CN112062802A - Chenodeoxycholic acid butyl acetate extracting solution and preparation method thereof, and chenodeoxycholic acid ammonium salt and chenodeoxycholic acid preparation method - Google Patents
Chenodeoxycholic acid butyl acetate extracting solution and preparation method thereof, and chenodeoxycholic acid ammonium salt and chenodeoxycholic acid preparation method Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
Abstract
The invention discloses a preparation method of a chenodeoxycholic acid butyl acetate extracting solution, which comprises the following steps: dissolving the bile in sodium hydroxide aqueous solution, adding butyl acetate and hydrogen peroxide, and stirring to obtain a decolorized solution; taking the decolorized solution, adjusting the pH value to 5.90-6.10, standing for liquid separation, and taking an organic phase to obtain a chenodeoxycholic acid extract; adding sodium hydroxide aqueous solution into the chenodeoxycholic acid extract, standing for liquid separation, and taking an organic layer to obtain impurity washing liquid; and adding a sodium hydroxide aqueous solution into the impurity washing solution, standing for liquid separation, and taking an organic layer to obtain a chenodeoxycholic acid butyl acetate extracting solution. The chenodeoxycholic acid tert-butylammonium salt prepared by the preparation method is high in purity, and chenodeoxycholic acid is further prepared by the chenodeoxycholic acid tert-butylammonium salt with high purity, so that the purity of the product can be improved, the income of an enterprise is increased, and the preparation method provided by the invention is simple, is easy to operate and is suitable for large-scale production of the enterprise.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a chenodeoxycholic acid butyl acetate extracting solution, a preparation method thereof, and a preparation method of chenodeoxycholic acid ammonium salt and chenodeoxycholic acid.
Background
Chenodeoxycholic acid is mainly extracted or semi-synthesized from animal bile, the animal is mainly selected from chicken, goose, pig and/or duck gall, the chenodeoxycholic acid has the function of reducing the saturation degree of cholesterol in the bile, so that the chenodeoxycholic acid has a better treatment effect on the cholelithiasis, the chenodeoxycholic acid is also an important raw material for chemically synthesizing ursodeoxycholic acid, the ursodeoxycholic acid is a main effective component of bear gall, and the chenodeoxycholic acid has a definite curative effect on various diseases such as liver, gall and the like.
The existing preparation method for preparing chenodeoxycholic acid has more technical problems, for example, the existing preparation method has complicated steps and long refining process period, and some methods can involve toxic heavy metal barium salt during salification, so that the method is not convenient for large-scale production.
Disclosure of Invention
The invention provides a preparation method of chenodeoxycholic acid, which is used for solving the problems in the prior art that: the prior art has the technical problems of complex preparation method and long preparation period.
The invention aims to provide a method for preparing chenodeoxycholic acid butyl acetate extracting solution, which comprises the following steps:
s1 preparation of decolored solution
Dissolving the bile in sodium hydroxide aqueous solution, adding butyl acetate and hydrogen peroxide, and stirring to obtain a decolorized solution;
s2 preparing extract
Taking the decolorized solution, adjusting the pH value to 5.90-6.10, standing for liquid separation, and taking an organic phase to obtain a chenodeoxycholic acid extract;
s3 preparation of impurity washing liquid
Adding sodium hydroxide aqueous solution into the chenodeoxycholic acid extract, standing for liquid separation, and taking an organic layer to obtain impurity washing liquid;
s4 preparation of chenodeoxycholic acid butyl acetate extract
And adding a sodium hydroxide aqueous solution into the impurity washing solution, standing for liquid separation, and taking an organic layer to obtain a chenodeoxycholic acid butyl acetate extracting solution.
In order to better realize the method, in the process of preparing the extraction liquid by S2, the pH value is adjusted to 5.95-6.05.
In order to better realize the method, the step of preparing the extraction liquid by the S2 further comprises the steps of retreating the water phase after the refined liquid separation, adding butyl acetate into the water phase, adjusting the pH value to be 6.00-6.10, standing for liquid separation, taking the organic phase to obtain chenodeoxycholic acid extraction liquid, and combining the two obtained chenodeoxycholic acid extraction liquids.
The chenodeoxycholic acid butyl acetate extracting solution is prepared by the method.
When preparing a chenodeoxycholic acid butyl acetate extracting solution, the adopted bile paste is selected from chicken and/or goose bile paste, impurities in the bile paste are cholic acid, the content of the cholic acid is 20-40%, the amount of other impurities is relatively small, researchers firstly use a sodium hydroxide aqueous solution to dissolve and decolor the cholic acid through long-term research aiming at the characteristics of cholic acid impurities, the subsequent treatment is convenient, a decolored solution is obtained after dissolution and decoloration, in the dissolving process, 1 part of the bile paste is mixed with 1-2 parts of the sodium hydroxide aqueous solution by weight, 1.5-3 parts of butyl acetate by weight and 0.1-0.2 part of hydrogen peroxide by weight are stirred and decolored, and the decolored solution is obtained.
The pH value of the obtained decolored solution is adjusted, the pH value range is selected according to the characteristics of the decolored solution in the process, in the prior art, the adjustment range of the pH value is wide, long-term research shows that the pH value range is too wide to facilitate extraction, so that the extraction cannot meet the requirements, and the subsequent extraction solution is inconvenient to efficiently separate.
In the extraction process, most of the chenodeoxycholic acid butyl acetate solution stays in the organic layer, a small part of the chenodeoxycholic acid butyl acetate solution stays in the extracted water phase, butyl acetate is added into the water phase again, the pH value is adjusted again, the pH value is preferably 6.00-6.10, the organic layer is obtained again through standing and liquid separation, extraction liquid is obtained, and the two extraction liquid are combined to obtain more chenodeoxycholic acid extraction liquid.
In an extraction system, the concentrations of the chenodeoxycholic acid corresponding to the two extractions are different, so that the pH value in a range is selected, and the chenodeoxycholic acid is obtained through long-term experimental research, thereby being beneficial to the extraction.
And then adding a sodium hydroxide aqueous solution into the obtained chenodeoxycholic acid extract to wash the chenodeoxycholic acid extract, wherein the weight part of the sodium hydroxide aqueous solution is 0.8-2, the sodium hydroxide aqueous solution contains 0.0037-0.0057 weight part of sodium hydroxide, the impurity cholic acid in the chenodeoxycholic acid extract is effectively remained in a water layer with specific pH value through the washing, most of the chenodeoxycholic acid is remained in a butyl acetate layer in the washing process, then, simple liquid separation and standing are carried out, an organic phase and a water phase are separated, and the chenodeoxycholic acid butyl acetate impurity washing liquid is obtained through the organic phase.
And adding a sodium hydroxide aqueous solution into the chenodeoxycholic acid butyl acetate impurity washing liquid again, further washing, wherein the weight part of the sodium hydroxide aqueous solution is 0.8-2, the amount of sodium hydroxide in the sodium hydroxide aqueous solution is 0.0020-0.0043, and obtaining a purer chenodeoxycholic acid extracting solution through washing again with the sodium hydroxide aqueous solution.
And (3) detecting cholic acid in the obtained chenodeoxycholic acid extracting solution, wherein when the cholic acid content is higher than 2%, the chenodeoxycholic acid extracting solution needs to be washed by a sodium hydroxide aqueous solution again.
The chenodeoxycholic acid butyl acetate extracting solution is obtained by the preparation method, and the preparation method is simple to operate, low in cost and beneficial to large-scale production of enterprises.
The second purpose of the invention is to provide a preparation method of chenodeoxycholic acid amine salt, which comprises the step of carrying out salt forming reaction on an organic alkali and the chenodeoxycholic acid butyl acetate extracting solution of claim 4 to obtain the sec-butyl ammonium salt of chenodeoxycholic acid.
To better implement the present invention, further, the organic base is selected from tert-butylamine and sec-butylamine.
In order to better realize the method, the temperature in the salt forming reaction process is 0-70 ℃.
The second purpose of the invention is to carry out salt-forming reaction on the extract of butyl acetate chenodeoxycholate by using organic base, wherein the organic base is selected from sec-butylamine and tert-butylamine, preferably tert-butylamine, mainly because the tert-butylamine has high yield and strong selectivity after the salt-forming reaction, and particularly has good impurity removal effect.
The third purpose of the invention is to provide a preparation method of chenodeoxycholic acid, which comprises the steps of adding sodium hydroxide aqueous solution into the prepared chenodeoxycholic acid tert-butyl ammonium salt, stirring, heating, adjusting pH, separating out slurry-like solid, heating, stirring for 1h, filtering, washing with water, and drying to obtain the chenodeoxycholic acid.
Or adding sodium hydroxide aqueous solution into the prepared sec-butyl ammonium chenodeoxycholate, stirring, heating, adjusting pH, precipitating slurry solid, heating, stirring for 1h, filtering, washing with water, and drying to obtain chenodeoxycholic acid.
In order to better realize the invention, the temperature is further heated to 28-32 ℃, the pH value is adjusted to 3.5-4.5, and the temperature is raised to 38-45 ℃.
The temperature is increased to 28-32 ℃ before the acid is adjusted, the solid particles are small mainly because of low temperature, oily substances are easy to form when the temperature is high, the PH is adjusted to 3.5-4.5 after the temperature is increased, the temperature is increased for the next time, and the temperature is increased to 38-45 ℃, so that the operation is beneficial to increasing the size of the solid particles and improving the filtering speed.
The chenodeoxycholic acid prepared by the method has high yield and high purity.
Adding an organic solvent and water into a crude product of chenodeoxycholic acid obtained after salification for dissolving, then adding tert-butylamine, stirring, cooling to room temperature, filtering, leaching a filter cake with butyl acetate, and drying to obtain the tert-butylamine salt of chenodeoxycholic acid, wherein the molar ratio yield is usually higher than 98.8%, and long-term experimental research shows that: only a butyl acetate organic solvent is adopted, the yield is highest, and the salification yield sequence of the organic solvent is as follows: butyl acetate > ethyl acetate > acetone > ethanol, so butyl acetate is preferred.
The embodiment of the invention has the beneficial effects that: the method for preparing the chenodeoxycholic acid butyl acetate extracting solution has the advantages of simple preparation process, easy operation and low cost, and particularly the obtained chenodeoxycholic acid butyl acetate extracting solution has less impurities, so that more refined chenodeoxycholic acid ammonium salt and chenodeoxycholic acid can be obtained conveniently in the follow-up process. According to the invention, through the salt formation reaction of the chenodeoxycholic acid butyl acetate extract and an organic base, the organic base is preferably tert-butylamine, and the product obtained through the salt formation reaction has high yield and better impurity removal effect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments.
Thus, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a preparation method of sec-butylamine salt of chenodeoxycholic acid comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 20-30 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 20-30 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 20-30 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
And adding 12g of tert-butylamine into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, stirring for 1h, cooling to room temperature, stirring for 30min, filtering, and leaching a filter cake with 20ml of butyl acetate. The filter cake was air dried. Obtaining chenodeoxycholic acid tert-butylamine salt, wherein the molar yield of the salt forming step is 98.9 percent, and the purity of the obtained chenodeoxycholic acid tert-butylamine salt is 92.1 percent.
Example 2:
a preparation method of chenodeoxycholic acid tert-butylamine salt comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 20-30 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 20-30 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 20-30 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
And adding 12g of tert-butylamine into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, stirring for 1h, cooling to room temperature, stirring for 30min, filtering, and leaching a filter cake with 20ml of butyl acetate. The filter cake was air dried. The mole yield of the sec-butylamine salt of chenodeoxycholic acid is 95.1 percent in the salt forming step, and the purity of the sec-butylamine salt of chenodeoxycholic acid is 91.1 percent.
Comparative example 1:
a preparation method of chenodeoxycholic acid triethylamine salt comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 20-30 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 20-30 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 20-30 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
And adding 12g of triethylamine into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, stirring for 1h, cooling to room temperature, stirring for 30min, filtering, and leaching a filter cake with 20ml of butyl acetate. The filter cake was air dried. In the preparation process, crystallization is not generated, and the requirements are not met.
Comparative example 2:
a preparation method of n-butylamine chenodeoxycholic acid salt comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 20-30 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 20-30 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 20-30 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
And adding 12g of n-butylamine into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, stirring for 1h, cooling to room temperature, stirring for 30min, filtering, and leaching a filter cake with 20ml of butyl acetate. The filter cake was air dried. Sticky solids are obtained during the preparation process, which is not satisfactory.
Comparative example 3:
a preparation method of chenodeoxycholic acid diethylamine salt comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 20-30 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 20-30 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 20-30 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
And adding 12g of diethylamine into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, stirring for 1h, cooling to room temperature, stirring for 30min, filtering, and leaching a filter cake with 20ml of butyl acetate. The filter cake was air dried. Does not crystallize and does not meet the requirement.
Example 3:
the t-butylamine chenodeoxycholate salt obtained in example 2 was dissolved in 250ml of water with 4g of sodium hydroxide, and the mixture was stirred to dissolve, heated to 30 ℃ and adjusted to pH 4 with 60% sulfuric acid to precipitate a slurry solid. The temperature in the reactor is increased by gradient and is increased by 41 ℃ until the reactor is stood still and can quickly and obviously delaminate, the reactor is stirred for 1h, and the system is filtered and washed to be neutral by tap water. The solid obtained was dried in a forced air drying oven, the temperature being set at 50 ℃. The chenodeoxycholic acid is obtained, and the content of the chenodeoxycholic acid is 92.1 percent.
Example 4:
a preparation method of sec-butylamine salt of chenodeoxycholic acid comprises the following steps:
s1 preparation of decolored solution
20g of sodium hydroxide is weighed and dissolved in 200g of water, and 150g of chicken bile paste (the content of cholic acid is 25 percent) is added. Heating to above 70 deg.C, and stirring until the bile paste is completely dissolved. When the system is completely dissolved, the system is cooled to the internal temperature of 40 ℃. 320ml (282g) of butyl acetate was added thereto, and 20g of 30% hydrogen peroxide was added dropwise. And after the dropwise addition, stirring for 3 hours at the temperature of 20-30 ℃ to obtain a decolorized solution.
S2 preparing extract
And (3) adjusting the pH of the decolorized solution obtained in the step (S1) to 5.95-6.05 by using 60% sulfuric acid, and stirring for 0.5h at the temperature of 50-60 ℃ after the pH is adjusted. Standing and separating liquid; separating an aqueous phase and an organic phase, wherein the organic phase is chenodeoxycholic acid butyl acetate extract, adding 80ml (70.5g) of butyl acetate into the aqueous phase, and adjusting and controlling the pH value to be 6.00-6.10 by using 60% sulfuric acid. And after the adjustment is finished, stirring for 0.5h at the normal temperature of 20-30 ℃, standing and separating to obtain an organic layer, and combining the two organic layers to obtain the chenodeoxycholic acid extract.
S3 preparation of impurity washing liquid
The chenodeoxycholic acid extract was added to a prepared sodium hydroxide solution (0.8g in 150g of water). Stirring for 0.5h at the normal temperature of 50-60 ℃. And (5) standing and separating. Collecting the upper organic layer to obtain the impurity washing liquid.
S4 preparation of chenodeoxycholic acid butyl acetate extract
Adding the impurity washing solution into a prepared sodium hydroxide solution (0.5g is dissolved in 150g of water), stirring for 0.5h at the normal temperature of 50-60 ℃, standing for liquid separation, and separating an organic phase and an aqueous phase. The organic layer is a butyl acetate layer, and the prepared sodium hydroxide solution (0.4g is dissolved in 150g of water) is added into the obtained organic phase and stirred for 0.5h at the normal temperature of 20-30 ℃. Standing, separating, collecting organic phase again to obtain chenodeoxycholic acid butyl acetate extract solution, and detecting the peak area ratio of cholic acid to chenodeoxycholic acid to be 1.6% by HPLC.
S5 salt-forming reaction
8g of tert-butylamine is added into the obtained chenodeoxycholic acid butyl acetate extraction solution at 60 ℃, the mixture is stirred for 1h, then the temperature is reduced to room temperature, the mixture is stirred for 30min, the filtration is carried out, and a filter cake is leached by 20ml of butyl acetate. The filter cake was air dried. Obtaining chenodeoxycholic acid tert-butylamine salt, wherein the molar yield of the salt forming step is 98.8 percent, and the purity of the obtained chenodeoxycholic acid tert-butylamine salt is 92.1 percent.
The chenodeoxycholic acid tert-butylammonium salt prepared by the preparation method is high in purity, and chenodeoxycholic acid is further prepared by the chenodeoxycholic acid tert-butylammonium salt with high purity, so that the purity of the product can be improved, the income of an enterprise is increased, and the preparation method provided by the invention is simple, is easy to operate and is suitable for large-scale production of the enterprise.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A method for preparing chenodeoxycholic acid butyl acetate extract is characterized by comprising the following steps: the method comprises the following steps:
s1 preparation of decolored solution
Dissolving the bile in sodium hydroxide aqueous solution, adding butyl acetate and hydrogen peroxide, and stirring to obtain a decolorized solution;
s2 preparing extract
Taking the decolorized solution, adjusting the pH value to 5.90-6.10, standing for liquid separation, and taking an organic phase to obtain a chenodeoxycholic acid extract;
s3 preparation of impurity washing liquid
Adding sodium hydroxide aqueous solution into the chenodeoxycholic acid extract, standing for liquid separation, and taking an organic layer to obtain impurity washing liquid;
s4 preparation of chenodeoxycholic acid butyl acetate extract
And adding a sodium hydroxide aqueous solution into the impurity washing solution, standing for liquid separation, and taking an organic layer to obtain a chenodeoxycholic acid butyl acetate extracting solution.
2. The method of claim 1, wherein: and in the process of preparing the extraction liquid by S2, adjusting the pH value to 5.95-6.05.
3. The method of claim 1, wherein: and in the process of preparing the extraction liquid by S2, the water phase after the fine liquid separation is treated again, butyl acetate is added into the water phase, the pH value is adjusted to be 6.00-6.10, the mixture is kept stand for liquid separation, an organic phase is taken to obtain chenodeoxycholic acid extraction liquid, and the two chenodeoxycholic acid extraction liquids are combined.
4. The preparation method of claims 1-3, wherein the chenodeoxycholic acid butyl acetate extract is prepared.
5. A preparation method of chenodeoxycholic acid amine salt is characterized by comprising the following steps: comprises the step of carrying out salt forming reaction on organic alkali and the butyl chenodeoxycholate acetate extracting solution of claim 4 to obtain sec-butyl ammonium chenodeoxycholate.
6. The method of claim 5, wherein: the organic base is selected from tert-butylamine and sec-butylamine.
7. The production method according to claim 5 or 6, characterized in that: the temperature in the salification reaction process is 0-70 ℃.
8. A preparation method of chenodeoxycholic acid is characterized by comprising the following steps: adding sodium hydroxide aqueous solution into the chenodeoxycholic acid ammonium salt prepared in the claim 5, stirring, heating, adjusting pH, separating out a slurry solid, heating, stirring for 1h, filtering, washing with water, and drying to obtain the chenodeoxycholic acid.
9. The method of claim 8, wherein: heating to 28-32 ℃, adjusting the pH value to 3.5-4.5, and heating to 38-45 ℃.
10. The method for preparing chenodeoxycholic acid according to claim 8.
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