A method for separating a bile acid compound

Abstract

The present application relates to a method for separating bile acid compounds. The method comprises the step of fractionating and extracting a raw material liquid containing bile acid compounds and a raw material solvent using an extractant containing a biocompatible ionic liquid; wherein the bile acid compounds include stercobilin and any one or both of hyodeoxycholic acid and chenodeoxycholic acid, and the raw material solvent includes a polar hydrophobic organic solvent. The method of the present application has the advantages of good separation selectivity, large processing capacity, low energy consumption and environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of chemical separation technology, specifically relating to a method for separating bile acid compounds, particularly porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid. Background Technology

[0002] Bile acids are a class of naturally occurring organic steroidal compounds widely found in animal bodies, which have functions such as regulating lipid metabolism, promoting the absorption of fat-soluble vitamins, and lowering cholesterol. Porcine deoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), and lithocholic acid (LCA) are three bile acid compounds that coexist in porcine bile. Deoxycholic acid (DOA) is a secondary bile acid that promotes lipid and cholesterol metabolism and treats cholecystitis. Chedeoxycholic acid (CHEO) is the main bile acid found in animal bile, belonging to the primary bile acid category. It has effects such as dissolving gallstones, antibacterial and anti-inflammatory properties, and is also a precursor for the synthesis of steroidal drugs such as ursodeoxycholic acid. Lithocholic acid is the most hydrophobic secondary bile acid, produced in vivo by the 7α-dehydroxylation of CHEO by intestinal bacteria. Its low critical micelle concentration and strong hydrophobicity give it extremely high membrane permeability. In vivo, it can act as a signaling molecule to activate enzymes and regulate physiological functions. However, excessive lithocholic acid can lead to mitochondrial damage and apoptosis, and is closely related to diseases such as liver damage, colitis, and even colorectal cancer. The lithocholic acid content in porcine bile-derived DOA and CHEO products needs to be strictly controlled. Effective separation of DOA, CHEO, and lithocholic acid is of great significance for preparing monomers of these three bile acid compounds from porcine bile.

[0003] Porcine deoxycholic acid and chenodeoxycholic acid are isomers, differing only in the position of the hydroxyl group on the steroid nucleus. Lithocholic acid, on the other hand, differs in the number of hydroxyl groups on the steroid nucleus, having a hydroxyl group only on the 3rd carbon atom. The three are similar in physicochemical properties, making the separation of the three components challenging.

[0004] Currently, the common method for separating porcine deoxycholic acid and chenodeoxycholic acid from porcine bile is heavy metal ion precipitation. For example, patents CN118496295A, CN117801045A, and CN1869044A involve treating saponified and decolorized porcine bile with alkali, adding magnesium or barium salts, and adjusting the pH of the solution. This allows for the separate extraction of magnesium / barium salts of porcine deoxycholic acid and magnesium / barium salts of chenodeoxycholic acid from the alkali-treated solution and the mother liquor, respectively. Further acid treatment yields pure porcine deoxycholic acid and chenodeoxycholic acid. However, this separation method introduces heavy metals into the separation system, easily causing metal residues and environmental pollution. Patent CN116375784A discloses a method for separating porcine deoxycholic acid and chenodeoxycholic acid using an extractant containing ionic liquids. However, the ionic liquid used in this patent has poor biocompatibility, and the patent does not address the separation of lithocholic acid from porcine deoxycholic acid and / or chenodeoxycholic acid. Patent CN115716857A reports a method for refining lithocholic acid using crude lithocholic acid as raw material, through alkali treatment, dichloromethane extraction of impurities, and acid precipitation. Although this method can increase the purity of lithocholic acid to over 99%, the recovery rate is less than 90%.

[0005] Therefore, it is necessary to develop separation media that have good separation effect, low toxicity and good biocompatibility to achieve efficient separation of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides a method for separating bile acid compounds. The method uses an extractant containing a biocompatible ionic liquid for fractional extraction, which can separate any two or three substances from bile acid compounds, including porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, with good separation effect and environmentally friendly process.

[0007] The technical solution of the present invention is as follows: A method for separating bile acid compounds includes a step of fractional extraction of a feed liquid containing bile acid compounds and a feed solvent using an extractant containing a biocompatible ionic liquid, wherein the bile acid compounds include lithocholic acid and any one or two selected from porcine deoxycholic acid and chenodeoxycholic acid; and the feed solvent includes a polar hydrophobic organic solvent.

[0008] According to some embodiments of the present invention, the bile acid compounds include porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, and the method includes the following steps: (S1) The raw material liquid is subjected to a first fractional extraction using an extractant containing a biocompatible ionic liquid to obtain a first extract containing porcine deoxycholic acid and a first raffinate containing chenodeoxycholic acid and lithocholic acid. (S2) The first raffinate is subjected to a second fractional extraction with a second extractant containing a biocompatible ionic liquid to obtain a second extract containing chenodeoxycholic acid and a second raffinate containing lithocholic acid.

[0009] According to some embodiments of the present invention, the bile acid compound includes lithocholic acid and porcine deoxycholic acid or lithocholic acid and chenodeoxycholic acid, and the method includes the following steps: (T1) The raw material liquid is fractionally extracted with an extractant containing a biocompatible ionic liquid to obtain an extract containing porcine deoxycholic acid or chenodeoxycholic acid and a raffinate containing lithocholic acid.

[0010] Liquid-liquid extraction is a method of separation that utilizes the difference in partition coefficients between two immiscible phases to achieve the separation of target components. Ionic liquids are substances composed of cations and anions that are liquid at room temperature and are structurally designable separation media. Compared with traditional organic solvents, ionic liquids have advantages such as good thermal and chemical stability, high cohesive energy, and strong designability. Using ionic liquids as extractants allows for structural modulation targeting minute differences in the molecules being separated, adjusting the interaction mode and intensity with the target molecules to achieve ideal separation results. Furthermore, in the separation of natural products, biocompatibility and non-toxicity are crucial. Since porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid are all pharmaceutical molecules or precursors for drug production, when using ionic liquids as separation media, it is essential to use ionic liquids with high biocompatibility while maintaining high processing capacity and separation efficiency to minimize the impact of residual separation media in the sample on product quality.

[0011] According to some embodiments of the present invention, the biocompatible ionic liquid is composed of cations and anions.

[0012] In some embodiments, the cation is selected from one or more of the following substituted or unsubstituted cations: imidazole cation, pyridine cation, pyrrolidine cation, quaternary ammonium cation, quaternary phosphorus cation, choline cation, guanidine cation, and betaine cation.

[0013] In some embodiments, the anion is selected from one or more of the following substituted or unsubstituted anions: amino acid anions, malate anions, citrate anions, and lactate anions.

[0014] In some embodiments, when the cation and anion contain substituents, the substituents are one or more, each independently selected from amino, hydroxyl, cyano, thiocyano, C1-C4 straight-chain alkyl, C1-C4 straight-chain alkenyl, phenyl, C1-C5 straight-chain alkyl substituted with cyano, C1-C4 straight-chain alkyl substituted with thiocyano, C1-C4 straight-chain alkyl containing sulfone, or C1-C4 straight-chain alkyl containing carbonyl. In this invention, when there are multiple substituents, each substituent may be the same or different.

[0015] In some embodiments, the cation is selected from one or more of tetraalkylguanidine cations, choline cations, N-alkylpyridine cations, pyrrolidine cations, tetraalkylammonium cations, tetraalkylphosphine cations, and betaine cations; wherein the alkyl group is selected from C1-C4 straight-chain alkyl groups, C1-C5 straight-chain alkyl groups substituted with cyano groups, C1-C4 straight-chain alkyl groups substituted with thiocyano groups, C1-C4 straight-chain alkyl groups containing sulfone groups, or C1-C4 straight-chain alkyl groups containing carbonyl groups.

[0016] In some embodiments, the cation is selected from one or more of tetramethylguanidine cations, choline cations, N-ethylpyridine cations, pyrrolidine cations, tetramethylammonium cations, tetrabutylphosphine cations, and betaine cations.

[0017] In some embodiments, the anion is selected from one or more of proline ion, malate ion, citrate ion, lactate ion, proline ion, leucine ion, alanine ion, valine ion, tryptophan ion, lysine ion, and arginine ion.

[0018] In some embodiments, the biocompatible ionic liquid is selected from one or more of tetramethylguanidine proline, tetramethylguanidine malic acid, tetramethylguanidine citrate, tetramethylguanidine lactic acid, choline proline, N-ethylpyridine leucine, pyrrolidine alanine, tetramethylammonium valine, tetrabutylphosphine tryptophan, choline lysine, and betaine arginine.

[0019] In some embodiments, the biocompatible ionic liquid in the extractant has a mass percentage content of 0.01%-100%, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any value between therewith. In some preferred embodiments, the biocompatible ionic liquid in the first extractant has a mass percentage content of 0.5%-90%. In some preferred embodiments, the biocompatible ionic liquid in the first extractant has a mass percentage content of 1%-50%. In some preferred embodiments, the biocompatible ionic liquid in the first extractant has a mass percentage content of 1%-20%. In some preferred embodiments, the biocompatible ionic liquid in the first extractant has a mass percentage content of 1%-10%.

[0020] In this invention, in steps (S1) and (S2), the mass percentage of the biocompatible ionic liquid in the extractant may be the same or different.

[0021] In some embodiments, the extractant also contains a diluent. In this invention, the diluent serves to reduce the viscosity of the ionic liquid and adjust the properties of the extractant. In some embodiments, the diluent is water.

[0022] In some preferred embodiments, the extractant is a mixture of the biocompatible ionic liquid and water.

[0023] In this invention, the types of extractants used in steps (S1) and (S2) can be the same or different. In some preferred embodiments, the types of extractants are the same.

[0024] In some preferred embodiments, the extractant is selected from one or more of the following: tetramethylguanidine proline, tetramethylguanidine malic acid, tetramethylguanidine citric acid, tetramethylguanidine lactic acid, choline proline, N-ethylpyridine leucine, pyrrolidine alanine, tetramethylammonium valine, tetrabutylphosphine tryptophan, choline lysine, betaine arginine, a mixture of tetramethylguanidine proline and water, a mixture of tetramethylguanidine malic acid and water, a mixture of tetramethylguanidine citric acid and water, a mixture of tetramethylguanidine lactic acid and water, a mixture of choline proline and water, a mixture of N-ethylpyridine leucine and water, a mixture of pyrrolidine alanine and water, a mixture of tetramethylammonium valine and water, a mixture of tetrabutylphosphine tryptophan and water, a mixture of choline lysine and water, and a mixture of betaine arginine and water.

[0025] In some embodiments, the total concentration of bile acid compounds in the feed solution is 0.1 g / L-100 g / L, for example, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, or any value between them. In some embodiments, the total concentration of bile acid compounds in the feed solution is 5-80 g / L. In some embodiments, the total concentration of bile acid compounds in the feed solution is 20-50 g / L.

[0026] In some embodiments, the total weight percentage of the bile acid compounds in the raw material liquid is 90% or more. The raw materials used in this invention, porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, can be refined from porcine gallbladder through well-known steps such as saponification, acidification precipitation, and crystallization.

[0027] In some embodiments, the mass ratio of porcine deoxycholic acid to lithocholic acid in the raw material solution is (10-20):1, for example, 10:1, 12:1, 15:1, 18:1, 20:1, etc.

[0028] In some embodiments, the mass ratio of chenodeoxycholic acid to lithocholic acid in the raw material solution is (1-10):1, for example, 1:1, 2:1, 5:1, 8:1, 10:1, etc.

[0029] In some embodiments, the mass ratio of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid in the raw material liquid is (10-20):(1-10):1.

[0030] In some embodiments, the raw material solvent is selected from one or more of the following hydrophobic polar solvents: C4-C8 straight-chain or branched alcohols (such as n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctanol, isoamyl alcohol, etc.), hydrophobic polar esters (preferably C3-C8 esters, such as one or more of methyl acetate, ethyl acetate, propyl acetate or butyl acetate), hydrophobic ethers (preferably C3-C8 ethers, such as isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, etc.), and hydrophobic ketones (preferably C5-C8 ketones, such as cyclohexanone, 2-pentanone, 3-pentanone, acetophenone, etc.).

[0031] In this invention, the fractional extraction includes an extraction section and a washing section. The extractant enters the fractional extraction system from the first stage of the extraction section, the feed liquid enters the fractional extraction system from the last stage of the extraction section, and the detergent enters the fractional extraction system from the first stage of the washing section. The feed liquid and the detergent are combined and enter the extraction section together in the last stage of the extraction section, and the extraction phase and the washing phase undergo multi-stage countercurrent contact.

[0032] According to some embodiments of the present invention, the extraction stage of the fractional extraction is 2-20 stages, for example, 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, 12 stages, 14 stages, 16 stages, 18 stages, 20 stages, etc., preferably 5-10 stages; the washing stage of the fractional extraction is 2-20 stages, for example, 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, 12 stages, 14 stages, 16 stages, 18 stages, 20 stages, etc., preferably 2-10 stages.

[0033] In some embodiments, in step (S1), the extraction stages of the first fractionation extraction are 2-20 stages, for example, 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, 12 stages, 14 stages, 16 stages, 18 stages, 20 stages, etc. In some embodiments, the extraction stages of the first fractionation extraction are 5-10 stages.

[0034] In some embodiments, in step (S1), the washing stage of the first fractionation extraction has 2-20 stages, such as 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, 12 stages, 14 stages, 16 stages, 18 stages, 20 stages, etc. In some embodiments, the washing stage of the first fractionation extraction has 5-10 stages.

[0035] In some embodiments, in step (S2), the extraction stages of the second fractionation extraction are 2-20 stages, for example, 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, 12 stages, 14 stages, 16 stages, 18 stages, 20 stages, etc. In some embodiments, the extraction stages of the second fractionation extraction are 6-10 stages.

[0036] In some embodiments, in step (S2), the washing stage of the second fractionation extraction has 2-10 stages, for example, 2 stages, 3 stages, 4 stages, 5 stages, 6 stages, 7 stages, 8 stages, 9 stages, 10 stages, etc. In some embodiments, the washing stage of the second fractionation extraction has 2-5 stages.

[0037] According to some embodiments of the present invention, in the fractional extraction, the flow ratio of the extractant to the feed liquid is (0.1-20):1, for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.8:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0038] According to some embodiments of the present invention, in the fractionation extraction, the flow ratio of detergent to feed liquid is (0.1-10):1, for example, 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0039] According to some embodiments of the present invention, in the fractional extraction, the flow ratio of extractant, detergent, and feed liquid is (0.1-20):(0.1-10):1. In some embodiments, in the fractional extraction, the flow ratio of extractant, detergent, and feed liquid is (2-8):(1-4):1.

[0040] In some embodiments, in step (S1), the flow ratio of extractant to feed liquid in the first fractionation extraction is (0.1-10):1, for example, 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0041] In some embodiments, in step (S1), the flow ratio of detergent to feed liquid in the first fractionation extraction is (0.1-10):1, for example, 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0042] In some embodiments, in step (S1), the flow ratio of extractant, detergent and feed liquid in the first fractionation extraction is (3-5):(2-4):1.

[0043] In some embodiments, in step (S2), the flow ratio of extractant to feed liquid in the second fractionation extraction is (0.5-20):1, for example, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0044] In some embodiments, in step (S2), during the second fractionation extraction, the flow ratio of detergent to feed liquid is (0.1-10):1, for example, 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0045] In some embodiments, in step (S2), the flow ratio of extractant, detergent and feed liquid in the second fractionation extraction is (2-8):(1-3):1.

[0046] In this invention, unless otherwise specified, the term "flow ratio" refers to the volume ratio.

[0047] In some embodiments, the detergent used for the fractional extraction is selected from one or more of the following hydrophobic polar solvents: C4-C8 straight-chain or branched alcohols (such as n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctanol, isoamyl alcohol, etc.), hydrophobic polar esters (preferably C3-C8 esters, such as one or more of methyl acetate, ethyl acetate, propyl acetate or butyl acetate), hydrophobic ethers (preferably C3-C8 ethers, such as isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, etc.), and hydrophobic ketones (preferably C5-C8 ketones, such as cyclohexanone, 2-pentanone, 3-pentanone, acetophenone, etc.).

[0048] In some preferred embodiments, the detergent used in the fractional extraction is of the same type as the raw material solvent, which has good solubility for cholic acid and can form a liquid-liquid two-phase system with low miscibility with the extractant.

[0049] According to some embodiments of the present invention, the operating temperature of the fractional extraction is 20°C-70°C, for example, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, or any value between them. According to some embodiments of the present invention, the operating temperature of the fractional extraction is 30°C-50°C.

[0050] In some embodiments, in step (S1), the operating temperature of the first fractionation extraction is 30°C-50°C. In some embodiments, in step (S2), the operating temperature of the second fractionation extraction is 20°C-70°C, for example, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, or any value between them. In some embodiments, the operating temperature of the second fractionation extraction is 30°C-50°C. If the extraction temperature is too low, the viscosity of the extractant is high, the mass transfer rate is reduced, the throughput is small, which is not conducive to production operation; if the temperature is too high, the solvent evaporates severely, which will reduce the distribution ratio and selectivity of the fractionation extraction.

[0051] According to some embodiments of the present invention, the method further includes: back-extracting the extract after fractional extraction, concentrating, washing with water and drying the obtained back-extracted phase to obtain porcine deoxycholic acid and / or chenodeoxycholic acid; and / or The raffinate after fractional extraction was concentrated and dried to obtain lithocholic acid.

[0052] According to some embodiments of the present invention, the method further includes: back-extracting the extract containing porcine deoxycholic acid or chenodeoxycholic acid, concentrating, washing with water and drying the obtained back-extracted phase to obtain porcine deoxycholic acid or chenodeoxycholic acid; and / or The raffinate containing lithocholic acid was concentrated and dried to obtain lithocholic acid.

[0053] In some embodiments, the bile acid compounds include porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, and the method further includes: (S3) The first extract containing porcine deoxycholic acid is back-extracted, and the resulting back-extracted phase is concentrated, washed with water, and dried to obtain porcine deoxycholic acid; and / or (S4) The second extract containing chenodeoxycholic acid is back-extracted, and the resulting back-extracted phase is concentrated, washed with water, and dried to obtain chenodeoxycholic acid; and / or (S5) In some embodiments, the method further includes: concentrating and drying the second raffinate containing lithocholic acid to obtain lithocholic acid.

[0054] In some embodiments, the bile acid compounds include porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. The method includes fractional extraction in a first and second fractional extraction tower connected in series. Specifically, the extract enriched with porcine deoxycholic acid flows out from the first stage of the washing section of the first fractional extraction tower. The extract is collected and subjected to back-extraction, vacuum concentration, water washing, and drying to obtain porcine deoxycholic acid. The extract enriched with chenodeoxycholic acid flows out from the first stage of the washing section of the second fractional extraction tower. This extract is then back-extracted, vacuum concentrated, water washing, and dried to obtain chenodeoxycholic acid. The raffinate enriched with lithocholic acid is collected from the first stage of the extraction section of the second fractional extraction tower and subjected to vacuum concentration, water washing, and drying to obtain lithocholic acid. The absolute purity of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid obtained by the separation method of this invention can reach over 95%, over 95%, and over 97%.

[0055] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention uses an extractant containing a biocompatible ionic liquid for fractional extraction, which has a good selective separation effect on any two- or three-component system of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid with very similar molecular structures. It can obtain three high-purity bile acid products in one step. The process is simple, easy to scale up and has low energy consumption.

[0056] 2. The ionic liquid phase used in this invention has better biocompatibility than conventional ionic liquids, reducing the impact on human health and the environment, and is more environmentally friendly.

[0057] 3. The method of the present invention can achieve a high throughput even when the amount of ionic liquid used is low, thereby reducing the viscosity of the extractant and production costs. Attached Figure Description

[0058] Figure 1 This is a process flow diagram of the separation of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid using a dual-tower series fractional distillation extraction method in an embodiment of the present invention.

[0059] Figure 2 The results are HPLC test results of the raw materials and the HDCA, CDCA and LCA products obtained after separation and purification in Example 1. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0061] This invention provides a method for efficiently separating any two or three components of a mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, characterized by good biocompatibility, high throughput, simple process, and high efficiency. This method employs fractional extraction using an extractant containing a biocompatible ionic liquid. Compared to conventional ionic liquids, the biocompatible ionic liquid selected in this invention possesses advantages such as high biodegradability, safety, and environmental friendliness. Furthermore, it can still form intermolecular forces such as hydrogen bonds with the solute. The interaction mode and intensity between the ionic liquid and the separated components can be adjusted by designing the anionic and cation structures of the ionic liquid, achieving molecular recognition and separation.

[0062] As one specific implementation method, such as Figure 1 As shown, the method for separating bile acid compounds provided by this invention is a method for separating porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid. The method is carried out in a series of fractionation extraction columns 1 and 2, and specifically includes the following steps: (1) A mixture containing porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid is prepared with a raw material solvent to form a raw material solution. In fractionation extraction tower 1, a binary mixed solvent composed of a biocompatible ionic liquid and a diluent is used as the extractant, and a solvent identical to the raw material solvent is used as the washing agent to perform the first fractionation extraction to obtain a first extract phase and a first raffinate phase. In fractionation extraction tower 2, the first raffinate phase is used as the raw material solution, a binary mixed solvent composed of a biocompatible ionic liquid and a diluent is used as the extractant, and a solvent identical to the raw material solvent is used as the washing agent to perform the second fractionation extraction to obtain a second extract phase and a second raffinate phase. The fractionation extraction is divided into an extraction section and a washing section. The extractant enters the fractionation extraction system from the first stage of the extraction section, the raw material solution enters the fractionation extraction system from the last stage of the extraction section, and the washing agent enters the fractionation extraction system from the first stage of the washing section. The raw material solution is combined with the washing solution in the last stage of the washing section and enters the extraction section together. The extract phase and the washing phase are subjected to multi-stage countercurrent extraction. (2) The first extract phase obtained in fractionation extraction tower 1 is back-extracted, vacuum concentrated, washed with water and dried to obtain chenodeoxycholic acid; the second extract phase obtained in fractionation extraction tower 2 is back-extracted with the same solvent as the raw material solvent and detergent, and the solvent-enriched phase is vacuum concentrated, washed with water and dried to obtain chenodeoxycholic acid; the raffinate phase obtained in fractionation extraction tower 2 is vacuum concentrated, washed with water and dried to obtain lithocholic acid.

[0063] In this invention, the biocompatible ionic liquid enriched phase can be recycled after vacuum concentration.

[0064] The molecular structures of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid are very similar. Porcine deoxycholic acid and chenodeoxycholic acid are isomers, differing only in the position of the hydroxyl group attached to the steroid nucleus, while lithocholic acid has only one hydroxyl group at the C-3 position. They also differ from the other two substances in the number of hydrogen bonding sites. Based on the structural characteristics and differences of the three bile acid molecules, ionic liquids with strong hydrogen bonding basicity are used as extractants. This enhances the extractant's ability to recognize bile acid molecules, increasing throughput and selectivity, while also reducing the amount of ionic liquid used in the extractant, thus controlling production costs. The ionic liquid-containing extractant used in this invention enables high-throughput selective separation of the three-component bile acid system at a relatively low ionic liquid concentration.

[0065] For example, for a raw material containing porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, using ethyl acetate as the raw material solvent and a 1% (w / w) aqueous solution of tetramethylguanidinyl glycine as the extractant, when performing single-stage extraction at 30°C, the partition coefficients of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid are 2.21, 0.86, and 0.007, respectively. The selectivity coefficients are porcine deoxycholic acid / chenodeoxycholic acid = 2.6, porcine deoxycholic acid / lithocholic acid = 315.7, and chenodeoxycholic acid / lithocholic acid = 121.4. It can be seen that adding only 1% (w / w) of tetramethylguanidinyl glycine can maintain good separation selectivity for the molecules in the three-component system. The preparation of three high-purity bile acid products can be achieved through multi-stage fractionation extraction.

[0066] The present invention will be further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0067] Unless otherwise specified, all reagents used in the following experiments of this invention are commercially available products or reagents prepared according to conventional methods. Unless otherwise specified, all methods used in the experiments are conventional experimental methods. Unless otherwise specified, all instruments used in the experiments are commercially available.

[0068] In the following examples, high-performance liquid chromatography (HPLC) was used to analyze the concentrations of three bile acid molecules. The specific HPLC analysis conditions were as follows: Waters Sunfire C18 column (4.6 mm ID × 250 mm, particle size 5 μm), column temperature 30°C, mobile phase: methanol:acetonitrile:0.1% formic acid aqueous solution = 50:30:20 (v / v / v), flow rate 0.9 mL / min. Because the ultraviolet response of bile acid molecules is weak, a general-purpose electro-fogging detector (Thermo Fisher Scientific) was used.

[0069] In the following embodiments, the yield and purity are calculated as follows: Yield = (Mass of bile acids in product / Mass of bile acids in feed) × 100%; Purity = Mass of bile acids in the product / Total mass of the product × 100%.

[0070] Example 1 A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 20 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (tetramethylguanidine-proline mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 99.63%, and the yield was 98.47%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0071] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A tetramethylguanidine-proline-water mixed solvent (tetramethylguanidine-proline ionic liquid mass fraction of 1%) is used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.70%, and the yield is 98.57%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 99.63%, and the yield was 98.47%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled. The HPLC test results of the raw materials used in this example and the HDCA, CDCA, and LCA products obtained after separation and purification are as follows: Figure 2 As shown.

[0072] Example 2

[0073] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 20 g / L. In the first fractionation extraction column, a tetramethylguanidine malic acid-water mixed solvent (tetramethylguanidine malic acid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.01%, and the yield was 97.30%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0074] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A tetramethylguanidine malic acid-water mixed solvent is used as the extractant (tetramethylguanidine malic acid ionic liquid mass fraction is 1%), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.10%, and the yield is 98.32%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.14%, and the yield was 98.40%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0075] Example 3

[0076] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 20 g / L. In the first fractionation extraction column, a tetramethylguanidine citric acid-water mixed solvent (tetramethylguanidine citric acid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.23%, and the yield was 97.58%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0077] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A tetramethylguanidine citric acid-water mixed solvent is used as the extractant (tetramethylguanidine citric acid ionic liquid mass fraction is 1%), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.06%, and the yield is 98.49%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.37%, and the yield was 98.01%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0078] Example 4

[0079] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 20 g / L. In the first fractionation extraction column, a tetramethylguanidine lactic acid-water mixed solvent (tetramethylguanidine lactic acid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.06%, and the yield was 97.03%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and can be recycled.

[0080] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A tetramethylguanidine lactic acid-water mixed solvent is used as the extractant (tetramethylguanidine lactic acid ionic liquid mass fraction is 1%), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the fractionation extraction is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.02%, and the yield is 98.59%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.19%, and the yield was 98.18%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0081] Example 5

[0082] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a choline-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.06%, and the yield was 98.50%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and can be recycled.

[0083] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A choline-proline-water mixed solvent (1% ionic liquid by mass) is used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.15%, and the yield is 98.13%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.64%, and the yield was 98.89%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0084] Example 6

[0085] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, an N-ethylpyridine-leucine-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.90%, and the yield was 98.96%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0086] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. N-ethylpyridine-leucine-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.37%, and the yield is 98.10%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The back-extracted ionic liquid-aqueous phase was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0087] Example 7

[0088] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a pyrrolidine alanine-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.61%, and the yield was 98.06%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0089] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A pyrrolidine alanine-water mixed solvent (1% ionic liquid by mass) is used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.46%, and the yield is 97.38%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.38%, and the yield was 98.09%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0090] Example 8

[0091] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylammonium valine-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.64%, and the yield was 99.10%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0092] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylammonium valine-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.57%, and the yield is 97.07%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.16%, and the yield was 98.32%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0093] Example 9

[0094] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, tetrabutylphosphine tryptophan-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.39%, and the yield was 98.91%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0095] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetrabutylphosphine-tryptophan-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.60%, and the yield is 97.02%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.78%, and the yield was 98.10%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0096] Example 10

[0097] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a choline-lysine-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.47%, and the yield was 98.85%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0098] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A choline-lysine-water mixed solvent (1% ionic liquid by mass) is used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.72%, and the yield is 96.93%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.08%, and the yield was 98.56%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0099] Example 11

[0100] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a betaine-arginine-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.08%, and the yield was 98.87%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0101] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A betaine-arginine-water mixed solvent (1% ionic liquid by mass) is used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.54%, and the yield is 97.09%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.53%, and the yield was 98.67%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0102] Example 12

[0103] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 100 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.02%, and the yield was 98.06%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and can be recycled.

[0104] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 99.12%, and the yield is 97.15%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.03%, and the yield was 98.01%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0105] Example 13

[0106] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 3:2:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.01%, and the yield was 98.02%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0107] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 2:1:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.90%, and the yield is 97.56%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.04%, and the yield was 98.40%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0108] Example 14

[0109] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 50°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 50°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.03%, and the yield was 98.46%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0110] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 50°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.21%, and the yield is 97.92%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 50°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.03%, and the yield was 98.59%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0111] Example 15

[0112] The raw materials, a mixture of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid and lithocholic acid 70:25:5), were dissolved in ethyl acetate to prepare a raw material solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent was used as the extractant (1% ionic liquid by mass), and ethyl acetate was used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, and the fractionation extraction consisted of an extraction section and a washing section (5 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in deoxycholic acid (DOCA) flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain deoxycholic acid (DOCA). The purity of DOCA in the product was 97.06%, and the yield was 98.52%. The back-extracted ionic liquid-aqueous phase is concentrated to increase the concentration of the ionic liquid, and can be recycled and reused.

[0113] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (6 stages in the extraction section and 3 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.47%, and the yield is 98.20%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.39%, and the yield was 98.03%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0114] Example 16

[0115] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 10%) was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.15%, and the yield was 98.66%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0116] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (10% ionic liquid mass fraction), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.79%, and the yield is 98.60%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.46%, and the yield was 98.50%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0117] Example 17

[0118] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in n-butanol to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and n-butanol as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total for both sections). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected and diluted 10-fold. It was then back-extracted three times at 30°C with 1 / 10 volume of n-butanol. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.02%, and the yield was 98.05%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, allowing for recycling.

[0119] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and n-butanol is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.16%, and the yield is 98.15%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of n-butanol at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 97.16%, and the yield was 98.32%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0120] Example 18

[0121] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in isopropyl ether to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and isopropyl ether as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of isopropyl ether. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.54%, and the yield was 98.31%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid and could be recycled.

[0122] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and isopropyl ether is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.73%, and the yield is 98.09%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of isopropyl ether. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.03%, and the yield was 98.42%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0123] Example 19

[0124] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in cyclohexanone to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a tetramethylguanidine-proline-water mixed solvent (ionic liquid mass fraction 1%) was used as the extractant, and cyclohexanone as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected and diluted 10-fold. It was then back-extracted three times with 1 / 10 volume of cyclohexanone at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 97.80%, and the yield was 98.10%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, allowing for recycling.

[0125] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidine-proline-water mixed solvent is used as the extractant (1% ionic liquid by mass), and cyclohexanone is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.52%, and the yield is 98.13%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of cyclohexanone at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.14%, and the yield was 98.06%. The ionic liquid-aqueous phase after back-extraction was concentrated to a concentration consistent with the extractant from the first extraction stage and then recycled.

[0126] Example 20

[0127] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, ratio 70:25:5) was dissolved in cyclohexanone to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, tetramethylguanidine-proline was used as the extractant, and ethyl acetate as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. The first stage of the washing section discharged an extract rich in porcine deoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 98.22%, and the yield was 98.50%. The ionic liquid after back-extraction can be recycled.

[0128] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Tetramethylguanidineproline is used as the extractant, and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and the extraction process is divided into an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 98.14%, and the yield is 98.06%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times at 30°C with 1 / 10 volume of ethyl acetate. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 98.27%, and the yield was 98.11%. The ionic liquid after back-extraction can be recycled.

[0129] Comparative Example 1 A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, water was used as the extractant and ethyl acetate as the washing agent, with a flow ratio of extractant, washing agent, and feed solution of 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 85.01%, and the yield was 50.69%.

[0130] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. Water is used as the extractant, and ethyl acetate as the washing agent, with a flow ratio of extractant, washing agent, and feed liquid of 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, and consists of an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 87.19%, and the yield is 56.04%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 80.64%, and the yield was 52.39%.

[0131] Comparative Example 2

[0132] A mixture of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid (total bile acid mass percentage 99%, mass ratio of porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid 70:25:5) was dissolved in ethyl acetate to prepare a feed solution with a total concentration of 50 g / L. In the first fractionation extraction column, a mixed solvent of 1-ethyl-3-methylimidazolium tetrafluoroborate-water was used as the extractant (ionic liquid mass fraction 1%), and ethyl acetate was used as the washing agent. The flow ratio of extractant, washing agent, and feed solution was 5:4:1 (v / v / v). Fractionation extraction was carried out at 30°C, consisting of an extraction section and a washing section (10 stages in total). The extractant entered the fractionation extraction system from the first stage of the extraction section, the feed solution entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. An extract rich in porcine deoxycholic acid flowed out from the first stage of the washing section. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain porcine deoxycholic acid. The purity of porcine deoxycholic acid in the product was 88.43%, and the yield was 90.06%. The ionic liquid-aqueous phase after back-extraction was concentrated to increase the concentration of the ionic liquid, and could be recycled.

[0133] The raffinate from the first stage of the extraction section of the first fractionation extraction tower is pumped into the second fractionation extraction tower as feed for the separation of the remaining two components. A mixed solvent of 1-ethyl-3-methylimidazolium tetrafluoroborate and water is used as the extractant (1% ionic liquid by mass), and ethyl acetate is used as the washing agent. The flow ratio of extractant, washing agent, and feed liquid is 8:3:1 (v / v / v). Fractional extraction is carried out at 30°C, consisting of an extraction section and a washing section (10 stages in the extraction section and 2 stages in the washing section). The extractant enters the fractionation extraction system from the first stage of the extraction section, the feed liquid enters from the last stage of the extraction section, and the washing agent enters from the first stage of the washing section. The raffinate from the first stage of the extraction section is concentrated under vacuum, washed with water, and dried to obtain lithocholic acid. The purity of lithocholic acid in the product is 91.26%, and the yield is 89.06%. The first stage of the washing section yields an extract rich in chenodeoxycholic acid. The extract was collected, diluted 10-fold, and back-extracted three times with 1 / 10 volume of ethyl acetate at 30°C. The organic phases were combined, concentrated under vacuum, washed with water, and dried to obtain chenodeoxycholic acid. The purity of chenodeoxycholic acid in the product was 90.64%, and the yield was 87.09%.

[0134] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A method for separating bile acid compounds, comprising the step of fractional extraction of a feed liquid containing bile acid compounds and a feed solvent using an extractant containing a biocompatible ionic liquid; in, The bile acid compounds include lithocholic acid and any one or two selected from porcine deoxycholic acid and chenodeoxycholic acid, and the raw material solvent includes a polar hydrophobic organic solvent.

2. The method according to claim 1, characterized in that, The bile acid compounds include porcine deoxycholic acid, chenodeoxycholic acid, and lithocholic acid, and the method includes the following steps: (S1) The raw material liquid is subjected to a first fractional extraction using an extractant containing a biocompatible ionic liquid to obtain a first extract containing porcine deoxycholic acid and a first raffinate containing chenodeoxycholic acid and lithocholic acid. (S2) The first raffinate is subjected to a second fractional extraction with an extractant containing a biocompatible ionic liquid to obtain a second extract containing chenodeoxycholic acid and a second raffinate containing lithocholic acid. Alternatively, the bile acid compounds include lithocholic acid and deoxycholic acid, or lithocholic acid and chenodeoxycholic acid, and the method includes the following steps: (T1) The raw material liquid is fractionally extracted with an extractant containing a biocompatible ionic liquid to obtain an extract containing porcine deoxycholic acid or chenodeoxycholic acid and a raffinate containing lithocholic acid.

3. The method according to claim 1 or 2, characterized in that, The biocompatible ionic liquid is composed of cations and anions, wherein the cations are selected from one or more of the following substituted or unsubstituted cations: imidazole cations, pyridine cations, pyrrolidine cations, quaternary ammonium cations, quaternary phosphorus cations, choline cations, guanidine cations, and betaine cations; and the anions are selected from one or more of the following substituted or unsubstituted anions: amino acid anions, malate anions, citrate anions, and lactate anions; when the cations and anions contain substituents, the substituents are one or more and each independently selected from amino, hydroxyl, cyano, thiocyano, C1-C4 straight-chain alkyl, C1-C4 straight-chain alkenyl, phenyl, C1-C5 straight-chain alkyl substituted with cyano, C1-C4 straight-chain alkyl substituted with thiocyano, C1-C4 straight-chain alkyl containing sulfone, or C1-C4 straight-chain alkyl containing carbonyl. Preferably, the cation is selected from one or more of tetraalkylguanidine cations, choline cations, N-alkylpyridine cations, pyrrolidine cations, tetraalkylammonium cations, tetraalkylphosphine cations, and betaine cations; wherein the alkyl group is selected from C1-C4 straight-chain alkyl groups, C1-C5 straight-chain alkyl groups substituted with cyano groups, C1-C4 straight-chain alkyl groups substituted with thiocyano groups, C1-C4 straight-chain alkyl groups containing sulfone groups, or C1-C4 straight-chain alkyl groups containing carbonyl groups; more preferably, the cation is selected from one or more of tetramethylguanidine cations, choline cations, N-ethylpyridine cations, pyrrolidine cations, tetramethylammonium cations, tetrabutylphosphine cations, and betaine cations; Preferably, the anion is selected from one or more of proline ion, malate ion, citrate ion, lactate ion, proline ion, leucine ion, alanine ion, valine ion, tryptophan ion, lysine ion, and arginine ion. Preferably, the biocompatible ionic liquid is selected from one or more of tetramethylguanidine proline, tetramethylguanidine malic acid, tetramethylguanidine citric acid, tetramethylguanidine lactic acid, choline proline, N-ethylpyridine leucine, pyrrolidine alanine, tetramethylammonium valine, tetrabutylphosphine tryptophan, choline lysine, and betaine arginine.

4. The method according to any one of claims 1-3, characterized in that, The biocompatible ionic liquid has a mass percentage content of 0.01%-100% in the extractant, preferably 0.5%-90%, more preferably 1%-50%, and even more preferably 1%-10%; and / or In steps (S1) and (S2), the mass percentage of the biocompatible ionic liquid in the extractant may be the same or different; and / or In steps (S1) and (S2), the types of biocompatible ionic liquids in the extractant may be the same or different, preferably the same.

5. The method according to any one of claims 1-4, characterized in that, The extractant also contains a diluent, preferably water; more preferably, the extractant is a mixture of the biocompatible ionic liquid and water; and / or The extractant is selected from one or more of the following: tetramethylguanidine proline, tetramethylguanidine malic acid, tetramethylguanidine citric acid, tetramethylguanidine lactic acid, choline proline, N-ethylpyridine leucine, pyrrolidine alanine, tetramethylammonium valine, tetrabutylphosphine tryptophan, choline lysine, betaine arginine, a mixture of tetramethylguanidine proline and water, a mixture of tetramethylguanidine malic acid and water, a mixture of tetramethylguanidine citric acid and water, a mixture of tetramethylguanidine lactic acid and water, a mixture of choline proline and water, a mixture of N-ethylpyridine leucine and water, a mixture of pyrrolidine alanine and water, a mixture of tetramethylammonium valine and water, a mixture of tetrabutylphosphine tryptophan and water, a mixture of choline lysine and water, and a mixture of betaine arginine and water.

6. The method according to any one of claims 1-5, characterized in that, The raw material solvent is selected from one or more of C4-C8 straight-chain or branched alcohols, C3-C8 esters, C3-C8 ethers, and C5-C8 ketones, preferably from one or more of n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctanol, isoamyl alcohol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, cyclohexanone, 2-pentanone, 3-pentanone, and acetophenone, more preferably from one or more of ethyl acetate, n-butanol, isopropyl ether, and cyclohexanone; and / or In the raw material solution, the mass ratio of porcine deoxycholic acid to lithocholic acid is (10-20):1; and / or In the raw material solution, the mass ratio of chenodeoxycholic acid to lithocholic acid is (1-10):1; and / or The total concentration of bile acid compounds in the raw material solution is 0.1 g / L-100 g / L, preferably 5-80 g / L, and more preferably 20-50 g / L.

7. The method according to any one of claims 1-6, characterized in that, The fractional extraction has 2-20 extraction stages, preferably 5-10 stages; the fractional extraction has 2-20 washing stages, preferably 2-10 stages. Preferably, in step (S1), the extraction stage of the first fractionation extraction has 2-20 stages, more preferably 5-10 stages; the washing stage of the first fractionation extraction has 2-20 stages, more preferably 5-10 stages. Preferably, in step (S2), the extraction stage of the second fractionation extraction has 2-20 stages, more preferably 6-10 stages; the washing stage of the second fractionation extraction has 2-10 stages, more preferably 2-5 stages.

8. The method according to any one of claims 1-7, characterized in that, The extraction temperature for the fractional extraction is 20℃-70℃, preferably 30℃-50℃; and / or In the fractional extraction, the flow ratio of extractant, detergent and feed liquid is (0.1-20):(0.1-10):1, preferably (2-8):(1-4):1; Preferably, in step (S1), the flow ratio of extractant, detergent and feed liquid in the first fractionation extraction is (0.1-10):(0.1-10):1, more preferably (3-5):(2-4):1; Preferably, in step (S2), the flow ratio of extractant, detergent and feed liquid in the second fractionation extraction is (0.5-20):(0.1-10):1, more preferably (2-8):(1-3):

1.

9. The method according to any one of claims 1-8, characterized in that, The detergent used in the fractionation extraction is selected from one or more of C4-C8 straight-chain or branched-chain alcohols, C3-C8 esters, C3-C8 ethers, and C5-C8 ketones, preferably from one or more of n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctanol, isoamyl alcohol, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, isopropyl ether, methyl tert-butyl ether, cyclopentyl methyl ether, cyclohexanone, 2-pentanone, 3-pentanone, and acetophenone, more preferably from one or more of ethyl acetate, n-butanol, isopropyl ether, and cyclohexanone; Preferably, the type of detergent is the same as the type of raw material solvent.

10. The method according to any one of claims 1-9, characterized in that, The method further includes: The extract after fractional extraction is back-extracted, and the resulting back-extracted phase is concentrated, washed with water, and dried to obtain porcine deoxycholic acid and / or chenodeoxycholic acid; and / or The raffinate after fractional extraction was concentrated and dried to obtain lithocholic acid.

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