Method for extracting bile acid from pig bile

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for extracting bile acid from pig bile, which comprises the following steps: (1) taking the pig bile, adjusting the pH value of the pig bile to be alkaline, heating the pig bile to 80-90 DEG C, stirring at constant temperature for 25-35 minutes, then cooling to room temperature, and adjusting the pH value of the system to be neutral to obtain pretreated pig bile; (2) filtering the pretreated pig bile in the step (1) by using a hyperbranched polymer film, concentrating filtrate under reduced pressure, adding an ethanol solution, heating to reflux, stirring for 25-35 minutes, and then filtering to obtain a crude bile acid product; and (3) adding a hydrochloric acid solution into the crude bile acid product in the step (2), adjusting the pH value to 2-3, centrifuging, filtering, washing and drying the obtained solid, adding ethyl acetate, heating to reflux until the solid is dissolved, cooling to room temperature, stirring for 2-4 hours, recrystallizing, separating and drying to obtain the bile acid. The extraction method is simple, and the extraction purity is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a method for extracting bile acids from pig bile. BACKGROUND

[0002] Pig bile, as an important raw material in the field of biopharmaceuticals, contains bilirubin, hyodeoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) which have significant medicinal value. Traditional processes mostly use single component extraction mode, but there are problems such as low resource utilization, serious environmental pollution, and high production cost. For example, the early extraction of bilirubin mainly relies on calcium salt method, which precipitates calcium ions and separates by acidification, but the process has a yield of only 1.5 per million, and needs multiple acid-base adjustments, which is complicated to operate; resin method improves the purity, but the cost of adsorption resin is high, and a large amount of wastewater is generated in the regeneration process; enzyme hydrolysis method has high efficiency in theory, but the extraction of special enzyme preparation is difficult, and its industrial application is limited.

[0003] For bile acid extraction, early processes simply simmered bile paste, resulting in high impurity content and serious loss of effective components. Subsequently, researchers proposed a saponification-esterification-step crystallization method to extract bile acids. For example, CN104041678B discloses "a bile acid composition and a preparation method thereof", which first uses pig bile powder as raw material, adds alkali solution for saponification, then adds hydrogen peroxide for decolorization, and finally adds hydrochloric acid for acidification, and then purifies to obtain bile acid. For another example, CN117801045A discloses "a separation and purification method of three kinds of bile acids in pig bile", which first uses pig bile without bilirubin as raw material, adds alkali and hydrogen peroxide for saponification and bleaching, then adds inorganic magnesium salt for filtration to obtain pig deoxycholic acid magnesium salt crude product, adds sodium hydroxide for ion exchange, then adjusts the pH to be acidic, and purifies to obtain pig deoxycholic acid product.

[0004] However, the above-mentioned bile acid extraction process is complicated and has low extraction purity, which is not suitable for industrial production. Therefore, it is urgent to develop a bile acid extraction method with simple extraction process and high extraction purity. SUMMARY

[0005] The main purpose of the present application is to provide a method for extracting bile acids from pig bile, which has a simple process and higher extraction purity.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] In one aspect, the present application provides a method for extracting bile acids from pig bile, comprising the following steps:

[0008] (1) taking pig gall, adjusting the pH of the pig gall to alkaline, heating the pig gall to 80-90℃, stirring at constant temperature for 25-35 min, then cooling to room temperature, adjusting the pH of the system to neutral, to obtain pretreated pig gall;

[0009] (2) the pretreated pig gall in step (1) is filtered by using a hyperbranched polymer membrane, the filtrate is concentrated under reduced pressure, ethanol solution is added and heated to reflux, stirred for 25-35 min, then filtered to obtain crude bile acid;

[0010] (3) hydrochloric acid solution is added to the crude bile acid in step (2), the pH is adjusted to 2-3, centrifuged, filtered, the obtained solid is washed with water and dried, then ethyl acetate is added and heated to reflux until the solid is dissolved, then cooled to room temperature, stirred for 2-4 h for recrystallization, separated and dried to obtain bile acid.

[0011] The extraction process of bile acid is currently relatively mature, and common bile acid extraction processes include salt method, resin method, rapid method, enzyme method, etc. However, how to extract high-purity bile acid through a simple process is a problem to be solved at present.

[0012] In the past, a large amount of acid and alkali agents were required in the extraction process of bile acid, resulting in a large amount of water and increased production difficulty, which made the process difficult to implement. In the present application, the saponified pig gall is filtered by using a hyperbranched polymer membrane, and then further acidified and purified to further improve the purity of the bile acid. The hyperbranched polymer membrane of the present application can be applied to the bile acid extraction process. The membrane can intercept substances with a molecular weight of 1000 and pass substances with a molecular weight of less than 900. Based on this, most of the impurities in the pretreated pig gall can be removed by membrane filtration, saving the cumbersome steps of the past, and improving the purity of the finally extracted bile acid.

[0013] In some embodiments, in step (1), the pH value of the alkaline is not less than 11.

[0014] In some embodiments, in step (2), the hyperbranched polymer membrane can intercept proteins, while passing bilirubin and bile acids.

[0015] In some embodiments, the preparation method of the hyperbranched polymer membrane comprises the following steps:

[0016] S1, mixing HPEI with acrylonitrile, heating to 35-45℃, then adding 4-carboxyphenylboronic acid, constant temperature stirring for 6-9 h, to obtain cyanoethyl-modified hyperbranched polyethyleneimine;

[0017] S2, mixing the aminated mesoporous silica with the cyanoethylated modified hyperbranched polyethylene imine in step S1, adding a cationic surfactant, and ultrasonic treatment for 30-50 min to obtain a composite sol;

[0018] S3, cooling the copper plate to -25 to -15 DEG C, smearing the composite sol in step S2, then naturally recovering to room temperature, mixing dipentaerythritol hexaacrylate, ethylene glycol, acetone and water uniformly to prepare a mixture, immersing the copper plate smeared with the composite sol into the mixture for 20-40 s, and irradiating under ultraviolet light for 10-20 min after taking out, and then peeling to obtain a hyperbranched polymer film.

[0019] In some embodiments, in step S1, the mass ratio of the HPEI to acrylonitrile is 1:(0.2-0.5).

[0020] The present application can avoid excessive crosslinking to cause the three-dimensional grid structure to have excessively high rigidity and thus cause the film elongation at break to decrease by regulating the mass ratio of the HPEI to acrylonitrile.

[0021] In some embodiments, in step S1, the mass of the 4-carboxyphenylboronic acid is 0.5-1% of the total mass of the HPEI and acrylonitrile.

[0022] The present application can make the film pore shrink moderately, and thus enable the film to retain substances with a molecular weight greater than 1000 and permeate substances with a molecular weight less than 900, and further prevent the film from having excessively high crosslinking density and thus having increased rigidity to cause the elongation at break to decrease by regulating the amount of the 4-carboxyphenylboronic acid added.

[0023] In some embodiments, in step S2, the mass ratio of the aminated mesoporous silica to the cyanoethylated modified hyperbranched polyethylene imine is (0.2-0.5):1.

[0024] In some embodiments, in step S3, the smearing thickness is 150-200 µm.

[0025] In some embodiments, in step S3, the mass ratio of the dipentaerythritol hexaacrylate, ethylene glycol, acetone and water is 1:1:(5-6):(12-15).

[0026] In some embodiments, in step (3), the concentration of the hydrochloric acid solution is 8-12 wt%.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] (1) The bile acid extraction method of the present application first pretreats pig bile by saponification, then filters it through a specific hyperbranched polymer film to remove impurities with a molecular weight greater than 1000, and then further purifies the crude bile acid obtained after filtration through acid washing, centrifugation and recrystallization to obtain bile acid. This method is simple to operate, suitable for process production, and the obtained bile acid is relatively pure.

[0029] (2) The hyperbranched polymer film of the present application can trap substances with a molecular weight greater than 1000 while allowing substances with a molecular weight less than 900 to pass through, and the hyperbranched polymer film has a high elongation at break. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0031] In the following preparation examples and examples, HPEI is purchased from Wuhan Kemik Biomedicine Technology Co., Ltd.; aminated mesoporous silica is purchased from Shaanxi Xingbei Aike Biological Technology Co., Ltd.

[0032] Preparation Example 1

[0033] The preparation method of the hyperbranched polymer film comprises the following steps:

[0034] S1, mix 100g HPEI with 35g acrylonitrile, heat to 40℃, then add 1g 4-carboxyphenylboronic acid, constant temperature stirring for 7.5h, to obtain cyanoethyl-modified hyperbranched polyethyleneimine;

[0035] S2, mix 35g aminated mesoporous silica with 100g cyanoethyl-modified hyperbranched polyethyleneimine in step S1, add 0.67g CTAB, ultrasonic treatment for 40min, to obtain a composite sol;

[0036] S3, cool the copper plate to -20℃, apply the composite sol in step S2 to a thickness of 170μm, then naturally restore to room temperature, mix 100g dipentaerythritol hexaacrylate, 100g ethylene glycol, 550g acetone and 1300g water uniformly to prepare a mixture, immerse the copper plate coated with the composite sol into the mixture for 30s, take it out and irradiate under ultraviolet light for 15min, after peeling, a hyperbranched polymer film is obtained.

[0037] Preparation Example 2

[0038] A method for preparing a hyperbranched polymer film, the specific embodiment being the same as in Preparation Example 1, except that acrylonitrile is 60 g.

[0039] Preparation Example 3

[0040] A method for preparing a hyperbranched polymer film, the specific embodiment being the same as in Preparation Example 1, except that 4-carboxyphenylboronic acid is 2.5 g.

[0041] Preparation Example 4

[0042] A method for preparing a hyperbranched polymer film, the specific embodiment being the same as in Preparation Example 1, except that 4-carboxyphenylboronic acid is 0.3 g.

[0043] Example 1

[0044] A method for extracting bile acids from pig bile, comprising the following steps:

[0045] (1) Take 100 g of pig bile, add sodium carbonate to adjust the pH of the pig bile to 12, heat the pig bile to 85°C, stir for 30 min at constant temperature, then cool to room temperature, adjust the pH of the system to 7, and obtain pretreated pig bile;

[0046] (2) Filter 100 g of the pretreated pig bile in step (1) using a hyperbranched polymer film, concentrate the filtrate under reduced pressure, add 600 mL of 95 wt% ethanol solution to heat to reflux, stir for 30 min, then filter to obtain crude bile acids;

[0047] (3) Add 2 mol / L hydrochloric acid solution to the crude bile acids in step (2), adjust the pH to 2, centrifuge, filter, and dry the obtained solid, then add 600 mL of ethyl acetate to heat to reflux until the solid dissolves, then cool to room temperature, stir for 3 h to recrystallize, separate and dry to obtain bile acids. The purity of the bile acids is 98.9%, and the yield is 85.7%.

[0048] The hyperbranched polymer film is prepared by Preparation Example 1.

[0049] Example 2

[0050] A method for extracting bile acids from pig bile, comprising the following steps:

[0051] (1) Take 100 g of pig bile, add sodium carbonate to adjust the pH of the pig bile to 11, heat the pig bile to 80°C, stir for 35 min at constant temperature, then cool to room temperature, adjust the pH of the system to 6, and obtain pretreated pig bile;

[0052] (2) The pretreated pig bile in step (1) is filtered by using the hyperbranched polymer membrane, the filtrate is concentrated under reduced pressure, 600 mL of ethanol solution with a concentration of 95 wt% is added, and the temperature is raised to reflux, stirred for 35 min, and then filtered to obtain a crude bile acid;

[0053] (3) The crude bile acid in step (2) is added with 2 mol / L hydrochloric acid solution, the pH is adjusted to 3, centrifuged, filtered, and the obtained solid is washed with water and dried, then 600 mL of ethyl acetate is added and the temperature is raised to reflux until the solid is dissolved, then cooled to room temperature, stirred for 2 h, recrystallized, separated and dried to obtain bile acid. The purity of the bile acid is 99.3%, and the yield is 84.1%.

[0054] The hyperbranched polymer membrane is prepared by the preparation example 1.

[0055] Example 3

[0056] A method for extracting bile acid from pig bile, comprising the following steps:

[0057] (1) 100 g of pig bile is taken, sodium carbonate is added to adjust the pH of the pig bile to 12, the pig bile is heated to 90℃, stirred at constant temperature for 25 min, then cooled to room temperature, and the pH of the system is adjusted to 8 to obtain pretreated pig bile;

[0058] (2) The pretreated pig bile in step (1) is filtered by using the hyperbranched polymer membrane, the filtrate is concentrated under reduced pressure, 600 mL of ethanol solution with a concentration of 95 wt% is added, and the temperature is raised to reflux, stirred for 25 min, and then filtered to obtain a crude bile acid;

[0059] (3) The crude bile acid in step (2) is added with 2 mol / L hydrochloric acid solution, the pH is adjusted to 3, centrifuged, filtered, and the obtained solid is washed with water and dried, then 600 mL of ethyl acetate is added and the temperature is raised to reflux until the solid is dissolved, then cooled to room temperature, stirred for 4 h, recrystallized, separated and dried to obtain bile acid. The purity of the bile acid is 97.3%, and the yield is 85.9%.

[0060] The hyperbranched polymer membrane is prepared by the preparation example 1.

[0061] Example 4

[0062] A method for extracting bile acid from pig bile, the specific implementation manner is the same as that of example 1, except that the hyperbranched polymer membrane is prepared by the preparation example 2. The purity of the bile acid is 98.9%, and the yield is 83.5%.

[0063] Example 5

[0064] A method for extracting bile acid from pig bile, the specific embodiment being the same as that of Example 1, except that the hyperbranched polymer membrane is prepared according to Preparation Example 3. The purity of the bile acid is 98.9%, and the yield is 77.9%.

[0065] Example 6

[0066] A method for extracting bile acid from pig bile, the specific embodiment being the same as that of Example 1, except that the hyperbranched polymer membrane is prepared according to Preparation Example 4. The purity of the bile acid is 91.4%, and the yield is 79.6%.

[0067] Comparative Example 1 Comparative Example 2

[0068] A method for extracting bile acid from pig bile, comprising the following steps:

[0069] (1) Take 100 g of pig bile, add sodium carbonate to adjust the pH of the pig bile to 12, heat the pig bile to 90°C, stir for 25 min at constant temperature, then cool to room temperature, adjust the pH of the system to 8, and obtain the pretreated pig bile;

[0070] (2) Add 2 mol / L hydrochloric acid solution to the crude bile acid in step (1), adjust the pH to 3, centrifuge, filter, and obtain the solid. After washing with water and drying, add 600 mL of ethyl acetate to warm to reflux until the solid dissolves, then cool to room temperature, stir for 4 h to recrystallize, and after separation and drying, obtain the bile acid. The purity of the bile acid is 70.7%, and the yield is 76.6%.

[0071] Results analysis:

[0072] The bile acid extracted by the methods of Examples 1-4 has high purity and yield; the purity of the bile acid extracted by the method of Example 5 is relatively high, but the yield is reduced; the purity and yield of the bile acid extracted by the method of Example 6 are both decreased; and the purity and yield of the bile acid in Comparative Example 1 are significantly decreased due to the absence of membrane filtration.

[0073] Performance test:

[0074] (1) Elongation at break: tested according to test standard ASTM D 882.

[0075] The hyperbranched polymer membranes of each preparation example are tested according to the above test method (1), and the results are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] According to the data in Table 1, the prepared hyperbranched polymer films of Preparation Examples 1 and 4 have higher elongation at break; the mass ratio of HPEI and acrylonitrile is changed in Preparation Example 2, which leads to the increase of crosslinking density of the film, thus the elongation at break of the film decreases; the addition amount of 4-carboxyphenylboronic acid is changed in Preparation Example 3, which also leads to the increase of crosslinking density of the film, thus the elongation at break of the film decreases.

[0080] Finally, it should be noted that the above detailed description is only used to illustrate the technical solutions of the present application, not to limit the present application. Although the present application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, which should be covered by the scope of the claims of the present application.

Claims

1. A method for extracting bile acids from pig bile, characterized by, The method comprises the following steps: (1) taking pig bile, adjusting the pH of the pig bile to be alkaline, heating the pig bile to 80-90℃, stirring for 25-35 min under constant temperature, then cooling to room temperature, adjusting the pH of the system to be neutral, and obtaining pretreated pig bile; (2) filtering the pretreated pig bile in step (1) by using an ultrabranched polymer membrane, concentrating the filtrate under reduced pressure, adding an ethanol solution, heating to reflux, stirring for 25-35 min, then filtering, and obtaining crude bile acid; (3) adding a hydrochloric acid solution to the crude bile acid in step (2), adjusting the pH to 2-3, centrifuging, filtering, drying the obtained solid after washing with water, adding ethyl acetate, heating to reflux until the solid is dissolved, then cooling to room temperature, stirring for 2-4 h for recrystallization, and drying to obtain bile acid.

2. The method of claim 1, wherein the method is characterized by, In step (1), the pH value of the alkaline is not less than 11.

3. The method of claim 1, wherein the method is characterized by, In step (2), the ultrabranched polymer membrane can intercept proteins, but pass through bilirubin and bile acid.

4. The method of claim 1, wherein the method is characterized by, The preparation method of the ultrabranched polymer membrane comprises the following steps: S1, mixing HPEI and acrylonitrile, heating to 35-45℃, then adding 4-carboxyphenylboronic acid, stirring for 6-9 h under constant temperature, and obtaining cyanoethyl-modified hyperbranched polyethyleneimine; S2, mixing amino-functionalized mesoporous silica and the cyanoethyl-modified hyperbranched polyethyleneimine in step S1, adding a cationic surfactant, and ultrasonically treating for 30-50 min to obtain a composite sol; S3, cooling a copper plate to -25 to -15℃, smearing the composite sol in step S2, then naturally recovering to room temperature, uniformly mixing dipentaerythritol hexaacrylate, ethylene glycol, acetone and water to prepare a mixed material, immersing the copper plate smeared with the composite sol into the mixed material for 20-40 s, then taking it out and irradiating under ultraviolet light for 10-20 min, and peeling off to obtain an ultrabranched polymer membrane.

5. The method of claim 4, wherein the method is characterized by, In step S1, the mass ratio of HPEI to acrylonitrile is 1:(0.2-0.5).

6. The method of claim 4, wherein the method is characterized by, In step S1, the mass of 4-carboxyphenylboronic acid is 0.5-1% of the total mass of HPEI and acrylonitrile.

7. The method of claim 4, wherein the method is characterized by, In step S2, the mass ratio of amino-functionalized mesoporous silica to cyanoethyl-modified hyperbranched polyethyleneimine is (0.2-0.5):

1.

8. The method of claim 4, wherein the method is characterized by, In step S3, the smearing thickness is 150-200 μm.

9. The method of claim 4, wherein the method is characterized by, In step S3, the mass ratio of dipentaerythritol hexaacrylate, ethylene glycol, acetone and water is 1:1:(5-6):(12-15).

10. The method of claim 1, wherein the method is characterized by, In step (3), the concentration of the hydrochloric acid solution is 8-12 wt%.

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