CN101830956A - Preparation method for separating and purifying chenodeoxycholic acid in porcine bile paste or leftovers - Google Patents
Preparation method for separating and purifying chenodeoxycholic acid in porcine bile paste or leftovers Download PDFInfo
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- CN101830956A CN101830956A CN200810176801A CN200810176801A CN101830956A CN 101830956 A CN101830956 A CN 101830956A CN 200810176801 A CN200810176801 A CN 200810176801A CN 200810176801 A CN200810176801 A CN 200810176801A CN 101830956 A CN101830956 A CN 101830956A
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- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 title claims abstract description 68
- 229960001091 chenodeoxycholic acid Drugs 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 210000000941 bile Anatomy 0.000 title claims abstract description 17
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 title claims abstract description 11
- 235000021190 leftovers Nutrition 0.000 title abstract 3
- DGABKXLVXPYZII-SIBKNCMHSA-N hyodeoxycholic acid Chemical compound C([C@H]1[C@@H](O)C2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 DGABKXLVXPYZII-SIBKNCMHSA-N 0.000 claims abstract description 29
- DGABKXLVXPYZII-UHFFFAOYSA-N Hyodeoxycholic acid Natural products C1C(O)C2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 DGABKXLVXPYZII-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 22
- 159000000007 calcium salts Chemical class 0.000 claims abstract description 17
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims abstract description 12
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 12
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- SMEROWZSTRWXGI-HVATVPOCSA-N lithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 SMEROWZSTRWXGI-HVATVPOCSA-N 0.000 claims abstract description 10
- SMEROWZSTRWXGI-UHFFFAOYSA-N Lithocholsaeure Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 SMEROWZSTRWXGI-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- 229920005989 resin Polymers 0.000 claims abstract description 6
- 239000011347 resin Substances 0.000 claims abstract description 6
- 244000144977 poultry Species 0.000 claims abstract description 5
- 238000007670 refining Methods 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- 239000001110 calcium chloride Substances 0.000 claims description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 10
- 230000003647 oxidation Effects 0.000 claims description 9
- 238000007254 oxidation reaction Methods 0.000 claims description 9
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 238000002425 crystallisation Methods 0.000 claims description 8
- 230000008025 crystallization Effects 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 7
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004380 Cholic acid Substances 0.000 claims description 6
- 235000019219 chocolate Nutrition 0.000 claims description 6
- 229960002471 cholic acid Drugs 0.000 claims description 6
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 6
- 235000019416 cholic acid Nutrition 0.000 claims description 6
- 238000005352 clarification Methods 0.000 claims description 6
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 239000006071 cream Substances 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 238000007127 saponification reaction Methods 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000008395 clarifying agent Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 235000017550 sodium carbonate Nutrition 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- OKTJSMMVPCPJKN-YPZZEJLDSA-N carbon-10 atom Chemical class [10C] OKTJSMMVPCPJKN-YPZZEJLDSA-N 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- VNWKDIUSXQCPGN-UHFFFAOYSA-J dicalcium tetrachloride Chemical compound [Cl-].[Cl-].[Cl-].[Cl-].[Ca+2].[Ca+2] VNWKDIUSXQCPGN-UHFFFAOYSA-J 0.000 claims description 2
- 229910001882 dioxygen Inorganic materials 0.000 claims description 2
- 238000006073 displacement reaction Methods 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 239000012452 mother liquor Substances 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 231100000956 nontoxicity Toxicity 0.000 abstract 1
- 230000001590 oxidative effect Effects 0.000 abstract 1
- 239000002699 waste material Substances 0.000 abstract 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- 235000012000 cholesterol Nutrition 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 4
- 241000272814 Anser sp. Species 0.000 description 3
- 241000282894 Sus scrofa domesticus Species 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 229960001661 ursodiol Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 230000001741 anti-phlogistic effect Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 159000000009 barium salts Chemical class 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 230000001989 choleretic effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method for separating and purifying chenodeoxycholic acid in porcine bile paste or leftovers, relating to an industrial preparation method for extracting and purifying chenodeoxycholic acid from poultry and animal bile or waste leftovers for extracting bilirubin hyodeoxycholic acid. The method comprises the following production processes of: saponifying, oxidizing to decolorize, clarifying, preparing primary calcium salt and secondary calcium salt, adsorbing by using macroporous absorption resin, resolving and crystallizing, refining, and the like. The whole process has environmental protection, safety, no toxicity and low cost, and is easy to realize the industrial scale production of the chenodeoxycholic acid. By using a high performance liquid chromatography (HPLC) method, it is found that the content of the chenodeoxycholic acid in an obtained product is larger than or equal to 98 percent, the content of the hyodeoxycholic acid is smaller than 0.5 percent, and the content of lithocholic acid is smaller than 0.1 percent. By using a thin-layer chromatography (TLC) method, a main chenodeoxycholic acid spot and a slight hyodeoxycholic acid spot can be seen.
Description
Technical field:
Invention relates to a kind of Chenodiol (3 α, 7 α-dihydroxyl-5 β-cholanic acid) preparation method for separating and purifying, relate generally to from poultry and animal bile, particularly extract in Fel Sus domestica unguentum (juice) or the tankage (extracting bilirubin or Hyodeoxycholic Acid tankage afterwards), and the preparation method of separation and purification Chenodiol.
Background technology:
Chenodiol (3 α, 7 α-dihydroxyl-5 β-cholanic acid) ChenodeoxycholicAcid (being called for short CDCA), found first in goose bile in 1824, nineteen twenty-four, wieland has found Chenodiol with windaus in different courage (people and goose), it is the most molten a kind of bile acide, is named as Chenodiol.
Chenodiol has antibechic and the antiphlogistic effect of reducing phlegm of relievining asthma, and its choleretic effect is particularly remarkable, and it is by reducing the absorption of cholesterol, synthetic, the bile inner cholesterol is descended, thereby prevent cholesterol calculus to form and short its dissolving, and effective reducing cholesterol saturation ratio.
Chenodiol is also as producing the ursodesoxycholic acid intermediate of (3 α, 7 β-dihydroxyl-5 β-cholanic acid are called for short UDCA).Ursodesoxycholic acid is the contained main component of rare Chinese medicine bear gall, is used for the treatment of various courage diseases and digestive tract diseases clinically.At present, traditional famous and precious medicinal animal bile is very in short supply, seeks medicinal animal bile and synthetic product that DEVELOPMENT PROSPECT is arranged and is popular research project.Further excavate the pharmaceutical use of animal bile, the biliary medicinal ingredients of analyzing animal also discloses its mechanism of action, makes animal bile obtain comprehensive development and utilization.Therefore, the preparation Chenodiol has very important significance for further preparation ursodesoxycholic acid also tool.
Chenodiol mainly contains in the bile of poultry or the animal bile and extracts, and is not easy to collect because the bile amount of poultry, particularly goose is few, and people have just turned to Fel Sus domestica, particularly extracts in the tankage after bilirubin or the Hyodeoxycholic Acid and extracts.Traditional extracting and preparing technique process complexity, yield is low, and particularly purity does not increase, and is less than 98% (HPLC), and other material (Hyodeoxycholic Acid, lithocholic acid is higher) content can not satisfy clinical demand; And use a large amount of unsafe organic solvents.
Technology is mainly divided following a few class:
1. be the silica gel column chromatography of representative with the Zhou Weishan academician of Shanghai organic chemistry institute of the Chinese Academy of Sciences." medicine industry ", 1988,19 (12): 529-30;
2. be the synthesis method of representative with Shenyang Institute of Chemical Technology and Zhou Weishan academician, (Chinese patent CN1869043A etc.)
3. with Shandong Boulder biotech firm the Amberlyst process (Chinese patent CN1775798A) of representative
4. be the barium salt of representative with Changde Yun Gang biotech firm, calcium salt method (Chinese patent CN1830996A, CN1869044A etc.)
5. be the process for refining of representative with U.S. Pat P4213911.4022806.4163017.4316849 etc.
Since be subjected to the places of origin of raw materials, commercial production conditions, the ripening degree of environmental requirement, particularly Technology has caused the extraction of Chenodiol, the separation and purification difficulty, and suitability for industrialized production can not up to standardly go up amount.
Summary of the invention:
The object of the present invention is to provide a kind of method that large-scale industry prepares high-purity chenodeoxy cholic acid that is used for, avoided the defective of prior art.Adopt safety and low toxicity, simple and practical method obtains content more than or equal to 98% (HPLC method, high performance liquid phase detects), Hyodeoxycholic Acid＜0.5%, the Chenodiol of lithocholic acid＜0.1% (CDCA).
The purpose of invention is achieved through the following technical solutions:
1. saponification is handled;
2. oxide treatment;
3. clarifying treatment;
4. the preparation of Hyodeoxycholic Acid crude product;
5. collecting of tankage, oxidation and clarifying treatment;
6. calcium salt;
7. secondary calcium salt;
8. macroporous resin adsorption;
9. the refining highly purified Chenodiol (CDCA) that obtains, content be more than or equal to 98% (HPLC), other material: Hyodeoxycholic Acid HDCA＜0.5% (HPLC), lithocholic acid LCA＜0.1%.
Detailed process is as follows:
(1) saponification .10%-15%NaOH, 100-120 ℃ of temperature.18-26 hours time; (2) oxidation, industrial hydrogen peroxide 0.5-2% is as the criterion to reach off-white color; (3) clarification, with 1+1 natural clarifying agent 0.5-5/ ten thousand, 80-100 ℃ of temperature, PH9-14,0.5-2 hours, filter press got clear filtrate; (4) transfer PH2-3 with dilute hydrochloric acid, get thick cholic acid, with 8 times of dissolvings of ethyl acetate, 1 times of activated carbon refluxed 2 hours, and filtrate is concentrated to 1/5 crystallization, got Hyodeoxycholic Acid.Get the Hyodeoxycholic Acid (HDCA) of fusing point more than 198 ℃ with re-crystallizing in ethyl acetate; (5) the ethyl acetate recovery with the secondary Hyodeoxycholic Acid finishes, and the courage paste of remaining chocolate adds hydrogen peroxide oxidation, and the clarification of 1+1 natural clarifying agent gets clear filtrate; (6) calcium salts; (the content high performance liquid chromatography detects to requiring 1 times of amount of courage paste industrial calcium chloride of adding chocolate.The mole ratio Chenodiol: 60-80 ℃ of the temperature of calcium chloride=1:1.1), 2 hours time, add yellow soda ash displacement Chenodiol after the filter press, adding mole ratio is 1.1 of calcium chloride, 90-100 ℃ of temperature, 1-3 hours time; (7) secondary calcium salt with the ratio row of (6), after processing condition become calcium salt, with 300-1000 times deionized water wash, is finished, and transfers PH2.0-3.5 to get the crude product Chenodiol; (8) the crude product Chenodiol is dissolved in buck, PH6.0-8.0, and after last weakly alkaline or strong basicity macroporous resin adsorption were saturated, impurity was removed in washing, and sig water is resolved, and judges the starting point of reception with thin-layer chromatography, and terminating point is transferred PH3 vacuum-drying; (9) Chenodiol is dissolved in the acetonitrile mixed solvent and refluxed 2 hours, add activated carbon 10%, heat filter, crystallization.Recrystallization gets Chenodiol elaboration content more than or equal to 98% (HPLC), contains Hyodeoxycholic Acid＜0.5% (HPLC), lithocholic acid＜0.1%, thin-layer chromatography (TLC): a Chenodiol principal spot, a slighter Hyodeoxycholic Acid spot.
Advantage of the present invention and effect:
Whole technology has easy and simple to handle, and safety is with low cost, is easy to suitability for industrialized production, is easy to obtain highly purified Chenodiol, and content contains Hyodeoxycholic Acid＜0.5% (HPLC) more than or equal to 98% (HPLC), lithocholic acid＜0.1% (HPLC).
Description of drawings:
Fig. 1 is high-efficient liquid phase chromatogram＜23.67% of tankage behind the extraction Hyodeoxycholic Acid 〉. gather date and time: 2007-6-15 14:11:32; User name: Admin; Sample title: trial-product; Injection rate: 20 μ l; The method filename: C: ChromatoSolution-Light Data Project1 no title .gcm; Report name: C: ChromatoSolution-Light Sample template .gcr; Storing path: D: Data project1 CDCA 12.gcd; Use the instrument type: liquid chromatography; Gradient mode: constant current; Instrument model: LC-10ATvp; Detector: ultraviolet; Column temperature (℃): 40; Wavelength (nm): 205; Post model: C18
Fig. 2 is Chenodiol finished product high-efficient liquid phase chromatogram＜98.80% 〉. gather date and time: 2007-6-27 21:30:53; User name: Admin; Sample title: CDCA; Injection rate: 20 μ l; The method filename: C: Chromato Solution-Light Data Project1 no title .gcm; Report name: C: Chromato Solution-Light Sample template .gcr; Storing path: D: Data project1 CDCA 17.gcd; Use the instrument type: liquid chromatography; Gradient mode: constant current; Instrument model: LC-10ATvp; Detector: ultraviolet; Column temperature (℃): 40; Wavelength (nm): 205; Post model: C18
Fig. 3 is Chenodiol finished product high-efficient liquid phase chromatogram＜98.50% 〉. gather date and time: 2007-6-27 20:22:43; User name: Admin; Sample title: CDCA; Injection rate: 20 μ l; The method filename: C: Chromato Solution-Light Data Project1 no title .gcm; Report name: C: Chromato Solution-Light Sample template .gcr; Storing path: D: Data project1 CDCA 14.gcd; Use the instrument type: liquid chromatography; Gradient mode: constant current; Instrument model: LC-10ATvp; Detector: ultraviolet; Column temperature (℃): 40; Wavelength (nm): 205; Post model: C18
Embodiment:
1. in separating the Chenodiol process, saponification 12%NaOH, 110 ℃ of temperature, 24 hours time; Oxidation dioxygen water consumption 1.5%, finings consumption 5/0,000,100 ℃ of temperature, PH10,1 hour time, crystallization PH3 obtains thick cholic acid.Obtain dystectic Hyodeoxycholic Acid (more than 198 ℃) with re-crystallizing in ethyl acetate.
2 reclaim the chocolate paste after the ethyl acetate, need further oxidation, clarification, hydrogen peroxide consumption 1.0%-3.0%, finings consumption 0.5-5/ ten thousand.
3. a calcium salt calcium chloride temperature is 65 ℃, 2 hours time, the add-on mole ratio is 1:1.1, with the high performance liquid chromatography detection computations, Chenodiol: calcium chloride=1:1.1. adds 95 ℃ of yellow soda ash temperature, 2 hours time, the add-on mole ratio is 1:1.05, calculates to add calcium chloride.
4. secondary calcium salt with a calcium salt technology, becomes behind the calcium salt with the washing of 500 times of amount deionized waters, to the calcium saline solution till the no thick Chenodiol, and accent PH3.0.
5. after Chenodiol dissolves, last weakly alkaline or strong basicity macroporous resin adsorption, Chenodiol mother liquor PH is 6.5.
6. in the Chenodiol, when resolving, judge the starting point of reception, terminating point with thin-layer chromatography with sig water.
7. when refining, the gac add-on is a Chenodiol 10%.Refluxed 2 hours.
According to the impurity spot situation of thin-layer chromatography selectively selective solvent come crystallization, to remove the impurity cholic acid, make its Chenodiol more than or equal to 98% (HPLC) Hyodeoxycholic Acid＜0.5% (HPLC), lithocholic acid＜0.1%.
9. in extracting the purifying chenodeoxycholic acid process, raw material can be a bird gall courage cream, can be animal class bile courage cream or its extraction bilirubin or Hyodeoxycholic Acid tankage afterwards, and prerequisite is to never degenerate, and goes mouldy.
Claims (10)
1. Chenodiol (3 α, 7 α-dihydroxyl-5 β-cholanic acid, CDCA) preparation method, it is characterized in that, used raw material is poultry and animal bile, and courage cream or its extract bilirubin, the tankage after the Hyodeoxycholic Acid, may further comprise the steps: (1) saponification .10%-15%NaOH, 100-120 ℃ of temperature.18-26 hours time; (2) oxidation, industrial hydrogen peroxide 0.5-2% is as the criterion to reach off-white color; (3) clarification, with 1+1 natural clarifying agent 0.5-5/ ten thousand, 80-100 ℃ of temperature, PH9-14,0.5-2 hours, filter press got clear filtrate; (4) transfer PH2-3 with dilute hydrochloric acid, get thick cholic acid, with 8 times of dissolvings of ethyl acetate, 1 times of activated carbon refluxed 2 hours, and filtrate is concentrated to 1/5 crystallization, got Hyodeoxycholic Acid.Get the Hyodeoxycholic Acid (HDCA) of fusing point more than 198 ℃ with re-crystallizing in ethyl acetate; (5) the ethyl acetate recovery with the secondary Hyodeoxycholic Acid finishes, and the courage paste of remaining chocolate adds hydrogen peroxide oxidation, and the clarification of 1+1 natural clarifying agent gets clear filtrate; (6) calcium salts; (the content high performance liquid chromatography detects to requiring 1 times of amount of courage paste industrial calcium chloride of adding chocolate.The mole ratio Chenodiol: 60-80 ℃ of the temperature of calcium chloride=1:1.1), 2 hours time, add yellow soda ash displacement Chenodiol after the filter press, add mole ratio and be 90-100 ℃ of 1.1 temperature of calcium chloride, 1-3 hours time; (7) secondary calcium salt with the ratio row of (6), after processing condition become calcium salt, with 300-1000 times deionized water wash, is finished, and transfers PH2.0-3.5 to get the crude product Chenodiol; (8) the crude product Chenodiol be dissolved on the buck PH6.0-8.0 weakly alkaline or strong basicity macroporous resin adsorption saturated after, impurity is removed in washing, sig water is resolved, and judges the starting point of reception, terminating point with thin-layer chromatography.Transfer PH3 vacuum-drying; (9) Chenodiol is dissolved in the acetonitrile mixed solvent and refluxed 2 hours, add activated carbon 10%, heat filter, crystallization.Recrystallization gets Chenodiol elaboration content more than or equal to 98% (HPLC), contains Hyodeoxycholic Acid＜0.5% (HPLC), lithocholic acid＜0.1%, thin-layer chromatography (TLC): a Chenodiol principal spot, a slighter Hyodeoxycholic Acid spot.
2. according to the preparation method of claim 1 narration, it is characterized in that, in separating the Chenodiol process, saponification 12%NaOH, 110 ℃ of temperature, 24 hours time; Oxidation dioxygen water consumption 1.5%, finings consumption 5/0,000,100 ℃ of temperature, PH10,1 hour time, crystallization PH3 obtains thick cholic acid.Obtain dystectic Hyodeoxycholic Acid (more than 198 ℃) with re-crystallizing in ethyl acetate.
3. according to the preparation method of claim 1 narration, it is characterized in that in extraction separation Chenodiol process, the chocolate paste after the recovery ethyl acetate needs further oxidation, clarification, hydrogen peroxide consumption 1.0%-3.0%, finings consumption 0.5-5/ ten thousand.
4. according to the preparation method of claim 1 narration, it is characterized in that, in extraction separation Chenodiol process, one time calcium salt calcium chloride temperature is 65 ℃, 2 hours time, the add-on mole ratio is 1:1.1, with the high performance liquid chromatography detection computations, Chenodiol: calcium chloride=1:1.1. adds 95 ℃ of yellow soda ash temperature, 2 hours time, the add-on mole ratio is 1:1.05, calculates to add calcium chloride.
5. according to the preparation method of claim 1 narration, it is characterized in that the secondary calcium salt with a calcium salt technology, becomes behind the calcium salt with the washing of 500 times of amount deionized waters, to the calcium saline solution till the no thick Chenodiol, accent PH3.0.
6. according to the preparation method of claim 1 narration, it is characterized in that, after the Chenodiol dissolving, last weakly alkaline or strong basicity macroporous resin adsorption, Chenodiol mother liquor PH is 6.5.
7. according to the preparation method of claim 1 narration, it is characterized in that, in the Chenodiol, when resolving, judge the starting point of reception, terminating point with thin-layer chromatography with sig water.
8. according to the preparation method of claim 1 narration, it is characterized in that when refining, the gac add-on is a Chenodiol 10%.Refluxed 2 hours.
9. according to the preparation method of claim 1 narration, it is characterized in that, according to the impurity spot situation of thin-layer chromatography selectively selective solvent come crystallization, to remove the impurity cholic acid, make its Chenodiol more than or equal to 98% (HPLC) Hyodeoxycholic Acid＜0.5% (HPLC), lithocholic acid＜0.1%.
10. according to the preparation method of claim 1 narration, it is characterized in that, in extracting the purifying chenodeoxycholic acid process, raw material can be a bird gall courage cream, can be animal class bile courage cream or its extraction bilirubin or Hyodeoxycholic Acid tankage afterwards, prerequisite be to never degenerate, and goes mouldy.
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