CN110903156A - Method for separating and purifying β -elemene, gamma-elemene and delta-elemene - Google Patents
Method for separating and purifying β -elemene, gamma-elemene and delta-elemene Download PDFInfo
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- CN110903156A CN110903156A CN201911126285.2A CN201911126285A CN110903156A CN 110903156 A CN110903156 A CN 110903156A CN 201911126285 A CN201911126285 A CN 201911126285A CN 110903156 A CN110903156 A CN 110903156A
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- C07C7/005—Processes comprising at least two steps in series
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- C07—ORGANIC CHEMISTRY
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- C07C7/04—Purification; Separation; Use of additives by distillation
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- C07C7/10—Purification; Separation; Use of additives by extraction, i.e. purification or separation of liquid hydrocarbons with the aid of liquids
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- C07—ORGANIC CHEMISTRY
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- C07C7/00—Purification; Separation; Use of additives
- C07C7/12—Purification; Separation; Use of additives by adsorption, i.e. purification or separation of hydrocarbons with the aid of solids, e.g. with ion-exchangers
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- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
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- C07B2200/07—Optical isomers
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Abstract
The invention adopts a complete set of technology taking semi-preparative liquid chromatography as a core for the first time, β -elemene, gamma-elemene and delta-elemene are separated and purified from the elemene extract, a process route for separating and purifying β -elemene, gamma-elemene and delta-elemene from the elemene extract is established, the average purity of the delta-elemene is not less than 90 percent, the process is simple, the solvent recovery is convenient, and the method has strong practical value.
Description
Technical Field
The invention belongs to the technical field of extraction and separation of traditional Chinese medicines, and particularly relates to a method for separating and purifying β -elemene, gamma-elemene and delta-elemene.
Background
Elemene (elemene) is a sesquiterpene natural anticancer drug which is firstly extracted from curcuma zedoaria of Zingiberaceae and has anticancer activity in China, elemene emulsion injection taking β -elemene as a main component is two new anti-tumor drugs which are researched and developed successfully in China, and is widely used for treating malignant serosal cavity effusion, lung cancer, digestive tract tumor, brain tumor and other superficial tumors clinically at present.
The elemene has three isomers, β -elemene, gamma-elemene, delta-elemene (the molecular formula is C)15H24) β -elemene, gamma-elemene and delta-elemene are double bond position isomers, the physicochemical properties are very similar, the gamma-elemene is unstable and is easy to be converted into β -elemene and other compounds at normal temperature, and β -elemene, the gamma-elemene and the delta-elemene are effective components for resisting tumors.
At present, column chromatography is mostly adopted for separating a large number of elemene extract samples, the method has the advantage of large sample loading amount, but the separation degree is poor, the separation speed is slow, and the method is not suitable for separating 3 isomers of β -elemene, gamma-elemene and delta-elemene, so that the research on an extraction and separation route which has high transfer rate of β -elemene, gamma-elemene and delta-elemene, simple process, small solvent toxicity and convenient recovery has important practical and economic significance.
Disclosure of Invention
In order to overcome the defects, the invention provides a method for separating and purifying β -elemene, gamma-elemene and delta-elemene, which effectively improves the extraction efficiency, purity and yield of β -elemene, gamma-elemene and delta-elemene.
The technical scheme of the invention is realized by the following steps that a method for separating and purifying β -elemene, gamma-elemene and delta-elemene comprises the following steps:
(1) adding acetonitrile with 2-30 times of volume of the extract into the elemene extract for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by using A semi-preparative liquid chromatography, adopting one of YMC-Pack ODS-A, XSelectt HSS, Sharpsil-U C18100A, Kromasil 100A C18 and other semi-preparative columns with the inner diameter of 4.6-30 mm, feeding the sample in an amount of 1-5 mL, taking an acetonitrile water solution with the mass fraction of 5-90% as an eluent, collecting the eluent at A flow rate of 0.5-8 mL/min according to the collection time of A detector, and respectively collecting the effluent of 77-85 min, 85-91 min and 107-114 min, which are respectively β -elemene, gammA-elemene and deltA-elemene target eluents.
(3) And (3) respectively placing the three target eluents at the temperature of 10 ℃ below zero to 10 ℃ under reduced pressure, carrying out rotary evaporation until no liquid drops drop, transferring the three target eluents to a separating funnel, respectively adding n-hexane with the volume of 2-5 times of the eluent in the separating funnel for extraction, and placing the collected n-hexane extract at the temperature of 10 ℃ below zero to 10 ℃ under reduced pressure for drying to obtain β -elemene, gamma-elemene and delta-elemene single products respectively.
Preferably, in the method for separating and purifying β -elemene, gamma-elemene and delta-elemene, in the step (1), the content of β -elemene, gamma-elemene and delta-elemene in the extract is more than 0.5 percent.
Preferably, in the method for separating and purifying β -elemene, gamma-elemene and delta-elemene, in the step (1), 9 times of acetonitrile is added into the extract for dissolving.
Preferably, in the method for separating and purifying β -elemene, gamma-elemene and delta-elemene, in the step (2), the semi-preparative column is Sharpsil-U C18100A.
Preferably, in the method for separating and purifying β -elemene, gamma-elemene and delta-elemene, the inner diameter of the semi-preparation column is 21.2 mm.
Preferably, in the method for separating and purifying β -elemene, gamma-elemene and delta-elemene, the sample volume of the elemene extract solution is 3 mL.
Preferably, the semi-preparative liquid chromatography separation method for separating and purifying β -elemene, gamma-elemene and delta-elemene adopts acetonitrile with the mass fraction of 75% as an eluent.
Preferably, the method for separating and purifying β -elemene, gamma-elemene and delta-elemene adopts a semi-preparative liquid chromatography separation method, and the flow rate of an eluent is 5 mL/min.
Preferably, the method for separating and purifying β -elemene, gamma-elemene and delta-elemene comprises the step of respectively placing target eluent at the temperature of-10 ℃ and carrying out reduced pressure rotary evaporation until no liquid drops drop.
Preferably, the method for separating and purifying β -elemene, gamma-elemene and delta-elemene comprises the step of adding 3 times of volume of n-hexane into rotary evaporation liquid respectively for extraction.
Preferably, the method for separating and purifying β -elemene, gamma-elemene and delta-elemene is characterized in that collected n-hexane extract is dried under reduced pressure at the temperature of-10 ℃.
The invention has the beneficial effects that a complete set of technology taking a semi-preparative liquid chromatography as a core is adopted for the first time, β -elemene, gamma-elemene and delta-elemene are separated and purified from the elemene extract, a process route for separating and purifying β -elemene, gamma-elemene and delta-elemene from the elemene extract is established, the average purity of the β -elemene, gamma-elemene and delta-elemene is separated and purified by the process, the process is simple, the solvent is convenient to recover, and the process has strong practical value.
Drawings
FIG. 1 is a liquid chromatogram of the product of example 1.
FIG. 2 is a liquid chromatogram of the product of example 2.
FIG. 3 is a liquid chromatogram of the product of example 3.
FIG. 4 is a liquid chromatogram of the product of example 4.
Detailed Description
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.
Example 1
(1) Adding 9 times of acetonitrile into elemene extract (β -elemene content 38.9%, gamma-elemene content 0.7%, delta-elemene content 12.6%) for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by semi-preparative liquid chromatography, adopting Sharpsil-U C18100A semi-preparative columns with the inner diameter of 21.2mm, the sample injection amount is 3mL, taking acetonitrile with the mass fraction of 75% as an eluent, and collecting target eluents of β -elemene, gamma-elemene and delta-elemene at the flow rate of 5 mL/min;
(3) and (3) placing the target eluent at the temperature of minus 10 ℃ respectively, performing reduced pressure rotary evaporation until no liquid drops, transferring the target eluent to a separating funnel, adding n-hexane with the volume being 3 times of that of the target eluent respectively for extraction, and placing the collected n-hexane extract at the temperature of minus 10 ℃ for reduced pressure drying to obtain β -elemene, gamma-elemene and delta-elemene products.
Example 2
(1) Adding 20 times of acetonitrile into elemene extract (β -elemene content 40.1%, gamma-elemene content 0.8%, delta-elemene content 13.2%) for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by semi-preparative liquid chromatography, adopting Sharpsil-U C18100A semi-preparative column with the inner diameter of 10mm, using acetonitrile with the mass fraction of 75% as eluent and the flow rate of 3mL/min, wherein the sample volume is 1mL, and collecting target eluent of β -elemene, gamma-elemene and delta-elemene.
(3) And (3) respectively placing the target eluent at the temperature of minus 10 ℃ for reduced pressure rotary steaming until no liquid drops, transferring the target eluent to a separating funnel, respectively adding 5 times of n-hexane for extraction, placing the collected n-hexane extract at the temperature of minus 10 ℃ for reduced pressure drying, and obtaining β -elemene, gamma-elemene and delta-elemene products.
Example 3
(1) Adding 15 times of acetonitrile into elemene extract (β -elemene content 42.5%, gamma-elemene content 0.97%, delta-elemene content 14.1%) for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by semi-preparative liquid chromatography, adopting Sharpsil-U C18100A semi-preparative columns with the inner diameter of 21.2mm, the sample injection amount is 3mL, acetonitrile with the mass fraction of 80% is used as an eluent, the flow rate is 5mL/min, and collecting target eluents of β -elemene, gamma-elemene and delta-elemene.
(3) And (3) placing the target eluent at the temperature of minus 10 ℃ respectively, performing reduced pressure rotary evaporation until no liquid drops, transferring the target eluent to a separating funnel, adding n-hexane with the volume being 3 times of that of the target eluent respectively for extraction, and placing the collected n-hexane extract at the temperature of minus 10 ℃ for reduced pressure drying to obtain β -elemene, gamma-elemene and delta-elemene products.
Example 4
(1) Adding 13 times of acetonitrile into elemene extract (β -elemene content 40.5%, gamma-elemene content 0.79%, delta-elemene content 13.7%) for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by semi-preparative liquid chromatography, adopting Sharpsil-U C18100A semi-preparative columns with the inner diameter of 21.2mm, the sample injection amount is 3mL, acetonitrile with the mass fraction of 90% is used as an eluent, the flow rate is 5mL/min, and collecting target eluents of β -elemene, gamma-elemene and delta-elemene.
(3) And (3) respectively placing the target eluent at 10 ℃ and carrying out reduced pressure rotary evaporation until no liquid drops, transferring the target eluent to a separating funnel, respectively adding 3 times of n-hexane for extraction, and placing the collected n-hexane extract at 10 ℃ and carrying out reduced pressure drying to obtain β -elemene, gamma-elemene and delta-elemene products.
Comparative example 1
(1) Adding 9 times of acetonitrile into elemene extract (β -elemene content 38.9%, gamma-elemene content 0.7%, delta-elemene content 12.6%) for dissolving;
(2) separating and purifying the extract solution obtained in the step (1) by semi-preparative liquid chromatography, adopting Sharpsil-U C18100A semi-preparative columns with the inner diameter of 21.2mm, the sample injection amount is 3mL, taking acetonitrile with the mass fraction of 75% as an eluent, and collecting target eluents of β -elemene, gamma-elemene and delta-elemene at the flow rate of 5 mL/min;
(3) and (3) respectively placing the target eluent at 30 ℃ and carrying out reduced pressure rotary evaporation until no liquid drops drop, transferring the target eluent to a separating funnel, respectively adding 3 times of n-hexane for extraction, placing the collected n-hexane extract at 30 ℃ and carrying out reduced pressure drying, and obtaining only a very small amount of β -elemene products, wherein gamma-elemene and delta-elemene products are not obtained.
The purity results of the semi-preparative liquid chromatography separation and purification processes of examples 1 to 4 and comparative example 1 are shown in table 1.
TABLE 1 purification rate after purification of elemene by semi-preparative liquid chromatography
The invention uses semi-preparative liquid chromatography to separate and purify β -elemene, gamma-elemene and delta-elemene in the elemene extract, and reduces the influence of other impurity components in an elution solvent as much as possible, as can be seen from table 1, the invention uses semi-preparative liquid chromatography to prepare β -elemene, gamma-elemene and delta-elemene components of the elemene extract, and the average purity of each component obtained by purification through an extraction process is not less than 90%, which indicates that the technical method is feasible.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A method for separating and purifying β -elemene, gamma-elemene and delta-elemene is characterized by comprising the following steps:
s1, adding acetonitrile into the extract to dissolve;
s2, separating and purifying the extract solution obtained in the step S1 through semi-preparative liquid chromatography, eluting with one of semi-preparative columns such as YMC-PackODS-A, XSelect HSS, Sharpsil-U C18100A, Kromasil 100A C18 and the like by using an eluent, and collecting β -elemene, gammA-elemene and deltA-elemene target eluents flowing through the semi-preparative columns;
s3, placing the target eluent at the temperature of-10 ℃ for decompression rotary steaming until no liquid drops, transferring the target eluent into a separating funnel, respectively adding n-hexane with the volume being 2-5 times of that of the eluent in the separating funnel for extraction, and placing the collected n-hexane extract at the temperature of-10 ℃ for decompression drying to obtain β -elemene, gamma-elemene and delta-elemene products.
2. The method as claimed in claim 1, wherein the extract obtained in step S1 contains more than 0.5% of each of β -elemene, γ -elemene, and δ -elemene, and acetonitrile is added in an amount of 2-30 times by volume.
3. The method as claimed in claim 1, wherein the inner diameter of the semi-preparative column in step S2 is 4.6-30 mm, the sample volume is 1-5 mL, the eluent is acetonitrile aqueous solution with the mass fraction of 5-90%, the flow rate is 0.5-8 mL/min, the effluent is collected according to the collection time of the detector, and the effluent is collected for 77-85 min, 85-91 min and 107-114 min, respectively, and respectively is β -elemene, γ -elemene and δ -elemene target eluents.
4. The method for separating and purifying β -elemene, gamma-elemene and delta-elemene as claimed in claim 1, wherein the amount of n-hexane added in step S3 is 2-5 times of the volume of the eluate in a separating funnel.
5. The method for separating and purifying β -elemene, gamma-elemene and delta-elemene as claimed in claim 2, wherein acetonitrile is added in an amount of 9 times by volume of the extract.
6. The method as claimed in claim 1, wherein the semi-preparative column in step S2 is sharp-U C18100A.
7. The method as claimed in claim 3, wherein the semi-preparative column has an inner diameter of 21.2mm, a sample volume of 3mL, and eluent of 75% acetonitrile water solution with a flow rate of 5 mL/min.
8. The method as claimed in claim 4, wherein the target eluent is placed at-10 ℃ and is reduced pressure and steamed until no liquid drops, the target eluent is transferred to a separating funnel, n-hexane with 3 times volume of the eluent in the separating funnel is added respectively for extraction, and the collected n-hexane extract is placed at-10 ℃ and is reduced pressure and dried to obtain β -elemene, gamma-elemene and delta-elemene products.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1200266A (en) * | 1997-04-14 | 1998-12-02 | 中国科技开发院医药科技开发所 | Method for preparing anti-cancer medicine using beta-elemene as main effective ingredient, and components of the medicine |
| CN101492339A (en) * | 2009-03-06 | 2009-07-29 | 北京联合大学应用文理学院 | Process and apparatus for the extraction separation of beta-elemene, and process for producing stuffing |
| CN102992941A (en) * | 2012-12-19 | 2013-03-27 | 石药集团远大(大连)制药有限公司 | Extraction method of Gamma-elemene |
| CN102992940A (en) * | 2012-12-19 | 2013-03-27 | 石药集团远大(大连)制药有限公司 | Method for extracting delta-elemene |
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2019
- 2019-11-18 CN CN201911126285.2A patent/CN110903156A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1200266A (en) * | 1997-04-14 | 1998-12-02 | 中国科技开发院医药科技开发所 | Method for preparing anti-cancer medicine using beta-elemene as main effective ingredient, and components of the medicine |
| CN101492339A (en) * | 2009-03-06 | 2009-07-29 | 北京联合大学应用文理学院 | Process and apparatus for the extraction separation of beta-elemene, and process for producing stuffing |
| CN102992941A (en) * | 2012-12-19 | 2013-03-27 | 石药集团远大(大连)制药有限公司 | Extraction method of Gamma-elemene |
| CN102992940A (en) * | 2012-12-19 | 2013-03-27 | 石药集团远大(大连)制药有限公司 | Method for extracting delta-elemene |
Non-Patent Citations (2)
| Title |
|---|
| 冯彬彬 等: "《全国高职高专药学类专业规划教材 第二轮 天然药物化学 第2版》", 31 August 2019, 中国医药科技出版社 * |
| 国家药典委员会: "《中国药典分析检测技术指南》", 31 August 2017, 中国医药科技出版社 * |
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