A method for preparing high-purity cholic acid

Abstract

This invention relates to the field of separation and purification, specifically a method for preparing high-purity cholic acid. The method involves adding bovine bile powder to an alkaline solution, heating and stirring, and adjusting the pH to 8-9 to obtain a bovine bile powder solution. A magnesium salt solution is then added to the bovine bile powder solution, and the mixture is heated and stirred to react. The mixture is then filtered to obtain filtrate A. An acidic solution is added to filtrate A to adjust the pH to acidic, and the mixture is filtered again to obtain precipitate A. Precipitate A is first washed with anhydrous ethanol, then washed with pure water and dried to obtain crude cholic acid. The crude cholic acid is then added to glacial acetic acid, heated to dissolve, cooled to crystallize, filtered, washed with water, and dried to obtain high-purity cholic acid. This method requires only one washing and one recrystallization step, ensuring a high yield of high-purity cholic acid.

Description

Technical Field

[0001] This invention relates to the field of separation and purification, and in particular to a method for preparing high-purity cholic acid. Background Technology

[0002] Bile acids are mainly found in the bile of animals such as cattle and sheep. They are hydrophobic, endogenous bile acids and are natural choleretic drugs. Bile acids have antipyretic, detoxifying, antibacterial, and antiviral properties. Modern traditional Chinese medicines such as Xiaozhiling capsules and Qingkailing tablets contain large amounts of bile acids. At the same time, bile acids are also substrates for valuable drugs such as deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, lithocholic acid, and taurocholic acid. To address the contradiction between the demand for and the scarcity of sources of drugs such as bezoar, snake bile, and bear bile in pharmacology, the research and development of bile acids is of great significance.

[0003] Currently, there are no reports of successful artificial synthesis of bile acid monomers, either domestically or internationally. Industrially, bile acids are generally obtained from bovine or ovine bile through ethanol crystallization. The ethanol crystallization method involves adding sodium hydroxide solution to bovine or ovine bile, heating to boiling, then adjusting the solution to acidity with hydrochloric acid to precipitate the precipitate. The precipitate is then boiled in water, washed, dried, and ground to obtain crude bile acid. The crude bile acid is first washed with ethanol until the filtrate is colorless, then dissolved in ethanol upon heating, filtered while hot, concentrated, cooled to crystallize, filtered again, and dried to obtain pure bile acid.

[0004] In the ethanol crystallization method, crude cholic acid is purified by washing with ethanol followed by recrystallization. While this removes some deoxycholic acid impurities, the purity of the resulting cholic acid is low because ethanol has low solubility for cholic acid but high solubility for deoxycholic acid impurities. Multiple washing and recrystallization operations are necessary to obtain high-purity cholic acid, but these repeated operations reduce the yield. Therefore, when preparing cholic acid using the ethanol crystallization method, it is impossible to simultaneously achieve both high purity and high yield. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing high-purity cholic acid, which requires only one washing and one recrystallization to obtain high-purity cholic acid while ensuring the yield.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] (1) Add the ox bile powder to the alkaline solution, heat and stir, and adjust the pH to 8-9 to obtain the ox bile powder solution;

[0008] (2) Add magnesium salt solution to bovine bile powder solution, heat and stir to react, and filter to obtain filtrate A;

[0009] (3) Add an acidic solution to filtrate A, adjust the pH value to acidic, and filter to obtain precipitate A;

[0010] (4) The precipitate A was first washed with anhydrous ethanol, then washed with pure water and dried to obtain crude cholic acid;

[0011] (5) Add crude cholic acid to glacial acetic acid, heat to dissolve, filter to remove impurities, cool to crystallize, and obtain suspension B. After filtration, washing with water and drying, high-purity cholic acid is obtained.

[0012] Preferably, in step (1), the alkaline solution is an aqueous solution of sodium hydroxide or an aqueous solution of potassium hydroxide; the heating time is 2 to 4 hours.

[0013] Preferably, in step (2), the magnesium salt solution is an aqueous solution of magnesium chloride or magnesium sulfate; the heating temperature is 80~90℃, and the heating and stirring time is 2~4 hours.

[0014] Preferably, in step (3), the acidic solution is hydrochloric acid or sulfuric acid, with a pH value of 2 to 5.

[0015] Preferably, in step (4), the mass ratio of anhydrous ethanol to precipitate A is 2~10:1.

[0016] Preferably, in step (5), the mass ratio of glacial acetic acid to crude cholic acid is 1~3:1.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] Traditional ethanol crystallization methods, which involve washing with ethanol followed by recrystallization, yield cholic acid with low purity. The method disclosed in this invention first reacts a magnesium salt solution with deoxycholic acid impurities to form a precipitate. After filtration to remove impurities, the precipitate A is washed with anhydrous ethanol to further remove deoxycholic acid impurities, taking advantage of the low solubility of cholic acid in anhydrous ethanol and the high solubility of deoxycholic acid impurities in ethanol. Finally, cholic acid is recrystallized from glacial acetic acid to remove deoxycholic acid impurities. This three-step purification process yields a high-purity cholic acid product.

[0019] The inventors discovered that the pH of the ox bile powder solution before the addition of magnesium salt, the amount of anhydrous ethanol used, and the amount of glacial acetic acid used have a significant impact on the purity and yield of cholic acid. The optimal pH of the ox bile powder solution before the addition of magnesium salt is maintained at 8-9. Too high a pH will cause the added magnesium salt to produce magnesium hydroxide precipitate, which cannot react with deoxycholic acid impurities to form a precipitate. Too low a pH will affect the impurity removal effect of the magnesium salt. The optimal mass ratio of anhydrous ethanol to precipitate A is 2-10:1. Too high a mass ratio will cause some cholic acid to be washed away, affecting the yield. Too low a mass ratio will result in incomplete washing of impurities, affecting purity. The optimal mass ratio of glacial acetic acid to crude cholic acid is 1-3:1. Too high a mass ratio will make it difficult for some cholic acid to crystallize out, affecting the yield. Too low a mass ratio will result in incomplete recrystallization of cholic acid.

[0020] 3. This invention uses bovine bile powder as the raw material for preparing bile acids. Compared with bovine bile as the raw material for preparing bile acids, it is more convenient to transport and facilitates the establishment of factories away from pastoral areas or slaughterhouses. Attached Figure Description

[0021] Figure 1 TLC comparison chart of bile acid standard and bile acid obtained in Example 1;

[0022] Figure 2 This is a graph showing the maximum absorption wavelength of bile acid standards.

[0023] Figure 3 This is a standard curve for bile acid standards. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments, which will help to understand the present invention. However, the present invention is not limited to the following embodiments. Example 1

[0025] Weigh 20g of ox bile powder into a flask, add 200ml of 30% NaOH solution, heat and stir for 4 hours until the ox bile powder is completely dissolved, then adjust the pH of the solution to 8. Next, add 400ml of 30% magnesium chloride solution to the ox bile powder solution, heat to 90℃, and stir thoroughly for 4 hours to obtain suspension A. Filter to obtain filtrate A. Then add hydrochloric acid to filtrate A and adjust the pH of the solution to approximately 2 to obtain suspension B. Filter to obtain precipitate A. Wash with anhydrous ethanol (10:10) and water thoroughly, dry in an oven, then dissolve in glacial acetic acid (3:3) at 60℃. Filter while hot, cool the filtrate to crystallize, and after complete crystallization, filter, wash with water, and dry thoroughly to obtain bile acid.

[0026] TLC spotting: Place 0.1 g of cholic acid product and standard into separate beakers, add a small amount of methanol to dissolve completely, transfer to a 100 mL volumetric flask and dilute to the mark. Shake well and let stand, then take a small amount into a beaker for later use. Take a thin-layer plate and draw a horizontal line 1.5 cm away from the plate with a pencil. Use a capillary tube to apply the prepared solution to the horizontal line on the plate. After the solvent has completely evaporated, place the plate in a developing cell containing methanol:dichloromethane = 1:4 developing solution. After the samples have fully developed, remove the plate and evaporate the developing solvent. Spray a layer of prepared 10% sulfuric acid-ethanol solution as a colorimetric reagent onto the plate, then heat in an 80℃ oven for 10 min. The results are shown in the appendix. Figure 1 .

[0027] As shown in the figure, point 1 is the purchased bile acid standard, and point 2 is the sample prepared in Example 1. The climbing plate heights of points 1 and 2 are the same, indicating that the sample prepared in Example 1 is bile acid.

[0028] Determination of bile acid purity by ultraviolet-visible spectrophotometry

[0029] Determination of the maximum absorption wavelength: Accurately weigh 20 mg of cholic acid standard, dissolve it in methanol, transfer it to a 100 mL volumetric flask and dilute to the mark. Transfer 0.5 mL of the solution to a test tube, then transfer 4.5 mL of 70% sulfuric acid solution to the test tube. Heat in a 70°C water bath for 30 minutes, then quickly transfer to an ice-water bath. After 2 minutes, using the corresponding reagent as a blank control, perform a wavelength scan. The scanning results are as follows: Figure 2 As shown, the maximum absorption wavelength of bile acid is 320 nm.

[0030] Construction of the standard curve: Accurately weigh 20 mg of cholic acid standard, dissolve it in methanol, transfer it to a 100 mL volumetric flask and dilute to the mark. Transfer 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of this solution to test tubes, respectively. Then transfer 4.8, 4.7, 4.6, 4.5, 4.4, and 4.3 mL of 70% sulfuric acid solution to test tubes, respectively. Heat in a 70°C water bath for 30 minutes, then quickly transfer to an ice-water bath. After 2 minutes, using the corresponding reagents as blank controls, measure the absorbance at 320 nm. Plot the measured absorbance on the x-axis and the concentration on the y-axis, as shown below. Figure 3 Linear fitting analysis was performed on the data, and the regression equation for bile acid in the range of 8.0~28.0 μg / mL was y=0.0298x+0.0168, R=0.9991.

[0031] Purity determination: Accurately weigh 20 mg of cholic acid sample, dissolve it in methanol, transfer it to a 100 mL volumetric flask and dilute to the mark. Transfer 0.5 mL of the solution to a test tube, then transfer 4.5 mL of 70% sulfuric acid solution to the test tube. Heat in a 70°C water bath for 30 minutes, then quickly transfer to an ice-water bath. After 2 minutes, using the corresponding reagent as a blank control, measure the absorbance at 320 nm. The purity of the sample is obtained by substituting the measured absorbance into a standard curve. Example 2

[0032] Weigh 30g of ox bile powder into a flask, add 400ml of 30% NaOH solution, heat and stir for 2 hours until the ox bile powder is completely dissolved, then adjust the pH of the solution to 9. Next, add 300ml of 30% magnesium chloride solution to the ox bile powder solution, heat to 80℃, and stir thoroughly for 2 hours to obtain suspension A. Filter to obtain filtrate A. Then add hydrochloric acid to filtrate A and adjust the pH of the solution to approximately 5 to obtain suspension B. Filter to obtain precipitate A. Wash with anhydrous ethanol (6 parts by mass), then thoroughly wash with water, and dry in an oven. Then dissolve in glacial acetic acid (3 parts by mass) by heating to 60℃, filter while hot, cool the filtrate to crystallize, and after complete crystallization, filter, wash with water, and dry thoroughly to obtain bile acid. Example 3

[0033] Weigh 20g of ox bile powder into a flask, add 400ml of 30% NaOH solution, heat and stir for 3 hours until the ox bile powder is completely dissolved, then adjust the pH of the solution to 8. Next, add 300ml of 30% magnesium chloride solution to the ox bile powder solution, heat to 85℃, and stir thoroughly for 3 hours to obtain suspension A. Filter to obtain filtrate A. Then add hydrochloric acid to filtrate A and adjust the pH of the solution to approximately 3 to obtain suspension B. Filter to obtain precipitate A. Wash with anhydrous ethanol (2 parts by mass) and then thoroughly with water, dry in an oven, then dissolve in glacial acetic acid (3 parts by mass) at 60℃, filter while hot, cool the filtrate to crystallize, and after complete crystallization, filter, wash with water, and dry thoroughly to obtain bile acid. Example 4

[0034] Weigh 20g of ox bile powder into a flask, add 400ml of 30% KOH solution, heat and stir for 4 hours until the ox bile powder is completely dissolved, then adjust the pH of the solution to 8. Next, add 300ml of 30% magnesium sulfate solution to the ox bile powder solution, heat to 90℃, and stir thoroughly for 4 hours to obtain suspension A. Filter to obtain filtrate A. Then add sulfuric acid to filtrate A and adjust the pH of the solution to approximately 2 to obtain suspension B. Filter to obtain precipitate A. Wash with anhydrous ethanol (6 parts by mass), then thoroughly wash with water, and dry in an oven. Then dissolve in glacial acetic acid (2 parts by mass) by heating to 60℃, filter while hot, cool the filtrate to crystallize, and after complete crystallization, filter, wash with water, and dry thoroughly to obtain bile acid. Example 5

[0035] Weigh 30g of ox bile powder into a flask, add 400ml of 30% NaOH solution, heat and stir for 4 hours until the ox bile powder is completely dissolved, then adjust the pH of the solution to 8. Next, add 300ml of 30% magnesium chloride solution to the ox bile powder solution, heat to 90℃, and stir thoroughly for 4 hours to obtain suspension A. Filter to obtain filtrate A. Then add hydrochloric acid to filtrate A and adjust the pH of the solution to approximately 2 to obtain suspension B. Filter to obtain precipitate A. Wash with anhydrous ethanol (6 parts by mass) and then thoroughly with water, dry in an oven, then dissolve in glacial acetic acid (1 part by mass) at 60℃. Filter while hot, cool the filtrate to crystallize, and after complete crystallization, filter, wash with water, and dry thoroughly to obtain bile acid. Comparative Example 1

[0036] The operation is the same as in Example 4, except that after adding the alkali solution in step (1), the pH is adjusted to 13, and the rest of the steps are the same. Comparative Example 2

[0037] The operation is the same as in Example 2, except that anhydrous ethanol is used for recrystallization in step (5), and the other steps are the same. Comparative Example 3

[0038] The operation is the same as in Example 4, except that in step (4), the mass ratio of anhydrous ethanol to precipitate A is 20:1. Comparative Example 4

[0039] The operation is the same as in Example 5, except that in step (5), the mass ratio of glacial acetic acid to crude cholic acid is 4:1.

[0040] Table 1 shows the yield and purity of cholic acid measured in Examples 1 to Comparative Example 4. The total cholic acid content in the bovine bile powder used in the experiments was 42%. Cholic acid yield refers to the ratio of the final mass of cholic acid to the mass of the raw bovine bile powder.

[0041]

[0042] As shown in the table, the purity of cholic acid prepared by the method disclosed in this invention is 92.4%~98.7%, and the yield is 28.0%~31.0%. In Comparative Example 1, adjusting the pH to 13 after adding alkali significantly reduced the purity of the resulting cholic acid. This was because the high pH caused the added magnesium salts to precipitate as magnesium hydroxide, preventing the formation of magnesium salt precipitates of deoxycholic acid complexes, thus hindering the removal of deoxycholic acid substances. Consequently, the purity of the obtained cholic acid was only 85.3%. In Comparative Example 2, recrystallization with anhydrous ethanol was problematic. Anhydrous ethanol has low solubility for cholic acid but high solubility for deoxycholic acid impurities, resulting in low purity and yield: 86.4% purity and only 14.0% yield. In Comparative Example 3, excessive anhydrous ethanol washing led to cholic acid loss, resulting in a low yield of only 21.5%. In Comparative Example 4, excessive glacial acetic acid prevented some dissolved cholic acid from crystallizing, further reducing the yield to only 21.3%. Therefore, in the preparation method disclosed in this invention, the pH of the bovine bile powder solution before adding magnesium salt, and the amount of anhydrous ethanol and glacial acetic acid used are key limiting factors that determine the purity and yield of bile acids.

Claims

1. A method for preparing high-purity cholic acid, characterized in that, Includes the following steps: (1) Add the ox bile powder to the alkaline solution, heat and stir, and adjust the pH to 8-9 to obtain the ox bile powder solution; (2) Add magnesium salt solution to bovine bile powder solution, heat and stir to react, filter to obtain filtrate A, wherein the heating temperature is 80~90℃ and the heating and stirring time is 2~4 hours; (3) Add an acidic solution to filtrate A, adjust the pH value to acidic, and filter to obtain precipitate A; (4) The precipitate A is first washed with anhydrous ethanol, then washed with pure water and dried to obtain crude cholic acid. The ratio of the amount of anhydrous ethanol used to the mass of the precipitate is 2~10:

1. (5) Add crude cholic acid to glacial acetic acid and heat to dissolve. Then cool and crystallize, filter, wash with water and dry to obtain high-purity cholic acid. The mass ratio of glacial acetic acid to crude cholic acid is 1~3:

1.

2. The preparation method according to claim 1, characterized in that, In step (1), the alkaline solution is an aqueous solution of sodium hydroxide or potassium hydroxide; the heating time is 2 to 4 hours.

3. The preparation method according to claim 1, characterized in that, In step (2), the magnesium salt solution is an aqueous solution of magnesium chloride or an aqueous solution of magnesium sulfate.

4. The preparation method according to claim 1, characterized in that, In step (3), the acidic solution is hydrochloric acid or sulfuric acid, with a pH value of 2 to 5.

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