CN114456138B - Method for separating three coumarin compounds from fingered citron extract - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 125000000332 coumarinyl group Chemical class O1C(=O)C(=CC2=CC=CC=C12)* 0.000 title 1
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- MTSYQYCIPWMRNP-UHFFFAOYSA-N 7-methoxy-5-(3-methylbut-3-enoxy)chromen-2-one Chemical compound C1=CC(=O)OC2=CC(OC)=CC(OCCC(C)=C)=C21 MTSYQYCIPWMRNP-UHFFFAOYSA-N 0.000 claims abstract description 12
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Abstract
The invention belongs to the technical field of traditional Chinese medicine extraction, in particular to a method for separating three coumarin compounds from a fingered citron extract, and particularly relates to a method for separating 5, 7-dimethoxy coumarin and isomers of 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and 7-methoxy-5 isopentenyloxycoumarin from a fingered citron extract rich in coumarin compounds.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for separating three coumarin compounds from a fingered citron extract.
Background
The fingered citron is a fruit with homology of medicine and food, can be used fresh and dried, can be eaten as fruit, is also a precious traditional Chinese medicine, mainly contains various bioactive substances such as flavonoid, volatile oil, coumarin and the like, and has very high practical value. The golden flower area is called as the golden flower with the color and luster orange and aromatic flavor of the fingered citron cultivated in the climates with clear four seasons, warmth, humidity and sufficient sunlight.
Coumarin components are reported to be widely existing in fingered citron plants, and coumarin compounds have various biological activities such as antibacterial, antitumor, anti-HIV, antioxidant and the like, have small molecular weight, relatively simple synthesis and high bioavailability, and have become hot points of research of domestic and foreign scholars.
The reference literature finds that the literature for separating the fingered citron extract is very few, most of the literature adopts the technologies such as silica gel column chromatography, macroporous resin adsorption separation and the like, the cost is high, the preparation amount is small, the process is complex, the irreversible adsorption of the stationary phase can cause the loss of a sample, and the method is not suitable for large-scale separation and preparation. Therefore, the separation technology of the fingered citron is not comprehensive in development, and the advantages and disadvantages of the technical means are not clear, so that the research on the efficacy components of the fingered citron is hindered.
Disclosure of Invention
The invention aims to provide a method for separating three coumarin compounds from a bergamot extract, which is used for rapidly extracting and separating 5, 7-dimethoxy coumarin, isomer 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and 7-methoxy-5 isopentenyl oxy coumarin from bergamot, and has the advantages of simple process, stronger purpose, rapidness and high efficiency.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A method for separating three coumarin compounds from fructus Citri Sarcodactylis extract comprises the following steps:
(1) Pulverizing fingered citron, adding the pulverized fingered citron into an ethanol solution with the dry weight of 2-10 times of that of the fingered citron, carrying out ultrasonic extraction, cooling, filtering and vacuum recovery on an extracting solution to obtain an ethanol crude extract;
Continuously degrading the fingered citron matrix by an ultrasonic method, leaching the effective components of the fingered citron in an ethanol solvent, and separating impurities with higher content and larger polarity by a petroleum ether extraction method to obtain a fingered citron sample mainly containing coumarin compounds;
(2) Extracting the obtained ethanol crude extract with petroleum ether of equal volume for 3-4 times, concentrating under reduced pressure to obtain petroleum ether layer extract;
(3) Mixing ethyl acetate, petroleum ether, water and absolute ethyl alcohol according to the volume ratio of 4:6:5:5, standing and separating to obtain an upper phase and a lower phase respectively, wherein the upper phase is used as a stationary phase of high-speed countercurrent chromatography, and the lower phase is used as a mobile phase; weighing petroleum ether layer extract, dissolving with upper phase and lower phase, introducing sample, separating by high-speed countercurrent chromatography, detecting by ultraviolet detector, collecting corresponding effluent according to peak shape of ultraviolet detector spectrogram, concentrating and drying effluent containing coumarin compound to obtain pure 5, 7-dimethoxy coumarin, pure 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and pure 7-methoxy-5-isopentenyloxycoumarin, wherein 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and 7-methoxy-5 isopentenyloxycoumarin are isomers.
Further, in the step (1), the mass fraction of the ethanol solution is 68-70%.
Further, in the step (1), the conditions of ultrasonic extraction are: the temperature is 40-60 ℃, and the ultrasonic frequency is 300-500W.
Further, in the step (1), ultrasonic extraction is performed for 1 to 1.5 hours.
Further, in the step (3), the volume ratio of the upper phase to the lower phase used for dissolving the petroleum ether layer extract is 1:1-1.2.
In the step (3), the column temperature of the high-speed countercurrent chromatography is 15-30 ℃.
Further, in the step (3), the rotating speed of the high-speed countercurrent chromatograph is 500-1000 r/min.
Further, in the step (3), the flow rate of the mobile phase is 0.5-5 mL/min.
Further, in the step (3), the ultraviolet detection wavelength of the sample is 254-360 nm.
The method preferably operates as follows: fully mixing ethyl acetate, petroleum ether, water and absolute ethyl alcohol according to the volume ratio of 4:6:5:5, standing and separating to obtain an upper phase and a lower phase respectively, wherein the upper phase is used as a stationary phase of high-speed countercurrent chromatography, and the lower phase is used as a mobile phase; the method comprises the steps of filling a multi-layer coil separation column of a high-speed countercurrent chromatograph with a stationary phase, setting the high-speed countercurrent chromatograph, enabling the column temperature to be 15 ℃ -30 ℃ (preferably 25 ℃), rotating anticlockwise at the rotating speed of 500-1000 r/min (preferably 800 r/min), injecting a mobile phase at the rotating speed of 0.5-5 mL/min (preferably 1.5 mL/min), detecting by an ultraviolet detector with the wavelength of 254-360 nm (preferably 280-326 nm), when the mobile phase flows out of the tail end of the column obviously, weighing an oil-ether layer extract, dissolving and injecting the upper phase and the lower phase according to the volume ratio of 1:1, collecting corresponding effluent according to the peak shape of a spectrogram of the ultraviolet detector, concentrating and drying the effluent containing coumarin compounds, and obtaining pure products of 5, 7-dimethoxy coumarin and pure products of the isomer 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and 7-methoxy-5 isopentenyloxycoumarin.
Preferably, 100-1000 mg of fingered citron extract is dissolved by using 15-20 mL of mixed solvent of upper phase and lower phase in a volume ratio of 1:1, and then the mixture is injected.
The invention has the following beneficial effects:
(1) The method adopts liquid-liquid chromatographic separation, can realize sample and reagent recovery, and has the advantages of high recovery rate up to 80%, simple operation, mild reagent and low cost;
(2) The invention has the advantages of large sample injection amount through liquid-liquid chromatographic separation, obviously better separation effect than the traditional separation method, can obtain the coumarin compound with high purity, has important effect on the full utilization of fingered citron medicinal materials, and has high economic benefit.
Drawings
FIG. 1 is a liquid-liquid chromatogram of the extract of example 1 obtained by separating and purifying bergamot petroleum ether to obtain pure 5, 7-dimethoxy coumarin, pure 5-methoxy-7 (3, 3-dimethylallyloxy) coumarin, and pure 7-methoxy-5-isopentenyl oxy coumarin;
FIG. 2 shows high performance liquid chromatograms of the petroleum ether extract (A) of the fingered citron of example 1, and the pure products (B) of No. I peak 5, 7-dimethoxy coumarin, the pure products (C) of No. II peak 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and the pure products (D) of No. III peak 7-methoxy-5 isopentenyloxy coumarin obtained by liquid-liquid chromatography separation.
In FIGS. 1 to 2, peak I represents 5, 7-dimethoxycoumarin, peak II represents 5-methoxy-7 (3, 3-dimethylallyloxy) coumarin, and peak III represents 7-methoxy-5-isopentenyloxycoumarin.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated. In the following examples, the water used was deionized water.
Example 1
(1) Pulverizing 1.0kg of golden bergamot silk, extracting with 6000mL of 70% ethanol solution by ultrasonic wave for 3 times, extracting with ultrasonic wave for 1h at 50deg.C, mixing extractive solutions, cooling, filtering, and vacuum recovering solvent to obtain brown viscous extract 196.0g;
(2) Dissolving the obtained extract in 1.0L of water, extracting with petroleum ether of equal volume for 3 times, and concentrating under reduced pressure to obtain petroleum ether layer extract 3.2g;
(3) Preparing a two-phase solvent comprising ethyl acetate, petroleum ether, water and absolute ethyl alcohol, and placing the two-phase solvent in a separating funnel according to a volume ratio of 4:6:5:5, fully shaking, standing for layering, wherein an upper phase is used as a stationary phase, and a lower phase is used as a mobile phase; weighing 100.0mg of bergamot petroleum ether layer extract, and dissolving with 7.5mL of mixed solution of stationary phase and 7.5mL of mobile phase to obtain liquid-liquid chromatographic separation sample solution;
The sample solution was separated by preparative liquid-liquid chromatograph, the column volume of the separation column being 190mL. Firstly, filling a stationary phase into a separation column at a flow rate of 10mL/min, then starting a speed controller to enable the separation column to rotate forwards, adjusting the rotating speed to 800r/min, setting the flow rate of a mobile phase to be 2mL/min, starting pumping the mobile phase, waiting until the mobile phase flows out of the tail end of the column, indicating that the two phases are balanced, and then injecting a liquid-liquid chromatographic separation sample solution into the separation column through a six-way valve; the eluent is received by an automatic part collector at the speed of 4 minutes/pipe, the eluent is tracked and detected by high performance liquid chromatography until no component exists in the eluent, collection is stopped, fig. 1 shows a liquid-liquid chromatogram of the pure 5, 7-dimethoxy coumarin, the pure 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and the pure 7-methoxy-5-isopentenyloxycoumarin obtained by separating and purifying bergamot petroleum ether extract, peak 5, 7-dimethoxy coumarin is eluted firstly, peak 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin is eluted secondly, and peak III 7-methoxy-5-isopentenyloxycoumarin is eluted finally.
FIG. 2 shows the high performance liquid chromatograms of bergamot petroleum ether extract, and liquid-liquid chromatography separation to obtain No. I peak 5, 7-dimethoxy coumarin pure product, no. II peak 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin pure product, and No. III peak 7-methoxy-5 isopentenyloxycoumarin pure product.
Finally, 52.6mg (purity: 99.2%) of 5, 7-dimethoxy coumarin, 4.9mg (purity: 98.1%) of isomer 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin, and 6.7mg (purity: 98.7%) of 7-methoxy-5-isopentenyloxycoumarin were isolated.
The conditions of the high performance liquid chromatography are as follows: the column was H & E SP ODS-A C 18 column (250 mm. Times.4.6 mm,5 μm). The liquid phase analysis conditions were: the mobile phase consists of acetonitrile (A) and 0.1% formic acid aqueous solution (B), the gradient elution mode is ：15-70％(A)in 0-20min,70-90％(A)in 20-21min,90-90％(A)in 21-30min,90-15％(A)in 30-35min,15-15％(A)in 35-45min.The flow rate was 1.0mL/min, columns at 30 ℃, the flow rate is 1mL/min, the detection wavelength is 326nm, and the sample injection amount is 20 mu L.
Comparative example 1:
Garcia [1] et al reported a method for separating coumarin compounds from plant Loricaria ferruginea using column chromatography, and finally 28.1mg of 5, 7-dimethoxy coumarin was separated from 5.5g of plant n-hexane extract. In contrast, the yield of 5, 7-dimethoxycoumarin in the method reported in the invention is estimated to be 103 times as high, and the time and reagent consumption are also greatly saved.
Comparative example 2
Yang Xinbao [2] et al repeatedly separating and purifying radix Saposhnikoviae dry root by silica gel column chromatography and preparative HPLC, and finally obtaining 10mg of 5-methoxy-7- (3, 3-dimethylallyloxy) -coumarin from 147g of radix Saposhnikoviae cyclohexane extract. In contrast, the yield of 5, 7-dimethoxycoumarin in the method reported in the invention is estimated to be 720 times as high, and the time and reagent consumption are also greatly saved.
Comparative example 3
Cui Gonghua [3] et al use silica gel atmospheric column chromatography, recrystallization refining and other modes to separate and purify the petroleum ether part in the ethanol extract of the Buddha's hand, and finally obtain the experimental result from 103.5g petroleum ether part by separating 21.3mg of 5-isopentenyloxy-7-methoxycoumarin. In contrast, the yield of 5-isopentyloxy-7-methoxycoumarin in the method reported in the invention is estimated to be 326 times as high, and the time and reagent consumption are also greatly saved.
Reference to the literature
[1]Garcia,G.R.M.,Hennig,L.,Rodriguez,E.F.R,Bussmann,R.W.(2016),Coumarins of Loricaria ferruginea,Rev.Bras.Farmacogn.,26(4):471-473.
[2] Zhao Bo, yang Xinbao, yang Xiuwei, et al, research on the chemical composition of Fangfenghua [ J ]. J.Chinese J. 2010,35 (12): 1569-1572.
[3] Cui Gonghua, gao Youheng, liang Chenglin, et al, chemical composition of bergamot, study (I) [ J ]. Chinese herbal medicine, 2007, (09): 1304-1306.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. A method for separating three coumarin compounds from fingered citron extract, comprising the steps of:
(1) Pulverizing fingered citron, adding the pulverized fingered citron into an ethanol solution with the dry weight of 2-10 times of that of the fingered citron, carrying out ultrasonic extraction, cooling, filtering and vacuum recovery on an extracting solution to obtain an ethanol crude extract;
(2) Extracting the obtained ethanol crude extract with petroleum ether of equal volume for 3-4 times, concentrating under reduced pressure to obtain petroleum ether layer extract;
(3) Mixing ethyl acetate, petroleum ether, water and absolute ethyl alcohol according to the volume ratio of 4:6:5:5, standing and separating to obtain an upper phase and a lower phase respectively, wherein the upper phase is used as a stationary phase of high-speed countercurrent chromatography, and the lower phase is used as a mobile phase; weighing petroleum ether layer extract, dissolving upper phase and lower phase, introducing sample, separating by high-speed countercurrent chromatography, ultraviolet detecting, collecting corresponding effluent according to peak shape of ultraviolet spectrogram, concentrating and drying effluent containing coumarin compound to obtain 5, 7-dimethoxy coumarin pure product, 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin pure product and 7-methoxy-5 isopentenyloxycoumarin pure product, wherein 5-methoxy-7 (3, 3-dimethyl allyloxy) coumarin and 7-methoxy-5 isopentenyloxycoumarin are isomers.
2. The method for separating three coumarin compounds from fingered citron extract according to claim 1, wherein in the step (1), the mass fraction of the ethanol solution is 68-70%.
3. The method for separating three coumarin compounds from fingered citron extract according to claim 2, wherein in step (1), the conditions of ultrasonic extraction are: the temperature is 40-60 ℃, and the ultrasonic frequency is 300-500W.
4. A method for separating three coumarin compounds from fingered citron extract according to claim 3, wherein in step (1), the ultrasonic extraction is performed for 1 to 1.5 hours.
5. The method for separating three coumarin compounds from fingered citron extract according to claim 4, wherein in step (3), the volume ratio of the upper phase to the lower phase used for dissolving petroleum ether layer extract is 1:1-1.2.
6. The method for separating three coumarin compounds from fingered citron extract according to claim 5, wherein in step (3), the column temperature of high-speed countercurrent chromatography is 15-30 ℃.
7. The method for separating three coumarin compounds from fingered citron extract according to claim 6, wherein in step (3), the rotation speed of the high-speed countercurrent chromatograph is 500-1000 r/min.
8. The method for separating three coumarin compounds from fingered citron extract according to claim 7, wherein the flow rate of the mobile phase in step (3) is 0.5-5 mL/min.
9. The method for separating three coumarin compounds from fingered citron extract according to claim 8, wherein in step (3), the ultraviolet detection wavelength of the sample is 254-360 nm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102010392A (en) * | 2010-10-27 | 2011-04-13 | 浙江工业大学 | Method for separating coumarin compound by using high-speed countercurrent chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102010392A (en) * | 2010-10-27 | 2011-04-13 | 浙江工业大学 | Method for separating coumarin compound by using high-speed countercurrent chromatography |
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