CN113185406A - Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis - Google Patents
Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis Download PDFInfo
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- 150000007965 phenolic acids Chemical class 0.000 title claims abstract description 39
- 239000004480 active ingredient Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 241000687938 Salvia yunnanensis Species 0.000 title claims description 21
- 235000017508 Salvia yunnanensis Nutrition 0.000 title claims description 21
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 claims abstract description 52
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims abstract description 52
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 51
- IBGBGRVKPALMCQ-UHFFFAOYSA-N 3,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1O IBGBGRVKPALMCQ-UHFFFAOYSA-N 0.000 claims abstract description 50
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- 239000000284 extract Substances 0.000 claims abstract description 29
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- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 claims abstract description 26
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- 239000002904 solvent Substances 0.000 claims abstract description 24
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- YMGFTDKNIWPMGF-UCPJVGPRSA-N Salvianolic acid A Chemical class C([C@H](C(=O)O)OC(=O)\C=C\C=1C(=C(O)C(O)=CC=1)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-UCPJVGPRSA-N 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 244000132619 red sage Species 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
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- WTPPRJKFRFIQKT-UHFFFAOYSA-N 1,6-dimethyl-8,9-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione;1-methyl-6-methylidene-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(=C)C3=CC=C2C2=C1C(C)=CO2.O=C1C(=O)C2=C3CCC=C(C)C3=CC=C2C2=C1C(C)=CO2 WTPPRJKFRFIQKT-UHFFFAOYSA-N 0.000 description 1
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- YMGFTDKNIWPMGF-QHCPKHFHSA-N Salvianolic acid A Natural products OC(=O)[C@H](Cc1ccc(O)c(O)c1)OC(=O)C=Cc2ccc(O)c(O)c2C=Cc3ccc(O)c(O)c3 YMGFTDKNIWPMGF-QHCPKHFHSA-N 0.000 description 1
- CASZJDFDIAIAGM-UHFFFAOYSA-N acetonitrile;ethyl acetate;hydrate Chemical compound O.CC#N.CCOC(C)=O CASZJDFDIAIAGM-UHFFFAOYSA-N 0.000 description 1
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- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-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
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/80—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/48—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses a high-efficiency preparation method of phenolic acid active ingredients in Yunnan red sage root, which comprises the steps of extracting Yunnan red sage root to obtain a Yunnan red sage root total phenolic acid extract, and separating and purifying the Yunnan red sage root total phenolic acid extract by adopting pH zone countercurrent chromatography to obtain rosmarinic acid, caffeic acid and/or protocatechualdehyde; the solvent system of the pH zone countercurrent chromatography comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of the petroleum ether to the ethyl acetate to the acetonitrile to the water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4. The method provided by the invention can be used for more simply and conveniently separating and extracting the rosmarinic acid, the caffeic acid and the protocatechuic aldehyde in the salvia miltiorrhiza bunge, and the rosmarinic acid, the caffeic acid and the protocatechuic aldehyde which are separated and extracted have higher purity without further subsequent treatment.
Description
Technical Field
The invention relates to a high-efficiency preparation method of phenolic acid active ingredients in salvia yunnanensis.
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.
Rosmarinic acid is a natural antioxidant with strong antioxidant activity, and helps to prevent cell damage caused by free radicals, thus reducing the risk of cancer and arteriosclerosis. The rosmarinic acid has strong anti-inflammatory activity, and also has antibacterial, antiviral and antitumor activities. Caffeic acid is an organic acid, can be safely used in cosmetics, has wide antibacterial and antiviral activities, and has the capacity of absorbing ultraviolet rays; the caffeic acid with low concentration can be used as adjuvant for inhibiting skin type hair dye, and is beneficial to enhancing color intensity. Protocatechualdehyde not only has obvious protective effect on myocardial damage caused by acute myocardial ischemia and anoxia, but also has the effect of dilating cardiac and cerebral vessels, and also has the effects of improving microcirculation, reducing blood viscosity, resisting bacteria and inflammation, antagonizing calcium and the like.
According to research, the salvia miltiorrhiza bunge contains the three compounds, but the inventor finds that the existing method for extracting and purifying the three compounds has the problems of repeated chromatographic separation and purification, complex operation and the like due to more compound components in the salvia miltiorrhiza bunge.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a high-efficiency preparation method of phenolic acid active ingredients in salvia miltiorrhiza bunge, which can more simply and conveniently separate and extract rosmarinic acid, caffeic acid and protocatechualdehyde in salvia miltiorrhiza bunge.
In order to reduce the times of chromatographic separation, the invention adopts pH zone countercurrent chromatography (pH-ZRCCC) to treat the total phenolic acid extract of the Yunnan salvia miltiorrhiza bunge, however, researches show that the total phenolic acid extract of the Yunnan salvia miltiorrhiza bunge contains a large amount of salvianolic acid compounds, and the polarity of the compounds is close to that of caffeoylquinic acid compounds, so that rosmarinic acid, caffeic acid and protocatechuic aldehyde are difficult to extract or the purity of the extracted compounds is low.
In order to achieve the purpose, the technical scheme of the invention is as follows:
on the one hand, the pH zone countercurrent chromatography solvent system for extracting rosmarinic acid, caffeic acid and/or protocatechuic aldehyde from the salvia miltiorrhiza bunge comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of the petroleum ether to the ethyl acetate to the acetonitrile to the water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4.
On the other hand, an efficient preparation method of phenolic acid active ingredients in the Yunnan red sage root comprises the steps of extracting the Yunnan red sage root to obtain a Yunnan red sage root total phenolic acid extract, and separating and purifying the Yunnan red sage root total phenolic acid extract by adopting pH zone countercurrent chromatography to obtain rosmarinic acid, caffeic acid and/or protocatechuic aldehyde; the solvent system for pH zone countercurrent chromatography is the above-described solvent system.
Therefore, the invention improves the solvent system of pH zone countercurrent chromatography for the total savianolic acid extract, and experiments prove that when the solvent system is used for separating and purifying the pH zone countercurrent chromatography of the total savianolic acid extract, rosmarinic acid, caffeic acid and protocatechuic aldehyde can be separated and extracted, and the purity of the extracted compounds is more than 96 percent.
The invention has the beneficial effects that:
1. the method adopts petroleum ether, ethyl acetate, acetonitrile and water as a solvent system of pH zone countercurrent chromatography, can completely separate rosmarinic acid, caffeic acid and protocatechuic aldehyde in the salvia miltiorrhiza bunge, and has good separation effect.
2. The method provided by the invention can be used for extracting the rosmarinic acid, the caffeic acid and the protocatechuic aldehyde in the extract by only adopting one-time chromatographic separation, and because the pH zone countercurrent chromatography takes liquid as a stationary phase, the irreversible adsorption of a solid fixed relative sample is avoided, the recovery rate of the sample is greatly improved, and the yield of pure components is improved. Therefore, the rosmarinic acid, the caffeic acid and the protocatechuic aldehyde with high purity (more than 96 percent) can be obtained without subsequent purification treatment, the operation steps are greatly simplified, the method is simple, and the purification cost is lower.
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 Performance Liquid Chromatography (HPLC) analysis chart of the total phenolic acid extract of Salvia yunnanensis with pure components separated by pH-ZRCCC according to the present invention;
FIG. 2 is a diagram showing the separation of pH-ZRCCC in the example 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.
The chemical structural formula of the rosmarinic acid is shown as
The chemical structural formula of caffeic acid is shown as
The chemical structural formula of protocatechualdehyde is
In view of the fact that the prior chromatographic technology needs to adopt a plurality of chromatographic separation and purification steps to extract rosmarinic acid, caffeic acid and protocatechuic aldehyde from the salvia miltiorrhiza bunge, the invention provides a high-efficiency preparation method of phenolic acid active ingredients in the salvia miltiorrhiza bunge.
The invention provides a solvent system for pH zone countercurrent chromatography for extracting rosmarinic acid, caffeic acid and/or protocatechuic aldehyde from Salvia miltiorrhiza Bunge, which comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of the petroleum ether to the ethyl acetate to the acetonitrile to the water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4.
In some embodiments, petroleum ether, ethyl acetate, acetonitrile and water are mixed uniformly, and the mixture is subjected to static liquid separation, wherein an upper phase after liquid separation is a stationary phase, and a lower phase after liquid separation is a mobile phase.
The stationary phase needs to be acidified and the mobile phase needs to be alkalified and used, in one or more embodiments, trifluoroacetic acid is added to the stationary phase and ammonia is added to the mobile phase. After the ultrasonic degassing treatment is carried out on the stationary phase and the mobile phase, the separation and extraction effect is better. When the concentration of trifluoroacetic acid in the stationary phase is 9-11 mM and the concentration of ammonia water in the mobile phase is 29-31 mM, the separation and extraction effect is more excellent.
Another embodiment of the invention provides a high-efficiency preparation method of phenolic acid active ingredients in Yunnan red sage root, which comprises the steps of extracting Yunnan red sage root to obtain a Yunnan red sage root total phenolic acid extract, and separating and purifying the Yunnan red sage root total phenolic acid extract by adopting pH zone countercurrent chromatography to obtain rosmarinic acid, caffeic acid and/or protocatechuic aldehyde; the solvent system for pH zone countercurrent chromatography is the above-described solvent system.
The invention aims at the total phenolic acid extract of the Yunnan salvia miltiorrhiza, and improves the components of a counter-current chromatographic solvent system in a pH zone according to the principle of 'similarity and compatibility', so that when the extract is subjected to counter-current chromatographic separation and extraction in the pH zone, the extract can be used for separating and extracting the rosmarinic acid, the caffeic acid and the protocatechuic aldehyde in one step, the operation is simple and convenient, and the purity of the extracted compound is higher.
In some embodiments, the process for obtaining the total phenolic acid extract of the salvia yunnanensis from the salvia yunnanensis comprises the following steps: extracting the salvia miltiorrhiza bunge by adopting an ethanol extraction method, carrying out primary extraction on the extracted liquid by adopting petroleum ether, adjusting the pH of the extracted water phase to 1.5-2.5, then adding ethyl acetate for secondary extraction, and concentrating the ethyl acetate phase obtained by the secondary extraction to obtain the total phenolic acid extract of the salvia miltiorrhiza bunge.
The 'primary' and 'secondary' in the primary extraction and the secondary extraction only indicate different extraction processes, and are not limited to the extraction times.
In one or more embodiments, the salvia yunnanensis is added into an ethanol water solution, reflux extraction is carried out at 65-75 ℃, an extracting solution is concentrated, water is added for redissolution, and then petroleum ether is adopted for primary extraction. Wherein the concentration of the ethanol water solution is 55-65% by volume fraction. The material-liquid ratio of the salvia yunnanensis to the ethanol water solution is 1: 7.5-8.5, kg: and L. The reflux extraction times are 2-4 times, and the time of each reflux extraction is 1.5-2.5 h.
In one or more embodiments, the number of secondary extractions is 3-7.
In some embodiments, the pH zone countercurrent chromatographic separation extraction process is: conveying the stationary phase to a separation column, injecting the solution of the total phenolic acid extract of the Yunnan red sage root into the separation column, and then conveying the mobile phase to the separation column continuously for separation and extraction.
In one or more embodiments, the stationary phase is delivered to the separation column at a flow rate of 25 to 35 mL/min.
In one or more embodiments, the flow rate of the mobile phase is 1.5 to 2.5 mL/min. The time to collect the fractions was 6min per collection (vial).
In some embodiments, the pH zone countercurrent chromatographic separation and extraction has a detection wavelength of 252-256 nm by an ultraviolet detector.
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.
The reagents used in the following examples are all commercially available unless otherwise specified. The Yunnan Danshen root is purchased from Xinping county of Yunnan province.
Examples
Extracting and preparing a sample:
weighing 3kg of Yunnan red sage root, crushing, and performing reflux extraction for 3 times at 70 ℃ by using 60% ethanol, wherein the material-liquid ratio is 1:8(w/v) and each time is 2 hours. The extract was filtered, concentrated to about 800mL at 50 ℃ under reduced pressure, and reconstituted to 1000mL with water. Extracting the extractive solution with petroleum ether for three times, removing the upper layer, adding hydrochloric acid into the water phase to adjust the pH of the extractive solution to 2, and extracting with ethyl acetate for 5 times. Mixing ethyl acetate phases, and concentrating under reduced pressure to obtain 136g of total phenolic acid extract of Saviae Miltiorrhizae radix.
Preparation of two-phase solvent system and sample solution:
according to a two-phase solvent system, the solvents are respectively weighed according to a proportion and are sequentially added into a separating funnel. After shaking, the mixture was allowed to stand for 12 hours, and the upper and lower phases were separated. Trifluoroacetic acid with a certain concentration is added into the upper phase as a stationary phase, and ammonia water with a certain concentration is added into the lower phase as a mobile phase. Ultrasonic degassing the upper and lower phases for 2min to obtain solvent system for pH-ZRCCC separation.
And (3) putting the sample into a test tube, and ultrasonically dissolving the sample by using the acidified upper phase solution and the equivalent non-alkalized lower phase solution, wherein the total volume is not more than 20mL, so as to prepare a sample solution for separating the pH-ZRCCC.
Separation by pH-zone countercurrent chromatography
Pumping the upper phase solution at the flow rate of 30mL/min until the separation column is filled, injecting the sample solution into the separation column through a sample injection ring, adjusting the separation column to rotate at the positive rotation speed of 800rpm, and simultaneously pumping the lower phase solution at the flow rate of 2 mL/min. The detection wavelength of the ultraviolet detector was adjusted to 254nm, fractions were collected at 6 min/vial intervals, and the pH of each vial fraction was measured using a pH meter. After the separation process is finished, the residual liquid in the column is blown into a measuring cylinder by using an air compressor, and the retention rate of the stationary phase is calculated. And finally, sequentially cleaning the separation column by using water and ethanol.
HPLC analysis and structural identification
HPLC analysis conditions of the total phenolic acid extract of Salvia yunnanensis and the pure components separated by pH-ZRCCC are Compass C18 chromatographic column (250X 4.6mm,5 μm), and the mobile phase is acetonitrile (A) -0.1% formic acid water solution (B): 0-10 min, 14% A; 10-20 min, 14% -17% A; 20-35 min, 17% -30% A. The flow rate is 1.0mL/min, the detection wavelength is 280nm, and the sample injection amount is 10 mu L.
The pure fraction isolated from pH-ZRCCC was purified using HPLC-ESI-QTOF-MS and13C-NMR、1H-NMR structural identification was carried out on the pure components.
Results of HPLC analysis
FIG. 1 shows HPLC analysis chart of total phenolic acid extract of Salvia Yunnanensis and pure components separated by pH-ZRCCC, and the peak area ratios of compounds I-III in the crude extract of total phenolic acid are 5.5% (rosmarinic acid), 1.29% (caffeic acid) and 0.77% (protocatechualdehyde) respectively, calculated by peak area normalization at 280 nm.
Optimization of two-phase solvent systems
Firstly, ethyl acetate-acetonitrile-water (4:1:5, v/v/v) is used as a separation solvent system, trifluoroacetic acid (10mM) is added into the upper phase, and ammonia water (10mM) is added into the lower phase to separate 2g of total Yunnan red sage root samples, but the compound is difficult to elute and has longer retention time. Considering that the total phenolic acid of the salvia yunnanensis contains a large amount of salvianolic acid compounds, the polarity of the salvianolic acid compounds is slightly lower than that of caffeoylquinic acid compounds, according to the principle of similar compatibility, a solvent system is changed into petroleum ether-ethyl acetate-acetonitrile-water (1.5:2.5:1:5, v/v/v/v) with lower polarity, meanwhile, the concentration of lower phase ammonia water is increased to 30mM, trifluoroacetic acid is added into an upper phase, and a 2g sample is separated. Combined with HPLC analysis, it was found that rosmarinic acid, caffeic acid and protocatechuic aldehyde were successfully separated within 4 h. Subsequently, the amount of the sample was gradually increased to 4g, and it was found that rosmarinic acid, caffeic acid and protocatechuic aldehyde still gave good separation effect, and the pH-ZRCCC separation pattern did not change much, as shown in FIG. 2. Through one-time separation, 55.6mg of rosmarinic acid, 69.0mg of caffeic acid and 18.9mg of protocatechuic aldehyde are obtained from 4g of crude extract of the Yunnan salvianolic acid, and the purity is 98.4%, 96.1% and 96.6% respectively.
Structural identification of compounds
According to ESI-MS,1H-NMR and13C-NMR carries out structural identification on the compounds I to III, and specific data are as follows:
rosmarinic acid: the peak of the excimer ion is M/z 359[ M-H]–The suggested relative molecular weight is 360, and the fragment of the second-order mass spectrum is m/z 161[ caffeoyloxy-H ]2O]–。1H-NMR(DMSO-d6,400MHz)δ(ppm):7.06(1H,s,H-2),6.77(1H,d,J＝8.0Hz,H-5),6.99(1H,d,J＝8.0Hz,H-6),7.43(1H,d,J＝16.0Hz,H-7),6.23(1H,d,J＝16.0Hz,H-8),6.68(1H,s,H-2’),6.63(1H,d,J＝8.0Hz,H-5’),6.52(1H,d,J＝8.0Hz,H-6’),3.02-2.98(1H,m,H-7’a),2.88-2.82(1H,m,H-7’b),4.97(1H,dd,J＝4.0,12.0Hz,H-8’).13C-NMR(DMSO-d6,100MHz)δ(ppm):125.9(C-1),115.4(C-2),146.2(C-3),149.1(C-4),116.3(C-5),121.8(C-6),145.8(C-7),114.3(C-8),166.5(C-9),128.6(C-1’),117.1(C-2’),145.4(C-3’),144.3(C-4’),115.9(C-5’),120.4(C-6’),37.0(C-7’),74.3(C-8’),171.9(C-9’)。
Caffeic acid: the peak of the excimer ion is M/z 179[ M-H]–The suggested relative molecular weight is 180, and the fragment of the second-order mass spectrum is m/z 135[ caffeoyloxy-CO ]2]–。1H-NMR(DMSO-d6,400MHz)δ(ppm):7.02(1H,s,H-2),6.75(1H,d,J＝8.0Hz,H-5),6.96(1H,d,J＝8.0Hz,H-6),7.41(1H,d,J＝16.0Hz,H-7),6.16(1H,d,J＝16.0Hz,H-8).13C-NMR(DMSO-d6,100MHz)δ(ppm):126.2(C-1),115.6(C-2),145.0(C-3),148.6(C-4),115.1(C-5)121.6(C-6),146.0(C-7),116.2(C-8),168.3(C-9)。
Protocatechualdehyde: the peak of the excimer ion is M/z 137[ M-H ]]–The relative molecular weight is indicated to be 138.1H-NMR(DMSO-d6,400MHz)δ(ppm):10.10(1H,s,4-OH),9.70(1H,s,-CHO),9.54(1H,s,3-OH),7.23(1H,s,H-2),6.91(1H,d,J＝8.0Hz,H-5),7.27(1H,d,J＝8.0Hz,H-6).13C-NMR(DMSO-d6,100MHz)δ(ppm):129.3(C-1),116.0(C-2),146.4(C-3),152.6(C-4),114.9(C-5),124.9(C-6),191.5(C-7)。
The invention uses a solvent system of petroleum ether-ethyl acetate-acetonitrile-water (1.5:2.5:1:5, v/v/v/v) (trifluoroacetic acid (10mM) is added on the upper phase, and ammonia water (30mM) is added on the lower phase) to separate 55.6mg of rosmarinic acid, 69.0mg of caffeic acid and 18.9mg of protocatechualdehyde from 4g of crude extract of total phenolic acid of Yunnan salvia miltiorrhiza, and the purity is higher than 96%.
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 (10)
1. A solvent system for extracting rosmarinic acid, caffeic acid and/or protocatechuic aldehyde in Salvia miltiorrhiza Bunge with pH zone countercurrent chromatography comprises petroleum ether, ethyl acetate, acetonitrile and water, wherein the volume ratio of the petroleum ether to the ethyl acetate to the acetonitrile to the water is 1.46-1.54: 2.46-2.54: 1: 4.6-5.4.
2. The solvent system according to claim 1, wherein petroleum ether, ethyl acetate, acetonitrile and water are mixed uniformly, and the mixture is subjected to static liquid separation, wherein an upper phase after liquid separation is a stationary phase, and a lower phase after liquid separation is a mobile phase.
3. The solvent system of claim 2, wherein trifluoroacetic acid is added to the stationary phase and aqueous ammonia is added to the mobile phase; preferably, the concentration of trifluoroacetic acid in the stationary phase is 9-11 mM, and the concentration of ammonia in the mobile phase is 29-31 mM.
4. An efficient preparation method of phenolic acid active ingredients in Yunnan red sage root is characterized in that the Yunnan red sage root total phenolic acid extract is obtained by extracting the Yunnan red sage root, and the Yunnan red sage root total phenolic acid extract is separated and purified by adopting pH zone countercurrent chromatography to obtain rosmarinic acid, caffeic acid and/or protocatechuic aldehyde; the solvent system for pH zone countercurrent chromatography is the solvent system as claimed in any one of claims 1 to 3.
5. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 4, wherein the total phenolic acid extract of Salvia yunnanensis is obtained by extracting Salvia yunnanensis with the following steps: extracting the salvia miltiorrhiza bunge by adopting an ethanol extraction method, carrying out primary extraction on the extracted liquid by adopting petroleum ether, adjusting the pH of the extracted water phase to 1.5-2.5, then adding ethyl acetate for secondary extraction, and concentrating the ethyl acetate phase obtained by the secondary extraction to obtain the total phenolic acid extract of the salvia miltiorrhiza bunge.
6. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 5, wherein the Salvia yunnanensis is added into ethanol water solution, reflux extraction is carried out at 65-75 ℃, the extract is concentrated, water is added for redissolution, and then petroleum ether is adopted for primary extraction; preferably, the concentration of the ethanol water solution is 55-65% by volume fraction; preferably, the material-liquid ratio of the salvia yunnanensis to the ethanol water solution is 1: 7.5-8.5; preferably, the reflux extraction times are 2-4 times, and the time of each reflux extraction is 1.5-2.5 h.
7. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 5, wherein the number of secondary extraction is 3-7.
8. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 4, wherein the pH zone countercurrent chromatography separation and extraction process comprises: conveying the stationary phase to a separation column, injecting the solution of the total phenolic acid extract of the Yunnan red sage root into the separation column, and then conveying the mobile phase to the separation column continuously for separation and extraction.
9. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 8, wherein the flow rate of the stationary phase conveyed to the separation column is 25-35 mL/min;
or the flow rate of the mobile phase is 1.5-2.5 mL/min.
10. The method for efficiently preparing phenolic acid active ingredients in Salvia yunnanensis of claim 4, wherein the detection wavelength of the ultraviolet detector is 252-256 nm in the pH zone countercurrent chromatography separation and extraction.
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