Method for extracting quercetin from oak bark

Abstract

The application provides a method for extracting quercetin from oak bark, and belongs to the technical field of extraction and separation. The method comprises the following steps: first, extracting oak bark with an alcohol-water solution and concentrating to obtain an alcohol-water extract; then, mixing the alcohol-water extract with water, performing petroleum ether extraction to obtain an aqueous phase; performing ethyl acetate extraction on the aqueous phase to obtain an extract; then, performing silica gel column separation on the extract, using a dichloromethane-methanol mixture to perform elution, and obtaining a quercetin-containing component; finally, performing gel exclusion chromatography separation on the obtained quercetin-containing component, using methanol to perform elution, and obtaining quercetin. The method provided by the application realizes the extraction of quercetin from oak bark, fills the blank of extracting and separating quercetin from oak bark, and realizes the efficient extraction of quercetin. Moreover, the method provided by the application is simple, has small environmental pollution, and is suitable for industrialized production.

Description

Technical Field

[0001] This invention belongs to the field of extraction and separation technology, specifically relating to a method for extracting quercetin from oak bark. Background Technology

[0002] Quercetin is a flavonoid compound that acts as an anti-apoptotic and antioxidant agent, protecting against damage to various organs. It also exhibits anti-cancer, anti-inflammatory, antibacterial, antiviral, hypoglycemic, hypotensive, immune-regulating, and cardiovascular protective effects. Quercetin is primarily extracted from plant branches and leaves. For example, existing technologies disclose methods for extracting quercetin from water chestnut peel, pomegranate peel, Bidens pilosa, tea oil, Lysimachia christinae, and Bourbon twigs. However, no methods for extracting quercetin from oak bark have been reported. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a method for extracting quercetin from oak bark, which can achieve efficient extraction of quercetin from oak bark.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] This invention provides a method for extracting quercetin from oak bark, comprising the following steps:

[0006] The oak bark and an alcohol-water solution were mixed, extracted, and then concentrated to obtain an alcohol-water extract; the volume fraction of alcohol in the alcohol-water solution was 63-67%.

[0007] The alcohol-water extract was mixed with water and extracted with petroleum ether to obtain an aqueous phase;

[0008] The aqueous phase was extracted with ethyl acetate to obtain an ethyl acetate extract;

[0009] The ethyl acetate extract was subjected to silica gel column separation to obtain a quercetin-containing fraction; the eluent used in the silica gel column separation included a dichloromethane-methanol mixture; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture was 20-30:1;

[0010] The quercetin-containing component was separated by gel size exclusion chromatography to obtain quercetin; the eluent used in the gel size exclusion chromatography included methanol.

[0011] Preferably, the dry weight of the oak bark and the volume ratio of the alcohol-water solution used for a single extraction are 1 g: 29 mL.

[0012] The alcohol in the aqueous solution includes methanol and / or ethanol.

[0013] Preferably, the extraction temperature is 60-70℃; the extraction is performed 2-3 times, and the extraction time for each extraction is 85-95 minutes.

[0014] Preferably, the petroleum ether extraction is performed 3 times; the extraction time for each petroleum ether extraction is 110-140 min.

[0015] Preferably, the ethyl acetate extraction is performed 6 times; the extraction time for each ethyl acetate extraction is 110-140 min.

[0016] Preferably, the elution method for silica gel column separation includes gradient elution, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 30:1 and 20:1 respectively during the gradient elution process.

[0017] Preferably, the flow rate of the eluent during the silica gel column separation process is 50–100 mL / h.

[0018] Preferably, the chromatographic column used for the gel size exclusion chromatography separation includes a Sephadex LH-20 column.

[0019] Preferably, the gel size exclusion chromatography separation process further includes thin-layer chromatography detection of the obtained eluent;

[0020] The developing solvent used in the thin-layer chromatography detection includes a mixed solution of dichloromethane, methanol, and acetic acid;

[0021] The volume ratio of dichloromethane, methanol, and acetic acid in the dichloromethane-methanol-acetic acid mixed solution is 15:1:0.4.

[0022] This invention provides a method for extracting quercetin from oak bark. The method involves first extracting oak bark with an aqueous alcohol solution and then concentrating the extract to obtain an aqueous alcohol extract. The aqueous alcohol extract is then mixed with water and extracted with petroleum ether to obtain an aqueous phase. This aqueous phase is then extracted with ethyl acetate to obtain an extract. The extract is then separated using a silica gel column chromatography with dichloromethane-methanol mixture as elution to obtain the quercetin-containing fraction. Finally, the quercetin-containing fraction is separated by gel size exclusion chromatography with methanol as elution to obtain quercetin. This method achieves efficient extraction of quercetin from oak bark, filling a gap in the extraction and separation of quercetin from oak bark. Furthermore, the method is simple, has minimal environmental pollution, and is suitable for industrial production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The image shows the thin-layer chromatographic results of the ethyl acetate extract, n-butanol extract, and quercetin standard obtained in Example 1.

[0025] Figure 2 The image shows the thin-layer chromatograms of the eluent containing quercetin and the quercetin standard obtained in Example 1.

[0026] Figure 3 The image shows the thin-layer chromatograms of the eluent of quercetin obtained in Example 1 and the quercetin standard.

[0027] Figure 4 The liquid chromatography-mass spectrum of quercetin extracted in Example 1;

[0028] Figure 5 The image shows the ultra-high performance liquid chromatogram of quercetin extracted in Example 1. Detailed Implementation

[0029] This invention provides a method for extracting quercetin from oak bark, comprising the following steps:

[0030] The oak bark and an alcohol-water solution were mixed, extracted, and then concentrated to obtain an alcohol-water extract; the volume fraction of alcohol in the alcohol-water solution was 63-67%.

[0031] The alcohol-water extract was mixed with water and extracted with petroleum ether to obtain an aqueous phase;

[0032] The aqueous phase was extracted with ethyl acetate to obtain an ethyl acetate extract;

[0033] The ethyl acetate extract was subjected to silica gel column separation to obtain a quercetin-containing fraction; the eluent used in the silica gel column separation included a dichloromethane-methanol mixture; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture was 20-30:1;

[0034] The quercetin-containing component was separated by gel size exclusion chromatography to obtain quercetin; the eluent used in the gel size exclusion chromatography included methanol.

[0035] In this invention, unless otherwise specified, all required materials are commercially available products well known to those skilled in the art.

[0036] This invention involves mixing oak bark with an alcohol-water solution, extracting the extract, and then concentrating the solution to obtain an alcohol-water extract.

[0037] In this invention, the volume fraction of alcohol in the aqueous alcohol solution is 63-67%, preferably 64-66%, and more preferably 64-65%.

[0038] In this invention, the oak bark is preferably dried and pulverized sequentially before use to obtain oak bark powder. The present invention does not specifically limit the drying method; any drying method well known to those skilled in the art can be used, such as air drying. In this invention, the particle size of the oak bark powder is preferably ≥60 mesh, more preferably 60–90 mesh.

[0039] In this invention, the preferred volume ratio of the dry weight of the oak bark to the alcohol-water solution used for a single extraction is 1 g: 29 mL. In this invention, the alcohol in the alcohol-water solution includes methanol and / or ethanol, more preferably ethanol.

[0040] In this invention, the extraction temperature is preferably 60-70℃, more preferably 62-68℃, and most preferably 64-66℃; the number of extractions is preferably 2-3 times, more preferably 2 times; and the extraction time for a single extraction is preferably 85-95 min, more preferably 87-92 min, and most preferably 88-90 min.

[0041] In this invention, the concentration is preferably achieved by concentrating the extract obtained from the extraction process to a constant weight. This invention does not specifically limit the concentration method; any conventional concentration method in the art can be used. Specifically, rotary evaporation followed by vacuum filtration is preferred.

[0042] After obtaining the alcohol-water extract, the present invention mixes the alcohol-water extract with water and performs petroleum ether extraction to obtain an aqueous phase.

[0043] In this invention, the mixing is preferably performed using ultrasonic oscillation. In this invention, the solid-liquid ratio of the alcohol-water extract and the water used for mixing is preferably 1g:8-15mL, more preferably 1g:9-12mL, and most preferably 1g:10-11mL.

[0044] In this invention, the petroleum ether extraction is preferably performed three times; the extraction time for each petroleum ether extraction is preferably 110–140 min, more preferably 110–130 min, and most preferably 120–125 min. In this invention, the volume ratio of the mixed water to the petroleum ether used for the single petroleum ether extraction is preferably 1:0.8–1.2, more preferably 1:0.9–1.1, and most preferably 1:0.9–1.0.

[0045] After obtaining the aqueous phase, the present invention extracts the aqueous phase with ethyl acetate to obtain an ethyl acetate extract.

[0046] In this invention, the ethyl acetate extraction is preferably performed 6 times; the extraction time for each ethyl acetate extraction is preferably 110–140 min, more preferably 110–130 min, and most preferably 120–125 min. In this invention, the volume ratio of the aqueous phase to the ethyl acetate used for the single ethyl acetate extraction is preferably 1:1.

[0047] After completing the ethyl acetate extraction, the present invention preferably further includes concentrating the ethyl acetate extract phase obtained from the ethyl acetate extraction to a constant weight to obtain an ethyl acetate extract. The present invention does not specifically limit the concentration method; any concentration method conventional in the art can be used, and rotary evaporation is preferred.

[0048] After obtaining the ethyl acetate extract, the present invention performs silica gel column separation on the ethyl acetate extract to obtain a component containing quercetin; the eluent used for the silica gel column separation includes a dichloromethane-methanol mixture; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 20-30:1.

[0049] In this invention, the silica gel used for silica gel column separation preferably has a particle size of 100-200 mesh. In this invention, the elution method for silica gel column separation is preferably gradient elution, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture during the gradient elution process is preferably 30:1 and 20:1, respectively. In this invention, the flow rate of the eluent during silica gel column separation is preferably 50-100 mL / h, specifically preferably 50 mL / h or 100 mL / h.

[0050] In this invention, the silica gel column separation process preferably further includes thin-layer chromatography detection of the obtained eluent, and collection of the target analyte based on the thin-layer chromatography detection results; the developing solvent used in the thin-layer chromatography detection is preferably a dichloromethane-methanol-acetic acid mixed solution, and the volume ratio of dichloromethane, methanol and acetic acid in the dichloromethane-methanol-acetic acid mixed solution is preferably 15:1:0.4.

[0051] After completing the silica gel column separation, the present invention preferably further includes concentrating the obtained eluent containing quercetin to a constant weight to obtain the quercetin-containing component. The present invention does not specifically limit the concentration method; conventional concentration methods in the art can be used.

[0052] After obtaining the quercetin-containing component, the present invention performs gel size exclusion chromatography to separate the quercetin-containing component to obtain quercetin; the eluent used in the gel size exclusion chromatography includes methanol.

[0053] In this invention, the chromatographic column used for the gel size exclusion chromatography separation preferably includes a Sephadex LH-20 column.

[0054] In this invention, the gel size exclusion chromatography separation process preferably further includes thin-layer chromatography detection of the obtained eluent, and collection of the target analyte based on the thin-layer chromatography detection results. In this invention, the developing solvent used for the thin-layer chromatography detection preferably includes a dichloromethane-methanol-acetic acid mixed solution; the volume ratio of dichloromethane, methanol, and acetic acid in the dichloromethane-methanol-acetic acid mixed solution is preferably 15:1:0.4.

[0055] After completing the gel size exclusion chromatography separation, the present invention preferably further includes concentrating the obtained quercetin eluent and drying it to constant weight to obtain quercetin. The present invention does not have any particular limitation on the concentration and drying methods; conventional concentration and drying methods in the art can be used.

[0056] To further illustrate the present invention, the following detailed description of a method for extracting quercetin from oak bark, in conjunction with the accompanying drawings and embodiments, is provided by the present invention, but should not be construed as limiting the scope of protection of the present invention.

[0057] In all embodiments and comparative examples of this invention, all chemical reagents involved were of analytical grade.

[0058] The preparation method of Sephadex LH-20 size exclusion chromatography column is as follows: Weigh 17g of Sephadex LH-20 packing material and place it in a clean beaker. Add 50mL of methanol, seal with plastic wrap, and soak in the refrigerator for 24 hours. Remove it one hour before loading onto the column and allow it to reach room temperature. Take another clean chromatography column, first wet the entire column with methanol, then slowly stir the Sephadex LH-20 / methanol solution until the solid and liquid are evenly mixed, and pour it into the column all at once. Let the entire column stand until the Sephadex LH-20 no longer settles. Open the column stopcock, and when the methanol is just tangent to the upper surface of the packing material, close the column stopcock to complete the column packing.

[0059] Example 1

[0060] Dried oak bark was pulverized and passed through a 60-mesh sieve; the portion passing through the sieve was oak bark powder. The oak bark powder was mixed with an ethanol-water solution and extracted twice at 64°C. The two extracts were combined, cooled, and concentrated to constant weight to obtain the ethanol-water extract. The volume fraction of ethanol in the ethanol-water solution was 65%; the extraction time for each extraction was 90 min; and the solid-liquid ratio of oak bark powder to ethanol-water solution was 1 g: 29 mL.

[0061] The total flavonoid content in the obtained alcohol-water extract was determined by ultraviolet-visual spectrophotometry. Calculated with rutin, the total flavonoid extraction rate in the alcohol-water extract was 0.647%, and the total flavonoid purity was 7.16%.

[0062] Weigh 290g of the alcohol-water extract, add 3000mL of water, and sonicate to dissolve, obtaining a total extract solution of 290g. Extract the extract solution three times with petroleum ether, and combine the aqueous phases from the petroleum ether extractions for subsequent extractions. The volume ratio of water to petroleum ether used in each extraction was 1:1, and the extraction time for each extraction was 2 hours.

[0063] The aqueous phase from the petroleum ether extraction was extracted six times with ethyl acetate. The ethyl acetate extract phase and the aqueous phase from the ethyl acetate extraction were combined separately. The ethyl acetate extract phase was then rotary evaporated to constant weight to obtain the ethyl acetate extract. The volume ratio of the aqueous phase from the petroleum ether extraction to the ethyl acetate used in each ethyl acetate extraction was 1:1, and the extraction time for each ethyl acetate extraction was 2 hours.

[0064] To verify that quercetin was enriched in the ethyl acetate extract phase after extraction, the aqueous phase extracted with ethyl acetate was further extracted with n-butanol to obtain the n-butanol extract phase. The ethyl acetate extract phase, the n-butanol extract phase, and the quercetin standard were then analyzed by thin-layer chromatography using dichloromethane:methanol:acetic acid = 15:1:0.003 (volume ratio). Figure 1 The image shows the thin-layer chromatographic results of the ethyl acetate extract, n-butanol extract, and quercetin standard obtained in Example 1. From left to right, the images show the ethyl acetate extract, quercetin standard, and n-butanol extract. Figure 1 It can be seen that quercetin has been enriched in the ethyl acetate extraction phase, and there is almost no quercetin in the aqueous phase.

[0065] Take 78g of ethyl acetate extract, add 100g of silica gel (silica gel particle size 100-200 mesh), and add an appropriate amount of ethyl acetate for dispersion. Rotary evaporate the resulting dispersion to constant weight to obtain silica gel containing ethyl acetate extract. Mix fresh silica gel with a dichloromethane-methanol mixture and stir continuously. After ultrasonic vibration for 30 min, pack the mixture into a column and allow it to settle for 12 h. The volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 30:1.

[0066] The ethyl acetate extract was dry-loaded onto silica gel, and eluted using the aforementioned dichloromethane-methanol mixture (eluent flow rate 50–100 mL / h). Thin-layer chromatography (TLC) was then employed for detection. After impurities in the TLC were eluted, elution was continued using a dichloromethane-methanol mixture with a volume ratio of 20:1 (dichloromethane:methanol), and TLC was again used for detection. Figure 2 The image shows the thin-layer chromatograms of the eluent containing quercetin and the quercetin standard obtained in Example 1, where numbers 311 to 331 represent the eluent containing quercetin. The eluents containing quercetin were combined and rotary evaporated to constant weight to obtain 2.3498 g of the quercetin-containing component.

[0067] The quercetin-containing component was dissolved in methanol. The resulting quercetin-containing methanol solution was filtered through a filter membrane and then separated using a Sephadex LH-20 column. Methanol was used for elution, and thin-layer chromatography was used for detection. The developing solvent was dichloromethane:methanol:glacial acetic acid = 15:1:0.4 (volume ratio). Figure 3 The image shows the thin-layer chromatograms of the eluent and standard of quercetin obtained in Example 1. Images 2 to 12 are all eluents of quercetin. The eluents of quercetin were concentrated and dried to constant weight to obtain quercetin.

[0068] Figure 4 The liquid chromatography-mass spectrum of quercetin extracted in Example 1 is shown below. Figure 4 It is known that the product extracted in this invention is quercetin. The purity of the quercetin eluent, which showed results consistent with those of the quercetin standard in thin-layer chromatography, was determined by ultra-high performance liquid chromatography (UHPLC). The UHPLC conditions were as follows: the chromatographic column was C200000. 18 The chromatographic column (250×4.6mm, 5μm; Waters ACQUITY UPLC BEH) used methanol as mobile phase A and ultrapure water as mobile phase B. Gradient elution was employed with the following program: 0–5 min, mobile phase A volume fraction 50%; 5–6 min, mobile phase A volume fraction increased from 50% to 100%; 6–15 min, mobile phase A volume fraction 100%. The mobile phase flow rate was 1 mL / min. The column temperature was 30℃, and the detection wavelength was 360 nm. The chromatograph was a UtiMate 3000 dual-gradient liquid chromatograph (Thermo Fisher) equipped with a diode array detector, dual ternary gradient pump, degasser, and RSLC system.

[0069] Figure 5 This is the ultra-high performance liquid chromatogram of quercetin extracted in Example 1, obtained by... Figure 5 It can be seen that the purity of the quercetin extracted by this invention exceeds 90%.

[0070] In this embodiment, a total of 33.2 mg of quercetin was extracted, with a yield of 5.15%.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting quercetin from oak bark, characterized in that, The steps are as follows: Oak bark was mixed with an alcohol-water solution, extracted, and then concentrated to obtain an alcohol-water extract; the volume fraction of alcohol in the alcohol-water solution was 65%; the mass-to-volume ratio of the dry weight of the oak bark to the alcohol-water solution used for a single extraction was 1 g: 29 mL; the extraction temperature was 64 °C; the extraction was performed twice, with each extraction lasting 90 min; the alcohol in the alcohol-water solution was ethanol. The alcohol-water extract was mixed with water and then extracted with petroleum ether to obtain an aqueous phase; the mass-to-volume ratio of the alcohol-water extract to the mixed water was 1 g: 8-15 mL; the petroleum ether extraction was performed 3 times; and the extraction time for each petroleum ether extraction was 120 min. The aqueous phase was extracted with ethyl acetate to obtain an ethyl acetate extract; the ethyl acetate extraction was performed 6 times; the extraction time for each ethyl acetate extraction was 120 min. The ethyl acetate extract was subjected to silica gel column chromatography to obtain a quercetin-containing fraction. The eluent used in the silica gel column chromatography was a dichloromethane-methanol mixture. The elution method used in the silica gel column chromatography was gradient elution, in which the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture was 30:1 and 20:1, respectively. The flow rate of the eluent during the silica gel column chromatography was 50-100 mL / h. The quercetin-containing component was separated by gel size exclusion chromatography to obtain quercetin; the eluent used in the gel size exclusion chromatography was selected from methanol; the chromatographic column used in the gel size exclusion chromatography was a Sephadex LH-20 column. The gel size exclusion chromatography separation process also includes thin-layer chromatography detection of the obtained eluent; The developing solvent used in the thin-layer chromatography detection is a mixed solution of dichloromethane, methanol, and acetic acid; The volume ratio of dichloromethane, methanol, and acetic acid in the dichloromethane-methanol-acetic acid mixed solution is 15:1:0.4.

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