CN115260032B - Method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by high-speed counter-current chromatography - Google Patents
Method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by high-speed counter-current chromatography Download PDFInfo
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- 238000010262 high-speed countercurrent chromatography Methods 0.000 title claims abstract description 32
- DHNGCHLFKUPGPX-RMKNXTFCSA-N ethyl trans-p-methoxycinnamate Chemical compound CCOC(=O)\C=C\C1=CC=C(OC)C=C1 DHNGCHLFKUPGPX-RMKNXTFCSA-N 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 23
- KBEBGUQPQBELIU-CMDGGOBGSA-N Ethyl cinnamate Chemical compound CCOC(=O)\C=C\C1=CC=CC=C1 KBEBGUQPQBELIU-CMDGGOBGSA-N 0.000 title abstract description 29
- KBEBGUQPQBELIU-UHFFFAOYSA-N cinnamic acid ethyl ester Natural products CCOC(=O)C=CC1=CC=CC=C1 KBEBGUQPQBELIU-UHFFFAOYSA-N 0.000 title abstract description 29
- DHNGCHLFKUPGPX-UHFFFAOYSA-N ethyl 4-methoxycinnamate Natural products CCOC(=O)C=CC1=CC=C(OC)C=C1 DHNGCHLFKUPGPX-UHFFFAOYSA-N 0.000 title abstract description 28
- XTZZULGXHUQOEN-UHFFFAOYSA-N ethyl p-methoxycinnamate Natural products CCOC1=CC=C(C=CC(=O)OC)C=C1 XTZZULGXHUQOEN-UHFFFAOYSA-N 0.000 title abstract description 28
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
- 244000062241 Kaempferia galanga Species 0.000 claims abstract description 25
- 238000000926 separation method Methods 0.000 claims abstract description 23
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 22
- HZQXXYJHLCSUGQ-UHFFFAOYSA-N ethyl acetate hexane methanol hydrate Chemical compound O.OC.CCCCCC.CCOC(C)=O HZQXXYJHLCSUGQ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000605 extraction Methods 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 45
- 239000000287 crude extract Substances 0.000 claims description 18
- 230000005526 G1 to G0 transition Effects 0.000 claims description 8
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- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004185 countercurrent chromatography Methods 0.000 description 2
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- 235000013305 food Nutrition 0.000 description 2
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- HESVSJPNPVYUIJ-UHFFFAOYSA-L [Na+].[Na+].OC.CC#N.OP([O-])([O-])=O Chemical compound [Na+].[Na+].OC.CC#N.OP([O-])([O-])=O HESVSJPNPVYUIJ-UHFFFAOYSA-L 0.000 description 1
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- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by using high-speed counter-current chromatography, belonging to the technical field of natural products. The method adopts a high-speed counter-current chromatography separation method, takes an n-hexane-ethyl acetate-methanol-water system as a two-phase solvent system, can efficiently prepare the kaempferia galanga extract, and has the advantages of large separation amount, no loss, high yield and high purity; moreover, the extraction method provided by the invention has the characteristics of simple and convenient operation, good reproducibility and mild separation environment. Further, the invention can obtain high-purity p-methoxy ethyl cinnamate and/or ethyl cinnamate by controlling the volume ratio of n-hexane, ethyl acetate, methanol and water.
Description
Technical Field
The invention relates to the technical field of natural products, in particular to a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by utilizing high-speed counter-current chromatography.
Background
Kaempferia galanga (A)kaempferia galangaL.), rhizoma Kaempferiae, rhizoma\34302, rhizoma Kaempferiae, fructus Kaempferiae, moschus, fructus kaempferiae, is prepared from Kaempferia galanga belonging to Zingiberaceae, distributed in Taiwan,Guangdong, guangxi, yunnan provinces, etc. The rhizoma kaempferiae has good clinical pharmacological action and drug effect, the rhizome of the rhizoma kaempferiae is a common traditional Chinese medicine, has the effects of promoting qi circulation, warming spleen and stomach, helping digestion and relieving pain, and is mainly used for clinically treating fullness and distention of chest and diaphragm, abdominal psychroalgia and indigestion. The kaempferia galanga volatile oil has high content and aromatic smell, and can be widely used in food industry as flavoring and spice in food. The present research shows that the kaempferia galanga extract has multiple functions of better oxidation resistance, inflammation resistance, cancer resistance, bacteria resistance, virus resistance and the like.
In the prior art, a research on an extraction process of ethyl p-methoxycinnamate in a Tibetan medicine rhizoma kaempferiae [ J ] Anhui agricultural science, 2008, 36 (33): 14585-14586' researches on an extraction process of ethyl p-methoxycinnamate in rhizoma kaempferiae medicinal materials, and the ethyl p-methoxycinnamate is obtained by firstly extracting by a Soxhlet extraction method, a thermal reflux method or an ultrasonic method and then performing high performance liquid chromatography separation. In the prior art, in the high performance liquid chromatography separation process, the sample volume of the ethyl p-methoxycinnamate is in a good linear relation within the range of 0.1 to 0.8 mu g, and the separation volume of the ethyl p-methoxycinnamate is small.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by high-speed counter-current chromatography, wherein the method provided by the present invention can separate ethyl p-methoxycinnamate and/or ethyl cinnamate from rhizoma kaempferiae efficiently and in large batch.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an application of n-hexane-ethyl acetate-methanol-water system in preparation of a kaempferia galanga extract by high-speed counter-current chromatography.
Preferably, the volume ratio of the n-hexane, the ethyl acetate, the methanol and the water in the n-hexane-ethyl acetate-methanol-water system is (3 to 4): (1 to 1.5): (3 to 4): (1 to 1.5).
The invention provides a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by using high-speed counter-current chromatography, which comprises the following steps:
mixing rhizoma kaempferiae with ethanol solution, and extracting with ethanol to obtain rhizoma kaempferiae crude extract;
performing high-speed counter-current chromatography separation on the rhizoma kaempferiae crude extract to obtain ethyl p-methoxycinnamate and/or ethyl cinnamate; the two-phase solvent system adopted by the high-speed countercurrent chromatography is a normal hexane-ethyl acetate-methanol-water system.
Preferably, the volume ratio of the n-hexane, the ethyl acetate, the methanol and the water in the n-hexane-ethyl acetate-methanol-water system is (3 to 4): (1 to 1.5): (3 to 4): (1 to 1.5).
Preferably, the volume fraction of the ethanol in the ethanol solution is 50 to 95 percent.
Preferably, the temperature of the alcohol extraction is 50 to 80 ℃, the number of the alcohol extraction is 2 to 4, and the time of single alcohol extraction is 1 to 4 hours.
Preferably, the n-hexane-ethyl acetate-methanol-water system is degassed before use.
Preferably, the high-speed counter-current chromatography separation conditions comprise: the temperature is 20 to 30 ℃; the rotating speed of the host is 500 to 1000r/min; the flow rate of the mobile phase is 5 to 20mL/min.
The invention provides a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by using high-speed counter-current chromatography, which comprises the following steps: mixing rhizoma Kaempferiae with ethanol solution, and extracting with ethanol to obtain rhizoma Kaempferiae crude extract; performing high-speed counter-current chromatography separation on the rhizoma kaempferiae crude extract to obtain ethyl p-methoxycinnamate and/or ethyl cinnamate; the two-phase solvent system adopted by the high-speed countercurrent chromatography is a normal hexane-ethyl acetate-methanol-water system. The method adopts a high-speed counter-current chromatography (HSCCC) method, takes an n-hexane-ethyl acetate-methanol-water system as a two-phase solvent system, takes an upper phase as a stationary phase and takes a lower phase as a mobile phase, can realize the efficient separation of ethyl p-methoxycinnamate and/or ethyl cinnamate from rhizoma kaempferiae, and has the advantages of large separation amount of ethyl cinnamate and ethyl methoxycinnamate, no loss and high yield of ethyl p-methoxycinnamate and/or ethyl cinnamate. Moreover, the extraction method provided by the invention has the characteristics of simple operation, good reproducibility and mild separation environment, and is suitable for industrial production.
Further, the invention can obtain high-purity p-methoxy ethyl cinnamate and/or ethyl cinnamate by controlling the volume ratio of n-hexane, ethyl acetate, methanol and water in an n-hexane-ethyl acetate-methanol-water system.
Drawings
FIG. 1 is a high performance liquid chromatogram of crude Kaempferia galanga extract prepared in example 1;
FIG. 2 is a high-speed countercurrent chromatography (HSCCC) chart of crude kaempferia galanga extract prepared in example 1;
FIG. 3 is a high performance liquid chromatogram of Compound a prepared in example 1;
FIG. 4 is a high performance liquid chromatogram of Compound b prepared in example 1;
FIG. 5 shows Compound a prepared in example 1 13 A C-NMR spectrum;
FIG. 6 shows Compound a prepared in example 1 1 H-NMR spectrum;
FIG. 7 is a mass spectrum of Compound a prepared in example 1;
FIG. 8 shows the preparation of Compound b in example 1 13 A C-NMR spectrum;
FIG. 9 shows the preparation of Compound b in example 1 1 H-NMR spectrum;
FIG. 10 is a mass spectrum of Compound b prepared in example 1.
Detailed Description
The invention provides a method for separating and purifying ethyl p-methoxycinnamate and/or ethyl cinnamate by using high-speed counter-current chromatography, which comprises the following steps:
mixing rhizoma Kaempferiae with ethanol solution, and extracting with ethanol to obtain rhizoma Kaempferiae crude extract;
performing high-speed counter-current chromatography separation on the rhizoma kaempferiae crude extract to obtain ethyl p-methoxycinnamate and/or ethyl cinnamate; the two-phase solvent system adopted by the high-speed countercurrent chromatography is a normal hexane-ethyl acetate-methanol-water system.
In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
The invention mixes rhizoma kaempferiae with ethanol solution, and carries out ethanol extraction to obtain rhizoma kaempferiae crude extract.
In the present invention, the rhizome of kaempferia galanga is preferably dried rhizome of kaempferia galanga, and the rhizome of kaempferia galanga is preferably pulverized before use, but the pulverization is not particularly limited in the present invention, and the pulverization may be carried out by a pulverization method known to those skilled in the art to a particle size of 0.5cm or less.
In the present invention, the volume fraction of ethanol in the ethanol solution is preferably 50 to 95%, more preferably 60 to 80%, and still more preferably 70 to 75%. In the present invention, the ratio of the mass of the rhizome (dry weight) of kaempferia galanga to the volume of the ethanol solution is preferably 1g: (1 to 4) mL, more preferably 1g: (2 to 3) mL.
In the invention, the temperature of the alcohol extraction is preferably 50 to 80 ℃, and more preferably 60 to 70 ℃; the number of times of alcohol extraction is preferably 2 to 4 times, and more preferably 3 times; the time of single alcohol extraction is preferably 1 to 4 hours, and more preferably 2 to 3 hours.
After the alcohol extraction is completed, the invention preferably further comprises concentrating the obtained alcohol extract to obtain a crude kaempferia galanga extract. The concentration in the present invention is not particularly limited, and the concentration may be carried out by a concentration method known to those skilled in the art to a constant weight.
After a rhizoma kaempferiae crude extract is obtained, performing high-speed countercurrent chromatography separation on the rhizoma kaempferiae crude extract to obtain p-methoxy ethyl cinnamate and/or ethyl cinnamate; the two-phase solvent system adopted by the high-speed countercurrent chromatography is a normal hexane-ethyl acetate-methanol-water system.
In the invention, the volume ratio of n-hexane, ethyl acetate, methanol and water in the n-hexane-ethyl acetate-methanol-water system is preferably (3 to 4): (1 to 1.5): (3 to 4): (1 to 1.5), more preferably (3.1 to 4): (1 to 1.4): (3.1 to 3.9): (1.1 to 1.4), more preferably (3.2 to 4): (1.2 to 1.4): (3.2 to 3.8): (1.2 to 1.5). In the present invention, the n-hexane-ethyl acetate-methanol-water system is preferably subjected to degassing treatment before use. In the invention, the degassing treatment is preferably ultrasonic degassing treatment, and the temperature of the ultrasonic degassing treatment is preferably 10 to 30 ℃, and more preferably 20 to 25 ℃; the time of the ultrasonic degassing treatment is preferably 10 to 30min, and more preferably 15 to 20min. In the invention, the n-hexane-ethyl acetate-methanol-water system is preferably prepared in a liquid storage tank, wherein the upper phase of the n-hexane-ethyl acetate-methanol-water system is a stationary phase, and the lower phase of the n-hexane-ethyl acetate-methanol-water system is a mobile phase.
In the present invention, before the separation by high-speed counter-current chromatography, the method preferably further comprises balancing the high-performance counter-current chromatography column, wherein the balancing is preferably: the chromatographic column of the high-efficiency countercurrent chromatography is filled with the stationary phase, and the mobile phase is injected until the two-phase solvent system reaches an equilibrium state (upper and lower layering) in the chromatographic column. In the present invention, the balance parameters preferably include: the temperature is preferably 20 to 30 ℃, and more preferably 25 ℃; the host machine preferably adopts a forward rotation mode, and the rotation speed of the host machine is preferably 500 to 1000r/min, and more preferably 700 to 800r/min; the flow rate of the mobile phase is preferably 5 to 20mL/min, more preferably 10 to 15mL/min.
In the invention, the kaempferia galanga crude extract is preferably subjected to high-speed counter-current chromatography separation: for example, dissolving rhizoma Kaempferiae crude extract in stationary phase, injecting rhizoma Kaempferiae crude extract solution, and injecting into mobile phase for high speed countercurrent chromatography. The invention does not specially limit the dosage of the stationary phase for dissolving, and can dissolve the kaempferia galanga crude extract.
In the present invention, the high-speed counter-current chromatography separation conditions preferably include: the temperature is preferably 20 to 30 ℃, and more preferably 25 ℃; the host machine preferably adopts a forward rotation mode, and the rotation speed of the host machine is preferably 500 to 1000r/min, and more preferably 700 to 800r/min; the flow rate of the mobile phase (namely the lower phase of the n-hexane-ethyl acetate-methanol-water system) is preferably 5 to 20mL/min, and more preferably 10 to 15mL/min; the detection wavelength is preferably 270nnm.
In the invention, the peak position of the high-speed counter-current chromatography of the ethyl p-methoxycinnamate is preferably 30 to 41min; the peak position of the ethyl cinnamate is preferably 43 to 54min.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
(1) Pulverizing dried rhizoma Kaempferiae to particle size of 0.5cm or less, extracting 1000g of dried rhizoma Kaempferiae with 3000mL of 95% ethanol water solution under reflux for 2h for 3 times, and concentrating the obtained ethanol extractive solution to constant weight to obtain rhizoma Kaempferiae crude extract (88 g).
(2) Placing n-hexane, ethyl acetate, methanol and water (3.5. Taking 1g of crude kaempferia galanga extract, and dissolving the crude kaempferia galanga extract in 20mL of stationary phase completely by ultrasonic wave to obtain a crude kaempferia galanga extract solution.
(3) Starting a circulating water bath, setting the temperature to be 25 ℃, pumping a stationary phase into a main machine at a flow rate of 30mL/min and filling a separation spiral tube, starting a power supply of the main machine to rotate forwards at 800r/min, pumping a mobile phase at a flow rate of 10mL/min after the rotation speed is stable, injecting a kaempferia galanga crude extract solution obtained in the step (2) into a sample injection ring by a sample injector after the mobile phase flows out from an outlet of the separation spiral tube and a base line is stable and is adjusted to zero, continuously detecting the effluent at the outlet of the separation spiral tube at 270nm, and manually collecting each chromatographic component according to a countercurrent chromatogram; the target components having peak receiving times of 30 to 41min were denoted as compound a (140 mg), and the target components having peak receiving times of 43 to 54min were denoted as compound b (15 mg).
The high performance liquid chromatogram of the crude extract of Kaempferia galanga is shown in FIG. 1, partial peak data is shown in Table 1, and the retention time of two main peaks is 11.13min and 11.26min respectively as can be seen from Table 1 and FIG. 1.
TABLE 1 data of partial peaks of HPLC of crude Kaempferia galanga extract
FIG. 2 is a high-speed countercurrent chromatography (HSCCC) chart of crude Kaempferia galanga extract.
The purity of the obtained compound a and compound b was measured by high performance liquid chromatography. The HPLC analysis conditions were as follows: agilent1260 ZORBAX SB-C18 column (5 μm,4.6 mm. Times.150, I.D.), DAD detector 270nm, mobile phase flow rate of 1.0mL/min, mobile phase acetonitrile-water (0 to 100, v/v).
The high performance liquid chromatogram of compound a is shown in fig. 3, and the peak data is shown in table 2:
TABLE 2 high Performance liquid chromatography data for Compound a
As can be seen from FIG. 3 and Table 2, the purity of Compound a was 99.89%, and the retention time was 11.12min.
The high performance liquid chromatogram of compound b is shown in fig. 4, and the peak data is shown in table 3:
TABLE 3 high Performance liquid chromatography data for Compound b
As can be seen from fig. 4 and table 3, the purity of compound b was 99.47%, and the retention time was 11.26min.
Structural identification of targeted components: carrying out the reaction on the compound a and the compound b 1 H-NMR、 13 C-NMR and mass spectrometric analysis. The carbon spectrum, the hydrogen spectrum and the mass spectrum of the compound a are shown in the following figures 5, 6 and 7 respectively, and the structural characterization data of the compound a are as follows: 1 H-NMR (CD 3 OD): 7.62 (1H, d, J = 15.9 Hz, H-7), 7.54 (2H, d, J = 7.6 Hz, H-2 and H-6), 6.94 (2H, d, J = 7.6 Hz, H-3 and H-5), 6.36 (1H, d, J = 15.9 Hz, H-8), 4.22 (2H, q, J = 7.1 Hz, H-10), 1.31 (3H, t, J = 7.1 Hz, H-11); 13 C-NMR (CD 3 OD): 169.1 (s, C-9), 163.2 (s, C-4), 145.9 (d, C-7), 130.9 (d, C-2 and C-6), 128.3 (s, C-1), 116.3 (d, C-8), 115.4 (d, C-3 and C-5), 61.5 (t, C-10), 55.9 (q, OCH 3 ), 14.6 (q, C-11)。
as shown in FIGS. 5 to 7 and the above structural characterization data, compound a is p-methoxy ethyl cinnamate ((R))E)-1-(4-Methoxyphenyl)pent-1-en-3-one）。
The carbon spectrum, the hydrogen spectrum and the mass spectrum of the compound b are respectively shown in fig. 8, 9 and 10, and the structural characterization data of the compound b are as follows: 1 H-NMR (CDCl 3): 7.69 (1H, d, J = 16.0 Hz, H-7), 7.52 (2H, m, H-2 and H-6), 7.38 (3H, m, H-3, H-4 and H-5), 6.44 (1H, d, J = 16.0 Hz, H-8), 4.27 (2H, q, J = 7.1 Hz, H-10), 1.34 (3H, t, J = 7.1 Hz, H-11), 13C-NMR (CDCl 3): 167.0 (s, C-9), 144.6 (d, C-7), 130.2 (d, C-4), 134.4 (s, C-1), 128.8 (d, C-3 and C-5), 128.0 (d, C-2 and C-6), 118.2 (d, C-8), 60.5 (t, C-10), 14.11 (q-11). As shown in FIGS. 8 to 10 and the above structural characterization data, compound b is ethyl cinnamate ((R))E)-1-Phenylpent-1-en-3-one）。
Example 2
Ethyl p-methoxycinnamate and ethyl cinnamate were prepared according to the method of example 1, differing from example 1 only in that: the volume ratio of n-hexane, ethyl acetate, methanol and water =3.5:1.25:3.5:1.25; the purity of the ethyl p-methoxycinnamate is 97.25%, and the purity of the ethyl cinnamate is 97.28%.
Example 3
Ethyl p-methoxycinnamate and ethyl cinnamate were prepared according to the method of example 1, differing from example 1 only in that: the volume ratio of n-hexane, ethyl acetate, methanol and water =4:1:3.5:1.5; the purity of the ethyl p-methoxycinnamate is 97.32%, and the purity of the ethyl cinnamate is 97.27%.
Comparative example 1
Ethyl p-methoxycinnamate and ethyl cinnamate were prepared according to the method of example 1, differing from example 1 only in that: the volume ratio of n-hexane, ethyl acetate, methanol and water =4.5:1:4:1; and continuously detecting the effluent liquid at the outlet of the column at 270nm, wherein the p-methoxy ethyl cinnamate and the ethyl cinnamate are not detected.
Comparative example 2
Ethyl p-methoxycinnamate and ethyl cinnamate were prepared according to the method of example 1, differing from example 1 only in that: the volume ratio of n-hexane, ethyl acetate, methanol and water =4.2:1:4:1; the purity of the ethyl p-methoxycinnamate is 78.2%, and the purity of the ethyl cinnamate is 75.8%.
Comparative example 3
Kaempferia galanga extract was prepared according to CN 111166842A, example 1. However, a solvent system consisting of acetonitrile-methanol-disodium hydrogen phosphate solution (pH = 6.5) does not separate layers, and high-speed counter-current chromatography separation cannot be achieved, and ethyl p-methoxycinnamate and ethyl cinnamate cannot be separated.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (1)
1. A method for separating and purifying trans-ethyl p-methoxycinnamate by high-speed counter-current chromatography comprises the following steps:
mixing rhizoma kaempferiae with ethanol solution, and extracting with ethanol to obtain rhizoma kaempferiae crude extract; the volume fraction of ethanol in the ethanol solution is 50 to 95 percent; the temperature of the alcohol extraction is 50 to 80 ℃, the number of the alcohol extraction is 2 to 4, and the time of single alcohol extraction is 1 to 4h;
performing high-speed counter-current chromatography separation on the kaempferia galangal crude extract to obtain trans-ethyl p-methoxycinnamate; the two-phase solvent system adopted by the high-speed countercurrent chromatography is a normal hexane-ethyl acetate-methanol-water system, the upper phase of the two-phase solvent system is a stationary phase, and the lower phase of the two-phase solvent system is a mobile phase; in the n-hexane-ethyl acetate-methanol-water system, the volume ratio of n-hexane to ethyl acetate to methanol to water is 3.5:1.25:3.5:1.25;
the n-hexane-ethyl acetate-methanol-water system is subjected to degassing treatment before use;
the high-speed counter-current chromatography separation conditions comprise: the temperature is 20 to 30 ℃; the rotating speed of the host is 500 to 1000r/min; the flow rate of the mobile phase is 5 to 20mL/min.
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