阅读说明：本技术 超临界co2萃取曼地亚红豆杉枝条中的紫杉醇方法 (Supercritical CO2Method for extracting paclitaxel from taxus media branches ) 是由 王强 于 2021-09-30 设计创作，主要内容包括：本发明属于溶剂萃取技术领域,公开了一种超临界CO-(2)萃取曼地亚红豆杉枝条中紫杉醇方法,该方法为：称取6000g粉碎后的曼地亚红豆杉放置于超临界装置的萃取罐中；通过CO-(2)泵和夹带剂泵将CO-(2)和夹带剂打入各泵的腔体中,经流体混合器混合后,流入萃取釜中的集流腔中,达到萃取条件后进行萃取；萃取时,超临界CO-(2)经分离釜Ⅰ和Ⅱ减压排放,流体带出的萃取物分别从分离釜Ⅰ和Ⅱ收集；将收集物旋转蒸发浓缩,并于50℃真空干燥2h后检测其中的紫杉醇含量。本发明大幅提高了提取效率,降低长期运行的生产成本,提取物有效成分不易被破坏,其残留的有毒有害污染物质降到了零,更有利于对人员和环境的保护。(The invention belongs to the technical field of solvent extraction and discloses supercritical CO 2 The method for extracting taxol from taxus media branches comprises the following steps: 6000g of crushed Taxus media are weighed and placed in an extraction tank of a supercritical device; by CO 2 Pump and entrainer Pump CO 2 And entrainer are pumped into the cavity of each pump, mixed by a fluid mixer and then flow into a collector in the extraction kettle, and extraction is carried out after the extraction conditions are reached; during extraction, supercritical CO 2 The extract carried out by the fluid is respectively collected from the separation kettles I and II after being discharged by the separation kettles I and II under reduced pressure; the collected material was concentrated by rotary evaporation and dried under vacuum at 50 ℃ for 2 hours, and then the content of paclitaxel was measured. The invention greatly improves the extraction efficiency, reduces the production cost of long-term operation, ensures that the effective components of the extract are not easy to damage, reduces the residual toxic and harmful pollutants to zero, and is more favorable for protecting personnel and environment.)

1. Supercritical CO₂The method for extracting the paclitaxel from the taxus media branches is characterized in that the supercritical CO is adopted₂The method for extracting paclitaxel from taxus media branches comprises the following steps:

the method comprises the following steps: 6000g of crushed Taxus media are weighed and placed in an extraction tank of a supercritical device;

step two: by CO₂Pump and entrainer Pump CO₂And entrainer are pumped into the cavity of each pump, mixed by a fluid mixer and then flow into a collector in the extraction kettle, and extraction is carried out after the extraction conditions are reached;

step three: during extraction, supercritical CO₂The extract carried out by the fluid is respectively collected from the separation kettles I and II after being discharged by the separation kettles I and II under reduced pressure;

step four: the collected material was concentrated by rotary evaporation and dried under vacuum at 50 ℃ for 2 hours, and then the content of paclitaxel was measured.

2. The supercritical CO of claim 1₂The method for extracting the paclitaxel from the taxus media branches is characterized in that the extraction conditions are as follows: the extraction is started when the pressure reaches 30MPa and the temperature is 45 ℃, and the extraction time is 180 min.

3. The supercritical CO of claim 1₂The method for extracting the paclitaxel from the taxus media branches is characterized in that the entrainer is an ethanol solution with the mass fraction of 95 percent, and the dosage is 20 percent.

4. The supercritical CO of claim 1₂The method for extracting paclitaxel from taxus media branches is characterized in that CO is₂The volume flow of (2) is 284L/h.

5. The supercritical CO of claim 1₂The method for extracting paclitaxel from taxus media branches is characterized in that the temperature of the separation kettle I is 30 ℃, the pressure is 6.5MPa, and the temperature of the separation kettle II is 30 ℃, and the pressure is 5.5 MPa.

6. The supercritical CO of claim 1₂Extracting Taxus media branchesThe method for detecting the content of the paclitaxel is characterized in that the content of the paclitaxel in a sample is calculated by adopting a high performance liquid chromatograph according to an external standard method by peak area, and specifically comprises the following steps:

the method comprises the following steps: accurately weighing appropriate amount of paclitaxel, impurity I and impurity II reference substances, adding acetonitrile to dissolve and dilute to obtain solution containing paclitaxel 0.5mg, impurity I and impurity II 2.5 μ g per 1ml, and using as system applicability test solution;

step two: accurately weighing taxol reference substance about 12mg, placing into a 100ml measuring flask, adding acetonitrile, dissolving, diluting to scale, and shaking to obtain reference substance solution;

step three: dissolving the extract in acetonitrile, and diluting to obtain solution containing 0.5mg per 1ml as test solution;

step four: injecting 10 μ l of the system applicability test solution into a liquid chromatograph, wherein the separation degrees of the paclitaxel peak, the impurity I peak and the impurity II peak are both more than 1.0; precisely measuring 10 μ l of the sample solution, injecting into a liquid chromatograph, recording chromatogram, measuring the reference solution by the same method, and calculating by peak area according to external standard method.

7. The supercritical CO of claim 6₂The method for extracting the paclitaxel from the taxus media branches is characterized in that the impurity I is cephalomannine; impurity II is 10-deacetyl paclitaxel.

8. The supercritical CO of claim 6₂The method for extracting the paclitaxel from the taxus media branches is characterized in that the chromatographic conditions of the high performance liquid chromatograph are as follows:

a chromatographic column: AgilentZORBAX SB-C18(150mmx4.6mm, 5 um);

mobile phase: methanol: water: acetonitrile (23:41:36)

The flow rate is 1.0 mL/min; the sample injection amount is 10 uL; the ultraviolet detection wavelength is 227 nm.

Technical Field

The invention belongs to the technical field of solvent extraction, and particularly relates to supercritical CO₂A method for extracting paclitaxel from Taxus media branches is provided.

Background

Currently, the current state of the art commonly used in the industry is such that: in 1971, the American chemist Wani et al isolated paclitaxel, a taxane diterpene compound with high anticancer activity, from the bark of North American Taxus brevifolia (T. brevifolia), found that paclitaxel has unique antitumor effect. Clinical studies of paclitaxel began in 1982, and completed the clinical trial at stage I, II and 1990 the clinical trial at stage III was performed by 1989. The national Food and Drug Administration (FDA) of 29 Japanese and American countries officially approves the drugs as anticancer drugs for advanced ovarian cancer, lung cancer, uterine cancer and the like to be on the market in 1992 at 12 months. Paclitaxel was approved for marketing in china as a new drug in 1995.

In recent years, anticancer drugs have developed rapidly and emerge endlessly. However, the first major anticancer drug is paclitaxel, which is an old drug used for many years. Paclitaxel is the first-choice anti-tumor drug in hospitals in various countries in the world.

Paclitaxel extraction from Taxus chinensis is one of its major sources. It is characterized by the structural conformity with the paclitaxel approved by FDA to be on the market.

Paclitaxel is mainly derived from roots, bark, branches and leaves of Taxus chinensis. Paclitaxel is the main active ingredient in Taxus chinensis, and the chemical structure of the active ingredient is a tetracyclic diterpene compound.

Taxol in Taxus chinensis is a macromolecular and thermosensitive substance due to its low content and many structural analogs. Under the influence of temperature, organic solvent, acid, alkali and other environmental conditions, other taxane substances are easily degraded or isomerized during the extraction process. These characteristics bring great difficulties to extraction and separation.

At present, most of methods for extracting paclitaxel from taxus chinensis adopt organic solvents such as ethyl acetate, dichloromethane, acetone, chloroform and the like to extract taxus chinensis raw materials for multiple times, and extract liquor is recovered and then extracted by the organic solvents, and is separated to obtain extract containing paclitaxel. The method has the following outstanding problems:

1. the process route is long and the solvent consumption is large. The extraction efficiency is low, the production cost is high, and the problem of great environmental protection exists.

2. The heating time is long in the leaching and recovery processes, and the structure of the paclitaxel is easy to change.

3. And (4) residual organic solvent in the waste residue. Causing adverse effects on personnel and the environment.

Therefore, the method has important practical significance on how to explore and select a production process which is low in cost, more environment-friendly and free of long-time heating to extract the paclitaxel.

In summary, the problems of the prior art are as follows:

1. the process route is long and the solvent consumption is large. The extraction efficiency is low, the production cost is high, and the problem of great environmental protection exists.

2. The heating time is long in the leaching and recovery processes, and the structure of the paclitaxel is easy to change.

3. And (4) residual organic solvent in the waste residue. Causing adverse effects on personnel and the environment.

Disclosure of Invention

The technical problem solved by the invention is to provide a method for extracting paclitaxel from taxus media, which effectively solves the problems of low extraction efficiency, high production cost, and avoidance of influence on environment caused by long-time heating.

The invention is realized by the following steps of₂A method for extracting paclitaxel from Taxus media branches, said supercritical CO₂The method for extracting paclitaxel from taxus media branches comprises the following steps:

the method comprises the following steps: 6000g of crushed Taxus media are weighed and placed in an extraction tank of a supercritical device;

step two: by CO₂Pump and entrainer Pump CO₂And entrainer are pumped into the cavity of each pump, mixed by a fluid mixer and then flow into a collector in the extraction kettle, and extraction is carried out after the extraction conditions are reached;

step three: during extraction, supercritical CO₂The extract carried out by the fluid is respectively collected from the separation kettles I and II after being discharged by the separation kettles I and II under reduced pressure;

step four: the collected material was concentrated by rotary evaporation and dried under vacuum at 50 ℃ for 2 hours, and then the content of paclitaxel was measured.

Further, the extraction conditions are as follows: the extraction is started when the pressure reaches 30MPa and the temperature is 45 ℃, and the extraction time is 180 min.

Further, the entrainer is an ethanol solution with the mass fraction of 95%, and the using amount is 20%.

Further, the CO is₂The volume flow of (2) is 284L/h.

Further, the temperature of the separation kettle I is 30 ℃, the pressure is 6.5MPa, and the temperature of the separation kettle II is 30 ℃, and the pressure is 5.5 MPa.

The invention also aims to provide a method for detecting the content of paclitaxel, which adopts a high performance liquid chromatograph to calculate the content of paclitaxel in a sample by peak area according to an external standard method, and specifically comprises the following steps:

the method comprises the following steps: accurately weighing appropriate amount of paclitaxel, impurity I and impurity II reference substances, adding acetonitrile to dissolve and dilute to obtain solution containing paclitaxel 0.5mg, impurity I and impurity II 2.5 μ g per 1ml, and using as system applicability test solution;

step two: accurately weighing taxol reference substance about 12mg, placing into a 100ml measuring flask, adding acetonitrile, dissolving, diluting to scale, and shaking to obtain reference substance solution;

step three: dissolving the extract in acetonitrile, and diluting to obtain solution containing 0.5mg per 1ml as test solution;

step four: injecting 10 μ l of the system applicability test solution into a liquid chromatograph, wherein the separation degrees of the paclitaxel peak, the impurity I peak and the impurity II peak are both more than 1.0; precisely measuring 10 μ l of the sample solution, injecting into a liquid chromatograph, recording chromatogram, measuring the reference solution by the same method, and calculating by peak area according to external standard method.

Further, the impurity I is cephalomannine; impurity II is 10-deacetyl paclitaxel.

Further, the chromatographic conditions of the high performance liquid chromatograph are as follows:

a chromatographic column: AgilentZORBAX SB-C18(150mmx4.6mm, 5 um);

mobile phase: methanol: water: acetonitrile (23:41:36)

The flow rate is 1.0 mL/min; the sample injection amount is 10 uL; the ultraviolet detection wavelength is 227 nm.

In summary, the advantages and positive effects of the invention are: the invention adopts supercritical CO₂The extraction efficiency of the taxol in the taxus media branches is greatly improved, the production cost of long-term operation is reduced, the taxol extraction method is particularly suitable for extracting heat-sensitive substances, the effective components of the extract are not easy to damage, and compared with the method for extracting the taxol from taxus media raw materials by using organic solvents which are mostly adopted at present, the taxol extraction method has the advantages that the residual toxic and harmful pollutants are reduced to zero, and the taxol extraction method is more beneficial to the protection of personnel and the environment.

The invention passes supercritical CO₂The extraction effect of the paclitaxel applied to the taxus media branches is remarkable, the solvent is easy to be immersed into the raw materials, the mass transfer process is strengthened, and the extraction efficiency is high; the regulation of the entrainer improves the selectivity of the solvent, and the mass fraction of the paclitaxel in the extract is higher.

Drawings

FIG. 1 shows supercritical CO provided by an embodiment of the present invention₂A flow chart of a method for extracting paclitaxel from taxus media branches;

FIG. 2 is a flow chart of a method for detecting paclitaxel according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following 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.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

As shown in FIG. 1, the supercritical CO provided by the embodiment of the invention₂The method for extracting paclitaxel from taxus media branches comprises the following steps:

s101: 6000g of crushed Taxus media are weighed and placed in an extraction tank of a supercritical device;

s102: by CO₂Pump and entrainer Pump CO₂And entrainer are pumped into the cavity of each pump, mixed by a fluid mixer and then flow into a collector in the extraction kettle, and extraction is carried out after the extraction conditions are reached;

s103: during extraction, supercritical CO₂The extract carried out by the fluid is respectively collected from the separation kettles I and II after being discharged by the separation kettles I and II under reduced pressure;

s104: the collected material was concentrated by rotary evaporation and dried under vacuum at 50 ℃ for 2 hours, and then the content of paclitaxel was measured.

The extraction conditions provided by the embodiment of the invention are as follows: the extraction is started when the pressure reaches 30MPa and the temperature is 45 ℃, and the extraction time is 180 min.

The entrainer provided by the embodiment of the invention is an ethanol solution with the mass fraction of 95%, and the using amount is 20%.

CO provided by the embodiment of the invention₂The volume flow of (2) is 284L/h.

The temperature of the separation kettle I provided by the embodiment of the invention is 30 ℃, the pressure is 6.5MPa, and the temperature of the separation kettle II is 30 ℃, and the pressure is 5.5 MPa.

As shown in fig. 2, the method for detecting paclitaxel content provided in the embodiment of the present invention is to calculate the content of paclitaxel in a sample by peak area according to an external standard method by using a high performance liquid chromatograph, and specifically includes:

s201: accurately weighing appropriate amount of paclitaxel, impurity I and impurity II reference substances, adding acetonitrile to dissolve and dilute to obtain solution containing paclitaxel 0.5mg, impurity I and impurity II 2.5 μ g per 1ml, and using as system applicability test solution;

s202: accurately weighing taxol reference substance about 12mg, placing into a 100ml measuring flask, adding acetonitrile, dissolving, diluting to scale, and shaking to obtain reference substance solution;

s203: dissolving the extract in acetonitrile, and diluting to obtain solution containing 0.5mg per 1ml as test solution;

s204: injecting 10 μ l of the system applicability test solution into a liquid chromatograph, wherein the separation degrees of the paclitaxel peak, the impurity I peak and the impurity II peak are both more than 1.0; precisely measuring 10 μ l of the sample solution, injecting into a liquid chromatograph, recording chromatogram, measuring the reference solution by the same method, and calculating by peak area according to external standard method.

The impurity I provided by the embodiment of the invention is cephalomannine; impurity II is 10-deacetyl paclitaxel.

The chromatographic conditions of the high performance liquid chromatograph provided by the embodiment of the invention are as follows:

a chromatographic column: AgilentZORBAX SB-C18(150mmx4.6mm, 5 um);

mobile phase: methanol: water: acetonitrile (23:41:36)

The flow rate is 1.0 mL/min; the sample injection amount is 10 uL; the ultraviolet detection wavelength is 227 nm.

The invention is further described with reference to specific examples.

The extraction method of the invention is compared with the process flow of the existing using method.

Supercritical CO₂Paclitaxel extraction process (paclitaxel extraction content can be more than 0.10% in the propanone)

Taxus media branch → fresh material cleaning → drying → crushing → supercritical CO₂And (4) extracting → detecting the content of the extract.

Extraction conditions are as follows: the pressure of the extraction kettle is 30MPa, the temperature of the extraction kettle is 45 ℃, the extraction time is 180min, and CO is added₂The volume flow of (2) is 284L/h, the entrainer is C₂H₂An alcoholic solution of OH (concentration: 95%, amount: 20%); the temperature of the separation kettle I is 30 ℃, the pressure is 6.5MPa, the temperature of the separation kettle II is 30 ℃, and the pressure is 5.5 MPa.

Each batch can be finished after 180 min.

Second, the production process flow of the current organic solvent extraction method (taking the extraction paclitaxel content more than or equal to 0.08 Hada as an example)

Drying Taxus chinensis raw material until water content is less than or equal to 15%, pulverizing, extracting with methanol under thermal cycle for 3 times, each time for 1 hr, mixing extractive solutions, and vacuum concentrating.

Adding 2 times (V/V) of ethyl acetate of the crude product A, stirring to fully dissolve effective components in the crude product A in the ethyl acetate, adding 1 time (V/V) of drinking water of the crude product A, stirring for 10 times at intervals, and standing for 6 hours; the extraction with ethyl acetate in the aqueous phase was repeated 2 times. And combining the first extraction liquid and the second extraction liquid, and concentrating to dryness at the temperature of 50-60 ℃ and the vacuum degree of more than or equal to-0.04 MPa.

Each batch was completed in at least 19 hours.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.