CN102212045A - Method for simultaneously extracting taxol and 10-DABIII (10-deacetyl baccatine III) from branches and leaves of Taxus media - Google Patents
Method for simultaneously extracting taxol and 10-DABIII (10-deacetyl baccatine III) from branches and leaves of Taxus media Download PDFInfo
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- CN102212045A CN102212045A CN2010101459261A CN201010145926A CN102212045A CN 102212045 A CN102212045 A CN 102212045A CN 2010101459261 A CN2010101459261 A CN 2010101459261A CN 201010145926 A CN201010145926 A CN 201010145926A CN 102212045 A CN102212045 A CN 102212045A
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Abstract
The invention relates to a method for simultaneously extracting taxol and 10-DABIII (10-deacetyl baccatine III) from branches and leaves of Taxus media, which separately employs ultrasonic extraction, solvent lixiviation and Soxhlet extraction and comprises the following steps: concentrating an extracted methanol solution to be dry while depressurizing, extracting with n-hexane to remove pigment and other fat-soluble impurities, extracting with dichloromethane to obtain a final extraction liquid of taxol and 10-DABIII, and detecting the contents by HPLC (high-performance liquid chromatography). The method provided by the invention fully takes advantages of the different extraction methods to simultaneously extract taxol and 10-DABIII, improves the utilization rate of resources, and has the characteristics of simplicity, rapid extraction and high efficiency.
Description
Technical field
The present invention relates to a kind of from plant leaf the method for separation and Extraction natural organic-compound, specifically a kind of method of from the Taxus x media branches and leaves, extracting taxol and 10-DAB III simultaneously.
Background technology
Taxol and 10-DAB III (10-deacetylate baccatin III) all are isolated natural organic-compounds from Ramulus et folium taxi cuspidatae.Taxol is a kind of compound of tetracyclic diterpene, is the natural drug of carrying out property of a kind of treatment ovarian cancer, mammary cancer, leukemia and other cancer, also is simultaneously the natural new antitumoral specifics of unique promotion tubulin cohesion and irreversible depolymerization.10-DAB III is the precursor of taxol.China has just begun the research work of Ramulus et folium taxi cuspidatae and taxol preparation from 1984; be from the Himalaya heavy foliage Ramulus et folium taxi cuspidatae in Tibet, to extract to obtain taxol the earliest; and obtain the peaceful alkali of the antitumour activity new alkaloids suitable three class China firs with taxol; also have 10-deacetylate taxol, 10-deacetylate Cephalomannine, 1q-hydroxyl crust card fourth III and 1B-hydroxyl crust card I III etc. in addition, antitumour activity is all arranged.But separate in Ramulus et folium taxi cuspidatae in the many taxanes diterpene-kind compounds and alkaloid that obtain, what activity was the strongest is taxol.But this compound content in Chinese yew is extremely low, greatly between 0.001%-0.076%, and extracts difficulty.The preparation taxol method of from taxus, extracting comparatively commonly used for a long time, its operational path is generally: leach (or ultrasonic wave or microwave extraction), and------extraction obtains crude extract, and---------recrystallization obtains product to bromination---normal-phase chromatography separation---, and has developed many improvement technologies on this basis in crystallization in 2-3 industrial chromatography separation in degreasing.But all there is a defective in these existing technologies, promptly once extract and can only obtain a product, as after obtaining taxol or obtaining 10-DAB III, obtain taxol by semisynthetic method.
Summary of the invention
Purpose of the present invention: invent a kind of method of from the Taxus x media branches and leaves, extracting taxol and 10-DAB III simply, quickly and accurately simultaneously.
Purpose of the present invention can be achieved through the following technical solutions:
(1) with behind 50 ℃ of bake dryings of Taxus x media branches and leaves, be ground into 80 orders---120 purpose powder, per 5 gram dry powder extract with the 80mL-120mL ml methanol, and triplicate filters, and merges 3 times methanol extract liquid;
(2) with the methanol extract liquid concentrating under reduced pressure, after the n-hexane extraction removal of impurities, use dichloromethane extraction again, last combined dichloromethane extraction liquid, methylene dichloride partly is exactly the solution that contains taxol and 10-DAB III;
(3) with behind the dichloromethane extraction liquid concentrating under reduced pressure, fixed molten with chromatographic grade methyl alcohol, and carry out the HPLC assay.
The present invention utilizes the branches and leaves resource of Taxus x media, has both extracted taxol, extracts 10-DABIII again, has improved extraction rate, and has improved extraction effect, and method is simple and reliable.
Embodiment
Below in conjunction with embodiment, invention is further described.
The test material therefor: the branches and leaves of Taxus x media, 50 ℃ of oven dry samples are ground into 100 purpose powder.
Test reagent: analytical pure methyl alcohol, normal hexane, methylene dichloride, (big forever chemical reagent development centre, Tianjin), chromatographically pure methyl alcohol and acetonitrile, water are ultrapure water.
The test instrument: KS-600E I type Ultrasonic Cleaners is by (ultrasonic device company limited is given birth to by sea, Ningbo daybreak section), Buchi R-210 Rotary Evaporators, HPLC detects and uses Waters 2487 dual wavelength detectors, Waters 515 pumps, the 7725I sampling valve, HS 2000 chromatographic data process softwares, Milli-Q ultrapure water instrument.The HPLC chromatographic condition: symmetry C 18 reversed-phase columns, 250mm * 4.6mm granularity 5 μ m, column temperature: 30 ℃, moving phase: methyl alcohol: water=65: 35, acetonitrile: water=30: 70, flow velocity: 0.8mLmin
-130 ℃ of column temperatures; Detect wavelength UV:227nm and 234nm; Sampling volume: 10 μ L.
Embodiment one
After gathering the Taxus x media branches and leaves, place 50 ℃ of drying in oven, be ground into 100 purpose dry powder, take by weighing 5g, add 100mL methyl alcohol, supersound process 40min, leave standstill filtration, filter residue so repeats 3 times again with the ultrasonic 40min of the methyl alcohol of 100mL, merges 3 times methanol extract liquid, in Rotary Evaporators, 50 ℃ of water-baths, under the vacuum of-0.09Mpa, reclaim under reduced pressure methyl alcohol is to doing.
Taxol in the HPLC detection methanol extract liquid and the content of 10-DAB III: reclaim under reduced pressure methyl alcohol is to the methyl alcohol of doing back adding 18mL and the water of 2mL, with oil-soluble impuritieses such as n-hexane extraction pigments for several times, after discarding hexane solution, methanol solution is concentrated into dried, use methylene dichloride and water extracted several times again, combined dichloromethane extraction liquid, 40 ℃ of water-baths in Rotary Evaporators,-0.03Mpa vacuum tightness, the evaporated under reduced pressure methylene dichloride.Residuum dissolves with hplc grade methanol, fixed molten 25mL, and the content that HPLC detects taxol and 10-DAB III is 0.177mgg
-1And 0.121mgg
-1
Embodiment two
Test prepares with the extracting solution of Taxus x media branches and leaves solvent lixiviate: after gathering the Taxus x media branches and leaves, place 50 ℃ of drying in oven, be ground into 100 purpose dry powder, take by weighing 5g, add 100mL methyl alcohol, place 50 ℃ of water-baths, 2h filters, and filter residue is again with the methyl alcohol water-bath 2h of 100mL, so repeat 3 times, merge 3 times methanol extract liquid, in Rotary Evaporators, 50 ℃ of water-baths, under the vacuum of-0.09Mpa, reclaim under reduced pressure methyl alcohol is to doing.
Taxol in the HPLC detection methanol extract liquid and the content of 10-DAB III: reclaim under reduced pressure methyl alcohol is to the methyl alcohol of doing back adding 18mL and the water of 2mL, with oil-soluble impuritieses such as n-hexane extraction pigments for several times, after discarding hexane solution, methanol solution is concentrated into dried, use methylene dichloride and water extracted several times again, combined dichloromethane extraction liquid, 40 ℃ of water-baths in Rotary Evaporators,-0.03Mpa vacuum tightness, the evaporated under reduced pressure methylene dichloride.Residuum dissolves with hplc grade methanol, fixed molten 25mL, and the content that HPLC detects taxol and 10-DAB III is 0.151mgg
-1And 0.097mgg
-1
Embodiment three
Test prepares with Taxus x media branches and leaves Soxhlet extracting solution: after gathering the Taxus x media branches and leaves, place 50 ℃ of drying in oven, be ground into 100 purpose dry powder, take by weighing 5g, place apparatus,Soxhlet's, add 150mL methyl alcohol, 80 ℃,, extract 6h with the speed of siphon per hour 3 times.Methanol solution in Rotary Evaporators, 50 ℃ of water-baths, under the vacuum of-0.09Mpa, reclaim under reduced pressure methyl alcohol is to doing.
Taxol in the HPLC detection methanol extract liquid and the content of 10-DAB III: reclaim under reduced pressure methyl alcohol is to the methyl alcohol of doing back adding 18mL and the water of 2mL, with oil-soluble impuritieses such as n-hexane extraction pigments for several times, after discarding hexane solution, methanol solution is concentrated into dried, use methylene dichloride and water extracted several times again, combined dichloromethane extraction liquid, 40 ℃ of water-baths in Rotary Evaporators,-0.03Mpa vacuum tightness, the evaporated under reduced pressure methylene dichloride.Residuum dissolves with hplc grade methanol, fixed molten 25mL, and the content that HPLC detects taxol and 10-DAB III is 0.097mgg
-1And 0.086mgg
-1
Claims (3)
1. from the Taxus x media branches and leaves, extract the method for taxol and 10-DABIII simultaneously, it is characterized in that:
(1) with behind 50 ℃ of bake dryings of Taxus x media branches and leaves, be ground into 80 orders-120 purpose powder, per 5 gram dry powder extract with 80mL-120mL methyl alcohol, and triplicate filters, and merges 3 times methanol extract liquid;
(2) with the methanol extract liquid concentrating under reduced pressure, after the n-hexane extraction removal of impurities, use dichloromethane extraction again, last combined dichloromethane extraction liquid, methylene dichloride partly is exactly the solution that contains taxol and 10-DAB III;
(3) with behind the dichloromethane extraction liquid concentrating under reduced pressure, fixed molten with chromatographic grade methyl alcohol, and carry out the HPLC assay.
2. according to the right 1 described method of extracting taxol and 10-DABIII from the Taxus x media branches and leaves, it is characterized in that: above-mentioned extracting with methyl alcohol is to adopt ultrasonic extraction.
3. according to the right 1 described method of extracting taxol and 10-DABIII from the Taxus x media branches and leaves, it is characterized in that: above-mentioned extracting with methyl alcohol can adopt conventional solvent lixiviate or Soxhlet to extract.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101459261A CN102212045A (en) | 2010-04-08 | 2010-04-08 | Method for simultaneously extracting taxol and 10-DABIII (10-deacetyl baccatine III) from branches and leaves of Taxus media |
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| CN2010101459261A CN102212045A (en) | 2010-04-08 | 2010-04-08 | Method for simultaneously extracting taxol and 10-DABIII (10-deacetyl baccatine III) from branches and leaves of Taxus media |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653528A (en) * | 2012-04-19 | 2012-09-05 | 重庆市碚圣农业科技股份有限公司 | Method for separating purified paclitaxel and 10 diaminobenzidine (DAB) III by utilizing macroporous resin |
| CN103145653A (en) * | 2013-04-12 | 2013-06-12 | 江苏斯威森生物医药工程研究中心有限公司 | High-efficiency extraction and purification process for 10-deacetyl baccatin (10-DAB) III |
| CN103804324A (en) * | 2012-11-15 | 2014-05-21 | 刘胜远 | Method for separating and purifying baccatin III from baccatin III containing extract |
| CN104529951A (en) * | 2014-12-10 | 2015-04-22 | 宁波绿之健药业有限公司 | Preparation method for natural paclitaxel |
| CN105669602A (en) * | 2016-03-03 | 2016-06-15 | 重庆市碚圣医药科技股份有限公司 | Method for efficiently and quickly extracting paclitaxel, cephalomannine and 10-DAB III from Taxus chinensis |
| CN110845450A (en) * | 2019-11-28 | 2020-02-28 | 云南皓瑞逸生物科技有限公司 | Method for producing 10-DAB semi-finished product by using crude paste or crystallization process product |
| CN113588846A (en) * | 2021-09-13 | 2021-11-02 | 江苏省中国科学院植物研究所 | HPLC method for simultaneously detecting contents of four components in Taxus media |
| CN113896695A (en) * | 2021-05-26 | 2022-01-07 | 江西省科学院应用化学研究所 | Method for simultaneously extracting cephalomannine and paclitaxel from taxus media |
-
2010
- 2010-04-08 CN CN2010101459261A patent/CN102212045A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102653528A (en) * | 2012-04-19 | 2012-09-05 | 重庆市碚圣农业科技股份有限公司 | Method for separating purified paclitaxel and 10 diaminobenzidine (DAB) III by utilizing macroporous resin |
| CN102653528B (en) * | 2012-04-19 | 2014-07-16 | 重庆市碚圣农业科技股份有限公司 | Method for separating purified paclitaxel and 10 diaminobenzidine (DAB) III by utilizing macroporous resin |
| CN103804324A (en) * | 2012-11-15 | 2014-05-21 | 刘胜远 | Method for separating and purifying baccatin III from baccatin III containing extract |
| CN103145653A (en) * | 2013-04-12 | 2013-06-12 | 江苏斯威森生物医药工程研究中心有限公司 | High-efficiency extraction and purification process for 10-deacetyl baccatin (10-DAB) III |
| CN104529951A (en) * | 2014-12-10 | 2015-04-22 | 宁波绿之健药业有限公司 | Preparation method for natural paclitaxel |
| CN105669602A (en) * | 2016-03-03 | 2016-06-15 | 重庆市碚圣医药科技股份有限公司 | Method for efficiently and quickly extracting paclitaxel, cephalomannine and 10-DAB III from Taxus chinensis |
| CN110845450A (en) * | 2019-11-28 | 2020-02-28 | 云南皓瑞逸生物科技有限公司 | Method for producing 10-DAB semi-finished product by using crude paste or crystallization process product |
| CN113896695A (en) * | 2021-05-26 | 2022-01-07 | 江西省科学院应用化学研究所 | Method for simultaneously extracting cephalomannine and paclitaxel from taxus media |
| CN113896695B (en) * | 2021-05-26 | 2023-12-01 | 四川杉宝生物科技有限公司 | Method for simultaneously extracting cephalomannine and taxol from Taxus media |
| CN113588846A (en) * | 2021-09-13 | 2021-11-02 | 江苏省中国科学院植物研究所 | HPLC method for simultaneously detecting contents of four components in Taxus media |
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Application publication date: 20111012 |