CN112694458A - Extraction and purification method of 10-deacetylbaccatin III - Google Patents
Extraction and purification method of 10-deacetylbaccatin III Download PDFInfo
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 5
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- 229960003668 docetaxel Drugs 0.000 description 4
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
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- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Epoxy Compounds (AREA)
Abstract
The invention discloses a method for extracting and purifying 10-deacetylbaccatin III, which comprises the steps of processing branches and leaves of taxus chinensis, extracting, filtering, concentrating, extracting, washing and concentrating to obtain a crude product A, carrying out freezing and suction filtration on the crude product A to obtain a crude product B, concentrating, freezing, filtering and washing the crude product B to obtain a crude product C, and concentrating, freezing and filtering the crude product C to obtain a final product of extracting and purifying 10-deacetylbaccatin III. After the taxus chinensis branches and leaves are extracted and extracted, the acidic impurities and the pigments in the materials are removed by washing with a sodium bicarbonate aqueous solution, so that the content of 10-deacetylbaccatin III in an extraction concentrate is more than or equal to 10 percent, the standard of acetonitrile washing is achieved, the whole process removes a chromatographic separation step, the production efficiency is improved, the production time is shortened, and the crude product B is washed by an acetone-normal hexane mixed solvent after being crystallized to remove the pigments in the crude product B. The content of the finally obtained 10-deacetylbaccatin III reaches up to 99.23 percent, the purity reaches up to 99.37 percent, and the product yield reaches 75.99 percent.
Description
Technical Field
The invention relates to the technical field of compound separation and purification, in particular to a method for extracting and purifying 10-deacetylbaccatin III from branches and leaves of southern taxus chinensis.
Background
Paclitaxel and docetaxel are currently important taxane anticancer drugs for treating advanced or metastatic breast cancer which fails in advanced chemotherapy, advanced or metastatic non-small cell lung cancer which fails in chemotherapy mainly by using cisplatin, treating breast cancer which can be operated and has positive lymph nodes by combining adriamycin and cyclophosphamide, treating locally advanced or metastatic non-small cell lung cancer at first line by combining cisplatin, treating androgen-independent metastatic prostate cancer by combining prednisone and treating advanced gastric cancer at first line by combining cisplatin and 5-fluorouracil. The taxol is naturally present in the yew, the docetaxel is obtained by modifying and synthesizing the structure of a taxane compound, and an important precursor substance 10-deacetylbaccatin III of the taxol biologically synthesized by the yew plant is a diterpenoid taxane derivative and is a main raw material for semi-synthesizing and producing the taxol and the docetaxel at present.
The 10-deacetylbaccatin III is mainly distributed in branches and leaves of annual yew, the development of Chinese yew breeding and planting technology in nearly 20 years cultivates a plurality of excellent yew varieties, the excellent southern yew variety bred and planted in large scale at present has the 10-deacetylbaccatin III content of 0.2-0.4 percent, the branches and leaves of the yew are renewable resources, and the large-scale breeding and planting of the 10-deacetylbaccatin III high-content excellent variety yew enables the semi-synthesis of taxol and docetaxel to become a research hotspot, and meanwhile, the 10-deacetylbaccatin III extraction and purification process optimization and improvement are more and more emphasized.
The application number is CN201510603454, the patent name is a method for preparing 10-deacetylbaccatin III by taking branches and leaves of Taxus media as raw materials, macroporous resin adsorption is carried out, different single solvents are adopted to dissolve a crude product, a large amount of insoluble impurities are removed, and then the 10-deacetylbaccatin III product is obtained through multiple crystallization. Application No. CN1473821A, entitled method for extracting and purifying 10-deacetylbaccatin III (10-DAB III), 10-deacetylbaccatin III (10-DAB III) is purified by silica gel chromatography.
The existing industrial production of 10-deacetylbaccatin III has the main defects that most processes adopt a silica gel chromatography separation method and a resin adsorption separation method in the purification process, the two production processes are long in time consumption and low in production efficiency, so that the product cost is increased, and the due profits of enterprises are occupied.
Disclosure of Invention
In view of the above disadvantages, the present invention aims to provide a method for extracting and purifying 10-deacetylbaccatin iii, which can improve the production efficiency and save the production cost.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a method for extracting and purifying 10-deacetylbaccatin III comprises the following steps:
s1 treatment of branches and leaves of yew: drying branches and leaves of taxus chinensis until the water content is less than or equal to 15%, and crushing;
s2 extraction and filtration: adding methanol-water solution, extracting for multiple times at 50-60 ℃, filtering to obtain an extracting solution, and combining the extracting solutions;
s3 concentration and extraction: under the vacuum condition of 55-65 ℃, concentrating until the volume of the residual concentrate is 0.3-0.6 times of the weight of the taxus chinensis branches and leaves, adding dichloromethane into the concentrate for extracting for multiple times, and combining the extract liquor;
s4 washing and concentrating: adding a sodium bicarbonate aqueous solution into the extract, slowly stirring and washing for 20-40 min, removing acidic impurities and pigments in the material, and concentrating the washed extract at 55-65 ℃ under a vacuum condition until the extract is dried to obtain a 10-deacetylbaccatin III crude product A;
s5 crude product A is subjected to freezing and suction filtration: adding acetonitrile into the 10-deacetylbaccatin III crude product A, uniformly stirring at room temperature, freezing and standing at low temperature for 15-20 hours, carrying out suction filtration, adding acetonitrile into the obtained filter cake, uniformly stirring at room temperature, freezing and standing at low temperature for 15-20 hours, carrying out suction filtration, and drying the filter cake under vacuum at 55-65 ℃ for 20-30 hours under reduced pressure to obtain a 10-deacetylbaccatin III crude product B;
s6: and (3) concentrating, freezing, filtering and washing the crude product B: adding acetone into the 10-deacetylbaccatin III crude product B, heating at 50-60 ℃, stirring and dissolving completely, then concentrating at 55-65 ℃ under a vacuum condition until 8 times of volume of saturated feed liquid is remained, stopping concentrating when a small amount of crystallized particles appear, cooling to room temperature, freezing and crystallizing at a low temperature for 15-20 hours, filtering, adding an acetone-n-hexane mixed solution into a filter cake, heating, stirring and washing for 40-80 minutes, cooling to room temperature, filtering, and drying the filter cake under a vacuum condition at 55-65 ℃ for 15-20 hours under a reduced pressure condition to obtain a 10-deacetylbaccatin III crude product C;
concentrating, freezing and filtering the S7 crude product C: adding acetone into the 10-deacetylbaccatin III crude product C, heating at 50-60 ℃, stirring to dissolve completely, then concentrating at 55-65 ℃ under a vacuum condition until 8 times of volume of saturated feed liquid is remained, stopping concentrating when a small amount of crystalline particles appear, cooling to room temperature, freezing and crystallizing at a low temperature for 15-20 hours, filtering, and decompressing a filter cake at 55-65 ℃ under a vacuum condition for 48 hours to obtain a 10-deacetylbaccatin III product with the content and the purity meeting the standard.
Preferably, in the step S1, 100kg of the branches and leaves of the taxus mairei are weighed, dried until the water content is 9.65%, and then crushed, wherein the particle size of the crushed powder is 10-20 meshes.
Preferably, in step S2, 80% methanol-water solution is added, and the extraction is performed for 3 times at 55 ℃, and a methanol-water mixed solvent with a volume 5 times of the weight of the branches and leaves is added for the first time, and the extraction is performed for 8 hours; adding a methanol-water mixed solvent with the volume 3 times of the weight of the branches and leaves for the second time, extracting for 8 hours, and adding a methanol-water mixed solvent with the volume 2 times of the weight of the branches and leaves for the third time, extracting for 4 hours.
Preferably, in the step S3, the mixture is concentrated at 60 ℃ and a vacuum degree of-0.060 to-0.085 MPa until the volume of the remaining concentrate is 0.5 times of the weight of the yew branches and leaves, the extraction is performed for 2 times, dichloromethane with the volume 2 times of that of the concentrate is extracted for the first time, and dichloromethane with the volume 1 time of that of the concentrate is extracted for the second time.
Preferably, in step S4, 5% sodium bicarbonate aqueous solution with 0.6 times volume of the extract is added, slowly stirred and washed for 30min, and then concentrated to dryness at 60 ℃ and vacuum degree of-0.020 to-0.085 MPa.
Preferably, in the step S5, acetonitrile with a volume 4.5 times of the weight of the crude product a is added, the mixture is frozen and stood at a low temperature of-25 ℃ for 16 hours, acetonitrile with a volume 2 times of the weight of the crude product a is added to the filter cake after suction filtration, the mixture is frozen and stood at a low temperature of-25 ℃ for 16 hours, and the filter cake after suction filtration is dried under vacuum at a temperature of 60 ℃ and a vacuum degree of-0.085 MPa for 24 hours under reduced pressure.
Preferably, in step S7, acetone with a volume 30 times of the weight of the crude product B is added, the crude product B is heated and stirred at 55 ℃ to be completely dissolved, the crude product B is concentrated at 60 ℃ and a vacuum degree of-0.03 to-0.04 MPa by using a rotary evaporator, the crude product B is frozen and crystallized at a low temperature of-25 ℃ for 16 hours, acetone-n-hexane with a volume 2 times of the weight of the crude product B is added to a filter cake, and the acetone: n-hexane 3:7, heating, stirring and washing at 55 ℃ for 1 hour, and drying the filter cake at 60 ℃ under vacuum of-0.085 MPa for 16 hours.
Preferably, in step S8, acetone with a volume 30 times of the weight of the crude product C is added, the mixture is heated and stirred at 55 ℃ to be completely dissolved, the mixture is concentrated by a rotary evaporator at 60 ℃ and a vacuum degree of-0.03 to-0.04 MPa, the mixture is frozen and crystallized at a low temperature of-25 ℃ for 16 hours, and the filter cake is dried under vacuum at 60 ℃ and a vacuum degree of-0.085 MPa for 48 hours under reduced pressure.
The invention has the beneficial effects that:
1. the invention takes the branches and leaves of taxus chinensis as raw materials, after extraction and extraction, the extraction is washed by sodium bicarbonate aqueous solution to remove acidic impurities and pigments in the materials, so that the content of 10-deacetylbaccatin III in the extract concentrate is more than or equal to 10 percent, thereby achieving the standard of washing acetonitrile, eliminating the step of chromatographic separation in the whole process, improving the production efficiency and shortening the production time;
2. after the crude product B is crystallized, washing the crude product B by using an acetone-n-hexane mixed solvent to remove pigments in the crude product B;
3. in the 10-deacetylbaccatin III product extracted and purified by the method, the content of the 10-deacetylbaccatin III is up to 99.23 percent, the purity is up to 99.37 percent, and the product yield is up to 75.99 percent.
Detailed Description
In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given in conjunction with the embodiments.
Example 1:
weighing 100kg of southern taxus chinensis branches and leaves (the content of 10-deacetylbaccatin III is 0.327 percent), drying the southern taxus chinensis branches and leaves until the water content is less than or equal to 15 percent, crushing the southern taxus chinensis branches and leaves into particles with the particle size of 10-20 meshes, adding 80 percent methanol-water solution, carrying out thermal cycle extraction at 55 ℃ for 3 times:
adding 500L of methanol-water mixed solvent for the first extraction, and extracting for 8 hours;
adding 300L of methanol-water mixed solvent for the second extraction, and extracting for 8 hours;
adding 200L methanol-water mixed solvent for the third extraction, and extracting for 4 hr;
discharging extractive solution from the bottom of the extraction tank, mixing the extractive solutions for 3 times to obtain about 820L extractive solution, concentrating at 60 deg.C and vacuum degree of-0.060-0.085 MPa to obtain about 50L (weight of 51.7kg), extracting the concentrate with dichloromethane for 2 times:
100L of dichloromethane is added for the first extraction;
adding 50L of dichloromethane for the second extraction;
and combining about 160L of the extract liquor obtained in 2 times, adding 96L of 5% sodium bicarbonate aqueous solution, slowly stirring and washing for 30min, concentrating the washed extract liquor to be dry at the temperature of 60 ℃ and the vacuum degree of-0.020-0.085 MPa to obtain 3.1kg of 10-deacetylbaccatin III crude product A, wherein the content of 10-deacetylbaccatin III in the crude product A is 12.04%, and the content of dry matter is 80.65%.
Adding 11.3L of acetonitrile into 3.1kg of 10-deacetylbaccatin III crude product A, uniformly stirring at room temperature, freezing and standing for 16 hours at a low-temperature freezer of-25 ℃, carrying out suction filtration, adding 5L of acetonitrile into a filter cake, uniformly stirring at room temperature, freezing and standing for 16 hours at a low-temperature freezer of-25 ℃, carrying out suction filtration, carrying out vacuum reduced pressure drying on the filter cake at the temperature of 60 ℃ and the vacuum degree of-0.085 MPa for 24 hours to obtain 640g of 10-deacetylbaccatin III crude product B, wherein the 10-deacetylbaccatin III is only slightly soluble in the acetonitrile, and the acetonitrile is well dissolved in other impurities, so that most of impurities are removed, the amount of the removed impurities is about 70%, and the content of the 10-deacetylbaccatin III in the obtained crude product.
Adding 19.2L of acetone into 640g of 10-deacetylbaccatin III crude product B, heating at 55 ℃, stirring and dissolving completely, concentrating by using a rotary evaporator at 60 ℃ and under the vacuum degree of-0.03 to-0.04 MPa until 5.5L of feed liquid is remained, stopping concentrating, cooling to room temperature when a small amount of crystal particles appear, putting into a low-temperature freezer at-25 ℃ for freeze crystallization for 16 hours, filtering, adding 1.3L of acetone-n-hexane (acetone: n-hexane: 3:7) into a filter cake, heating at 55 ℃, stirring and washing for 1 hour, cooling to room temperature, filtering, drying the filter cake at 60 ℃ and under the vacuum degree of-0.085 MPa for 16 hours in vacuum, and obtaining 310.8g of 10-deacetylbaccatin III crude product C, wherein the content of 10-deacetylbaccatin III in the crude product C is 84.86%.
Adding 9.3L of acetone into 310.8g of 10-deacetylbaccatin III crude product C, heating at 55 ℃, stirring for dissolving completely, concentrating by using a rotary evaporator at the temperature of 60 ℃ and the vacuum degree of-0.03 to-0.04 MPa until about 2.5L of feed liquid is remained, stopping concentrating when a small amount of crystal particles appear, cooling to room temperature, putting into a low-temperature freezer at-25 ℃ for freezing and crystallizing for 16 hours, filtering, and drying a filter cake at the temperature of 60 ℃ and the vacuum degree of-0.085 MPa for 48 hours under vacuum reduced pressure to obtain 248.5g of white 10-deacetylbaccatin III product.
In the finally obtained 10-deacetylbaccatin III product, the content of the 10-deacetylbaccatin III is up to 99.23 percent, the purity is up to 99.37 percent, and the product yield is up to 75.99 percent.
Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention.
Claims (8)
1. A method for extracting and purifying 10-deacetylbaccatin III is characterized by comprising the following steps:
s1 treatment of branches and leaves of yew: drying branches and leaves of taxus chinensis until the water content is less than or equal to 15%, and crushing;
s2 extraction and filtration: adding methanol-water solution, extracting for multiple times at 50-60 ℃, filtering to obtain an extracting solution, and combining the extracting solutions;
s3 concentration and extraction: under the vacuum condition of 55-65 ℃, concentrating until the volume of the residual concentrate is 0.3-0.6 times of the weight of the taxus chinensis branches and leaves, adding dichloromethane into the concentrate for extracting for multiple times, and combining the extract liquor;
s4 washing and concentrating: adding a sodium bicarbonate aqueous solution into the extract, slowly stirring and washing for 20-40 min, removing acidic impurities and pigments in the material, and concentrating the washed extract at 55-65 ℃ under a vacuum condition until the extract is dried to obtain a 10-deacetylbaccatin III crude product A;
s5 crude product A is subjected to freezing and suction filtration: adding acetonitrile into the 10-deacetylbaccatin III crude product A, uniformly stirring at room temperature, freezing and standing at low temperature for 15-20 hours, carrying out suction filtration, adding acetonitrile into the obtained filter cake, uniformly stirring at room temperature, freezing and standing at low temperature for 15-20 hours, carrying out suction filtration, and drying the filter cake under vacuum at 55-65 ℃ for 20-30 hours under reduced pressure to obtain a 10-deacetylbaccatin III crude product B;
s6: and (3) concentrating, freezing, filtering and washing the crude product B: adding acetone into the 10-deacetylbaccatin III crude product B, heating at 50-60 ℃, stirring and dissolving completely, then concentrating at 55-65 ℃ under a vacuum condition until 8 times of volume of saturated feed liquid is remained, stopping concentrating when a small amount of crystallized particles appear, cooling to room temperature, freezing and crystallizing at a low temperature for 15-20 hours, filtering, adding an acetone-n-hexane mixed solution into a filter cake, heating, stirring and washing for 40-80 minutes, cooling to room temperature, filtering, and drying the filter cake under a vacuum condition at 55-65 ℃ for 15-20 hours under a reduced pressure condition to obtain a 10-deacetylbaccatin III crude product C;
concentrating, freezing and filtering the S7 crude product C: adding acetone into the 10-deacetylbaccatin III crude product C, heating at 50-60 ℃, stirring to dissolve completely, then concentrating at 55-65 ℃ under a vacuum condition until 8 times of volume of saturated feed liquid is remained, stopping concentrating when a small amount of crystalline particles appear, cooling to room temperature, freezing and crystallizing at a low temperature for 15-20 hours, filtering, and decompressing a filter cake at 55-65 ℃ under a vacuum condition for 48 hours to obtain a 10-deacetylbaccatin III product with the content and the purity meeting the standard.
2. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S1, 100kg of branches and leaves of the taxus mairei are weighed, dried until the water content is 9.65%, and then crushed, wherein the particle size of the crushed powder is 10-20 meshes.
3. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S2, adding 80% methanol-water solution, extracting for 3 times at 55 ℃ in a thermal cycle manner, adding a methanol-water mixed solvent with the volume 5 times of the weight of the branches and leaves for the first time, and extracting for 8 hours; adding a methanol-water mixed solvent with the volume 3 times of the weight of the branches and leaves for the second time, extracting for 8 hours, and adding a methanol-water mixed solvent with the volume 2 times of the weight of the branches and leaves for the third time, extracting for 4 hours.
4. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S3, the mixture is concentrated under the conditions of 60 ℃ and the vacuum degree of-0.060-0.085 MPa until the volume of the residual concentrate is 0.5 times of the weight of the taxus chinensis branches and leaves, the extraction is carried out for 2 times, dichloromethane with the volume 2 times of that of the concentrate is added in the first extraction, and dichloromethane with the volume 1 time of that of the concentrate is added in the second extraction.
5. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S4, 5% sodium bicarbonate aqueous solution with the volume 0.6 times of that of the extract is added, slowly stirred and washed for 30min, and then concentrated to be dry at the temperature of 60 ℃ and the vacuum degree of-0.020-0.085 MPa.
6. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S5, acetonitrile with the weight 4.5 times of the volume of the crude product A is added into the crude product A, the mixture is frozen and kept stand for 16 hours at the low temperature of minus 25 ℃, acetonitrile with the weight 2 times of the volume of the crude product A is added into a filter cake after suction filtration, the mixture is frozen and kept stand for 16 hours at the low temperature of minus 25 ℃, and the filter cake is dried for 24 hours under vacuum reduced pressure at the temperature of 60 ℃ and the vacuum degree of minus 0.085MPa after suction filtration.
7. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S7, acetone with the weight and the volume 30 times of the crude product B is added, the mixture is heated and stirred at the temperature of 55 ℃ to be completely dissolved, the mixture is concentrated by a rotary evaporator at the temperature of 60 ℃ and the vacuum degree of-0.03 to-0.04 MPa, the mixture is frozen and crystallized at the temperature of-25 ℃ for 16 hours, acetone-normal hexane with the weight and the volume 2 times of the crude product B is added into a filter cake, and the weight ratio of acetone: n-hexane 3:7, heating, stirring and washing at 55 ℃ for 1 hour, and drying the filter cake at 60 ℃ under vacuum of-0.085 MPa for 16 hours.
8. The method for extracting and purifying 10-deacetylbaccatin III according to claim 1, wherein: in the step S8, acetone with the weight and the volume being 30 times of the crude product C is added, the mixture is heated and stirred at the temperature of 55 ℃ to be completely dissolved, the mixture is concentrated by a rotary evaporator at the temperature of 60 ℃ and the vacuum degree of-0.03 to-0.04 MPa, the mixture is frozen and crystallized at the temperature of-25 ℃ for 16 hours, and a filter cake is dried at the temperature of 60 ℃ and the vacuum degree of-0.085 MPa for 48 hours under vacuum and reduced pressure.
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| CN113149935A (en) * | 2021-04-25 | 2021-07-23 | 迪庆顺源生物有限公司 | Extraction, separation and purification method of 10-DAB |
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