acetate or isopropyl acetate, and the molar ratio of alkali to cholic acid in organic layer C is 1-6:1.

[0019] S2, add water to organic layer B and mix, add alkali and stir until pH 8-12, let stand and separate, add acid water to aqueous layer B to acidify until crystallization is complete, filter to obtain chenodeoxycholic acid.

[0020] Furthermore, the acidic water is an aqueous solution of sulfuric acid or hydrochloric acid.

[0021] Furthermore, the alkaline solution is an aqueous solution of sodium hydroxide or potassium hydroxide; the alkali is sodium hydroxide or potassium hydroxide.

[0022] Furthermore, in S1, the weight ratio of chenodeoxycholate ammonium salt, organic solvent, and alkaline water is 1:6 to 16:0.5 to 5.

[0023] Furthermore, the weight ratio of chenodeoxycholate ammonium salt in S1 to water added in S2 is 1:3-12.

[0024] Furthermore, chenodeoxycholic acid ammonium salt is an ammonium salt formed by chenodeoxycholic acid with tert-butylamine, sec-butylamine and α-phenylethylamine, respectively.

[0025] In the technical solution of this application: the cholecystokinin is extracted with water A, and multiple extractions can be performed during the extraction and separation process using organic solvents.

[0026] In natural bile, chenodeoxycholic acid exists in a bound form (such as glycochenodeoxycholic acid). After enzymatic or chemical hydrolysis of the bile, a saponified aqueous solution of chenodeoxycholic acid is obtained. For ease of transportation and sale, this chenodeoxycholic acid aqueous solution is acidified to precipitate bile paste solids. After removing the water, only the solids are sold and transported. The first step of this invention involves extracting the purchased bile paste using water A and an organic solvent. Simply changing the method—using an organic solvent to extract the saponified aqueous solution of chenodeoxycholic acid—also falls within the scope of this invention.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. This application dissolves choline paste in two systems, an organic solvent and water, and controls it within a narrow pH range. This allows chenodeoxycholic acid to preferentially form a salt with an organic base, precipitating out as a solid ammonium chenodeoxycholic acid salt for purification. Meanwhile, very little cholic acid forms a salt with the organic base, and a large amount of cholic acid is removed from the solution. At the same time, under these conditions, impurities such as pigments and oils are more easily soluble in the solution and difficult to precipitate, thus eliminating the need for hydrogen peroxide decolorization and impurity removal steps. This allows for the production of a white chenodeoxycholic acid product with high content.

[0029] 2. The present invention has a chenodeoxycholic acid content of up to 98% or more, and significantly reduces cholic acid impurities and other maximum single impurities, which can meet the needs of a wider range of customers and has higher added value.

[0030] 3. The production steps of this invention are simplified, the number of washing and impurity removal times are reduced from 3 to 1, the salt decolorization effect is better, less wastewater is generated, and the corresponding costs and equipment investment are reduced. Attached Figure Description

[0031] Figure 1 This is the HPLC chromatogram of the butyl acetate extract in Example 7 of this invention;

[0032] Figure 2 This is the HPLC chromatogram of chenodeoxycholic acid tert-butylamine salt in Example 7 of the present invention;

[0033] Figure 3 This is the HPLC chromatogram of the chenodeoxycholic acid product in Example 7 of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0035] Therefore, the following descriptions only represent selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] This embodiment provides a method for preparing high-content chenodeoxycholic acid ammonium salt and high-content chenodeoxycholic acid. 200 kg of butyl acetate (twice the weight of chicken bile extract) and 50 kg of water (0.5 times the weight of chicken bile extract) are added to a reactor. 100 kg of chicken bile extract is added while stirring. The temperature is raised to 60-65℃, and the pH is adjusted to 2 to dissolve the material. The mixture is allowed to stand and separate. The aqueous layer is back-extracted with 50 kg of butyl acetate (0.5 times the weight of chicken bile extract), kept at 50-55℃ and stirred for 5 minutes. The mixture is then allowed to stand and separate. The two butyl acetate layers are combined, 100 kg of water is added, and the mixture is stirred for 5 minutes. The mixture is allowed to stand and separate. The butyl acetate layer is weighed, and a sample is sent for testing to determine the content of CDCA (chenodeoxycholic acid) and CA (cholic acid).

[0038] Add 100 kg of water B to the butyl acetate layer, adjust the pH to 6.0 with sodium hydroxide, add tert-butylamine at 50-65℃ until the pH reaches 9, stir for 1 hour, then adjust the pH to 6.9 with 50% sulfuric acid, cool to 25-30℃, continue stirring for 1 hour, centrifuge, wash the filter cake with butyl acetate and water to obtain 33.26 kg of crude wet product of tert-butylamine chenodeoxycholic acid salt (28.95 kg dry) and filtrate A.

[0039] 33.26 kg (28.95 kg dry weight) of crude wet chenodeoxycholic acid tert-butylamine salt was added to 200 kg of butyl acetate. Stirring was started, and 20% sulfuric acid was added to adjust the pH of the aqueous layer to 2. 30 kg of water was added and stirred. The mixture was allowed to stand and separated. Organic layer C was mixed with 100 kg of sodium hydroxide aqueous solution (the molar ratio of sodium hydroxide to cholic acid in organic layer C was 4:1) to obtain aqueous layer A and organic layer B. Organic layer B was then mixed with 120 kg of water and treated with hydrogen... The pH was adjusted to 10 with sodium oxide, and the mixture was separated. The aqueous layer was concentrated to 100 kg by distillation, and then 20 kg of water was added. The pH was adjusted to 4 with 50% sulfuric acid at 26°C, and a slurry solid precipitated out. The temperature was gradually increased until the internal temperature reached 41°C, until the mixture could quickly and clearly separate into layers after standing. The temperature was then lowered to 20-30°C and stirred for 1 hour. The system was filtered and washed with tap water until neutral. The mixture was then heated and dried with forced air to obtain chenodeoxycholic acid, yielding 18.12 kg of chenodeoxycholic acid with a purity of 98.0%.

[0040] Comparative Example 1 refers to patent application number CN201910502719.8:

[0041] A method for preparing chenodeoxycholic acid tert-butylamine salt includes the following steps:

[0042] S1 Preparation of Decolorizing Solution

[0043] Weigh 20g of sodium hydroxide and dissolve it in 200g of water. Add 150g of chicken bile extract (containing 25% bile acid). Heat to above 70℃ and stir until the bile extract is completely dissolved. Once the system is completely dissolved, cool it to an internal temperature of 40℃. Add 320ml (282g) of butyl acetate and dropwise add 20g of 30% hydrogen peroxide. After the addition is complete, stir for 3 hours at 20-30℃ to obtain the decolorized solution.

[0044] S2 Preparation of Extract

[0045] The decolorized solution obtained from S1 was adjusted to pH 5.95–6.05 with 60% sulfuric acid. After adjustment, the solution was stirred at 20–30°C for 0.5 h. The solution was allowed to stand and then separated into aqueous and organic phases. The organic phase was the chenodeoxycholic acid butyl acetate extract. 80 ml (70.5 g) of butyl acetate was added to the aqueous phase, and the pH was adjusted to 6.00–6.10 with 60% sulfuric acid. After adjustment, the solution was stirred at 20–30°C for 0.5 h, allowed to stand, and then separated to obtain the organic layer. The two organic layers were combined to obtain the chenodeoxycholic acid extract.

[0046] S3 prepares washing solution

[0047] The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8 g dissolved in 150 g water). The mixture was stirred at 20–30 °C for 0.5 h. After standing, the layers were separated. The upper organic layer was collected to obtain the washing liquid.

[0048] S4 Preparation of butyl chenodeoxycholic acid acetate extract

[0049] The washing solution was added to a prepared sodium hydroxide solution (0.5 g dissolved in 150 g water), and stirred at 20–30 °C for 0.5 h. After standing, the liquid was separated into organic and aqueous phases. The organic layer was a butyl acetate layer. A prepared sodium hydroxide solution (0.4 g dissolved in 150 g water) was added to the obtained organic phase, and the mixture was stirred at 20–30 °C for 0.5 h. After standing, the liquid was separated again, and the organic phase was collected to obtain a butyl acetate extract of chenodeoxycholic acid. HPLC analysis showed that the peak area ratio of cholic acid to chenodeoxycholic acid was 1.6%.

[0050] S5 salt formation reaction

[0051] The obtained chenodeoxycholic acid butyl acetate extract solution was treated with 12 g of tert-butylamine at 60 °C, stirred for 1 h, then cooled to room temperature and stirred for 30 min. The mixture was filtered, and the filter cake was washed with 20 ml of butyl acetate. The filter cake was then dried by forced air drying. Tert-butylamine chenodeoxycholic acid salt was obtained, with a molar yield of 95.1% for the salt formation step and a purity of 91.1%.

[0052] Preparation of chenodeoxycholic acid by salt decomposition: Chenodeoxycholic acid tert-butylamine salt was dissolved in 250 ml of water with 4 g of sodium hydroxide and stirred until dissolved. The solution was heated to 30°C and the pH was adjusted to 4 with 60% sulfuric acid, resulting in the precipitation of a slurry solid. The temperature was gradually increased to an internal temperature of 41°C until the mixture rapidly and clearly separated into layers after standing. The mixture was stirred for 1 hour, and the system was filtered and washed with tap water until neutral. The resulting solid was dried in a forced-air drying oven at 50°C. Chenodeoxycholic acid with a purity of 92.1% was obtained.

[0053] The advantages and disadvantages of Embodiment 1 of this application and Comparative Example 1 are shown in Table 1:

[0054] Table 1 Comparison of Example 1 of this application and Comparative Example 1

[0055]

[0056] As shown in Table 1, the product obtained by this invention has significantly increased content and significantly reduced impurities, which can satisfy a wider range of customers and has higher added value. Moreover, the production steps are simplified, the number of washing and impurity removal times is reduced from 3 to 1, the salt decolorization effect is better, and there is less wastewater, resulting in lower costs and equipment investment.

[0057] Example 2

[0058] Add 200 kg of butyl acetate (twice the weight of chicken bile extract) and 50 kg of water (0.5 times the weight of chicken bile extract) to the reactor, and add 100 kg of chicken bile extract while stirring. Heat to 60-65℃, stir, and adjust the pH to 2 to dissolve the material. Allow to stand and separate the layers. Back-extract the aqueous layer twice with 50 kg of butyl acetate (0.5 times the weight of chicken bile extract). Combine the three butyl acetate layers.

[0059] The combined butyl acetate layer was added to aqueous layer A in Example 1, and sodium hydroxide was added to adjust the pH to 5.98. Tert-butylamine was added at 50-65°C until the pH reached 8.5. The mixture was stirred for 1 hour, and then the pH was adjusted to 7.0 with 50% sulfuric acid. The mixture was then cooled to room temperature and stirred for another hour. After centrifugation, the filter cake was washed with butyl acetate and water to obtain 40.68 kg of crude wet product of tert-butylamine chenodeoxycholic acid salt (34.99 kg dry) and filtrate A1.

[0060] 40.68 kg (34.99 kg dry weight) of crude wet chenodeoxycholic acid tert-butylamine salt was added to 350 kg of butyl acetate and 50 kg of water. Concentrated hydrochloric acid was added while stirring to adjust the pH of the aqueous layer to 2. The mixture was allowed to stand and separate. Organic layer C was mixed with 150 kg of sodium hydroxide aqueous solution (the molar ratio of sodium hydroxide to cholic acid in the organic layer was 5:1) to obtain aqueous layer A and organic layer B. Organic layer B, 200 kg of water, and 2.3 kg of sodium hydroxide were mixed and stirred, allowed to stand and separate. Aqueous layer B was distilled and concentrated to 160 kg. The pH was adjusted to 3 with 50% sulfuric acid at 26℃, precipitating a slurry solid. The temperature was gradually increased until the internal temperature reached 41℃, until rapid and obvious separation occurred after standing. The temperature was then lowered to 20–30℃ and stirred for 1 hour. The system was filtered and washed with tap water until neutral. The mixture was then heated and dried with forced air to obtain 20.59 kg of chenodeoxycholic acid with a purity of 98.2%.

[0061] Example 3

[0062] Add 1200 kg of butyl acetate (twice the weight of chicken bile extract) and 400 kg of water (0.5 times the weight of chicken bile extract) to the reactor, and add 400 kg of chicken bile extract while stirring. Heat to 60-65℃, stir, and adjust the pH to 2 to dissolve the material. Allow to stand and separate the layers. Back-extract the aqueous layer with 400 kg of butyl acetate (0.5 times the weight of chicken bile extract). Combine the two butyl acetate layers.

[0063] The combined butyl acetate layer was divided into 4 equal parts:

[0064] Take one portion of the butyl acetate layer and add it to 100 kg of water. Add sodium hydroxide to adjust the pH to 6.0. Add tert-butylamine at 50-65℃ until the pH reaches 8.5. Stir for 1 hour, then adjust the pH to 6.9 with 50% sulfuric acid. Cool down to 25-30℃ and continue stirring for 1 hour. Centrifuge, wash the filter cake with butyl acetate and water, and dry to obtain crude tert-butylamine chenodeoxycholic acid salt.

[0065] Comparative Example 2

[0066] Take one portion of the butyl acetate layer, add tert-butylamine at 50-65℃ until the pH reaches 8.5, stir for 1 hour, then cool to 25-30℃ and continue stirring for 1 hour. Centrifuge, wash the filter cake with butyl acetate and water, and dry to obtain crude tert-butylamine chenodeoxycholic acid salt.

[0067] Comparative Example 3

[0068] Take one portion of the butyl acetate layer and add it to 100 kg of water. Add sodium hydroxide to adjust the pH to 5.0. Add tert-butylamine at 50-65℃ until the pH reaches 8.5. Stir for 1 hour, then adjust the pH to 6.9 with 50% sulfuric acid. Cool down to 25-30℃ and continue stirring for 1 hour. Centrifuge, wash the filter cake with butyl acetate and water, and dry to obtain crude tert-butylamine chenodeoxycholic acid salt.

[0069] Comparative Example 4

[0070] Take one portion of the butyl acetate layer and add it to 100 kg of water. Add sodium hydroxide to adjust the pH to 7.0. Add tert-butylamine at 50-65℃ until the pH reaches 8.5. Stir for 1 hour, then adjust the pH to 6.9 with 50% sulfuric acid. Cool down to 25-30℃ and continue stirring for 1 hour. A brown, viscous gel-like substance precipitates out and cannot be centrifuged or filtered.

[0071] The comparison results between Example 3 and Comparative Examples 2-4 are shown in Table 2:

[0072] Table 2 shows the comparison results between Example 3 and Comparative Examples 2-4.

[0073]

[0074] As shown in Table 2, the present invention can form salts within a specific pH range, thereby removing bile acid impurities, improving product color grade, and increasing product content.

[0075] Example 4

[0076] The pH of filtrate A obtained in Example 1 was adjusted to 6.1, and the mixture was allowed to stand and separated. The aqueous layer was back-extracted with 50 kg of butyl acetate, and the organic layers were combined to obtain a recovered chenodeoxycholic acid butyl acetate solution. The pH of the aqueous layer was adjusted to 2.0 with 50% butyl acetate, and a solid precipitated. Crude cholic acid was recovered.

[0077] The recovered chenodeoxycholic acid butyl acetate solution was concentrated under reduced pressure to 40 kg, tert-butylamine was added at 55°C, stirred for 1 hour, cooled to room temperature and stirred for 1 hour, filtered, and washed to obtain the wet product of recovered chenodeoxycholic acid tert-butylamine salt.

[0078] Example 5

[0079] This embodiment provides a method for preparing high-content chenodeoxycholic acid ammonium salt and high-content chenodeoxycholic acid. 300 kg of ethyl acetate (3 times the weight of chicken bile extract) and 100 kg of water (1 times the weight of chicken bile extract) are added to a reaction vessel. 100 kg of chicken bile extract is added while stirring. The temperature is raised to 60-65℃, and the pH is adjusted to 4 while stirring. The material is dissolved and allowed to stand for separation. The aqueous layer is back-extracted with 200 kg of ethyl acetate (2 times the weight of chicken bile extract), kept at 50-55℃ and stirred for 5 minutes. The layers are then allowed to stand for separation. The two ethyl acetate layers are combined, 100 kg of water is added, and the mixture is stirred for 5 minutes. The layers are then allowed to stand for separation. The ethyl acetate layer is weighed, and a sample is sent for testing to determine the content of CDCA (chenodeoxycholic acid) and CA (cholic acid).

[0080] Add 100 kg of water B to the ethyl acetate layer, adjust the pH to 5.8 with sodium hydroxide, add tert-butylamine at 50-65℃ until the pH reaches 8.5, stir for 1 hour, then adjust the pH to 7.2 with 50% sulfuric acid, cool to 25-30℃, continue stirring for 1 hour, centrifuge, wash the filter cake with ethyl acetate and water to obtain 33.06 kg of crude wet product of tert-butylamine chenodeoxycholic acid salt (28.76 kg dry equivalent) and filtrate A.

[0081] 33.06 kg (28.76 kg dry weight) of crude wet chenodeoxycholic acid tert-butylamine salt was added to 300 kg of ethyl acetate. Stirring was started, and 20% sulfuric acid was added to adjust the pH of the aqueous layer to 2.5. 60 kg of water was added and stirred, then allowed to stand and separate. Organic layer C was mixed with 150 kg of sodium hydroxide aqueous solution (the molar ratio of sodium hydroxide to cholic acid in organic layer C was 3:1) to obtain aqueous layer A and organic layer B. Organic layer B was then mixed with 300 kg of water, and the pH was adjusted to 10 with sodium hydroxide. The mixture was separated, and the aqueous layer was concentrated to 150 kg by distillation. The pH was adjusted to 4 with 50% sulfuric acid at 26°C, precipitating a slurry-like solid. The temperature was gradually increased to an internal temperature of 42°C, then decreased to 20–30°C and stirred for 1 hour. The system was filtered and washed with tap water until neutral. The system was then heated and dried with forced air to obtain 18.02 kg of chenodeoxycholic acid with a purity of 98.1%.

[0082] Example 6

[0083] This embodiment provides a method for preparing high-content chenodeoxycholic acid ammonium salt and high-content chenodeoxycholic acid. 300 kg of isopropyl acetate (3 times the weight of chicken bile extract) and 100 kg of water (1 times the weight of chicken bile extract) are added to a reaction vessel. 100 kg of chicken bile extract is added while stirring. The temperature is raised to 60-65℃, and the pH is adjusted to 4.5 while stirring. The material is dissolved and allowed to stand for separation. The aqueous layer is back-extracted with 100 kg of isopropyl acetate (1 times the weight of chicken bile extract), kept at 50-55℃ and stirred for 5 minutes. The mixture is then allowed to stand for separation. The two isopropyl acetate layers are combined, 100 kg of water is added, and the mixture is stirred for 5 minutes. The mixture is then allowed to stand for separation. The isopropyl acetate layer is weighed, and a sample is sent for testing to determine the content of CDCA (chenodeoxycholic acid) and CA (cholic acid).

[0084] Add 100 kg of water B to the isopropyl acetate layer, adjust the pH to 5.8 with sodium hydroxide, add tert-butylamine at 50-65℃ until the pH reaches 8.5, stir for 1 hour, then adjust the pH to 7.2 with 50% sulfuric acid, cool to 25-30℃, continue stirring for 1 hour, centrifuge, wash the filter cake with isopropyl acetate and water to obtain 33.16 kg of crude wet product of tert-butylamine chenodeoxycholic acid salt (28.85 kg dry) and filtrate A.

[0085] 33.06 kg (28.76 kg dry weight) of crude wet chenodeoxycholic acid tert-butylamine salt was added to 300 kg of isopropyl acetate. Stirring was started, and 20% sulfuric acid was added to adjust the pH of the aqueous layer to 2.5. 60 kg of water was added and stirred, then allowed to stand and separate. Organic layer C was mixed with 150 kg of sodium hydroxide aqueous solution (the molar ratio of sodium hydroxide to cholic acid in organic layer C was 3:1) to obtain aqueous layer A and organic layer B. Organic layer B was then mixed with 300 kg of water, and the pH was adjusted to 10 with sodium hydroxide. The mixture was separated, and the aqueous layer was concentrated to 150 kg by distillation. The pH was adjusted to 4 with 50% sulfuric acid at 26°C, precipitating a slurry-like solid. The temperature was gradually increased to an internal temperature of 42°C, then decreased to 20–30°C and stirred for 1 hour. The system was filtered and washed with tap water until neutral. The system was then heated and dried with forced air to obtain 18.10 kg of chenodeoxycholic acid with a purity of 98.0%.

[0086] Example 7

[0087] Add 250 kg of butyl acetate (2.5 times the weight of chicken bile extract) and 100 kg of water (1 times the weight of chicken bile extract) to the reactor, and add 100 kg of chicken bile extract while stirring. Heat to 60-65℃, stir, and adjust the pH to 2 to dissolve the material. Allow to stand and separate the layers. Back-extract the aqueous layer twice with 100 kg of butyl acetate (1 times the weight of chicken bile extract). Combine the three butyl acetate layers and take samples for HPLC analysis (see [link to HPLC analysis]). Figure 1 The peak area ratio in the liquid phase spectrum was: CA (24.779 min) / CDCA (50.576 min) = 27.4%.

[0088] Add 100 kg of water to the butyl acetate layer, adjust the pH to 6.1 with sodium hydroxide, add tert-butylamine dropwise at 50-65℃ until the pH reaches 9, stir for 1 hour, then adjust the pH to 7.0 with 50% sulfuric acid, cool to 25-30℃, continue stirring for 1 hour, centrifuge, wash the filter cake with butyl acetate and water to obtain 33.22 kg of crude wet product of tert-butylamine chenodeoxycholic acid salt (equivalent to 28.82 kg dry). Take a sample for HPLC analysis (see...). Figure 2 The peak area ratio in the liquid phase spectrum was: CA (24.930 min) / CDCA (48.917 min) = 6.0%.

[0089] 33.22 kg (28.82 kg dry weight) of crude wet chenodeoxycholic acid tert-butylamine salt was added to 450 kg of butyl acetate. Stirring was started, and 20% sulfuric acid was added to adjust the pH of the aqueous layer to 2.2. 90 kg of water was added and stirred, then allowed to stand and separate. The organic layer was mixed with 280 kg of sodium hydroxide aqueous solution (the molar ratio of sodium hydroxide to cholic acid in the organic layer was 6:1) to obtain aqueous layer A and organic layer B. Organic layer B was then mixed with 290 kg of water, and the pH was adjusted to 8.5 with sodium hydroxide. The mixture was separated, and the aqueous layer was concentrated to 250 kg by distillation. The pH was adjusted to 2 with 50% sulfuric acid at 28℃, precipitating a slurry solid. The temperature was gradually increased to 42℃, stirred for half an hour, then cooled to 20-30℃ and stirred for 1 hour. The system was filtered and washed with tap water until neutral. The system was then heated and dried with forced air to obtain chenodeoxycholic acid. 16.28 kg of chenodeoxycholic acid was obtained, and a sample was analyzed by HPLC (see [link to HPLC analysis]). Figure 3 The peak area ratio in the liquid phase spectrum was: CA (27.222 min) / CDCA (52.822 min) = 0.15%, with a purity of 99.15%.

[0090] Table 3 is... Figure 1 Peak table corresponding to butyl acetate extraction layer chromatogram

[0091]

[0092] Table 4 is... Figure 2 Peak table of HPLC chromatogram of chenodeoxycholic acid tert-butylamine salt

[0093]

[0094] Table 5 is... Figure 3 Peak table of HPLC chromatogram of chenodeoxycholic acid product

[0095]

[0096]

[0097] Figure 1 Table 3 shows the HPLC chromatogram and corresponding peaks of the butyl acetate extract. The peak with a retention time of 25.163 min is the elution peak of the impurity cholic acid, and the peak with a retention time of 51.755 min is the elution peak of chenodeoxycholic acid. The peak area ratio between the two is CA (25.163 min) / CDCA (51.755 min) = 27.4%. The purity of chenodeoxycholic acid is 74.6%.

[0098] Figure 2Table 4 shows the HPLC chromatogram and corresponding peaks of chenodeoxycholic acid tert-butylamine salt. The peak with a retention time of 24.930 min is the elution peak of the impurity cholic acid, and the peak with a retention time of 48.917 min is the elution peak of chenodeoxycholic acid. The ratio of the peak areas between the two is CA (24.930 min) / CDCA (48.917 min) = 6.0%, indicating a significant reduction in cholic acid impurities and an increase in the purity of chenodeoxycholic acid to 90.6%.

[0099] Figure 3 Table 5 shows the HPLC chromatogram and corresponding peaks of the finished product chenodeoxycholic acid. The peak with a retention time of 27.222 min is the elution peak of the impurity cholic acid, and the peak with a retention time of 52.822 min is the elution peak of chenodeoxycholic acid. The ratio of the peak areas between the two is CA (27.222 min) / CDCA (52.822 min) = 0.15%. The cholic acid impurity in the finished product is further reduced, and the purity of chenodeoxycholic acid is increased to 99.15%.

Claims

1. A method for preparing ammonium chenodeoxycholate, characterized in that, The process includes the following steps: Extraction of the bile paste with water (A) and an organic solvent is performed. Water (B) is added to the organic solvent layer. The pH is adjusted to 5.8-6.3 with an alkali. While stirring, an organic base is added to adjust the pH to 8-12. The pH is then adjusted to 6.0-7.2 with an acid. The mixture is filtered and washed to obtain ammonium chenodeoxycholate and filtrate A. The organic solvent is butyl acetate, ethyl acetate, or isopropyl acetate. The organic base includes tert-butylamine, sec-butylamine, or α-phenylethylamine. The base is sodium hydroxide or potassium hydroxide, and the acid is sulfuric acid or hydrochloric acid. The weight ratio of cholecystokinin, water A, organic solvent and water B is 1:0.5-3:2-5:0.5-2.

2. The method for preparing chenodeoxycholate ammonium salt according to claim 1, characterized in that, The pH of filtrate A was adjusted to 5.7-6.9, and after standing, it was separated to obtain an organic layer A1 and an aqueous layer C1. Aqueous layer C1 was back-extracted with an organic solvent to obtain an organic layer A2 and an aqueous layer C2. Organic layers A1 and A2 were combined to obtain a recovered chenodeoxycholic acid organic solvent solution. Aqueous layer C2 was acidified to precipitate a solid, and cholic acid was recovered.

3. The method for preparing an ammonium chenodeoxycholate salt according to claim 1 or 2, characterized in that, The ammonium chenodeoxycholate salt prepared by this method is used to prepare chenodeoxycholic acid, comprising the following steps: S1. Ammonium chenodeoxycholate salt is mixed with an organic solvent, acid water is added until the salt is completely dissolved, water is added until the solution is clear, the mixture is allowed to stand and separated, organic layer C is taken, alkaline water is added and stirred, and the mixture is allowed to stand and separated to obtain aqueous layer A and organic layer B. The organic solvent is butyl acetate, ethyl acetate or isopropyl acetate, and the molar ratio of alkali in alkaline water to cholic acid in organic layer C is 1-6:

1. S2, organic layer B is mixed with water, alkali is added and stirred until pH 8-12, the aqueous layer B obtained by standing and separation is acidified with acid water until crystallization is complete, and filtered to obtain chenodeoxycholic acid; the content of the chenodeoxycholic acid is ≥98%.

4. The preparation method according to claim 3, characterized in that, In S1, the weight ratio of chenodeoxycholate ammonium salt, organic solvent, and alkaline water is 1:6 to 16:0.5 to 5.

5. The preparation method according to claim 3, characterized in that, The weight ratio of chenodeoxycholate ammonium salt in S1 to water added in S2 is 1:3-12.

6. The preparation method according to claim 3, characterized in that, Chedeoxycholic acid ammonium salt is an ammonium salt formed by chedeoxycholic acid with tert-butylamine, sec-butylamine and α-phenylethylamine, respectively.

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