CN101560197B - Method for extracting paclitaxel from artificially cultivated yew branches and leaves - Google Patents

Abstract

The invention relates to an extraction method of taxol from branches and leaves of artificially cultivated yew. A plurality of methods, such as planting of stocky yew, cultivating of tissue and semi-synthesis and total synthesis of taxol, have been developed home and abroad so as to obtain taxol raw material; however, the tissue cultivating method has low yield and high broth price; and the total synthesis has extremely low yield, worse therapeutic effect than natural-source taxol, and a high cost; thereby being difficult for commercial application. The invention uses branches and leaves of artificially cultivated taxus media, Yunnan taxus, southern taxus or northeastern taxus as raw materials; the raw materials are marinated by carbinol and extracted by petroleum oil and propyl acetate insequence; and normal-pressure normal-phase silica gel column chromatography or reverse-phase high-pressure liquid chromatography is used to obtain high-purity taxol with a purity being higher than 99 .6%. The preparation method has simple process, low cost, recyclable solvent and eluant; therefore, the method is applicable to industrial production and protects plant resource.

Description

Method for extracting paclitaxel from artificially cultivated yew branches and leaves

technical field

The content of the invention belongs to the technical field of preparation of biochemical products, and relates to a method for extracting paclitaxel from branches and leaves of yew artificially cultivated.

Background technique

Paclitaxel (taxol) is an antineoplastic drug with complex chemical structure and unique mechanism of action extracted from the bark, branches and leaves of Taxus (Taxaceae) plants. The sources of natural paclitaxel are very limited, mainly from yew or yew bark (Wani et al., J. Am. Chem. Soc. 1971, 93, 2325). The growth of yew tree species is extremely slow. Generally, a century-old tree can only peel 25kg of bark. If 1kg of pure taxol is extracted to provide 12,000 cancer patients, it is equivalent to the bark of 30,000 yew trees. . In order to obtain paclitaxel, methods such as planting dwarf yew trees, tissue culture, semi-synthesis and total synthesis of paclitaxel have been carried out at home and abroad to obtain paclitaxel raw materials. However, the yield obtained by the tissue culture method is very small, the culture medium is expensive, the total synthesis yield is very small, and the curative effect is not as good as that of natural source paclitaxel, the cost is high, and it is difficult to commercialize it. The semi-synthetic method still requires paclitaxel semi-synthetic precursor 10-deacetylbaccatin III isolated from natural yew as starting materials. In order to protect the natural taxus resources and maintain the ecological environment, it is the most effective way to extract paclitaxel from the artificially planted taxus with fast growth and higher active ingredient content (Gao Jinming, etc., paclitaxel resource development research, Journal of Northwest Forestry University, 1997 , 12(3): 94; Gao Jinming et al., The Structure-Activity Relationship of New Anticancer Drug Paclitaxel, Journal of Northwest Agricultural University, 1997, 25(5): 96; Gao Jinming et al., Taxol Resources, Biological Activity and Chemical Synthesis, World Forestry Research, 1997, 10(6):38; Gragg et al., J. Nat. Prod., 1993, 56, 1657; Kingston, Phytochemistry, 2007, 68, 1844).

Miller (J.Org.Chem., 1981, 46, 1469) used (Taxus wallichiana) trunk and leaves, extracted with methanol, concentrated, distributed in hexane and water, extracted with chloroform, concentrated, and separated by silica gel column chromatography several times ; Secondary countercurrent distribution; finally separated and purified by HPLC to obtain 184 components, the resulting paclitaxel purity 99.1%. The whole extraction and purification process has eight steps. Senilh et al. (J.Nat.Prod., 1984, 47, 131) extracted the bark of European yew (T.baccata) with methanol, concentrated, distributed in dichloromethane and water, separated by silica gel column chromatography, and then oxidized Separated by aluminum column chromatography; then separated by medium pressure silica gel column chromatography; finally purified by preparative high pressure liquid chromatography.

Nalr (USP 5,279,949,1994) invented the extraction of paclitaxel and its analogues from the fresh branches and leaves of yew (T.media cv.Hicksii). The fresh branches and leaves are extracted with aqueous methanol, the water-soluble components are centrifuged and separated from the solid precipitation containing paclitaxel, the filtered filtrate is decolorized with activated carbon and filtered with diatomaceous earth, the filtrate is evaporated to dryness, and the product is obtained by liquid phase separation through a silica gel column. Alternatively, demethanolize the filtrate filtered through diatomaceous earth under vacuum, extract it with ethyl acetate, concentrate the extract, and purify it through a silica gel column to obtain the product. Nalr (USP 5,478,736,1995) extracts fresh branches and leaves with methanol and acetone, which can obtain higher paclitaxel production. Separation by secondary column chromatography, separation of crude paclitaxel by low-pressure silica gel column chromatography, and final purification by reverse phase chromatography.

Polysciences isolated paclitaxel from Pacific yew bark. First extract the bark with methanol or ethanol, concentrate; extract the concentrate with dichloromethane, concentrate and dry to powder; % propanol ligroin solution, filtered through a Florisil column; the paclitaxel component flowing out from the column was crystallized and purified twice; the crystallized paclitaxel was separated on a silica gel column, and the product cephalomannine with a similar structure was separated from paclitaxel out; paclitaxel flowing out from the column is recrystallized twice to obtain paclitaxel. Rao et al. (USP 5,380,916, 1992) introduced the use of ozone to treat the difficult-to-separate impurity component cephalomannine to obtain paclitaxel. After the crude product is separated by reverse-phase preparative chromatography, it still contains cephalomannine which is difficult to separate. After being oxidized by ozone, it is separated by reverse-phase preparative chromatography to obtain paclitaxel with high purity. The method uses dimethyl sulfide to treat residual ozone in the solution and recrystallizes to obtain pure paclitaxel. Kingston et al. (J. Nat. Prod., 1992, 55, 259) treated a mixture of paclitaxel and cephalomannine with the oxidizing agent osmium tetroxide. Paclitaxel does not react with osmium tetroxide, and the double bond at the end of the C-13 side chain of cephalomannine is oxidized, so it is easy to separate from paclitaxel. Durand (USP 5,723,635, 1998) used centrifugal partition chromatography to separate and purify paclitaxel. Paclitaxel was separated and purified from the crude product with a C ₁₈ reverse-phase chromatographic column, and the amount of treatment at one time was relatively large. There are four main steps: treatment of crude paclitaxel extract by reversed-phase chromatography at preparative scale; elution of paclitaxel and other products from the adsorbent; recovery of paclitaxel and analogues from the eluted column; treatment of the final product with ozone , thereby isolating and removing the analogs of paclitaxel.

Rao (USP 5,380,916, 1995) extracted the bark with 95% methanol, concentrated, extracted with chloroform and water. Continuous extraction for 3 times, concentration of chloroform phase, gradient elution with acetonitrile and water on the _C18 packed chromatography column, receiving fractions with higher paclitaxel in sections, diluting with hot water, collecting fractions with reversed-phase column, through High-purity paclitaxel and its analogues were obtained by recrystallization. Chen Jianmin etc. have invented a kind of method (ZL 1427002) of preparing paclitaxel from planting yew leaf branch. Soak in ethanol, extract with butyl acetate, concentrate to get 1% paclitaxel crude raw material, then go through silica gel column chromatography, elute with cyclohexane and ethyl acetate gradient, collect the fractions containing more than 10% paclitaxel, concentrate and go through reaction phase high-pressure liquid chromatography to obtain high-purity paclitaxel.

For the separation and purification of Taxus paclitaxel, the lower alcohol solvent is used to extract the bark, and the extract is first degreased and then extracted with chloroform; the chloroform layer is concentrated, and Paclitaxel and its analogs are obtained by column chromatography. Commonly used chromatographic column chromatography requires multiple forward and reverse phase liquid chromatography, centrifugal partition chromatography, etc., and also includes crystallization and oxidation to remove impurities. The steps of the separation process are cumbersome, and the primary yield of paclitaxel with a purity of more than 99% is low. Solvents, eluents, etc. are highly toxic and cause serious pollution. In addition, paclitaxel has similar properties to cephalomannine, 7-epi-paclitaxel and 7-epi-10-deacetyl paclitaxel, and it is difficult to separate them (Yang et al., J. Chromatogr. A, 1998, 813, 201). Effectively removing these structural analogues from paclitaxel samples is the key to extracting high-purity paclitaxel.

Contents of the invention

The object of the present invention is to provide a method for preparing paclitaxel from branches and leaves of yew artificially cultivated. The method is simple in process, easy to separate the structural analogs of paclitaxel, and the obtained paclitaxel has high purity.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A method for preparing paclitaxel from artificially cultivated yew branches and leaves, which comprises the steps in turn:

A. After the yew branches and leaves are crushed, soak them with 75% to 95% methanol for 3 times at 40 to 50°C, each time for 4 hours, and use a volume ratio of 4:1 to 1:1 petroleum ether/water after the filtrate is concentrated. Extraction; Separate the lower aqueous phase;

B. Extract the lower aqueous phase with propyl acetate; concentrate the propyl acetate extract to obtain a paclitaxel crude extract with a paclitaxel content of more than 2% to 5%;

C. Mix more than 2% paclitaxel crude extract with silica gel to prepare a dry sample, fill the dry sample to the upper end of the chromatographic column of silica gel (200-300 mesh), and use 4:1 petroleum ether-ethyl acetate, 1:1 respectively Petroleum ether-ethyl acetate, 1:5 petroleum ether-ethyl acetate and ethyl acetate were eluted, and the eluate fractions with paclitaxel content between 15% and 40% were collected in sections, concentrated and dried , to produce intermediate products;

D, the intermediate product obtained in step C is dissolved in acetonitrile and water containing 0.1% _{trifluoroacetic} acid with a volume ratio of 30:70, and separated by high-pressure liquid chromatography. Acetonitrile of alkylpyridinium quaternary ammonium salt and water containing 0.1% trifluoroacetic acid are used as mobile phases, the eluate is collected, concentrated, and a mixture of paclitaxel crystals and octadecylpyridinium trifluoroacetic acid quaternary ammonium salt solid is separated out.

The mixture is centrifuged, washed with water, centrifuged again, washed with water, filtered and freeze-dried to obtain paclitaxel with a purity of more than 99.6%.

The above eluent and the solvent in the mobile phase can be reused after rectification.

The yew artificially cultivated in the above step A is Taxus Mandia, Taxus Yunnan or Taxus chinensis.

The concentration of octadecylpyridinium quaternary ammonium trifluoroacetate in step D above is 2-15 mg/mL.

The volume ratio of the mobile phase acetonitrile-0.1% trifluoroacetic acid water in the above step D is 30:70.

The octadecylpyridinium trifluoroacetic acid quaternary ammonium salt obtained in the above step D can be reused after being washed with water, centrifugally filtered, and freeze-dried.

Compared with the prior art, the present invention has the following advantages:

The method has simple process, low cost, recyclable solvent and eluent, is suitable for industrial production, and protects plant resources.

Description of drawings:

Fig. 1 is the process flow diagram of extracting and purifying paclitaxel from the branches and leaves of Chinese yew;

Fig. 2 is the high-performance liquid chromatogram of the crude extract of yew branches and leaves;

Figure 3 is a high performance liquid chromatogram with paclitaxel content above 15%;

Fig. 4 is the high performance liquid phase chromatogram of paclitaxel crystal and octadecylpyridinium trifluoroacetate quaternary ammonium salt;

Fig. 5 is a high-performance liquid chromatogram of paclitaxel with an isolation purity of 99.6% or more.

Detailed ways

The present invention uses artificially cultivated branches and leaves of Taxus chinensis, Taxus yunnanensis, Taxus chinensis or Taxus chinensis as raw materials, soaks and extracts them in methanol, extracts them with petroleum ether and propyl acetate in sequence, and then uses normal pressure normal phase High-purity paclitaxel was obtained by silica gel column chromatography and reversed-phase high-pressure liquid chromatography.

The technical scheme adopted in the present invention is: a kind of method for extracting paclitaxel from the artificially cultivated yew branches and leaves, comprising the following steps successively:

(1) After the branches and leaves of the three-year yew artificially cultivated are crushed, soak them three times with 75-95% methanol at 40-50° C., each time for 4 hours, combine the filtrates, concentrate, and use a volume ratio of 4 : 1 petroleum ether/water extraction, separate the lower layer, and extract the lower layer aqueous phase with an equal volume of propyl acetate;

(2) concentrating the propyl acetate extract to obtain a paclitaxel concentrate with a paclitaxel content of more than 2%;

(3) Companion more than 2% paclitaxel concentrate with silica gel, and fill it on a silica gel column, elute with eluent gradient, add petroleum ether and ethyl acetate (4:1), petroleum ether and ethyl acetate Esters (1:1), petroleum ether and ethyl acetate (1:5), ethyl acetate, after the eluent flows out of the chromatographic column, the fractions are collected in sections, and the paclitaxel content between 15% and 40% is eluted in sections The liquid fractions were collected and combined, concentrated and dried to obtain an intermediate product;

(4) Dissolve the intermediate product obtained in step (3) and step (4) with acetonitrile and water (30:70) containing 0.1% trifluoroacetic acid, and separate with C ₁₈ reverse-phase high-pressure liquid chromatography to obtain trifluoroacetic acid-containing The acetonitrile of octadecylpyridinium quaternary ammonium salt and water (30:70) containing 0.1% trifluoroacetic acid are mobile phases, then the mobile phase containing paclitaxel after the separation is concentrated to a certain volume, and paclitaxel crystals and trifluoroacetic acid decaclitaxel are separated out. Mixture of octaalkylpyridine quaternary ammonium salts solid.

(5) the mixture of paclitaxel and octadecylpyridinium trifluoroacetate quaternary ammonium salt of step (4) gained, through

Centrifuge, wash with water, centrifuge again, filter, and freeze-dry to obtain paclitaxel with a purity of more than 99.6%.

The yew in the step (1) is Taxus mandia (T.media), Taxus yunnanensis (T.yunnanensis), or Taxus chinensis (T.cuspidata).

The process of the method for extracting paclitaxel of the present invention is shown in Figure 1. The process shown in Figure 1 is divided into three processes.

The first process: Utilize artificially cultivated fresh or dried yew branches and leaves to be pulverized, soak them with 75%-95% methanol three times at 40-50°C, each time for 4 hours, and the volume ratio of raw materials and methanol is 1:3-1 : 8 soaking solution filtered to remove the filter residue, the filtrate (HPLC analysis see Figure 2) is placed in a rotary evaporator or a concentration tank to concentrate, adding a volume ratio of 1:1 petroleum ether/water for extraction, stirring for 30 minutes, and standing for about 20 to 30 minutes to form The upper layer of light green clear liquid and the lower layer of dark green colloidal liquid were separated from the upper layer of petroleum ether phase and extracted continuously for 3 times. After the lower aqueous phase is extracted with the same volume of propyl acetate, the propyl acetate extract is placed for 0.5 to 1 hour, and the propyl acetate layer is separated from the water layer, and the propyl acetate layer is evaporated, concentrated and dried to obtain a dark green or brown powder , the obtained paclitaxel content is more than 2%.

The second process: the crude extract with a paclitaxel content above 2% is dissolved in acetone and mixed with silica gel (200-300 mesh) to prepare a dry sample. The dry sample was placed on the upper end of the silica gel column, and the mixed solvent of petroleum ether and ethyl acetate was used for gradient elution, and a container of the same volume was used to receive fractions 1-20 of the same volume. Fractions 9-15 are yellow or greenish-yellow solid powder after being evaporated to dryness, and the paclitaxel content is between 15.0% and 40.0% (Fig. 3). During this process, fractions with a paclitaxel content between 15.0% and 40.0% were combined and evaporated to dryness, and dissolved in acetonitrile and water containing 0.1% trifluoroacetic acid (30:70) for further purification of paclitaxel.

The third process: with C ₁₈ reverse phase high performance liquid chromatography column, inject 10 grams of acetonitrile aqueous solution of 25% paclitaxel crude extract, with the acetonitrile containing trifluoroacetic acid octadecyl pyridinium quaternary ammonium salt and containing 0.1% trifluoroacetic acid water (30:70) was the mobile phase, and fractions 1-35 were obtained. In this process, after the fraction 15-25 is vacuum-concentrated to a certain volume in a rotary evaporator, a mixture of paclitaxel and octadecylpyridinium trifluoroacetate quaternary ammonium salt is separated out (Figure 4), and then centrifuged, washed with water, and centrifuged again. Filtration and lyophilization yielded paclitaxel with a purity of over 99.9% ( FIG. 5 ).

Compared with the existing paclitaxel extraction process, the method of the present invention has fewer steps, simple process, and low production cost, is beneficial to the industrialized large-scale production of paclitaxel, does not destroy natural resources, and is beneficial to ecological environment protection; no toxic reagents, The solvent meets the requirements of environmental protection; the yield of paclitaxel is high, and the purity can reach more than 99.6%.

Petroleum ether, propyl acetate, ethyl acetate, methanol, trifluoroacetic acid, acetic acid, octadecylpyridinium trifluoroacetic acid quaternary ammonium salt etc. used in the present invention are analytically pure. Acetonitrile was HPLC grade and the water used was HPLC grade and deionized water.

The analysis of raw material, intermediate product and paclitaxel all adopts U.S. Waters high-pressure liquid chromatograph, and chromatographic column is U.S. Waters product SymmetryShield RP ₁₈ silica gel chromatographic column (50 * 4.6mm), installs the Warters product SymmetryShieldC ₁₈ protection column ( 3.9×20mm), the filler size is 3.5μm, Waters 996 diode array detector (210～500nm), the mobile phase is acetonitrile+water+0.1%TFA, the proportion of acetonitrile increases from 20% to 80%, and the flow rate is 1ml /min, automatic injection, the injection volume is 10μL. The chromatographic column uses an incubator, the column temperature is 20°C, the computer is operated, and the chromatographic software is Millennium ³² .

The high-purity paclitaxel was prepared using the Waters high-pressure liquid chromatography system in the United States. The chromatographic column was Shiseido Capcell Pak C18 produced by JM Science in the United States. 227nm, the mobile phase is acetonitrile + 0.1% TFA water (30+70) containing octadecylpyridinium trifluoroacetic acid quaternary ammonium salt, the flow rate is 1.0ml/min, automatic sampling, collected every 20min.

High-resolution mass spectrometry was performed on a Micromass WatersQ-TOF Global mass spectrometer produced by Micromass, UK, +ESI source.

Example 1:

As shown in Figure 1, the method for extracting paclitaxel from the artificially cultivated yew branches and leaves is:

(1) Take 1 kg of dry branches and leaves of Taxus chinensis artificially cultivated, crush them with a crusher, add 3 L of 90% methanol to a 5 L round bottom flask, stir well, soak three times at 40-50 ° C, each time 4-6 Hour. Stir 3 to 6 times during the standing process. After soaking, filter to remove waste residue, and concentrate the filtrate to a light green viscous liquid with a volume of about 50ml. Add 2L of petroleum ether to the solution, mix well, add 2L of water, stir for 30 minutes, and let it stand for about 25 minutes to form a light green clear liquid in the upper layer and a dark green gelatinous liquid in the lower layer. Soluble pigments and other impurities. Add 2 L of propyl acetate to the separated lower aqueous phase, stir for 30 min, let stand for about 25 min, extract continuously for 3 times, and combine the three propyl acetate extracts.

(2) The propyl acetate extract was evaporated to dryness under reduced pressure to form a brown powder with a weight of 12.6 grams. The paclitaxel content was determined to be 3.28%, and the suspension in the lower layer did not contain paclitaxel.

(3) Dissolve propyl acetate powder with a content of more than 3.28% in acetone, and mix it with silica gel to prepare a dry sample. The dry sample was placed on the upper end of the silica gel column, and 0.3L of petroleum ether-ethyl acetate (4:1), 0.3L of petroleum ether-ethyl acetate (1:1), and 0.5L of petroleum ether-ethyl acetate ( 1:5) and 0.2 L of ethyl acetate, receiving the same volume of fractions 1-20 in the same volume of vessel. Fractions 6-8 contain more 7-epi-10-deacetyl-paclitaxel and 7-epi-paclitaxel, and fractions 9-15 are yellow or green-yellow solid powder after evaporation to dryness. The content of paclitaxel in fractions 9-15 is between 15.0% and 35.0%.

(4) Take 10 grams of paclitaxel samples with a content of 27.5%, dissolve them with 50ml of acetonitrile and water (30:70) containing 0.1% trifluoroacetic acid, separate and purify by C ₁₈ reverse phase high pressure liquid chromatography, add 27.5% paclitaxel acetonitrile -Aqueous solution, with acetonitrile containing trifluoroacetic acid octadecylpyridinium quaternary ammonium salt (3mg/mL) and water (30:70) containing 0.1% trifluoroacetic acid as mobile phase, flow rate is 1ml/min, collect fraction 1- 35. Analysis of fractions 1-14 after evaporating to dryness showed that they were impurity components, containing a relatively large amount of cephalomannine, a small amount of paclitaxel (below 2.8%) and 7-epi-10-deacetyl paclitaxel. Fractions 15-25 were concentrated to a certain volume, and a mixture of paclitaxel crystals and octadecylpyridinium quaternary ammonium trifluoroacetate solid was precipitated.

(5) The mixture of paclitaxel and octadecylpyridinium trifluoroacetate quaternary ammonium salt obtained in step (4) was centrifuged, washed with water, centrifuged, filtered, and freeze-dried, and so repeated 3 times to obtain 2.82 grams of product. HPLC assay showed that the purity of paclitaxel was above 99.9% ( FIG. 5 ).

Solvents in the eluent and mobile phase can be reused after rectification. The obtained octadecylpyridinium trifluoroacetic acid quaternary ammonium salt was washed with water, concentrated, freeze-dried and reused.

The product is white crystal, optical rotation [α] _D ²⁵ = -54.6 (c = 0.2, MeOH), melting point is 236 ~ 237 ° C, 500MHz nuclear magnetic resonance spectrum verification is the same as paclitaxel standard sample, high resolution mass spectrometry (HR-ESI- MS) m/z 854.3397 ([M+H] ⁺ ), the corresponding molecular formula is C ₄₇ H ₅₁ NO ₁₄ . The retention time of the product is consistent with that of the standard sample in HPLC analysis.

Example 2:

(1) Take 100kg of fresh branches and leaves of the 3-year-old Taxus Mandina cultivated artificially, put them in a ^2m3 extraction tank after pulverizing with a crusher, then add 550L of 90% methanol, stir evenly, Soak with intermittent stirring for 4 hours. After soaking, release the solution in the extraction tank, and then soak 3 times in the same way. The released methanol solutions were combined and concentrated in a concentration tank to a volume of about 25L. In the extraction tank, add about 250L of petroleum ether to the solution, mix well, add 250L of water and stir for 1 hour, let it stand for about 30min to form two layers, and separate the lower layer. Add about 250L propyl acetate to the lower layer solution, mix evenly, stir for 1 hour, and let stand for about 30min to form two layers.

(2) The propyl acetate extract of the lower layer was released, evaporated to dryness under reduced pressure, and 1032 grams of brown powder was formed, and the paclitaxel content was determined to be 3.31%.

(3) Dissolve 1 kg of paclitaxel brown powder containing 3.31% in acetone, and mix it with 2 kg of silica gel to prepare a dry sample. The dry sample was placed on a 10kg silica gel column, and was washed with 30L of petroleum ether-ethyl acetate (4:1), 30L of petroleum ether-ethyl acetate (1:1), and 50L of petroleum ether-ethyl acetate ( 1:5) and 20L of ethyl acetate eluent, one fraction was collected per 10L, and the same volume of fractions 1-20 was received with the same volume container. Fraction 9-15 is yellow or greenish yellow solid powder after evaporation to dryness. The content of paclitaxel in fractions 9-15 is between 15.0% and 30.0%.

(4) Dissolve the sample with paclitaxel content of 20.3% in acetonitrile and water (30:70) containing 0.1% trifluoroacetic acid, and separate by C ₁₈ reverse phase high pressure liquid chromatography to obtain octadecane containing trifluoroacetic acid Acetonitrile based pyridinium quaternary ammonium salt (5 mg/mL) and water containing 0.1% trifluoroacetic acid (30:70) were used as the mobile phase at a flow rate of 1 ml/min, and fractions 1-35 were collected. Fractions 15-25 were concentrated to give a mixture of paclitaxel crystals and octadecylpyridinium trifluoroacetate as a solid.

(5) The paclitaxel and octadecylpyridinium trifluoroacetate quaternary ammonium salt mixture. Centrifuged, washed with water (300ml), centrifuged again, filtered, and so repeated 3 times, freeze-dried. Paclitaxel with a purity of over 99.7% was obtained with a yield of 91%.

Claims (6)

1. method for preparing taxol from tame Ramulus et folium taxi cuspidatae is characterized in that in turn including the following steps:

A, Ramulus et folium taxi cuspidatae pulverized after, soak 3 times with 75%～95% methyl alcohol down at 40～50 ℃, each 4 hours, it was that 4: 1～1: 1 sherwood oil/water extracts with volume ratio that filtrate is concentrated the back; Tell lower floor's water;

B, with propyl acetate extraction lower floor water; The propyl acetate extraction liquid concentrates, and obtains content of taxol at 2%～5% taxol crude extract;

C, 2% above taxol crude extract is mixed with dry sample with silica gel, filled dry sample to silica gel 200～300 purpose chromatographic columns upper end, use 4: 1 petroleum ether-ethyl acetate, 1: 1 petroleum ether-ethyl acetate, 1: 5 petroleum ether-ethyl acetate and eluent ethyl acetate respectively, by section the elutriant cut of content of taxol between 15%～40% put together, through concentrated, dry, make intermediates;

D, be 30: 70 acetonitrile with the intermediates volume ratio of step C gained and contain 0.1% trifluoracetic acid water dissolution that separate with high pressure liquid chromatography, chromatographic column is C ₁₈Silica filler is moving phase with the acetonitrile that contains trifluoracetic acid octadecyl pyridine quaternary ammonium salt with containing 0.1% trifluoracetic acid water, collects elutriant, concentrates, and separates out paclitaxel crystal and trifluoracetic acid octadecyl pyridine quaternary ammonium salt solid mixture;

This mixture is through centrifugal, and washing is centrifugal again, and washing is filtered, and lyophilize obtains purity and be the taxol more than 99.6%.

2. the method for preparing taxol from tame Ramulus et folium taxi cuspidatae according to claim 1 is characterized in that: the solvent in described elutriant and the moving phase is reused through after the rectifying.

3. the method for preparing taxol from tame Ramulus et folium taxi cuspidatae according to claim 1 is characterized in that: tame Ramulus et folium taxi cuspidatae is Taxus x media, taxusyunnanensis or taxus chinensis in northeast in the steps A.

4. the method for preparing taxol from tame Ramulus et folium taxi cuspidatae according to claim 1 is characterized in that: the trifluoracetic acid octadecyl pyridine quaternary ammonium salt concentration among the step D is 2～15mg/mL.

5. the method for preparing taxol from tame Ramulus et folium taxi cuspidatae according to claim 1 is characterized in that: the volume ratio of the moving phase acetonitrile-0.1% trifluoracetic acid water among the step D is 30: 70.

6. the method for preparing taxol from tame Ramulus et folium taxi cuspidatae according to claim 1 is characterized in that: the resulting trifluoracetic acid octadecyl of step D pyridine quaternary ammonium salt, and centrifugal through washing, filter, reuse after the lyophilize.

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