阅读说明：本技术 一种紫杉醇分离纯化工艺 (Paclitaxel separation and purification process ) 是由 周春 杨志平 孙伟 胥雯 于 2021-08-09 设计创作，主要内容包括：本发明涉及一种紫杉醇分离纯化工艺,涉及化合物分离纯化技术领域,解决了现有技术中紫杉醇分离提纯工艺中,提取纯度和生产效率低的问题；采用的有机溶剂品种相对较少,溶解剂与流动相中有相同的成分,且能够相互套用,操作灵活简便,降低了紫杉醇的分离提纯成本,且对紫杉醇的回收率较高,能够应用与大规模的紫杉醇分离提纯生产中；在层析柱底部设置流量阀,并控制流动相洗脱时的流速,便于紫杉醇的层析分离,提高提纯效果,既缩短了生产周期,简化了生产工艺,又减少了“溴加提纯工艺”中多次柱层析紫杉醇不可逆吸附造成的损失,提高了紫杉醇的收率,又大大降低了分离纯化成本。(The invention relates to a paclitaxel separation and purification process, relates to the technical field of compound separation and purification, and solves the problems of low extraction purity and low production efficiency in the paclitaxel separation and purification process in the prior art; the variety of the adopted organic solvents is relatively less, the dissolving agent and the mobile phase have the same components, the dissolving agent and the mobile phase can be mutually applied, the operation is flexible and simple, the separation and purification cost of the paclitaxel is reduced, the recovery rate of the paclitaxel is higher, and the method can be applied to large-scale separation and purification production of the paclitaxel; the flow valve is arranged at the bottom of the chromatographic column, and the flow rate of the mobile phase during elution is controlled, so that the chromatographic separation of the paclitaxel is facilitated, the purification effect is improved, the production period is shortened, the production process is simplified, the loss caused by the irreversible adsorption of the paclitaxel by multiple column chromatography in the bromine adding purification process is reduced, the yield of the paclitaxel is improved, and the separation and purification cost is greatly reduced.)

1. A process for separating and purifying paclitaxel comprises the following steps:

step 1, selecting a taxol crude product as a raw material, and dissolving the raw material by using a dissolving agent, wherein the weight ratio of the dissolving agent to the raw material is 1:5-12 to prepare a sample;

and 2, adopting silica gel as a stationary phase, wherein the weight ratio of the silica gel to the sample prepared in the step 1 is 10: 1-1.5;

step 3, adopting a mixed solvent of n-hexane, dichloromethane, acetone, triethylamine and ethyl acetate as a five-membered mobile phase;

step 4, separating and purifying the paclitaxel by adopting a column chromatography method, loading a sample from the top end of a chromatographic column,

step 5, eluting by adopting a mobile phase, controlling the flow rate of the mobile phase in the chromatographic column, and collecting effluent in hours;

step 6, concentrating and drying the effluent liquid in the step 5 to obtain a semi-finished product;

and 7, recrystallizing the semi-finished product in the step 6 for 2-4 times by adopting water and ethanol to obtain a paclitaxel finished product.

2. The process of claim 1, wherein the paclitaxel is prepared by the following steps: in the step 1, the content of the taxol in the taxol crude product is 2-10%.

3. The process of claim 1, wherein the paclitaxel is prepared by the following steps: chloroform or acetone is adopted as a dissolving agent in the step 1.

4. The process of claim 1, wherein the paclitaxel is prepared by the following steps: the mesh number of the silica gel in the step 2 is 220-280 meshes.

5. The process of claim 1, wherein the paclitaxel is prepared by the following steps: in the step 3, the volume ratio of n-hexane to dichloromethane to acetone to triethylamine to ethyl acetate is 6:3:1:1: 1.5.

6. The process of claim 1, wherein the paclitaxel is prepared by the following steps: the flow rate of the mobile phase in the step 5 is 10-15L/h.

7. The process of claim 1, wherein the paclitaxel is prepared by the following steps: and (3) detecting each group of eluent components obtained in the step (5) by adopting HPLC.

8. The process of claim 1, wherein the paclitaxel is prepared by the following steps: in the step 6, the drying temperature is 60-64 ℃, and the drying time is 36-38 hours.

9. The process of claim 1, wherein the paclitaxel is prepared by the following steps: the volume ratio of the water to the ethanol in the step 7 is 1: 1.1-1.3.

Technical Field

The invention relates to the technical field of compound separation and purification, in particular to a paclitaxel separation and purification process.

Background

Paclitaxel, a natural anticancer drug, with molecular formula C₄₇H₅₁NO₁₄Has been widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer clinically. Paclitaxel as a diterpene alkaloid compound with anticancer activity has a novel and complex chemical structure, wide and remarkable biological activity, a completely new and unique action mechanism and a scarce natural resource, so that the paclitaxel is subjected to phytologists and chemosynthesisThe great favor of scientists, pharmacologists and molecular biologists makes the research focus of the anticancer star which draws attention in the second half of the 20 th century.

Separation and purification means separating impurities from a mixture to improve the purity of the mixture. The separation and purification as an important chemical method not only has important function in chemical research, but also has very important function in chemical production. Many important chemical researches and chemical production are mainly based on separation and purification. Such as the oil industry, etc.

Paclitaxel (paclitaxel) is a diterpenoid compound isolated from the rare plant Taxus chinensis, has novel structure and unique anti-tumor mechanism, and can induce and stabilize microtubule polymerization, prevent microtubule from disintegrating, and stop cancer cells at G2/M until death. The product has wide anticancer range, particularly has special effect on breast cancer and ovarian cancer, and is proved by FAD in the United states in 1992, so that the product becomes an anticancer drug which has the best anticancer effect and the strongest activity and is generally accepted in the world today. However, there are still many problems associated with the scale preparation of paclitaxel in actual drug production. First, raw material resources are scarce. The taxol is mainly present in barks and needles of the taxus chinensis, and because wild taxus chinensis plants grow very slowly, rarely grow into forests, have very limited resources and have unique greening ornamental value, the taxol is listed as a first-level protection value object in China, and is forbidden to be felled and used for industrial production. Secondly, the content of paclitaxel in plants is very low, about 0.010% to 0.013%, and the chemical structure and polarity of paclitaxel is very similar to those of other taxane compounds, making their complete separation difficult. The pharmaceutical standard of the paclitaxel is more than 98 percent (content), but the current domestic separation and purification technology process can hardly meet the requirement, namely, the separation and purification products of part of production enterprises can meet the standard, but the separation and purification process is complex, the cost is very high, and the industrial production of the paclitaxel is also severely restricted.

The new species of Taxus media, which can replace wild Taxus chinensis, has been introduced from America and Japan in Beijing, Qingdao, Sichuan, Hunan and other provinces of China, and the taxol content of the Taxus media is far higher than that of wild Taxus media plants.

At present, the paclitaxel is extracted and separated by the most advanced carbon dioxide supercritical extraction technology in plant extraction in the world, and the effective ingredients of the Chinese herbal medicines can be extracted almost hundred percent by carbon dioxide due to different pertinence of the carbon dioxide to the selection of the substances in different pressure and temperature intervals. And has no harmful substance residue, and can completely kill oxygen-consuming microorganism in plant extract, and the bioactive substances are not destroyed, so that the extraction purity and production efficiency are high. However, the technology is still used for extracting paclitaxel in experimental research, the extraction process is not mature, or the process technology is mature, but the technology can only be used for crude extraction of paclitaxel products, and paclitaxel cannot be effectively separated from cephalomannine in the purification process.

Disclosure of Invention

In view of the problems in the prior art, the invention discloses a paclitaxel separation and purification process, which comprises the following steps:

step 1, selecting a taxol crude product as a raw material, and dissolving the raw material by using a dissolving agent, wherein the weight ratio of the dissolving agent to the raw material is 1:5-12 to prepare a sample;

and 2, adopting silica gel as a stationary phase, wherein the weight ratio of the silica gel to the sample prepared in the step 1 is 10: 1-1.5;

step 3, adopting a mixed solvent of n-hexane, dichloromethane, acetone, triethylamine and ethyl acetate as a five-membered mobile phase;

step 4, separating and purifying the paclitaxel by adopting a column chromatography, loading a sample from the top end of the chromatographic column, arranging a flow valve at the bottom of the chromatographic column, and controlling the flow rate of a mobile phase during elution, thereby facilitating the chromatographic separation of the paclitaxel and improving the purification effect;

step 5, eluting by adopting a mobile phase, controlling the flow rate of the mobile phase in the chromatographic column, and collecting effluent in hours;

step 6, concentrating and drying the effluent liquid in the step 5 to obtain a semi-finished product;

and 7, recrystallizing the semi-finished product in the step 6 for 2-4 times by adopting water and ethanol to prepare a taxol finished product, wherein the adopted organic solvents have relatively few varieties, the dissolving agent and the mobile phase have the same components and can be mutually applied, the operation is flexible and simple, the separation and purification cost of the taxol is reduced, the recovery rate of the taxol is higher, and the taxol can be applied to large-scale taxol separation and purification production.

As a preferred embodiment of the present invention, the paclitaxel content in the crude paclitaxel in step 1 is 2-10%.

As a preferable scheme of the invention, chloroform or acetone is used as the dissolving agent in the step 1; chloroform of the formula CHCl₃The liquid is colorless and transparent liquid, has special smell, sweet taste, high refraction, non-inflammability, heavy weight and easy volatilization; acetone, also known as dimethyl ketone, is an organic substance with molecular formula C₃H₆O, is the simplest saturated ketone. Is a colorless transparent liquid with slight fragrance; is easily soluble in water and organic solvent such as methanol, ethanol, diethyl ether, chloroform, pyridine, etc. Is inflammable and volatile, and has active chemical property.

As a preferable scheme of the invention, the mesh number of the silica gel in the step 2 is 220-280 meshes, and the silica gel and the substance to be separated have an effect under certain conditions, wherein the effect mainly comprises two effects of physical effect and chemical effect. The physical effect comes from van der waals forces between the surface of the silica gel and the solute molecules. The chemical action is mainly the hydrogen bonding between the silicon hydroxyl on the surface of the silica gel and the substance to be separated.

As a preferable scheme of the invention, in the step 3, the volume ratio of n-hexane, dichloromethane, acetone, triethylamine and ethyl acetate is 6:3:1:1: 1.5; the material carrying the component to be detected to move forward in the chromatographic process is called as a mobile phase; the other phase is in a balanced state with the stationary phase and drives the sample to move forwards; liquid chromatography is the mass exchange of sample components between column packing and a mobile phase for separation purposes.

As a preferable scheme of the invention, the flow rate of the mobile phase in the step 5 is 10-15L/h.

In a preferred embodiment of the present invention, the fractions of the plurality of eluates obtained in step 5 are detected by HPLC, which is also called high pressure liquid chromatography, high performance liquid chromatography, high resolution liquid chromatography, modern column chromatography, etc. High performance liquid chromatography is an important branch of chromatography, liquid is used as a mobile phase, a high-pressure infusion system is adopted, mobile phases such as single solvents with different polarities or mixed solvents, buffer solutions and the like with different proportions are pumped into a chromatographic column filled with a stationary phase, and after components in the column are separated, the mobile phases enter a detector for detection, so that analysis of a sample is realized.

In a preferable embodiment of the present invention, in the step 6, the drying temperature is 60-64 ℃, and the drying time is 36-38 hours.

As a preferred scheme of the invention, the volume ratio of the water to the ethanol in the step 7 is 1:1.1-1.3, so that the production period is shortened, the production process is simplified, the loss caused by irreversible adsorption of the paclitaxel by multiple column chromatography in a bromine and purification process is reduced, the yield of the paclitaxel is improved, and the separation and purification cost is greatly reduced.

The invention has the beneficial effects that: the method has the advantages of relatively few varieties of organic solvents, the same components in the dissolving agent and the mobile phase, mutual application, flexible and simple operation, reduction of the separation and purification cost of the paclitaxel, higher recovery rate of the paclitaxel and application in large-scale separation and purification production of the paclitaxel.

Furthermore, the flow valve is arranged at the bottom of the chromatographic column, and the flow rate of the mobile phase during elution is controlled, so that the chromatographic separation of the paclitaxel is facilitated, the purification effect is improved, the production period is shortened, the production process is simplified, the loss caused by irreversible adsorption of the paclitaxel by multiple column chromatography in the bromine and purification process is reduced, the yield of the paclitaxel is improved, and the separation and purification cost is greatly reduced.

Detailed Description

Example 1

The invention discloses a paclitaxel separation and purification process, which comprises the following steps:

step 1, selecting a taxol crude product as a raw material, and dissolving the raw material by using a dissolving agent, wherein the weight ratio of the dissolving agent to the raw material is 1:5 to prepare a sample; the taxol crude product is a raw material subjected to primary extraction, and the taxol content in the raw material is low, so that the taxol in the raw material needs to be further extracted;

and 2, adopting silica gel as a stationary phase, wherein the weight ratio of the silica gel to the sample prepared in the step 1 is 10: 1; adsorbing impurities in the paclitaxel raw material by utilizing the adsorbability of silica gel to realize the separation between paclitaxel and impurities;

step 3, adopting a mixed solvent of n-hexane, dichloromethane, acetone, triethylamine and ethyl acetate as a five-membered mobile phase, wherein the mobile phase has a certain dissolving capacity relative to a sample, and ensuring that components of the sample cannot be precipitated in a chromatographic column; the mobile phase also did not chemically react with paclitaxel;

step 4, separating and purifying paclitaxel by adopting a column chromatography, loading a sample from the top end of the chromatographic column, arranging a flow valve below the chromatographic column, and controlling the flow rate of a mobile phase in the chromatographic column by adjusting the opening degree of the flow valve; the bottom of the chromatographic column is provided with a flow valve, and the flow rate of the mobile phase during elution is controlled, so that the chromatographic separation of the paclitaxel is facilitated, and the purification effect is improved;

step 5, eluting by adopting a mobile phase, controlling the flow rate of the mobile phase in the chromatographic column, and collecting effluent in hours; storing the collected mobile phase fractions, detecting the content of the paclitaxel in each group of fractions by HPLC, separating out fractions with the paclitaxel content reaching the standard, and performing the next procedure; introducing the flow part with insufficient paclitaxel content from the top end of the chromatographic column, and continuously eluting the chromatographic column;

step 6, concentrating and drying the effluent liquid in the step 5 to obtain a semi-finished product; the paclitaxel content in the effluent liquid reaches the preset value of separation and purification;

and 7, recrystallizing the semi-finished product in the step 6 for 2 times by adopting water and ethanol to prepare a paclitaxel finished product, so that the production period is shortened, the production process is simplified, the loss caused by irreversible adsorption of paclitaxel by multiple column chromatography in a bromine and purification process is reduced, the yield of paclitaxel is improved, and the separation and purification cost is greatly reduced.

As a preferred technical scheme of the invention, the content of the taxol in the taxol crude product in the step 1 is 2%.

In a preferred embodiment of the present invention, chloroform or acetone is used as the solvent in step 1, acetone is a representative compound of aliphatic ketones, and has a typical reaction of ketones, and acetone is an active chemical.

As a preferred technical scheme of the present invention, the mesh number of the silica gel in the step 2 is 220 meshes, the larger the mesh number of the silica gel is, the better the separation effect is, but the flow rate of the mobile phase will be reduced, and the suitable mesh number of the silica gel is selected, so that the separation effect is improved, the flow rate of the mobile phase can be ensured, and the separation and purification efficiency is also improved.

As a preferred technical scheme of the invention, in the step 3, the volume ratio of n-hexane, dichloromethane, acetone, triethylamine and ethyl acetate is 6:3:1:1:1.5, the viscosity of the mobile phase is low, and a good separation effect is obtained; meanwhile, the pressure drop of the chromatographic column is reduced, the service life is prolonged, the variety of the adopted organic solvents is relatively less, the dissolving agent and the mobile phase have the same components, the dissolving agent and the mobile phase can be mutually applied, the operation is flexible and simple, the separation and purification cost of the paclitaxel is reduced, the recovery rate of the paclitaxel is higher, and the method can be applied to large-scale separation and purification production of the paclitaxel.

As a preferable technical scheme of the invention, the flow rate of the mobile phase in the step 5 is 10L/h.

As a preferred technical scheme of the invention, the plurality of groups of eluent fractions obtained in the step 5 are detected by HPLC, and HPLC are adopted, and the advantages of repeated use of chromatographic columns, no damage to samples, easy recovery and the like are achieved.

As a preferable technical scheme of the present invention, in the step 6, the drying temperature is 60 ℃, and the drying time is 36 hours.

As a preferred technical scheme of the invention, the volume ratio of the water to the ethanol in the step 7 is 1:1.1, so that the production period is shortened, the production process is simplified, the loss caused by irreversible adsorption of the paclitaxel by multiple column chromatography in a bromine and purification process is reduced, the yield of the paclitaxel is improved, and the separation and purification cost is greatly reduced.

Example 2

This example differs from example 1 in that: step 1, selecting a taxol crude product as a raw material, and dissolving the raw material by using a dissolving agent, wherein the weight ratio of the dissolving agent to the raw material is 1:12 to prepare a sample;

and 2, adopting silica gel as a stationary phase, wherein the weight ratio of the silica gel to the sample prepared in the step 1 is 10: 1.5;

and 7, recrystallizing the semi-finished product in the step 6 for 4 times by adopting water and ethanol to obtain a paclitaxel finished product.

As a preferred technical scheme of the invention, the content of the taxol in the taxol crude product in the step 1 is 10%.

As a preferred technical scheme of the invention, the mesh number of the silica gel in the step 2 is 280 meshes.

As a preferable technical scheme of the present invention, in the step 6, the drying temperature is 64 ℃, and the drying time is 38 hours.

In a preferred embodiment of the present invention, the volume ratio of water to ethanol in step 7 is 1: 1.3.

Example 3

This example differs from example 1 in that: step 1, selecting a taxol crude product as a raw material, and dissolving the raw material by using a dissolving agent, wherein the weight ratio of the dissolving agent to the raw material is 1:8.5 to prepare a sample;

and 2, adopting silica gel as a stationary phase, wherein the weight ratio of the silica gel to the sample prepared in the step 1 is 10: 1.25; and 7, recrystallizing the semi-finished product in the step 6 for 3 times by adopting water and ethanol to obtain a paclitaxel finished product.

As a preferred technical scheme of the invention, the content of the taxol in the taxol crude product in the step 1 is 6%.

As a preferred technical scheme of the invention, the mesh number of the silica gel in the step 2 is 250 meshes.

As a preferable technical solution of the present invention, in the step 6, the drying temperature is 62 ℃, and the drying time is 37 hours.

In a preferred embodiment of the present invention, the volume ratio of water to ethanol in step 7 is 1: 1.2.

Components not described in detail herein are prior art.

Although the present invention has been described in detail with reference to the specific embodiments, the present invention is not limited to the above embodiments, and various changes and modifications without inventive changes may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.