CN113845496A - Method for synchronously and efficiently separating various taxanes - Google Patents
Method for synchronously and efficiently separating various taxanes Download PDFInfo
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- CN113845496A CN113845496A CN202111188746.6A CN202111188746A CN113845496A CN 113845496 A CN113845496 A CN 113845496A CN 202111188746 A CN202111188746 A CN 202111188746A CN 113845496 A CN113845496 A CN 113845496A
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- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
Abstract
The invention belongs to the technical field of separation and purification, and particularly relates to a method for synchronously and efficiently separating various taxanes. The invention firstly extracts the branches and leaves of the taxus chinensis by an organic reagent, adds water and alcohols after the extract is concentrated, stands and deposits after being mixed evenly, obtains the supernatant fluid of alcohol precipitation by filtration, then directly carries out adsorption and gradient elution to the supernatant fluid of alcohol precipitation by a macroporous resin chromatographic column, and respectively concentrates to obtain the crude products of the taxanes with different high contents after HPLC detection and combination of the same components. The method can rapidly improve the purity of more than ten important taxane products in the taxus chinensis extract, has short production period, high purification efficiency, simple and easy operation, less solvent use and is suitable for industrial application and market popularization.
Description
Technical Field
The invention belongs to the technical field of separation and purification, and particularly relates to a method for synchronously and efficiently separating various taxanes.
Background
Paclitaxel (Paclitaxel) is a diterpenoid compound, is used as a natural anticancer drug with high efficiency, broad spectrum, strong activity and unique action mechanism, especially for ovarian cancer, breast cancer, non-small cell lung cancer and the like, and is gradually popularized to more cancer treatments. However, the content of taxol in branches and leaves of taxus chinensis is very low, and accounts for about 0.005-0.06% of the dry weight of the plant, and a large amount of taxane compounds similar to the taxol in structure exist, and the compounds are small molecular compounds with complex structures. At present, the method has a plurality of chemical semi-synthesis ways, and can convert taxane compounds which have a small amount of groups different from paclitaxel and have consistent parent-nucleus structures into a plurality of medicaments with good anti-tumor effects, such as paclitaxel, docetaxel or cabazitaxel. Patent document CN200910010554.9 discloses a semi-synthesis method of paclitaxel, which uses 10-deacetyl-7-xylosylpaclitaxel as starting material, firstly, the xylosyl at the 7-position is oxidized, then acetyl is introduced to the hydroxyl at the 10-position and the 2' -position, and finally, the target product paclitaxel is obtained by hydrazinolysis; patent document CN200710173713.8 discloses a semi-synthesis method of docetaxel, which has the application effects of carrying out condensation reaction on protected 10-DAB and synthesized C-13 side chain hydrolysate, then opening oxazoline ring, grafting tert-butyloxycarbonyl on nitrogen of side chain, and finally removing protection of 7 and 10 sites to obtain docetaxel. There are still a large number of potential compounds which, together with structural analogues of taxanes, can be converted into taxol, docetaxel or cabazitaxel.
At present, the methods for synchronously separating and purifying paclitaxel and various taxanes reported in the literature mainly focus on the pretreatment of the sample at the early stage, and the types and the contents of the taxanes obtained after the sample is treated are not high. Patent document CN201910960435.3 discloses an industrial method for primarily separating natural paclitaxel and taxane compounds, which mainly combines extraction, silica gel column chromatography and macroporous resin column chromatography to realize effective separation of natural paclitaxel, 10-deacetyl paclitaxel and 10-deacetyl baccatin iii synchronously, and proves that the macroporous resin can effectively improve the content of various taxanes, but only the above 3 target taxane compounds are limited, the comprehensive utilization rate of taxus chinensis resources is not high, and simultaneously, the extraction process can generate a large amount of wastewater, and the wastewater treatment cost is very high. Patent document CN201110167134.9 discloses a method for separating paclitaxel and related taxane substances, which can obtain crude products rich in 10-DAB, 10-deacetyl-7-xylosylpaclitaxel and paclitaxel by mixing various macroporous resins for column chromatography, with few product types and little purity improvement after sample purification. Therefore, there is an urgent need to further develop a technology for efficiently separating and purifying paclitaxel and its precursor substances, so as to improve the separation and purification efficiency of taxane and improve the comprehensive utilization rate of taxus resources.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synchronously and efficiently separating various taxanes, which can quickly remove water-soluble impurities in a sample under the condition of better retaining the contents of various taxanes by using an alcohol precipitation process and reduce the column chromatography pressure of macroporous resin; by combining a macroporous resin column chromatography gradient elution process, taxane products are collected in batches according to the polarity, the content of various important taxanes is rapidly improved, and the technical problems of large wastewater amount, low separation efficiency, low purity, small number of separated taxanes and the like in the method for synchronously separating various taxanes in the prior art are solved.
In order to achieve the above object, the present invention provides a method for efficiently separating multiple taxanes simultaneously, comprising the following steps:
(1) pulverizing branches and leaves of taxus chinensis, extracting by using an organic reagent, and concentrating an extracting solution until the density of the extracting solution is more than 1 to obtain a concentrated solution;
(2) mixing the concentrated solution obtained in the step (1) with water, adding C1-C4 alcohol, stirring uniformly, continuously adding inorganic salt, standing for several hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, carrying out sample loading treatment, then sequentially selecting mixed solvents of C1-C4 alcohols and water in different proportions for gradient elution, carrying out HPLC detection, combining samples with the same components, concentrating, and drying to obtain a plurality of taxane crude products.
Preferably, the organic reagent in step (1) is one or more of methanol, ethanol, dichloromethane and ethyl acetate.
Preferably, step (1) is specifically: crushing the taxus chinensis branches and leaves to 40-300 meshes, adding an organic reagent according to the mass volume ratio of 1: 1-1: 10, extracting for 1-3 hours at 30-65 ℃, filtering, continuously adding the organic reagent into filter residues, repeatedly extracting for 2-4 times, collecting and combining filtrate, and concentrating the filtrate at 30-65 ℃ to obtain a concentrated solution.
Preferably, the density of the concentrated solution in the step (1) is controlled to be 1.2-1.4.
Preferably, the C1-C4 alcohol is one or more of methanol, ethanol and butanol.
Preferably, the step (2) is specifically: adding pure water into the concentrated solution obtained in the step (1), adding pure water according to the volume ratio of the concentrated solution to the pure water of 1: 5-1: 20, then adding C1-C4 alcohols according to the volume ratio of the pure water to the C1-C4 alcohols of 1: 0.5-1: 2, stirring uniformly, continuing to add inorganic salt, standing for 3-8 hours at room temperature, and filtering to obtain a supernatant.
Preferably, the inorganic salt in the step (2) is a calcium salt, preferably one of calcium hydrogen phosphate, calcium lactate and calcium chloride, and the ratio of the added mass of the inorganic salt to the total volume of the pure water and the alcohols from C1 to C4 added in the step (2) is 1 g: 0.1L to 10L, more preferably 1 g: 1L to 3L.
Preferably, the macroporous resin in the step (3) is one of D101, HZ818, D4020, AB-8, XAD-8 and LSA-40; the ratio of the actual sample dry weight in the supernatant to the macroporous resin weight is controlled to be 1: 1-1: 20; the sampling flow rate of the macroporous resin chromatographic column is controlled to be 1-4 BV/h, and the preferable flow rate is 2.5-3 BV/h.
Preferably, the amount of an elution solvent used in the macroporous resin chromatographic column in the step (3) is 2-5 times of the column volume, and the elution solvent is a mixed solvent prepared by mixing C1-C4 alcohols and water according to a volume ratio of 1: 4-1: 0 and in different proportions.
Preferably, the plurality of taxanes of step (3) comprises one or more of 10-deacetylbaccatin III, baccatin III, 7-xylose-10-deacetyl paclitaxel C, 7-epi-10-deacetyl paclitaxel C, 10-deacetylcephalomannine, 7-epi-10-deacetyl paclitaxel C, 7-epi-10-deacetyl paclitaxel, cephalomannine, paclitaxel, and N-methyl paclitaxel.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) the method for synchronously separating various taxanes provided by the invention comprises the steps of firstly concentrating an extracting solution extracted by an organic reagent, carrying out alcohol precipitation after concentration, removing fat-soluble impurities in a sample, then adopting macroporous resin column chromatography for adsorption, and then eluting to obtain various taxanes. The method can rapidly remove fat-soluble impurities in a sample by using an alcohol precipitation process under the condition of better retaining the content of various taxanes, and reduce the column chromatography pressure of macroporous resin; combining with macroporous resin column chromatography gradient elution process, collecting taxane products in batches according to polarity, and rapidly increasing the content of various important taxanes.
(2) The traditional paclitaxel extraction method usually adopts extraction steps, and the amount of wastewater produced is about 20 tons per day according to the production line of 90kg paclitaxel produced annually. The method for synchronously separating various taxanes provided by the invention omits the extraction step in the traditional extraction method, and correspondingly cooperates with the alcohol precipitation process and the macroporous resin column chromatography, so that the generation of residual water phase solvent in the traditional process is avoided, and the generation amount of wastewater is almost 0; before the traditional macroporous resin sample is treated, the solvent in the extracting solution is completely recovered, and then the macroporous resin is separated and recovered, but in the extraction method, the concentration of the alcohol precipitation liquid can directly pass through the macroporous resin, alcohol precipitation is directly carried out according to the specific addition of the C1-C4 alcohol solvent and the addition of water, and the alcohol precipitation supernatant can directly become a sampling raw material of macroporous resin column chromatography.
(3) The prior method for separating and recovering the taxol can only recover about 3 kinds of taxane molecules, and the improvement degree of the purity of the taxane molecules is limited. The method for synchronously separating various taxanes provided by the invention can synchronously separate various important taxane molecules such as 10-deacetyl baccatin III, 7-xylose-10-deacetyl taxol C, 7-epi-10-deacetyl taxol C, 10-deacetyl cephalomannine, 7-epi-10-deacetyl taxol C, 7-epi-10-deacetyl taxol, cephalomannine, taxol, N-methyl taxol and the like, and particularly can improve the purity of the taxol from 1.02 percent to 10.87 percent.
(4) Firstly, extracting the polarity of taxus chinensis branches and leaves by an organic reagent, concentrating an extracting solution to a certain volume and density, adding a certain volume of water, alcohols and inorganic salts, uniformly mixing, standing for precipitation, filtering to obtain an alcohol precipitation supernatant, performing gradient elution by using a macroporous resin chromatographic column, detecting by HPLC, combining the same components, and respectively concentrating to obtain crude taxanes with different high contents. The method can rapidly improve the purity of various important taxane products such as 10-deacetylbaccatin III, baccatin III, 7-xylose-10-deacetyltaxol C, 7-epi-10-deacetyltaxol C, 10-deacetylcephalomannine, 7-epi-10-deacetyltaxol C, 7-epi-10-deacetyltaxol, cephalomannine, taxol, N-methyl taxol and the like in the taxus chinensis extract, and has the advantages of short production cycle, high purification efficiency, simple and easy operation method, less solvent use and suitability for industrial application and market popularization. The resin used in the method is common cheap resin, and the organic reagent is common solvent with low toxicity and easy recovery.
Drawings
FIG. 1 is a schematic flow chart of the method for efficiently separating a plurality of important taxanes simultaneously according to the present invention;
FIG. 2 is a chemical formula of several important taxane products isolated in example 1 of the present invention;
FIG. 3 shows the HPLC analysis results of example 1 of the present invention after combining the same taxane fractions.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In some embodiments of the present invention, a method for efficiently separating multiple taxanes simultaneously is provided, as shown in fig. 1, comprising the following steps:
(1) pulverizing branches and leaves of taxus chinensis, extracting by using an organic reagent, and concentrating an extracting solution to obtain a concentrated solution until the density of the concentrated solution is more than 1;
(2) mixing the concentrated solution obtained in the step (1) with water, adding C1-C4 alcohol, stirring uniformly, continuously adding inorganic salt, standing for several hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, carrying out sample loading treatment, then sequentially selecting mixed solvents of C1-C4 alcohols and water in different proportions to carry out gradient elution, detecting by means of HPLC (high performance liquid chromatography), merging samples with the same components, concentrating, and drying to obtain various crude taxane products.
In some embodiments, the organic reagent of step (1) is one or more of methanol, ethanol, dichloromethane, and ethyl acetate. Preferably methanol or ethanol. In the step (1), the taxane substances in the taxus chinensis are extracted by adopting an organic reagent, and impurities such as ester derivatives and the like are also contained. And (2) concentrating the extracting solution in the step (1) until the density of the concentrated solution is controlled to be 1.2-1.4. Part of the organic reagents used in the extraction process can be recovered during the concentration process.
In some embodiments, step (1) is specifically: crushing the branches and leaves of the taxus chinensis to 40-300 meshes, adding an organic reagent, extracting for 1-3 hours at the temperature of 30-65 ℃, wherein the ratio of the powder raw materials of the branches and leaves of the taxus chinensis to the added organic reagent is 100 g: (100-1000) mL; filtering, continuously adding an organic reagent into filter residues, repeatedly extracting for 2-3 times, collecting and combining filtrate, and concentrating the filtrate at 30-65 ℃ to obtain a concentrated solution. Wherein the ratio of the taxus chinensis branch and leaf powder raw material to the total volume of the added organic reagent is 100 g: (400-4000) mL.
The concentrated taxus chinensis liquid is directly subjected to alcohol precipitation treatment to remove impurities such as ester derivatives and the like in the concentrated taxus chinensis liquid, and in some embodiments, C1-C4 alcohol adopted in the alcohol precipitation process is one or more of methanol, ethanol and butanol. It was found in the experiment that the alcohol solvent such as methanol in the alcohol precipitation step is too high in proportion, the impurity removal rate is lowered, and paclitaxel is lost in the alcohol precipitation step when the methanol ratio is too low. When the macroporous resin is used for sampling, if the proportion of methanol is higher, part of taxane can not be retained on the column and can be eluted together with the elution waste liquid, so that the product yield is influenced. The ratio of the alcohol solvent to water in the alcohol precipitation process step is strictly controlled, so that the separation and recovery results of the taxane are greatly influenced.
In some embodiments, the alcohol precipitation in step (2) is specifically: adding pure water into the concentrated solution obtained in the step (1), adding water according to the volume ratio of the concentrated solution to the pure water of 1: 5-1: 20, adding C1-C4 alcohols according to the volume ratio of the water to the C1-C4 alcohols of 1: 0.5-1: 2, stirring uniformly, continuing to add a certain amount of inorganic salt, standing for 3-8 hours at room temperature, and filtering to obtain a supernatant.
In order to realize rapid sedimentation of alcohol precipitation residue in the alcohol precipitation process in the step (2), a certain amount of inorganic salt is added, in some embodiments, the inorganic salt in the step (2) is a calcium salt, preferably one of calcium hydrogen phosphate, calcium lactate and calcium chloride, and further preferably calcium chloride, and the mass-to-volume ratio of the added mass of the inorganic salt to the total volume of the pure water and the alcohols from C1 to C4 added in the step (2) is 1 g: 0.1L to 10L, preferably 1 g: 1L to 3L. Other inorganic salts, such as sodium salts, were tried in the experiment, but the alcohol sediment was found to settle at a much lower rate after the addition of sodium salts than when calcium salts were added, while the calcium chloride was found to settle at the fastest rate when different calcium salts, such as calcium lactate, calcium carbonate, calcium hydrogen phosphate and calcium chloride, were also compared.
The macroporous resin in the step (3) can be one or more of D101, HZ818, D4020, AB-8, XAD-8 and LSA-40; in some embodiments, the ratio of the dry weight of the actual sample in the supernatant to the weight of the macroporous resin is controlled to be between 1:1 and 1:20, preferably between 1:2 and 1: 5; the sampling flow rate of the macroporous resin chromatographic column is controlled to be 1-4 BV/h, and in the preferred embodiment, the sampling flow rate is 2.5-3 BV/h.
In some embodiments, the amount of the elution solvent used in the macroporous resin chromatography column in step (3) is 2 to 5BV (BV represents the volume of the column), one part of the elution solvent is collected in each third of the volume of the column, and the elution solvent is a mixed solvent prepared by mixing C1 to C4 alcohols and water according to a volume ratio of 1:4 to 1:0 and in different proportions. In some examples, enriched 10-deacetylbaccatin III may be obtained by eluting 3 column volumes using a ratio of C1-C4 alcohols to water by volume of 1:2, enriched 4 column volumes using a ratio of C1-C4 alcohols to water by volume of 1:1, enriched mixtures of baccatin III, 7-xylose-10-deacetyltaxol C, 7-epi-10-deacetyltaxol C, 10-deacetylcephalomannine, 7-epi-10-deacetyltaxol C, and the like, enriched mixtures of components of 7-epi-10-deacetyltaxol, cephalomannine, taxol, and the like, obtained by eluting 3 column volumes using a ratio of C1-C4 alcohols to water by volume of 1:0.65, enriched mixtures of 2 column volumes using a ratio of C1-C4 alcohols to water by volume of 1:0.25, obtaining a fraction enriched in N-methyl paclitaxel;
the plurality of taxanes of step (3) of the present invention comprise one or more of 10-deacetylbaccatin III, baccatin III, 7-xylose-10-deacetyltaxol C, 7-epi-10-deacetyltaxol C, 10-deacetylcephalomannine, 7-epi-10-deacetyltaxol C, 7-epi-10-deacetyltaxol, cephalomannine, taxol and N-methyl taxol.
The following are specific examples:
example 1
A method for synchronously and efficiently separating a plurality of taxanes is characterized by comprising the following steps:
(1) pulverizing 100g ramulus et folium taxi Cuspidatae to 40 mesh, adding 500ml methanol, extracting at 65 deg.C for 3h, filtering to obtain extractive solution, adding 500ml methanol to extract residue, repeating for 2 times, and concentrating the extractive solution at 30 deg.C to density of 1.4 to obtain concentrated solution;
(2) mixing the concentrated solution obtained in the step (1) with 100ml of water, adding 200ml of methanol, stirring uniformly, continuously adding 0.3g of calcium chloride, standing for 3 hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, and carrying out sample loading treatment, wherein the ratio of the actual sample dry weight in the supernatant to the weight of the macroporous resin is controlled to be 1: 2; controlling the sample loading flow rate of the macroporous resin chromatographic column at 2BV/h, then sequentially selecting mixed solvents of ethanol and water according to different proportions of 1:2, 1:1.5, 1:0.8 and 1:0.1 to perform gradient elution, wherein the volume of each eluent is 2 column volumes, one part of eluent is collected in each third column volume, detecting by means of HPLC, merging samples with the same components, concentrating and drying to obtain a plurality of taxane crude products.
The molecular structural formula of the obtained taxane is shown in fig. 2, and fig. 3 is an HPLC detection result of the embodiment after combining the same taxane components. FIG. 2 and FIG. 3, 1 is 10-deacetyl kiosk III; 2 is baccatin III; 3 is 7-xylose-paclitaxel; 4 is 7-xylose-10-deacetyl taxol C; 5 is 10-deacetylcephalomannine; 6 is 7-epi-10-deacetyl paclitaxel C; 7 is 7-epi-10-deacetyl paclitaxel; 8 is cephalomannine; 9 is 7-epi-10-deacetyl paclitaxel; 10 is paclitaxel; 11 is N-methyl paclitaxel. The concentration of paclitaxel, a representative compound, was increased from 1.02% to 8.95% with a yield of 96%.
Example 2
A method for synchronously and efficiently separating a plurality of taxanes is characterized by comprising the following steps:
(1) pulverizing 100g ramulus et folium taxi Cuspidatae to 200 mesh, adding 100ml methanol, extracting at 55 deg.C for 1h, filtering to obtain extractive solution, adding 100ml methanol to extract residue, repeating for 2 times, and concentrating the extractive solution at 55 deg.C to density of 1.2 to obtain concentrated solution;
(2) mixing the concentrated solution obtained in the step (1) with 50ml of water, adding 100ml of methanol, stirring uniformly, continuously adding 0.45g of calcium hydrophosphate, standing for 5 hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, and carrying out sample loading treatment, wherein the ratio of the actual sample dry weight in the supernatant to the weight of the macroporous resin is controlled to be 1: 3.5; controlling the sampling flow rate of the macroporous resin chromatographic column at 2.5BV/h, then sequentially selecting mixed solvents of ethanol and water according to different proportions of 1:85, 1:1.25, 1:0.9 and 1:0.25 for gradient elution, wherein the volume of each eluent is 3 column volumes, one third of the eluent is collected into one part, detecting by means of HPLC, merging samples with the same components, concentrating and drying to obtain a plurality of taxane crude products, wherein the concentration of paclitaxel is increased from 1.02% to 9.56%, and the yield is 95%.
Example 3
A method for synchronously and efficiently separating a plurality of taxanes is characterized by comprising the following steps:
(1) pulverizing 100g ramulus et folium taxi Cuspidatae to 400 mesh, adding 1000ml methanol, extracting at 30 deg.C for 2h, filtering to obtain extractive solution, adding 1000ml methanol to extract residue, repeating for 2 times, and concentrating the extractive solution at 30 deg.C to density of 1.3 to obtain concentrated solution;
(2) mixing the concentrated solution obtained in the step (1) with 100ml of water, adding 100ml of methanol, stirring uniformly, continuously adding 0.4g of calcium lactate, standing for 8 hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, and carrying out sample loading treatment, wherein the ratio of the actual sample dry weight in the supernatant to the weight of the macroporous resin is controlled to be 1: 5; controlling the sampling flow rate of the macroporous resin chromatographic column at 3BV/h, then sequentially selecting mixed solvents of ethanol and water according to different proportions of 1:75, 1:1.15, 1:1 and 1:0.05 for gradient elution, wherein the volume of each eluent is 5 column volumes, one part of eluent is collected in each third column volume, detecting by means of HPLC, merging samples with the same components, concentrating, drying to obtain a plurality of taxane crude products, wherein the concentration of the paclitaxel is increased from 1.02% to 10.2%, and the yield is 97%.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for synchronously and efficiently separating a plurality of taxanes is characterized by comprising the following steps:
(1) pulverizing branches and leaves of taxus chinensis, extracting by using an organic reagent, and concentrating an extracting solution until the density of the extracting solution is more than 1 to obtain a concentrated solution;
(2) mixing the concentrated solution obtained in the step (1) with water and C1-C4 alcohols, stirring uniformly, continuously adding inorganic salt, standing for several hours at room temperature, and filtering to obtain a supernatant;
(3) injecting the supernatant obtained in the step (2) from a liquid inlet of a macroporous resin chromatographic column, carrying out sample loading treatment, then selecting mixed solvents of C1-C4 alcohols and water in different proportions to carry out gradient elution, carrying out HPLC detection, combining samples with the same components, concentrating, and drying to obtain various crude taxane products.
2. The method of claim 1, wherein the organic reagent in step (1) is one or more of methanol, ethanol, dichloromethane, and ethyl acetate.
3. The method according to claim 2, wherein step (1) is specifically: crushing the taxus chinensis branches and leaves to 40-300 meshes, adding an organic reagent according to the mass volume ratio of 1: 1-1: 10, extracting for 1-3 hours at 30-65 ℃, filtering, continuously adding the organic reagent into filter residues, repeatedly extracting for 2-4 times, collecting and combining filtrate, and concentrating the filtrate at 30-65 ℃ to obtain a concentrated solution.
4. The method of claim 1, wherein the density of the concentrate in step (1) is controlled to be between 1.2 and 1.4.
5. The method of claim 1, wherein the C1-C4 alcohol is one or more of methanol, ethanol, and butanol.
6. The method according to claim 1, wherein step (2) is specifically: adding pure water into the concentrated solution obtained in the step (1), adding pure water according to the volume ratio of the concentrated solution to the pure water of 1: 5-1: 20, then adding C1-C4 alcohols according to the volume ratio of the pure water to the C1-C4 alcohols of 1: 0.5-1: 2, stirring uniformly, continuing to add inorganic salt, standing for 3-8 hours at room temperature, and filtering to obtain a supernatant.
7. The method according to claim 6, wherein the inorganic salt in step (2) is a calcium salt, preferably one of calcium hydrogen phosphate, calcium lactate and calcium chloride, and the ratio of the added mass of the inorganic salt to the total volume of the pure water and the C1-C4 alcohols added in step (2) is 1 g: 0.1L to 10L.
8. The method of claim 1, wherein the macroporous resin of step (3) is D101, HZ818, D4020, AB-8, XAD-8 or LSA-40; the ratio of the actual sample dry weight in the supernatant to the macroporous resin weight is controlled to be 1: 1-1: 20; and the sampling flow rate of the macroporous resin chromatographic column is controlled to be 1-4 BV/h.
9. The method of claim 1, wherein the amount of the eluting solvent used in the macroporous resin chromatography column in the step (3) is 2-5 times of the column volume, and the eluting solvent is a mixed solvent prepared by mixing C1-C4 alcohols and water according to a volume ratio of 1: 4-1: 0 and in different proportions.
10. The method of claim 1, wherein the plurality of taxanes of step (3) comprises one or more of 10-deacetyl baccatin III, 7-xylose-10-deacetyl paclitaxel C, 7-epi-10-deacetyl paclitaxel C, 10-deacetyl cephalomannine, 7-epi-10-deacetyl paclitaxel C, 7-epi-10-deacetyl paclitaxel, cephalomannine, paclitaxel, and N-methyl paclitaxel.
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