CN114957362B - Method for separating anti-inflammatory active ingredients of coreopsis tinctoria - Google Patents
Method for separating anti-inflammatory active ingredients of coreopsis tinctoria Download PDFInfo
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- CN114957362B CN114957362B CN202210579348.5A CN202210579348A CN114957362B CN 114957362 B CN114957362 B CN 114957362B CN 202210579348 A CN202210579348 A CN 202210579348A CN 114957362 B CN114957362 B CN 114957362B
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Abstract
The invention belongs to the technical field of plant extract separation, and particularly relates to a method for separating anti-inflammatory active ingredients of coreopsis tinctoria. The flavonoid content in coreopsis tinctoria is high, but the types of flavonoid compounds in coreopsis tinctoria found at present are relatively few, and the coreopsis tinctoria needs to be further developed. The invention separates the compound gossypetin-5- (6' - (E) -caffeoyl) -beta-D-glucide from coreopsis tinctoria, and the specific technical scheme is as follows: 1) Grading and extracting the coreopsis tinctoria ethanol extract with petroleum ether and ethyl acetate, collecting ethyl acetate extract, and removing solvent to obtain ethyl acetate extract; 2) Gradient eluting the ethyl acetate part extractum by using a macroporous resin column, wherein the gradient solvent is 0% -40% ethanol solution; taking a 40% ethanol elution part to obtain an active part Fr2; standing the active site Fr2 to separate out precipitate to obtain an active site Fr2-1; the active site Fr2-1 is subjected to semi-preparative liquid chromatography to obtain an active site Fr2-1-2; the active site Fr2-1-2 is subjected to semi-preparative liquid chromatography to obtain a compound gossypetin-5- (6' - (E) -caffeoyl) -beta-D-glucoside. The invention provides reliable theoretical and experimental basis for future development and research of coreopsis tinctoria drug resources.
Description
Technical Field
The invention belongs to the technical field of plant extract separation, and particularly relates to a method for separating anti-inflammatory active ingredients of coreopsis tinctoria.
Background
Coreopsis tinctoria, the name of coreopsis tinctoria (Coreopsis tinctoria nutt.). The plant is also called as a perennial herb of the genus Coreopsis (Coreopsis) of the family Compositae (Compositae), and is mainly distributed in Xinjiang Hetian and Yunnan part of China. The sun-dried stamen and petals preserve complete organic components and have high nutritional and medicinal values. Modern pharmacological studies show that coreopsis tinctoria has various activities of reducing blood sugar, resisting oxidation, resisting inflammation, resisting cardiovascular diseases, protecting nerves, resisting tumors and the like.
Li and the like are separated from the dried inflorescences of coreopsis tinctoria to obtain various phenolic compounds such as phenolic acids, chalcones, flavonoids, flavonols and the like, and the obtained compounds are acted on N9 cells induced by LPS, so that the phenolic acids can obviously inhibit the generation of NO in the N9 cells. Zhang et al identified 4 novel C14-polyacetylene glycosides from the snow chrysanthemum head, designated nucleosides A-D, found that each compound was found at 1X 10 -6 At a concentration of mol/L (IC 50 ：0.22～8.8×10 -2 Mu mol/L), has stronger inhibition effect on cyclooxygenase-2 (COX-2). Cao Fenghua it has been found by studies that ethyl acetate extract of coreopsis tinctoria (1-100 mu moL/L) and the main component of the extract of coreopsis tinctoria can significantly inhibit proliferation of CD4+ T cells (an important T lymphocyte in the human immune system) of a C57BL/6 mouse, and are concentration-dependent. From the above studies, it can be seen that the current evaluation of the anti-inflammatory effect of coreopsis tinctoria is not comprehensive, and only a few documents report the inhibition of a single inflammatory factor by a compound in vitro, and further more experiments are required to confirm the anti-inflammatory effect of coreopsis tinctoria and the molecular mechanism thereof.
In addition, it has been reported that snow chrysanthemum contains 37 kinds of flavone. The flavone in the coreopsis tinctoria is abundant in quantity, and the coreopsis tinctoria flavonoid compounds still need to be further developed, so that separation and identification of the flavonoid compounds in the coreopsis tinctoria are necessary, and reliable theoretical and experimental basis is provided for future development and research of coreopsis tinctoria drug resources.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for separating active ingredients of coreopsis tinctoria Huang Kangyan, and to investigate the specific mechanism of action of these anti-inflammatory active ingredients.
After flavonoid compounds in coreopsis tinctoria are separated by the experimental scheme, a compound 5 (gosypetin-5- (6' - (E) -caffeoyl) -beta-D-glucose) is discovered from coreopsis tinctoria for the first time, and the structural formula is as follows:
specifically, the invention is realized by the following technical scheme:
a method for isolating anti-inflammatory active ingredients of coreopsis tinctoria, comprising the following:
1) Grading and extracting the coreopsis tinctoria ethanol extract with petroleum ether and ethyl acetate, collecting ethyl acetate extract, and removing solvent to obtain ethyl acetate extract;
2) Gradient eluting the ethyl acetate part extractum by using a macroporous resin column, wherein the gradient solvent is 0% -40% ethanol solution; taking a 40% ethanol elution part to obtain an active part Fr2; standing the active site Fr2 to separate out precipitate to obtain an active site Fr2-1;
3) The active site Fr2-1-2 is obtained by preparative chromatography;
wherein the mobile phase A is methanol, and the mobile phase B is water; the elution conditions were: 30% -80% of A; time: 50min;
4) The active site Fr2-1-2 is subjected to preparation chromatography to obtain a compound 5;
wherein the mobile phase A is methanol, and the mobile phase B is water; the elution condition is 40-41% A; time: 60min; the retention time of the Fr2-1-2 part on the semi-preparative chromatography is 7.5-12.5 min, and the retention time of the compound 5 in the semi-preparative chromatography is 12.5min.
In the invention, the coreopsis tinctoria ethanol extract is an ethanol extract extracted by conventional extraction methods such as heating extraction, normal temperature extraction, leaching or thermal reflux, and the like, the alcohol is one or two of methanol and ethanol, and in the specific experimental scheme of the invention, the alcohol is 80% ethanol.
In the technical scheme of the invention, the macroporous resin column used in the step 2) is an AB-8 macroporous resin column.
In the specific technical scheme of the invention, in the step 2), when the ethyl acetate part is subjected to gradient elution, firstly, 10VB water is used for removing impurities, then 15VB 20% ethanol is used for eluting to obtain an active part Fr1, and then 10BV 40% ethanol is used for eluting to obtain an active part Fr2.
In the technical method of the present invention, fr2 is a precipitate which is precipitated after the active site Fr1 is left to stand, and is obtained by filtration. The Fr2 active site of the invention can also be obtained by conventional separation methods such as centrifugation, filtration and the like.
In the technical scheme of the invention, the semi-preparative chromatographic conditions in the step 3) further comprise any one or more of the following i-v:
i flow rate: 10-20 mL/min
ii column temperature: 25-30 DEG C
iii sample injection amount: 100-500 mu L
iv detection wavelength: 280-360 nm;
v chromatography column: c18 chromatographic column
Further, the semi-preparative chromatographic conditions in step 3) may further comprise any one or more of the following i-v:
i flow rate: 15mL/min
ii column temperature: 25 DEG C
iii sample injection amount: 500 mu L
iv detection wavelength: 280nm to 360nm
v chromatography column: a KromasilC18 column, 21.2X105 mm,5 μm in size.
In the technical scheme of the invention, the semi-preparative chromatographic conditions in the step 4) further comprise any one or more of the following i-v:
i flow rate: 10-20 mL/min
ii column temperature: 25-30 DEG C
iii sample injection amount: 100-300 mu L
iv detection wavelength: 280-360 nm;
v chromatography column: c18 chromatographic column
Further, the semi-preparative chromatographic conditions in step 4) may further comprise any one or more of the following i-v:
i flow rate: 15mL/min
ii column temperature: 25 DEG C
iii sample injection amount: 300 mu L
iv detection wavelength: 280nm to 360nm
v chromatography column: a KromasilC18 column, 21.2X105 mm,5 μm in size.
In the experimental scheme of the invention, the ethanol content is the volume percentage content.
The invention also provides application of the active sites Fr2, fr2-1-2 or the compound 5 extracted from the coreopsis tinctoria by the method in anti-inflammatory products, wherein the products are medicines, in particular, any one or more of the coreopsis tinctoria active sites Fr2, fr2-1-2 and the compound 5 are used as active ingredients, and various preparation formulations of medicines are prepared according to any pharmaceutically acceptable carrier.
The invention has the beneficial effects that:
1. the flavonoid compound 5 is separated from coreopsis tinctoria for the first time, and anti-inflammatory activity research shows that the compound 5 has a certain anti-inflammatory activity, and reliable theoretical and experimental basis is provided for future development and research of coreopsis tinctoria drug resources.
2. The experimental scheme of the invention also separates compounds 1-4 and 6-12 from coreopsis tinctoria, wherein the compound 1 is isococaine-7-O-beta-D-glucopyranoside; the compound 1 is isooxycarnine-7-O-beta-D-glucopyranoside; compound 2 is protocatechuic acid; the compound 3 is butein-7-O-beta-D-glucopyranoside; compound 4 is quercitrin-7-O-beta-D-glucopyranoside; compound 6 is quercitin; compound 7 is butein-4' -O-beta-D-glucopyranoside; compound 8 is ocaine; compound 9 is isoocaine; compound 10 is quercetin-7-O-beta-D-glucopyranoside; compound 11 is ocanin-4' -O-beta-D-glucopyranoside; compound 12 is 5,7,3',5' -tetrahydroxyflavanonol.
Anti-inflammatory activity researches show that the compounds 3, 4, 6, 8, 9, 11 and 12 have certain anti-inflammatory activity effects. The method provides a theoretical basis for further researching and developing medicines with anti-inflammatory action for anti-inflammatory mechanism of coreopsis tinctoria flavonoids.
Drawings
FIG. 1 is a schematic diagram of a scheme for separating ethyl acetate sites of coreopsis tinctoria;
FIG. 2 is an analysis and preparation profile of Compound 1;
FIG. 3 is a Fr1-2 analysis and preparation map;
FIG. 4 is a Fr1-2-1 analysis and preparation profile;
FIG. 5 is a Fr1-2-3 analysis and preparation profile;
FIG. 6 is a Fr2-1 analysis and preparation map;
FIG. 7 is a Fr2-1-2 analysis and preparation map;
FIG. 8 is a Fr2-2 analysis and preparation map;
FIG. 9 is a Fr2-2-3 analysis and preparation map;
FIG. 10 is a Fr2-2-4 analysis and preparation map;
FIG. 11 is a Fr3 analysis and preparation map;
FIG. 12 is a Fr3-2 analysis and preparation map;
FIG. 13 is a HPLC chromatogram of Compounds 1-12
FIG. 14 is a graph showing the effect of flavonoid monomer compounds on RAW264.7 cell viability;
FIG. 15 is a graph of NO content of RAW264.7 cell supernatants;
FIG. 16 is a graph showing TNF- α, IL-6 content of RAW264.7 cells;
FIG. 17 is a graph of COX-2 relative protein expression levels of RAW264.7 cells;
FIG. 18 is a graph showing the relative protein expression levels of TLR-4 from RAW264.7 macrophages;
FIG. 19 is a graph showing NF-. Kappa.B relative protein expression levels of RAW264.7 macrophages;
FIG. 20 is a graph showing the relative protein expression levels of IκBα from RAW264.7 macrophages.
Detailed Description
The technical scheme of the present invention is further defined below in conjunction with the specific embodiments, but the scope of the claimed invention is not limited to the description.
EXAMPLE 1 enrichment and extraction of active ingredients of coreopsis tinctoria
3kg of coreopsis tinctoria raw material is subjected to diacolation extraction by 80% ethanol, and 786.4g of ethanol extract is obtained after decompression concentration, and the extraction rate is about 26.2%. The ethanol extract is subjected to fractional extraction and reduced pressure concentration to obtain 94.0g of petroleum ether extract, 89.2g of ethyl acetate extract, 197.8g of n-butanol extract and 406.4g of water extract.
EXAMPLE 2 preparation of coreopsis tinctoria flavonoid monomer Compound
1. Experimental instrument
The relevant instrumentation used in the present invention is shown in table 1.
Table 1 instrument and apparatus
In the separation and preparation link of the invention, an Agilent 1260 high performance liquid chromatograph is adopted for sample analysis; the preparation adopts Hanbang N7010 semi-preparation liquid phase; the analytical chromatographic columns finally used are all Kromasil C18,4.6 mm. Times.250 mm,5 μm; the preparation chromatographic columns are all Kromasil C18, 21.2X105 mm,5 μm; the analysis flow rates are all 1mL/min; the preparation flow rates are all 15mL/min; the analysis detection wavelength is 254nm, 280nm and 360nm; the detection wavelength is 280nm and 360nm.
3000mL of activated AB-8 macroporous resin is added into a glass chromatographic column, the macroporous resin bed is pretreated, 70.0g of the ethyl acetate part in the example 1 is dissolved in 200mL of deionized water, and the mixture is slowly dripped on the surface of the resin bed in a circle by a glass dropper, and then the mixture is statically adsorbed for 24 hours. After the adsorption is completed, the resin bed is washed by 10BV deionized water to remove water-soluble impurities such as saccharides and the like. The elution was performed sequentially with 20% (15 BV), 40% (10 BV), 60% (8 BV), 80% (6 BV) ethanol solution. The eluents with the concentrations are combined and concentrated under reduced pressure to obtain 14.34g of a 20% ethanol elution part (Fr 1) (comprising 4.70g of precipitation mass), 14.89g of a 40% ethanol elution part (Fr 2) (comprising 4.27g of precipitation mass), 3.20g of a 60% ethanol elution part (Fr 3) and 1.53g of an 80% ethanol elution part (Fr 4).
The 20% ethanol elution part (Fr 1) was allowed to stand to obtain a precipitate Fr1-1, 0.05g of the Fr1-1 part was dissolved in 2mL of methanol and 200. Mu. LDMSO was added to assist dissolution, and HPLC analysis was performed under the following chromatographic conditions: chromatographic column: kromasil C18,4.6 mm. Times.250 mm,5 μm; sample injection amount: 5. Mu.L; flow rate: 1mL/min; mobile phase: acetonitrile (a) -0.1% formic acid water; elution conditions: 10% -60% of A; time: 60min; wavelength: 254nm, 280nm and 360nm. Sample 4.50g and 20.00g C18 powder were mixed, loaded into pre-column and loaded under the following conditions: chromatographic column: kromasil C18, 21.2×250mm,5 μm; sample loading amount: about 0.5g of mix C18 powder at a time; flow rate: 15mL/min; mobile phase: methanol (a) -water (B); elution conditions: 25% -60% of A; time: 60min; wavelength: 254nm, 280nm, and the analysis and preparation patterns are shown in FIG. 2. Compound Fr1-1-1 (compound 1:126.2mg, retention time: 12.3 min) was obtained.
Standing 20% ethanol eluting part (Fr 1) to obtain supernatant Fr1-1, dissolving 4.52g of Fr1-2 part in 200mL methanol to obtain 22mg/mL solution, and performing HPLC analysis under the following chromatographic conditions: sample injection amount: 5. Mu.L; mobile phase: acetonitrile (a) -0.1% trifluoroacetic acid water (B); elution conditions: 10% -35% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 300. Mu.L; mobile phase: acetonitrile (a) -water (B); elution conditions: 10% -35% of A; time: and 40min. And (3) carrying out sectional peak grafting to obtain Fr1-2-1, fr1-2-2, fr1-2-3 and Fr1-2-4, and analyzing and preparing the patterns as shown in figure 3.
The Fr1-2-1 fraction was dissolved in 10mL of methanol and analyzed by HPLC under the following chromatographic conditions: chromatographic column: sample injection amount: 5. Mu.L; mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 20% -22% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 300. Mu.L; mobile phase: methanol (a) -water (B); elution conditions: 18-19% of A; time: and 60min. The compound Fr1-2-1-1 (compound 2:27.2mg, retention time: 7.6 min) was obtained, and the analysis and preparation patterns were as shown in FIG. 4.
The Fr1-2-3 fraction was dissolved in 20mL of methanol and analyzed by HPLC under the following chromatographic conditions: sample injection amount: 5. Mu.L; mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 30-36% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 300. Mu.L; mobile phase: methanol (a) -water (B); elution conditions: 30% -34% of A; time: and 40min. The analysis and preparation patterns of the obtained compounds Fr1-2-3-1 (compound 3:121.5mg, retention time: 10.6 min) and Fr1-2-3-2 (compound 4:25.1mg, retention time: 11.1 min) are shown in FIG. 5.
Standing 40% ethanol eluting part (Fr 2) to obtain precipitate Fr2-1, dissolving 2.08g of Fr2-1 part in 100mL of methanol to obtain 20mg/mL solution, and performing HPLC analysis under the following chromatographic conditions: sample injection amount: 5. Mu.L; mobile phase: acetonitrile (a) -0.1% formic acid water (B); elution conditions: 20% -35% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 500. Mu.L; mobile phase: methanol (a) -water (B); elution conditions: 30% -80% of A; time: 50min. The analysis and preparation pattern is shown in FIG. 6.
1.21g of Fr2-1-2 fraction (retention time of 7.5-12.5 min on semi-preparative chromatography) was dissolved in 20mL of methanol to give a solution with a concentration of about 60mg/mL, and the solution was subjected to HPLC analysis using a 0.22 μm filter membrane under the following chromatographic conditions: sample injection amount: 3 μL; mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 40% -41% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 300. Mu.L; flow rate: 15mL/min; mobile phase: methanol (a) -water (B); elution conditions: 40% -41% of A; time: and 60min. The peak was spiked to give the compound Fr2-1-2-1 (compound 5:8.2mg, retention time: 12.7 min), fr2-1-2-2 (compound 6:10.6mg, retention time: 13.2 min), fr2-1-2-3 (compound 7:21.7mg, retention time: 15.0 min), fr2-1-2-4 (compound 8:820.0mg, retention time: 18.0 min), and the analytical and preparative patterns were as shown in FIG. 7.
Standing 40% ethanol eluting part (Fr 2) to obtain supernatant Fr2-2, taking 10.02g of Fr2-2 part, dissolving in 200mL methanol to obtain solution with concentration of about 50mg/mL, and performing HPLC analysis under the following chromatographic conditions: mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 20% -50% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 250 μl; mobile phase: methanol (a) -water (B); elution conditions: 20% -50% of A; time: and 40min. The obtained Fr2-2-1, fr2-2-2, fr2-2-3, fr2-2-4 are shown in figure 8.
1.96g of Fr2-2-3 part was taken and dissolved in 40mL of methanol to give a solution with a concentration of about 50mg/mL, and the solution was subjected to HPLC analysis with a 0.22 μm filter membrane under the following chromatographic conditions: sample injection amount: 3 μL; mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 36-37% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 500. Mu.L; mobile phase: methanol (a) -water (B); elution conditions: 32% -33% of A; time: and 60min. The monomer compound Fr2-2-3-1 (compound 9:73.5mg, retention time: 19.7 min) was obtained, fr2-2-3-3 (compound 10:25.9mg, retention time: 22.6 min). The analysis and preparation pattern is shown in FIG. 9.
1.26g of Fr2-2-4 part was dissolved in 60mL of methanol to obtain a solution with a concentration of about 20mg/mL, and the solution was subjected to HPLC analysis through a 0.22 μm filter membrane under the following chromatographic conditions: sample injection amount: 3 μL; mobile phase: methanol (a) -0.1% trifluoroacetic acid water (B); elution conditions: 35% -42% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 500. Mu.L; mobile phase: methanol (a) -water (B); elution conditions: 35% -40% of A; time: and 60min. The monomer compound Fr2-2-4-1 (compound 11:25.9mg, retention time: 11.2 min) was obtained. The analysis and preparation pattern is shown in FIG. 10.
1.0g of Fr3 part was dissolved in 40mL of methanol to obtain a solution with a concentration of about 25mg/mL, and the solution was subjected to HPLC analysis through a 0.22 μm filter membrane under the following chromatographic conditions: sample injection amount: 3 μL; mobile phase: acetonitrile (a) -0.1% trifluoroacetic acid water (B); elution conditions: 20% -45% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 400. Mu.L; mobile phase: acetonitrile (a) -water (B); elution conditions: 20% -45% of A; time: and 40min. The analysis and preparation pattern is shown in FIG. 11.
0.5g of Fr3-2 part was dissolved in 20mL of methanol to obtain a solution with a concentration of about 25mg/mL, and the solution was filtered through a 0.22 μm filter membrane for HPLC analysis under the following chromatographic conditions: sample injection amount: 3 μL; mobile phase: acetonitrile (a) -0.1% trifluoroacetic acid water; elution conditions: 40% -70% of A; time: and 40min. The preparation conditions are as follows: sample injection amount: 300. Mu.L; mobile phase: acetonitrile (a) -water (B); elution conditions: 47% -53% of A; time: for 40min; the monomer compound Fr3-2-1 (compound 12:27.1mg, retention time: 12.5 min) was obtained. The analysis and preparation pattern is shown in FIG. 12.
The specific isolation method of the compounds of the present invention is shown in FIG. 1.
Example 3 analytical identification of coreopsis flavonoid monomeric Compounds
The prepared monomer purities were analyzed by Agilent 1260 high performance liquid chromatograph, and the final purities were determined by both peak area and UV spectra. Each monomer compound was prepared by MS, 1 H-NMR 13 C-NMR and other techniques.
1. Purity detection
12 flavonoid compounds were detected using a high performance liquid chromatograph, UV280nm, see FIG. 13.
The purity of each compound was calculated according to the area normalization method, and the results are shown in table 2.
Table 2 purity of flavonoid Table 43Thepurity of flavonoids
2. Structural identification
The flavonoid compounds in coreopsis tinctoria are enriched through column chromatography, then the monomer compounds are separated from the target crude extract through semi-preparative liquid chromatography, the obtained compounds are characterized, the relationship between the compound structure and the anti-inflammatory activity is conveniently and deeply discussed, and a material basis and compound structure guidance are provided for subsequent activity experiments.
A large number of active ingredients in coreopsis tinctoria have been isolated in previous studies, such as polyacetylenes, flavonoids, polysaccharides, alkaloids, etc. Based on the basic research of the prallethrin by the former, systematic separation and purification of chemical components of the prallethrin are carried out, and partial repeatedly separated compounds are removed, so that 12 monomer compounds are obtained. Through MS (MS), 1 H-NMR 13 The compound was analyzed by C-NMR and other analysis techniques, and the results were as follows:
Compound 1: isooxycarnine-7-O-beta-D-glucopyranoside (Huang Nuoma glycoside), white powder with molecular formula of C 21 H 22 O 11 The molecular weight was 450. Compound 1 1 H-NMR was (DMSO, 600 Hz) δ:7.38 (1H, dd, j=9.0, 3.6hz, H-5), 6.92 (1H, d, j=9.0 hz, H-6), 6.98 (1H, d, j=2.4 hz, H-2 '), 6.78 (1H, d, j=7.8, 1.8hz, H-6'), 6.74 (1H, d, j=7.8 hz, H-5), 5.39 (1H, dd, j=12.6, 3.0hz, H-2), 2.68 (1H, dd, j=16.8, 3.6hz, H-3 a), 3.10 (1H, dd, j=16.8, 12.6H)z，H-3β)，4.93(1H，d，J＝7.2Hz，H-1’)； 13 The C-NMR data were: (CD) 3 OD,600 Hz) δ:79.2 (C-2), 43.4 (C-3), 191.1 (C-4), 118.0 (C-5), 109.0 (C-6), 150.7 (C-7), 135.1 (C-8), 150.5 (C-9), 114.5 (C-10), 129.9 (C-1 '), 115.2 (C-2'), 145.1 (C-3 '), 145.2 (C-4'), 116.5 (C-5 '), 116.5 (C-6'), 101.5 (C-1 "), 73.2 (C-2"), 77.3 (C-3 "), 69.5 (C-4"), 75.7 (C-5 "), 60.6 (C-6"). In comparison, the compound was identified as Huang Nuoma glycoside (isococaine-7-O-beta-D-glucopyranoside).
Compound 2: protocatechuic acid, white to brown crystalline powder, molecular formula C 7 H 6 O 4 The molecular weight was 154. Compound 2 1 H-NMR was (CD 3 OD，600MHz)δ：7.45(d，1H，J＝6.0Hz，H-2)，7.44(dd，1H，J＝6.0Hz，H-6)，6.81(d，1H，J＝6.0Hz，H-5)； 13 The C-NMR data were: (CD) 3 OD,600 MHz) data were δ:121.7 (C-1), 116.3 (C-2), 144.7 (C-3), 150.2 (C-4), 114.4 (C-5), 122.5 (C-6), 168.8 (COOH). In the literature, compound 2 was identified as protocatechuic acid.
Compound 3: butein-7-O-beta-D-glucopyranoside is pale yellow powder with molecular formula of C 21 H 22 O 10 The molecular weight is 434. Of compounds 1 H-NMR data were (CD 3 OD，600MHz)δ：5.22(1H，dt，J＝12.9，2.9Hz，H-2)，2.93(1H，ddd，J＝16.9，12.9，2.0Hz，H-3α)，2.61(1H，ddd，J＝16.9，4.8，3.0Hz，H-3β)，7.67(1H，dd，J＝8.8，1.7Hz，H-5)，5.22(1H，dt，J＝12.1，5.4，3.8Hz，H-6)，6.58(1H，dd，J＝3.7，2.3Hz，H-8)，6.83(1H，d，J＝1.6Hz，H-2’)，6.68(1H，d，J＝2.0Hz，H-5’)，6.65(1H，dd，J＝8.8，2.3Hz，H-6’)，4.90(1H，dd，J＝7.4，5.2Hz，H-1”)，3.39～3.24(6H，m，J＝5.9Hz，H-2”～6”)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:79.9 (C-2), 43.6 (C-3), 192.3 (C-4), 128.1 (C-5), 110.8 (C-6), 163.6 (C-7), 103.9 (C-8), 164.2 (C-9), 114.9 (C-10), 130.4 (C-1 '), 113.4 (C-2'), 145.1 (C-3 '), 145.5 (C-4'), 115.6 (C-5 '), 118 (C-6'), 100.1 (C-1 "), 73.3 (C-2"), 76.4 (C-3 "), 69.8 (C-4"), 76.8 (C-5 "), 60.9 (C-6"). In the comparison literature, compound 3 was identified as butein-7-O-beta-D-glucopyranoside.
Compound 4: quercetin-7-O-beta-D-glucopyranoside, light yellow powder, and molecular formula C 21 H 20 O 13 The molecular weight is 480. Compound 4 1 H-NMR data were (CD 3 OD，600MHz)δ：7.76(1H，d，J＝2.2Hz，H-2’)，6.88(1H，d，J＝8.4Hz，H-5’)，7.67(1H，dd，J＝8.5，2.2Hz，H-6’)，6.95(1H，s，H-3)，5.04(1H，d，J＝7.6Hz，H-1”)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:148.3 (C-2), 136.1 (C-3), 176.6 (C-4), 145.8 (C-5), 130.1 (C-6), 152.0 (C-7), 94.0 (C-8), 148.6 (C-9), 105.6 (C-10), 122.5 (C-1 '), 115.9 (C-2'), 145.5 (C-3 '), 147.9 (C-4'), 116 (C-5 '), 120.4, (C-6'), 101.4 (C-1 "), 73.6 (C-2"), 76.2 (C-3 "), 70.1 (C-4"), 77.7 (C-5 "), 61.09 (C-6"). In comparison, compound 4 was identified as quercitrin-7-O-beta-D-glucopyranoside.
Compound 5: gosypetin-5- (6' - (E) -caffeoyl) -beta-D-glucide as pale yellow powder with molecular formula C 30 H 23 O 16 The molecular weight was 642. Compound 5 1 H-NMR data were (CD 3 OD，600MHz)δ：7.71(1H，d，J＝2.4Hz)，7.61(1H，dd，J＝8.4，2.4Hz)，7.43(1H，d，J＝15.6Hz)，6.82(2H，d，J＝7.8Hz)，6.73(1H，d，J＝2.4Hz)，6.56(1H，dd，J＝8.4，2.4Hz)，6.52(1H，d，J＝8.4Hz)，6.52(1H，d，J＝8.4Hz)，6.18(1H，d，J＝15.6Hz)，5.08(1H，d，J＝7.2Hz)，3.49(1H，dd，J＝10.8Hz)，3.85(1H，m)，3.63(1H，t，9.1Hz)，3.59(1H，t，9.1Hz)，4.65(1H，dd，J＝11.9，2.2Hz)，4.34(1H，dd，J＝11.9，7.1Hz)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:148.81 (C-2), 137.1 (C-3), 177.3 (C-4), 152.5 (C-5), 94.9 (C-6), 150.2 (C-7), 130.8 (C-8), 146.8 (C-9), 106.5 (C-10), 124.1 (C-1 '), 116.2 (C-2 '), 146.0 (C-3 '), 148.8 (C-4 '), 116.2 (C-5 '), 121.9 (C-6 '), 102.1 (C-1 "), 74.6 (C-2"), 75.6 (C-3 "), 72.1 (C-4"), 77.4 (C-5 "), 64.6 (C-6"), 127.4 (C-1 '), 115.6 (C-2 '), 146.4 (C-3 '), 149.3 (C-4 '), 116.5 (C-5 '), 122.3 (C-6 '), 147.6 (C-7 '), 114.5 (C-8 '), 169.1 (C-9 '). By literature comparison, it was determined that compound 5 was gospypetin-5- (6 "- (E) -caffeoyl) - β -D-glucoside, which was first discovered in coreopsis tinctoria.
Compound 6: quercetin (quercetin), light yellow powder, and molecular formula C 15 H 10 O 8 The molecular weight was 318. Compound 6 1 H-NMR data were (CD 3 OD，600MHz)δ：7.62(dd，J＝2.0Hz，H-2)，7.72(d，J＝2.0Hz，8.4Hz，H-6)，6.88(d，J＝8.4Hz，H-5)，6.50(s，H-9)，8.69，9.22，9.31，9.58，10.52，12.28(6×OH)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:145.5 (C-2, s), 135.5 (C-3, s), 176.0 (C-4, s), 145.4 (C-5,s), 128.4 (C-6, s), 153.2 (C-7,s), 92.9 (C-8, d), 149.7 (C-9,s), 103.4 (C-10, s), 122.9 (C-1 ', s), 114.6 (C-2', d), 144.8 (C-3 ', s), 147.3 (C-4', s), 114.8 (C-5 ', d), 120.3 (C-6', d) . By literature comparison, compound 6 was determined to be quercetin (quercitin).
Compound 7: butein-4' -O-beta-D-glucopyranoside, orange-yellow amorphous powder, and its molecular formula is C 21 H 22 O 10 The molecular weight is 434. Compound 7 1 H-NMR data were (CD 3 OD,600 MHz) data were δ:8.04 (1 h, d, j=9.0 hz, h-6 '), 7.76 (1 h, d, j=15.0 hz, h- β), 7.56 (1 h, d, j=15.6 hz, h- α), 7.19 (1 h, d, j=2.4 hz, h-2), 7.13 (1 h, dd, j=7.8, 1.8hz, h-6), 6.82 (1 h, d, j=8.4 hz, h-5), 6.69 (1 h, dd, j=9.0, 2.4hz, h-5'), 6.62 (1 h, dd, j=2.4 hz, h-3 "), 5.03 (1 h, d, j=6.9 hz, h-1"); 13 C-NMR(CD 3 OD,600 MHz) δ:126.92 (C-1), 115.24 (C-2), 145.47 (C-3), 148.78 (C-4), 116.32 (C-5), 122.46 (C-6), 114.52 (C-1 '), 165.46 (C-2'), 103.79 (C-3 '), 163.7 (C-4'), 107.94 (C-5 '), 131.51 (C-6'), 192.5 (C=O), 145.5 (C-beta), 99.95 (C-1 "), 73.35 (C-2"), 76.93 (C-3 "), 69.8 (C-4"), 76.5 (C-5 "), 60.99 (C-6"). Compared with the literature, the compound 7 is butein-4' -O-beta-D-glucopyranoside.
Compound 8: acanine, orange powder, molecular weight 288, molecular formula C 15 H 12 O 6 . Nuclear magnetic data of 1 H-NMR(CD 3 OD，600MHz)δ：7.18(1H，d，J＝1.8Hz，H-2)，7.72(1H，d，J＝15.0Hz，H-β)，7.52(1H，d，J＝9.0Hz，H-6’)，7.11(1H，dd，J＝8.4，1.8Hz，H-6)，7.54(1H，d，J＝6.6Hz，H-α)，6.82(1H，d，J＝8.4Hz，H-5)，6.47(1H，d，J＝8.4Hz，H-5’)； 13 C-NMR(CD 3 OD，600Hz)δ：192.7(C＝O)，153.0(C-2’)，151.9(C-4’)，148.5(C-4) 144.6 (C-. Beta.), 145.5 (C-3), 132.4 (C-3 '), 127.0 (C-1), 122.2 (C-6), 116.9 (C-6'), 121.8 (C-. Alpha.), 115.2 (C-5), 114.4 (C-2), 113.8 (C-1 '), 107.1 (C-5'). The comparison determines that compound 8 is ocaine.
Compound 9: isooxycarnine, pale yellow crystal and molecular formula C 15 H 12 O 6 The molecular weight is 288. Nuclear magnetic data of 1 H-NMR(CD 3 OD，600MHz)δ：7.3(1H，d，J＝8.4Hz，H-6’)，6.99(1H，d，J＝2.4Hz，H-2’)，6.86(1H，dd，J＝7.8，1.8Hz，H-5’)，6.79(1H，d，J＝7.8Hz，H-5)，6.50(1H，d，J＝8.7Hz，H-6)，5.36(1H，dd，J＝12.6，3.0Hz，H-2)，3.07(1H，dd，J＝16.80，12.0Hz，H-3α)，2.73(1H，dd，J＝16.8，3.0Hz，H-3β)。 13 C-NMR(CD 3 OD,600 MHz) δ:192.67 (C-4), 152.61 (C-7), 151.33 (C-9), 145.52 (C-4 '), 145.08 (C-3'), 132.6 (C-8), 130.53 (C-1 '), 118.21 (C-6'), 117.97 (C-5), 114.85 (C-5 '), 114.3 (C-10), 113.63 (C-2'), 109.51 (C-6), 80.17 (C-2), 43.65 (C-3). In the comparative literature, compound 9 was identified as isooxycodone.
Compound 10: quercetin-7-O-beta-D-glucopyranoside, pale yellow powder, molecular weight: 464, molecular formula: c (C) 21 H 20 O 12 . Nuclear magnetic data of 1 H-NMR(CD 3 OD，600MHz)δ：7.74(1H，d，J＝2.4Hz，H-2’)，7.65(1H，dd，J＝8.4，1.8Hz，H-6’)，6.88(1H，d，J＝8.4Hz，H-5’)，6.72(1H，d，J＝1.8Hz，H-8)，6.44(1H，d，J＝2.4Hz，H-6)，5.05(1H，d，J＝7.2Hz，glc-1-H)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:147.6 (C-2), 136.2 (C-3), 176.1 (C-4), 160.7 (C-5), 94.1 (C-6), 163.0 (C-7), 98.8 (C)-8), 156.3 (C-9), 104.9 (C-10), 122.5 (C-1 '), 114.8 (C-2'), 147.3 (C-3 '), 144.8 (C-4'), 114.7 (C-5 ') and 120.5 (C-6') carbon signals; glycosyl moiety carbon signal δ:100.3 (C-1 "), 73.3 (C-2"), 76.9 (C-3 "), 69.9 (C-4"), 76.5 (C-5 "), 61.1 (C-6"). In comparison, compound 10 was identified as quercetin-7-O-beta-D-glucopyranoside.
Compound 11: okanin-4' -O-beta-D-glucopyranoside (martin), yellow powder, and molecular formula C 21 H 22 O 11 The molecular weight was 450. Compound 11 1 H-NMR data were (CD 3 OD，600MHz)δ：7.19(1H，d，J＝1.8Hz，H-2)，7.77(1H，d，J＝15.3Hz，H-β)，7.55(1H，d，J＝15.3Hz，H-α)，7.62(1H，d，J＝9.1Hz，H-6’)，7.12(1H，dd，J＝8.2Hz，2.1Hz，H-6)，6.83(1H，d，J＝8.2Hz，H-5)，6.84(1H，d，J＝9.1Hz，H-5’)，4.98(1H，d，J＝7.2Hz，H-1”)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:126.9 (C-1), 115.98 (C-2), 145.58 (C-3), 148.86 (C-4), 115.25 (C-5), 122.54 (C-6), 193.23 (c=o), 145.52 (C- β), 116.7 (C- α), 114.47 (C-1 '), 152.49 (C-2'), 134.45 (C-3 '), 150.31 (C-4'), 106.7 (C-5 '), 121.17 (C-6'), 101.24 (C-1 "), 73.35 (C-2"), 11.09 (C-3 "), 69.89 (C-4"), 76.14 (C-5 "), 61.09 (C-6"). In comparison, compound 11 was identified as oxcarbanin-4' -O- β -D-glucopyranoside (martin).
Compound 12:5,7,3',5' -tetrahydroxyflavanonol, pale yellow powder, and its molecular formula is C 15 H 12 O 6 The molecular weight is 288. Nuclear magnetic data of 1 H-NMR(CD 3 OD，600MHz)δ：5.3(1H，dd，J＝12.72，3.00Hz，H-2)，3.09(1H，dd，J＝17.16，12.84Hz，H-3α)，2.71(1H，dd，J＝17.16，3.06Hz，H-3β)，5.92(1H，d，J＝2.16Hz，H-6)，5.91(1H，d，J＝2.16Hz，H-8)，6.81(1H，s，H-2’)，6.94(1H，s，H-4’)，6.81(1H，s，H-6’)； 13 C-NMR(CD 3 OD,600 MHz) data were δ:79.11 (C-2), 42.71 (C-3), 196.37 (C-4), 164.06 (C-5), 95.61 (C-6), 166.96 (C-7), 94.75 (C-8), 163.45 (C-9), 101.95 (C-10), 130.38 (C-1 '), 113.3 (C-2'), 145.49 (C-3 '), 114.84 (C-4'), 145.11 (C-5 '), 117.85 (C-6'). By comparison in the literature, compound 12 was 5,7,3',5' -tetrahydroxyflavanonol.
The invention adopts column chromatography and high performance liquid chromatography to systematically separate and purify the ethyl acetate part of coreopsis tinctoria, 12 monomer compounds are obtained by total separation, and MS is utilized, 1 H-NMR 13 H-NMR techniques reference literature data for structural identification of isolated compounds containing 3 flavones (alcohols) (quercetin, quercetin-7-O-beta-D-glucopyranoside), 5 flavonols (isokulanine, huang Nuoma glycoside, butein-7-O-beta-D-glucopyranoside, gosypetin-5- (6 '- (E) -caffeoyl) -beta-D-glucoside, 5,7,3',5 '-tetrahydroxydihydroflavone), 3 chalcones (ocaine, butein-4' -O-beta-D-glucopyranoside, malide), and 1 phenolic acids (protocatechuic acid). Wherein isoquinine-7-O-beta-D-glucopyranoside (Huang Nuoma glycoside) and ocaine-4 '-O-beta-D-glucopyranoside (martin) are isomers with each other, butein-7-O-beta-D-glucopyranoside and butein-4' -O-beta-D-glucopyranoside are isomers with each other, and ocaine, isoquinine and 5,7,3',5' -tetrahydroxy dihydroflavonol are isomers with each other; gossypetin-5- (6 "- (E) -caffeoyl) -beta-D-glucide was first isolated from coreopsis tinctoria.
EXAMPLE 4 anti-LPS-induced macrophage inflammatory injury action of flavonoid
Researches show that coreopsis tinctoria contains various bioactive components, wherein flavonoid compounds are used as main components of coreopsis tinctoria, and have various pharmacological activities. The existing research on anti-inflammatory activity of coreopsis tinctoria adopts crude extract, and the verification on inflammatory factors or related proteins is single, and the coreopsis tinctoria is lack of systematicness and comprehensiveness.
According to the invention, an RAW264.7 cell inflammation model is established to examine the anti-inflammatory activity of the separated 12 monomer compounds, and the optimal action concentration of the monomer compounds is screened through an MTT experiment; detecting the NO content in the cell supernatant by a Griess experiment, and screening a compound with obvious anti-inflammatory effect; ELISA experiments are carried out on 8 compounds screened, the expression levels of IL-6 and TNF-alpha in cells are detected, and Western Blot is utilized to detect the expression levels of inflammation-related proteins COX-2, TLR-4 and NF-kappa B, I kappa B alpha. The invention systematically discusses the inflammatory protection effect and the mechanism of the coreopsis tinctoria flavonoid compound, and aims to provide theoretical basis for developing coreopsis tinctoria related anti-inflammatory drugs.
1. Experimental materials, reagents and instruments
Source of sample to be tested
12 flavonoids were isolated and extracted in example 2, which were: protocatechuic acid (compound 1), huang Nuoma glycoside (compound 2), butein-7-O- β -D-glucopyranoside (compound 3), quercetin-7-O- β -D-glucopyranoside (compound 4), gossypetin-5- (6 "- (E) -caffeoyl) - β -D-glucoside (compound 5), quercetin (compound 6), butein-4 ' -O- β -D-glucopyranoside (compound 7), oxybutynin (compound 8), isoquinine (compound 9), quercetin-7-O- β -D-glucopyranoside (compound 10), maliside (compound 11), 5,7,3',5' -tetrahydroxydihydroflavone (compound 12).
(II), reagents and materials
The relevant materials and reagents used in this experiment are shown in table 3.
TABLE 3 materials and reagents
(III), instruments and devices
The relevant instruments and equipment used in this experiment are shown in table 4.
Table 4 instrument and apparatus
2. Reagent preparation
Preparation of monomer Compound stock solution
The 12 flavonoid monomer compounds are weighed respectively, dissolved by corresponding amount of DMSO, prepared into stock solution with concentration of 100mM, stored in a refrigerator with the temperature of-20 ℃, and diluted according to the experimental use amount.
(II) 5mg/mL MTT configuration
The MTT powder is fully dissolved in PBS (phosphate buffer solution) according to a certain proportion, and after the sterilization and split charging of the microporous filter membrane, the MTT powder is preserved in a dark place at the temperature of minus 20 ℃. Note that: MTT is easy to decompose by visible light, and light is protected from the experiment and the storage process; the preparation process should avoid bacteria contamination; it is available at present and can not be repeatedly frozen and dissolved.
(III) LPS mother liquor configuration
Accurately weighing 10mg of LPS powder, dissolving in 1mL of sterile PBS, subpackaging as required, and preserving at-20deg.C. Note that: can not be repeatedly frozen and dissolved.
The reagents required for Western blot experiments are shown in Table 5.
TABLE 5 preparation of reagents required for Western blot experiments
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3. Experimental method
(one), LPS acting on RAW264.7 macrophage concentration screening
Taking RAW264.7 cells with growth density of 80%, counting the cells according to the density after counting, and counting the cells by 1×10 5 The density of each/mL is inoculated into a 96-well plate according to 100 mu L of each well, after incubation is carried out for 24 hours in a cell culture box, the culture is continued for 24 hours by changing into a DMEM culture medium (containing 2% FBS) containing LPS with different concentrations, 10 mu L of MTT solution is added into each well, incubation is carried out for 4 hours in the cell culture box, finally, the original culture medium is discarded, 100 mu L of DMSO is added, shaking and dissolving are carried out for 10 minutes at 37 ℃ on a micro-pore plate oscillator, and the absorbance value at the 490nm wavelength is measured. The cell viability calculation formula is as follows:
Wherein ODs are sample group, OD blank Is blank, OD control Is a control group.
(II) detecting NO content of cell supernatant by Griess method
1) Drawing a standard curve: 1mol/LNaNO was performed with pre-formulated complete medium (DMEM+10% FBS) 2 The standard solutions were serially diluted to 0, 1, 2, 5, 10, 20, 40, 60, 100 μm series of standard solutions.
2) Sample detection: cell plating and administration method same "(one)", after 24 hours of culture, griess Reagent I and II were removed and allowed to return to room temperature. The procedure was as per NO kit instructions.
(III) MTT detection of RAW264.7 macrophages
The reference literature determines the concentrations of each monomer compound acting on RAW264.7 cells to be 3.125 μm,6.25 μm,12.5 μm,25 μm,50 μm,100 μm, respectively. After 24h incubation, the cells were incubated for a further 24h with DMEM medium (containing 2% FBS) containing different concentrations of each monomeric compound.
(IV) detection of cell supernatant NO by Griess method
MTT experiments showed that the optimal concentration of each monomer compound was determined by cell plating, incubating for 24 hours, and then culturing in DMEM medium (containing 2% FBS) containing the selected concentration of each extraction site and 5. Mu.g/mL LPS for 24 hours, and detecting the NO content of the supernatant by Griess method. Based on the NO detection results of each monomer compound, a subsequent experiment of anti-inflammatory activity of the monomer compound was performed.
(V) detection of inflammatory factors such as cell supernatant TNF-alpha, IL-6 and the like by Elisa kit
Cell culture and plating, incubation for 24h after plating, changing into DMEM medium (containing 2% FBS) containing each monomer compound with set concentration and LPS with concentration of 5 mug/mL, and continuously culturing for 24h, wherein the content of TNF-alpha and IL-6 inflammatory factors in cell supernatant is detected by an Elisa method. Reference and preliminary experiments, cell culture supernatants were diluted 4-fold for determination of TNF- α inflammatory factor; in the determination of IL-6 inflammatory factor, cell culture supernatant does not need dilution. The inflammatory factors were measured according to the Elisa kit instructions.
(VI) Western blot analysis
RAW264.7 cells at 1X 10 5 The cells were inoculated into 6-well plates at a density of one/mL, incubated in a cell culture incubator for 24 hours, and then replaced with DMEM medium (containing 2% FBS) containing each monomer compound at a predetermined concentration and LPS at a concentration of 5. Mu.g/mL, and cultured for a specific time to extract proteins. The specific operation method is as follows:
(1) RAW264.7 cell protein extraction:
1) Preparation: the ice box was prepared in advance, the EP tube was marked, a cell lysate was prepared (RIPA lysate was mixed with PMSF at 100:1), PBS was stored in a refrigerator pre-chilled at 4 ℃.
2) Cleaning: the culture medium was aspirated as much as possible without residue. The cold PBS was slowly spun into a 6-well plate to wash the cells twice, and after washing, the PBS was thoroughly blotted.
3) Cell lysis: adding 80 μl of cell lysate into each well, shaking 6-well plate, mixing, and placing on ice for 5min for lysis.
4) Protein extraction: proteins were scraped in the same direction with a cell scraper and collected into 1.5mL centrifuge tubes.
5) And (3) centrifuging: the centrifuge tube was placed in an ice bin and placed in a refrigerator at 4 ℃ for further lysis for 30min. After completion of the lysis, the mixture was centrifuged at 12000r/min at 4℃for 15min, and the supernatant was taken out in a fresh EP tube for use.
(2) BCA assay for cellular protein concentration:
1) Preparation: the EP tube containing the protein was placed in an ice bin.
2) Drawing a protein standard curve: the standard protein formulation is shown in table 6.
Table 6BCA method protein quantitative Table
(3) Protein concentration was determined by BCA method and specific procedures were performed according to BCA kit instructions. After calculation from BCA assay, all protein samples were diluted to the same concentration (equal volume loading in Western blot experiments) with PBS and 5 x loading buffer.
(4) Protein denaturation: the diluted protein sample was denatured in a metal bath at 100℃for 15min. After the denatured protein is cooled, the protein is stored at the temperature of-80 ℃ for standby. In use, the protein sample is centrifuged and the bottom of the EP tube is flicked to allow the sample to be melted thoroughly.
(5) SDS-PAGE electrophoresis: and selecting corresponding lower glue (separating glue) according to the molecular weight of the protein for configuration. Before preparing the electrophoresis gel, cleaning the gel-making glass plate and drying; the solutions are placed at room temperature in advance, so that gas is prevented from being separated out by heat generated in the process of separating and gelling the solid to generate bubbles. And then, putting the two glass plates in an aligned manner into a glue making frame, clamping and vertically fixing the two glass plates on the glue making frame, and pouring UP water for leak detection (standing for 10min until the water surface is not lowered, so that the glue making plates are correctly placed). After the UP water was poured out, the residual water in the glue plate was sucked with a water absorbing paper, and a release glue was prepared, and the release glue was prepared with reference to tables 7 and 8.
TABLE 7 separation ranges for SDS-PAGE separating gels at different concentrations
TABLE 8 preparation of the volumes of the components required for different volumes of 8% SDS-PAGE separating gel
After about 4.5mL of separation gel was filled, isopropanol was again filled to flatten the liquid surface and remove air bubbles. After 30min, the separating gel is solidified (refer to the separating gel which is not filled into the gel-making plate), the upper isopropanol layer is poured out, the residue is sucked by using absorbent paper, the prepared concentrated gel is filled, and a proper comb is inserted. After 40min, the concentrated gel is solidified (the concentrated gel which is not filled into the gel preparation plate can be referred), the prepared electrophoresis gel is horizontally clamped on an electrophoresis tank, electrophoresis liquid is filled for leakage detection (the leakage or the unbalance of an electrode can lead to the deviation or the slow movement of a strip, a comb is horizontally pulled out to keep a sample feeding hole in a vertical state as far as possible, a sample is required to be centrifuged until the sample is completely melted before protein is extracted, 20-30 mug of the sample is fed, marker is required to be added at two ends and/or in the middle of the sample feeding, electrophoresis liquid is replenished, electrophoresis is started, constant pressure is firstly used for 80V, the sample is concentrated in the concentrated gel for about 30min, after the electrophoresis of the sample is carried out until the gel separation, the sample is changed into constant pressure 120V, the continuous separation is carried out for 50-90 min, and the electrophoresis is stopped when the electrophoresis is carried out until the blue strip is 5-10 mm away from the lowest end of the gel preparation plate.
(6) Transferring: first, a PVDF membrane of a suitable size (depending on the desired molecular weight of the protein and the range of molecular weights in the different separation gels) is cut off and a corner is cut off as a marker (distinguishing front and back in subsequent experiments). And sequentially soaking the PVDF film, the sponge and the filter paper which are subjected to methanol activation for 1min in the precooled film transferring liquid. The sponge and the filter paper are sequentially placed on one black surface of the transfer film clamp. Taking out the glass plate in the electrophoresis tank, prying the glass plate by a glue cutter, cutting the electrophoresis gel where the target protein is positioned according to a Marker mark, placing the electrophoresis gel on the filter paper of the black film transferring clamp, taking care to avoid generating bubbles between the electrophoresis gel and the filter paper, placing the PVDF film on the electrophoresis gel, and meanwhile taking care to remove the bubbles between the PVDF film and the electrophoresis gel. Finally, filter paper and sponge are covered. After the installation, the clamp is placed in a film transfer instrument, the black clamp faces the black surface of the electrophoresis tank, the constant current is 250mA, the setting time is 30-90 min different according to the molecular weight (an ice bag is needed to be placed in the film transfer instrument, and the film transfer instrument is placed in an ice-water mixture after being electrified, so that protein degradation caused by overheating is avoided).
(7) Closing: the sealing liquid is 5% skimmed milk. After the film transfer is finished, opening a film transfer clamp, checking whether the Marker is completely transferred to the PVDF film, if the Marker still remains on the electrophoresis gel, the protein may not be completely transferred, and the current or the time needs to be properly increased in the next experiment. If the Marker is transferred to the back of the film, the time is reduced in the next experiment. After the membrane transfer is finished, the PVDF membrane is put into an antibody incubation box, skimmed milk is added to permeate the PVDF membrane, and the PVDF membrane is placed into a room temperature shaking table and slowly shaken for 1.5-2 h or overnight at 4 ℃.
(8) Incubating primary antibodies: the blocking solution was rinsed off with TBST and the incubation cassette was placed on a 4℃shaker, slowed, and incubated overnight.
(9) Incubating a secondary antibody: the primary antibody was recovered, and PVDF membrane was washed 3 times with TBST at room temperature for 8min each, after which the secondary antibody was incubated at room temperature for 1h at a slow speed. After incubation, the PVDF membrane was washed 3 times with TBST at room temperature for 8min each.
(10) Developing: ECL luminescence was applied drop wise to PVDF film using ECL chemistry and imaged with gel.
(seventh), statistical analysis
All data were statistically analyzed using GraphPad Prism analysis software, the comparison between groups was made using One Way ANOVA (One Way ANOVA), P < 0.05 indicated that the results had a statistical difference, P < 0.01 indicated that the results had a significant statistical difference, and P < 0.001 indicated that the results had a very significant statistical difference.
4. Experimental results
First, influence of flavonoid compounds on RAW264.7 cell viability
The effect of each test on RAW264.7 cell viability under LPS induction at a concentration of 5. Mu.g/mL was examined by MTT assay. The test for each of the monomeric compound test subjects in the experiment was set up with 6 sets of concentration gradients (3.125. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M) respectively. As can be seen from fig. 14, after compounds 1 to 7 with different concentrations act on RAW264.7 cells, the cell viability is above 80%; when the concentration of the compounds 8 to 10 and 12 is more than 12.5 mu M, the cell viability is reduced, the concentration is lazy, and the reduction is large. We selected a concentration of less than 12.5 μm as the effect concentration of the monomeric compound, and finally we selected 10 μm as the dose concentration of the flavonoid monomeric compound.
(II) Effect of flavonoid Compounds on NO level of RAW264.7 cells
The monomer compounds with the concentration of 10 mu M and 5 mu g/mL of LPS are jointly acted on RAW264.7 cells for 24 hours, and the NO content in the cell culture solution is detected by a Griess method. As shown in fig. 15, it can be seen that the LPS treated group at a concentration of 5 μg/mL had a very significant increase in NO content (P < 0.001) compared to the normal cell control group; compounds 3, 4, 5, 6, 8, 9, 11, 12 have various degrees of inhibition of NO production after acting on RAW264.7 macrophages. Based on this we selected compounds 3, 4, 5, 6, 8, 9, 11, 12 for subsequent experiments.
(III) Effect of flavonoid Compounds on TNF-alpha and IL-6 levels in RAW264.7 cells
The effect of each monomer compound on TNF- α, IL-6 levels in RAW264.7 cells at 5 μg/mL LPS induction was examined by the Elisa method. As shown in fig. 16, it can be found that the LPS-treated group at a concentration of 5 μg/mL has a very significant increase in the content of TNF- α and IL-6 inflammatory factors (P < 0.001) compared to the normal cell control group; the differences in production of TNF- α and IL-6 inflammatory factors in groups 3, 4, 5, 6 compared to the LPS model control group were not statistically significant (P > 0.05). The differences between the groups 8, 9, 11 and 12 and the LPS model control group are very obvious (P < 0.001), and the production of TNF-alpha and IL-6 inflammatory factors can be obviously inhibited.
(IV) influence of flavonoid compounds on expression of RAW264.7 cell inflammation-related proteins
(1) Effect of flavonoids on the COX-2 expression level of RAW264.7 cells
During inflammation, cyclooxygenase-2 (COX-2) plays a key role in amplifying inflammatory responses. Cyclooxygenase-2 (COX-2) is an inducible enzyme that is rarely expressed under normal physiological conditions and is increased in inflammatory or neoplastic pathological conditions.
As shown in FIG. 17, the COX-2 expression level was significantly increased in the LPS-treated group at a concentration of 5. Mu.g/mL compared to the normal cell control group (P < 0.001), the statistical difference was found between the compounds 3 and 5 compared to the LPS model control group (P < 0.05), and the difference was significantly found between the compounds 8, 9, 11 and 12 compared to the LPS model control group (P < 0.001), so that the COX-2 expression level was significantly reduced.
(2) Effect of flavonoids on expression level of TLR-4 in RAW264.7 cells
TLRs contain a cytoplasmic domain homologous to the IL-1 receptor signaling domain, and activation of TLRs results in activation of NF-. Kappa.B and induction of cytokines required to activate adaptive immune responses.
As shown in fig. 18, the COX-2 expression level of the LPS-treated group at a concentration of 5 μg/mL was significantly increased (P < 0.001) compared to the normal cell control group, the statistical difference (P < 0.05) was found between the compound 4 and the LPS model control group, and the difference (P < 0.001) was significantly found between the compound 8, 9, 11, 12 groups compared to the LPS model control group, so that the TLR-4 expression level could be significantly reduced.
(3) Effect of flavonoids on NF- κB expression level in RAW264.7 cells
Nuclear factor kappa B (NF-kappa B) regulates the expression of a number of genes involved in immune and inflammatory responses and in cell proliferation and differentiation. NF-. Kappa.B is activated by cytokines, inflammatory lesions, infections, and other stress conditions requiring rapid reprogramming of gene expression in response to various stimuli [94] 。
As shown in fig. 19, the expression level of the LPS-treated group at a concentration of 5 μg/mL was significantly increased (P < 0.001) compared to the normal cell control group, the statistical difference was present between the compound 5 and the LPS model control group (P < 0.05), the NF- κb relative protein expression level was significantly different between the compound 8, 9, 11, 12 and the LPS model control group (P < 0.001), and the NF- κb expression level was significantly reduced.
(4) Effect of flavonoids on expression level of IκBα in RAW264.7 cells
In the resting state, ikB (inhibitor of NF- κB) is present in the cytoplasm in an inactive state, and when IKK (IkB kinase) is activated by the upstream TLR-4/NF- κB signaling pathway, it phosphorylates and degrades IkBα, initiating inflammatory cytokine transcription and ultimately inducing inflammation.
As shown in fig. 20, the expression level of iκbα relative protein was significantly increased in the LPS-treated group at a concentration of 5 μg/mL compared to the normal cell control group, (P < 0.001), and there was a statistical difference (P < 0.05) in compound 3 compared to the LPS model control group, and the differences were significantly significant (P < 0.001) in compound 8, 9, 11, 12 compared to the LPS model control group, so that the expression level of iκbα relative protein was significantly reduced.
Further research through Westerm Blot experiments shows that flavonoid compounds can inhibit the LPS-induced phosphorylation level of RAW264.7 cells NF-kappa B, p-Ikappa Ba to different degrees, and can simultaneously down regulate the release of Toll-like receptors (TLR-4), pro-inflammatory mediators (COX-2) and pro-inflammatory cytokines (TNF-alpha and IL-6), which indicates that flavonoid compounds in coreopsis tinctoria have anti-inflammatory activity on the whole, so that macrophage inflammatory response is inhibited. However, the anti-inflammatory activity of 8 compounds screened by the NO experiment has variability, and the activity intensity sequence is 12 & gt11 & gt9 & gt8 & gt3, 4, 5 and 6 in sequence. This difference in activity results, on the one hand, from the difference in cellular level of action on the compound and, on the other hand, from the structural specificity of the compound itself.
In summary, the invention evaluates the anti-inflammatory activity of the in vitro cell level of 12 compounds separated and purified from the ethyl acetate part of coreopsis tinctoria, and researches show that 8 flavonoids have better NO inhibition effect and 4 flavonoids have good anti-inflammatory activity, which provides a theoretical basis for further researching anti-inflammatory mechanism of coreopsis tinctoria flavonoids and developing medicines with anti-inflammatory effect.
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to those specific embodiments. It will be apparent to those skilled in the art that several deductions or substitutions may be made without departing from the spirit of the invention, and these shall be considered to be within the scope of the invention.
Claims (9)
1. A method for isolating anti-inflammatory active ingredients of coreopsis tinctoria, comprising the following:
1) Grading and extracting the coreopsis tinctoria ethanol extract with petroleum ether and ethyl acetate, collecting ethyl acetate extract, and removing solvent to obtain ethyl acetate extract;
2) Gradient elution is carried out on the ethyl acetate part extractum by adopting a macroporous resin column, wherein the gradient solvent is 0% -40% ethanol solution; taking a 40% ethanol elution part to obtain an active part Fr2; standing the active site Fr2 to separate out precipitate to obtain an active site Fr2-1;
3) The active site Fr2-1 is subjected to semi-preparative liquid chromatography to obtain an active site Fr2-1-2;
wherein the mobile phase A is methanol, and the mobile phase B is water; the elution conditions were: 30% -80% of A; time: 50min; the retention time of the active site Fr2-1-2 in the semi-preparative chromatography ranges from 7.5 to 12.5min;
4) The active site Fr2-1-2 is subjected to semi-preparative liquid chromatography to obtain a compound 5;
wherein the mobile phase A is methanol, and the mobile phase B is water; the elution condition is 40% -41% A; time: 60min; the retention time of compound 5 in semi-preparative chromatography was 12.7min;
the structural formula of the compound 5 is as follows:
。
2. the method of claim 1, wherein the coreopsis tinctoria ethanol extract is an ethanol or methanol extract.
3. The method of claim 1, wherein the coreopsis tinctoria ethanol extract is an 80% ethanol extract.
4. The separation method according to claim 1, wherein the macroporous resin column used in step 2) is an AB-8 macroporous resin column.
5. The separation method according to claim 1, wherein in step 2), the impurity is removed with 10VB water and then eluted with 10VB 40% ethanol to obtain the active site Fr2.
6. The separation method according to any one of claims 1 to 5, wherein the semi-preparative chromatography conditions in step 3) further comprise any one or more of the following i to v:
i flow rate: 10-20 mL/min
ii column temperature: 25-30 DEG C
iii sample injection amount: 100-500 mu L
iv detection wavelength: 280-360 nm
v chromatography column: c18 column.
7. The separation method according to claim 6, wherein the semi-preparative chromatography conditions in step 3) further comprise any one or more of the following i to v:
i flow rate: 15 mL/min
ii column temperature: 25 DEG C
iii sample injection amount: 500 mu L
iv detection wavelength: 280 nm or 360 nm
v chromatography column: a KromasilC18 column, 21.2X105 mm,5 μm in size.
8. The separation method according to claim 1, wherein the semi-preparative chromatography conditions in step 4) further comprise any one or more of the following i to v:
i flow rate: 10-20 mL/min
ii column temperature: 25-30 DEG C
iii sample injection amount: 100-300 mu L
iv detection wavelength: 280-360 nm
v chromatography column: c18 column.
9. The separation method according to claim 8, wherein the semi-preparative chromatography conditions in step 4) further comprise any one or more of the following i to v:
i flow rate: 15 mL/min
ii column temperature: 25 ℃ or 30 DEG C
iii sample injection amount: 300 mu L
iv detection wavelength: 280 nm or 360 nm
v chromatography column: a KromasilC18 column, 21.2X105 mm,5 μm in size.
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| Flavanone and Diphenylpropane Glycosides and Glycosidic Acyl Esters from Viscum;Kuo et al.;《J. Nat. Prod.》;第73卷(第2期);第109-114页 * |
| Junli Li,et al.Two New Phenolic Glycosides from Gnaphalium affine D. Don and Their Anti-Complementary Activity.《Molecules》.2013,第18卷第7751-7760页. * |
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