CN101391989B - Method for preparing polyhydroxy taxone and paclitaxel - Google Patents
Method for preparing polyhydroxy taxone and paclitaxel Download PDFInfo
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- CN101391989B CN101391989B CN2008101968245A CN200810196824A CN101391989B CN 101391989 B CN101391989 B CN 101391989B CN 2008101968245 A CN2008101968245 A CN 2008101968245A CN 200810196824 A CN200810196824 A CN 200810196824A CN 101391989 B CN101391989 B CN 101391989B
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Abstract
The invention relates to a method for preparing polyhydroxy taxane and paclitaxel, which pertains to the manufacturing method of natural medical chemical substances or drug raw materials, aims at the tight source of paclitaxel, and selectively converts the multi-acetyl taxane with high abundance in the alcohol extract of yew plant material or cell culture into corresponding polyhydroxy taxane while separating the paclitaxel. The invention comprises the steps of resin column separation, alumina chromatographic column separation, hydrolysis reaction, the solid phase extraction of polyhydroxy taxane, the elution of polyhydroxy taxane, the purification of paclitaxel and the purification of polyhydroxy taxane, thus obtaining the paclitaxel with the purity approaching to 99.5 percent and the polyhydroxy taxane with the purity being more than 98 percent. The invention selectively converts the multi-acetyl taxane with high abundance into corresponding polyhydroxy taxane, produces raw materials which can be used in the pharmaceuticals industry, significantly reduces the loss of taxane compounds in the raw materials and improves the availability of biological resources.
Description
Technical field
The invention belongs to the manufacture method of natural product exploitation or pharmaceutical raw material, the alcohol medicinal extract that is specifically related to Chinese yew material or cell culture is raw material, transforms, prepares the method for polyhydroxy taxone and separated in synchronization taxol.
Background technology
Taxol (taxol), it is a kind of natural antitumor medicine evident in efficacy, aspect the relevant Kaposi sarcoma of treatment AID (Kaposi ' s Sarcoma) good prospects for application is being arranged also, it and the s-generation taxol drug taxotere (taxotere) that has gone on the market at present, remove acetyl crust Ka Ting (ortataxel) with the third generation taxol 14-β-hydroxyl that enters the clinical II phase, has identical mother nucleus structure, difference is the difference of indivedual substituted radicals on parent nucleus only, is referred to as bearing taxanes.Taxol is the diterpenes secondary metabolite that Ramulus et folium taxi cuspidatae produces, structure is very complicated, very unrealistic by the complete synthesis approach production of chemistry, now mainly from bark of Ramulus et folium taxi cuspidatae or branches and leaves, direct extraction separation from taxus chinensis cell culture on a small quantity, perhaps separating earlier purifies is present in 10 in the Ramulus et folium taxi cuspidatae medicinal extract-remove acetyl baccatin III (10-deaceyl-baccatin III) and baccatin III (baccatin III) simultaneously, is that raw material carries out chemical semi-synthetic acquisition taxotere (taxotere) and 14-β-hydroxyl goes acetyl to cling to Ka Ting (ortataxel) with these precursor compounds again.But taxol and semi-synthetic precursor 10 thereof-go acetyl baccatin III (10-deaceyl-baccatinIII) and the content of baccatin III (baccatin III) in plant tissue or cell cultures restricted by kind and many complicated factors, in most cases, their content is not high.For example, taxol in reproducible Ramulus et folium taxi cuspidatae content on average below 0.02%; 10-remove acetyl baccatin III (10-deaceyl-baccatin III) and baccatin III (baccatin III) though in berry Ramulus et folium taxi cuspidatae (T.baccata) branches and leaves content up to 0.1%, but in Chinese Ramulus et folium taxi cuspidatae (T.chinensis), content is but very low, the general order of magnitude at ppm, low levels causes a large amount of plant resourceses to be felled on the one hand, aggravate the imbalance between supply and demand of taxol, strengthened the difficulty of extraction and separation process on the other hand.
In fact, the Taxan that also contains a large amount of other kinds in Ramulus et folium taxi cuspidatae tissue and the cell medicinal extract, Israel and Palestine card booth VI (baccatin VI) and Yunnan peaceful (taxuyunnanine C) for many acetyl Taxan of representative Chinese Ramulus et folium taxi cuspidatae, taxus chinensis in northeast, Taxus x media, Xizang Taxus chinensis, taxusyunnanensis, content is quite high in these kinds such as T. canadensis, and described many acetyl Taxan is meant to have as chin card booth VI (baccatin VI), one of peaceful (taxuyunnanine C) structural framework feature in Yunnan, at C7, C9, C10, C13, C14, C5 these any 3 or above sites have the Japanese yew diterpenes material that acetyl replaces.Though these many acetyl Taxans have the same with taxol or similar mother nucleus structure, itself does not show tangible anti-tumor activity, and they are present in the alcohol medicinal extract of Chinese yew or cell culture.Existing is that the technology of extraction separation target is seen (1) U.S. Pat P5 with taxol or 10-go acetyl baccatin III (10-deaceyl-baccatin III) and baccatin III (baccatin III), 412,116 (1995) oxidation of glycoside substituted taxanes to taxolor taxo l precursors and new taxane compounds formed asintermediates; (2) publication number CN1096820, the method for producing taxol by means of cell culture; (3) publication number CN1534032, a kind of method of efficient production taxol; (4) publication number CN101128448 prepares the method for taxol; (5) publication number CN1158126 prepares the method for taxol and derivative thereof; (6) publication number CN1197796, a kind of novel method of taxol preparation.In these methods, many acetyl Taxan is used as " impurity " usually and is removed, and causes the significant wastage of valuable natural resource.Therefore, in separating and purifying taxol, through structure of modification, the taxol that " turning waste into wealth " generation is useful or the precursor of its pharmacy are significant with these high yield Taxans in the medicinal extract.
Summary of the invention
The invention provides a kind of method for preparing polyhydroxy taxone and taxol; at the nervous problem in the source of taxol; from the alcohol medicinal extract of Chinese yew material or cell culture isolation of taxol the time; with wherein abundant many acetylizes Taxan; optionally be converted into corresponding polyhydroxy taxone; the raw material that production can be utilized by pharmaceutical industry transforms into polyhydroxy taxone compound four at many acetyl taxane compounds crust card booth VI especially and removes acetyl crust card booth VI; many acetyl taxane compounds Yunnan is rather transformed into polyhydroxy taxone compound four goes to acetyl Yunnan peaceful.
A kind of method for preparing polyhydroxy taxone and taxol of the present invention comprises:
(1) resin column separating step: the methyl alcohol or the ethanol extract of Chinese yew tissue or cell culture are loaded on the macroporous resin column, with the aqueous solution flushing that contains methyl alcohol 20%～40%, remove water-soluble impurity or pigment, again with the aqueous solution flushing that contains methyl alcohol 50%～80%, collect the elutriant of 3 times of column volumes, with the methyl alcohol evaporate to dryness in the elutriant, add extraction solvent isopyknic and that water is immiscible and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness, obtain macroporous resin column and separate crude product, described per-cent is volume percent;
(2) alumina chromatographic column separating step: separate crude product with methylene dichloride mixed solution dissolving macroporous resin column with methyl alcohol, be splined on the alumina column, the mass ratio that macroporous resin column is separated crude product and aluminum oxide is 1/60～1/100, carry out the mesolow normal-phase chromatography, with methyl alcohol and methylene dichloride mixed solution wash-out, thin-layer chromatography detects flow point in conjunction with efficient liquid-phase chromatography method, the flow point of many acetyl Taxan is rich in acquisition, and the flow point that is rich in taxol, the evaporated under reduced pressure solvent obtains to contain many acetyl Taxan crude product and taxol crude product;
(3) hydrolysis reaction step: in containing many acetyl Taxan crude product, add polar organic solvent, add basic solution or suspension again, making whole pH value of solution value is 9～11, polar organic solvent accounts for 30%～60% of whole liquor capacity, stirring reaction 0.5～1 hour, with in the thin-layer chromatography method detection reaction liquid during no many acetyl Taxan, reaction is finished, whole solution is filtered, and filtrate being is rich in the polyhydroxy taxone solution of polyhydroxy taxone;
(4) the Solid-Phase Extraction step of polyhydroxy taxone: the pH value of polyhydroxy taxone solution is transferred to 3.5～6 with hydrochloric acid, add the polymeric amide polymeric adsorbent of handling well, stirred 3～5 hours, stir with stopping during no polyhydroxy taxone in the thin-layer chromatography method test solution, filter, the polymeric amide polymeric adsorbent is packed in the glass chromatography column;
(5) elution step of polyhydroxy taxone: with the pH value is 2.5～4.5 aqueous citric acid solution washing polyamide column, removes wherein pigment and alkaloids impurity; Volumetric concentration with 2 times of column volumes is the methanol aqueous solution wash-out of 40%-60%, collect the wash-out flow point, reclaim alcohol wherein, in raffinate, add isopyknic methylene dichloride, fully concussion back extraction, organic solvent layer is separated, and the solid matter of evaporate to dryness organic solvent gained is and contains the polyhydroxy taxone crude product;
(6) purification step of taxol: with the taxol crude product that step (2) obtains, press anti-phase liquid phase chromatography column to separate in using, recrystallize obtains purity and separates product near the 99.5% taxol degree of depth;
(7) purification step of polyhydroxy taxone: contain the polyhydroxy taxone crude product with what obtain in the step (5), with in press anti-phase liquid phase chromatography column to separate, recrystallize obtains purity and separates product greater than the 98% polyhydroxy taxone degree of depth.
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the described resin column separating step, the macroporous resin of filling in the described macroporous resin column is nonpolar macroporous vinylbenzene polymeric adsorbent; Described extraction solvent is a kind of in chloroform, methylene dichloride, the ethyl acetate.
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the described alumina chromatographic column separating step, described alumina column filler is the chromatography alumina packing, particle diameter is 100 orders, it is the chromatography column of 15:1～20:1 that wet-filling becomes aspect ratio, use 2 column volumes of elutriant wash-out of volume ratio methylene dichloride: methyl alcohol=97:3～99:1, use 2 column volumes of elutriant wash-out of volume ratio methylene dichloride: methyl alcohol=95:5～90:10, chromatography column pressure is 5～20bar, flow velocity is 1～5ml/min, with the concentration of high-performance liquid chromatography method elutriant.
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the described hydrolysis reaction step, described polar organic solvent is a kind of in acetone, acetonitrile, the methyl alcohol; The basic solution that adds is a kind of in mass percent 10%～20% wet chemical, 10%～20% aqueous sodium carbonate or the calcium hydroxide suspension; Stir speed (S.S.) is 120r/min～60r/min.
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the Solid-Phase Extraction step of described polyhydroxy taxone, described polyamide resin is that particle diameter is 30～60 purpose chromatography polyamide resin filler powder, wet-filling is the glass column of 0.6cm～1.5cm to diameter, and the height of filler is 10:1～20:1 with the ratio of column diameter; Every gram dried resin applied sample amount is 10mg～50mg, and elution flow rate is 2BV.h
-1
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the purification step of described taxol,
Press anti-phase liquid phase chromatography column separating process to be in described: with taxol crude product dissolve with methanol, last C18 reverse phase filler, in 20～40bar pressure range, use volume ratio methyl alcohol: water is the 4:1 wash-out, collects positive component, extremely does in 30 ℃～50 ℃ underpressure distillation;
Described crystallisation process is: to the positive component of reversed phase chromatography evaporate to dryness, add volumetric concentration again and be 80%～95% methyl alcohol hexane solution, 30 ℃～40 ℃ fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃～6 ℃ freezing and crystallizings, and crystal is purity near 99.5% taxol elaboration;
The described method for preparing polyhydroxy taxone and taxol is characterized in that:
In the purification step of described polyhydroxy taxone,
Press anti-phase liquid phase chromatography column separating process to be in described: with polyhydroxy taxone crude product dissolve with methanol, last C18 reverse phase filler in 20～40bar pressure range, is used volume ratio methyl alcohol: water=3:2 wash-out, collect positive component, extremely do in 30 ℃～50 ℃ underpressure distillation;
Described crystallisation process is: to the positive component of reversed phase chromatography evaporate to dryness, add volumetric concentration again and be 70%～90% methanol ethyl acetate solution, 30 ℃～40 ℃ fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃～6 ℃ freezing and crystallizings, and crystal is purity and separates product greater than 98% the polyhydroxy taxone degree of depth.
Analyze with efficient liquid-phase chromatography method (HPLC) and liquid-matter coupling (LC-MS) through the applicant, the content of finding many acetyl taxane compounds crust card booth VI (bacctin VI) accounts for 0.2% of Chinese branch of Ramulus et folium taxi cuspidatae leaf dry weight, exceeds 2 orders of magnitude than taxol0.007%; Adopt existing cell culture processes, Yunnan peaceful (taxuyunnanine C) can reach 400mg/L in taxus chinensis cell culture system output, and the mean yield of taxol (taxol) only has 20mg/L; The present invention optionally is converted into corresponding polyhydroxy taxone with these abundant many acetyl Taxans, the raw material that production can be utilized by pharmaceutical industry, significantly reduce raw material cost, improve the availability of Biological resources.Described polyhydroxy taxone is meant that having four removes acetyl crust card booth VI (7,9,10,13-deacetyl-bacctin VI) or four go to acetyl Yunnan peaceful (2,5,10,14-deacetyl-taxuyunnanine C) one of structural framework feature, have the Japanese yew diterpenes material that hydroxyl replaces in C7, C9, C10, C13, C14, C5 these any 3 or above sites.VI carries out the hydrolysis of selectivity acetyl to many acetyl taxane compounds crust card booth, remove the ethanoyl of its C7, C9, C10, C13 position exactly, the polyhydroxy taxone compound four that makes it to become respective configuration removes acetyl crust card booth VI, but the ethanoyl on the reservation C4 position and the benzoyl of C2 position, the polyhydroxy taxone compound four that generates removes acetyl crust card booth VI, can modify easily becomes 10 again-remove acetyl baccatin III (, becoming carbonyl) with hydroxyl dehydrogenation on the C9 position; Equally; the hydrolysis of selectivity acetyl is rather carried out in Yunnan; ethanoyl on its C2, C5, C10, the C14 position is changed into compound four go to acetyl Yunnan peaceful (2; 5; 10; 14-deacetyl-taxuyunnanine C), will shorten the synthetic route that 14-β-hydroxyl removes acetyl crust Ka Ting (ortataxel).
The superiority that the present invention had is:
(1) utilize hydrolysis reaction step of the present invention, transforming many acetyl Taxan is polyhydroxy taxone, significantly reduces the loss of bearing taxanes in the raw material, has enlarged the source of Taxan raw material.If need the taxane compounds in 1 mole of biosynthesizing source to calculate by generating every mole of taxol (the perhaps s-generation, three generations's taxol), utilize the present invention can obtain more Taxans that can be utilized by pharmaceutical industry, being equivalent to has increased by 1320 times taxol raw material sources than existing technology, utilization of resources degree significantly increases.
(2) utilize the polyhydroxy taxone of technology preparation of the present invention to can be used as the useful raw material of pharmacy; because the hydroxyl on the polyhydroxy taxone parent nucleus is equivalent to introduce a plurality of " rivets " on parent nucleus; utilization can be carried out modifications such as follow-up radical protection, substituting group elimination or acidylate easier, more easily in these " rivets " good reactivity and binding ability on corresponding site.Utilize particularly that the present invention prepares four remove acetyl crust card booth VI and just had originally that C14 replaces four go to acetyl Yunnan rather to set out, than remove acetyl crust Ka Ting from baccatin III to third generation taxanes medicine 14-β-hydroxyl, synthetic route is shorter, technical easier realization.
(3) utilize the present invention; not only can be that many acetylizes of high abundance Taxan in the medicinal extract is selectively converted to corresponding polyhydroxy taxone with Chinese yew tissue or cell cultures; the taxol that exists in the medicinal extract can also be separated and purifying; the yield of taxol and prior art maintain an equal level or are slightly high, have realized taxol and polyhydroxy taxone synchronous production.
Description of drawings
Fig. 1 is taxol (taxol) molecular structure;
Fig. 2 is taxotere (taxotere) molecular structure;
Fig. 3 is that 14-β-hydroxyl removes acetyl crust Ka Ting (ortataxel) molecular structure;
Fig. 4 is 10-remove acetyl baccatin III (10-deacetyl-bacctin III) molecular structure;
Fig. 5 is baccatin III (bacctin III) molecular structure;
Fig. 6 is crust card booth VI (bacctin VI) molecular structure;
Fig. 7 is peaceful (taxuyunnanine C) molecular structure in Yunnan;
Fig. 8 four removes acetyl crust card booth VI (7,9,10,13-deacetyl-bacctin VI) molecular structure;
Fig. 9 be four go acetyl Yunnan peaceful (2,5, the molecular structure of 10,14-deacetyl-taxuyunnanineC);
Figure 10 goes acetyl to cling to the reaction formula of card booth VI (7,9,10,13-deacetyl-bacctin VI) for the hydrolysis of crust card booth VI (bacctin VI) selectivity acetyl generates four;
Figure 11 removes the rather reaction formula of (2,5,10,14-deacetyl-taxuyunnanine C) of acetyl Yunnan for peaceful (taxuyunnanine C) the selectivity acetyl hydrolysis in Yunnan generates four.
Embodiment
(1) resin column separating step: get the medicinal extract 10g after Chinese Ramulus et folium taxi cuspidatae methanol extraction concentrates, analyze through HPLC and to contain taxol 0.2% (20mg), contain crust card booth VI and be about 5.7% (570mg), use dissolve with methanol, be loaded on the resin column of nonpolar macroporous vinylbenzene polymeric adsorbent XAD16, with the aqueous solution flushing that contains methyl alcohol 30%, flush away water-soluble impurity or pigment; The aqueous solution wash-out that contains methyl alcohol 50% again with 3 times of column volumes, collect this elutriant, to wherein methyl alcohol evaporate to dryness, add isopyknic ethyl acetate and carry out liquid-liquid extraction as extraction solvent, collect ethyl acetate layer, evaporate to dryness obtains the 1.45g macroporous resin column and separates crude product, analyzes through HPLC to contain taxol 1.3% (18.8mg), contain crust card booth VI and be about 39% (565mg); The taxol yield is 94%; Crust card booth VI yield, 99%.
(2) alumina chromatographic column separating step: getting particle diameter is 100 purpose chromatography alumina packings, becoming aspect ratio with wet-filling is the chromatography column of 16:1, above-mentioned 1.45g macroporous resin column is separated methyl alcohol and the methylene dichloride mixed solution dissolving of crude product with volume ratio 1:99, be splined on the alumina column, the mass ratio that macroporous resin column is separated crude product and aluminum oxide is 1/100; Earlier use methylene dichloride: the methyl alcohol volume ratio is 2 column volumes of elutriant wash-out of 97:3, obtains the flow point 340ml of rich taxol; Use methylene dichloride again: the methyl alcohol volume ratio is the elutriant wash-out of 95:5, obtains to be rich in the flow point 340ml of many acetyl Taxan; Chromatography column pressure is 5bar, and flow velocity is 1ml/min, and 2 part flow points of the above-mentioned collection of evaporated under reduced pressure detect in conjunction with efficient liquid-phase chromatography method with thin-layer chromatography respectively, obtains to contain the crude product 32mg of taxol 59.3%; The crude product 706mg that contains crust card booth VI80%;
(3) hydrolysis reaction step: contain interpolation methyl alcohol 20ml in the crude product that clings to card booth VI to above-mentioned 706mg, the wet chemical 37ml of mass ratio 20%, methyl alcohol accounts for 30% of overall solution volume, pH is 10, and 120r/min rotating speed stirring reaction is after about 1 hour, detect nothing crust card booth VI in the Indicator Reaction liquid with the TLC method, with finishing reaction, whole solution to be filtered, filtrate being is rich in four and gone the acetyl crust to block the solution of booth VI;
(4) the Solid-Phase Extraction step of polyhydroxy taxone: the pH value of above-mentioned polyhydroxy taxone solution is transferred to 5 with hydrochloric acid, adding the particle diameter of handling well is 30～60 purpose chromatography polyamide resin filler powder, stirred 3 hours, stop to stir when removing acetyl crust card booth VI with nothing four in the thin-layer chromatography method test solution, filter, with the polymeric amide polymeric adsorbent diameter of packing into is in the 0.6cm glass chromatography column, and the height of filler is 20:1 with the ratio of column diameter; Every gram dried resin volume containing the sample is 10mg, and elution flow rate is 2BV.h
-1
(5) elution step of polyhydroxy taxone: with pH is 3.5 aqueous citric acid solution washing polyamide column, remove wherein pigment and alkaloids impurity, it with the volumetric concentration of 2 times of column volumes (34ml) 60% methanol aqueous solution wash-out, collect the wash-out flow point, reclaim wherein methyl alcohol, in raffinate, add isopyknic methylene dichloride, fully concussion back extraction, dichloromethane layer is separated, the evaporate to dryness organic solvent, obtain the solid matter of 300mg, analyzing four through HPLC, to remove the content of acetyl crust card booth VI be 82%, and transformation efficiency is 60%, separation yield is 95%;
(6) purification step of taxol: with the crude product 0.5ml dissolve with methanol of 32mg59.3% (HPLC analysis) taxol of above-mentioned acquisition, at pressure is under the 20bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 4:1, collect positive component, extremely do in 30 ℃ of underpressure distillation, the adding volume percent is 80% methyl alcohol hexane solution 2ml, 30 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃ of freezing and crystallizing, and obtaining the 15.8mg crystal purity is 99.5% taxol elaboration; Taxol total recovery 79%;
(7) purification step of polyhydroxy taxone: four of the 300mg82% of above-mentioned acquisition is removed acetyl crust card booth VI crude product 1ml dissolve with methanol, at pressure is under the 40bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 3:2, collect positive component, extremely do in 30 ℃ of underpressure distillation, the adding volume percent is 80% methanol ethyl acetate 4ml, 40 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃ of freezing and crystallizing, and 98% crystal four that obtains 192mg removes acetyl crust card booth VI, and Taxan utilization of resources degree is 19 (doubly).
(1) resin column separating step: the medicinal extract 5g after getting the taxus chinensis cell methanol extraction and concentrating, analyze through HPLC and to contain taxol 1% (50mg), contain Yunnan and rather be about 16% (800mg).Use dissolve with methanol, be loaded on the resin column of nonpolar macroporous vinylbenzene polymeric adsorbent XAD1180, contain 20% aqueous solution flushing, flush away water-soluble impurity or pigment with methyl alcohol; The aqueous solution wash-out that contains methyl alcohol 80% again with 3 times of column volumes, collect this elutriant, to wherein methyl alcohol evaporate to dryness, add isopyknic chloroform and carry out liquid-liquid extraction, collect chloroform layer, evaporate to dryness obtains the 2.65g macroporous resin column and separates crude product, analyzes through HPLC to contain taxol 1.8% (47mg), contain Yunnan and rather be about 30% (795mg); The taxol yield is 94%; The peaceful yield in Yunnan, 99%.
(2) alumina chromatographic column separating step: getting particle diameter is 100 purpose chromatography alumina packings, becoming aspect ratio with wet-filling is the chromatography column of 20:1, above-mentioned 2.65g macroporous resin column is separated methyl alcohol and the dissolving of methylene dichloride mixed solution of crude product volume ratio 1:99, be splined on the alumina column, the mass ratio that macroporous resin column is separated crude product and aluminum oxide is 1/60; Earlier use methylene dichloride: the methyl alcohol volume ratio is 2 column volumes of elutriant wash-out of 99:1, obtains the flow point of rich taxol; Use methylene dichloride again: the methyl alcohol volume ratio is the elutriant wash-out of 96:4, obtains to be rich in the flow point of many acetyl Taxan; Chromatography column pressure is 20bar, and flow velocity is 5ml/min, and 2 part flow points of the above-mentioned collection of evaporated under reduced pressure detect in conjunction with efficient liquid-phase chromatography method with thin-layer chromatography respectively, obtains to contain the crude product 78mg of taxol 60%; The crude product 910mg that contains Yunnan peaceful 83%;
(3) hydrolysis reaction step: contain interpolation acetone 25ml in the peaceful crude product in Yunnan to above-mentioned 910mg, mass ratio 10% aqueous sodium carbonate 25ml, acetone accounts for 50% of overall solution volume, pH is 9, and 60r/min rotating speed stirring reaction is after about 0.5 hour, detect nothing crust card booth VI in the Indicator Reaction liquid with the TLC method, with finishing reaction, whole solution to be filtered, filtrate being is rich in four and gone the acetyl crust to block the solution of booth VI;
(4) the Solid-Phase Extraction step of polyhydroxy taxone: the pH value of above-mentioned polyhydroxy taxone solution is transferred to 3.5 with hydrochloric acid, adding the particle diameter of handling well is 30～60 purpose chromatography polyamide resin filler powder, stirred 5 hours, do not stop to stir when going to acetyl Yunnan peaceful with having four in the thin-layer chromatography method test solution, filter, with the polymeric amide polymeric adsorbent diameter of packing into is in the 1.5cm glass chromatography column, and the height of filler is 10:1 with the ratio of column diameter; Every gram dried resin applied sample amount is 30mg, and elution flow rate is 2BV.h
-1
(5) elution step of polyhydroxy taxone: with pH is 4.5 aqueous citric acid solution washing polyamide column, remove wherein pigment and alkaloids impurity, it with 2 times of column volumes, volumetric concentration 50% methanol aqueous solution wash-out, collect the wash-out flow point, reclaim wherein methyl alcohol, in raffinate, add isopyknic methylene dichloride again, fully concussion back extraction, dichloromethane layer is separated, the evaporate to dryness organic solvent obtains the solid matter of 299mg, and analyzing four through HPLC, to remove the peaceful content in acetyl Yunnan be 80%, transformation efficiency is 50%, and separation yield is 95%;
(6) purification step of taxol: with the crude product 0.5ml dissolve with methanol of 78mg60% (HPLC analysis) taxol of above-mentioned acquisition, at pressure is under the 40bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 4:1, collect positive component, extremely do in 50 ℃ of underpressure distillation, the adding volume percent is 95% methyl alcohol hexane solution 2ml, 40 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 6 ℃ of freezing and crystallizings, and obtaining the 37mg crystal purity is 99.5% taxol elaboration;
(7) purification step of polyhydroxy taxone: the 299mg80% four of above-mentioned acquisition is removed acetyl Yunnan peaceful crude product 1.5ml dissolve with methanol, at pressure is under the 40bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 3:2, collect positive component, extremely do in 50 ℃ of underpressure distillation, the adding volume percent is 90% methanol ethyl acetate 4ml, 30 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 4 ℃ of freezing and crystallizings, and the crystal four that obtains 233.4mg98% goes to acetyl Yunnan peaceful.
(1) resin column separating step: the medicinal extract 1Kg after getting southerm yew branches and leaves methanol extraction and concentrating, analyze through HPLC and to contain taxol 0.18% (1.8g), contain crust card booth VI (B VI) and be about 5.5% (55g).Use dissolve with methanol, be loaded on the resin column of nonpolar macroporous vinylbenzene polymeric adsorbent XAD16, with the aqueous solution flushing that contains methyl alcohol 40%, flush away water-soluble impurity or pigment; The aqueous solution wash-out that contains methyl alcohol 70% again with 3 times of column volumes, collect this elutriant, to wherein methyl alcohol evaporate to dryness, add isopyknic methylene dichloride and carry out liquid-liquid extraction as extraction solvent, collect dichloromethane layer, evaporate to dryness obtains the 1.43g macroporous resin column and separates crude product, analyzes through HPLC to contain taxol 1.2% (1.717), contain crust card booth VI and be about 40% (53.9g); The taxol yield is 95%; Crust card booth VI yield, 98%.
(2) alumina chromatographic column separating step: getting particle diameter is 100 purpose chromatography alumina packings, becoming aspect ratio with wet-filling is the chromatography column of 15:1, above-mentioned 143g macroporous resin column is separated methyl alcohol and the methylene dichloride mixed solution dissolving of crude product with volume ratio 2:98, be splined on the alumina column, the mass ratio that macroporous resin column is separated crude product and aluminum oxide is 1/80; Earlier use methylene dichloride: the methyl alcohol volume ratio is 2 column volumes of elutriant wash-out of 98:2, obtains the flow point of rich taxol; Use methylene dichloride again: the methyl alcohol volume ratio is the elutriant wash-out of 90:10, obtains to be rich in the flow point of many acetyl Taxan; Chromatography column pressure is 15bar, and flow velocity is 3ml/min, and 2 part flow points of the above-mentioned collection of evaporated under reduced pressure detect in conjunction with efficient liquid-phase chromatography method with thin-layer chromatography respectively, obtains to contain the crude product 3.399g of taxol 49.5%; The crude product 62.445g that contains crust card booth VI82%;
(3) hydrolysis reaction step: contain interpolation acetonitrile 300ml in the crude product that clings to card booth VI to above-mentioned 62.445g, calcium hydroxide suspension 200ml, acetonitrile accounts for 60% of overall solution volume, pH is 11, and 100r/min rotating speed stirring reaction is behind about 45min, detect nothing crust card booth VI in the Indicator Reaction liquid with the TLC method, with finishing reaction, whole solution to be filtered, filtrate being is rich in four and gone the acetyl crust to block the solution of booth VI;
(4) the Solid-Phase Extraction step of polyhydroxy taxone: the pH value of above-mentioned polyhydroxy taxone solution is transferred to 6 with hydrochloric acid, adding the particle diameter of handling well is 30～60 purpose chromatography polyamide resin filler powder, stirred 4 hours, stop to stir when removing acetyl crust card booth VI with nothing four in the thin-layer chromatography method test solution, filter, with the polymeric amide polymeric adsorbent diameter of packing into is in the 1.4cm glass chromatography column, and the height of filler is 15:1 with the ratio of column diameter; Every gram dried resin volume containing the sample is 50mg, and elution flow rate is 2BV.h
-1
(5) elution step of polyhydroxy taxone: with pH is 2.5 aqueous citric acid solution washing polyamide column, remove wherein pigment and alkaloids impurity, it with the volumetric concentration of 2 times of column volumes 40% methanol aqueous solution wash-out, collect the wash-out flow point, reclaim wherein methyl alcohol, in raffinate, add isopyknic methylene dichloride, fully concussion back extraction, dichloromethane layer is separated, the evaporate to dryness organic solvent, obtain the solid matter of 26.41g, analyzing four through HPLC, to remove the content of acetyl crust card booth VI be 85%, and transformation efficiency is 58.5%, separation yield is 98%;
(6) purification step of taxol: with the crude product dissolve with methanol of the 3.399g of above-mentioned acquisition (HPLC analyzes and contains taxol 49.5%) taxol, at pressure is under the 30bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 4:1, collect positive component, extremely do in 40 ℃ of underpressure distillation, the adding volume percent is 90% methyl alcohol hexane solution 100ml, 35 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 4 ℃ of freezing and crystallizings, and obtaining the 1.353g crystal purity is 99.5% taxol elaboration;
(7) purification step of polyhydroxy taxone: four of the 26.41g85% of above-mentioned acquisition is removed acetyl crust card booth VI crude product dissolve with methanol, at pressure is under the 20bar, prepare the anti-phase liquid phase chromatography column separation of pressure in the type with C18, volume ratio with methyl alcohol and water is the moving phase wash-out of 3:2, collect positive component, extremely do in 40 ℃ of underpressure distillation, the adding volume percent is 70% methanol ethyl acetate 4ml, 40 ℃ of fully dissolvings down, filtered while hot, filtrate is placed into 6 ℃ of freezing and crystallizings, and the crystal four that obtains 20.653g98% removes acetyl crust card booth VI, and Taxan utilization of resources degree is 20 (doubly).
The analytical procedure of polyhydroxy taxone: HPLC/MS and NMR structural confirmation, quantitative with HPLC, with TLC method coarse analysis.
The calculating of transformation efficiency: (actual output/theoretical yield) * 100%;
The calculating of Taxan utilization of resources degree:
Taxan utilization of resources degree=(A * B)/(C * D);
Wherein, A four goes acetyl crust card booth VI (or four go acetyl Yunnan peaceful) actual output in the step (7); B is a taxol relative molecular mass 853; C is a taxol actual output in the step (6); D four removes acetyl crust card booth VI (or four go acetyl Yunnan peaceful) relative molecular mass.
Claims (7)
1. method for preparing polyhydroxy taxone and taxol comprises:
(1) resin column separating step: the methyl alcohol or the ethanol extract of Chinese yew tissue or cell culture are loaded on the macroporous resin column, with the aqueous solution flushing that contains methyl alcohol 20%～40%, remove water-soluble impurity or pigment, again with the aqueous solution flushing that contains methyl alcohol 50%～80%, collect the elutriant of 3 times of column volumes, with the methyl alcohol evaporate to dryness in the elutriant, add extraction solvent isopyknic and that water is immiscible and carry out liquid-liquid extraction, collect organic solvent layer, evaporate to dryness, obtain macroporous resin column and separate crude product, described per-cent is volume percent;
(2) alumina chromatographic column separating step: separate crude product with methylene dichloride mixed solution dissolving macroporous resin column with methyl alcohol, be splined on the alumina column, the mass ratio that macroporous resin column is separated crude product and aluminum oxide is 1/60～1/100, carry out the mesolow normal-phase chromatography, with methyl alcohol and methylene dichloride mixed solution wash-out, thin-layer chromatography detects flow point in conjunction with efficient liquid-phase chromatography method, the flow point of many acetyl Taxan is rich in acquisition, and the flow point that is rich in taxol, the evaporated under reduced pressure solvent obtains to contain many acetyl Taxan crude product and taxol crude product;
(3) hydrolysis reaction step: in containing many acetyl Taxan crude product, add polar organic solvent, add basic solution or suspension again, making whole pH value of solution value is 9～11, polar organic solvent accounts for 30%～60% of whole liquor capacity, stirring reaction 0.5～1 hour, with in the thin-layer chromatography method detection reaction liquid during no many acetyl Taxan, reaction is finished, whole solution is filtered, and filtrate being is rich in the polyhydroxy taxone solution of polyhydroxy taxone;
(4) the Solid-Phase Extraction step of polyhydroxy taxone: the pH value of polyhydroxy taxone solution is transferred to 3.5～6 with hydrochloric acid, add the polymeric amide polymeric adsorbent of handling well, stirred 3～5 hours, stir with stopping during no polyhydroxy taxone in the thin-layer chromatography method test solution, filter, the polymeric amide polymeric adsorbent is packed in the glass chromatography column;
(5) elution step of polyhydroxy taxone: with the pH value is 2.5～4.5 aqueous citric acid solution washing polyamide column, removes wherein pigment and alkaloids impurity; Volumetric concentration with 2 times of column volumes is the methanol aqueous solution wash-out of 40%-60%, collect the wash-out flow point, reclaim alcohol wherein, in raffinate, add isopyknic methylene dichloride, fully concussion back extraction, organic solvent layer is separated, and the solid matter of evaporate to dryness organic solvent gained is and contains the polyhydroxy taxone crude product;
(6) purification step of taxol: with the taxol crude product that step (2) obtains, press anti-phase liquid phase chromatography column to separate in using, recrystallize obtains purity and separates product near the 99.5% taxol degree of depth;
(7) purification step of polyhydroxy taxone: contain the polyhydroxy taxone crude product with what obtain in the step (5), with in press anti-phase liquid phase chromatography column to separate, recrystallize obtains purity and separates product greater than the 98% polyhydroxy taxone degree of depth.
2. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the described resin column separating step, the macroporous resin of filling in the described macroporous resin column is nonpolar macroporous vinylbenzene polymeric adsorbent; Described extraction solvent is a kind of in chloroform, methylene dichloride, the ethyl acetate.
3. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the described alumina chromatographic column separating step, described alumina column filler is the chromatography alumina packing, particle diameter is 100 orders, it is the chromatography column of 15:1～20:1 that wet-filling becomes aspect ratio, use 2 column volumes of elutriant wash-out of volume ratio methylene dichloride: methyl alcohol=97:3～99:1, use 2 column volumes of elutriant wash-out of volume ratio methylene dichloride: methyl alcohol=95:5～90:10, chromatography column pressure is 5～20bar, flow velocity is 1～5ml/min, with the concentration of high-performance liquid chromatography method elutriant.
4. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the described hydrolysis reaction step, described polar organic solvent is a kind of in acetone, acetonitrile, the methyl alcohol; The basic solution that adds is mass percent 10%～20% wet chemical or aqueous sodium carbonate, and the alkaline suspension of adding is mass percent 10%～20% a calcium hydroxide suspension; Stir speed (S.S.) is 120r/min～60r/min.
5. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the Solid-Phase Extraction step of described polyhydroxy taxone, described polymeric amide polymeric adsorbent is that particle diameter is 30～60 purpose chromatography polyamide resin filler powder, wet-filling is the glass column of 0.6cm～1.5cm to diameter, and the height of filler is 10: 1～20: 1 with the ratio of column diameter; Every gram dried resin applied sample amount is 10mg～50mg, and elution flow rate is 2BVh
-1
6. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the purification step of described taxol,
Press anti-phase liquid phase chromatography column separating process to be in described: with taxol crude product dissolve with methanol, last C18 reverse phase filler, in 20～40bar pressure range, use volume ratio methyl alcohol: water is 4: 1 wash-outs, collects positive component, extremely does in 30 ℃～50 ℃ underpressure distillation;
Described crystallisation process is: to the positive component of reversed phase chromatography evaporate to dryness, add volumetric concentration again and be 80%～95% methyl alcohol hexane solution, 30 ℃～40 ℃ fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃～6 ℃ freezing and crystallizings, and crystal is purity and separates product near 99.5% the taxol degree of depth.
7. the method for preparing polyhydroxy taxone and taxol as claimed in claim 1 is characterized in that:
In the purification step of described polyhydroxy taxone,
Press anti-phase liquid phase chromatography column separating process to be in described: with polyhydroxy taxone crude product dissolve with methanol, last C18 reverse phase filler in 20～40bar pressure range, is used volume ratio methyl alcohol: water=3: 2 wash-outs, collect positive component, extremely do in 30 ℃～50 ℃ underpressure distillation;
Described crystallisation process is: to the positive component of reversed phase chromatography evaporate to dryness, add volumetric concentration again and be 70%～90% methanol ethyl acetate solution, 30 ℃～40 ℃ fully dissolvings down, filtered while hot, filtrate is placed into 0 ℃～6 ℃ freezing and crystallizings, and crystal is purity and separates product greater than 98% the polyhydroxy taxone degree of depth.
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| CN103804325A (en) * | 2012-11-15 | 2014-05-21 | 刘胜远 | Method for separating and purifying 10-deacetyltaxol from 10-deacetyltaxol-containing extract |
| CN104230858B (en) * | 2014-08-08 | 2016-04-20 | 中山大学 | A kind of method of separating and purifying taxol from Ramulus et folium taxi cuspidatae or bark |
| CN113717131B (en) * | 2021-08-27 | 2023-09-22 | 常熟纳微生物科技有限公司 | Separation and purification method of paclitaxel |
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| US5412116A (en) * | 1992-11-06 | 1995-05-02 | Hauser Chemical Research, Inc. | Oxidation of glycoside substituted taxanes to taxol or taxol precursors and new taxane compounds formed as intermediates |
| CN1534032A (en) * | 2004-02-18 | 2004-10-06 | 东北林业大学 | Method of high effect preparing rhoxadunol |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5412116A (en) * | 1992-11-06 | 1995-05-02 | Hauser Chemical Research, Inc. | Oxidation of glycoside substituted taxanes to taxol or taxol precursors and new taxane compounds formed as intermediates |
| CN1534032A (en) * | 2004-02-18 | 2004-10-06 | 东北林业大学 | Method of high effect preparing rhoxadunol |
Non-Patent Citations (1)
| Title |
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| Sheng-Hong Li,et al.Novel taxoids from the Chinese yew Taxus yunnanensis.《Tetrahedron》.2003,第59卷37-45. * |
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