CN117700391A - Method for simultaneously extracting apigenin and dihydromyricetin from vine tea and application of apigenin and dihydromyricetin in anti-inflammatory and antibacterial products - Google Patents
Method for simultaneously extracting apigenin and dihydromyricetin from vine tea and application of apigenin and dihydromyricetin in anti-inflammatory and antibacterial products Download PDFInfo
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- CN117700391A CN117700391A CN202311480852.0A CN202311480852A CN117700391A CN 117700391 A CN117700391 A CN 117700391A CN 202311480852 A CN202311480852 A CN 202311480852A CN 117700391 A CN117700391 A CN 117700391A
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- KJXSIXMJHKAJOD-LSDHHAIUSA-N (+)-dihydromyricetin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC(O)=C(O)C(O)=C1 KJXSIXMJHKAJOD-LSDHHAIUSA-N 0.000 title claims abstract description 150
- KQILIWXGGKGKNX-UHFFFAOYSA-N dihydromyricetin Natural products OC1C(=C(Oc2cc(O)cc(O)c12)c3cc(O)c(O)c(O)c3)O KQILIWXGGKGKNX-UHFFFAOYSA-N 0.000 title claims abstract description 75
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 title claims abstract description 72
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- FTODBIPDTXRIGS-UHFFFAOYSA-N homoeriodictyol Natural products C1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 FTODBIPDTXRIGS-UHFFFAOYSA-N 0.000 description 1
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- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of extraction methods of plant active ingredients, and particularly relates to a method for simultaneously extracting apigenin and dihydromyricetin from vine tea and application of the method in anti-inflammatory and antibacterial products. The method comprises the following steps; s1, drying and crushing vine tea, sieving, soaking vine tea powder in a solvent A, performing first ultrasonic treatment, heating and extracting in a water bath, filtering to obtain filtrate A and filter residues, soaking filter residues in a solvent B, performing second ultrasonic treatment, heating and extracting in a water bath, and filtering to obtain filtrate B; s2, mixing the filtrate A and the filtrate B, and separating and purifying to obtain dihydromyricetin and apigenin respectively; the solvent A consists of ethylene glycol and ethanol with the mass fraction of 75-85%; the solvent B consists of brominated 1-hexyl-3-methylimidazole, absolute ethyl alcohol and water. The invention utilizes the solubility of the solvents to synchronously improve the extraction rate and purity of apigenin and dihydromyricetin, and the combination of the apigenin and the dihydromyricetin has synergistic effect on inhibiting staphylococcus aureus and pseudomonas aeruginosa.
Description
Technical Field
The invention relates to the technical field of plant active ingredient extraction methods, in particular to a method for simultaneously extracting apigenin and dihydromyricetin from vine tea and application of the method in anti-inflammatory and antibacterial products.
Background
Ampelopsis grossedentata is tender stem and leaf of Ampelopsis grossedentata of Ampelopsis of Vitaceae, and is a special medicinal and edible vine plant in China. It is recorded that the drug has sweet, bland and cool taste, and has the effects of clearing heat and detoxicating, and is mainly used for treating icteric hepatitis, wind-heat type common cold, sore throat and the like. Researches show that the main secondary metabolites in the vine tea are mainly flavonoid compounds, and the flavonoid substances in the vine tea are various, wherein the vine tea contains higher dihydromyricetin, and also comprises myricetin, quercetin, apigenin, kaempferol, hesperetin and the like.
The content of the dihydromyricetin in the vine tea can be up to about 30%, and the dihydromyricetin has various effects of scavenging free radicals, resisting oxidation, resisting thrombus, resisting tumor, diminishing inflammation and the like, and has obvious inhibition effect on the growth of escherichia coli, staphylococcus aureus, bacillus dysenteriae and the like. The methods reported in the present literature and patent for extracting dihydromyricetin from Guan Teng tea mainly comprise an organic solvent extraction method, an alkaline extraction method, an enzyme extraction method, a microwave-assisted method and a water extraction method, wherein the most common methods are the organic solvent extraction method, however, the organic solvent has a plurality of defects such as environmental protection, flammability, limitation of the use of certain foods and the like, and enzyme and microwave increase the extraction cost.
Apigenin is also known as apigenin. A great deal of research at home and abroad finds that apigenin has various biological efficacy activities of scavenging free radicals, improving autoimmune power, resisting oxidation, resisting inflammation, resisting cancer, resisting tumor and the like. However, apigenin has low solubility in common solvents such as water and ethanol, which results in poor extraction of apigenin from plants.
There is no description in the literature about the simultaneous combined extraction of both apigenin and dihydromyricetin in vine tea. When extraction of various components is involved, the process is often complicated and the corresponding product purity and yield are also low. With the increase of the demand of natural functional components of plants, the exploration of a novel energy-saving and efficient extraction method for simultaneously extracting apigenin and dihydromyricetin from vine tea becomes a problem to be solved in the future.
Disclosure of Invention
The invention aims to provide a method for simultaneously extracting apigenin and dihydromyricetin in vine tea and application thereof, and the method increases the solubility of an extract in an extraction solvent, improves the biological safety and effectively increases the extraction efficiency and purity of apigenin and dihydromyricetin by exploring and researching conditions such as the extraction solvent, the extraction process and the like of the apigenin and the dihydromyricetin; meanwhile, the application also provides application of the combined synergy of apigenin and dihydromyricetin in anti-inflammatory and antibacterial products.
In order to achieve the above purpose, the specific technical scheme of the invention is as follows:
a method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
s1, drying and crushing vine tea, sieving, soaking vine tea powder in a solvent A, performing first ultrasonic treatment, heating and extracting in a water bath, filtering to obtain filtrate A and filter residues, soaking filter residues in a solvent B, performing second ultrasonic treatment, heating and extracting in a water bath, and filtering to obtain filtrate B;
s2, mixing the filtrate A obtained in the step S1 with the filtrate B, and separating and purifying to obtain dihydromyricetin and apigenin respectively;
the solvent A consists of ethylene glycol and 75-85% of ethanol by mass fraction;
the solvent B consists of brominated 1-hexyl-3-methylimidazole, absolute ethyl alcohol and water.
Preferably, the particle size of the vine tea crushed in the step S1 is 80-100 meshes, and the soaking time is 2-4 hours.
Preferably, the ratio of the vine tea powder to the solvent A in the step S1 is 1: 8-12 (g/mL), wherein the weight ratio of the solvent A to the solvent B is 2-5: 1.
preferably, the heating temperature of the first ultrasonic extraction in the step S1 is 40-60 ℃, and the extraction time is 40-60 minutes; the heating temperature of the second ultrasonic extraction is 30-40 ℃ and the extraction time is 30-50 minutes.
Preferably, in step S1, the weight ratio of ethylene glycol to ethanol in the solvent a is 1:10 to 15 percent; the mol ratio of the brominated 1-hexyl-3-methylimidazole to the absolute ethyl alcohol in the solvent B is 1:20, the volume percentage of water is 15-20%.
Preferably, in the step S2, macroporous resin is adopted for chromatographic treatment of the mixed solution, specifically, the mixed solution is loaded on a macroporous adsorption resin column, adsorbed at a flow rate of 0.5-1.0 mL/min, eluted with 20% ethanol, and the eluent is collected and concentrated to obtain dihydromyricetin; eluting with 80% ethanol and ethyl acetate mixture, collecting eluate, and concentrating to obtain apigenin.
The extraction efficiency and purity of apigenin and dihydromyricetin obtained by the extraction method are improved.
The second object of the invention is to provide the application of apigenin and dihydromyricetin extracted by the method in preparing antibacterial infection medicines, wherein the medicines comprise apigenin and dihydromyricetin extracted by the method and pharmaceutically acceptable carriers or excipients, and the apigenin and the dihydromyricetin have a synergistic effect, and particularly have a better antibacterial effect on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa.
Compared with the prior art, the invention has the following technical effects:
(1) According to the invention, through optimizing various parameters and steps such as an extraction solvent, an extraction process and the like in a method for jointly extracting apigenin and dihydromyricetin from vine tea, the solubility promotion between solvents is fully utilized, the extraction efficiency and purity of apigenin and dihydromyricetin are obviously improved, and the resource utilization of vine tea is realized.
(2) The extracted apigenin and dihydromyricetin are combined to have synergistic inhibition effects on important pathogenic bacteria for inhibiting traumatic infection, such as staphylococcus aureus and pseudomonas aeruginosa, and are expected to be applied to preparation of anti-inflammatory and antibacterial products with good effects.
Drawings
FIG. 1 is a graph showing the combination sterilization of apigenin and dihydromyricetin extracted in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with examples of the present invention and comparative examples, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
In order to reduce the error of different flavonoid component contents among different vine tea samples, the vine tea samples in the embodiment and the comparative example are prepared from the same batch of Guizhou Jiangkou origin by crushing, and then the subsequent operation is carried out.
Example 1
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 40 ℃ for 40min, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 40deg.C, extracting for 40min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of the following components in percentage by weight: 13 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:10 (g/mL); the mol ratio of the solvent B is 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20 percent; the weight ratio of the solvent A to the solvent B is 3:1, a step of;
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 0.8mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Example 2
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 40 ℃ for 40min, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 40deg.C, extracting for 40min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of the following components in percentage by weight: 15 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:9 (g/mL); the mol ratio of the solvent B is 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20 percent; the weight ratio of the solvent A to the solvent B is 5:1, a step of;
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 0.8mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Example 3
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 40 ℃ for 60 minutes, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 30deg.C, extracting for 50min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of the following components in percentage by weight: 10 of ethylene glycol and 80% of ethanol by mass percent, wherein the feed liquid ratio of vine tea powder to solvent A is 1:11 (g/mL); the mol ratio of the solvent B is 1:20, namely, brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 18%; the weight ratio of the solvent A to the solvent B is 4:1, a step of;
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 0.5mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Example 4
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 40 ℃ for 50 minutes, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 40deg.C, extracting for 40min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of the following components in percentage by weight: 13 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:8 (g/mL); the mol ratio of the solvent B is 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20 percent; the weight ratio of the solvent A to the solvent B is 3:1, a step of;
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 0.5mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Example 5
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 60 ℃ for 40 minutes, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 40deg.C, extracting for 30min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of the following components in percentage by weight: 13 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:10 (g/mL); the mol ratio of the solvent B is 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20 percent; the weight ratio of the solvent A to the solvent B is 3:1, a step of;
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 1.0mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Comparative example 1
The difference compared to example 1 is that in step (1) ethylene glycol is absent from solvent A and brominated 1-hexyl-3-methylimidazole is absent from solvent B, and other experimental steps and conditions are the same and will not be repeated here.
Comparative example 2
Compared with example 1, the difference is that in the solvent A in the step (1), the weight ratio of the solvent A is 1:17 and 80% ethanol, wherein choline chloride is used in the solvent B to replace brominated 1-hexyl-3-methylimidazole, and other experimental steps and conditions are the same and are not repeated here.
Comparative example 3
Compared with example 1, the difference is that the solvent A in the step (1) is prepared from the following components in weight ratio 1:6 and 80% ethanol, wherein the mol ratio of the solvent B is 1:15, namely brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20%; other experimental procedures and conditions were the same and will not be repeated here.
Comparative example 4
Compared with example 1, the difference is that the feed liquid ratio of the vine tea powder to the solvent A in the step (1) is 1:6 (g/mL), the weight ratio of solvent A to solvent B is 6:1, other experimental steps and conditions are the same and will not be repeated here.
Comparative example 5
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying Ampelopsis grossedentata, pulverizing, sieving with 80 mesh sieve to obtain Ampelopsis grossedentata powder, soaking Ampelopsis grossedentata powder in mixed solvent A+B for 4 hr, performing ultrasonic treatment (ultrasonic power is 350W), heating in water bath at 40deg.C for 80min, and filtering to obtain filtrate and residue;
wherein the solvent A consists of the following components in percentage by weight: 13 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:10 (g/mL); the mol ratio of the solvent B is 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent B is 20 percent; the weight ratio of the solvent A to the solvent B is 3:1.
(2) Subjecting the obtained filtrate to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at flow rate of 0.8mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Comparative example 6
A method for simultaneously extracting apigenin and dihydromyricetin from vine tea comprises the following steps:
(1) Drying vine tea, crushing, sieving with an 80-mesh sieve to obtain vine tea powder, adding the vine tea powder into a solvent A, soaking for 2 hours, performing first ultrasonic treatment, heating in a water bath at 40 ℃ for 40min, and filtering to obtain a filtrate A and filter residues; soaking the filter residue in the solvent B for 2h, performing a second ultrasonic treatment, heating in water bath at 40deg.C, extracting for 40min, and filtering to obtain filtrate B with ultrasonic power of 350W;
wherein the solvent A consists of a molar ratio of 1:20, namely 20 percent of brominated 1-hexyl-3-methylimidazole and absolute ethyl alcohol, wherein the volume percentage of water in the solvent A is 20 percent; the solvent B is prepared from the following components in percentage by weight: 13 and 80% ethanol, wherein the feed liquid ratio of vine tea powder to solvent A is 1:10 (g/mL); the weight ratio of the solvent A to the solvent B is 3:1.
(2) Mixing the obtained filtrate A and filtrate B, subjecting the mixed solution to chromatography with strong polarity macroporous adsorbent resin column (model ADS-7), loading the mixed solution onto macroporous adsorbent resin column, adsorbing at a flow rate of 0.8mL/min, eluting with 20% ethanol, collecting eluate, and concentrating to obtain dihydromyricetin; eluting with mixed solution of 80% ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio of 6:1), collecting eluate, and concentrating to obtain apigenin.
Analysis of results
1. Optimization of extraction solvent
In the screening aspect of the extraction solvent, the ionic liquid consisting of 3 anions with different ionic radii is adopted as the extraction liquid, and the difference of acting forces such as hydrogen bonds, charges and the like between phenolic hydroxyl groups and anions in target substances apigenin and dihydromyricetin is probably caused, as shown in the data of table 1, the formula [ C 4 MIm]Br＞[C 4 MIm]Cl＞[C 4 MIm]BF4, end [ C 4 MIm]Br showed better extraction than the other two ionic liquids.
According to the method, the dissolution capacity and the extraction rate of the target are also changed along with the growth of the alkyl chain in the ionic liquid, and experiments prove that the dissolution capacity of the target is not linearly improved along with the growth of the alkyl chain, the hydrophobicity of cations from butyl to hexyl is gradually increased, and the extraction efficiency of apigenin and dihydromyricetin is gradually improved along with the trend; however, from hexyl to octyl, extraction efficiency tends to decrease, which may be due to the steric effect impairing the van der Waals forces between the ionic liquid and the target. The relative increase in solution viscosity may be caused by the increase in molecular chain, which hinders diffusion into ionic liquid, and is unfavorable for dissolution of target substances, and the final extraction effect is shown as [ C ] as shown in the data of Table 1 6 MIm]Br＞[C 4 MIm]Br＞[C 8 MIm]Br. The brominated 1-hexyl-3 methylimidazole adopted by the application shows better performance of jointly extracting apigenin and dihydromyricetin.
TABLE 1
2. Optimization of extraction process conditions
The contents and purities of apigenin and dihydromyricetin obtained by the extraction methods in examples 1 to 5 and comparative examples 1 to 6 of the present application were examined, and the data are shown in Table 2.
TABLE 2
As is clear from Table 2, the content and purity of dihydromyricetin and apigenin obtained by the extraction methods described in examples 1 to 5 of the present application were higher than those of the comparative example group. When the extraction solvent, the feed-liquid ratio or the extraction process is changed in the extraction process, the combined extraction content and purity of the dihydromyricetin and the apigenin are reduced.
3. Combined antibacterial effect of apigenin and dihydromyricetin extracted by the application
(1) Preparation work
(1) The autoclaved MH broth was cooled for later use, and apigenin and dihydromyricetin obtained by the extraction method of example 1 of the present application were prepared as 16, 4mg/mL stock solutions, respectively, for later use, with reference to the CLSI micro broth dilution method.
(2) Inoculating staphylococcus aureus ATCC 6538 and pseudomonas aeruginosa ATCC 9027 to LB solid medium, and performing activating culture at 37 ℃ for 24 hours; single colony is taken and inoculated into LB liquid medium, and cultured for 18-24h in a shaking table at 37 ℃; the broth was diluted with MH broth to give a test broth (about 10 6 CFU/mL), ready for use.
(2) Determination of minimum inhibitory concentration of apigenin and dihydromyricetin
(1) Taking a sterilized 96-well plate, adding 100 mu L of MH broth culture medium and 100 mu L of to-be-tested liquid into each 1 st well, uniformly mixing, taking out 100 mu L of to-be-tested liquid from each first well, adding 100 mu L of MH broth culture medium into each 2 nd well, uniformly mixing, sequentially diluting apigenin and dihydromyricetin to 8-0.03125 mg/mL and 2-0.00781 mg/mL according to the 2-fold dilution method, and simultaneously performing blank culture medium control.
(2) Each well was added with 100. Mu.L (about 10 6 CFU/mL), placing the inoculated 96-well plate into a 37 ℃ incubator for incubation for 24 hours, observing the growth state of bacteria, and detecting the OD (600 nm) value of each well by using an enzyme-labeled instrument; the test was repeated 3 times, 3 duplicate wells each time, and the MIC values of apigenin and dihydromyricetin for the standard strain Staphylococcus aureus and Pseudomonas aeruginosa were determined by taking the average.
(3) Determination of combination index of apigenin and dihydromyricetin
(1) Determining antibacterial effect of combination application of apigenin and dihydromyricetin by micro chessboard dilution method
The dilution of the combined assay was determined from the MIC values obtained, 6-8 dilutions were selected, the two active substances were applied in combination at a mass concentration of 2 XMIC-1/32 XMIC, 50. Mu.L of the test substance was added to each well, 100. Mu.L (about 10 6 CFU/mL), after incubation in an incubator at 37 ℃ for 24 hours, the growth state of the bacteria is observed, and the OD (600 nm) value of each well is detected by an enzyme-labeled instrument; the test was repeated 3 times, 3 duplicate wells per time.
(2) Calculating a combined bacteriostasis concentration index FIC according to the measurement result and the following formula
FIC index = MIC at MIC/a alone at mic+b combination at MIC/B alone at MIC/B combination
Judgment standard: 1) FIC index less than or equal to 0.5 is synergistic; 2) FIC index more than 0.5 and less than or equal to 1 is added; 3) FIC index is more than 1 and less than or equal to 2, and has no relation effect; 4) FIC index > 2 is antagonism.
(4) Combined sterilization effect of apigenin and dihydromyricetin
(1) Inoculating a standard strain staphylococcus aureus ATCC 6538 and pseudomonas aeruginosa ATCC 9027 into an LB liquid culture medium, and culturing for 18-24 hours in a shaking table at 37 ℃; the broth was diluted with MH broth to give a test broth (about 10 6 CFU/mL), ready for use.
(2) And (3) adding apigenin and dihydromyricetin with sub-antibacterial concentration into a MH broth centrifuge tube for use singly or in combination, then adding 4mL of diluted bacterial solutions obtained by diluting in the step (1), and mixing uniformly by vortex 5 s. A blank control group was set up as a quality control, and the control group was kept consistent with the test group except for the absence of drug. Placing in a shaking table at 37 ℃ for 180-turn culture, wherein the bacterial liquid system is 8mL;
(3) Taking bacterial liquid at the time of 0, 3, 6, 9 and 24 hours of culture time, absorbing 100 mu L of bacterial liquid, adding the bacterial liquid into a 2mL centrifuge tube filled with 900 mu L of 0.85% physiological saline for gradient dilution, absorbing 25 mu L of bacterial liquid after dilution, dripping the bacterial liquid on MH agar culture medium, incubating the bacterial liquid in an incubator at 37 ℃ for 18 hours, counting, and carrying out statistical analysis after experimental results are subjected to three biological repetitions. Counting the colony number at each time point, and drawing a sterilization curve graph.
Analysis of results
The combined action results of the apigenin and the dihydromyricetin extracted by the application show that under the action of 1/4 xMIC apigenin-1/16 xMIC dihydromyricetin, 1/8 xMIC apigenin-1/8 xMIC dihydromyricetin and 1/16 xMIC apigenin-1/4 xMIC dihydromyricetin, the combined administration starts to show obvious effect of inhibiting staphylococcus aureus. Under the action of 1/4 xMIC apigenin-1/8 xMIC dihydromyricetin and 1/8 xMIC apigenin-1/4 xMIC dihydromyricetin, the combined administration starts to show obvious effect of inhibiting pseudomonas aeruginosa.
MIC values and FIC values of apigenin and dihydromyricetin on staphylococcus aureus and pseudomonas aeruginosa are shown in tables 3 and 4. As can be seen, the FIC values of the combination of apigenin and dihydromyricetin are smaller than 0.5 specified by a judgment standard, and the combination of apigenin and dihydromyricetin has a synergistic effect on inhibiting staphylococcus aureus and pseudomonas aeruginosa.
TABLE 3 Table 3
TABLE 4 Table 4
Fig. 1 is a graph showing the combination sterilization of apigenin and dihydromyricetin extracted in the application, showing that the bacterial load is significantly reduced (more than 100 times) compared with that of single components after the apigenin and dihydromyricetin are combined for 24 hours, and showing that the apigenin and dihydromyricetin have excellent sterilization effect.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (8)
1. A method for simultaneously extracting apigenin and dihydromyricetin from vine tea, which is characterized by comprising the following steps:
s1, drying and crushing vine tea, sieving, soaking vine tea powder in a solvent A, performing first ultrasonic treatment, heating and extracting in a water bath, filtering to obtain filtrate A and filter residues, soaking filter residues in a solvent B, performing second ultrasonic treatment, heating and extracting in a water bath, and filtering to obtain filtrate B;
s2, mixing the filtrate A obtained in the step S1 with the filtrate B, and separating and purifying to obtain dihydromyricetin and apigenin respectively;
the solvent A consists of ethylene glycol and 75-85% of ethanol by mass fraction;
the solvent B consists of brominated 1-hexyl-3-methylimidazole, absolute ethyl alcohol and water.
2. The extraction method according to claim 1, wherein the particle size of the vine tea crushed in the step S1 is 80-100 meshes, and the soaking time is 2-4 hours.
3. The extraction method according to claim 1, wherein the ratio of the vine tea powder to the solvent a in step S1 is 1: 8-12, wherein the weight ratio of the solvent A to the solvent B is 2-5: 1.
4. the extraction method according to claim 1, wherein the heating temperature of the first ultrasonic extraction in step S1 is 40-60 ℃ and the extraction time is 40-60 minutes; the heating temperature of the second ultrasonic extraction is 30-40 ℃ and the extraction time is 30-50 minutes.
5. The extraction method according to claim 1, wherein the weight ratio of ethylene glycol to ethanol in the solvent a in step S1 is 1: 8-10; the mol ratio of the brominated 1-hexyl-3-methylimidazole to the absolute ethyl alcohol in the solvent B is 1:20, the volume percentage of water is 15-20%.
6. The extraction method according to claim 1, wherein in the step S2, the separation and purification are performed by adopting polar macroporous resin to perform chromatography treatment on the mixed solution, specifically, the mixed solution is loaded on a macroporous adsorption resin column, adsorbed at a flow rate of 0.5-1.0 mL/min, eluted with 20% ethanol, and the eluent is collected and concentrated to obtain dihydromyricetin; eluting with 80% ethanol and ethyl acetate mixture, collecting eluate, and concentrating to obtain apigenin.
7. A combination synergistic antibacterial agent comprising apigenin and dihydromyricetin extracted by the method of any one of claims 1-6 and a pharmaceutically acceptable carrier or excipient.
8. The medicament according to claim 7, wherein the bacteria are staphylococcus aureus, pseudomonas aeruginosa.
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