CN111948319B - Efficient separation and analysis method for active ingredients in enteromorpha - Google Patents
Efficient separation and analysis method for active ingredients in enteromorpha Download PDFInfo
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Abstract
The invention discloses a high-efficiency separation and analysis method of active ingredients in enteromorpha, which comprises the steps of carrying out ethyl acetate extraction on an alcohol extract of enteromorpha to obtain an enteromorpha extract, carrying out separation and purification on the enteromorpha extract by high-speed countercurrent chromatography to obtain a plurality of components, and carrying out liquid chromatography-mass spectrometry detection on each component; the high-speed countercurrent chromatography adopts two phase systems, wherein the first phase system is tert-butyl methyl ether, acetonitrile and water according to the volume ratio of 3.5-4.5: 1: 4.5-5.5, the second phase system is tert-butyl methyl ether, n-butyl alcohol, acetonitrile and water according to the volume ratio of 2.5-3.5: 0.9-1.1: 1: 4.5-5.5, when in use, the lower phase of the first two phase system is used as a stationary phase, and the upper phase of the first two phase system and the upper phase of the second two phase system are both used as mobile phases. The method can be used for separating and analyzing the polyphenol active ingredients in the enteromorpha.
Description
Technical Field
The invention relates to a high-efficiency analysis method of active ingredients of enteromorpha, in particular to a high-efficiency separation analysis method of active ingredients in enteromorpha.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Enteromorpha (Enteromorpha) belongs to Chlorophyta (chlorephyta), ulva (Ulvales) and Ulvaceae (Ulvaceae) in taxonomy, and mainly comprises 5 Enteromorpha (Enteromorpha clathrata), Enteromorpha (e.intestinalis), Enteromorpha oblata (e.compressa), Enteromorpha (e.prolifra) and Enteromorpha linza (e.linza), and grows in intertidal zone. The enteromorpha prolifera is green tide algae, has algae resources with high utilization value, and researches show that the enteromorpha prolifera contains rich nutritional ingredients. The components of the enteromorpha are required to be excavated in detail and analyzed deeply.
According to the research of the inventor, the research of the seaweed at present mainly focuses on the research of polysaccharide, the research on polyphenol is less, and the traditional separation method is mainly used for the separation and purification of the polyphenol. The traditional separation and purification method mainly comprises the following steps: separating with macroporous resin and preparing liquid phase. The disadvantages of macroporous resin separation are mainly: (1) high organic residue and high pretreatment difficulty; (2) the strength is poor, the damage is serious in the using process, and the service life is short; (3) wide particle size distribution and poor separation effect. The disadvantages of preparative liquid phase separations are mainly: low efficiency, low yield, and unsatisfactory purification effect for excessively complex mixtures.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a high-efficiency separation and analysis method for active ingredients in enteromorpha, which can be used for separating and analyzing small molecular compounds containing polyphenol active ingredients in enteromorpha.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a high-efficiency separation and analysis method for active ingredients in Enteromorpha prolifera comprises the steps of carrying out ethyl acetate extraction on an alcohol extract of Enteromorpha prolifera to obtain an Enteromorpha prolifera extract, carrying out separation and purification on the Enteromorpha prolifera extract by high-speed countercurrent chromatography to obtain a plurality of components, and carrying out liquid chromatography-mass spectrometry detection on each component;
the high-speed countercurrent chromatography adopts two phase systems, wherein the first phase system is tert-butyl methyl ether, acetonitrile and water according to the volume ratio of 3.5-4.5: 1: 4.5-5.5, the second phase system is tert-butyl methyl ether, n-butyl alcohol, acetonitrile and water according to the volume ratio of 2.5-3.5: 0.9-1.1: 1: 4.5-5.5, when in use, the lower phase of the first two phase system is used as a stationary phase, and the upper phase of the first two phase system and the upper phase of the second two phase system are both used as mobile phases.
Experiments show that the two-phase system of high-speed countercurrent chromatography is adopted to influence the separation of active ingredients in the enteromorpha, the two-phase system provided by the invention can realize the preliminary separation of the active ingredients in the enteromorpha, and then the liquid chromatography-mass spectrometry is adopted to further detect and separate the preliminarily separated active ingredients.
The invention has the beneficial effects that:
according to the invention, the high-speed counter-current chromatography is adopted to primarily separate the polyphenol active ingredients in the enteromorpha, and then the liquid chromatography-mass spectrometry is adopted to detect each of the primarily separated components, so that the components of the enteromorpha can be excavated and deeply analyzed in detail.
The invention adopts a specific two-phase system to realize the preliminary separation of the micromolecule compounds containing the polyphenol active ingredients in the enteromorpha, thereby obtaining a plurality of components which can be used for LC-MS detection.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a high-speed countercurrent chromatogram for crude Enteromorpha prolifera separation in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of example 1 of the present invention;
FIG. 3 is a high-speed countercurrent chromatogram of example 2 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the difficulty in separating and detecting small molecular compounds containing polyphenol active ingredients in enteromorpha in the prior art, the invention provides a high-efficiency separation and analysis method for active ingredients in enteromorpha.
The invention provides a typical implementation mode and provides a high-efficiency separation analysis method of active ingredients in enteromorpha, wherein the alcohol extract of enteromorpha is subjected to ethyl acetate extraction to obtain an enteromorpha strip extract, the enteromorpha strip extract is subjected to high-speed countercurrent chromatography for separation and purification to obtain a plurality of components, and each component is subjected to liquid chromatography-mass spectrometry detection;
the high-speed countercurrent chromatography adopts two phase systems, wherein the first phase system is tert-butyl methyl ether, acetonitrile and water according to the volume ratio of 3.5-4.5: 1: 4.5-5.5, the second phase system is tert-butyl methyl ether, n-butyl alcohol, acetonitrile and water according to the volume ratio of 2.5-3.5: 0.9-1.1: 1: 4.5-5.5, when in use, the lower phase of the first two phase system is used as a stationary phase, and the upper phase of the first two phase system and the upper phase of the second two phase system are both used as mobile phases.
Experiments show that the two-phase system of high-speed countercurrent chromatography is adopted to influence the separation of active ingredients in the enteromorpha, the two-phase system provided by the invention can realize the preliminary separation of micromolecule compounds containing polyphenol active ingredients in the enteromorpha, and then the preliminary separation of the active ingredients can be further detected and separated by adopting liquid chromatography-mass spectrometry.
In some embodiments of the embodiment, the alcohol extract of Enteromorpha prolifera is extracted by ethanol with volume fraction of 55-65%.
In one or more embodiments, the alcohol extract of the Enteromorpha prolifera is extracted by refluxing Enteromorpha prolifera powder with 55-65% ethanol for 2-4 times, each time for 2-4 days, filtering after extraction, and concentrating until no alcohol smell exists to obtain the alcohol extract of the Enteromorpha prolifera.
In some examples of this embodiment, the raw materials of the two-phase system are mixed and allowed to stand for layering, with the upper layer being the upper phase of the two-phase system and the lower layer being the lower phase of the two-phase system.
In some examples of this embodiment, when performing the separation and purification by high-speed countercurrent chromatography, the lower phase of the first two-phase system is used as the stationary phase, the upper phase of the first two-phase system is used as the mobile phase for performing the chromatographic separation, and after the separation is completed, the upper phase of the second two-phase system is used as the mobile phase for continuing the chromatographic separation.
In one or more embodiments, the upper phase of the first two-phase system is used as the mobile phase for chromatographic separation, and after the 5 th component is separated, the upper phase of the second two-phase system is used as the mobile phase for chromatographic separation.
In some examples of the embodiment, the column volume of the high-speed countercurrent chromatography is 250-350 mL, the sample loading amount is 150-250 mg, the rotation speed is 700-900 r/min, the flow rate is 1.5-2.5 mL/min, and the detection wavelength is 254 +/-2 nm.
In some embodiments of this embodiment, the chromatography conditions for LC-MS are: the chromatographic column is C18Column, ultraviolet detection wavelength 280 ± 2nm, flow rate: 0.5-1.5 mL/min, sample injection amount: 5-15 mu L of the mixture, wherein the mobile phase A is acetonitrile, and the mobile phase B is 0.2% formic acid solution.
In one or more embodiments, C18The particle size of the filler of the column is 5 +/-1 mu m, the inner diameter of the column is 4.6 +/-0.1 mm, and the length of the column is 245-255 mm.
In one or more embodiments, the gradient elution procedure for liquid chromatography-mass spectrometry is: 0min, 10% A; 0-5 min, 10-25% of A; 5-15 min, 25-25% A; 15-40 min, 25-45% A; 40-45 min, 45-45% A; 45-46 min, 45-10% A; 46-51 min, 10-10% A. % is volume percent.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Sample extraction:
completely pulverizing dried Enteromorpha prolifera (1kg) into powder, extracting with 3L 60% ethanol for 3 times, 3 days each time, and filtering the extract with vacuum filtration device. All extracts were combined and concentrated to no alcohol taste, then the concentrate was extracted with equal amount of ethyl acetate. The ethyl acetate fractions were evaporated to dryness by rotation at 55 ℃ to give 10g of crude sample, which was stored in a refrigerator at 4 ℃ for further HSCCC purification.
Applying gradient countercurrent chromatography to enrich polyphenol compounds in the segmented Enteromorpha prolifera:
preparing a two-phase solvent system of tert-butyl methyl ether/n-butanol/acetonitrile/water (4:0:1:5, v/v) and tert-butyl methyl ether/n-butanol/acetonitrile/water (3:1:1:5, v/v) according to the solvent ratio respectively, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, wherein the upper phase is a mobile phase, and the lower phase is a stationary phase. 200mg of Enteromorpha prolifera ethyl acetate extract is dissolved in 5mL of the upper phase and 5mL of the lower phase for standby. A high-speed counter-current chromatograph is adopted and comprises a plunger pump, a sample injection valve, an ultraviolet detector, a recorder, a chromatographic separation column (a spiral pipe column formed by winding polytetrafluoroethylene pipes in multiple layers and with the capacity of 300mL) and the like, wherein the sample injection valve is firstly in a sample injection state, the chromatographic separation column is filled with a lower phase (stationary phase) of tert-butyl methyl ether/n-butyl alcohol/acetonitrile/water (4:0:1:5, v/v) at a certain flow rate by using the pump, and the pump is stopped. Starting a speed controller to enable a chromatographic separation column of a high-speed flow chromatograph to reversely rotate, setting the flow rate of a mobile phase to be 5.0mL/min when the rotation speed reaches 800 revolutions/min, starting pumping an upper phase of the mobile phase of tert-butyl methyl ether/n-butanol/acetonitrile/water (4:0:1:5, v/v) into the counter-current chromatograph for balancing, and injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector after the hydrodynamic balance is achieved, so that the sample enters the chromatographic separation column. Setting the flow rate of the mobile phase to be 5.0mL/min, starting pumping the upper phase of the mobile phase of tert-butyl methyl ether/n-butanol/acetonitrile/water (4:0:1:5, v/v), and then receiving the components a-e in the figure 1 according to the ultraviolet spectrogram of a detector. After e is taken out, the gradient elution of the upper phase of the mobile phase tert-butyl methyl ether/n-butanol/acetonitrile/water (3:1:1:5, v/v) is started, and then the components f-k in the figure 1 are received according to the ultraviolet spectrogram of a detector.
And (3) analyzing the separated substances by using high performance liquid chromatography, wherein the liquid phase conditions are as follows: waters-RP C18Column (4.6X 250mm), ultraviolet detection wavelength 280nm, column temperature: 25 ℃, flow rate: 0.8mL/min, sample size: 20 μ L, mobile phase was eluted with a gradient of methanol (a) and 0.2% aqueous formic acid (B) under the following conditions: 0min, 10% A; 0-5 min, 10-25% of A; 5-15 min, 25-25% A; 15-40 min, 25-45% A; 40-45 min, 45-45% A; 45-46 min, 45-10% A; 46-51 min, 10-10% A.
The result of high performance liquid chromatography is shown in fig. 2, the uppermost graph in fig. 2 is the high performance liquid chromatogram of the ethyl acetate extract, and from the graph, only 7 obvious chromatographic peaks are shown, while the high performance liquid chromatogram of the gradient countercurrent chromatography fractional collection of the components a-k in the embodiment can be seen, 24 compounds can be separated and detected, and the high performance liquid chromatogram of the ethyl acetate extract can show the chromatographic peaks of other 15 compounds only after being amplified. This example shows that more compound fractions in the ethyl acetate extract can be detected.
And (3) structural identification: and (3) respectively measuring the primary mass spectrum and the secondary mass spectrum of the polyphenol obtained by separation by using an Agilent 5973N mass spectrometer.
Primary mass spectrum conditions: full scan mode (full MS); nitrogen flow rate: 8L/min; voltage: 3.5 KV; drying temperature: 200 ℃; the scanning range m/z is 50-1500.
Secondary mass spectrum conditions: the ionization source is in an electrospray negative ion mode (ESI-); and (3) monitoring mode: a full scan mode; nitrogen flow rate: 8L/min; voltage: 3 KV; drying temperature: 200 ℃; the scanning range m/z is 50-1500.
24 compounds were detected in total, and 14 of them were identified as polyphenols (7), flavonoids (6), alkaloids (1), and coumarins (1), respectively, as shown in table 1.
Table 1 Mass spectrum analysis table for separation of crude enteromorpha prolifera products
Example 2
The difference from example 1 is that the upper phase is directly eluted and enriched using a solvent system of tert-butyl methyl ether/n-butanol/acetonitrile/water (3:1:1:5, v/v), and the rest of the procedure is the same as example 1, with the results shown in FIG. 3.
As can be seen from FIG. 3, the use of only one two-phase solvent system results in no separation between the effective components.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A high-efficiency separation and analysis method of active ingredients in Enteromorpha prolifera is characterized in that ethanol extract of the Enteromorpha prolifera is extracted by ethyl acetate to obtain Enteromorpha prolifera extract, the Enteromorpha prolifera extract is separated and purified by high-speed countercurrent chromatography to obtain a plurality of components, and each component is subjected to liquid chromatography-mass spectrometry detection;
the high-speed countercurrent chromatography adopts two phase systems, wherein the first phase system is tert-butyl methyl ether, acetonitrile and water according to the volume ratio of 3.5-4.5: 1: 4.5-5.5, the second phase system is tert-butyl methyl ether, n-butyl alcohol, acetonitrile and water according to the volume ratio of 2.5-3.5: 0.9-1.1: 1: 4.5-5.5, when in use, the lower phase of the first phase system is used as a stationary phase, and the upper phase of the first phase system and the upper phase of the second phase system are both used as mobile phases;
taking the upper phase of the first two-phase system as a mobile phase for chromatographic separation, separating out the 5 th component, and then taking the upper phase of the second two-phase system as the mobile phase for chromatographic separation;
the column volume of the high-speed countercurrent chromatograph is 250-350 mL, the sample loading amount is 150-250 mg, the rotating speed is 700-900 r/min, the flow rate is 1.5-2.5 mL/min, and the detection wavelength is 254 +/-2 nm;
the chromatographic conditions for LC-MS are as follows: the chromatographic column is a C18 column, the ultraviolet detection wavelength is 280 +/-2 nm, and the flow rate is as follows: 0.5-1.5 mL/min, sample injection amount: 5-15 mu L of mobile phase A, acetonitrile and 0.2% formic acid solution;
the gradient elution procedure for LC-MS chromatography was: 0min, 10% A; 0-5 min, 10-25% of A; 5-15 min, 25-25% A; 15-40 min, 25-45% A; 40-45 min, 45-45% A; 45-46 min, 45-10% A; 46-51 min, 10-10% A.
2. The efficient separation and analysis method for the active ingredients in the enteromorpha prolifera as claimed in claim 1, wherein the alcohol extract of the enteromorpha prolifera is extracted by ethanol with the volume fraction of 55-65%.
3. The method for efficiently separating and analyzing active ingredients in Enteromorpha prolifera according to claim 2, wherein the alcohol extract of Enteromorpha prolifera is obtained by extracting Enteromorpha prolifera powder with 55-65% ethanol under reflux for 2-4 times, each time for 2-4 days, filtering after extraction, and concentrating until no alcohol smell exists to obtain the alcohol extract of Enteromorpha prolifera.
4. The method for efficiently separating and analyzing active ingredients in enteromorpha according to claim 1, wherein the raw materials of the two-phase system are mixed and allowed to stand for layering, and the upper layer is the upper phase of the two-phase system and the lower layer is the lower phase of the two-phase system.
5. The method for separating and analyzing active ingredients in enteromorpha according to claim 1, wherein when the separation and purification are performed by high-speed countercurrent chromatography, the lower phase of the first two-phase system is used as a stationary phase, the upper phase of the first two-phase system is used as a mobile phase for chromatographic separation, and after the separation is finished, the upper phase of the second two-phase system is used as a mobile phase for further chromatographic separation.
6. The efficient separation and analysis method for active ingredients in enteromorpha as claimed in claim 1, wherein the filler particle size of the C18 column is 5 +/-1 μm, the inner diameter of the column is 4.6 +/-0.1 mm, and the length of the column is 245-255 mm.
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