CN101033218A - Method of extracting Paclitaxel and 9-dihydro-13-acetyl baccatin III from taxus genus - Google Patents
Method of extracting Paclitaxel and 9-dihydro-13-acetyl baccatin III from taxus genus Download PDFInfo
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Abstract
This invention provides a method for picking up perilla alcohol organic solution by a load and lock axial compression column purification technology in industrial scale HPLC, in which, the load and lock axial compression column is the one that uses a piston to fill and take out the chromatographic bed and eliminates spacings in the bed basically to keep the compression state of the column when using, which prevents form of spacings in the bed basically so as to increase the process efficiency, and the chromatographic column can be filled by any kind of fillings including medium of small particles and large tower plates can be generated with this mode and the length of the column bed can be adjusted by controlling the filling volume used in preparing the column.
Description
Technical field
The present invention relates to from naturally occurring Cercocarpus separation and purification Taxane derivative.
Background technology
Taxol is separated from the yew yewtree of west in 1971 by Wani etc. at first and obtains.Taxol is a member of Taxan family in the diterpene, and Taxan is the diterpene compound that using value is arranged in the pharmacy field, and Taxan can find in plant, and separates from plant.Taxol is a kind of well-known chemicals of controlling new various metastatic cancers, and approved is used for the treatment of ovary and mammary cancer.
Taxol and various Taxane derivative are highly Cytotoxic, and some leukemia and cancer system are had strong activity in vivo.Taxol is considered to a kind of cancer Chemo-Therapy new drug likely especially.At present unique source of taxol be several growths ten minutes slowly Japanese yew (Cercocarpus Taxaceae) taxol be a kind of natural product, mainly the bark from Pacific yew tree yewtree extracts, and also is found in weeping branch european yew, Xizang Taxus chinensis, taxusyunnanensis and T. canadensis.The separable programming of existing use is pretty troublesome and yield poorly, use liquid methanol from the existing report of Cercocarpus tree extracting taxol, but the content of Taxan in vegetable material is common very low, consequently, the act taxol is an example, may be destructive as its source with a large amount of slowly yew trees of growth, more very it, using conventional liquid extraction method need use the organic solvent of enormous amount, also is very time-consuming.
The concentration of taxol in all starting material generally is very low, as in the Pacific yew bark, the quantity of its concentration is between 0.0004 and 0.01% (w/w), it is very challenging that lower concentration like this makes the material that extracts and be purified to pharmaceutical grade from starting material, thereby be unpractical on commercial size, the phase chromatographic technique of having set up various routines is used for from raw-material crude extract purification of paclitaxel.
The characteristic of post is depended in the success of low pressure chromatographic technique to a great extent.The variety of issue all use with silica gel and alchlor is relevant.All these materials all be in the distribution system as the carrier of the classics of stationary phase, they have constituted stable static phases to most of solvent systemss.But absorptive character are strong, might participate in sepn process to a certain extent, chromatography behavior and sample are reclaimed be affected.
Chromatography method developed be used for analyze with the preparation level on detect and separate the taxol that derives from various Cercocarpus kinds.Its sepn process is mainly carried out on little laboratory scale, and have low selectivity, the low recovery and expensive problem, therefore need method a kind of economy, feasible to be used for valuable antineoplastic compound taxol being separated especially from the analogue cephalotaxin very close and other closely-related Taxan with taxol.
Using educable and renewable plant strain part should be to increase the most practical and attractive approach that taxol is supplied with as the leaf (needle) and the branch of Japanese yew, existing several Japanese yew kinds comprise that the needle of T. canadensis done research, and the content of taxol of finding them is suitable with yewtree.
T. canadensis is a kind of evergreen filling basis that mainly is found in Canada and Northeastern United States; the Taxan that this kind contains is unique; the Taxan that its needle mainly contains is 9 one dihydro-13-acetyl baccatin (q-DHABIII; 4) together with taxol (0.009-0.05%); 10-deacetylate Ba Kading (10-DABIII, 6), Ba Kading (5); cephalotaxin (3) also contains a small amount of other Taxan.It was reported that the concentration of 9-DHABIII in needle is 7-10 times of taxol.It seems that 9-DHABIII may become the important as precursors of the water-soluble novel semi-synthetic chemotherapeutic agent that increases of a class.
The method of prior art discloses the use of the various chromatographic techniques of isolation of taxol and relevant Taxan, be included in forward and reverse chromatography on silicagel column or the bonded silica gel post, the method of prior art ends in low yield, expensive or comprise a plurality of steps, makes it be difficult to be amplified to large-scale commercial production.
From viewing and admiring the needle of Japanese yew, it is uneconomic that bark and root are produced taxol at present, Gu in leaching process, extract has been brought very a high proportion of unwanted impurity into, (40-50% that is about the material plant dry weight).View and admire in the solvent extraction liquid of Japanese yew needle unusual a high proportion of foreign matter content and make and expend very highly, very uneconomical from this raw material sources purification of paclitaxel and Taxan, dry in addition needle also needs with high investment.
The program of prior art is tediously long, and is expensive, perhaps only can be limited to analytical scale in practice.Because of taxol contents level in needle is low, needle does not contain a large amount of wax.Cause and separate pure taxol from needle and reach clinical receptible degree and proposed more challenge.A maximum difficult problem be with the separating of close closely analogue cephalotaxin, cephalotaxin is present in needle and the bark, these two analogues can adopt optionally from the mixture that contains both, and chemical transformation separates, the shortcoming of this method comprises owing to use expensively, is toxic reagent and the extra cost that causes sometimes; Need additional chromatography owing to the cephalotaxin that transforms will be separated from taxol; Cephalotaxin goes to pot in the process, and taxol also can go to pot sometimes; Must carry out extra chemical conversion for reclaiming cephalotaxin.
Maximally related prior art is considered to the United States Patent (USP) NO.5 of 1999.10.19 issue, 969,156, this patent provides and obtained seemingly a kind of method of thing of high yield, high-purity taxone from the source thing that contains Taxan, this method is used the polymeric resin absorbent material, under low pressure separating analogous thing, complicated in the technology that need not to provide now, tediously long purification procedures.This separates from the source thing that contains Taxan and the method for purifying 10-deacetyltaxol comprises, the first step is with organic extraction agent such as dichloromethane extraction, second step contacted a kind of absorbing medium with extraction agent, with this medium dress post, this post contains certain absorption agent such as aluminum chloride then.Next procedure is to use ORGANIC SOLVENT MIXTURES.As mixture wash-out under about 10-20psi pressure of hexane and acetone, make to produce each component contain bearing taxanes, next step is to make each component crystallization so that a kind of solid-state Taxan material and mother liquor to be provided again, and concentrates said mother liquor.Next step is to make the mixture of polar solvent and mother liquor carry out wash-out so that at least the second taxane compounds to be provided by the polymeric resin again.
Like this, this technology has continued to obtain Taxan in search even method more effective and more environmental safety, uses plant material and organic solvent to reduce.
Description is from the publication and cumulative the adding of patent a few days of the separation of Japanese yew kind and purification of paclitaxel and Taxan, but very complicated, difficulty of the program of being known and productive rate are low now, outline typical this class patent below, these patents state fold to the various technology of anti-phase layer folding from the positive layer, from Cercocarpus not the productive rate of isolating Paclitaxel of the same race from 0.005-0.017%.
Part in these patents is listed as follows:
The U.S. Patent No. 5,019.504 of the Chrisen of promulgation in 1991;
The U.S. Patent No. 5,380,916 of the Rao of promulgation on January 10 nineteen ninety-five;
The U.S. Patent No. 5,380,916 of the Rao of promulgation on January 10th, 1998;
The U.S. Patent No. 5,407,674 of the Gabetta of promulgation on April 18 nineteen ninety-five etc.;
The U.S. Patent No. 5,620,875 of the Hoffman of promulgation on April 15th, 1997 etc.;
The U.S. Patent No. 8,670,673 of the Rao of promulgation on September 23rd, 1997;
The U.S. Patent No. 6,503,396 of the Kim of promulgation on January 7th, 2003 etc.;
The Canadian Patent No.2 of the Nair of promulgation on November 18th, 1993,126,698;
The Canadian Patent No.2 of the Durand of promulgation on March 18th, 1994 etc., 157,905; And
The Canadian Patent No.2 of the Caron of promulgation on June 15th, 1999,213,952
The present invention will be at first with the summary formal description, describes the enforcement of concrete scheme then in detail with reference to the accompanying drawing of back.These embodiments are intended to illustrate the principle of this invention, the mode of enforcement.Then, the widest implication of this invention and more specifically form will in each of each claim of concluding this specification sheets, be further described and define.
Summary of the invention
It is useful that the minimizing use plant material that environmental safety more effectively and is more arranged of an acquisition Taxan and the method for organic solvent are provided
The present invention has realized above-mentioned desired result, provides from the biomass that are easy to obtain to separate commercial important natural product, provide from the mixture of other compound synthetic materials obtain an effective means of Taxan; Provide applicable to industrial-scale production, from the bark needle of Cercocarpus kind or cell culture, extracted the program of taxol and other Taxan; From vegetable material, preferably from viewing and admiring the fresh material in yew source, with high yield extraction and isolation of taxol and other Taxan; A kind of method of remarkable reduction Taxane derivative production cost is provided, the method that obtains the above mentioned natural product on the business demand amount from the renewable material source of easy acquisition is provided.
1. Fa Ming statement
First aspect, the present invention has briefly provided improving one's methods from the material source separation and purification Taxan that contains 10-deacetyltaxol, comprise the solution that will contain Taxane derivative step by chromatography column, this kind improvement comprises the filled with medium in the hold-down column, the space that holds the organic solution that contains impure Taxane derivative with eliminating, chromatography column keeps under high pressure, as about 250-400Psi or even up to about 5000Psi.
Second aspect, the invention provides a method, this method comprises and will contain the primary step of the organic solution of impure Taxan by a chromatography column, filled with medium in this chromatography column has been forced into has got rid of space wherein, and keep under high pressure 300-400Psi or even up to 1000Psi according to appointment.
The third aspect, the invention provides a method, thereby this method comprises the slurries that will extract the organic solvent of taxol and compressed the space of having got rid of in this tightly by the filled with medium in this post of chromatography column, and remain on can be to the pressure of about 1000Psi, according to appointment 30-50Psi.
Fourth aspect, the invention provides a kind of method, this method comprises with organic solvent extracting Japanese yew biomaterial, and extract is distributed between hexane and water, and then between methylene dichloride or chloroform and water, distribute, reclaim the methane dioxide solution or the chloroformic solution of impure taxol.
2. other characteristics of the present invention
Characteristics of a first aspect of the present invention comprise carries out an additional post step of crossing, be about to contain the organic solution of impure Taxane derivative by a chromatography column, filled with medium in said post has been forced into the space of having removed in this post, keeps then under the pressure of about 400Psi
Another characteristics of a first aspect of the present invention comprise carries out the additional post step of crossing, be about to contain extracted be called the organic solution of impure Taxane derivative by a chromatography column, filled with medium in said post has been pressed onto the space of having removed in the said post, be kept at then under the pressure of about 400Psi, provide thus by the taxol of extracting in organic solvent, and with this said extracting the organic agent slurries of taxol by a chromatography column, filled with medium in this post has been pressed onto the space of having removed in the said post, and remains under the pressure of about 50Psi;
Another characteristics of a first aspect of the present invention comprise uses a kind of organic solvent that the Japanese yew biomass are carried out extractive primary step, and said solution distributed between hexane and water, between methylene dichloride or chloroform and water, reallocate then, reclaim the dichloromethane solution or the chloroformic solution of thick taxol at last.
Another characteristics of first aspect present invention comprise that selecting organic solution is methyl alcohol, ethanol, methyl alcohol and alcoholic acid mixture, methylene dichloride and methanol mixture or methylene dichloride and alcoholic acid mixture.
Another characteristics of first aspect present invention comprise that selecting filled with medium is the silica gel of phenylbenzene bonding or the silica gel of C-18 bonding.
Another characteristics of first aspect present invention comprise the gradient solution wash-out pillar with acetone, methanol or acetonitrile/water.
Another characteristics of first aspect present invention comprise that selecting flow velocity is about 350ml/min, and selective pressure is maintained at about 600-800Psi.
Another characteristics of first aspect present invention comprise crystallization and reclaim the step of the taxol of purifying basically.
Characteristics of second aspect present invention comprise that selecting organic solution is acetone, ethanol/water or isopropanol.
Another characteristics of second aspect present invention comprise that the filled with medium of selecting post is polystyrene-DVB resin, or the resin of polymethacrylate resin or polyaromatic.
Another characteristics of second aspect present invention comprise the gradient solvent with acetone, methanol or acetonitrile/water, carry out the post wash-out with the flow velocity of for example about 1L/min.
Another characteristics of second aspect present invention comprise from the step of the unpurified taxol of post elutriant crystallization.
Characteristics of third aspect present invention comprise that selecting filled with medium is the resin of polystyrene-DVB resin or polymethacrylate resin or polyaromatic.
Another characteristics of third aspect present invention comprise selects the stage gradient liquid of acetone, methanol or acetonitrile/water to carry out the post wash-out.
Another characteristics of third aspect present invention comprise selects the post elution flow rate at about 350ml/min, and pressure is at about 600-800Psi or even up to 1000Psi.
Another characteristics of third aspect present invention comprise from the additional step of the part effluent liquid crystallization 9-dihydro-1.3-acetyl baccatin III of post.
The characteristics of fourth aspect present invention comprise selects the solution of 10-deacetyltaxol solution as coarse crystallization taxol in ethanol.
Put it briefly briefly and more, " loading and the locking axial compression column " (the Load and LockAxial Compression Column) that provided by adopting the senior liquid chromatography (LC) of technical scale preparation property in the present invention extracts the purifying process of the organic solution of taxol.
The successful use in the senior liquid chromatography (LC) of preparation property of medium and major diameter post needs suitable hard equipment." loading and the locking axial compression column " is a kind of like this device, and its piston is used for filling and takes out the chromatography bed, and in use keeps post bed pressure.This has just prevented to form the space in the post bed effectively basically, and pillar can be used any filling material, comprises small-particle (about 10um) medium, and can produce very high stage number, and post bed length can be controlled by the filling material amount of control preparation pillar.
This by using " loading and the locking axial compression column " the invention provides a kind of commercial program of extracting the economy of taxol from Japanese yew, this program relates to a series of procedural steps, the unpurified paclitaxel solution that last steps necessary can be got the third step preparation carries out, and also desirable solution without the third step preparation carries out.Similarly, the unpurified paclitaxel solution of desirable second step preparation of third step carries out, the solution that also can get without the preparation of second step carries out, the second step impure paclitaxel solution that can get first step preparation carries out in addition, or the solution that also can get without the first step preparation carries out.
In a preferred embodiment, first step relates to from vegetable material Cercocarpus kind, T. canadensis (ground hemlock) preferably, extract any part such as bark that contains one or more Taxans in the plant, the tree rod, leaf or needle, branch, tender tree, seed or full seedling all can be utilized, preferably cut short needle and spray, dry also grinding, vegetable material grinds available ordinary method and finishes, as use trimming machine or pulverizing mill, Taxan can be by whole plant or plant part such as stem from separating, root, leaf (needle), seed or their mixture extract, being used to extract material can be fresh or exsiccant, preferably uses needle.
Plant material is preferably dry earlier, is ground to appropriate size, and the general size scope is from about 0.001～about 10mm
3Plant material uses organic solution extracting such as methyl alcohol, ethanol, methyl alcohol and alcoholic acid mixture, methylene dichloride and methanol mixture, or methylene dichloride and alcoholic acid mixture.Mix with water by the filtrate that extracting obtains, concentrate reducing liquid volume, concentrated solution divides to be equipped with between hexane and water defatting step is worked, and water is again at organic solution such as methylene dichloride, reallocate between chloroform or vinyl acetic monomer and water, and shorten thick slurry deeply into.
In a preferred embodiment, second step relates to underflow and crosses post, to this step ion-exchange, best solution is to use organic solvent, underflow as the preparation of the first step of acetone, ethanol or isopropanol, but, the organic solution of any taxol that contains extraction suitably all can be used as the parent material of second step
The slurries that prepare in methylene dichloride/acetone for example are by impouring, and after by one the pressure column of absorption resin as medium of certain poly being housed, can be divided into several components, polymeric absorbs resin, polystyrene-DVB resin for example, the resin of polymethacrylate resin or any other suitable polyaromatic.The most normally used resin is the poly styrene polymer that has crosslinked divinylbenzene in polymeric absorption resin, the absorption resin of this polymkeric substance should have suitable granular size, be adapted to the size of post and the operating pressure and the flow velocity of setting again, the example of an indefiniteness is that particle size is about the 5-100 order.
The suitable water soluble solvent of operational phase gradient is crossed the post wash-out as acetone, methanol, ethanol/water or second eyeball/water, and flow velocity is variable, can change according to different operational conditions, and the example of an indefiniteness is the about 2L/min of flow velocity.
Each component that contains until about 45% acetone can be used for generating 9-two chloro-1, the crystallization of 3-acetyl crust card fourth III.
Contain the component that surpasses about 45% acetone and concentrate, use suitable organic solvent then to remove most of acetone, as acetyl triethyl, CH
3Cl or CH
2Cl
2Extracting, and concentrate is to provide the solid slag of rough taxol.
Third step relates to the purifying of rough solid taxol, and preferably the solid shape slag of this rough taxol is taken from the product of second step, but the rough solid shape taxol that anyway makes can be as the starting material of third step.
Rough solid shape taxol is dissolved in the mixed solvent of suitable water soluble, as acetone, purpose is that defatting step is worked, organic solvent/aqueous solution such as acetone solution that the water soluble that produces like this mixes, method elution by axial compression column, this system is the high performance liquid chromatography system, and it has provides and keep for example about 250-400Psi or even up to the setting of about 5000Psi pressure.
As mentioned above, with sample on the Pacific yew alcoholic solution to the post that contains suitable ion exchange resin, use for example acetone of gradient under aforesaid high pressure, to carry out wash-out then, make the acetone component that contains taxol carry out crystallization, and be dried to semipurified taxol.
Final step relates to the purifying of half purification of paclitaxel, and this semipurified taxol is the product of third step preferably, but the half pure taxol of anyway producing all can be used as the parent material of final step.
It is identical that the pillar that final step uses and third step use, and ion exchange resin in post or filled media can be that the silica gel of the silica gel of phenylbenzene bonding or C-18 bonding or any other anti-phase are loaded and expected.
Half pure taxol is dissolved in the solvent that suitable water soluble mixes, for example acetonitrile, ethanol or methyl alcohol, get sample filling post on the lysate, be mixed with the machine solvent with water soluble then, for example the gradient solvent of acetonitrile/water is with for example about 350ml/min of flow velocity and remain under the suitable pressure, for example about 600-800Psi or even up to about 1000Psi, carry out wash-out.
Get the component that contains pure taxol and carry out crystallization, get this crystallization recrystallize from acetone/ethane then, obtain the white powder of pure taxol.
Above outline the invention principal feature and more selectable aspects thereof, this invention can be able to further understanding by the preferred embodiment of narrating below.
Embodiment
Embodiment 1
The extracting of biomass:
Vegetable material (ground hemlock, T. canadensis Taxus canadenisi) gathers in the crops from Canadian P.E.I province and New Brunswick autumn in 2002, contain 0.032% taxol of having an appointment, the fresh chips of ground hemlock is put drying oven in 60-70 ℃ of drying, grind then.
Needle that 600Kg is ground and spray are placed into 3000L and are equipped with in the extractor of reflux exchanger, add 2400L methyl alcohol then, biomaterial about 3 hours with methanol eddy leaches solvent, collect first batch of filtrate, add 2000L methyl alcohol to the biomass slag again after leaching, refluxed about 2 hours, refilter, collect second batch of filtrate and discard biomass, merge first and second batch of filtrate, add about 10% water, this aqueous solution places the vaporizer vacuum concentration to about 15% of original volume.
Concentrated solution two sub-distribution between hexane and water make degreasing, collect water layer, twice reallocation between methylene dichloride (or chloroform) and water again, and collected organic layer is put evaporator for decompression and concentration to underflow, reclaims hexane layer, discards water layer.
Can be able to nearly 45Kg underflow from the 600Kg biomass, the methylene dichloride underflow contains the taxol of nearly 1-1.3%.
Embodiment 2: the preliminary purification of taxol
The 23Kg methylene dichloride underflow that derives from embodiment 1 places and is equipped with churned mechanically container, dilute with 10L water, also can heat if desired, stirring and adjusting is changeed to per second 1.5, little by little in 10min, add the 3L deionized water, the aqueous solution of methylene dichloride underflow is divided into several components by the pressure medium post of plant-scale stainless steel, and this post is equipped with the surge pump of band pressure medium table.
In the post of the methanol slurry impouring 300mm diameter of 120Kg polystyrene-DVB resin (Rohm-Hass XAD-1600), 3000 length, resin particle size is about the 50-100 order.After post is installed, under 30-50Psi pressure, pump into solvent in, replace methyl alcohol up to column equilibration with the aqueous solution that contains 30% acetone.
The underflow sample transfer of dilution is arrived the column top,, underflow is transferred on the post with the water wash container that several L contain 30% acetone.Seal pillar then, begin logistics, use 35,45 then along with eluting solvent pumps into post, 50,60,65,70 and the stage gradient of 80% acetone carry out the post wash-out, flow rate control is collected and is flowed out component at about 2L/min, the about 20L of every component, and pressure is maintained at about 30-50Psi.
Pillar again with the pure acetone washing, is used the mixture washing of acetone/ethyl acetate (1: 1) first with after the washing of 80% acetone subsequently, becomes colourless up to effluent liquid from initial very dark color.
After the washing, post is again with the water balance that contains 30% acetone, and at this trim point, pillar obtains regeneration, prepares the usefulness of operation next time.
The component of collecting is monitored it in uv of 227nm absorption with analyzing HPLC, or monitors with the alcoholic acid thin-layer chromatography that contains 10%H2O of spraying.Merge and contain 9-dihydro-1; the component of 3-acetyl baccatin III (great majority are in the component of 45% acetone); concentrate and remove most of acetone; putting Fume Hoods spends the night and finishes up to crystallization; leach crystal, put recrystallize in the methyl alcohol, obtain the pure 9-dihydro-1 of 240 grams; the white pin sample crystal of 3 ethanol crust card fourth III, productive rate 0.08%.
Merging the component (great majority are in 63% acetone) that contains taxol also concentrates to remove most of acetone, the aqueous solution is used the ethyl acetate extracting again, collecting organic phase is concentrated into dried, residue is analyzed with analyzing HPLC, its (rough taxol raw material) contains the taxol of the 10-15% that has an appointment, taxol from the rate of recovery of methylene dichloride slurries 90～100%.
Embodiment 3:
Taxol secondarily purified:
The high performance liquid chromatography system of secondarily purified use preparation property, this system are called " axial compression column chromatography ".The post of this use is a Varian Load and Lock axial compression column (the long 1000mm of diameter 250mm), this chromatographic system is a Varian ST-4000 system, it is equipped with two high-pressure diaphragm pumps, a sample input aperture, a UV monitor and a Varian control software system, chromatography column is equipped with a hydraulic piston that is used for loading and taking out post, and sealed adapter, be suspended in alcoholic acid polystyrene-DVB resin (Rohm-HassCG-161m with 50L, the about 75 μ m of granular size) behind the filling post, open hydraulic piston, this piston is slowly outer to be moved, the medium that loads in the pressure compression post with about 3000Psi.Be compressed simultaneously at post, lock is turned to lock-bit post is pinned, and post just can produce very high stage number like this.Post is installed and is finished, and when pumping under the pressure of solvent at about 250-300Psi, the water that ethanol is contained about 30% acetone replaces up to column equilibration.
From the solid shape slag that embodiment 2 obtains, approximate 1.5Kg is dissolved in 4L acetone and all dissolves up to all solid substances, under agitation adds 2L water gradually, if desired, can filter.The aqueous solution pumps into post by the sample input aperture then,
Ladder with acetone crosses solvent (water 100% acetone that contains 50% acetone) wash-out post then, and the control flow rate keeps the pressure of about 300-400Psi at 1L/min, collects and flows out component, every part of about 5L.
The component of collecting detects the uv absorption with analyzing HPLC in 227mm, and the ethanol that contains 10% water of spraying is analysed detection as thin layer, merging the component (in most of 70% propanol/water) that contains taxol concentrates to remove most of acetone, putting stink cupboard spends the night to finish up to crystallization and leaches crystal, recrystallization from the water that contains 70% methyl alcohol, put straight empty stove drying and obtain the albescent needle crystal of 235 grams, this half pure taxol purity is about 70-80%, is about 90-95% from the yield of rough taxol.
Embodiment 4
The final purifying of taxol
Final purifying uses preparation property high performance liquid chromatography system and axial compression chromatography column, the post that is adopted is Varian Load and lock axial compression high-pressure post (diameter 100mm, long 1000mm), used chromatographic system is a Varian SD-2 preparation property HPLC system, this system disposition has a sample input aperture, a UV detector and a Varian control software.High-pressure column is equipped with hydraulic piston and sealed adapter that is used to load and take out post.The filled with medium of using is the silica gel of phenylbenzene bonding or the silica gel of C-18 bonding.
6L is suspended in after the alcoholic acid filled with medium all is packed into post, open hydraulic piston, this piston is slowly outer to be moved with medium in the pressure compression post of about 3000Psi, in the compressed while of post, lock is diverted lock-bit post is pinned, before using post to make purifying, replace ethanol until column equilibration with the water that contains about 30% acetonitrile.
With about 50 grams of the rough taxol that derives from embodiment 3, be dissolved in the 200ml acetonitrile and all dissolve up to used solid substance, the limit is stirred in the 100ml waterside progressively add, if desired, can filter, then the aqueous solution is pumped into post by sample inlet.
Ladder with acetonitrile/water crosses solvent (water-100% acetonitrile that contains 30% acetonitrile) wash-out post subsequently, and the about 350ml/min of control flow velocity keeps post and is pressed in about 600-800Psi, collects the stripping component, every part of about 1L.
The component of collecting is used the UV detector to survey the 227nm absorption and is monitored, and the ethanol that contains about 10% water with spraying is done the TLC monitoring simultaneously.Merge the component contain pure taxol, removes most of acetonitrile, put logical cupboard and spend the night and finish up to crystallization through concentrating.Leach crystal,,, obtain 36.5 gram white powders then at straight empty stove inner drying again from acetone hexane (1: 1) recrystallization.The taxol purity of purifying is greater than 99%, productive rate: be about 85-90% from the yield of the thick pure taxol of the about 70-80% of purity, overall yield is greater than about 73%
As mentioned above, the method that all respects of the present invention are used all is simple, use the present invention can be with highly purified taxol of produced in high yields and purple refined alkane on technical scale, therefore the method for all respects of the present invention is from plant origin or from tissue culture, especially from T. canadensis, the relevant Taxan with other of mass production of taxinol provides the method for high investment repayment.
The method of all respects of the present invention provides taxol and the relevant Taxan mass production from Cercocarpus (Taxaceae) plant.
Separation and purification process that all respects of the present invention are used make the Taxane derivative that can reclaim purifying from naturally occurring Cercocarpus expeditiously.
Improvements over the prior art of the present invention be reflected in Taxan high total yield and on the purity of isolating taxol.
Conclusion
Narrated some specific embodiments above, shown how the present invention can operate and drop into application.These embodiments only are illustrations, and the present invention the most widely and more specifically aspect will do further statement and define in claim subsequently.
These claims and the language Ying Yiyi that uses in this state the aspect of the variant scheme of invention and go to understand.But they should not be subject to these different schemes, but should be interpreted as covering four corner that is implied among the present invention and the present invention and the disclosed content that provides in this.
Claims (13)
1 one kinds are separated from the thing source of containing said 10-deacetyltaxol and the method for purifying 10-deacetyltaxol, comprise the organic solution that will contain said Taxane derivative step by chromatography column, this step comprises the said filled with medium of compression, basically removed the space in the post, the organic solution that contains impure Taxane derivative is provided thus; And keep said chromatography column to be in pressure until about 5000psi.
2 methods according to claim 1, comprise the said organic solution that will contain said impure Taxane derivative additional step by chromatography column, in this chromatography column, said filled with medium is compressed, removing the space in the said post, and keep the pressure of post to be in until about 400psi.
3 according to claim 1 or the described method of claim 2, comprise and carry out some additional steps, the organic solution that is about to contain the said impure Taxane derivative that has extracted is passed through chromatography column, in this chromatography column, filled with medium is compressed, to remove the space in the said post, keep post to be in pressure then until about 400psi; Prepared the taxol that is extracted in the organic solvent thus; Be extracted in taxol slurries in the organic solvent by chromatography column with said, the filled with medium in this post is compressed, removing the space in the said post, and keeps post to be in pressure until about 50psi.
4 according to claim 1, and the described method of claim 2 or claim 3 comprises the primary step with organic solvent extraction Japanese yew biomass; Said solution is distributed between hexane and water; Then this solution is reallocated between methylene dichloride or chloroform and water; Reclaim the dichloromethane solution or the chloroformic solution of rough taxol at last.
5 according to each described method in the claim 1 to 4, wherein said 10-deacetyltaxol solution comprises that the coarse crystallization taxol is at methyl alcohol, ethanol, methyl alcohol and alcohol mixture, methylene dichloride and carbinol mixture or the solution in methylene dichloride and alcohol mixture.
6 according to each described method in the claim 1 to 5, and wherein said filled with medium is the silica gel of phenylbenzene bonding, or the silica gel of C-18 bonding; Or polystyrene-DVB resin; Or polymethacrylate resin; Or polyaromatic resin.
7 according to each described method in the claim 1 to 6, wherein said post acetone, and the gradient solution of methanol or acetonitrile/water carries out wash-out.
8 according to each described method in the claim 1 to 7, and wherein flow velocity is about 350ml/min, and wherein column pressure is maintained at about 600-800psi.
9 according to each described method in the claim 1 to 8, and wherein flow velocity is about about 1L/min to about 2L/min.
10 according to each described method in the claim 1 to 9, and the organic solvent in the wherein said first step comprises methylene dichloride, and in said third step, the precipitation liquid to be washed by said post comprises acetone, methanol or acetonitrile/water.
11 according to each described method in the claim 1 to 10, and wherein organic solvent is a methylene dichloride, and further, the elute soln by said post is an acetone; Or methanol; Or ethanol/water; Or isopropanol; Or acetonitrile/water.
12 according to each described method in the claim 1 to 11, comprises the additional step of the impure taxol step of crystallization from the elutriant of said post.
13 according to each described method in the claim 1 to 12, comprises the additional step of crystallization 9-dihydro-1,3 acetyl baccatin III step from the part elutriant of said post.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102552338A (en) * | 2012-02-13 | 2012-07-11 | 林树芳 | Paclitaxel oral anticancer preparation obtained by nontoxic extraction from complete stool of Chinese yew and preparation method thereof |
| CN105511507A (en) * | 2015-12-11 | 2016-04-20 | 苏州泽达兴邦医药科技有限公司 | Liquid level stability control method in traditional Chinese medicine percolation process |
| CN108101869A (en) * | 2017-12-20 | 2018-06-01 | 上海金和生物制药有限公司 | A kind of extracting method of natural Japanese yew alcohol |
-
2006
- 2006-03-10 CN CN 200610067303 patent/CN101033218A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102552338A (en) * | 2012-02-13 | 2012-07-11 | 林树芳 | Paclitaxel oral anticancer preparation obtained by nontoxic extraction from complete stool of Chinese yew and preparation method thereof |
| CN105511507A (en) * | 2015-12-11 | 2016-04-20 | 苏州泽达兴邦医药科技有限公司 | Liquid level stability control method in traditional Chinese medicine percolation process |
| CN108101869A (en) * | 2017-12-20 | 2018-06-01 | 上海金和生物制药有限公司 | A kind of extracting method of natural Japanese yew alcohol |
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