CN1244566C - Method of preparing taxadol from leaf and twing of planted taxus chinensis - Google Patents
Method of preparing taxadol from leaf and twing of planted taxus chinensis Download PDFInfo
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- CN1244566C CN1244566C CN 01131862 CN01131862A CN1244566C CN 1244566 C CN1244566 C CN 1244566C CN 01131862 CN01131862 CN 01131862 CN 01131862 A CN01131862 A CN 01131862A CN 1244566 C CN1244566 C CN 1244566C
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Abstract
The present invention relates to a method for preparing taxol from planted yew leaves and branches. The leaves and the branches of yews which are planted for 2 to 4 years are used; after the branches the diameter of which is smaller than 0.5cm are sheared, the leaves and the branches are crushed and soaked by 5% to 95% of ethanol for 24 to 48 hours; after the filtration is carried out, waste residues are removed; after filter liquor is concentrated, the dispersion extraction is carried out by 1 to 1 of butyl acetate and water; 1% of taxol crude raw materials are obtained by concentrating and drying butyl acetate extraction liquid; the gradient elution of ethyl acetate and cyclohexane is carried out by the 1% of taxol raw materials and normal pressure positive phase silica gel column chromatography, and eluate is collected piecewise; cut fraction the taxol content of which is from 10% to 30% is separated by reversed phase C#-[18] high pressure liquid phase chromatography after being concentrated, and taxol the purity of which is more than 99.5% is obtained. The method has the advantages of simple manufacturing process, low cost, and no nontoxic reagents or solvents, and can not destroy natural resources. The present invention is favorable to ecological protection. The method is especially suitable for producing taxol by adopting planted yews as raw materials.
Description
Technical field
The present invention relates to a kind of method that from plantation Ramulus et folium taxi cuspidatae leaf branch, prepares taxol.
Background technology
The present invention is a kind of method for preparing taxol from plantation Ramulus et folium taxi cuspidatae leaf branch.
Taxol is a kind of natural organic-compound of finding in the Ramulus et folium taxi cuspidatae seeds, and it has strong lethal effect to mammary cancer, lung cancer with from the sick cell of blood etc.(J.Amer.Chem.Soc., 1971,93,2325) reported first such as Wani in 1971 and Wall the separation and the physicochemical characteristics of taxol, and determined the antitumour activity that it is good, caused that people pay close attention to widely.Nineteen eighty-three U.S. food drug control administration (FDA) approval is classified taxol as treatment mammary cancer and uterus carcinoma medicine, enters the I clinical trial phase.Based on many clinical trial phases result, come into the market as treatment advanced ovarian cancer medicine by the U.S. FDA official approval the end of the year 1992, be approved for treatment mammary cancer the end of the year 1993 again.Because the taxol side effect is little, drug effect is remarkable, is called as the PTS of hottest point behind Zorubicin and cis-platinum, and demand increases sharply.
The source of natural Japanese yew alcohol is very limited, and it is mainly derived from bark of Ramulus et folium taxi cuspidatae.Chinese yew genus plants, the whole world has 11 kinds, in state-owned 4 kinds and 1 subspecies, be respectively taxus chinensis in northeast (Taxus cuspid-ata Sieb.Et Zucc.), southerm yew (Taxus mairei), taxusyunnanensis (Taxus yunna-nensis), Xizang Taxus chinensis (Taxus willichiana Zucc.) and beautiful Ramulus et folium taxi cuspidatae (Taxus chinensis).The growth of Ramulus et folium taxi cuspidatae seeds is extremely slow, and the century-old veteran of growth generally can only strip the 25kg bark, provides 10,000 2 thousand routine carninomatosis patients to use if extract the pure taxol of 1kg, is equivalent to the bark of needs 30,000 strain Chinese yews.In order to obtain taxol, carried out methods such as the Chinese yew of planting short strongization, tissue culture, semi-synthetic and complete synthesis taxol abroad and obtained the taxol raw material.The U.S. uses Hybrid Taxus x media (Taxus media cv.Hicksii), plant back its branches and leaves of 3-5 and extract taxol and semi-synthetic precursor thereof, (the Gragg etc. that supply raw materials in a large number of Shi Guibao company when taxol manufacturer hundred, J.Natural Prod., 1993,56,1657-1668), but this seeds poor growth.The output that tissue culture method obtains is very little, and nutrient solution costs an arm and a leg, and complete synthesis yield is very little, and cost is huge, is difficult to commercial applications.Semisynthesis still needs isolated taxol concomitant product of natural Ramulus et folium taxi cuspidatae such as 10-deacetylate crust card fourth III etc. as initial feed.In order to protect natural taxus resource, safeguard ecotope, extracting taxol with growth plantation Ramulus et folium taxi cuspidatae fast, that active constituent content is higher is valid approach the most.
Recently, China in Shandong, ground such as northeast, Jiangxi, Fujian planted a certain amount of Ramulus et folium taxi cuspidatae woods, southerm yew quantity maximum with the plantation of area, Sanming City, Fujian Province, planting southerm yew amount about 30 ten thousand strains of plantation more than 3 years, plantation about 1,300,000 strains more than 3 years, calendar year 2001 has been planted 1,500,000 strains again.In this area, the southerm yew growth is very fast, and 2 years living southerm yew branches and leaves dry weights of every strain can reach more than 1.2 kilograms, and content of taxol has very high utility value between 0.016%-0.025%.
The separation and purification of extracting taxol for Ramulus et folium taxi cuspidatae mostly is uses 95% methanol solution leaching bark extracting solution, extracting solution to carry out chloroform extraction after the degreasing earlier; Extraction liquid water-dispersion, chloroform layer water layer separate the back and concentrate, and obtain taxol and concomitant product thereof through column chromatography.Column chromatography method commonly used is through needs column chromatography repeatedly, and how filler separately fills not stationary chromatographic post, the sepn process complexity, and toxicity such as solvent, leacheate are big, cause serious pollution.
Senilh (J.Natural Prod., 1984,47,131) etc. extract taxol from European China fir (Taxul bacata) and use earlier the alcohol extraction bark, after concentrating, distribute in water and methylene dichloride, separate through " filtration " chromatography; Silica gel column chromatography separates; Then through the aluminum oxide chromatographic separation; Separate through the silica gel medium pressure column chromatography again; Adopt the preparation high pressure liquid chromatography to separate at last.Miller (J.Org.Chem., 1981,46,1469) is with Taxus wallichiana tree root, trunk and leaf, through methanol extraction, be concentrated into solid, distributes in water and hexane, use chloroform extraction, and is concentrated then, separates with silicagel column; The secondary silica gel column chromatography separates again; Distribute through adverse current, second adverse current distributes; Obtain 184 components with the HPLC separation and purification at last, the taxol purity about 99.1% that makes.Whole extraction purge process mainly contains eight steps, comprising twice normal-phase chromatography column chromatography.
Caster etc. (usp.5,440,055,1995) utilize supercritical methanol technology really to connect and handle Chinese fir leaf tissue.Because the Chinese fir leaf is a renewable resources, utilize leaf to protecting rare seeds more useful.This method is used N
2O, CO
2, C
3H
8Or CHClF
2Deng as supercritical gas, handle the China fir leaf that drying is pulverized, leacheate extracts and C in system
18The reverse chromatograms post separates, and obtains pure product of paclitaxel, and this method is handled and is about each 30mg, and the taxol purity that obtains at last is about 99.4%.
Polysciences Inc. is isolation of taxol from the bark of Ramulus et folium taxi cuspidatae of the Pacific Ocean.Elder generation's methyl alcohol or alcohol extraction bark concentrate behind the hybrid extraction and remove most of solvent; The concentrated solution dichloromethane extraction, extraction matter concentrate drying is to powder; Powder dissolves with propyl alcohol and ligroin (1: 1), and indissolvable component removes by filter; The filtrate of not containing taxol concentrates, and is dissolved in 30% propyl alcohol filtration naphtha, filters through the Florisil post; Effusive taxol component crystallization purifying secondary from post; A nearly step of crystalline taxol separates in silicagel column, and product Cephalomannine the most similar in this step is separated from taxol; Effusive taxol recrystallize secondary from post; Unsegregated mixture and other solute, circular treatment obtains taxol.
(usp.5,380,916 such as Rao, 1992) get pure taxol after the most difficult isolating impurity composition Cephalomannine of use ozonize separates, leacheate uses ethanol, chloroform etc., after last reverse preparative chromatography is separated thick product, still contains a certain amount of Cephalomannine separately that is difficult to.After the mixture of taxol and Cephalomannine feeds ozone oxidation, separate to obtain the higher taxol of purity through reverse preparative chromatography, this method need use that residual ozone and recrystallize obtain pure product of paclitaxel in the dimethyl sulphide treatment soln.Kingston etc. (J.Natural Prod., 1992,55,259) also use oxygenant O
sO
4The mixture of oxidation taxol and Cephalomannine.Taxol does not react with perosmic anhydride, and the two keys of the C-13 side chain terminal of Cephalomannine are oxidized, easily separate with taxol.
Durand (usp.5,723,635,1998) utilizes centrifugal partition chromatograph method separating and purifying taxol, uses a C
18Reverse-phase chromatographic column is separating and purifying taxol from crude product well, and the primary treatment amount is bigger.Mainly contain four steps: under the preparative column scale, handle taxol bulk processing thing with reverse-phase chromatography; Wash-out taxol and other product from the sorbent material; In the pillar of wash-out, reclaim taxol and resemblance; Use ozone purifying final product, thereby separate the concomitant product of removing taxol.
Use the fresh branches and leaves of Ramulus et folium taxi cuspidatae, also can directly extract taxol as raw material.Nalr (usp.5,279,949,1994) uses the normal phase silica gel column chromatography method from inferior (Taxus media cv.Hicksii) fresh Chinese fir separate tissue taxol and the homologue thereof in graceful ground.The fresh Chinese fir tissue mixture extraction Taxanes of second alcohol and water, extraction matter concentrates removes most of organic solvent, water soluble ingredient is centrifugal to be separated with the solid precipitation that contains taxol, filtrate filtered is with the activated carbon decolorizing diatomite filtration, filtrate evaporates into dried, vacuum was descended silica gel chromatographic column, and liquid phase separation obtains product.After perhaps taking off ethanol under the filtrate vacuum with diatomite filtration, use ethyl acetate extraction, extraction liquid concentrates, and obtains product with the vacuum liquid phase chromatography through the silicagel column purifying.Nalr (usp.5,478,736,1995) by changing extraction agent, promptly with methyl alcohol and the fresh Chinese fir tissue of acetone extract, can obtain higher taxol output again.The secondary column chromatography is separated, and the low pressure silica gel column chromatography separates thick taxol component, reverse-phase chromatography final purification.
Directly utilize reversed-phase preparative chromatography, the method for separating and purifying taxol is comparatively numerous and diverse.Rao (usp.5,380,916,1995) concentrated with the dried bark of 95% alcohol extraction 3-5 time, ethanolic extract, with 1: 1 chloroform dispersion extraction.Chloroform separates, and uses the chloroform extraction water again 2 times, chloroform evaporated is concentrated into dried, forms solid at C
18With acetonitrile and water gradient elution, segmentation is accepted and is contained the higher cut of taxol and concentrate on the filler chromatography column, and after the hot water dilution, reversed-phase column regathers cut, obtains highly purified taxol and concomitant product product thereof through recrystallization.
Use bigger chloroform, the methylene dichloride equal solvent of toxicity in above-mentioned many methods mostly, sepn process comprises crystallization process, oxidation impurities method, repeatedly normal phase chromatography, repeatedly reverse phase liquid chromatography, centrifugal partition chromatograph method and supercritical extraction etc., it is tediously long to cross post, and the yield of taxol that reaches 99% above purity is lower.Similar performance (Chen Jianmin etc., the J.Liq.Chrom.﹠amp of taxol (a) and Cephalomannine (b) and 7-table-10-deacetylate taxol (c); R.T., 2000,23,2499), its structure is as follows respectively:
(a) taxol (b) Cephalomannine
(c) 7-table-10-deacetylate taxol
At above three kinds of compounds, set up a kind of comparatively easy, method of being easy to separate taxol concomitant product b, c, be the key that taxol prepares suitability for industrialized production.
Summary of the invention
The present invention aims to provide a kind of method for preparing taxol from plantation Ramulus et folium taxi cuspidatae leaf branch, and this method technology is simple, is easy to separate the taxol concomitant product, the taxol purity height of gained.
The present invention is based on such design concept: branch, the leaf of southerm yew, taxus chinensis in northeast, taxusyunnanensis and Taxus x media with plantation is raw material, through soaking the pre-treatment of extraction, n-butyl acetate extraction, obtain high-purity taxol with normal pressure purification on normal-phase silica gel column chromatography chromatography and high pressure liquid chromatography partition method then.
The technical solution adopted in the present invention is: a kind of method for preparing taxol from plantation Ramulus et folium taxi cuspidatae leaf branch in turn includes the following steps:
(1) will plant the leaf of the Ramulus et folium taxi cuspidatae more than 2 years of having grown and diameter after the branch below the 0.5cm is pulverized, at room temperature use 75%～95% alcohol immersion 24～72 hours, it is that 4: 1～1: 4 butylacetate and water extracts with volume ratio that filtrate is concentrated the back;
(2) n-butyl acetate extraction liquid is concentrated, obtain content of taxol at the taxol concentrated solution more than 1%;
(3) concentrated solution is mixed with dry sample with silica gel, dry sample is placed the upper end of the chromatographic column silica gel of filling gel, the silica gel chromatography column length is 80～1800cm, and column diameter is 8～220cm, and the silica gel particle diameter is 10-50 μ m, and surface-area is 450m
2/ g;
(4) will fill the chromatographic column leacheate gradient elution of dry sample and silica gel, leacheate is divided into 5 gradients, addition sequence is respectively hexanaphthene, hexanaphthene and ethyl acetate are pressed 1: 4 proportioning by mixed solution, hexanaphthene and the ethyl acetate of 1: 1 proportioning by mixed solution, hexanaphthene and the ethyl acetate of 4: 1 proportionings mixed solution, ethyl acetate, Fractional Collections cut after the leacheate outflow chromatographic column, by section the leacheate cut of content of taxol between 10%～30% put together, through concentrated, dry, make intermediates;
(5) content of taxol is lower than 10% taxol leacheate cut centralized collection, after concentrating, repeating step (3), step (4);
(6) intermediates with step (4) gained dissolve with acetonitrile, separate with high pressure liquid chromatography, and chromatographic column is C
18Silica filler, moving phase are acetonitrile and water, and the moving phase that contains taxol after then will separating is evaporated, drying, obtain purity and reach taxol more than 99.5%.
The preparation method of dry sample obtains adsorbing the silica gel dry sample of 1% taxol for the silica gel of 10-50 μ m and content of taxol with volume are mixed at the taxol concentrated solution more than 1% behind the evaporate to dryness in the step (3).
The weight ratio of dry sample and silica gel is 1: 2～1: 20 in the middle chromatographic column of step (3).
Described used silica gel is treated reusable, and method is 280～380 ℃ of following roastings 24～36 hours.
Solvent in described leacheate and the moving phase is through reusable after the rectifying.
Ramulus et folium taxi cuspidatae in the step (1) is southerm yew, taxusyunnanensis, taxus chinensis in northeast, Xizang Taxus chinensis, beautiful Ramulus et folium taxi cuspidatae or Taxus x media.
The branches and leaves of Ramulus et folium taxi cuspidatae can be fresh in the step (1).
The branches and leaves of Ramulus et folium taxi cuspidatae are through natural airing or 30～60 ℃ of heat dryings in the cool in the step (1).
The procedure that the present invention prepares taxol as shown in Figure 1.Technological process shown in Figure 1 is divided into three processes.First process is for utilizing the fresh or dry pre-treatment process of Ramulus et folium taxi cuspidatae of plantation, be about to fresh or exsiccant leaf, several pulverizing after, at 20～40 ℃ with 80%～95% alcohol immersion 24～72 hours, raw material and the alcoholic acid volume ratio that feeds intake is 1: 1～1: 5, and soak solution filters removes filter residue, and it is dried that filtrate places Rotary Evaporators or concentration tank to be concentrated into, form sap green or brown ceramic powder, after the butylacetate dissolving, add water-dispersion with volume, upper strata butylacetate solution is extraction liquid.0.5 after～2 hours, butylacetate layer and water layer are separated, get butylacetate layer evaporating, concentrating and drying and obtain sap green or dark-brown powder.The content of taxol of this process of preparing is in 1% above (see figure 2).
Second process is the powder that butylacetate layer evaporating, concentrating and drying obtains, and uses acetic acid ethyl dissolution, is mixed with dry sample with silica gel.Dry sample places the mixed solvent gradient elution of silicagel column upper end with hexanaphthene and ethyl acetate, accepts the cut 1-15 that obtains equal volume with the container of equal volume.Be faint yellow gluey thing behind cut 1,2 evaporates to dryness, detect less than taxol; It behind the cut 3-5 evaporate to dryness the little colloidal solid of light brown; the taxol that wherein contains minute quantity in the cut 5; among the cut 6-8; contain more 7-table-10-deacetylate taxol and 7-table-taxol; wherein content of taxol is 0.6% in the cut 6, and cut 7 contains taxol 3.5% approximately, contains taxol 7.3% in the cut 8 approximately; behind the cut 9-13 evaporate to dryness, be yellow or green-yellow pressed powder.Wherein cut 9 contains taxol and is about 8.8%, and content of taxol is between 10.0% ~ 30.0% among the cut 10-13, and the HPLC collection of illustrative plates of cut 12 as shown in Figure 3.As seen from Figure 3, by just being separated, 7-table-10-deacetylate taxol is partly removed, and content of taxol is 27.5%.Cephalomannine exceeds about 3～5% than the content of taxol in the cut 13; Be light green (using fresh branches and leaves) or pale brown look pressed powder (using dry branches and leaves) behind the cut 14-15 evaporate to dryness, content of taxol is between 7.5～0.8%.The cut merging evaporate to dryness of content of taxol between 10.0 ~ 30.0% dissolves with acetonitrile in this process, and the 3rd process of carrying out is further purified taxol.
In the 3rd process, using performance liquid chromatographic column, inject 27.5% taxol, 10 gram acetonitrile solutions, as moving phase, is 25% from 0min to 5min acetonitrile ratio with acetonitrile and water; Rise to 45% from 5min to 35min acetonitrile ratio from 25%, and keep 30min; Rise to 80% from 65min to 120min acetonitrile ratio from 45%, and keep 30min.Use above-mentioned gradient that the taxol acetonitrile solution is carried out separation and purification, preparation HPLC process as shown in Figure 4.Branch is accepted for three sections and is obtained cut 16-18.Retention time the moving phase 0-80min between of cut 16 for collecting, cut 17 is the moving phase of 80-92min, cut 18 is the moving phase of 92-150min.Cut 16 evaporate to dryness post analysis are indicated as impurity composition, contain the more amount Cephalomannine; Cut 17 is taxol, and purity is up to 99.6%, and HPLC analyzes collection of illustrative plates as shown in Figure 5; More impurity behind a small amount of taxol and the taxol peak is arranged in the cut 18, be mainly 7-table-10-deacetylate taxol, content of taxol is below 3.0%.In this process, cut 17 is vacuum, careful evaporate to dryness below 40 ℃ in Rotary Evaporators, can obtain 99.6% high-purity taxol.This product is the white solid powder, and specific rotation is-56 ℃ (in methanol solutions), and fusing point is 236-238 ℃, and the checking of 400MZ nuclear magnetic resonance map is identical with the taxol standard model, liquid one matter coupling stratographic analysis M/Z
+Be 853.6.This product is consistent with standard model retention time in HPLC analyzes.The Fourier infrared spectrum of product as shown in Figure 6.
The inventive method compares with existing taxol extraction process that step is few, technology is simple, production cost is low, helps the industrialized production of taxol; Do not use the bigger reagent of toxicity, solvent, compliance with environmental protection requirements; A yield height of taxol, purity can reach more than 99.5%.
Description of drawings
Fig. 1 is the preparation flow figure of taxol;
Fig. 2 is the high-efficient liquid phase chromatogram that obtains cut (content of taxol is 1.32%) from branches and leaves through separation;
Fig. 3 is the high-efficient liquid phase chromatogram of cut 12 (content of taxol is 27.5%);
Fig. 4 is the preparative high performance liquid chromatography figure of separation purity 27.5% taxol;
Fig. 5 is the high-efficient liquid phase chromatogram of cut 17 (product);
Fig. 6 is the Fourier infrared spectrum figure of product.
Embodiment
(1) gather the fresh southerm yew leaf in 2 years of plantation and diameter less than the branch 1kg of 0.5cm, be ground into the viscosity pasty state with crusher, be transferred in the 5L beaker, add 95% ethanol 3L, stir, normal temperature was placed 48 hours.After 48 hours, use the sand core funnel suction filtration, remove waste residue, filtrate is concentrated into viscous fluid, this moment, this viscous fluid was a deep green, and volume is about 90ml.Add the 2L butylacetate to this solution, mix the back and add 2L water, stir 30min, place about 20min, form upper strata light green clear liquid and lower floor's deep green Colloidal fluid.
(2) after supernatant liquid separates with the lower floor Colloidal fluid, it is dried to place Rotary Evaporators to be evaporated to the supernatant liquid solvent, forms the sap green powder, and weight is 14.3 grams, content of taxol is (to see high-efficient liquid phase chromatogram shown in Figure 2) more than 1.32% after measured, does not contain taxol in lower floor's colloidal solution.
(3) take by weighing powder 1kg, the silica gel with adding 2kg behind the acetic acid ethyl dissolution obtains the silica gel dry sample behind the rotary evaporation.
(4) dry sample is inserted the glass chromatography column upper end that 10kg silica gel is housed, the silica gel chromatography column length is 80～1800cm, and column diameter is 8～220cm, and the silica gel particle diameter is 10～50 μ m, and surface-area is 450m
2/ g; Use hexanaphthene+ethyl acetate (4: 1) of 20L hexanaphthene, 30L, hexanaphthene+ethyl acetate (1: 1) of 30L, hexanaphthene+ethyl acetate (1: 4) and the drip washing of 20L ethyl acetate of 50L respectively, every 10L collects a cut, accepts 15 cuts altogether.
Evaporate to dryness 1-5 cut, weight are about 120 grams, for light brown jelly or little viscose glue solid, wherein only contain the minute quantity taxol in the cut 5, with its whole removals; Contain more 7-table-10-deacetylate taxol and 7-table-taxol among the cut 6-8, wherein content of taxol is 0.6% in the cut 6, contain taxol 3.5% in the cut 7 approximately, contain taxol 7.3% in the cut 8 approximately, cut 9 contains taxol and is about 8.8%, to get red-brown powder 225 grams behind the cut 6-9 merging evaporate to dryness, content of taxol is 6.8%; Content of taxol is between 10.0%-30.0% among the cut 10-13, the HPLC collection of illustrative plates of cut 12 as shown in Figure 3, as seen by just being separated, 7-table-10-deacetylate taxol is partly removed, content of taxol is 27.5%; Behind the cut 14-15 evaporate to dryness is the light green pressed powder, and content of taxol is between 7.5～0.8%.With cut 10-13 centralized collection, through concentrate, dry, obtain the taxol average content and be 27.5% powder, this part weight is total up to 641 grams.
(5) powder collection behind cut 6-9 and the cut 14-15 evaporate to dryness is got up, repeat above-mentioned steps (3), step (4).
(6) powder (content of taxol is 27.5%) that obtains behind the cut 10-13 evaporate to dryness is taken by weighing 10 grams, be dissolved in the 100ml acetonitrile, separate with anti-phase preparative high-performance liquid chromatographic method, chromatographic column is C
18Silica filler, moving phase are acetonitrile and water.Inject 27.5% taxol acetonitrile solution, as moving phase, flow velocity is 140ml/min with acetonitrile and water.From 0min to 5min acetonitrile ratio is 25%; Rise to 45% from 5min to 35min acetonitrile ratio from 25%, and keep 30min; Rise to 80% from 65min to 120min acetonitrile ratio from 45%, and keep 30min.Use above-mentioned gradient that the taxol acetonitrile solution is carried out separation and purification, preparation HPLC process as shown in Figure 4.Before accepting 80min, the moving phase of 80-92min and 92-150min, obtain three cuts 16,17,18 respectively.Cut 16 evaporate to dryness post analysis are indicated as impurity composition, contain the more amount Cephalomannine; More impurity behind a small amount of taxol and the taxol peak is arranged in the cut 18, be mainly 7-table-10-deacetylate taxol, content of taxol is below 3.0%.Is that careful rotary evaporation removes moving phase in the Rotary Evaporators of 0.01MPA to cut 17 at 40 ℃, vacuum tightness, product 2.66 grams, HPLC measures and shows that taxol purity is 99.6% (see figure 5); The rate of recovery is about 96.3%.This product is the white solid powder, and specific rotation is-56 ° (in methanol solutions), and fusing point is 236-238 ℃, and the checking of 400MZ nuclear magnetic resonance map is identical with the taxol standard model, liquid-matter coupling stratographic analysis M/Z
+Be 853.6.The Fourier infrared spectrum of product is seen Fig. 6.Under the KBr substrate at the bottom of the taxol infrared absorption be respectively 2925,1724,1243,1070,712cm-1, be the infrared absorption peak of water at the infrared absorption peak of 3433cm-1.
Used butylacetate, ethyl acetate, hexanaphthene etc. are analytical pure in the above-mentioned steps, use after distilling.Acetonitrile is the HPLC level, and water is for to prepare hplc grade water through Millipore produced in USA.Day island proper Tianjin 1120 type high pressure liquid chromatographs, the C that chromatographic column is produced for Sweden Kromasil are used in the analysis of raw material and intermediates
18Silica gel chromatographic column, filler size are 5 μ m, 4.6 * 250mm, and the mensuration wavelength is 227nm, and moving phase is acetonitrile+water (40+60), and flow velocity is 1ml/min, and hand sampling, sample size are 10 μ m.U.S. Waters Breeze high pressure liquid chromatograph is used in the high-purity taxol analysis, the C that chromatographic column is produced for Sweden Kromasil
18Filler size is 5 μ m, 4.6 * 250mm chromatographic column, and analytical wavelengths is 227+2nm, and moving phase is acetonitrile+water (40+60), flow velocity is 1.0ml/min, automatic sampling, sample size are 10 μ m, the chromatographic column thermostat container, column temperature is 35 ℃, PI II computer control, computer operation, chromatogram software is Millium2000.The LCQAD-30000SPCL type LC/ (MS) that liquid one mass spectroscopy is produced in U.S. Finnigan company
nCarry out, the APCI/ESI source, liquid phase analysis is diode square row (PDA) monitor, Zorbax2.1 * 100mmC
18Chromatographic column, filler size are 5 μ m, and flow velocity is 0.3ml/min, and sample size is 5 μ m, and voltage is 15KV, and sweep limit is that M/Z is between 30～2000.The centrifugal freeze drier of KLG-II type.The 5L-20L Rotary Evaporators that Shanghai Shen Sheng company produces.The 21011V001R type Rotary Evaporators that pure product use U.S. Bush to produce.The super digital display thermostatted of CS501-SP, W22-2A automatic ultrasonic instrument, WKS-1A digital display fusing point instrument.The PrepLC 4000 type preparative high performance liquid chromatographies that U.S. Waters company produces, chromatogram software is Millium2000
32, preparative column is a stainless steel chromatogram post, it is maximum that to fill length be 370cm, diameter is 30cm, detections wavelength is 227 ± 2nm, acetonitrile and water mixed flow mutually, the acetonitrile ratio rises to 80% from 20%, the preparation manipulation time is about 160min at every turn.Fourier infrared spectrum is determined on the Avatar360 Fourier infrared spectrograph that U.S. Nicolet company produces and carries out, Digital PII robot calculator control spectrum software OmicZ.S.P. gathers Fourier infrared spectrum, and the ratio of sample and KBr is 1: 160.
Used silica gel was 280～550 ℃ of following roastings 24～36 hours, and it is identical to reuse effect.The silica gel that adopts this method to handle is reusable at least more than 12 times.
Solvent in leacheate and the moving phase is through reusable after the rectifying.
Embodiment 2, step (1), (2) can for:
(1) get the southerm yew cured leaf in 2 years of plantation and the diameter dried branch 1kg less than 0.5cm, be crushed to below the 1.2mm with crusher, remove big branches and leaves and be placed in the 5L beaker, add 90% ethanol 3L, stir, normal temperature was placed 72 hours.Stir 3-4 time in the put procedure, use the sand core funnel suction filtration after immersion is finished, remove waste residue, filtrate being concentrated into is the light green viscous fluid, volume is about 100ml.Add the 2L butylacetate to solution, mix the back and add 2L water, stir 30min, place about 20min, form upper strata nattierblue clear liquid and lower floor's deep green Colloidal fluid.
(2) upper strata liquid and subnatant are separated, upper strata liquid is positioned over is evaporated to driedly in the Rotary Evaporators, form the dark-brown powder, weight is 15.6 grams, and content of taxol is more than 1.27% after measured, does not contain taxol in lower floor's colloidal solution.
Step (3)～(6) are identical with embodiment 1.
Embodiment 3, step (1), (2) can for:
(1) get plantation growth more than 2 years the southerm yew cured leaf and diameter less than the dried branch 100kg of 0.2cm, pulverize with crusher and to be placed on 2m
3Extractor in, add 90% ethanol 500L then, stir soak at room temperature 24 hours.Stir with the built-in agitator of extractor in the immersion process.After immersion is finished, emit the solution (the core filter of dismounting is equipped with in the extractor bottom) in the extractor, use then with quadrat method and soak 3 times.The waste residue that more than obtains reclaims residual ethanolic soln in the waste residue by centrifugation, and with extractor in the ethanolic soln of emitting concentrate to be placed in the concentration tank and concentrate, be about 20L to volume.In the extractor, divide 3 flushing backs to concentrate, add about 250L butylacetate, mix the back and add 250L water, stirred 1 hour, place about 30min and form two-layer to this solution with about 20L butylacetate.
(2) emit lower floor's Colloidal fluid, upper strata liquid is evaporated to dried, form brown ceramic powder 1374 grams, content of taxol is more than 1.31% after measured.
Step (3)～(6) are identical with embodiment 1.
Claims (8)
1, a kind of method for preparing taxol from plantation Ramulus et folium taxi cuspidatae leaf branch is characterized in that in turn including the following steps:
(1) will plant the leaf of the Ramulus et folium taxi cuspidatae more than 2 years of having grown and diameter after the branch below the 0.5cm is pulverized, at room temperature use 75%～95% alcohol immersion 24～72 hours, it is that 4: 1～1: 4 butylacetate and water extracts with volume ratio that filtrate is concentrated the back;
(2) n-butyl acetate extraction liquid is concentrated, obtain content of taxol at the taxol concentrated solution more than 1%;
(3) concentrated solution is mixed with dry sample with silica gel, dry sample is placed the upper end of the chromatographic column silica gel of filling gel, the silica gel chromatography column length is 80～1800cm, and column diameter is 8～220cm, and the silica gel particle diameter is 10-50 μ m, and surface-area is 450m
2/ g;
(4) will fill the chromatographic column leacheate gradient elution of dry sample and silica gel, leacheate is divided into 5 gradients, addition sequence is respectively hexanaphthene, hexanaphthene and ethyl acetate are pressed 1: 4 proportioning by mixed solution, hexanaphthene and the ethyl acetate of 1: 1 proportioning by mixed solution, hexanaphthene and the ethyl acetate of 4: 1 proportionings mixed solution, ethyl acetate, Fractional Collections cut after the leacheate outflow chromatographic column, by section the leacheate cut of content of taxol between 10%～30% put together, through concentrated, dry, make intermediates;
(5) content of taxol is lower than 10% taxol leacheate cut centralized collection, after concentrating, repeating step (3), step (4);
(6) intermediates with step (4) gained dissolve with acetonitrile, separate with high pressure liquid chromatography, chromatographic column is the C18 silica filler, and moving phase is acetonitrile and water, the moving phase that contains taxol after then will separating is evaporated, drying, obtains purity and reaches taxol more than 99.5%.
2, method according to claim 1, it is characterized in that: the preparation method of dry sample obtains adsorbing the silica gel dry sample of 1% taxol for the silica gel of 10-50 μ m and content of taxol with volume are mixed at the taxol concentrated solution more than 1% behind the evaporate to dryness in the step (3).
3, method according to claim 1 and 2 is characterized in that: the weight ratio of dry sample and silica gel is 1: 2～1: 20 in the middle chromatographic column of step (3).
4, method according to claim 1 and 2 is characterized in that: used silica gel is treated reusable, and method is 280～380 ℃ of following roastings 24～36 hours.
5, method according to claim 1 and 2 is characterized in that: the solvent in leacheate and the moving phase is through reusable after the rectifying.
6, method according to claim 1 is characterized in that: the Ramulus et folium taxi cuspidatae in the step (1) is southerm yew, taxusyunnanensis, taxus chinensis in northeast, Xizang Taxus chinensis, beautiful Ramulus et folium taxi cuspidatae or Taxus x media.
7, according to claim 1 or 6 described methods, it is characterized in that: the branches and leaves of Ramulus et folium taxi cuspidatae can be fresh in the step (1).
8, according to claim 1 or 6 described methods, it is characterized in that: the branches and leaves of Ramulus et folium taxi cuspidatae are through natural airing or 30～60 ℃ of heat dryings in the cool in the step (1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01131862 CN1244566C (en) | 2001-12-18 | 2001-12-18 | Method of preparing taxadol from leaf and twing of planted taxus chinensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01131862 CN1244566C (en) | 2001-12-18 | 2001-12-18 | Method of preparing taxadol from leaf and twing of planted taxus chinensis |
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| Publication Number | Publication Date |
|---|---|
| CN1427002A CN1427002A (en) | 2003-07-02 |
| CN1244566C true CN1244566C (en) | 2006-03-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01131862 Expired - Lifetime CN1244566C (en) | 2001-12-18 | 2001-12-18 | Method of preparing taxadol from leaf and twing of planted taxus chinensis |
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| CN (1) | CN1244566C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103058961B (en) * | 2013-01-28 | 2015-04-29 | 普洱市红宝生物科技有限公司 | Method for extracting paclitaxel from taxus yunnanensis |
| CN104211667A (en) * | 2014-07-31 | 2014-12-17 | 大生祥(武汉）中医投资管理有限公司 | Plant extract applied in taxol preparation and preparation method thereof |
| CN105153077A (en) * | 2015-10-19 | 2015-12-16 | 丁玉琴 | Method for extracting paclitaxel from Taxus chinensis leaf |
| CN107137434B (en) * | 2017-06-22 | 2021-04-30 | 无锡紫杉药业有限公司 | Pharmaceutical composition, preparation method thereof and application thereof in preparation of coxsackie virus resistant medicine |
| CN113831305A (en) * | 2020-06-24 | 2021-12-24 | 北京创新通恒科技有限公司 | Separation equipment and process method for purifying high-purity paclitaxel from paclitaxel extract |
-
2001
- 2001-12-18 CN CN 01131862 patent/CN1244566C/en not_active Expired - Lifetime
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