A method for separating bile acid and deoxycholic acid using a biocompatible two-phase system

Abstract

The invention discloses a method for extracting and separating bile acid and deoxycholic acid in a liquid-liquid two-phase system with good biocompatibility, low toxicity and high efficiency, the method comprising fractionating and extracting a raw material liquid containing bile acid and deoxycholic acid with an extractant containing an amino acid ionic liquid, and obtaining high-purity bile acid and deoxycholic acid from the extract and the raffinate, respectively. The amino acid ionic liquid extractant used in the present invention has advantages such as better degradability, higher biocompatibility and higher separation efficiency than conventional ionic liquids and organic solvent extractants, has the characteristics of large processing capacity, simple process, low toxicity and environmental protection, low cost, etc., and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical separation, and specifically relates to a method for extracting and separating bile acid and deoxycholic acid, which uses an amino acid ionic liquid with high biocompatibility as a medium and has the advantages of biocompatibility, low ionic liquid dosage and high processing capacity. Background Art

[0002] Cholic acid and deoxycholic acid are steroid derivatives with medical value in regulating cholesterol metabolism, dissolving lipids, and treating symptoms such as cholecystitis and indigestion. They can also be used in biochemical research such as cell lysis and nuclear protein extraction, and have broad application prospects. In nature and chemical production, cholic acid and deoxycholic acid often exist in the form of a mixture. Their molecular structures are very similar, differing only by a hydroxyl group at the 7th carbon atom, so it is challenging to achieve efficient separation of the two.

[0003] Ionic liquids are substances composed of anions and cations that are liquid at or near room temperature. They have attracted attention in the field of separation as a new type of "green" and efficient separation medium. Compared with traditional organic solvents as extractants, ionic liquids as extractants have some unique properties, such as good thermal and chemical stability, high cohesive energy and easy formation of a two-phase system, multiple intermolecular interactions with solutes and high solubility, and strong designability of structural properties. They can adjust the mode and intensity of interaction between ionic liquids and solutes by designing the anion and cation structure of ionic liquids to target the slight differences in the structure and properties of homologous compounds, thereby achieving a specific separation effect.

[0004] Although ionic liquids have been considered "green" solvents, more and more studies have shown that the potential toxicity of ionic liquids cannot be ignored. On the one hand, the acute toxicity of ionic liquids is closely related to their structure. On the other hand, the toxicity of ionic liquids is also reflected in the fact that they are not easily degraded in the environment and accumulate along the food chain, thus affecting the human body. In addition, compared with conventional solvents, ionic liquids are expensive and have higher costs in industrial production. In summary, the amount of ionic liquids used in fractionation extraction needs to be strictly controlled.

[0005] Cholic acid and deoxycholic acid are preparations with medical value. In the technical route of separating cholic acid and deoxycholic acid using ionic liquid as a medium, it is necessary to comprehensively consider the toxicity and cost of ionic liquids, and to use ionic liquids with good biocompatibility, large processing capacity and high separation efficiency as much as possible for the separation of this system. It can be seen that there is still a lot of room for improvement in the method of separating cholic acid and deoxycholic acid. Summary of the invention

[0006] Based on the deficiencies in the prior art, the present invention proposes a method for separating high-purity cholic acid and deoxycholic acid from a mixture of cholic acid and deoxycholic acid by multi-stage fractionation extraction using an extractant containing an amino acid ionic liquid. The amino acid ionic liquid used in the present invention has better degradability and biocompatibility than conventional ionic liquids, has the characteristics of low toxicity, low dosage and high processing capacity, and is suitable for industrial production.

[0007] In order to achieve the above object, the present invention provides a method for separating cholic acid and deoxycholic acid, which comprises fractionating and extracting a raw material solution containing cholic acid and deoxycholic acid using an extractant containing an amino acid ionic liquid.

[0008] In some embodiments, the ionic liquid consists of cations and amino acid anions.

[0009] In some embodiments, the cation is selected from at least one of an imidazolium cation, a quinolinium cation, a pyridinium cation, an isoquinolinium cation, a benzimidazolium cation, a quaternary ammonium cation, a quaternary phosphonium cation, a triazolium cation, a betaine cation, a guanidinium cation, a morphazolium cation, an oxazolidine cation, a choline cation, or a sulfonium salt cation, and the cation is optionally substituted with one or more substituents.

[0010] In some embodiments, the substituent is selected from amino, hydroxyl, alkyl, olefin, phenyl, cyano, thiocyano, sulfone or carbonyl. In some preferred embodiments, the alkyl is a C1-C4 straight-chain alkyl, the olefin is a C1-C4 straight-chain olefin, the cyano is a C1-C4 straight-chain cyano, the thiocyano is a C1-C4 straight-chain thiocyano, the sulfone is a C1-C4 straight-chain sulfone, and the carbonyl is a C1-C4 straight-chain carbonyl. The number of substituents can be monosubstituted, disubstituted or trisubstituted, and the substituents at different sites can be the same or different when multiple substitutions are used.

[0011] In some embodiments, the amino acid anion is selected from carboxylic acid anions containing amino groups, secondary amino groups and / or tertiary amino groups in the molecule. In some embodiments, the amino acid anion is selected from at least one of neutral amino acid anions, basic amino acid anions or acidic amino acid anions. In some specific embodiments, the amino acid anions include but are not limited to neutral amino acids such as glycine anion, proline anion, alanine anion, leucine anion, valine anion, phenylalanine anion, tryptophan anion, asparagine anion, glutamine anion, lysine anion, arginine anion, and other basic amino acids, cysteine ​​anion, methionine anion, and other sulfur-containing amino acids and glutamate anion. At least one of the acidic amino acids.

[0012] In some embodiments, the amino acid anion is optionally substituted with one or more substituents.

[0013] In some embodiments, the substituent is selected from amino, hydroxyl, alkyl, olefin, phenyl, cyano, thiocyano, sulfone or carbonyl. In some preferred embodiments, the alkyl is a C1-C4 straight-chain alkyl, the olefin is a C1-C4 straight-chain olefin, the cyano is a C1-C4 straight-chain cyano, the thiocyano is a C1-C4 straight-chain thiocyano, the sulfone is a C1-C4 straight-chain sulfone, and the carbonyl is a C1-C4 straight-chain carbonyl. The number of substituents can be monosubstituted, disubstituted or trisubstituted, and the substituents at different sites can be the same or different when multiple substitutions are used.

[0014] In some specific embodiments, the ionic liquid is selected from at least one of 1-ethyl-3-methylimidazole amino acid ionic liquid, choline amino acid ionic liquid, tert-butylammonium amino acid ionic liquid, 1-ethyl-1-methylpiperidinyl phosphate amino acid ionic liquid, 1-ethyl-1-methylpyrrolidine amino acid ionic liquid, 1-hydroxyethyl-3-methylimidazole amino acid ionic liquid, betaine amino acid ionic liquid or 1-butyl-2,3-dimethylimidazole amino acid ionic liquid.

[0015] In some specific embodiments, the ionic liquid is selected from at least one of a betaine alanine ionic liquid, a choline proline ionic liquid, a choline serine ionic liquid or a choline arginine ionic liquid.

[0016] Amino acids are the smallest units that make up proteins in living organisms. Most of them come from organisms and are of various types, low in price, and have good biodegradability. Therefore, amino acid ionic liquids have good biocompatibility. In addition, since amino acid anions contain carboxylate anions and amino groups, their ability to bind hydrogen atoms as hydrogen bond receptors is stronger than that of carboxylate anions, and they have more action sites, which has significant advantages in the separation of systems with different intermolecular hydrogen bond interactions. Cholic acid and deoxycholic acid molecules have different abilities to form hydrogen bonds due to the difference in the number of hydroxyl groups. Therefore, amino acid ionic liquids as a medium have both high selectivity and good biocompatibility in the separation of cholic acid and deoxycholic acid.

[0017] In some embodiments, the method further comprises concentrating and drying the raffinate of the fractionation extraction to obtain deoxycholic acid. In some specific embodiments, the raffinate of the fractionation extraction is vacuum concentrated, washed with water and dried to obtain deoxycholic acid.

[0018] In some embodiments, the method further comprises back-extracting the extract of the fractionation extraction and concentrating and drying the back-extracted phase to obtain bile acid. In some specific embodiments, the extract of the fractionation extraction is back-extracted, and the back-extracted phase is vacuum concentrated, washed with water and dried to obtain bile acid.

[0019] In some embodiments, when fractional extraction is performed, taking into account factors such as product quality and production cost, the flow ratio of the extractant, detergent and raw material liquid is 1: (0.5-5): (0.1-10), preferably 1: (0.75-1): (0.25-5). In some specific embodiments, the flow ratio of the extractant, detergent and raw material liquid is 4:3:1 or 1:1:5.

[0020] In some embodiments, the mass fraction of the ionic liquid in the extractant is 0.01%-100%, for example, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any value therebetween. In some preferred embodiments, the mass fraction of the ionic liquid in the extractant is 0.01%-20%, more preferably 0.05%-5%, and further preferably 0.05%-2%.

[0021] In some embodiments, the total concentration of bile acid and deoxycholic acid in the raw material solution is 0.1g / L-100g / L, for example, 0.2g / L, 0.5g / L, 1g / L, 10g / L, 50g / L, 100g / L or any value therebetween. In the mixture of bile acid and deoxycholic acid in the raw material solution of the present invention, the total weight percentage of bile acid and deoxycholic acid is 90% or more. The mixture of bile acid and deoxycholic acid can be purchased directly from the market, or an animal bile acid extract with a low bile acid content can be refined by well-known extraction, precipitation and other steps to obtain a raw material with a total mass percentage of bile acid and deoxycholic acid of 90% or more.

[0022] In some embodiments, the raw material solution further comprises a raw material solvent, and preferably, the raw material solvent is selected from a C4-C8 straight-chain or branched alcohol compound. In some preferred embodiments, the raw material solvent is selected from at least one of n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctyl alcohol or isopentanol.

[0023] In some embodiments, the extractant further comprises a diluent to reduce viscosity and adjust the properties of the extractant. In some embodiments, the diluent is selected from a polar solvent or a non-polar solvent. In some preferred embodiments, for hydrophilic ionic liquids, the diluent is selected from polar solvents such as water and dimethyl sulfoxide. In some preferred embodiments, for hydrophobic ionic liquids, the diluent is selected from non-polar solvents such as n-hexane.

[0024] In some embodiments, the mass fraction of the diluent in the extractant is 0.01%-99.9%, for example, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, 100% or any value therebetween. In some preferred embodiments, the mass fraction of the ionic liquid in the extractant is 0.01%-20%, more preferably 0.05%-5%, and further preferably 0.05%-2%.

[0025] In some embodiments, the detergent is selected from C4-C8 straight-chain or branched alcohol compounds. In some preferred embodiments, the detergent is selected from at least one of n-butanol, 2-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctyl alcohol or isopentanol. In a further preferred embodiment, the detergent is the same as the raw material solvent, has good solubility for bile acid, and can form a liquid-liquid two-phase system with a small mutual solubility with the extractant.

[0026] In some embodiments, 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 therebetween. If the temperature is too low, the viscosity of the extractant is high, the mass transfer rate is reduced, the processing volume is small, and it is not conducive to production operation; if the temperature is too high, the solvent evaporates seriously, which will reduce the distribution ratio and selectivity of the fractional extraction.

[0027] In some embodiments, the fractionation extraction is multi-stage fractionation extraction.

[0028] In some embodiments, the fractionation extraction equipment used in the fractionation extraction includes 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 liquid enters the fractionation extraction system from the last stage of the extraction section, the detergent enters the fractionation extraction system from the first stage of the washing section, and the raw material liquid is combined at the last stage of the extraction section to enter the extraction section together. The extraction phase and the washing phase are subjected to multi-stage countercurrent contact, and the raffinate enriched in deoxycholic acid flows out from the first stage of the extraction section. The raffinate is collected and vacuum concentrated-washed-dried to obtain deoxycholic acid. The extract enriched in bile acid flows out from the first stage of the washing section, and bile acid is obtained through back extraction-vacuum concentration-washed-dried. The absolute purity of bile acid obtained by the separation method of the present invention is >90%; the relative content of bile acid:deoxycholic acid is >95:5 (mass ratio, w:w).

[0029] In some embodiments, the method comprises the following steps:

[0030] (1) preparing a mixture containing bile acid and deoxycholic acid with a raw material solvent to form a raw material liquid, using a binary mixed solvent composed of an amino acid ionic liquid and a diluent as an extractant, and using a solvent that is the same as the raw material liquid solvent as a detergent, and performing fractional extraction; the fractional extraction is divided into an extraction section and a washing section, the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, the raw material liquids are combined at the last stage of the washing section and enter the extraction section together, the extraction phase and the washing phase are subjected to multi-stage countercurrent extraction, an extract rich in bile acid flows out from the first stage of the washing section, and a raffinate enriched in deoxycholic acid flows out from the first stage of the extraction section;

[0031] (2) The raffinate in step (1) is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract in step (1) is back-extracted with a solvent that is the same as the raw material solvent and the detergent, the solvent-enriched phase is vacuum concentrated, washed with water and dried to obtain cholic acid, and the ionic liquid-enriched phase is vacuum concentrated and recycled.

[0032] The advantages of the present invention are:

[0033] 1. The present invention uses an extractant containing an amino acid ionic liquid for fractional extraction, and has good selective separation ability for bile acid and deoxycholic acid systems whose molecular structures differ by only one hydroxyl group. The process flow is simple, the energy consumption is low, and it is safe and environmentally friendly;

[0034] 2. The amino acid ionic liquid used in the present invention has good degradability and biocompatibility, reduces the impact on human body and environment, and has broad green application prospects;

[0035] 3. The amino acid ionic liquid used in the present invention has a low dosage but a high processing capacity, which can reduce the viscosity of the extractant and the production cost, and has industrial application value. DETAILED DESCRIPTION

[0036] The present invention provides a method for separating bile acid and deoxycholic acid with low toxicity, good biocompatibility, large processing capacity, simple process and high efficiency. The method uses an extractant containing an amino acid ionic liquid with amino acid roots as anions for fractional extraction. Amino acid ionic liquids have attracted attention as a new type of green separation medium in the field of separation. Compared with conventional ionic liquids, amino acid ionic liquids have some unique properties as extractants, such as good biocompatibility, strong degradability, safety and environmental protection, etc.; in addition, amino acid ionic liquids have amino and carboxylate anions, and there are more sites for forming intermolecular forces such as hydrogen bonds with solutes, and the effect is stronger. By designing the anion and cation structure of the ionic liquid to adjust the mode and intensity of action between the ionic liquid and the separation object, the purpose of molecular recognition and separation is achieved.

[0037] The molecular structures of bile acid and deoxycholic acid are very similar, the difference is that bile acid has a hydroxyl group at the C-7 position, while deoxycholic acid does not, so the two have differences in the number of sites that can form hydrogen bonds. According to the structural characteristics and differences of the structures of bile acid and deoxycholic acid, an amino acid ionic liquid with strong hydrogen bond alkalinity is used as an extractant, which can not only strengthen the interaction strength and difference between bile acid and the extractant, improve the processing volume and selectivity, but also reduce the amount of ionic liquid in the extractant and control production costs. If the concentration of ionic liquid in the extractant is too high, the amount of ionic liquid consumed in the production of unit mass of product will increase, the cost will increase, and the viscosity of the extractant will be large and the mass transfer rate will be low. The extractant containing amino acid ionic liquid used in the present invention can perform high-processing selective separation of bile acid-deoxycholic acid system at a lower ionic liquid concentration.

[0038] For a mixture containing bile acid and deoxycholic acid, single-stage extraction was performed at 30°C using n-butanol as solvent. When pure water was used as the extractant, the distribution coefficients of bile acid and deoxycholic acid were only 0.0257 and 0.0117, which were not suitable for industrial production. When a 1% mass fraction choline glycine aqueous solution was used as the extractant, the distribution coefficients of bile acid and deoxycholic acid could be increased to 1.36 and 0.58, which were suitable for industrial production. At the same time, the selectivity coefficient of bile acid / deoxycholic acid could reach 2.4. It can be seen that compared with pure water as the extractant, the addition of only 1% mass fraction of choline glycine can significantly improve the distribution coefficients of bile acid and deoxycholic acid while maintaining a high separation selectivity.

[0039] Based on the above research results, the present invention proposes a method for separating high-purity bile acid and deoxycholic acid from a mixture containing bile acid and deoxycholic acid by multi-stage fractionation extraction using a mixed solvent consisting of an amino acid ionic liquid and a diluent as an extractant.

[0040] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention, rather than to limit the scope of protection of the present invention.

[0041] In the following examples, the concentrations of cholic acid and deoxycholic acid were analyzed by high performance liquid chromatography (HPLC), and the specific analysis conditions of HPLC were: Waters Sunfire C18 column (4.6 mm 250 mm, particle size 5 μm), column temperature 30° C., mobile phase methanol: acetonitrile: 0.1% formic acid aqueous solution = 63: 17: 20 (v / v / v), flow rate 0.8 mL / min. Since the ultraviolet response of cholic acid and deoxycholic acid is weak, the detector adopts a general-purpose charged aerosol detector (Thermo Fisher).

[0042] In the following examples, the calculation method of yield and purity is as follows:

[0043] Yield = mass of bile acid in the product / mass of bile acid in the raw material × 100%;

[0044] Purity = mass of bile acid in the product / total mass of the product × 100%.

[0045] Example 1

[0046] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material solution with a total concentration of 20 g / L. A choline proline ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the choline proline ionic liquid is 1%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material solution is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material solution enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected respectively.

[0047] The raffinate is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract is diluted 10 times, back-extracted with 1 / 10 volume of n-butanol at 30°C for 3 times, the n-butanol phases are combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product is 89.35%, the yield is 93.57%, and the purity of deoxycholic acid is 85.06%, the yield is 93.65%. The ionic liquid-water phase after back-extraction is concentrated to increase the concentration of the ionic liquid, which can be recycled for reuse.

[0048] Example 2

[0049] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material liquid with a total concentration of 20 g / L. A betaine alanine ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the betaine alanine ionic liquid is 1%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0050] The raffinate is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract is diluted 10 times, back-extracted 3 times with 1 / 10 volume of n-butanol at 30°C, the n-butanol phases are combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product is 91.36%, the yield is 91.31%, and the purity of deoxycholic acid is 91.31%, the yield is 91.37%. The ionic liquid-water phase after back extraction is concentrated to increase the concentration of the ionic liquid, which can be recycled and reused.

[0051] Example 3

[0052] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-heptanol to prepare a raw material liquid with a total concentration of 20 g / L. A betaine alanine ionic liquid-dimethyl sulfoxide mixed solvent is used as an extractant (the mass fraction of the betaine alanine ionic liquid is 1%), n-heptanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0053] The raffinate is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract is diluted 10 times, back-extracted 3 times with 1 / 10 volume of n-heptanol at 30°C, the n-heptanol phases are combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product is 79.53%, the yield is 90.35%, and the purity of deoxycholic acid is 65.38%, the yield is 76.98%. The ionic liquid-diluent phase after back extraction is concentrated to increase the concentration of the ionic liquid, which can be recycled and reused.

[0054] Example 4

[0055] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material liquid with a total concentration of 20 g / L. A betaine alanine ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the betaine alanine ionic liquid is 0.08%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0056] The raffinate is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract is diluted 10 times, back-extracted 3 times with 1 / 10 volume of n-butanol at 30°C, the n-butanol phases are combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product is 85.69%, the yield is 93.10%, and the purity of deoxycholic acid is 87.94%, the yield is 92.65%. The ionic liquid-water phase after back extraction is concentrated to increase the concentration of the ionic liquid, which can be recycled for reuse.

[0057] Example 5

[0058] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in 2-heptanol to prepare a raw material solution with a total concentration of 20 g / L. A choline serine ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the choline serine ionic liquid is 1%), 2-heptanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material solution is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material solution enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0059] The raffinate was vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract was diluted 10 times, back-extracted with 1 / 10 volume of 2-heptanol at 30°C for 3 times, and the 2-heptanol phases were combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product was 87.21%, the yield was 93.87%, and the purity of deoxycholic acid was 86.48%, and the yield was 90.79%. The ionic liquid-enriched phase after back-extraction was concentrated to increase the ionic liquid concentration to the initial concentration for recycling.

[0060] Example 6

[0061] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in 2-heptanol to prepare a raw material solution with a total concentration of 20 g / L. A choline serine ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the choline serine ionic liquid is 1%), 2-heptanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material solution is 4:3:1 (v / v / v), and fractional extraction is performed at 50° C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material solution enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0062] The raffinate was vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract was diluted 10 times, back-extracted with 1 / 10 volume of 2-heptanol at 30°C for 3 times, and the 2-heptanol phases were combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product was 75.64%, the yield was 86.79%, and the purity of deoxycholic acid was 70.80%, the yield was 81.65%. The ionic liquid-enriched phase after back-extraction was concentrated to increase the ionic liquid concentration to the initial concentration for recycling.

[0063] Example 7

[0064] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material liquid with a total concentration of 20 g / L. A choline arginine ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the choline arginine ionic liquid is 1%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 1:1:5 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0065] The raffinate is vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract is diluted 10 times, back-extracted 3 times with 1 / 10 volume of n-butanol at 50°C, the n-butanol phases are combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product is 97.32%, the yield is 60.37%, and the purity of deoxycholic acid is 71.42%, the yield is 93.58%. The ionic liquid-enriched phase after back-extraction is concentrated to increase the ionic liquid concentration to the initial concentration for recycling.

[0066] Comparative Example 1

[0067] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material liquid with a total concentration of 20 g / L. A 1-ethyl-3-methylimidazolium chloride-water mixed solvent is used as an extractant (the mass fraction of 1-ethyl-3-methylimidazolium chloride is 1%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material liquid is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is reserved from the first stage of the extraction section, and the extract and the raffinate are collected.

[0068] The raffinate was vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract was diluted 10 times, back-extracted 3 times with 1 / 10 volume of n-butanol at 30°C, the n-butanol phases were combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product was 66.58%, the yield was 4.21%, and the purity of deoxycholic acid was 43.97%, the yield was 96.48%. The ionic liquid-enriched phase after back-extraction was concentrated to increase the ionic liquid concentration to the initial concentration for recycling.

[0069] Comparative Example 2

[0070] A mixture of raw bile acid and deoxycholic acid (wherein the total mass percentage of bile acid and deoxycholic acid is 99%) is dissolved in n-butanol to prepare a raw material solution with a total concentration of 20 g / L. A tetramethylammonium chloride ionic liquid-water mixed solvent is used as an extractant (the mass fraction of the tetramethylammonium chloride ionic liquid is 1%), n-butanol is used as a detergent, the flow ratio of the extractant, the detergent and the raw material solution is 4:3:1 (v / v / v), and fractional extraction is performed at 30°C. The fractional extraction is divided into an extraction section and a washing section (the extraction section has 6 stages and the washing section has 3 stages), the extractant enters the fractional extraction system from the first stage of the extraction section, the raw material solution enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, an extract rich in bile acid flows out from the first stage of the washing section, a raffinate rich in deoxycholic acid is retained from the first stage of the extraction section, and the extract and the raffinate are collected.

[0071] The raffinate was vacuum concentrated, washed with water and dried to obtain deoxycholic acid; the extract was diluted 10 times, back-extracted with 1 / 10 volume of n-butanol at 30°C for 3 times, the n-butanol phases were combined, vacuum concentrated, washed with water and dried to obtain cholic acid. The purity of cholic acid in the product was 60.58%, the yield was 6.87%, and the purity of deoxycholic acid was 41.60%, the yield was 98.71%. The ionic liquid-enriched phase after back-extraction was concentrated to increase the ionic liquid concentration to the initial concentration for recycling.

[0072] The above embodiments are only used to illustrate the technical solution of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solution of the present application.

Claims

1. A method for separating cholic acid and deoxycholic acid, comprising fractionating and extracting a raw material solution containing cholic acid and deoxycholic acid using an extractant containing an amino acid ionic liquid, The amino acid ionic liquid is composed of cations and amino acid anions. in, The cation is selected from at least one of an imidazolium cation, a pyridinium cation, a benzimidazolium cation, a quaternary ammonium cation, a quaternary phosphonium cation, a betaine cation, a guanidine cation or a choline cation, and the cation is optionally substituted by one or more substituents selected from a C1-C4 straight-chain alkyl group; The amino acid anion is selected from at least one of a neutral amino acid anion, a basic amino acid anion or an acidic amino acid anion. The neutral amino acid anion includes at least one of a glycine anion, a proline anion, an alanine anion, a leucine anion, a valine anion or a phenylalanine anion; the basic amino acid includes at least one of a tryptophan anion, an asparagine anion, a glutamine anion, a lysine anion or an arginine anion; the acidic amino acid includes at least one of a cysteine ​​anion, a methionine anion or a glutamate anion, The amino acid anion is optionally substituted by one or more substituents selected from hydroxyl groups, C1-C4 straight chain olefin groups; The mass fraction of the ionic liquid in the extractant is 0.01%-2%; The total concentration of cholic acid and deoxycholic acid in the raw material solution is 10 g / L-20 g / L.

2. The method according to claim 1, characterized in that The amino acid ionic liquid is selected from at least one of 1-ethyl-3-methylimidazole amino acid ionic liquid, choline amino acid ionic liquid, tert-butylammonium amino acid ionic liquid, betaine amino acid ionic liquid or 1-butyl-2,3-dimethylimidazole amino acid ionic liquid.

3. The method according to claim 1, characterized in that The amino acid ionic liquid is selected from at least one of betaine alanine ionic liquid, choline proline ionic liquid, choline serine ionic liquid or choline arginine ionic liquid.

4. The method according to any one of claims 1 to 3, characterized in that The method further comprises: Concentrating and drying the raffinate from the fractionation extraction to obtain deoxycholic acid; and / or The extract of the fractionation extraction is subjected to back extraction, and the back extraction phase is concentrated and dried to obtain bile acid.

5. The method according to any one of claims 1 to 3, characterized in that: The fractional extraction is divided into an extraction section and a washing section, wherein the flow ratio of the extractant, the washing agent and the raw material liquid is 1: (0.5-5): (0.1-10).

6. The method according to claim 5, characterized in that In the fractional extraction, the flow ratio of the extractant, the detergent and the raw material liquid is 1:(0.75-1):(0.25-5).

7. The method according to claim 5, characterized in that The raw material liquid also includes a raw material solvent, The raw material solvent is selected from C4-C8 straight-chain or branched alcohol compounds.

8. The method according to claim 7, characterized in that The raw material solvent is selected from at least one of n-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctyl alcohol and isopentanol.

9. The method according to any one of claims 1 to 3, characterized in that: The extractant further comprises a diluent, and the diluent is selected from a polar solvent or a non-polar solvent.

10. The method according to claim 9, characterized in that The polar solvent is selected from water and / or dimethyl sulfoxide, and the non-polar solvent is selected from n-hexane.

11. The method according to claim 5, characterized in that The detergent is selected from C4-C8 straight-chain or branched-chain alcohol compounds.

12. The method according to claim 11, characterized in that The detergent is selected from at least one of n-butanol, isobutanol, n-pentanol, n-hexanol, 2-hexanol, n-heptanol, isooctyl alcohol and isopentanol.

13. The method according to claim 7, characterized in that The detergent is the same as the raw material solvent.

14. The method according to any one of claims 1 to 3, characterized in that The operating temperature of the fractional extraction is 20°C-70°C.

15. The method according to claim 14, characterized in that The operating temperature of the fractional extraction is 30°C-50°C.