Preparation method of high-purity cholic acid

Abstract

The invention relates to the field of separation and purification, in particular to a preparation method of high-purity cholic acid, which comprises the following steps: adding ox gall powder into alkali liquor, heating and stirring, and adjusting the pH value to 8-9 to obtain an ox gall powder solution; adding a magnesium salt solution into the ox gall powder solution, heating and stirring for reaction, and filtering to obtain filtrate A; adding an acidic solution into the filtrate A, adjusting the pH value to be acidic, and filtering to obtain a precipitate A; washing the precipitate A with absolute ethyl alcohol, then washing with pure water, and drying to obtain crude cholic acid; and adding the crude cholic acid into glacial acetic acid, heating and dissolving, cooling, crystallizing, filtering, washing with water, and drying to obtain the high-purity cholic acid. According to the method, high-purity cholic acid can be obtained only through one-time washing and one-time recrystallization on the basis of ensuring the yield.

Description

Technical Field

[0001] The present invention relates to the field of separation and purification, in particular to a method for preparing high-purity bile acid. Background Art

[0002] Bile acid, primarily found in the bile of animals like cattle and sheep, is a hydrophobic, endogenous bile acid and a natural choleretic. It possesses antipyretic, detoxifying, antibacterial, and antiviral properties. Modern Chinese patent medicines such as Xiaozhiling Capsules and Qingkailing Tablets contain significant amounts of bile acid. Furthermore, bile acid is a substrate for valuable drugs such as deoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, lithocholic acid, and taurocholic acid. Research and development of bile acid is crucial to address the pharmacological challenge of the limited availability of drugs like bezoar, snake gall, and bear bile, while also addressing the demand for these drugs.

[0003] Currently, there are no reports of successful artificial synthesis of bile acid monomers, either domestically or internationally. Industrially, bile acid is typically obtained from bovine or ovine bile using ethanol crystallization. This method involves adding sodium hydroxide solution to bovine or ovine bile, heating and boiling, then acidifying the solution with hydrochloric acid to precipitate. The precipitate is then boiled, rinsed, 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 by heating, hot-filtered, concentrated, cooled, crystallized, filtered, and dried to obtain pure bile acid.

[0004] In the ethanol crystallization method, crude bile acid is purified by washing with ethanol and then recrystallizing with ethanol. Although this method can remove some deoxycholic acid impurities from the crude bile acid, the resulting bile acid is of low purity because ethanol has a low solubility for bile acid and a high solubility for deoxycholic acid impurities. High-purity bile acid can only be obtained through multiple washing and recrystallization operations, but these multiple washing and recrystallization operations reduce the bile acid yield. Therefore, when preparing bile acid using the ethanol crystallization method, its purity and yield cannot be simultaneously achieved. Summary of the Invention

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

[0006] To achieve the above object, the present invention provides the following solutions: (1) Add ox bile powder to alkali solution, heat and stir, adjust the pH to 8-9, and obtain ox bile powder solution; (2) Add magnesium salt solution to the ox bile powder solution, heat and stir to react, and filter to obtain filtrate A; (3) Add an acidic solution to the filtrate A, adjust the pH 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 bile acid; (5) Add crude cholic acid to glacial acetic acid, heat to dissolve, filter to remove impurities, then cool down to crystallize to obtain suspension B, and obtain high-purity cholic acid after filtration, washing with water and drying.

[0007] Preferably, in the 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.

[0008] Preferably, in the step (2), the magnesium salt solution is an aqueous solution of magnesium chloride or an aqueous solution of magnesium sulfate; the heating temperature is 80 to 90 °C, and the heating and stirring time is 2 to 4 hours.

[0009] Preferably, in the step (3), the acidic solution is hydrochloric acid or sulfuric acid, and the pH value is 2 to 5.

[0010] Preferably, in the step (4), the mass ratio of absolute ethanol to precipitate A is 2 to 10:1.

[0011] Preferably, in the step (5), the mass ratio of the amount of glacial acetic acid used to the mass of crude cholic acid is 1 to 3:1.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The cholic acid obtained by the traditional ethanol crystallization method through ethanol washing and ethanol recrystallization process has a low purity; while the preparation method disclosed in the present invention first reacts the magnesium salt solution with deoxycholic acid impurities to form a precipitate, and after filtration to remove impurities, then utilizes the characteristics that cholic acid has a small solubility in absolute ethanol while deoxycholic acid impurities have a large solubility in ethanol, and uses absolute ethanol to wash precipitate A to further remove deoxycholic acid impurities. Finally, by using the characteristics that cholic acid has a large solubility in glacial acetic acid while deoxycholic acid impurities have a small solubility in glacial acetic acid, deoxycholic acid impurities in cholic acid are removed by recrystallization with glacial acetic acid. By adopting the above three-step purification process, a high-purity cholic acid product is obtained.

[0013] The inventor found that the pH of the bovine bile powder solution before adding the magnesium salt, the amount of absolute ethanol used, and the amount of glacial acetic acid used have a great impact on the purity and yield of cholic acid. It is optimal to maintain the pH of the bovine bile powder solution at 8 to 9 before adding the magnesium salt. If the pH is too high, magnesium hydroxide precipitation will be produced by the added magnesium salt, and it cannot react with deoxycholic acid impurities to form a precipitate. If it is too low, the impurity removal effect of the magnesium salt will be affected; the optimal mass ratio of the amount of absolute ethanol used to precipitate A is 2 to 10:1. If the mass ratio is too high, part of the cholic acid will be washed away, affecting the yield. If the mass ratio is too low, the impurities will not be washed clean, affecting the purity; the optimal mass ratio of the amount of glacial acetic acid used to the mass of crude cholic acid is 1 to 3:1. If the mass ratio is too high, part of the cholic acid is difficult to crystallize out, affecting the yield. If the mass ratio is too low, the recrystallization of cholic acid is incomplete.

[0014] 3. The present invention uses bovine bile powder as the raw material for preparing cholic acid. Compared with using bovine bile as the raw material, it is more convenient for transportation, which is conducive to establishing a factory away from pastoral areas or slaughterhouses. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a TLC plate comparison diagram of cholic acid standard product and cholic acid obtained in Example 1; Figure 2 It is the maximum absorption wavelength diagram of cholic acid standard product; Figure 3 It is the standard curve diagram of cholic acid standard product. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The following further illustrates the solution of the present invention with specific embodiments, which will help to understand the present invention, but the present invention is not limited to the following embodiments. Example 1

[0017] Weigh 20 g of bovine bile powder into a flask, add 200 ml of 30% NaOH solution by mass fraction, heat and stir for 4 hours. After the bovine bile powder is completely dissolved, adjust the pH of the solution to 8. Then add 400 ml of 30% magnesium chloride solution by mass fraction to the bovine bile powder solution, heat to 90 °C, and stir well for 4 hours to obtain suspension A. Filter to obtain filtrate A. Then add hydrochloric acid to filtrate A to adjust the pH of the solution to about 2 to obtain suspension B. Filter to obtain precipitate A. Then wash with absolute ethanol with a mass ratio of 10 and wash thoroughly with water, dry in an oven. Then use glacial acetic acid with a mass ratio of 3, heat to 60 °C to dissolve, filter while it is hot, cool the filtrate to crystallize. After the crystallization is complete, filter, wash with water, and dry thoroughly to obtain cholic acid.

[0018] TLC plate: Take 0.1 g of cholic acid finished product and standard product respectively and put them into beakers. After adding a small amount of methanol to completely dissolve them, transfer them to a 100 mL volumetric flask and make up to the scale. After shaking evenly and standing still, take a small amount and place it in a beaker for use. Take a thin-layer plate, draw a horizontal line with a pencil at a distance of 1.5 cm from one end of the thin-layer plate. Dip the prepared solution with a capillary tube and spot it on the horizontal line of the thin-layer plate. After the solvent has completely evaporated, place the thin-layer plate in a developing tank containing a developing solution of methanol:dichloromethane = 1:4 to develop. After the sample spots are completely developed, take out the thin-layer plate and volatilize the developing agent on the thin-layer plate. Spray a layer of prepared 10% sulfuric acid-ethanol solution developer on the thin-layer plate, then place it in an oven at 80 °C and heat for 10 min and then take it out. The result is shown in the attached Figure 1 .

[0019] As can be seen from the figure, point 1 is the purchased cholic acid standard product, point 2 is the sample prepared in Example 1, and the climbing heights of point 1 and point 2 on the plate are the same, indicating that the sample prepared in Example 1 is cholic acid.

[0020] Determination of the Purity of Cholic Acid by Ultraviolet-Visible Spectrophotometry Determination of the maximum absorption wavelength: Accurately weigh 20 mg of cholic acid standard. After dissolving it in methanol, transfer it to a 100 mL volumetric flask and make up to the mark. Pipette 0.5 mL into a test tube, and then pipette 4.5 mL of 70% sulfuric acid solution into the test tube. After heating in a 70 °C water bath for 30 minutes, quickly transfer it to an ice-water bath. After 2 minutes, using the corresponding reagent as a blank control, perform wavelength scanning. The scanning results are as Figure 2 shown. The maximum absorption wavelength of cholic acid is 320 nm.

[0021] Drawing of the standard curve: Accurately weigh 20 mg of cholic acid standard. After dissolving it in methanol, transfer it to a 100 mL volumetric flask and make up to the mark. Pipette 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL into test tubes respectively, and then pipette 4.8, 4.7, 4.6, 4.5, 4.4, and 4.3 mL of 70% sulfuric acid solution into the test tubes respectively. After heating in a 70 °C water bath for 30 minutes, quickly transfer it to an ice-water bath. After 2 minutes, using the corresponding reagent as a blank control, measure their respective absorbances at a wavelength of 320 nm. Plot the measured absorbance on the abscissa and the concentration on the ordinate, as Figure 3 . And perform linear fitting analysis on the data. The fitting regression equation for cholic acid in the range of 8.0 - 28.0 μg / mL is y = 0.0298x + 0.0168, R = 0.9991.

[0022] Purity determination: Accurately weigh 20 mg of cholic acid sample. After dissolving it in methanol, transfer it to a 100 mL volumetric flask and make up to the mark. Pipette 0.5 mL into a test tube, and then pipette 4.5 mL of 70% sulfuric acid solution into the test tube. After heating in a 70 °C water bath for 30 minutes, quickly transfer it to an ice-water bath. After 2 minutes, using the corresponding reagent as a blank control, measure its absorbance at a wavelength of 320 nm. According to the measured absorbance, substitute it into the drawn standard curve to obtain the purity of the sample to be tested. Example 2

[0023] Weigh 30 g of ox bile powder into a flask, add 400 ml of a 30% NaOH solution by mass fraction, heat and stir for 2 hours. After the ox bile powder is completely dissolved, adjust the pH of the solution to 9. Then add 300 ml of a 30% magnesium chloride solution by mass fraction to the ox bile powder solution, heat to 80 °C, and stir well 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 about 5 to obtain suspension B, and filter to obtain precipitate A. Then wash it with absolute ethanol with a mass ratio of 6 and wash it thoroughly with water, dry it in an oven. Then use glacial acetic acid with a mass ratio of 3, heat to 60 °C to dissolve it, filter while it is hot, cool the filtrate to crystallize. After the crystallization is complete, filter, wash with water, and dry thoroughly to obtain cholic acid. Example 3

[0024] Weigh 20 g of bovine bile powder into a flask, add 400 ml of 30% NaOH solution by mass fraction, heat and stir for 3 hours. After the bovine bile powder is completely dissolved, adjust the pH of the solution to 8. Then add 300 ml of 30% magnesium chloride solution by mass fraction to the bovine bile powder solution, heat to 85 °C, stir well for 3 hours to obtain suspension A, filter to obtain filtrate A, then add hydrochloric acid to filtrate A, adjust the pH of the solution to about 3 to obtain suspension B, and filter to obtain precipitate A. Then wash with absolute ethanol with a mass ratio of 2 and wash thoroughly with water, dry in an oven, then dissolve with glacial acetic acid with a mass ratio of 3 by heating to 60 °C, filter while it is hot, cool the filtrate to crystallize. After crystallization is complete, filter, wash with water, and dry thoroughly to obtain cholic acid. Example 4

[0025] Weigh 20 g of bovine bile powder into a flask, add 400 ml of 30% KOH solution by mass fraction, heat and stir for 4 hours. After the bovine bile powder is completely dissolved, adjust the pH of the solution to 8. Then add 300 ml of 30% magnesium sulfate solution by mass fraction to the bovine bile powder solution, heat to 90 °C, stir well for 4 hours to obtain suspension A, filter to obtain filtrate A, then add sulfuric acid to filtrate A, adjust the pH of the solution to about 2 to obtain suspension B, and filter to obtain precipitate A. Then wash with absolute ethanol with a mass ratio of 6 and wash thoroughly with water, dry in an oven, then dissolve with glacial acetic acid with a mass ratio of 2 by heating to 60 °C, filter while it is hot, cool the filtrate to crystallize. After crystallization is complete, filter, wash with water, and dry thoroughly to obtain cholic acid. Example 5

[0026] Weigh 30 g of bovine bile powder into a flask, add 400 ml of 30% NaOH solution by mass fraction, heat and stir for 4 hours. After the bovine bile powder is completely dissolved, adjust the pH of the solution to 8. Then add 300 ml of 30% magnesium chloride solution by mass fraction to the bovine bile powder solution, heat to 90 °C, stir well for 4 hours to obtain suspension A, filter to obtain filtrate A, then add hydrochloric acid to filtrate A, adjust the pH of the solution to about 2 to obtain suspension B, and filter to obtain precipitate A. Then wash with absolute ethanol with a mass ratio of 6 and wash thoroughly with water, dry in an oven, then dissolve with glacial acetic acid with a mass ratio of 1 by heating to 60 °C, filter while it is hot, cool the filtrate to crystallize. After crystallization is complete, filter, wash with water, and dry thoroughly to obtain cholic acid. Comparative Example 1

[0027] Perform the same operation as in Example 4, except that the pH is adjusted to 13 after adding the alkali solution in step (1), and the other steps are the same. Comparative Example 2

[0028] The operation was the same as that in Example 2, except that recrystallization was carried out with absolute ethanol in step (5), and the remaining steps were the same. Comparative Example 3

[0029] The operation was the same as that in Example 4, except that in step (4), the mass ratio of absolute ethanol to precipitate A was 20:1. Comparative Example 4

[0030] The operation was the same as that in Example 5, except that in step (5), the mass ratio of the amount of glacial acetic acid used to the mass of crude cholic acid was 4:1.

[0031] Table 1 shows the yields and purities of cholic acid measured in Examples 1 to Comparative Example 4. The total cholic acid content in the bovine bile powder used in the experiment was 42%. The cholic acid yield refers to the ratio of the mass of the finally prepared cholic acid to the mass of the raw bovine bile powder.

[0032]

[0033] As can be seen from the table, the purity of cholic acid prepared by the preparation method disclosed in the present invention is 92.4% - 98.7%, and the yield is 28.0% - 31.0%. In Comparative Example 1, the pH was adjusted to 13 after adding the alkali solution, and the purity of the finally prepared cholic acid was significantly reduced. Because the pH was too high, the added magnesium salts were all formed into magnesium hydroxide precipitates, and it was impossible to produce magnesium salt precipitates of deoxycholic acid complexes to achieve the purpose of removing deoxycholic acid substances. Therefore, the purity of the obtained cholic acid was only 85.3%; in Comparative Example 2, recrystallization was carried out with absolute ethanol. Since the solubility of absolute ethanol in cholic acid was low, while the solubility in deoxycholic acid impurities was high, the purity and yield of the obtained cholic acid were not high, the purity was 86.4%, and the yield was only 14.0%; in Comparative Example 3, the amount of absolute ethanol used for washing was too high, resulting in the loss of cholic acid, so the yield was low, only 21.5%; in Comparative Example 4, the amount of glacial acetic acid used was too high, which would cause part of the cholic acid dissolved in glacial acetic acid to be unable to crystallize, resulting in a low yield, only 21.3%. Therefore, the pH of the bovine bile powder solution before adding magnesium salts, and the amounts of absolute ethanol and glacial acetic acid used in the preparation method disclosed in the present invention are the key restrictive factors determining the purity and yield of cholic acid.

Claims

1. A method for preparing high-purity cholic acid, characterized in that, It includes the following steps: (1) Add bovine bile powder into an alkaline solution, heat and stir, and adjust the pH to 8 - 9 to obtain a bovine bile powder solution; (2) Add a magnesium salt solution into the bovine bile powder solution, heat and stir for reaction, and filter to obtain filtrate A; (3) Add an acidic solution into filtrate A, adjust the pH value to acidic, and filter to obtain precipitate A; (4) Wash precipitate A first with absolute ethanol, then with pure water, and dry to obtain crude cholic acid; (5) Add the crude cholic acid into glacial acetic acid, heat to dissolve, then cool down for crystallization, filter, wash with water, and dry to obtain high - purity cholic acid.

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

3. The preparation method according to claim 1, characterized in that In step (2), the magnesium salt solution is an aqueous magnesium chloride solution or an aqueous magnesium sulfate solution; the heating temperature is 80 - 90 °C, and the heating and stirring time is 2 - 4 hours.

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

5. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of absolute ethanol to precipitate A is 2 - 10:

1.

6. The preparation method according to claim 1, wherein In step (5), the mass ratio of the amount of glacial acetic acid used to the mass of crude cholic acid is 1 - 3:1.

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