CN103570647A - Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid - Google Patents
Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid Download PDFInfo
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- CN103570647A CN103570647A CN201310546214.4A CN201310546214A CN103570647A CN 103570647 A CN103570647 A CN 103570647A CN 201310546214 A CN201310546214 A CN 201310546214A CN 103570647 A CN103570647 A CN 103570647A
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Abstract
The invention relates to a method for preparing paclitaxel, and in particular relates to a method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid. The method comprises the following steps of filtering the cell culture fluid, performing adsorptive enrichment to obtain a crude extract by using macroporous resin, performing normal-pressure alkaline alumina column chromatography and C18 reversed-phase preparative high-performance liquid separation, collecting components stepwise, performing centrifugation to obtain precipitates, and freeze-drying the precipitates to obtain high-purity paclitaxel monomers. According to the method, the high-paclitaxel yield cell culture fluid is used as a raw material; compared with extracts of taxus chinensis branches, leaves, barks and the like, the cell culture fluid contains few impurities such as pigments and lipid; compared with fungal fermentation liquid and bacterial fermentation liquid, the cell culture fluid contains more paclitaxel, and is convenient to extract; in addition, the paclitaxel in the taxus chinensis cell culture fluid is enriched directly by using the macroporous resin, so that the use of a great amount of organic solvent is avoided; a process is simple, economical, energy-friendly and high in recovery rate, and the obtained paclitaxel is high in purity.
Description
Technical field
The present invention relates to the preparation method of taxol, especially relate to the method for preparing high-purity taxol from yew cell nutrient solution.
Background technology
Taxol is the secondary metabolite of a kind of complexity in Chinese yew genus plants, is also that understand at present only a kind of can promote microtubule polymerization and the stable medicine of polymerization microtubule.Taxol is global best-selling antitumor drug, is also the strong active anticancer medicine of wide spectrum of generally acknowledging in the world simultaneously, and along with the sharply expansion of clinical application, international medical market has huge demand to paclitaxel api.
In recent years, both at home and abroad to tissue extraction such as the many barks from plant of taxol preparation, branches and leaves, complex process, solvent energy expenditure is more, and cost is higher.After wild Ramulus et folium taxi cuspidatae is protected by the law, can only obtain bark from tame Ramulus et folium taxi cuspidatae, but growth of taxol is slow, has limited bark raw material sources.At present, also has certain methods: as prepared taxol by semisynthesis, but it must first obtain taxol prerequisite compound from vegetable material, so also can be subject to resource limit; Also as taxol total synthesis method, but its preparation process and complexity thereof, cost is very high, so far cannot suitability for industrialized production; For another example from fungus and bacterium broth extraction taxol, because its content is too low, do not realize at present suitability for industrialized production.The U.S. and Italy are utilizing the development of cell culture method direct production paclitaxel api very fast, up to now, the total scale that the whole world utilizes yew cell culture method to produce taxol still only has thousands of liters every year, in current global taxol raw material ultimate production, the taxol portion that cell culture method is produced is minimum.But because cell culture method can not consume valuable yew tree or Chinese yew resource, therefore this method belongs to " green production novel process " and representing future of paclitaxel api industry, have and have vast potential for future development and the market growth space.
At present, the distinguishing feature that vegetable cell large scale culturing is produced taxol is that nutrient solution is bulky, and the content of taxol in nutrient solution is lower, generally in 20mg/L left and right, both some was secreted in nutrient solution for it, also had most to remain in cell.In prior art, there is extracting the method for taxol in cell, it is that the cell of harvesting time is separated with nutrient solution, then after cell being dried, extract, in this method, those because of secretion or part cell rupture be lost in nutrient solution taxol will be dropped, cause the rate of recovery lower; The method that has also occurred enrichment extraction taxol from yew cell nutrient solution filtrate, it is, with a large amount of organic solvents, nutrient solution is carried out to the extraction of liquid-liquid.This method will inevitably cause a large amount of organic solvents to expend and environment suffering, also will drop into substantial contribution and be used for purchasing large-sized liquid-liquid extraction equipment; Also occur adopting membrane separation concentration technology from the method from yew cell suspending culture solution isolation of taxol, although the method can be saved solvent extraction, can remove a part of impurity, the method troublesome poeration, the rate of recovery is not high.
Summary of the invention
For above-mentioned technical problem, the object of the present invention is to provide a kind of novel method of preparing high-purity taxol from yew cell nutrient solution, the method not only technique is simple, and the rate of recovery is high.
The technical scheme that the present invention solves the problems of the technologies described above employing is: from yew cell nutrient solution, prepare the method for high-purity taxol, it comprises the following steps:
(1) yew cell nutrient solution is carried out to solid-liquid separation, obtain stoste and cell; Then will after cell wall breaking, filter, obtain filtrate and filter residue; Again described stoste is mixed with filtrate;
(2) above-mentioned mixing liquid is passed through to the enrichment of non-polar macroporous resin absorption chromatograph column, then use distilled water flushing, then use ethanolic soln wash-out, collect elutriant, and by the ethanol evaporate to dryness in elutriant;
(3) in the elutriant of above-mentioned evaporate to dryness ethanol, add extraction agent, carry out twice liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying, obtains macroporous resin column enrichment taxol crude product;
(4) above-mentioned crude product is reproduced in to the chromatography column of making filler of alkali alumina, with elute soln, carries out wash-out, fraction collection wash-out part, and vacuum-drying, obtain sample;
(5) above-mentioned sample is passed through to C18 performance liquid chromatographic column purifying, then with making moving phase wash-out containing methanol solution, fraction collection taxol wash-out part, is placed in ventilating kitchen by the component of collection, volatilization organic phase, after there is crystallization, go to 4 ℃ of refrigerators and place 24h, the a part of crystal of centrifugal collection, then continues placement by supernatant liquor and makes crystallization complete, centrifugal collection another part crystal, by whole crystal lyophilizes of collecting, obtain high-purity taxol monomer again.
As preferably, in step (1), by described medium centrifugal, obtain cell, then cell is used to refiner homogenate, then added isopyknic ethanol to carry out supersound extraction the pasty state fluid after homogenate, and after centrifuging, obtain described filtrate.
Preferred as another kind, in step (1) by described nutrient solution through sand core funnel suction filtration or through Plate Filtration, obtain cell, then cell is used to refiner homogenate, add isopyknic ethanol to carry out supersound extraction the pasty state fluid after homogenate again, and use sand core funnel suction filtration, obtain described filtrate.
Further, in step (2), adopting volumetric concentration is that 75 ~ 85% ethanolic solns carry out wash-out.
Further, in the elutriant of evaporate to dryness ethanol, add isopyknic extraction agent, the methylene dichloride that extraction agent is 1:1 and water or be the trichloromethane of 1:1 and water.
Further, step (4) adopts trichloromethane: the elutriant that methyl alcohol volume ratio is 96:4
The present invention compared with prior art, has the following advantages:
(1) the present invention selects taxol high yield clone nutrient solution as starting material, compares with extractions such as Ramulus et folium taxi cuspidatae, barks, and the impurity such as contained pigment, lipid still less, than fungus and bacterium fermented liquid, its contained taxol is higher, and this material is convenient to be extracted, and impurities still less.
(2) from yew cell nutrient solution, directly use macroporous resin enrichment taxol, with respect to solvent extraction, saved a large amount of organic solvents and used; With respect to membrane separation technique, operate easylier, the rate of recovery is higher; And a large amount of organic solvents adopt ethanol, Environmental Safety;
(3) total yield of the present invention, more than 90%, utilizes the fine control product purity of efficient preparation liquid phase energy, and the taxol purity of acquisition is high, can be greater than 99.5%;
(4) when efficiently preparing liquid phase separation taxol, can other bearing taxanes of fraction collection;
(5) whole technical process is easy, is easy to amplify industrialization and produces.
Accompanying drawing explanation
Fig. 1 is FB(flow block) of the present invention.
Embodiment
Below in conjunction with Fig. 1, method of the present invention is described in further detail:
Present method is: (1) carries out solid-liquid separation by yew cell nutrient solution, obtains stoste and cell, and solid-liquid separation can adopt by modes such as centrifugal, Plate Filtration, sand core funnel suction filtrations, and scale operation is generally by Plate Filtration or centrifugation; Then will after cell wall breaking, filter, obtain filtrate and filter residue; Again described stoste is mixed with filtrate; Taxol in cell culture fluid is almost completely dissolved in last mixed solution, by centrifugal or filtration, can remove broken cellular constituent, and gained filtrate impurities still less, is convenient to follow-up macroporous adsorbing resin for purification.
(2) above-mentioned mixing liquid is passed through to the enrichment of non-polar macroporous resin absorption chromatograph column, nonpolar macroporous adsorption resin can be D-101, HP-30, and D4020's is a kind of; Then use distilled water flushing, then use ethanolic soln wash-out, collect elutriant, and by the ethanol evaporate to dryness in elutriant; Like this can be from large quantity of fluid enrichment taxol, concentrated after content of taxol can reach 1～5%.
(3) in the elutriant of above-mentioned evaporate to dryness ethanol, add extraction agent, carry out twice liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying, obtains macroporous resin column enrichment taxol crude product; After extraction, can remove a part of impurity, and can remove moisture, be convenient to subsequent oxidation aluminium column chromatography.
(4) above-mentioned crude product is reproduced in to the chromatography column of making filler of alkali alumina, with elute soln, carries out wash-out, fraction collection wash-out part, and vacuum-drying, obtain sample; Alkali alumina chromatography column isolation of taxol simultaneously can catalysis 7-epi-taxol etc. bearing taxanes to taxol, transform, the impurity of removing more difficult separation improves the rate of recovery simultaneously.
(5) above-mentioned sample is passed through to C18 performance liquid chromatographic column purifying, then with making moving phase wash-out containing methanol solution, fraction collection taxol wash-out part, is placed in ventilating kitchen by the component of collection, volatilization organic phase, after there is crystallization, go to 4 ℃ of refrigerators and place 24h, the a part of crystal of centrifugal collection, then continues placement by supernatant liquor and makes crystallization complete, centrifugal collection another part crystal, by whole crystal lyophilizes of collecting, obtain high-purity taxol monomer again.Owing to utilizing C18 performance liquid chromatographic column, energy high efficiency separation taxol, reproducible, be convenient to control.Specific embodiment is as follows:
Embodiment 1
Yew cell suspending culture solution 300mL, detects in nutrient solution and in cell altogether containing taxol 27mg through HPLC.
(1) by the centrifugal 5min of nutrient solution 5000r/min, obtain 82mL cell solid, by refiner homogenate 2min under the rotating speed of 10000r/min for cell, pasty state fluid after homogenate is added to isopyknic 95% ethanol ultrasonic extraction 30min, after 5000r/min centrifuging 129mL, to obtain altogether 347mL with liquid mixing before.
(2) get domestic D-101 macroporous adsorbent resin, wet method is dressed up the chromatography column of 1.5 * 30cm, mixed solution is loaded into macroporous resin chromatography top (applied sample amount is about 3mg taxol/mL resin), with distilled water flushing, remove water-soluble impurity and pigment, aqueous solution wash-out by volume percent containing ethanol 75%, collect 30mL elutriant, by the ethanol evaporate to dryness in elutriant, add isopyknic trichloromethane: water (1:1) carries out liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying obtains 1.150g macroporous resin column enrichment taxol crude product, through HPLC, analyze containing taxol 2.3% (25.9mg), taxol yield is 95.9%.
(3) by the separated crude product of above-mentioned 1.150g macroporous resin column, be reproduced in the chromatography column of making filler of alkali alumina, trichloromethane: the elutriant that methyl alcohol volume ratio is 96:4 is done moving phase, fraction collection wash-out part, low-temperature vacuum drying obtains sample 0.116g; Through HPLC, analyze containing taxol 21.7% (25.2mg), the taxol rate of recovery is 97.3%.
(4) sample is by C18 performance liquid chromatographic column purifying, with the methanol aqueous solution containing methyl alcohol 55%, make moving phase wash-out, fraction collection taxol wash-out part, the component of collection is placed in to ventilating kitchen, and volatilization organic phase goes to 4 ℃ of refrigerators and places 24h after there is crystallization, the centrifugal crystal that obtains, supernatant liquor continues to place makes crystallization as far as possible completely, by after the crystal lyophilize of collecting, obtains high-purity taxol monomer.The smart isolation of taxol 24.8mg that acquisition purity is 99.6%, the rate of recovery is 98.4%, total yield is 91.5%.
Embodiment 2
Yew cell suspending culture solution 1L, detects in nutrient solution and in cell altogether containing taxol 92mg through HPLC.
(1) nutrient solution is obtained after sand core funnel suction filtration to 260mL cell solid, by refiner homogenate 2min under the rotating speed of 10000r/min for cell, pasty state fluid after homogenate is added to isopyknic 95% ethanol ultrasonic extraction 30min, after sand core funnel suction filtration 405mL, and after liquid mixing before, to obtain altogether 1145mL.
(2) get import HP-30 macroporous adsorbent resin, wet method is dressed up the chromatography column of 2 * 30cm, mixed solution is loaded into macroporous resin chromatography top (applied sample amount is about 3mg taxol/mL resin), with distilled water flushing, remove water-soluble impurity and pigment, aqueous solution wash-out by volume percent containing ethanol 80%, collect the elutriant of 100mL, by the ethanol evaporate to dryness in elutriant, add isopyknic methylene dichloride: water (1:1) carries out liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying obtains 3.351g macroporous resin column enrichment taxol crude product, through HPLC, analyze containing taxol 2.6% (88.5mg), the taxol rate of recovery is 96.2%.
(3) by the separated crude product of above-mentioned 3.351g macroporous resin column, be reproduced in the chromatography column of making filler of alkali alumina, trichloromethane: the elutriant that methyl alcohol volume ratio is 96:4 is done moving phase, fraction collection wash-out part, 50 ℃ of vacuum-dryings, obtain sample 0.349g; Through HPLC, analyze containing taxol 24.8% (86.7mg), the taxol rate of recovery is 98.0%.
(4) sample, by C18 performance liquid chromatographic column purifying, is made moving phase wash-out, fraction collection taxol wash-out part with the methanol aqueous solution containing methyl alcohol 55%, the component of collection is placed in to ventilating kitchen, volatilization organic phase goes to 4 ℃ of refrigerators and places 24h, the centrifugal crystal that obtains after there is crystallization, supernatant liquor continues to place makes crystallization as far as possible completely, by after the crystal lyophilize of collecting, obtain high-purity taxol monomer 85.63mg, purity is 99.5%, the rate of recovery is 98.3%, and total yield is 92.6%.
Embodiment 3
Yew cell suspending culture solution 3L, detects in nutrient solution and in cell altogether containing taxol 265mg through HPLC.
(1) nutrient solution is obtained after sand core funnel suction filtration to 775mL cell solid, by refiner homogenate 2min under the rotating speed of 10000r/min for cell, pasty state fluid after homogenate is added to isopyknic 95% ethanol ultrasonic extraction 30min, after filtration 1210mL, to obtain altogether 3435mL with liquid mixing before.
(2) get domestic D-101 macroporous adsorbent resin, wet method is dressed up the chromatography column of 3 * 30cm, mixed solution is loaded into macroporous resin chromatography top (applied sample amount is about 3mg taxol/mL resin), with distilled water flushing, remove water-soluble impurity and pigment, aqueous solution wash-out by volume percent containing ethanol 85%, collect the elutriant of 300mL, by the ethanol evaporate to dryness in elutriant, add isopyknic trichloromethane: water (1:1) carries out liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying obtains 12.130g macroporous resin column enrichment taxol crude product, through HPLC, analyze containing taxol 2.1% (251mg), taxol yield is 94.7%.
(3) by the separated crude product of above-mentioned 12.130g macroporous resin column, be loaded into the chromatography column of making filler of alkali alumina, trichloromethane: the elutriant that methyl alcohol volume ratio is 96:4 is done moving phase, fraction collection wash-out part, low-temperature vacuum drying, obtain sample 1.098g, through HPLC, analyze containing taxol 22.3% (245mg), the taxol rate of recovery is 97.6%.
(4) sample is by C18 performance liquid chromatographic column purifying, with the methanol aqueous solution containing methyl alcohol 50%, make moving phase wash-out, fraction collection taxol wash-out part, the component of collection is placed in to ventilating kitchen, and volatilization organic phase goes to 4 ℃ of refrigerators and places 24h after there is crystallization, the centrifugal crystal that obtains, supernatant liquor continues to place makes crystallization as far as possible completely, by after the crystal lyophilize of collecting, obtains high-purity taxol monomer.The smart isolation of taxol 242mg that acquisition purity is 99.5%, the rate of recovery is 98.5%, total yield is 90.9%
Above-mentioned embodiment is used for illustrative purposes only, and be not limitation of the present invention, the those of ordinary skill in relevant technologies field, without departing from the spirit and scope of the present invention, can also make various variations and modification, therefore all technical schemes that are equal to also should belong to category of the present invention.
Claims (6)
1. from yew cell nutrient solution, prepare the method for high-purity taxol, it comprises the following steps:
(1) yew cell nutrient solution is carried out to solid-liquid separation, obtain stoste and cell; Then will after cell wall breaking, filter, obtain filtrate and filter residue; Again described stoste is mixed with filtrate;
(2) above-mentioned mixing liquid is passed through to the enrichment of non-polar macroporous resin absorption chromatograph column, then use distilled water flushing, then use ethanolic soln wash-out, collect elutriant, and by the ethanol evaporate to dryness in elutriant;
(3) in the elutriant of above-mentioned evaporate to dryness ethanol, add extraction agent, carry out twice liquid-liquid extraction, collect organic solvent layer, low-temperature vacuum drying, obtains macroporous resin column enrichment taxol crude product;
(4) above-mentioned crude product is reproduced in to the chromatography column of making filler of alkali alumina, with elute soln, carries out wash-out, fraction collection wash-out part, and vacuum-drying, obtain sample;
(5) above-mentioned sample is passed through to C18 performance liquid chromatographic column purifying, then with making moving phase wash-out containing methanol solution, fraction collection taxol wash-out part, is placed in ventilating kitchen by the component of collection, volatilization organic phase, after there is crystallization, go to 4 ℃ of refrigerators and place 24h, the a part of crystal of centrifugal collection, then continues placement by supernatant liquor and makes crystallization complete, centrifugal collection another part crystal, by whole crystal lyophilizes of collecting, obtain high-purity taxol monomer again.
2. method according to claim 1, it is characterized in that: in step (1), by described medium centrifugal, obtain cell, then cell is used to refiner homogenate, add isopyknic ethanol to carry out supersound extraction the pasty state fluid after homogenate again, and after centrifuging, obtain described filtrate.
3. method according to claim 1, it is characterized in that: in step (1) by described nutrient solution through sand core funnel suction filtration or through Plate Filtration, obtain cell, then cell is used to refiner homogenate, add isopyknic ethanol to carry out supersound extraction the pasty state fluid after homogenate again, and use sand core funnel suction filtration, obtain described filtrate.
4. it is characterized in that according to the method in claim 2 or 3: in step (2), adopting volumetric concentration is that 75 ~ 85% ethanolic solns carry out wash-out.
5. method according to claim 4, is characterized in that: in step (3), in the elutriant of evaporate to dryness ethanol, add isopyknic extraction agent, the methylene dichloride that extraction agent is 1:1 and water or be the trichloromethane of 1:1 and water.
6. method according to claim 5, is characterized in that: step (4) adopts trichloromethane: the elutriant that methyl alcohol volume ratio is 96:4.
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Cited By (4)
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| CN104230858A (en) * | 2014-08-08 | 2014-12-24 | 中山大学 | Method for separating and purifying paclitaxel from taxus chinensis branches and leaves or bark |
| CN104529951A (en) * | 2014-12-10 | 2015-04-22 | 宁波绿之健药业有限公司 | Preparation method for natural paclitaxel |
| CN105418542A (en) * | 2015-11-17 | 2016-03-23 | 重庆臻源红豆杉发展有限公司 | Crystallization purification method for low-content and low-purity taxol crude product |
| CN112645906A (en) * | 2020-12-29 | 2021-04-13 | 重庆臻源红豆杉发展有限公司 | Method for separating and purifying paclitaxel by high-efficiency chromatography |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104230858A (en) * | 2014-08-08 | 2014-12-24 | 中山大学 | Method for separating and purifying paclitaxel from taxus chinensis branches and leaves or bark |
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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 |
| CN105418542A (en) * | 2015-11-17 | 2016-03-23 | 重庆臻源红豆杉发展有限公司 | Crystallization purification method for low-content and low-purity taxol crude product |
| CN112645906A (en) * | 2020-12-29 | 2021-04-13 | 重庆臻源红豆杉发展有限公司 | Method for separating and purifying paclitaxel by high-efficiency chromatography |
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Application publication date: 20140212 |