CN102286051A - Method for separating chenodeoxycholic acid from ursodesoxycholic acid - Google Patents
Method for separating chenodeoxycholic acid from ursodesoxycholic acid Download PDFInfo
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Abstract
The invention discloses a method for separating chenodeoxycholic acid from ursodesoxycholic acid, which belongs to the technical field of biochemical separation and is characterized by comprising the following steps in turn: (1) preparing column loading solution, namely dissolving mixture of ursodesoxycholic acid and chenodeoxycholic acid in alkaline aqueous solution according an equivalence ratio of 1:2.5, regulating the pH value of the solution to 7 to 8, and diluting deionized water till the solid content is 10 to 20 percent; (2) loading onto a column for absorption, namely pumping the column loading solution into a resin column filled with nonpolar porous synthetic absorbent; (3) desorbing, namely preparing eluting solution by using water soluble organic solvent, and pumping the eluting solution into the resin column for desorbing; and (4) performing post-treatment, namely concentrating under vacuum, recovering solvent, adding acid solution into the concentrated product to regulate the pH value to 2 to 3, precipitating, filtering, washing till the pH value is 7, drying, crystallizing and drying under vacuum to obtain a finished product. The method has the advantages that: the macroporous resin has high separation and purification performance and high accuracy; the ursodesoxycholic acid is separated from chenodeoxycholic acid with high yield; the process is simple; the production cost is low; and the industrial production efficiency is high.
Description
Technical field
The present invention relates to the separation method of a kind of Chenodiol and ursodesoxycholic acid, particularly a kind of method of separating Chenodiol and ursodesoxycholic acid with macroporous adsorbent resin belongs to the biochemical industry separation technology field.
Background technology
Ursodesoxycholic acid is a kind of avirulent hydrophilic cholic acid, and multiple disease is had therapeutic action.Ursodesoxycholic acid is used to treatment of diseases such as primary sclerosing cholangitis, cholesterol gall stone, bile reflux gastritis and courage source property film adenositis at present.In addition, ursodesoxycholic acid also is the choice drug of cholestasis hepatopathy, and is used to improve the surviving rate of liver transplantation postoperative.
Ursodesoxycholic acid is the distinctive bile acide composition of bear class, and structural formula is shown in general formula ⑴:
Ursodesoxycholic acid is mainly obtained by two kinds of methods at present.First kind is to culture bear, obtains the bile of bear then by the live body drainage, extracts ursodesoxycholic acid again from bile.The cycle of this method is longer, and efficient is not high, and obvious to the actual bodily harm of bear.Second kind is by other raw materials, as the synthetic ursodesoxycholic acid of Chenodiol.
Chenodiol is the isomers of ursodesoxycholic acid, and itself also can be used for the treatment of cholesterol gall stone.But compare ursodesoxycholic acid, Chenodiol treatment gallbladdergallstonecholetithiasis is easy to generate side effects such as diarrhoea, hepatic disorder, serum cholesterol concentration increase.The structural formula of Chenodiol is shown in general formula ⑵:
⑵
Because Chenodiol can obtain from chicken courage, duck courage and pig courage, it is abundanter to originate, so industrial common more application is from the synthetic ursodesoxycholic acid of Chenodiol.But the reaction conversion ratio by the synthetic ursodesoxycholic acid of Chenodiol can only reach 50%～75%, in order to reduce Side effects of pharmaceutical drugs and to improve utilization ratio of raw materials, Chenodiol must be separated with ursodesoxycholic acid with Chenodiol in the ursodeoxycholic acid mixture.Present industrial employing organic solvent optionally dissolves impurity such as removing Chenodiol from the mixture that contains ursodesoxycholic acid and Chenodiol.But the employing solvent method has shortcomings such as the throughput of unit solvent is lower, and solvent load is big, and solvent toxicity is big, and product yield is low.
Summary of the invention
The objective of the invention is to improve utilization ratio of raw materials, meet economy, environmental requirement, a kind of method of separating Chenodiol and ursodesoxycholic acid with macroporous adsorbent resin is provided for overcoming the shortcoming of solvent method.
For achieving the above object, the technical scheme of employing is: the separation method of a kind of Chenodiol and ursodesoxycholic acid is characterized in that comprising successively step:
⑴ preparation upper prop liquid: get the mixture of ursodesoxycholic acid and Chenodiol, be dissolved in the alkali aqueous solution, adjusting pH is 7-8, and adding deionized water then, to be diluted to solid content be 10%-20%;
⑵ upper prop absorption: step ⑴ gained upper prop liquid pump is gone into to load in the resin column of nonpolar porous synthetic adsorbent;
⑶ desorb: the water-miscible organic solvent that with the percent by volume is 20%-30% disposes first eluent, dispose second eluent with the pure water solubleness organic solvent, respectively three times of column volume first eluents and two times of column volume second eluents are pumped into desorb in the adsorption resin column, flow velocity be 1 times of column volume/hour;
⑷ aftertreatment:, reclaim solvent with the stripping liquid vacuum concentration of step ⑶ gained; Enriched material adds the inorganic acid fluid adjust pH to 2-3, separates out precipitation, filters, and washing is extremely neutral, drying, and crystallization, vacuum-drying can get finished product.
The described alkali aqueous solution of step ⑴ is sodium hydroxide, potassium hydroxide, or ammoniacal liquor.
The described nonpolar porous synthetic adsorbent of step ⑵ is macroporous adsorbent resin HZ816, HZ818, HZ830.
The described water-miscible organic solvent of step ⑶ is a methyl alcohol, ethanol, n-propyl alcohol, Virahol, the trimethyl carbinol, dioxan, tetrahydrofuran (THF), acetone, dimethyl formamide, methyl-sulphoxide etc.
The described inorganic acid fluid of step ⑷ is a hydrochloric acid, sulfuric acid, or nitric acid.
The invention has the beneficial effects as follows, macroporous adsorbent resin separation and purification performance is good, serially or off and on from the mixture of ursodeoxycholic hydrochlorate and CDC, high precision, separate ursodeoxycholic hydrochlorate and CDC with high yield, technology is simple, and production cost is low, industrial production efficient height.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, and the technical scheme that the present invention relates to is described, discloses preferred forms of the present invention, makes those of ordinary skill in the art can application implementation the present invention.But should be appreciated that the present invention is not limited to following embodiment, based on enlightenment of the present invention, any conspicuous conversion or equivalent substitution also should be considered to fall into protection scope of the present invention.
Among the embodiment, during preparation upper prop liquid, get the mixture of ursodesoxycholic acid and Chenodiol, in 1: 2.5 ratio of equivalence ratio, be dissolved in the excess base aqueous solution, regulating pH then is 7-8, and it is 10%-20% that the adding deionized water is diluted to solid content; The upper prop flow rate control 1.5-2 times of column volume/hour.
Glass column internal diameter 28mm, long 400mm, filling 200ml nonpolar macroporous adsorption resin HZ816, or HZ818 or HZ830, specific surface area is not less than 400m
2/ g, pore volume is not less than 1ml/g, commercially available Shanghai Huazhen Science and Technology Co., Ltd. product.
The water-miscible organic solvent that with the percent by volume is 20%-30% disposes first eluent, dispose second eluent with the pure water solubleness organic solvent, respectively three times of column volume first eluents and two times of column volume second eluents are pumped into desorb wash-out in the adsorption resin column, flow velocity be 1 times of column volume/hour; Can carry out continuously in the chromatographic column elution process, or batch process.
The testing conditions of high effective liquid chromatography for measuring product purity is: high performance liquid chromatography (Tianjin, island VP-ODS, C18 chromatographic column 250mmX 4. 6mm, 5 μ m), and moving phase is: acetonitrile: water: acetate is 55: 45: 1, and flow velocity is 1.0mL/min, and column temperature is 30
0C, sample size are 10uL; Detector: differential detector.
Embodiment 1:
Mixture containing 4.5g ursodesoxycholic acid and 6g Chenodiol is dissolved in the aqueous sodium hydroxide solution, and regulating pH is 8, adds the dilution of 75ml deionized water and forms upper prop liquid; Described upper prop liquid is injected the resin column of the nonpolar porous synthetic adsorbent macroporous adsorbent resin HZ816 of filling by peristaltic pump, using three times of column volumes (600ml) percent by volume then is first eluent of 30% methanol aqueous solution configuration, pump into desorb in the adsorption resin column, eluent is with 1 times of column volume/hour (200ml/h) flow velocity wash-out, merge and collect the elutriant (the ursodesoxycholic acid sodium salt is main in the elutriant) that obtains, again eluent is made in second of the pure methanol solution configuration of two times of column volumes (400ml), pump into desorb in the adsorption resin column, elution flow rate is 1 times of column volume/hour (200ml/h), merges to collect the elutriant (the Chenodiol sodium salt is main in the solution) that obtains; Concentrate elutriant that obtains by first eluent and the elutriant that obtains by second eluent at last respectively, and recovery methyl alcohol, concentrated solution adds hydrochloric acid respectively, transfers pH to 2-3, obtains ursodesoxycholic acid and Chenodiol precipitation respectively, be washed to neutrality, drying, the purity of employing high effective liquid chromatography for measuring products obtained therefrom is wherein in 30% meoh eluate, in massfraction, the ursodeoxycholic acid content is 87.3%, and Chenodiol content is 12.7%, in the pure meoh eluate, in massfraction, the ursodeoxycholic acid content is 9.8%, and Chenodiol content is 90.2%, wherein, the total yield of ursodesoxycholic acid is 90.1%, and the Chenodiol total yield is 85.3%.
Embodiment 2:
Except that replacing with nonpolar macroporous adsorption resin HZ818 among the embodiment 1 HZ816, other are with embodiment 1 operation, the result is after measured in 30% meoh eluate, in massfraction, the ursodeoxycholic acid content is 82.2%, and Chenodiol content is 17.8%, in the pure meoh eluate, in massfraction, the ursodeoxycholic acid content is 12.8%, and Chenodiol content is 87.2%.The total yield of ursodesoxycholic acid is 82%, and the Chenodiol total yield is 83%.
Embodiment 3:
Adopt nonpolar macroporous adsorption resin HZ830 to replace HZ816 among the embodiment 1, other conditions are with embodiment 1, the result is after measured in 30% meoh eluate, in massfraction, the ursodeoxycholic acid content is 83.2%, and Chenodiol content is 16.8%, in the pure meoh eluate, in massfraction, the ursodeoxycholic acid content is 12.3%, and Chenodiol content is 87.7%.The total yield of ursodesoxycholic acid is 85%, and the Chenodiol total yield is 81%.
Embodiment 4:
Upper prop liquid is regulated pH and is changed 7 into, other conditions are with embodiment 1, the result is after measured wherein in 30% meoh eluate, counting the ursodeoxycholic acid content with massfraction is 88.6%, Chenodiol content is 11.4%, in the pure meoh eluate, counting the ursodeoxycholic acid content with massfraction is 8.5%, and Chenodiol content is 91.5%.The total yield of ursodesoxycholic acid is 92%, and the Chenodiol total yield is 89%.
Embodiment 5:
Remove upper prop liquid adjusting pH and change 7.5 into, other conditions are with embodiment 1.Wherein in 30% meoh eluate, in massfraction, the ursodeoxycholic acid content is 82.6% after measured, and Chenodiol content is 17.4%, and in the pure meoh eluate, in massfraction, the ursodeoxycholic acid content is 14.5%, and Chenodiol content is 85.5%.The total yield of ursodesoxycholic acid is 86.3%, and the Chenodiol total yield is 83.7%.
Embodiment 6:
Get the mixture that contains 4.5g ursodesoxycholic acid and 6g Chenodiol, be dissolved in the aqueous sodium hydroxide solution, regulator solution pH is 8, adds the dilution of 75ml deionized water again and forms upper prop liquid; Described upper prop liquid is injected the resin column of the nonpolar porous synthetic adsorbent HZ816 of filling by peristaltic pump, be that 20% aqueous acetone solution disposes first eluent with three times of column volumes (600ml) percent by volume then, pump into desorb in the adsorption resin column, flow velocity be 1 times of column volume/hour, collect and merge elutriant (the ursodesoxycholic acid sodium salt is main in the solution), again with two times of column volumes (400ml) pure acetone solution allocation, second eluent, pump into desorb in the adsorption resin column, flow velocity be 1 times of column volume/hour; Collect and merging elutriant (the Chenodiol sodium salt is main in the solution); Concentrate above-mentioned secondary elutriant at last respectively and reclaim acetone, concentrated solution adds hydrochloric acid respectively and transfers pH to 2-3, obtain ursodesoxycholic acid and Chenodiol precipitation respectively, be washed to neutrality, drying, the purity of employing high effective liquid chromatography for measuring products obtained therefrom is wherein in the 20% acetone elutriant, in massfraction, the ursodeoxycholic acid content is 89.3%, and Chenodiol content is 10.7%, in the pure acetone elutriant, in massfraction, the ursodeoxycholic acid content is 9.8%, and Chenodiol content is 90.2%, wherein, the total yield of ursodesoxycholic acid is 90.1%, and the Chenodiol total yield is 85.3%.
Embodiment 7:
Mixture containing 6g ursodesoxycholic acid and 8g Chenodiol is dissolved in the potassium hydroxide aqueous solution, and regulating pH is 7, and other operations are all identical with embodiment 1.After measured in 30% meoh eluate, in massfraction, the ursodeoxycholic acid content is 86.6%, Chenodiol content is 13.4%, in the pure meoh eluate, in massfraction, the ursodeoxycholic acid content is 8.5%, Chenodiol content is 91.5%, and the total yield of ursodesoxycholic acid is 91%, and the Chenodiol total yield is 88%.
Embodiment 8:
Mixture containing 9g ursodesoxycholic acid and 12g Chenodiol is dissolved in the aqueous sodium hydroxide solution, and regulating pH is 7, and other operations are identical with embodiment 1.After measured in 30% meoh eluate, counting the ursodeoxycholic acid content with massfraction is 81.6%, Chenodiol content is 18.4%, in the pure meoh eluate, counting the ursodeoxycholic acid content with massfraction is 18.5%, and Chenodiol content is 81.5%, wherein, the total yield of ursodesoxycholic acid is 91%, and the Chenodiol total yield is 86%.
Claims (5)
1. the separation method of Chenodiol and ursodesoxycholic acid is characterized in that comprising successively step:
⑴ preparation upper prop liquid: get the mixture of ursodesoxycholic acid and Chenodiol, be dissolved in the alkali aqueous solution, adjusting pH is 7-8, and adding deionized water then, to be diluted to solid content be 10%-20%;
⑵ upper prop absorption: step ⑴ gained upper prop liquid pump is gone into to load in the resin column of nonpolar porous synthetic adsorbent;
⑶ desorb: the water-miscible organic solvent that with the percent by volume is 20%-30% disposes first eluent, dispose second eluent with the pure water solubleness organic solvent, respectively three times of column volume first eluents and two times of column volume second eluents are pumped into desorb in the adsorption resin column, flow velocity be 1 times of column volume/hour;
⑷ aftertreatment:, reclaim solvent with the elutriant vacuum concentration of step ⑶ desorb gained; Enriched material adds the inorganic acid fluid adjust pH to 2-3, separates out precipitation, filters, and washing is extremely neutral, drying, and crystallization, vacuum-drying gets finished product.
2. according to the separation purification method of described a kind of Chenodiol of claim 1 and ursodesoxycholic acid, it is characterized in that the described alkali aqueous solution of step ⑴ is a sodium hydroxide, potassium hydroxide, or ammoniacal liquor.
3. according to the separation method of described a kind of Chenodiol of claim 1 and ursodesoxycholic acid, it is characterized in that the described nonpolar porous synthetic adsorbent of step ⑵ is macroporous adsorbent resin HZ816, or HZ818, or HZ830.
4. according to the separation method of described a kind of Chenodiol of claim 1 and ursodesoxycholic acid, it is characterized in that the described water-miscible organic solvent of step ⑶ is a methyl alcohol, ethanol, n-propyl alcohol, Virahol, the trimethyl carbinol, dioxan, tetrahydrofuran (THF), acetone, dimethyl formamide, methyl-sulphoxide etc.
5. according to the separation method of described a kind of Chenodiol of claim 1 and ursodesoxycholic acid, it is characterized in that the described inorganic acid fluid of step ⑷ is a hydrochloric acid, or sulfuric acid, or nitric acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617689A (en) * | 2012-03-14 | 2012-08-01 | 江苏正大清江制药有限公司 | Purification method of hyodeoxycholic acid |
| CN102766185A (en) * | 2012-08-02 | 2012-11-07 | 苏州天绿生物制药有限公司 | Method for respectively recovering ursodesoxycholic acid and chenodeoxycholic acid from ursodesoxycholic acid waste mother liquor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528779A (en) * | 2003-09-29 | 2004-09-15 | 华东理工大学 | Method for preparing cheodexycholic acid |
| CN1775798A (en) * | 2005-12-13 | 2006-05-24 | 山东博尔德生物科技有限公司 | Method for producing high-purity chenodeoxy cholic acid from poultry and livestock bile |
| CN101830956A (en) * | 2008-11-19 | 2010-09-15 | 毕小升 | Preparation method for separating and purifying chenodeoxycholic acid in porcine bile paste or leftovers |
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2011
- 2011-08-15 CN CN2011102318602A patent/CN102286051B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528779A (en) * | 2003-09-29 | 2004-09-15 | 华东理工大学 | Method for preparing cheodexycholic acid |
| CN1775798A (en) * | 2005-12-13 | 2006-05-24 | 山东博尔德生物科技有限公司 | Method for producing high-purity chenodeoxy cholic acid from poultry and livestock bile |
| CN101830956A (en) * | 2008-11-19 | 2010-09-15 | 毕小升 | Preparation method for separating and purifying chenodeoxycholic acid in porcine bile paste or leftovers |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617689A (en) * | 2012-03-14 | 2012-08-01 | 江苏正大清江制药有限公司 | Purification method of hyodeoxycholic acid |
| CN102766185A (en) * | 2012-08-02 | 2012-11-07 | 苏州天绿生物制药有限公司 | Method for respectively recovering ursodesoxycholic acid and chenodeoxycholic acid from ursodesoxycholic acid waste mother liquor |
| CN102766185B (en) * | 2012-08-02 | 2015-04-22 | 苏州天绿生物制药有限公司 | Method for respectively recovering ursodesoxycholic acid and chenodeoxycholic acid from ursodesoxycholic acid waste mother liquor |
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