CN104892551A - Method for separation and purification of 10-deacetylbaccatin III from branches and leaves of taxus chinensis - Google Patents
Method for separation and purification of 10-deacetylbaccatin III from branches and leaves of taxus chinensis Download PDFInfo
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- CN104892551A CN104892551A CN201510357296.7A CN201510357296A CN104892551A CN 104892551 A CN104892551 A CN 104892551A CN 201510357296 A CN201510357296 A CN 201510357296A CN 104892551 A CN104892551 A CN 104892551A
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- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
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Abstract
The invention discloses a method for separation and purification of 10-deacetylbaccatin III from branches and leaves of taxus chinensis. The method includes the steps: 1) crushing and drying the branches and the leaves of taxus chinensis, adopting 80% ethyl alcohol for reflux extraction for 5 hours, continuously extracting for 3 times, and subjecting filtrate to vacuum concentration to obtain 10-DAB (deacetylbaccatin) III extracts; 2) dissolving the 10-DAB III extracts, performing dry method sampling to collect eluent containing 10-DAB III, and evaporating to remove solvents so as to obtain a semi-finished 10-DAB III product; 3) dissolving the semi-finished 10-DAB III product, adding imidazole compounds according to a mole ratio of (1:1.5)-4, stirring for 30min, standing, cooling at the temperature of 0 DEG C, filtering and drying to obtain a crystal complex of 10-DAB III and imidazole; 4) dissolving the crystal complex of 10-DAB III and imidazole in water, and extracting, drying and removing solvents to obtain a pure 10-DAB III product with 10-DAB III content being more than 99%.
Description
Technical field
The present invention relates to a kind of method of separating-purifying 10-deacetylate baccatin III (10-DAB III) from Ramulus et folium taxi cuspidatae, particularly utilize 10-DAB III crude product and glyoxaline compound crystallization purifying to obtain the separation method of high purity 10-DAB III.
Background technology
Docetaxel is considered to one of the most significant cancer therapy drug of curative effect so far, within 1996, confirms the clinical medicine started for aspects such as lung cancer, mammary cancer, colorectal carcinomas through FDA.Docetaxel leukemia and the anti entity tumour activity with wide spectrum the same as taxol (Taxol), complex structure, polyfunctional group and multichiral center etc., complete synthesis cost is high, difficulty is larger.When extracting higher 10-deacetyltaxol 10-deacetylate bar Ka Ting (the 10-deacetyl baccatin III of concentration in the needle finding european yew; being called for short 10-DAB III) after intermediate, synthesis and the large-scale production of Docetaxel just become possibility.The structural formula of 10-DAB III is as follows:
10-DAB III is mainly distributed in the branches and leaves of Ramulus et folium taxi cuspidatae, and its content can up to more than 0.1%, and minimum at perennial content of bark.Its distribution is just contrary with the distribution of taxol, and taxol is mainly distributed in perennial bark of Ramulus et folium taxi cuspidatae, and content is many below 0.03%, and content is extremely low in leaf, mostly not as good as 1/10th of content in bark.Branches and leaves are renewable resourcess, and therefore produce 10-DAB III and do not resemble production taxol to sacrifice limited taxus resource for cost, therefore, the Isolation and purification technique tool of research 10-DAB III is of great significance.
At present, the extraction and isolation about 10-DAB III has lot of documents and patent report, is substantially all to take extracting and developing, crystallization three steps, and wherein separation and crystallization are the committed steps affecting yield and purity.Conventional methyl alcohol or the alcohol of adopting carries out soak extraction, and extraction is concentrated, and then repeatedly silica gel column chromatography is separated, and recrystallization obtains the 10-DABIII of certain purity.Repeatedly column chromatography for separation and recrystallization process are not only wasted time and energy, and increase solvent cost, also can reduce yield.Thus how separation purifying technique is simplified, the least possible utilize organic solvent to obtain easily emphasis that high-quality 10-DABIII becomes this area research.
China Patent Publication No. CN1473821A discloses a kind of method extracting 10-DABIII, and adopt the work in-process 10-DABIII that the process of conventional silica gel column chromatography separation and Extraction goes out, the method comparatively expends time in, and solvent-oil ratio is also large, easily causes the loss of yield.China Patent Publication No. CN1398859A discloses a kind of from the plantation Ramulus et folium taxi cuspidatae method extracting 10-DABIII rear residue; the method relates to preparative liquid chromatography sepn process; the complex process but solvent for use system and gradient elution means seem in large-scale production, solvent-oil ratio is large.China Patent Publication No. CN101045719A discloses a kind of work in-process utilizing preparative liquid chromatography separation and purification 10-DABIII, be elution flow phase with analytical pure tetrahydrofuran (THF), normal hexane and methanol mixed solvent, preparation technology is comparatively complicated, is more suitable for laboratory small serial production.
Summary of the invention
Extraction purification for 10-DABIII needs to use repeatedly column chromatography and recrystallization, or use the present situation that complicated preparative chromatography technology realizes, the present invention proposes 10-DABIII crude product through primary column chromatography with once add the simplification separation method that glyoxaline compound carries out crystallization purifying acquisition high purity fine work.Consider in the structure of 10-DAB III containing poly-hydroxy, if add organic compounds containing nitrogen, then can form crystalline compounds by hydrogen bond between the two, we study discovery, 10-DAB III easily forms crystalline complex with imidazoles, and is not subject to other impurity effect, what is more important, after the crystalline complex formed adds water can easily with organic solvent extraction out, such Crystallizing process provides conveniently for obtaining highly purified 10-DAB III.
The object of the present invention is to provide a kind of simple and efficient separation and purification 10-DABIII crude product, obtain the method for high purity 10-DABIII.
Object of the present invention is achieved through the following technical solutions:
(1) after Ramulus et folium taxi cuspidatae being pulverized oven dry, in the aqueous ethanolic solution refluxing extraction 5 hours that the ratio volume fraction of solid-to-liquid ratio 1:10 is 80%, extract 3 times continuously, merging filtrate, obtain 10-DABIII extract after concentrating under reduced pressure, wherein the mass content of 10-DABIII is 8-20%;
(2) by dry method loading after 10-DABIII extract dissolution with solvents, with elution, collect the elutriant containing 10-DABIII, steam and desolventize to obtain 10-DABIII work in-process;
(3) after above-mentioned 10-DABIII work in-process methylene dichloride being dissolved, be 10-DABIII work in-process in molar ratio: the ratio of glyoxaline compound=1:1.5-4.0 adds glyoxaline compound, after stirring at room temperature 30min, leave standstill, 0 DEG C of cooling, gained crystalline solid filtered, vacuum-drying, obtains the crystalline complex of 10-DABIII and imidazoles;
(4) crystalline complex of above-mentioned 10-DABIII and imidazoles is dissolved in water, with dichloromethane extraction three times, merges organic phase, then use anhydrous sodium sulfate drying, to remove after solvent to obtain 10-DAB III sterling.
The described solvent dissolving 10-DABIII extract of step (2) is one or more in tetrahydrofuran (THF), methylene dichloride, methyl alcohol;
Elutriant described in step (2) be selected from following mixed solvent any one: ethyl acetate-light petrol, methyl alcohol-tetrahydrofuran (THF)-normal hexane, Ethyl acetate-cyclohexane;
Glyoxaline compound described in step (3) is one or more in imidazoles, 1-Methylimidazole, glyoxal ethyline.
The present invention has the following advantages:
(1) utilize 10-DAB III easily and glyoxaline compound form the feature of crystalline complex, achieve the object that 10-DAB III and other bearing taxanes carry out being separated easily;
(2), after 10-DAB III and glyoxaline compound form crystalline complex, can be separated with organic solvent extraction easily, thus obtain high purity product;
(3) sepn process only needs primary column chromatography and primary crystallization sepn process, avoids column chromatography and the recrystallization operation of continuous several times, greatly reduces human and material resources and expend;
(4) glyoxaline compound forming crystalline complex with 10-DAB III can reclaim, and recycles, the lower also more environmental protection of cost.
Embodiment
Embodiment 1:
(1) 100 object fine powders are ground into after being dried by Ramulus et folium taxi cuspidatae, get the aqueous ethanolic solution refluxing extraction 5 hours that 1kg fine powder in the ratio volume fraction of solid-to-liquid ratio 1:10 is 80%, continuous extraction 3 times, merging filtrate, obtains 10-DABIII extract 120g after concentrating under reduced pressure.
(2) dry method loading after 10-DABIII extract being dissolved with 300mL tetrahydrofuran (THF), with ethyl acetate-light petrol (volume ratio ethyl acetate: sherwood oil=2:1) wash-out, collect the elutriant containing 10-DABIII, steam and desolventize to obtain 10-DABIII work in-process 1.85g.
(3) after being dissolved by above-mentioned 10-DABIII work in-process 20mL methylene dichloride, add imidazoles 0.20g, after stirring at room temperature 30min, leave standstill, 0 DEG C of cooling, gained crystalline solid filtered, vacuum-drying, obtains the crystalline complex 1.08g of 10-DABIII and imidazoles.
(4) crystalline complex of above-mentioned 10-DABIII and imidazoles is dissolved in water, with dichloromethane extraction three times, merges organic phase, anhydrous sodium sulfate drying, to remove after solvent to obtain 10-DAB III sterling 0.896g, purity 99.2%.
Embodiment 2:
(1) 100 object fine powders are ground into after being dried by Ramulus et folium taxi cuspidatae, get the aqueous ethanolic solution refluxing extraction 5 hours that 10kg powder in the ratio volume fraction of solid-to-liquid ratio 1:10 is 80%, continuous extraction 3 times, merging filtrate, obtains 10-DABIII extract 1350g after concentrating under reduced pressure.
(2) repeatedly dry method loading after 10-DABIII extract being dissolved with 3000mL tetrahydrofuran (THF), with Ethyl acetate-cyclohexane (volume ratio ethyl acetate: hexanaphthene=2:1) wash-out, collect the elutriant containing 10-DABIII, steam and desolventize to obtain 10-DABIII work in-process 22.8g.
(3) after being dissolved by above-mentioned 10-DABIII work in-process 200mL methylene dichloride, add imidazoles 3.0g, after stirring at room temperature 30min, leave standstill, 0 DEG C of cooling, gained crystalline solid filtered, vacuum-drying, obtains the crystalline complex 11.9g of 10-DABIII and imidazoles.
(4) crystalline complex of above-mentioned 10-DABIII and imidazoles is dissolved in water, with dichloromethane extraction three times, merges organic phase, anhydrous sodium sulfate drying, to remove after solvent to obtain 10-DAB III sterling 9.35g, purity 99.4%.
Embodiment 3:
(1) 100 object fine powders are ground into after being dried by Ramulus et folium taxi cuspidatae, get the aqueous ethanolic solution refluxing extraction 5 hours that 5kg powder in the ratio volume fraction of solid-to-liquid ratio 1:10 is 80%, continuous extraction 3 times, merging filtrate, obtains 10-DABIII extract 680g after concentrating under reduced pressure.
(2) repeatedly dry method loading after 10-DABIII extract being dissolved with 1200mL methylene dichloride, with ethyl acetate-light petrol (volume ratio ethyl acetate: sherwood oil=2:1) wash-out, collect the elutriant containing 10-DABIII, steam and desolventize to obtain 10-DABIII work in-process 10.9g.
(3) after being dissolved by above-mentioned 10-DABIII work in-process 100mL methylene dichloride, add imidazoles 2.0g, after stirring at room temperature 30min, leave standstill, 0 DEG C of cooling, gained crystalline solid filtered, vacuum-drying, obtains the crystalline complex 5.8g of 10-DABIII and imidazoles.
(4) crystalline complex of above-mentioned 10-DABIII and imidazoles is dissolved in water, with dichloromethane extraction three times, merges organic phase, anhydrous sodium sulfate drying, to remove after solvent to obtain 10-DAB III sterling 4.38g, purity 99.5%.
Claims (6)
1. the method for high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae, is characterized in that comprising the steps:
(1) after Ramulus et folium taxi cuspidatae being pulverized oven dry, in the aqueous ethanolic solution refluxing extraction 5 hours that the ratio volume fraction of solid-to-liquid ratio 1:10 is 80%, extract 3 times continuously, merging filtrate, obtain 10-DABIII extract after concentrating under reduced pressure, wherein the mass content of 10-DABIII is 8-20%;
(2) by dry method loading after 10-DABIII extract dissolution with solvents, with elution, collect the elutriant containing 10-DABIII, steam and desolventize to obtain 10-DABIII work in-process;
(3) after above-mentioned 10-DABIII work in-process methylene dichloride being dissolved, be 10-DABIII work in-process in molar ratio: the ratio of glyoxaline compound=1:1.5-4.0 adds glyoxaline compound, after stirring at room temperature 30min, leave standstill, 0 DEG C of cooling, gained crystalline solid filtered, vacuum-drying, obtains the crystalline complex of 10-DABIII and imidazoles;
(4) crystalline complex of above-mentioned 10-DABIII and imidazoles is dissolved in water, with dichloromethane extraction three times, merges organic phase, then use anhydrous sodium sulfate drying, to remove after solvent to obtain 10-DAB III sterling.
2. the method for a kind of high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae as claimed in claim 1, is characterized in that: the solvent dissolving 10-DABIII extract in step (2) is one or more in tetrahydrofuran (THF), methylene dichloride, methyl alcohol.
3. the method for a kind of high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae as claimed in claim 1, is characterized in that: the elutriant in step (2) is selected from the one in following mixed solvent: ethyl acetate-light petrol, methyl alcohol-tetrahydrofuran (THF)-normal hexane, Ethyl acetate-cyclohexane.
4. the method for a kind of high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae as claimed in claim 1, is characterized in that: the glyoxaline compound in step (3) is one or more in imidazoles, 1-Methylimidazole, glyoxal ethyline.
5. the method for a kind of high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae as claimed in claim 1, is characterized in that: through primary column chromatography with once add glyoxaline compound and carry out crystallization purifying and obtain high purity 10-DABIII.
6. the method for a kind of high efficiency separation purification 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae as claimed in claim 1, is characterized in that: gained 10-DABIII adds water with the crystalline complex of imidazoles and is separated, and organic solvent extracting obtains sterling.
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| CN201510357296.7A CN104892551B (en) | 2015-06-25 | 2015-06-25 | A kind of method of separating-purifying 10-deacetylate baccatin III from Ramulus et folium taxi cuspidatae |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003145A (en) * | 2019-04-15 | 2019-07-12 | 云南汉德生物技术有限公司 | A method of extracting 10- deacetylate Bakating III from Chinese yew |
| CN114275769A (en) * | 2021-12-15 | 2022-04-05 | 井冈山大学 | Method for separating C from fullerene mixture by utilizing silver salt60And C70Method (2) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1398859A (en) * | 2002-08-27 | 2003-02-26 | 上海万古生物科技发展有限公司 | Process of preparing 10-deacetyl Bakatin III from taxad branch and leaf residue |
| CN1473821A (en) * | 2003-07-03 | 2004-02-11 | 上海金和生物技术有限公司 | Method for extracting and purifying 10-desacetyl bakading III (10-DABIII) |
| CN101045719A (en) * | 2007-04-30 | 2007-10-03 | 成都普思生物科技有限公司 | Method for high efficiency separating and purifying 1-deacetyl Baccatins III (10-DABIII) |
| CN102827106A (en) * | 2012-08-29 | 2012-12-19 | 宁波绿之健药业有限公司 | Method for extracting and purifying 10-DAB (diaminobenzidine) |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398859A (en) * | 2002-08-27 | 2003-02-26 | 上海万古生物科技发展有限公司 | Process of preparing 10-deacetyl Bakatin III from taxad branch and leaf residue |
| CN1473821A (en) * | 2003-07-03 | 2004-02-11 | 上海金和生物技术有限公司 | Method for extracting and purifying 10-desacetyl bakading III (10-DABIII) |
| CN101045719A (en) * | 2007-04-30 | 2007-10-03 | 成都普思生物科技有限公司 | Method for high efficiency separating and purifying 1-deacetyl Baccatins III (10-DABIII) |
| CN102827106A (en) * | 2012-08-29 | 2012-12-19 | 宁波绿之健药业有限公司 | Method for extracting and purifying 10-DAB (diaminobenzidine) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003145A (en) * | 2019-04-15 | 2019-07-12 | 云南汉德生物技术有限公司 | A method of extracting 10- deacetylate Bakating III from Chinese yew |
| CN114275769A (en) * | 2021-12-15 | 2022-04-05 | 井冈山大学 | Method for separating C from fullerene mixture by utilizing silver salt60And C70Method (2) |
| CN114275769B (en) * | 2021-12-15 | 2023-04-25 | 井冈山大学 | Separation of C from fullerene mixture using silver salt 60 And C 70 Is a method of (2) |
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