Method for preparing high-purity 7-ketolithocholic acid from pig bile

Abstract

The invention discloses a method for preparing high-purity 7-ketolithocholic acid from pig bile, which comprises the following steps: blanking the pig bile from which bilirubin is extracted, and saponifying and decolorizing; hyodeoxycholic acid in the decoloring solution is separated with magnesium salt, and chenodeoxycholic acid and hyocholic acid filtrate are obtained; neutralizing the filtrate, dissolving in a system of ethanol and water, and oxidizing to obtain a 7-ketolithocholic acid crude product; the method comprises the following steps: dissolving a 7-ketolithocholic acid crude product in ethanol water, and adding n-hexane for crystallization to obtain a 7-ketolithocholic acid primary crystal substance; and dissolving the 7-ketolithocholic acid primary crystal in ethanol water, and adding n-hexane for crystallization to obtain a 7-ketolithocholic acid refined product with the concentration of 95% or above. According to the method, the solubility of each bile acid after oxidation is different, the intermediate product 7-ketolithocholic acid is directly purified and separated from the pig bile, the three bile acids in the pig bile do not need to be completely separated, the process steps are relatively short, the adopted solvent is easy to treat, the product purity is high, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the preparation of 7-ketolithocholic acid, and in particular to a method for preparing high-purity 7-ketolithocholic acid from pig bile. Background Art

[0002] The main components of pig bile are hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid, and hyodeoxycholic acid accounts for 40%-50% of pig bile acid, hyocholic acid accounts for 10%-15% of pig bile acid, and chenodeoxycholic acid accounts for 25%-30% of bile acid. Among them, chenodeoxycholic acid is the first-generation litholytic drug used for cholesterol gallstones, and is also the main raw material of 7-ketolithocholic acid. Since the process of separating chenodeoxycholic acid from pig bile is relatively cumbersome and complicated, most of the 7-ketolithocholic acid on the market is obtained by oxidizing chenodeoxycholic acid derived from chicken or duck bile, resulting in a great waste of bile acid in pig bile.

[0003] 7-Ketolithocholic acid, whose chemical name is 3α-hydroxy-7-oxo-5β-cholestane-24-oic acid (7K-LCA for short), is an important intermediate for the synthesis of ursodeoxycholic acid and obeticholic acid. Ursodeoxycholic acid is a common litholytic drug that can inhibit the synthesis of cholesterol in the liver and has a good therapeutic effect on a variety of hepatobiliary diseases. Obeticholic acid can inhibit the synthesis of bile acid and is used to treat primary biliary cirrhosis (PBC) and non-alcoholic fatty liver disease. Therefore, the preparation of high-purity 7-ketolithocholic acid is of great significance and value to medical research. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a method for preparing high-purity 7-ketolithocholic acid in pig bile.

[0005] To achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing high-purity 7-ketolithocholic acid from pig bile, comprising the following process steps: (1) The pig bile from which bilirubin is extracted is fed and subjected to saponification and decolorization; (2) separating the chenodeoxycholic acid in the decolorizing solution of step (1) with magnesium salt to obtain chenodeoxycholic acid and chenodeoxycholic acid filtrate; (3) neutralizing the filtrate of step (2), dissolving it in a system of ethanol and water, and then oxidizing it to obtain a crude product of 7-ketolithocholic acid; (4) dissolving the crude 7-ketolithocholic acid obtained in step (3) in ethanol water, adding n-hexane for crystallization, and obtaining primary crystals of 7-ketolithocholic acid; (5) Repeat step (4), dissolve the primary crystals of 7-ketolithocholic acid in ethanol water, add n-hexane for crystallization, and obtain 7-ketolithocholic acid with a purity of more than 95%.

[0006] Furthermore, the saponification decolorization in step (1) is to add sodium hydroxide to the pig bile waste after bilirubin extraction, adjust the pH value to above 12, heat the saponification reflux, and then add hydrogen peroxide to react for 2 hours for decolorization.

[0007] Furthermore, the saponification temperature in step (1) is 95-100°C, the saponification reflux time is 24 hours, and the temperature is cooled to 70°C after 24 hours of saponification reflux. The amount of hydrogen peroxide added during decolorization is 0.1-0.2% of the amount of pig bile waste.

[0008] Furthermore, the preparation process of the chenodeoxycholic acid and hyodeoxycholic acid filtrate in step (2) is as follows: the decolorized solution obtained in step (1) is adjusted to a pH value of 7-10 with hydrochloric acid, and then a weak base is added to adjust the pH value of the system to 9-11, the temperature is controlled at 50-70°C, and an inorganic magnesium salt is added with stirring to prepare a 30% concentration aqueous solution. After stirring for 2 hours, the mixture is cooled and filtered to obtain a crude product of hyodeoxycholic acid magnesium salt and a primary magnesium salt mother liquor.

[0009] Furthermore, the inorganic magnesium salt in step (2) is magnesium sulfate or magnesium chloride, and the amount of the inorganic magnesium salt added is 1.5-2.0% of the pig bile waste.

[0010] Furthermore, the oxidation process of the crude 7-ketolithocholic acid in step (3) is as follows: take the primary magnesium salt mother liquor in step (2), adjust the pH value of the system to 2-3 with hydrochloric acid, filter to obtain crude chenodeoxycholic acid and hyocholic acid, add ethanol and water to dissolve, and then add inorganic acid and sodium hypochlorite for oxidation to obtain.

[0011] Furthermore, in step (3), the amount of ethanol added is 10-15 times that of the crude products of chenodeoxycholic acid and hyocholic acid; and the mass fraction of ethanol in the ethanol-water system is 60%-70%.

[0012] Furthermore, the inorganic acid in step (3) is hydrochloric acid or phosphoric acid, the amount of sodium hypochlorite added as an oxidant is 1.1-1.3 times that of the crude products of chenodeoxycholic acid and hyocholic acid, and the effective chlorine content of the sodium hypochlorite is 10%.

[0013] Furthermore, the amount of the ethanol-water solvent in step (4) is 15-20 times that of the crude 7-keto-lithocholic acid, and the mass fraction of ethanol in the ethanol-water solvent is 80%-90%; the temperature at which the ethanol-water solvent dissolves the crude 7-keto-lithocholic acid is 50°C-60°C.

[0014] Furthermore, in step (4), the amount of n-hexane added is 0.4-0.5 times that of ethanol, and the solvent temperature when n-hexane is added is 40°C-45°C, and the crystallization temperature is 0°C-5°C.

[0015] Compared with the prior art, the advantages of the present invention are: full use of pig bile scraps, reduced waste, and in the preparation process, it is not necessary to completely separate the three bile acids in the pig bile, and after purifying chenodeoxycholic acid through multiple purification processes, 7-keto-lithocholic acid is prepared. Instead, the different solubilities of the bile acids after oxidation are utilized to directly purify and separate the intermediate product 7-keto-lithocholic acid from the pig bile, and the solvent used in the entire process route is easy to handle, the process steps are relatively short, the product purity is high, and it is suitable for industrial production. DETAILED DESCRIPTION

[0016] The technical solution in the method of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0017] The method for preparing high-purity 7-ketolithocholic acid from pig bile of the present invention comprises the following process steps: (1) adding sodium hydroxide to the pig bile waste after bilirubin extraction, adjusting the pH value to above 12, and performing saponification. During the saponification process, the saponification temperature is controlled at 95-100°C. After saponification reflux for 24 hours, the mixture is cooled to 70°C, and hydrogen peroxide in an amount of 0.1-0.2% of the pig bile waste is added, and the reaction is performed for 2 hours to complete decolorization; (2) The decolorized liquid obtained in step (1) is adjusted to a pH value of 7-10 with hydrochloric acid, and then a weak base is added to adjust the pH value of the system to 9-11, the temperature is controlled at 50-70° C., and magnesium sulfate or magnesium chloride is added with stirring, wherein the amount of magnesium sulfate or magnesium chloride added is 1.5-2.0% of the amount of pig bile waste, and a 30% concentration aqueous solution is prepared. After stirring for 2 hours, the mixture is cooled and filtered to obtain a crude product of hyodeoxycholic acid magnesium salt and a primary magnesium salt mother liquor; (3) taking the primary magnesium salt mother liquor in step (2), adjusting the pH value of the system to 2-3 with hydrochloric acid, filtering to obtain crude products of chenodeoxycholic acid and hyodolic acid, adding ethanol and water thereto, wherein in the ethanol-water system, the amount of ethanol-water solvent is 10-15 times that of the crude products of chenodeoxycholic acid and hyodolic acid, and the mass fraction of ethanol is 60%-70%; after dissolution, adding hydrochloric acid or phosphoric acid, adding sodium hypochlorite in an amount of 1.1-1.3 times that of the crude products of chenodeoxycholic acid and hyodolic acid, and performing oxidation to obtain crude products of 7-keto-lithocholic acid; (4) dissolving the crude 7-keto-lithocholic acid product of step (3) in ethanol water, wherein the amount of ethanol water solvent in the ethanol water system is 15-20 times that of the crude 7-keto-lithocholic acid product, the mass fraction of ethanol is 80%-90%, and the temperature of the ethanol water solvent for dissolving the crude 7-keto-lithocholic acid product is 50°C-60°C; after dissolution, adding n-hexane for crystallization, the amount of n-hexane added is 0.4-0.5 times that of ethanol, and the solvent temperature when n-hexane is added is 40°C-45°C, and the crystallization temperature is 0°C-5°C, to obtain a primary crystal of 7-keto-lithocholic acid; (5) Repeat step (4) to dissolve the primary crystals of 7-ketolithocholic acid in ethanol water, wherein the amount of ethanol water solvent in the ethanol water system is 15-20 times that of the primary crystals of 7-ketolithocholic acid, the mass fraction of ethanol is 80%-90%, and the temperature of the ethanol water solvent for dissolving the primary crystals of 7-ketolithocholic acid is 50°C-60°C; after dissolution, n-hexane is added for crystallization, the amount of n-hexane added is 0.4-0.5 times that of ethanol, and the solvent temperature when n-hexane is added is 40°C-45°C, and the crystallization temperature is 0°C-5°C, thereby obtaining more than 95% of the fine product of 7-ketolithocholic acid. Embodiment 1

[0018] Take 1000g of pig bile after bilirubin extraction, add 200g of sodium hydroxide, adjust the pH value to above 12, control the temperature at 95°C, saponify and reflux for 24 hours, then cool to 70°C, add 10g of hydrogen peroxide, and react for 2 hours to complete decolorization; The temperature of the decolorizing solution was controlled at 70°C, the pH value of the system was adjusted to 10 with hydrochloric acid, the pH value of the system was adjusted to 11 with a weak base, 60g of a 30% magnesium sulfate and water mixed solution was added, and the mixture was stirred for reaction for 2 hours, and then cooled to room temperature and filtered to obtain a crude product of hyodeoxycholic acid magnesium salt and a primary magnesium salt mother liquor; The primary magnesium salt mother liquor was adjusted to a pH value of 2 with hydrochloric acid, and filtered to obtain crude chenodeoxycholic acid and crude hyocholic acid, which were then dried in vacuo to obtain 45 g of a sample; 450 g of a 60% ethanol aqueous solution was added to the above product, heated to 40°C to dissolve, cooled to 5°C, 100 ml of 15% hydrochloric acid was added, stirred for 5 min, 49.5 g of a 10% sodium hypochlorite solution was slowly added, the temperature was controlled to react for 6 h, the degree of reaction was detected by high performance liquid chromatography, 100 g of water was added, stirring was continued for 2 h, crystallization was fully allowed to proceed, filtered, and dried in vacuo to obtain 36.8 g of a crude 7-ketolithocholic acid; To the crude 7-ketolithocholic acid, 552 g of an ethanol aqueous solution with a mass fraction of 80% was added, the temperature was raised to 50°C to dissolve, and then the temperature was lowered to 40°C, 221 g of n-hexane was added, the temperature was continued to be lowered to 5°C, the mixture was stirred for crystallization for 2 hours, suction filtered, and vacuum dried to obtain 27.6 g of 7-ketolithocholic acid crystals; to the above 7-ketolithocholic acid crystals, 276 g of an ethanol aqueous solution with a mass fraction of 80% was added, the temperature was raised to 50°C to dissolve, the temperature was reduced to 40°C, 111 g of n-hexane was added, the temperature was continued to be lowered to 5°C, the mixture was stirred for crystallization for 2 hours, suction filtered, and vacuum dried to obtain 22.3 g of 7-ketolithocholic acid fine product, and its purity was detected by high performance liquid chromatography to be above 98%. Embodiment 2

[0019] Take 1000g of pig bile after bilirubin extraction, add 200g of sodium hydroxide, adjust the pH value to above 12, control the temperature at 100°C, saponify and reflux for 24 hours, cool to 70°C, add 20g of hydrogen peroxide, and react for 2 hours to complete decolorization; The temperature of the decolorizing solution was controlled at 60°C, the pH value of the system was adjusted to 10 with hydrochloric acid, the pH value of the system was adjusted to 11 with a weak base, 60g of a 30% magnesium chloride and water mixed solution was added, and the mixture was stirred for reaction for 2 hours, and then cooled to room temperature and filtered to obtain a crude product of hyodeoxycholic acid magnesium salt and a primary magnesium salt mother liquor; The primary magnesium salt mother liquor was adjusted to a pH value of 3 with hydrochloric acid, and filtered to obtain crude chenodeoxycholic acid and crude hog bile acid, which were vacuum dried to obtain 43.5 g of a sample; 652 g of a 70% ethanol aqueous solution was added to the above product, heated to 40°C to dissolve, cooled to 5°C, 100 ml of 15% phosphoric acid was added, stirred for 5 min, 56.5 g of a 10% sodium hypochlorite solution was slowly added, the temperature was controlled to react for 6 h, the reaction degree was detected by high performance liquid chromatography, 100 g of water was added, stirring was continued for 2 h, crystallization was fully allowed to proceed, filtered, and vacuum dried to obtain 35.2 g of a crude 7-ketolithocholic acid; To the crude 7-ketolithocholic acid, 704 g of 90% ethanol aqueous solution was added, the temperature was raised to 60°C to dissolve, and then the temperature was lowered to 45°C, 352 g of n-hexane was added, the temperature was continued to be lowered to 5°C, the mixture was stirred for crystallization for 2 hours, suction filtered, and vacuum dried to obtain 25.7 g of 7-ketolithocholic acid crystals; to the above 7-ketolithocholic acid crystals, 514 g of 90% ethanol aqueous solution was added, the temperature was raised to 60°C to dissolve, the temperature was reduced to 45°C, 257 g of n-hexane was added, the temperature was continued to be lowered to 5°C, the mixture was stirred for crystallization for 2 hours, suction filtered, and vacuum dried to obtain 20.1 g of 7-ketolithocholic acid fine product, and its purity was detected by high performance liquid chromatography to be above 98%.

[0020] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing high-purity 7-ketolithocholic acid from pig bile, characterized in that: The process steps include: (1) The pig bile from which bilirubin is extracted is fed and subjected to saponification decolorization; (2) separating the chenodeoxycholic acid in the decolorizing solution of step (1) with magnesium salt to obtain chenodeoxycholic acid and chenodeoxycholic acid filtrate; (3) neutralizing the filtrate of step (2), dissolving it in a system of ethanol and water, and then oxidizing it to obtain a crude product of 7-ketolithocholic acid; (4) dissolving the crude 7-ketolithocholic acid obtained in step (3) in ethanol water, adding n-hexane for crystallization, and obtaining primary crystals of 7-ketolithocholic acid; (5) Repeat step (4), dissolve the primary crystals of 7-ketolithocholic acid in ethanol water, add n-hexane for crystallization, and obtain more than 95% of 7-ketolithocholic acid fine product.

2. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The saponification decolorization described in step (1) is to add sodium hydroxide to the pig bile scraps after bilirubin extraction, adjust the pH value to above 12, heat the saponification reflux, and then add hydrogen peroxide to react for 2 hours for decolorization.

3. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 2, characterized in that: The saponification temperature in step (1) is 95-100°C, the saponification reflux time is 24 hours, and the temperature is cooled to 70°C after 24 hours of saponification reflux. The amount of hydrogen peroxide added during decolorization is 0.1-0.2% of the amount of pig bile waste.

4. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The preparation process of the chenodeoxycholic acid and hyodeoxycholic acid filtrate in step (2) is as follows: the decolorized solution obtained in step (1) is adjusted to a pH value of 7-10 with hydrochloric acid, and then a weak base is added to adjust the pH value of the system to 9-11, the temperature is controlled at 50-70° C., an inorganic magnesium salt is added with stirring to prepare a 30% concentration aqueous solution, and the mixture is stirred for reaction for 2 hours and then cooled and filtered to obtain a crude product of hyodeoxycholic acid magnesium salt and a primary magnesium salt mother liquor.

5. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 4, characterized in that: The inorganic magnesium salt described in step (2) is magnesium sulfate or magnesium chloride, and the amount of the inorganic magnesium salt added is 1.5-2.0% of the pig bile waste.

6. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The oxidation process of the crude 7-ketolithocholic acid in step (3) is as follows: take the primary magnesium salt mother liquor in step (2), adjust the pH value of the system to 2-3 with hydrochloric acid, filter to obtain crude chenodeoxycholic acid and hyocholic acid, add ethanol and water to dissolve, and then add inorganic acid and sodium hypochlorite for oxidation to obtain crude chenodeoxycholic acid and hyocholic acid.

7. A method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 6, characterized in that: In step (3), the amount of ethanol added is 10-15 times that of the crude products of chenodeoxycholic acid and hyodolic acid; and the mass fraction of ethanol in the ethanol-water system is 60%-70%.

8. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 7, characterized in that: The inorganic acid described in step (3) is hydrochloric acid or phosphoric acid, and the amount of sodium hypochlorite added as an oxidant is 1.1-1.3 times that of the crude products of chenodeoxycholic acid and hyocholic acid, and the effective chlorine content of the sodium hypochlorite is 10%.

9. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 1, characterized in that: The amount of the ethanol-water solvent in step (4) is 15-20 times that of the crude 7-keto-lithocholic acid, and the mass fraction of ethanol in the ethanol-water solvent is 80%-90%; the temperature at which the ethanol-water solvent dissolves the crude 7-keto-lithocholic acid is 50°C-60°C.

10. The method for extracting bilirubin, bile acid and chenodeoxycholic acid from chicken bile according to claim 9, characterized in that: In step (4), the amount of n-hexane added is 0.4-0.5 times that of ethanol, and the solvent temperature when n-hexane is added is 40°C-45°C, and the crystallization temperature is 0°C-5°C.