CN101314597A - Method for separating paclitaxel from yew cell suspending culture solution - Google Patents
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Abstract
The invention provides a method for separating paclitaxel from a yew cell suspension culture solution, belongs to a method for preparing natural drug chemical substances or drug raw materials, and aims to reduce paclitaxel extracellular loss, improve the utilization rate of the raw material and reduce cost and environmental pollution. The method comprises the steps as follows: (1) homogenizing cells, (2) performing the membrane separation, (3) extracting the paclitaxel, (4) enriching a macro-porous adsorptive resin, and (5) fine-separating to obtain the paclitaxel with the purity being higher than 98%. The modern membrane separation technology is introduced into the paclitaxel separation and the purification process. Therefore, the consumption of an organic solvent for extracting and the large-size extraction equipment are greatly reduced, and at the same time, the paclitaxel extracellular loss is reduced, the cost and the environmental pollution are reduced, and the utilization rate of the raw material is obviously improved.
Description
Technical field
The invention belongs to the manufacture method of Natural Medicine Chemistry material or pharmaceutical raw material, being specifically related to a kind of is the method for raw material isolation of taxol with the yew cell suspending culture solution.
Background technology
The active substance of the Kaposi sarcoma (Kaposi ' s Sarcoma) that taxol is a kind of natural antitumor evident in efficacy, anti-AID is relevant, belong to the diterpenes secondary metabolite that Chinese yew genus plants or cell produce, its structure is very complicated, and is very unrealistic by the complete synthesis production of chemistry.Be found to from the beginning now, people mainly extract taxol from bark of Ramulus et folium taxi cuspidatae or branches and leaves, but because the synthetic and cumulative process of taxol in Ramulus et folium taxi cuspidatae is very long and complicated, even in generally acknowledging the living bark of Ramulus et folium taxi cuspidatae of higher a century, the content of taxol strips bark in a large number and will produce destructive destruction to taxus resource also below 0.06%; What the tissue extraction method was mainly considered at present is to gather the reproducible branches and leaves of Ramulus et folium taxi cuspidatae partly to extract, although the pressure of taxol resource temporarily can be alleviated in this source, but the Ramulus et folium taxi cuspidatae regeneration period was generally 2～5 years, still can't fundamentally solve taxol according to this approach and stablize and come source problem.By contrast, utilize the large-scale cultivation method of yew plant cells not to be subjected to the restriction of season and land resources, can regulate and control on the synthetic capability theory of taxol, therefore, this method is thoroughly to solve the most promising approach that taxol comes source problem.
The distinguishing feature that the vegetable cell large scale culturing is produced taxol is that nutrient solution is bulky, and the content of taxol in nutrient solution is lower, and generally about 20mg/L, both some was secreted in the nutrient solution for it, also has most to remain in the cell.The method of the producing taxol by means of cell culture that Wang Junjian etc. propose, publication number is CN1096820, only at intracellular taxol, the cell of harvesting time is separated (centrifugal or filtration) with nutrient solution, then cell oven dry back is supplied to extract, in this method, those because of the secretion or the part cell rupture be lost in the nutrient solution taxol will be dropped.The method of taxol is extracted in the enrichment from yew cell nutrient solution filtrate that the bright grade of kingdom is proposed, and publication number is CN1305999; And Wu, J.; Lin, L.Enhancementof taxol production and release in Taxus chinensis cellcultures by ultrasound, methyl jasmonate and in situ solventextraction.Applied Microbiology and Biotechnology, 2003,62 (2-3): 151-155; Carry out resin concentration at the taxol that is secreted in the nutrient solution.Yu, Guang ' ao; Mei, Xingguo; Ke, Tie; Chen, Jing.Research ondifferent solvent extraction of taxol from Taxus cell.Huazhong Li gong Daxue Xuebao, 2000,28 (11): 83-84. carries out liquid-liquid extraction with a large amount of organic solvents to nutrient solution.These methods will inevitably cause a large amount of organic solvents to expend and the environment suffering, also will drop into substantial contribution and be used to purchase large-sized liquid-liquid extraction equipment.
Summary of the invention
The invention provides a kind of method from the yew cell suspending culture solution isolation of taxol, purpose is to reduce the loss of the outer taxol of born of the same parents, improves raw material availability, reduces cost and environmental pollution.
A kind of method from the yew cell suspending culture solution isolation of taxol of the present invention comprises:
(1) cell homogenates step: the yew cell suspending culture solution of harvesting time is filtered, and isolated cell and nutrient solution are homogenate with refiner with cytoclasis, add then in the original fluid, obtain yew cell suspension homogenate;
(2) membrane sepn step: yew cell is suspended homogenate earlier by the membrane filtration of aperture smaller or equal to 10 μ m, obtain cell relic filter residue and filtrate; Filtrate is 15000～25000 ultrafiltration membrance filter by the relative molecular mass cutoff value; Effusive filtrate is 1000～5000 ultrafiltration membrance filter again by the relative molecular mass cutoff value, back two times of ultrafiltration membrane filtration pressure is 0.1-0.4MPa, temperature is 20 ℃～30 ℃, back two times of ultrafiltration membrane filtration rear filtrate cycles of concentration 10-30 doubly, enriched material on cell relic filter residue and the back two times of ultrafiltration film is merged, obtain the membrane sepn trapped substance;
(3) taxol extraction step: with the membrane sepn trapped substance under 30 ℃～60 ℃ temperature, dry to water content less than 5%, grind into powder add to extract solvent, uses ultrasonic-assisted extraction at normal temperatures, filters, and reclaims the filtrate solvent, medicinal extract;
(4) macroporous adsorbent resin enriching step: with methyl alcohol or dissolve with ethanol medicinal extract, be loaded on the macroporous resin column, the aqueous solution that contains methyl alcohol 20%～40% with volume percent washes, and removes water-soluble impurity and pigment, contains the aqueous solution wash-out of methyl alcohol 60%～80% with volume percent, collect the elutriant of 3 times of column volumes, with the methyl alcohol evaporate to dryness in the elutriant, add isopyknic extraction solvent and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains macroporous resin column enrichment taxol crude product;
(5) smart separating step: combine with normal-phase chromatography and reversed phase chromatography the taxol crude product is separated, obtain purity greater than 98% taxol.
Described method from the yew cell suspending culture solution isolation of taxol, it is characterized in that, the process of described cell homogenates step is: with nutrient solution with sand core funnel or industrial Plate Filtration, isolated cell and nutrient solution, is homogenate with cytoclasis with refiner under the rotating speed of 5000～20000r/min, broken time 1～3min adds the pasty state fluid after the homogenate in the original fluid, obtains yew cell suspension homogenate.
Described method from the yew cell suspending culture solution isolation of taxol, it is characterized in that, in the described membrane sepn step, described molecular weight cutoff value is that the ultra-filtration membrane material of 15000～25000 ultra-filtration membrane and molecular weight cutoff value 1000～5000 adopts polypropylene hollow fiber membrane wire material or modification polysulfone membrane wire material.
Described method from the yew cell suspending culture solution isolation of taxol is characterized in that:
In the described taxol extraction step, described extraction solvent is a kind of in methyl alcohol, acetonitrile, ethanol, the methylene dichloride, or volume ratio is 9: 1 or 1: 9 the methyl alcohol and the mixture of methylene dichloride.
Described method from the yew cell suspending culture solution isolation of taxol is characterized in that, in the described macroporous adsorbent resin enriching step:
Described extraction solvent is a kind of in chloroform, methylene dichloride, ethyl acetate, the ether;
Polymeric adsorbent in the described macroporous resin column is homemade D-401, DK-110A, and HD-2's is a kind of;
Described macroporous resin column blade diameter length ratio is: be 1: 10～1: 20, applied sample amount is 60mg～80mg medicinal extract/g dried resin.
Described method from the yew cell suspending culture solution isolation of taxol is characterized in that, in the described smart separating step:
Describedly be integrated as the combination of alumina column chromatography and C18 reversed phase chromatography, normal-phase chromatography methylene dichloride with normal-phase chromatography and reversed phase chromatography: the methyl alcohol volume ratio is moving phase at 98: 2; Reversed phase chromatography adopts the C18 prefabricated post, makes moving phase with the alcohol solution that contains methyl alcohol 80%.
High purity can be further purified with the crystalline method if need more, finally arrives purity greater than 99.5% product.
The present invention separates cell by filter method earlier with nutrient solution, assisting broken down back to return in ultrasonic wave in cell is added in the original fluid, polymeric film with differing molecular quality cutoff value carries out the secondary membrane sepn, and trapped substance is used the macroporous adsorbent resin enriched product with after pure lixiviate and concentrating; Utilize conventional alkali alumina normal-phase chromatography to combine again enriched substance is further separated, obtain purity greater than 98% product with the C18 reversed phase chromatography; Key of the present invention is to have introduced modern membrane separation technique in the technology of separating and purifying taxol, can dwindle significantly that the organic solvent that is used to extract expends and the large size extraction equipment, reduced the loss of the outer taxols of born of the same parents simultaneously, reduce cost and environmental pollution, and obviously improve utilization ratio of raw materials.
Embodiment
Embodiment 1. yew cell suspending culture solution 1L contain taxol 25mg altogether in HPLC detection nutrient solution with in the cell,
(1) nutrient solution is filtered with sand core funnel, obtain about 300ml filter residue, filter residue with refiner homogenate 2min under the rotating speed of 5000r/min, is broken for homogenate, the pasty state fluid after the homogenate is added in the former filtrate, obtain yew cell suspension homogenate.
(2) with the millipore filtration of above-mentioned suspension homogenate by aperture 10 μ m, obtain cell relic filter residue and filtrate, filtrate earlier is the ultra-filtration membrane of 20000 polypropylene hollow fiber membrane wire material by the relative molecular mass cutoff value, effusive filtrate is the ultra-filtration membrane of 2000 polypropylene hollow fiber membrane wire material again by the relative molecular mass cutoff value, be operating as pressure 0.1MPa, service temperature is 20 ℃, 20 times of cycles of concentration, enriched material on cell relic filter residue and 2 ultra-filtration membranes is merged, and the solid substance of the about 86g of fresh weight is as extracting taxol usefulness;
(3) above-mentioned 86g solid substance is put into dry under 30 ℃ of temperature to water content less than 5%, weigh, obtain 20.6g oven dry product, grind into powder adds methyl alcohol, uses ultrasonic-assisted extraction at normal temperatures, filters, and reclaims the solvent in the filtrate, gets medicinal extract 1.6g;
(4) get homemade D-401 macroporous adsorbent resin, wet method is dressed up the chromatography column (blade diameter length ratio is 1: 20) of 1.5 * 30cm, with the above-mentioned medicinal extract of dissolve with methanol, sample is loaded into macroporous resin chromatography top (applied sample amount is 60mg medicinal extract/g dried resin), contain the aqueous solution flushing of methyl alcohol 20% with volume percent, remove water-soluble impurity and pigment, the aqueous solution wash-out that contains methyl alcohol 80% with volume percent, collect the elutriant of 90ml, with the methyl alcohol evaporate to dryness in the elutriant, add isopyknic methylene dichloride and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains 0.170g macroporous resin column enrichment taxol crude product; Contain taxol 14.0% (23.8mg) through the HPLC analysis, the taxol yield is 95.2%; (5) above-mentioned 0.170g macroporous resin column is separated crude product, do filler, methylene dichloride with aluminum oxide: the methyl alcohol volume ratio is the normal-phase chromatography that 98: 2 elutriant is done moving phase, preparation type C18 prefabricated post purifying, make the moving phase wash-out with the alcohol solution that contains methyl alcohol 80%, through solvent evaporated, acquisition purity is 98.3% smart isolation of taxol 20.6mg, yield, 85%; Should product 2ml methanol crystallization, obtain 18.2mg purity and be 99.5% taxol purifying product.
Embodiment 2. yew cell suspending culture solution 5L contain taxol 130mg. altogether in HPLC detection nutrient solution with in the cell
(1) nutrient solution is filtered with sand core funnel, obtain about 1.5L filter residue, filter residue with refiner homogenate 1min under the rotating speed of 20000r/min, is broken for homogenate, the pasty state fluid after the homogenate is added in the former filtrate, obtain yew cell suspension homogenate.
(2) with the millipore filtration of above-mentioned suspension homogenate by aperture 10 μ m, obtain cell relic filter residue and filtrate, filtrate is earlier 15000 modification polysulfone membrane wire material ultra-filtration membrane by the relative molecular mass cutoff value, effusive filtrate is 1000 modification polysulfone membrane wire material ultra-filtration membrane again by the relative molecular mass cutoff value, be operating as pressure 0.4MPa, service temperature is 30 ℃, 30 times of cycles of concentration, enriched material on cell relic filter residue and 2 ultra-filtration membranes is merged, and the solid substance of the about 370g of fresh weight is as extracting taxol usefulness;
(3) above-mentioned 370g solid substance is put into dry under 60 ℃ of temperature to water content less than 5%, weigh, obtain 24.5g oven dry product, grind into powder adds acetonitrile, uses ultrasonic-assisted extraction at normal temperatures, filters, and reclaims the solvent in the filtrate, gets medicinal extract 8.1g;
(4) get homemade DK-110A macroporous adsorbent resin, wet method is dressed up the chromatography column (blade diameter length ratio is 1: 10) of 3.0 * 30cm, with the above-mentioned medicinal extract of dissolve with ethanol, sample is loaded into the macroporous resin chromatography top, applied sample amount is 80mg medicinal extract/g dried resin, contain the aqueous solution flushing of methyl alcohol 30% with volume percent, remove water-soluble impurity and pigment, contain the aqueous solution wash-out of methyl alcohol 60% with volume percent, collect the elutriant of 600ml, the methyl alcohol evaporate to dryness in the elutriant, add isopyknic ethyl acetate and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains 0.960g macroporous resin column enrichment taxol crude product; Contain taxol 12.2% (117mg) through the HPLC analysis, the taxol yield is 90.2%;
(5) above-mentioned 0.960g macroporous resin column is separated crude product, do filler, methylene dichloride with alkali alumina: the methyl alcohol volume ratio is the normal-phase chromatography that 98: 2 elutriant is done moving phase, preparation type C18 prefabricated post purifying, make the moving phase wash-out with the alcohol solution that contains methyl alcohol 80%, through solvent evaporated, acquisition purity is 98.5% smart isolation of taxol 101.0mg, yield, 85%; Should product 2ml methanol crystallization, obtain 80.8mg purity and be 99.5% taxol purifying product.
Embodiment 3. yew cell suspending culture solution 10L contain taxol 200mg. altogether in HPLC detection nutrient solution with in the cell
(1) nutrient solution is filtered with sand core funnel, obtain about 3L filter residue, filter residue with refiner homogenate 2min under the rotating speed of 10000r/min, is broken for homogenate, the pasty state fluid after the homogenate is added in the former filtrate, obtain yew cell suspension homogenate.
(2) with the millipore filtration of above-mentioned suspension homogenate by aperture 10 μ m, obtain cell relic filter residue and filtrate, filtrate is earlier 25000 polypropylene hollow fiber membrane wire material ultra-filtration membrane by the relative molecular mass cutoff value, effusive filtrate is 5000 modified polypropene hollow-fibre membrane wire material ultra-filtration membrane again by the relative molecular mass cutoff value, be operating as pressure 0.3MPa, service temperature is 25 ℃, 25 times of cycles of concentration, enriched material on cell relic filter residue and 2 ultra-filtration membranes is merged, and the solid substance of the about 750g of fresh weight is as extracting taxol usefulness;
(3) above-mentioned 750g solid substance is put into dry under 50 ℃ of temperature to water content less than 5%, weigh, obtain 50.1g oven dry product, grind into powder adds ethanol, uses ultrasonic-assisted extraction at normal temperatures, filters, and reclaims the solvent in the filtrate, gets medicinal extract 16.7g;
(4) get homemade HD-2 macroporous adsorbent resin, wet method is dressed up the chromatography column (blade diameter length ratio is 1: 14) of 3.0 * 42cm, with the above-mentioned medicinal extract of dissolve with methanol, sample is loaded into the macroporous resin chromatography top, applied sample amount is 70mg medicinal extract/g dried resin, contain the aqueous solution flushing of methyl alcohol 40% with volume percent, remove water-soluble impurity and pigment, contain the aqueous solution wash-out of methyl alcohol 70% with volume percent, collect the elutriant of 700ml, the methyl alcohol evaporate to dryness in the elutriant, add isopyknic chloroform and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains 1.68g macroporous resin column enrichment taxol crude product; Contain taxol 11.5% (193.2mg) through the HPLC analysis, the taxol yield is 96.6%;
(5) above-mentioned 1.68g macroporous resin column is separated crude product, do filler, methylene dichloride with aluminum oxide: the methyl alcohol volume ratio is the normal-phase chromatography that 98: 2 elutriant is done moving phase, preparation type C18 prefabricated post purifying, make the moving phase wash-out with the alcohol solution that contains methyl alcohol 80%, through solvent evaporated, acquisition purity is 98.1% smart isolation of taxol 167.4mg, yield 85%.
Embodiment 4. yew cell suspending culture solution 200L contain taxol 5.0g. altogether in HPLC detection nutrient solution with in the cell
(1) nutrient solution is used industrial Plate Filtration, obtained about 67L filter residue, the filter residue refiner
Homogenate 3min is broken for homogenate under the rotating speed of 5000r/min, and the pasty state fluid after the homogenate is added
In the former filtrate, obtain yew cell suspension homogenate.
(2) with the millipore filtration of above-mentioned suspension homogenate by aperture 10 μ m, obtain cell relic filter residue and filtrate, filtrate is earlier 20000 modification polysulfone membrane wire material ultra-filtration membrane by the relative molecular mass cutoff value, effusive filtrate is 2000 modification polysulfone membrane wire material ultra-filtration membrane again by the relative molecular mass cutoff value, be operating as pressure 0.2MPa, service temperature is 30 ℃, 10 times of cycles of concentration, enriched material on cell relic filter residue and 2 ultra-filtration membranes is merged, and the solid substance of the about 14.8Kg of fresh weight is as extracting taxol usefulness;
(3) above-mentioned 14.8Kg solid substance is put into dry under 60 ℃ of temperature to water content less than 5%, weigh, obtain 1000.8g oven dry product, grind into powder, add volume ratio and be the mixture of 9: 1 methyl alcohol and methylene dichloride, use ultrasonic-assisted extraction at normal temperatures, filtration, reclaim the solvent in the filtrate, get medicinal extract 330g;
(4) get homemade D-401 macroporous adsorbent resin, wet method is dressed up the chromatography column (blade diameter length ratio is 1: 10) of 18cm * 180cm, with the above-mentioned medicinal extract of dissolve with methanol, sample is loaded into the macroporous resin chromatography top, applied sample amount is 80mg medicinal extract/g dried resin, contain the aqueous solution flushing of methyl alcohol 20% with volume percent, remove water-soluble impurity and pigment, contain the aqueous solution wash-out of methyl alcohol 80% with volume percent, collect the elutriant of 90ml, the methyl alcohol evaporate to dryness in the elutriant, add isopyknic methylene dichloride and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains 32.8g macroporous resin column enrichment taxol crude product; Contain taxol 12.9% (4.23g) through the HPLC analysis, the taxol yield is 85%;
(5) above-mentioned 32.8g macroporous resin column is separated crude product, do filler, methylene dichloride with aluminum oxide: the methyl alcohol volume ratio is the normal-phase chromatography that 98: 2 elutriant is done moving phase, preparation type C18 prefabricated post purifying, make the moving phase wash-out with the alcohol solution that contains methyl alcohol 80%, through solvent evaporated, acquisition purity is 98.0% smart isolation of taxol 3.67g, yield 85%.
Embodiment 5. yew cell suspending culture solution 500L contain taxol 13g. altogether in HPLC detection nutrient solution with in the cell
(1) nutrient solution is used industrial Plate Filtration, obtained about 170L filter residue, filter residue with refiner homogenate 2min under 5000 rotating speed, is broken for homogenate, the pasty state fluid after the homogenate is added in the former filtrate, obtain yew cell suspension homogenate.
(2) step (2) by embodiment 4 obtains the solid substance of the about 12.6Kg of fresh weight as extracting taxol usefulness;
(3) above-mentioned 12.6Kg solid substance is put into dry under 60 ℃ of temperature to water content less than 5%, weigh, obtain the oven dry product, grind into powder, add volume ratio and be the mixture of 1: 9 methyl alcohol and methylene dichloride, use ultrasonic-assisted extraction at normal temperatures, filtration, reclaim the solvent in the filtrate, get medicinal extract 840g;
(4) medicinal extract 840g is carried out the macroporous adsorbent resin enriching step by the step (4) of embodiment 4 at twice, obtain 94.5g macroporous resin column enrichment taxol crude product; Contain taxol 11.0% (10.4g) through the HPLC analysis, the taxol yield is 80.2%;
(5) above-mentioned 94.5g macroporous resin column is separated the step that crude product is pressed embodiment 4 (5), acquisition purity is 98.0% smart isolation of taxol 10.1g, yield, 85%; Should product 100ml methanol crystallization, obtain 8.1g purity and be 99.5% taxol purifying product; Taxol total recovery 62.0%.
Claims (6)
1. method from the yew cell suspending culture solution isolation of taxol comprises:
(1) cell homogenates step: the yew cell suspending culture solution of harvesting time is filtered, and isolated cell and nutrient solution are homogenate with refiner with cytoclasis, add then in the original fluid, obtain yew cell suspension homogenate;
(2) membrane sepn step: yew cell is suspended homogenate earlier by the membrane filtration of aperture smaller or equal to 10 μ m, obtain cell relic filter residue and filtrate; Filtrate is 15000～25000 ultrafiltration membrance filter by the relative molecular mass cutoff value; Effusive filtrate is 1000～5000 ultrafiltration membrance filter again by the relative molecular mass cutoff value, back two times of ultrafiltration membrane filtration pressure is 0.1-0.4MPa, temperature is 20 ℃～30 ℃, back two times of ultrafiltration membrane filtration rear filtrate cycles of concentration 10-30 doubly, enriched material on cell relic filter residue and the back two times of ultrafiltration film is merged, obtain the membrane sepn trapped substance;
(3) taxol extraction step: with the membrane sepn trapped substance under 30 ℃～60 ℃ temperature, dry to water content less than 5%, grind into powder add to extract solvent, uses ultrasonic-assisted extraction at normal temperatures, filters, and reclaims the filtrate solvent, medicinal extract;
(4) macroporous adsorbent resin enriching step: with methyl alcohol or dissolve with ethanol medicinal extract, be loaded on the macroporous resin column, the aqueous solution that contains methyl alcohol 20%～40% with volume percent washes, and removes water-soluble impurity and pigment, contains the aqueous solution wash-out of methyl alcohol 60%～80% with volume percent, collect the elutriant of 3 times of column volumes, with the methyl alcohol evaporate to dryness in the elutriant, add isopyknic extraction solvent and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness obtains macroporous resin column enrichment taxol crude product;
(5) smart separating step: combine with normal-phase chromatography and reversed phase chromatography the taxol crude product is separated, obtain purity greater than 98% taxol.
2. the method from the yew cell suspending culture solution isolation of taxol as claimed in claim 1, it is characterized in that, the process of described cell homogenates step is: with nutrient solution with sand core funnel or industrial Plate Filtration, isolated cell and nutrient solution, is homogenate with cytoclasis with refiner under the rotating speed of 5000～20000r/min, broken time 1～3min adds the pasty state fluid after the homogenate in the original fluid, obtains yew cell suspension homogenate.
3. the method from the yew cell suspending culture solution isolation of taxol as claimed in claim 1, it is characterized in that: in the described membrane sepn step, described molecular weight cutoff value is that the ultra-filtration membrane material of 15000～25000 ultra-filtration membrane and molecular weight cutoff value 1000～5000 adopts polypropylene hollow fiber membrane wire material or modification polysulfone membrane wire material.
4. the method from the yew cell suspending culture solution isolation of taxol as claimed in claim 1 is characterized in that:
In the described taxol extraction step, described extraction solvent is a kind of in methyl alcohol, acetonitrile, ethanol, the methylene dichloride, or volume ratio is 9: 1 or 1: 9 the methyl alcohol and the mixture of methylene dichloride.
5. the method from the yew cell suspending culture solution isolation of taxol as claimed in claim 1 is characterized in that, in the described macroporous adsorbent resin enriching step:
Described extraction solvent is a kind of in chloroform, methylene dichloride, ethyl acetate, the ether;
Polymeric adsorbent in the described macroporous resin column is homemade D-401, DK-110A, and HD-2's is a kind of;
Described macroporous resin column blade diameter length ratio is: be 1: 10～1: 20, applied sample amount is 60mg～80mg medicinal extract/g dried resin.
6. the method from the yew cell suspending culture solution isolation of taxol as claimed in claim 1 is characterized in that, in the described smart separating step:
Describedly be integrated as the combination of alumina column chromatography and C18 reversed phase chromatography, normal-phase chromatography methylene dichloride with normal-phase chromatography and reversed phase chromatography: the methyl alcohol volume ratio is moving phase at 98: 2; Reversed phase chromatography adopts the C18 prefabricated post, makes moving phase with the alcohol solution that contains methyl alcohol 80%.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260227A (en) * | 2011-06-21 | 2011-11-30 | 沈阳化工大学 | Method for separating paclitaxel and related taxane substances |
| CN103570647A (en) * | 2013-11-06 | 2014-02-12 | 湖南农业大学 | Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid |
| CN103819430A (en) * | 2014-02-25 | 2014-05-28 | 河南省科学院化学研究所有限公司 | Paclitaxel purifying method of paclitaxel crude product produced by Chinese yew cell culture |
| CN104450601A (en) * | 2014-12-04 | 2015-03-25 | 天津艾赛博生物技术有限公司 | Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain |
| CN104529951A (en) * | 2014-12-10 | 2015-04-22 | 宁波绿之健药业有限公司 | Preparation method for natural paclitaxel |
| CN105254597A (en) * | 2014-07-15 | 2016-01-20 | 福建南方制药股份有限公司 | Extraction method for 10-DAB III and/or paclitaxel in Taxus chinensis |
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| CN1096820A (en) * | 1993-06-25 | 1994-12-28 | 华中理工大学 | The method of producing taxol by means of cell culture |
| CN1054382C (en) * | 1996-03-05 | 2000-07-12 | 核工业北京化工冶金研究院 | Method for separating and purifying taxol by reversed-phase preparative liquid chromatography |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102260227A (en) * | 2011-06-21 | 2011-11-30 | 沈阳化工大学 | Method for separating paclitaxel and related taxane substances |
| CN102260227B (en) * | 2011-06-21 | 2013-10-30 | 沈阳化工大学 | Method for separating paclitaxel and related taxane substances |
| CN103570647A (en) * | 2013-11-06 | 2014-02-12 | 湖南农业大学 | Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid |
| CN103819430A (en) * | 2014-02-25 | 2014-05-28 | 河南省科学院化学研究所有限公司 | Paclitaxel purifying method of paclitaxel crude product produced by Chinese yew cell culture |
| CN105254597A (en) * | 2014-07-15 | 2016-01-20 | 福建南方制药股份有限公司 | Extraction method for 10-DAB III and/or paclitaxel in Taxus chinensis |
| CN105254597B (en) * | 2014-07-15 | 2019-08-30 | 福建南方制药股份有限公司 | The extracting method of 10-DAB III and/or taxol in Chinese yew |
| CN104450601A (en) * | 2014-12-04 | 2015-03-25 | 天津艾赛博生物技术有限公司 | Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain |
| 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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