CN112521349B - Method for purifying paclitaxel - Google Patents
Method for purifying paclitaxel Download PDFInfo
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- CN112521349B CN112521349B CN202011256738.6A CN202011256738A CN112521349B CN 112521349 B CN112521349 B CN 112521349B CN 202011256738 A CN202011256738 A CN 202011256738A CN 112521349 B CN112521349 B CN 112521349B
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 title claims abstract description 88
- 229930012538 Paclitaxel Natural products 0.000 title claims abstract description 87
- 229960001592 paclitaxel Drugs 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000002386 leaching Methods 0.000 claims abstract description 47
- 238000001179 sorption measurement Methods 0.000 claims abstract description 32
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000012141 concentrate Substances 0.000 claims abstract description 27
- 239000003960 organic solvent Substances 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 16
- 239000000843 powder Substances 0.000 claims abstract description 13
- 241001149649 Taxus wallichiana var. chinensis Species 0.000 claims abstract description 12
- 238000000746 purification Methods 0.000 claims abstract description 11
- 239000013078 crystal Substances 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 85
- 239000000243 solution Substances 0.000 claims description 62
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 54
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 45
- 239000000047 product Substances 0.000 claims description 41
- 238000006243 chemical reaction Methods 0.000 claims description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 239000007853 buffer solution Substances 0.000 claims description 33
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 30
- 235000019441 ethanol Nutrition 0.000 claims description 30
- 238000002156 mixing Methods 0.000 claims description 30
- 238000003756 stirring Methods 0.000 claims description 30
- 238000001035 drying Methods 0.000 claims description 26
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 23
- 239000000460 chlorine Substances 0.000 claims description 23
- 229910052801 chlorine Inorganic materials 0.000 claims description 23
- 238000001914 filtration Methods 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 18
- 239000000463 material Substances 0.000 claims description 15
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- VVNATJLDMWFTTM-UHFFFAOYSA-N ethane-1,2-diamine;triethoxy(propyl)silane Chemical compound NCCN.CCC[Si](OCC)(OCC)OCC VVNATJLDMWFTTM-UHFFFAOYSA-N 0.000 claims description 13
- YIVJZNGAASQVEM-UHFFFAOYSA-N Lauroyl peroxide Chemical compound CCCCCCCCCCCC(=O)OOC(=O)CCCCCCCCCCC YIVJZNGAASQVEM-UHFFFAOYSA-N 0.000 claims description 11
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 10
- 230000002378 acidificating effect Effects 0.000 claims description 10
- 238000004440 column chromatography Methods 0.000 claims description 10
- 239000012043 crude product Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 239000012149 elution buffer Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000005086 pumping Methods 0.000 claims description 10
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 7
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 229960001124 trientine Drugs 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 229920000936 Agarose Polymers 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 238000011049 filling Methods 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000003208 petroleum Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000002425 crystallisation Methods 0.000 claims description 4
- 230000008025 crystallization Effects 0.000 claims description 4
- 241001116500 Taxus Species 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000001953 recrystallisation Methods 0.000 abstract description 2
- 239000000872 buffer Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- -1 taxane compounds Chemical class 0.000 description 1
- MRIYLSBNRSGNBM-ARSSDYQWSA-N taxol cephalomannine Chemical compound O([C@@H]1[C@]2(O)C[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]3(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]31)OC(C)=O)C2(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)C(/C)=C/C)C=1C=CC=CC=1)C(=O)C1=CC=CC=C1.O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 MRIYLSBNRSGNBM-ARSSDYQWSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
Abstract
The invention relates to the technical field of medicine purification, in particular to a method for purifying paclitaxel; the invention firstly carries out primary extraction on the fine taxus chinensis powder through the leaching liquor, and then carries out impurity removal on the obtained leaching liquor in an electrophoretic adsorption mode, so that the taxol and other components mixed with the taxol can be effectively separated, and the purity of the taxol is improved; dissolving the paclitaxel concentrate after electrophoretic adsorption by an organic solvent; the three-dimensional topological structure of the surface of the alumina molecule is effectively improved by the modified alumina column, so that the adsorption capacity of the alumina molecule to the paclitaxel is greatly improved, and the efficiency of paclitaxel separation and purification is also effectively improved; then the obtained paclitaxel concentrated solution is sequentially subjected to the subsequent processes of extraction, secondary chromatography, crystal growing, recrystallization and the like, so that paclitaxel is fully dispersed with other components, and a high-purity paclitaxel product is finally obtained.
Description
Technical Field
The invention relates to the technical field of medicine purification, in particular to a method for purifying paclitaxel.
Background
Paclitaxel, alias taxol, violin, telin, chemical name 5 beta, 20-epoxy-1, 2 alpha, 4,7 beta, 10 beta, 13 alpha-hexahydroxy-taxane-11-en-9-one-4, 10-diacetate-2-benzoate-13 [ (2 'R,3' S) -N-benzoyl-3-phenylisoserine ester]Molecular weight 853.92, molecular formula C 47 H 51 NO 14 。
Paclitaxel is the most promising anticancer drug discovered in the world in the last two decades, has good curative effect on treating breast cancer, ovarian cancer, esophageal cancer, lung cancer, liver cancer and other cancers, has extremely strong killing power on cancer cells, and has extremely small toxic and side effects. Therefore, the method becomes the focus of the research of the pharmaceutical industry at present.
However, the purity of paclitaxel prepared in the prior art is low, the purification effect is not ideal, and the paclitaxel cannot directly reach the pharmaceutical standard, so that the method for purifying paclitaxel is provided, and the technical problem to be solved by the technical personnel in the field is urgently needed.
Disclosure of Invention
Aiming at the technical problems in the background art, the invention provides a method for purifying paclitaxel, and the paclitaxel purified by the method has higher purity, less impurity content and better quality; the paclitaxel purification process provided by the invention has wider market prospect and is more suitable for popularization.
Technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for purifying paclitaxel, comprising the steps of:
s1, accurately weighing 60-80 g of taxus chinensis fine powder with the granularity of 60-100 meshes, leaching for 3-4 times by using leaching liquor with the temperature of 30-40 ℃, wherein the leaching time is 30-40 min each time, and then collecting and combining the leaching liquor obtained after each leaching for later use;
s2, adding the leaching liquor combined in the step S1 into a proper amount of acidic buffer solution, and recording the obtained solution as mixed liquor; continuously conveying the mixed solution to the electrode chamber, the flushing chamber and the sampling chamber by using a pump; then starting the power supply of the electrophoresis apparatus; when the agarose affinity medium and the mixed solution reach the equilibrium, the adsorption buffer solution with the pH value of 6.4 is input into the sample chamber by a pump; starting electrophoretic adsorption, and carrying out electrophoretic leaching for 15-20 min after the electrophoretic adsorption process lasts for 50-60 min; after the electrophoretic leaching is finished, turning off the power supply, changing the adsorption buffer solution into an elution buffer solution with the pH of 8.5, turning on the power supply to carry out electrophoretic elution, collecting the active part, and concentrating to obtain a taxol concentrate;
s3, dissolving the paclitaxel concentrate obtained in the step S2 by using an organic solvent, adding modified alumina after the paclitaxel concentrate is fully dissolved, mixing and stirring uniformly, and drying to obtain a dry material sample; storing it for later use;
s4, filling the dry material sample obtained in the step S3 into a chromatographic column, eluting the dry material sample through flow matching at low pressure, and collecting an effective section in the elution; concentrating the collected effective sections, and storing the obtained concentrated solution for later use; wherein the mobile phase is prepared by mixing ethyl acetate and n-hexane; and the percentage of ethyl acetate in the mobile phase volume is 40-60%;
s5, adding an organic extracting agent formed by mixing ethyl acetate, acetone and the like in mass into the concentrated solution obtained in the step S4 to extract the concentrated solution, and then pumping the extract phase into a concentration tank to concentrate the extract; performing secondary column chromatography separation according to the steps, dissolving the product obtained after the secondary column chromatography separation by using acetone, and then transferring the product into a preparative high performance liquid chromatograph for separation and purification;
s6, concentrating and pumping the paclitaxel fraction obtained in the step S5 under reduced pressure, adding petroleum ether into the paclitaxel fraction while stirring, naturally cooling to room temperature, growing crystals, filtering, drying and the like to obtain a recrystallized crude product, repeating the operation for 2-3 times, adding acetone into the recrystallized crude product, dissolving the acetone at the temperature of 40-50 ℃, adding n-hexane with the same volume as that of the acetone while stirring, and performing crystallization, filtering and drying treatment at room temperature to finally obtain a purified paclitaxel finished product.
Further, the leaching solution used in step S1 is prepared by mixing methanol and dichloromethane in equal volume.
Further, the concentration of the ethanol solution used in the step S1 is 60-75%, and the dosage of the ethanol solution is 6-8 times of the fine powder of the taxus chinensis.
Furthermore, the acidic buffer in step S2 is 0.1mol/L citric acid-sodium citrate buffer, and the pH thereof is 6.4.
Further, the adsorption buffer solution in step S2 is 20mM citric acid-sodium citrate buffer solution, and has a ph of 4.5 and a current intensity I =60mA.
Further, the elution buffer in step S2 is 20mm tris-HCl, and the current intensity I =40mA.
Furthermore, the organic solvent used in step S3 is selected from any one of chloroform or acetone, and the amount of the organic solvent is 4-6 times of that of the paclitaxel concentrate.
Further, the preparation method of the modified alumina used in the step S3 comprises the following steps:
a. weighing a proper amount of chlorine balls, transferring the chlorine balls into a reaction device provided with a condensing device, and then respectively adding 5-8 times of methanol solution with the mass of 5-65% of the chlorine balls and 2-3 times of triethylene tetramine into the reaction device; after mixing and stirring uniformly, adding a proper amount of sodium carbonate and tetra-n-butylammonium bromide into a reaction device, electrically stirring for 5-8 h at 80-90 ℃, stopping reaction, filtering, washing with absolute ethyl alcohol, washing with water, drying in vacuum to constant weight, and storing for later use; wherein the dosage of the sodium carbonate is 40-60% of the chlorine ball, and the dosage of the tetra-n-butylammonium bromide is 8-12% of the sodium carbonate;
b. b, placing the product obtained in the step a into a reaction kettle containing an ethanol solution, then respectively adding alumina, ethylenediamine propyl triethoxysilane and lauroyl peroxide into the reaction kettle, and mixing and stirring uniformly; reacting the mixed components obtained in the kettle at a constant temperature of 40-50 ℃ for 12-15 h, and after the reaction is finished and the temperature in the kettle naturally drops to room temperature, sequentially filtering, washing with alcohol and drying in vacuum to constant weight to obtain a modified alumina finished product;
wherein, the mass ratio of the product obtained in the step a to the aluminum oxide to the ethylenediamine propyl triethoxysilane to the lauroyl peroxide is 1: 0.03 to 0.05;
further, the concentration of the ethanol solution used in the step b is 60 to 75 percent, and the dosage of the ethanol solution is 5 to 8 times of that of the product obtained in the step a.
Advantageous effects
Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:
the method comprises the step of primarily extracting the fine taxus chinensis powder through the leaching liquor, wherein the methanol in the leaching liquor is matched with dichloromethane to be used for efficiently extracting the taxus chinensis in the fine taxus chinensis powder. Then the obtained leaching liquor is subjected to 'impurity removal' in an electrophoresis adsorption mode, so that the paclitaxel and other components (such as lipid, pigment and taxane compounds) mixed with the paclitaxel can be effectively separated, and the purity of the paclitaxel is improved. The paclitaxel concentrate after electrophoretic adsorption is dissolved by organic solvent and passes through a modified alumina column. Under the coordination of chlorine spheres, triethylene tetramine, tetra-n-butyl ammonium bromide, ethylenediamine propyl triethoxysilane and lauroyl peroxide, the chlorine spheres and organic functional groups in the ethylenediamine propyl triethoxysilane molecules are subjected to chemical reaction to form bonds, and inorganic functional groups in the ethylenediamine propyl triethoxysilane molecules and alumina are subjected to reaction to form bonds. Thereby successfully grafting the chlorine ball to the surface of the alumina, effectively improving the three-dimensional topological structure of the surface of the alumina molecule, greatly improving the capability of adsorbing the paclitaxel, and effectively improving the efficiency of separating and purifying the paclitaxel. Then the obtained taxol concentrated solution is sequentially subjected to the subsequent processes of extraction, secondary chromatography, crystal growing, recrystallization and the like, so that the taxol is fully dispersed from other components, and a high-purity taxol product is finally obtained.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
The present invention will be further described with reference to the following examples.
Example 1
A method for purifying paclitaxel, comprising the following steps:
s1, accurately weighing 60g of fine taxus chinensis powder with the granularity of 60 meshes, leaching for 3 times by using an ethanol solution at the temperature of 40 ℃, wherein the leaching time is 30min each time, and then collecting and combining leaching liquor obtained after each leaching for later use;
s2, adding the leaching liquor combined in the step S1 into a proper amount of acidic buffer solution, and recording the obtained solution as mixed liquor; continuously conveying the mixed solution to the electrode chamber, the flushing chamber and the sampling chamber by using a pump; then starting the power supply of the electrophoresis apparatus; when the agarose affinity medium and the mixed solution reach balance, the adsorption buffer solution with the pH value of 6.4 is input into the sampling chamber by a pump; starting electrophoretic adsorption, and carrying out electrophoretic leaching for 15min after the electrophoretic adsorption process lasts for 50 min; after the electrophoretic leaching is finished, turning off the power supply, changing the adsorption buffer solution into an elution buffer solution with the pH value of 8.5, turning on the power supply to carry out electrophoretic elution, collecting active parts, and concentrating to obtain a paclitaxel concentrate;
s3, dissolving the paclitaxel concentrate obtained in the step S2 by using an organic solvent, adding modified alumina after the paclitaxel concentrate is fully dissolved, mixing and stirring uniformly, and drying to obtain a dry material sample; storing it for later use;
s4, filling the dry material sample obtained in the step S3 into a chromatographic column, eluting the dry material sample through flow matching at low pressure, and collecting an effective section in the elution; concentrating the collected effective sections, and storing the obtained concentrated solution for later use; wherein the mobile phase is prepared by mixing ethyl acetate and n-hexane; and the percentage of ethyl acetate in the volume of the mobile phase is 40%;
s5, adding an organic extracting agent formed by mixing ethyl acetate, acetone and the like in mass into the concentrated solution obtained in the step S4 to extract the concentrated solution, and then pumping the extract phase into a concentration tank to concentrate the extract; performing secondary column chromatography separation according to the steps, dissolving the product obtained after the secondary column chromatography separation by using acetone, and then transferring the product into a preparative high performance liquid chromatograph for separation and purification;
and S6, concentrating and pumping the paclitaxel fraction obtained in the step S5 under reduced pressure, adding petroleum ether into the paclitaxel fraction while stirring, naturally cooling to room temperature, performing crystal growing, filtering, drying and other processes to obtain a recrystallized crude product, repeating the operation for 2 times, adding acetone into the recrystallized crude product, dissolving the acetone at the temperature of 40 ℃, adding n-hexane with the same volume as that of the acetone while stirring, and performing crystallization, filtering and drying treatment at the room temperature to finally obtain a purified paclitaxel finished product.
The leaching liquor used in the step S1 is prepared by mixing methanol and dichloromethane in equal volume.
The concentration of the ethanol solution used in the step S1 is 60%, and the dosage of the ethanol solution is 6 times of that of the fine powder of the taxus chinensis.
The acidic buffer solution in step S2 is citric acid-sodium citrate buffer solution with 0.1mol/L, and the pH value is 6.4.
In step S2, the adsorption buffer solution is 20mM citric acid-sodium citrate buffer solution, and the ph thereof is 4.5, and the current intensity I =60mA.
In step S2, 20mM Tris-HCl is used as the elution buffer, and the current intensity I =40mA.
Chloroform is used as the organic solvent in step S3, and the dosage of the organic solvent is 4 times of that of the taxol concentrate.
The preparation method of the modified alumina used in the step S3 comprises the following steps:
a. weighing a proper amount of chlorine spheres, transferring the chlorine spheres into a reaction device provided with a condensing device, and then respectively adding a methanol solution with the mass being 5 times that of the chlorine spheres and the concentration being 55% and 2 times that of triethylene tetramine into the reaction device; after mixing and stirring evenly, adding a proper amount of sodium carbonate and tetra-n-butylammonium bromide into a reaction device, electrically stirring for 5 hours at the temperature of 80 ℃, stopping the reaction, filtering, washing with absolute ethyl alcohol, washing with water, drying in vacuum to constant weight, and storing for later use; wherein the dosage of the sodium carbonate is 40 percent of that of the chlorine ball, and the dosage of the tetra-n-butylammonium bromide is 8 percent of that of the sodium carbonate;
b. b, placing the product obtained in the step a into a reaction kettle containing an ethanol solution, then respectively adding alumina, ethylenediamine propyl triethoxysilane and lauroyl peroxide into the reaction kettle, and mixing and stirring uniformly; reacting the mixed components obtained in the kettle at a constant temperature of 40 ℃ for 12 hours, and filtering, washing with alcohol and drying in vacuum to constant weight in sequence when the reaction is finished and the temperature in the kettle naturally drops to room temperature to obtain a modified alumina finished product;
wherein the mass ratio of the product obtained in the step a to the aluminum oxide to the ethylenediamine propyltriethoxysilane to the lauroyl peroxide is 1: 0.03;
the concentration of the ethanol solution used in step b was 60% and the amount thereof was 5 times that of the product obtained in step a.
Example 2
A method for purifying paclitaxel, comprising the following steps:
s1, accurately weighing 70g of 80-mesh fine taxus chinensis powder, leaching for 3 times by using an ethanol solution at the temperature of 45 ℃, wherein the leaching time is 35min each time, and then collecting and combining leaching liquor obtained after each leaching for later use;
s2, adding the leaching liquor combined in the step S1 into a proper amount of acidic buffer solution, and recording the obtained solution as mixed liquor; continuously conveying the mixed solution to the electrode chamber, the flushing chamber and the sampling chamber by using a pump; then starting the power supply of the electrophoresis apparatus; when the agarose affinity medium and the mixed solution reach the equilibrium, the adsorption buffer solution with the pH value of 6.4 is input into the sample chamber by a pump; starting electrophoretic adsorption, and carrying out electrophoretic leaching for 18min after the electrophoretic adsorption process lasts for 55 min; after the electrophoretic leaching is finished, turning off the power supply, changing the adsorption buffer solution into an elution buffer solution with the pH value of 8.5, turning on the power supply to carry out electrophoretic elution, collecting active parts, and concentrating to obtain a paclitaxel concentrate;
s3, dissolving the paclitaxel concentrate obtained in the step S2 by using an organic solvent, adding modified alumina after the paclitaxel concentrate is fully dissolved, mixing and stirring uniformly, and drying to obtain a dry material sample; storing it for later use;
s4, filling the dry material sample obtained in the step S3 into a chromatographic column, eluting the dry material sample through flow matching at low pressure, and collecting an effective section in the elution; concentrating the collected effective sections, and storing the obtained concentrated solution for later use; wherein the mobile phase is prepared by mixing ethyl acetate and normal hexane; and the percentage of ethyl acetate in the volume of the mobile phase is 50%;
s5, adding an organic extracting agent formed by mixing ethyl acetate, acetone and the like in mass into the concentrated solution obtained in the step S4 to extract the concentrated solution, and then pumping the extract phase into a concentration tank to concentrate the extract; performing secondary column chromatography separation according to the steps, dissolving the product obtained after the secondary column chromatography separation by using acetone, and then transferring the product into a preparative high performance liquid chromatograph for separation and purification;
and S6, concentrating and pumping the paclitaxel fraction obtained in the step S5 under reduced pressure, adding petroleum ether into the paclitaxel fraction while stirring, naturally cooling to room temperature, performing crystal growing, filtering, drying and other processes to obtain a recrystallized crude product, repeating the operation for 2 times, adding acetone into the recrystallized crude product, dissolving the acetone at the temperature of 45 ℃, adding n-hexane with the same volume as that of the acetone while stirring, and performing crystallization, filtering and drying treatment at the room temperature to finally obtain a purified paclitaxel finished product.
The leaching liquor used in the step S1 is prepared by mixing methanol and dichloromethane in equal volume.
The concentration of the ethanol solution used in the step S1 is 65%, and the dosage of the ethanol solution is 7 times of that of the fine powder of the taxus chinensis.
The acidic buffer solution in step S2 is citric acid-sodium citrate buffer solution with a concentration of 0.1mol/L, and the pH value is 6.4.
In step S2, 20mM citric acid-sodium citrate buffer is used as the adsorption buffer solution, and the ph of the adsorption buffer solution is 4.5, and the current intensity I =60mA.
In step S2, 20mM Tris-HCl is used as the elution buffer, and the current intensity I =40mA.
The organic solvent used in step S3 is acetone, and the dosage of the organic solvent is 5 times of that of the taxol concentrate.
The preparation method of the modified alumina used in the step S3 comprises the following steps:
a. weighing a proper amount of chlorine balls, transferring the chlorine balls into a reaction device provided with a condensing device, and then respectively adding a methanol solution with the mass being 6 times that of the chlorine balls and the concentration being 60% and 2 times that of triethylene tetramine into the reaction device; after mixing and stirring evenly, adding a proper amount of sodium carbonate and tetra-n-butylammonium bromide into a reaction device, electrically stirring for 7 hours at 85 ℃, stopping the reaction, filtering, washing with absolute ethyl alcohol, washing with water, drying in vacuum to constant weight, and storing for later use; wherein the dosage of the sodium carbonate is 50 percent of that of the chlorine ball, and the dosage of the tetra-n-butylammonium bromide is 10 percent of that of the sodium carbonate;
b. b, placing the product obtained in the step a into a reaction kettle containing an ethanol solution, then respectively adding alumina, ethylenediamine propyl triethoxysilane and lauroyl peroxide into the reaction kettle, and mixing and stirring uniformly; carrying out constant-temperature reaction on the mixed components obtained in the kettle at the temperature of 45 ℃ for 14h, and after the reaction is finished and the temperature in the kettle naturally drops to room temperature, sequentially filtering, washing with alcohol and drying in vacuum to constant weight to obtain a modified aluminum oxide finished product;
wherein the mass ratio of the product obtained in the step a to the aluminum oxide to the ethylenediamine propyltriethoxysilane to the lauroyl peroxide is 1: 0.04;
the concentration of the ethanol solution used in step b was 70% and the amount thereof was 6 times that of the product obtained in step a.
Example 3
A method for purifying paclitaxel, comprising the steps of:
s1, accurately weighing 80g of taxus fine powder with the granularity of 100 meshes, leaching for 4 times by using an ethanol solution at the temperature of 50 ℃, wherein the leaching time is 40min each time, and collecting and combining leaching liquor obtained after each leaching for later use;
s2, adding the combined leaching liquor obtained in the step S1 into a proper amount of acidic buffer solution, and recording the obtained solution as mixed liquor; continuously conveying the mixed solution to the electrode chamber, the flushing chamber and the sampling chamber by using a pump; then starting the power supply of the electrophoresis apparatus; when the agarose affinity medium and the mixed solution reach balance, the adsorption buffer solution with the pH value of 6.4 is input into the sampling chamber by a pump; starting electrophoretic adsorption, and carrying out electrophoretic leaching for 20min after the electrophoretic adsorption process lasts for 60 min; after the electrophoretic leaching is finished, turning off the power supply, changing the adsorption buffer solution into an elution buffer solution with the pH of 8.5, turning on the power supply to carry out electrophoretic elution, collecting the active part, and concentrating to obtain a taxol concentrate;
s3, dissolving the paclitaxel concentrate obtained in the step S2 by using an organic solvent, adding modified alumina after the paclitaxel concentrate is fully dissolved, mixing and stirring uniformly, and drying to obtain a dry material sample; storing it for later use;
s4, filling the dry material sample obtained in the step S3 into a chromatographic column, eluting the dry material sample through flow matching at low pressure, and collecting an effective section in the elution; concentrating the collected effective sections, and storing the obtained concentrated solution for later use; wherein the mobile phase is prepared by mixing ethyl acetate and n-hexane; and the percentage of ethyl acetate to the volume of the mobile phase is 60%;
s5, adding an organic extracting agent formed by mixing ethyl acetate, acetone and the like in mass into the concentrated solution obtained in the step S4 to extract the concentrated solution, and then pumping the extract phase into a concentration tank to concentrate the extract; performing secondary column chromatography separation according to the steps, dissolving the product obtained after the secondary column chromatography separation by using acetone, and then transferring the product into a preparative high performance liquid chromatograph for separation and purification;
and S6, concentrating and pumping the paclitaxel fraction obtained in the step S5 under reduced pressure, adding petroleum ether while stirring, naturally cooling to room temperature, growing crystals, filtering, drying and the like to obtain a recrystallized crude product, repeating the operation for 3 times, adding acetone into the recrystallized crude product, dissolving at the temperature of 50 ℃, adding n-hexane with the same volume as that of the acetone while stirring, crystallizing at room temperature, filtering and drying to finally obtain a purified paclitaxel finished product.
The leaching liquor used in the step S1 is prepared by mixing methanol and dichloromethane in equal volume.
The concentration of the ethanol solution used in the step S1 is 75%, and the dosage of the ethanol solution is 8 times of the fine powder of the taxus chinensis.
The acidic buffer solution in step S2 is citric acid-sodium citrate buffer solution with a concentration of 0.1mol/L, and the pH value is 6.4.
In step S2, 20mM citric acid-sodium citrate buffer is used as the adsorption buffer solution, and the ph of the adsorption buffer solution is 4.5, and the current intensity I =60mA.
In step S2, 20mM Tris-HCl is used as the elution buffer, and the current intensity I =40mA.
Chloroform is used as the organic solvent in step S3, and the dosage of the organic solvent is 6 times of that of the taxol concentrate.
The preparation method of the modified alumina used in the step S3 comprises the following steps:
a. weighing a proper amount of chlorine balls, transferring the chlorine balls into a reaction device provided with a condensing device, and then respectively adding a methanol solution with the mass being 8 times that of the chlorine balls and the concentration being 65% and 3 times that of triethylene tetramine into the reaction device; after mixing and stirring uniformly, adding a proper amount of sodium carbonate and tetra-n-butylammonium bromide into a reaction device, electrically stirring for 8 hours at the temperature of 90 ℃, stopping the reaction, filtering, washing with absolute ethyl alcohol, washing with water, drying in vacuum to constant weight, and storing for later use; wherein the dosage of the sodium carbonate is 60 percent of that of the chlorine ball, and the dosage of the tetra-n-butylammonium bromide is 12 percent of that of the sodium carbonate;
b. b, placing the product obtained in the step a into a reaction kettle containing ethanol solution, then respectively adding alumina, ethylenediamine propyl triethoxysilane and lauroyl peroxide into the reaction kettle, and mixing and stirring uniformly; reacting the mixed components obtained in the kettle at a constant temperature of 50 ℃ for 15h, and filtering, washing with alcohol and drying in vacuum to constant weight in sequence when the reaction is finished and the temperature in the kettle naturally drops to room temperature to obtain a modified alumina finished product;
wherein the mass ratio of the product obtained in the step a to the aluminum oxide to the ethylenediamine propyltriethoxysilane to the lauroyl peroxide is 1: 0.05;
the ethanol solution used in step b had a concentration of 75% and was used in an amount 8 times the amount of the product obtained in step a.
Performance testing
Comparative example: a paclitaxel product purified according to the method for separating and purifying paclitaxel from a crude paclitaxel product provided in patent document CN 03135916.7;
example (b): paclitaxel products purified by examples 1 to 3 of the present invention (referred to as examples 1 to 3);
HPLC detection was performed on the paclitaxel products purified in comparative examples and examples 1-3, respectively, and the data obtained are reported in the following table:
| purity/% of paclitaxel | Cephalomannine content/%) | 7-epitaxol content/%) | |
| Example 1 | 99.36 | 0.053 | 0.038 |
| Example 2 | 99.65 | 0.068 | 0.042 |
| Example 3 | 99.73 | 0.062 | 0.045 |
| Comparative example | 84.68 | 4.27 | 2.29 |
As can be seen from the related data in the above table, compared with the method for isolating and purifying paclitaxel from the crude paclitaxel product provided by the comparative example, the paclitaxel purified by the present invention has higher purity, less impurity content and better quality. The paclitaxel purification process provided by the invention has wider market prospect and is more suitable for popularization.
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for purifying paclitaxel, comprising the steps of:
s1, accurately weighing 60-80 g of taxus chinensis fine powder with the granularity of 60-100 meshes, leaching for 3-4 times by using leaching liquor with the temperature of 30-40 ℃, wherein the leaching time is 30-40 min each time, and then collecting and combining the leaching liquor obtained after each leaching for later use;
s2, adding the combined leaching liquor obtained in the step S1 into a proper amount of acidic buffer solution, and recording the obtained solution as mixed liquor; continuously conveying the mixed solution to the electrode chamber, the flushing chamber and the sampling chamber by using a pump; then starting the power supply of the electrophoresis apparatus; when the agarose affinity medium and the mixed solution reach the equilibrium, the adsorption buffer solution with the pH value of 6.4 is input into the sample chamber by a pump; starting electrophoretic adsorption, and carrying out electrophoretic leaching for 15-20 min after the electrophoretic adsorption process lasts for 50-60 min; after the electrophoretic leaching is finished, turning off the power supply, changing the adsorption buffer solution into an elution buffer solution with the pH of 8.5, turning on the power supply to carry out electrophoretic elution, collecting the active part, and concentrating to obtain a taxol concentrate;
s3, dissolving the paclitaxel concentrate obtained in the step S2 by using an organic solvent, adding modified alumina after the paclitaxel concentrate is fully dissolved, mixing and stirring uniformly, and drying to obtain a dry material sample; storing it for later use;
s4, filling the dry material sample obtained in the step S3 into a chromatographic column, eluting the dry material sample through flow matching at low pressure, and collecting an effective section in the elution; concentrating the collected effective sections, and storing the obtained concentrated solution for later use; wherein the mobile phase is prepared by mixing ethyl acetate and normal hexane; and the percentage of ethyl acetate in the mobile phase volume is 40-60%;
s5, adding an organic extracting agent formed by mixing ethyl acetate, acetone and the like in mass into the concentrated solution obtained in the step S4 to extract the concentrated solution, and then pumping the extract phase into a concentration tank to concentrate the extract; performing secondary column chromatography separation according to the steps, dissolving the product obtained after the secondary column chromatography separation by using acetone, and then transferring the product into a preparative high performance liquid chromatograph for separation and purification;
and S6, after the paclitaxel fraction obtained in the step S5 is subjected to reduced pressure concentration and pumping drying, adding petroleum ether into the paclitaxel fraction while stirring, naturally cooling to room temperature, growing crystals, filtering, drying and the like to obtain a recrystallized crude product, repeating the operation for 2-3 times, adding acetone into the recrystallized crude product, dissolving the acetone at the temperature of 40-50 ℃, then adding n-hexane with the same volume as that of the acetone while stirring, and performing crystallization, filtering and drying treatment at room temperature to finally obtain a purified paclitaxel finished product.
2. The method of claim 1, wherein the paclitaxel is purified by: the leaching liquor used in the step S1 is prepared by mixing methanol and dichloromethane in equal volume.
3. The method of claim 1, wherein the paclitaxel is purified by: the concentration of the ethanol solution used in the step S1 is 60-75%, and the dosage of the ethanol solution is 6-8 times of the taxus fine powder.
4. The method of claim 1, wherein the paclitaxel is purified by: the acidic buffer solution in the step S2 is citric acid-sodium citrate buffer solution with 0.1mol/L, and the pH value is 6.4.
5. The method of claim 1, wherein the paclitaxel is purified by: the adsorption buffer solution in the step S2 is 20mM citric acid-sodium citrate buffer solution, and the ph thereof is 4.5, and the current intensity I =60mA.
6. The method of claim 1, wherein the paclitaxel is purified by: in the step S2, 20mM Tris-HCl is used as an elution buffer, and the current intensity I =40mA.
7. The method of claim 1, wherein the paclitaxel is purified by: the organic solvent used in the step S3 is any one of chloroform or acetone, and the dosage of the organic solvent is 4-6 times of that of the paclitaxel concentrate.
8. The method for purifying paclitaxel according to claim 1, wherein the modified alumina used in step S3 is prepared by the following steps:
a. weighing a proper amount of chlorine balls, transferring the chlorine balls into a reaction device provided with a condensing device, and then respectively adding 5-8 times of methanol solution with the mass of 5-65% of the chlorine balls and 2-3 times of triethylene tetramine into the reaction device; after mixing and stirring uniformly, adding a proper amount of sodium carbonate and tetra-n-butylammonium bromide into a reaction device, electrically stirring for 5-8 h at 80-90 ℃, stopping reaction, filtering, washing with absolute ethyl alcohol, washing with water, drying in vacuum to constant weight, and storing for later use; wherein the dosage of the sodium carbonate is 40-60% of the chlorine ball, and the dosage of the tetra-n-butylammonium bromide is 8-12% of the sodium carbonate;
b. b, placing the product obtained in the step a into a reaction kettle containing an ethanol solution, then respectively adding alumina, ethylenediamine propyl triethoxysilane and lauroyl peroxide into the reaction kettle, and mixing and stirring uniformly; reacting the mixed components obtained in the kettle at a constant temperature of 40-50 ℃ for 12-15 h, and after the reaction is finished and the temperature in the kettle naturally drops to room temperature, sequentially filtering, washing with alcohol and drying in vacuum to constant weight to obtain a modified alumina finished product;
wherein the mass ratio of the product obtained in the step a, the alumina, the ethylenediamine propyl triethoxysilane and the lauroyl peroxide is 1: 0.03 to 0.05.
9. The method of claim 8, wherein the paclitaxel is purified by: the concentration of the ethanol solution used in the step b is 60-75%, and the dosage of the ethanol solution is 5-8 times of that of the product obtained in the step a.
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| JP2003502414A (en) * | 1999-06-22 | 2003-01-21 | チャイケム・ファーマシューティカル・インターナショナル | Extraction and purification process of paclitaxel from natural resources |
| US6759539B1 (en) * | 2003-02-27 | 2004-07-06 | Chaichem Pharmaceuticals International | Process for isolation and purification of paclitaxel from natural sources |
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| CN104529951A (en) * | 2014-12-10 | 2015-04-22 | 宁波绿之健药业有限公司 | Preparation method for natural paclitaxel |
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| JP2003502414A (en) * | 1999-06-22 | 2003-01-21 | チャイケム・ファーマシューティカル・インターナショナル | Extraction and purification process of paclitaxel from natural resources |
| US6759539B1 (en) * | 2003-02-27 | 2004-07-06 | Chaichem Pharmaceuticals International | Process for isolation and purification of paclitaxel from natural sources |
| CN1526708A (en) * | 2003-09-22 | 2004-09-08 | 丁志坚 | Coarse taxol product separating and purifying process |
| WO2008087218A2 (en) * | 2007-01-19 | 2008-07-24 | Becton, Dickinson & Company | Stabilizing and separation media for electrophoresis applications |
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