CN112159443B - Alpha-glucosidase inhibitor extracted from bougainvillea spectabilis and preparation method thereof - Google Patents
Alpha-glucosidase inhibitor extracted from bougainvillea spectabilis and preparation method thereof Download PDFInfo
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Abstract
The application provides an alpha-glucosidase inhibitor, which comprises quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose and a composition thereof. Proved by glycosidase activity experiments, the alpha-glucosidase inhibitor has high inhibition rate, and particularly the composition with the mass ratio of 3:1:3 has the highest inhibition rate. The application relates to a medicine for reducing postprandial blood sugar of diabetics.
Description
Technical Field
The application relates to the technical field of traditional Chinese medicines, in particular to an alpha-glucosidase inhibitor extracted from golden plum, and an extraction method and application thereof.
Background
Type 2 diabetes (type 2 diabetes mellitus,T2DM) belongs to the category of "diabetes" in traditional Chinese medicine, which is called "diabetes" in the book of internal menstruation, and is considered to be a polygenic disease. Modern research has found that T2DM is a chronic metabolic disease that is mainly hyperglycemia due to insulin resistance (IR, insulin resistance) with relative hyposecretion of insulin. According to World Health Organization (WHO), so far, the number of patients suffering from T2DM may be higher than 85% -95% in countries with medium income and low income, the prevalence rate of the patients is higher in countries with high income, and the prevalence rate of T2DM in different countries and regions is gradually increased, and the prevalence rate of T2DM is higher in north america and asia than in europe. The increase or decrease of the incidence rate reflects the influence degree of the disease on the health of people, so that effective preventive and intervention measures are sought to delay the occurrence and development of diabetes complications, reduce the pain and disease burden of patients and improve the health level of the whole human, and become important health issues facing the world.
The golden plum (Potentilla fruticosa.L) family of shrubs, up to 0.51 meters, is multi-branched. The branches are reddish brown, and the young is long and soft. The feathered compound leaf has 5 (3) lobules, long, inverted egg-shaped long round or egg-shaped technical needle, is 0.72 cm long, has flat or slightly reversely rolled edges, has sharp tips or blunt tips at the front ends, has yellow wedge-shaped basal parts and has wide inverted oval shapes. The application discloses a method for preparing a Chinese medicinal composition, which is characterized in that a clematis fruit is nearly oval in shape, a cooked product is brown and has a length of about 15 mm, the Chinese medicinal composition is produced in grasslands, gravel slopes, irrigated places and the like, is produced in Qinghai, tibet Sichuan and the like, is a common Tibetan medicinal material, is recorded in the Tibetan medicine named Banna, and mainly treats dyspepsia and encephalopathy, charcoal of the Chinese medicinal composition can be yellow water, chemical components such as flavonoids, tannins, organic acids, phenolic acids and the like are mainly contained in the Chinese medicinal composition in modern researches, pharmacological researches prove that the Chinese medicinal composition has the effects of resisting bacteria, resisting oxidation, enhancing immunity and the like, and the extract of the Chinese medicinal composition has the high-efficiency alpha-glucosidase and aldose reductase inhibition effect and can effectively reduce the blood sugar of type 2 diabetes. The golden plum monomer compound has higher alpha-glucosidase inhibitory activity for the first time. Alpha-glucosidase (AG) is a class of disaccharide hydrolase distributed on the surface of the micro-chorion of the intestinal epithelium mucosa, including maltase, isomaltase, sucrase, trehalase, and the like. The enzyme can convert disaccharide taken into human body into monosaccharide easy to be absorbed by human body, and has important effect on catabolism of sugar. The alpha-glucosidase inhibitor is a medicament for treating diabetes mellitus, which can reduce postprandial blood sugar by competitive inhibition with AG.
The prior art has the problems that: the most commonly used alpha-glucosidase inhibitor hypoglycemic drug acarbose at present can effectively control diabetes and reduce blood sugar, but is applicable to people with limited crowds, is not applicable to people taking high-protein and high-fat foods such as meat, eggs and the like as staple foods, is slowly decomposed and absorbed in small intestines, prolongs the residence time, increases gas production through glycolysis of intestinal bacteria, can cause abdominal distension, abdominal pain, diarrhea and the like, and is not applicable to people with lower income due to higher price of acarbose. Other hypoglycemic drugs also have various degrees of side effects and social problems.
Disclosure of Invention
The key technical problem to be solved by the application is to provide an alpha-glucosidase inhibitor extracted from the golden plum and verify the inhibition activity of the alpha-glucosidase inhibitor.
In order to solve the technical problems, the application adopts the following technical scheme:
1. an alpha-glucosidase inhibitor comprising:
(1) quercetin-7-O-beta-D-glucuronic acid;
(2) Gallic acid;
(3) 3,4, 6-tri-O-galloyl- β -D-glucose;
(4) quercetin-7-O- β -D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl- β -D-glucose.
2. An alpha-glucosidase inhibitor comprising: quercetin-7-O-beta-D-glucuronic acid; gallic acid; 3,4, 6-tri-O-galloyl- β -D-glucose; the mass ratio of the three components is 3:1:3 or 3:2:2 or 2:1:2.
3. A method for preparing an alpha-glucosidase inhibitor, comprising the steps of:
(1) Extracting: weighing 500g of dried golden plum leaves in the shade, sieving with a 40-mesh sieve, carrying out reflux extraction with 70% ethanol for 3 times at 80 ℃ in a feed-liquid ratio of 1:20g/mL, each time for 2 hours, combining the extracting solutions, and concentrating to obtain 0.6L of crude extract.
(2) Separating: the crude extract is put on macroporous adsorption resin (AB-8), and is eluted by 10L volume of methanol with the mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%, and the collected eluent is concentrated and dried by rotary evaporation. High performance liquid chromatograph, column model: welch Ultimate MB-C18, column temperature 30 ℃, mobile phase: 99% ultrapure water, methanol, detection conditions: 5-30% methanol, 60min, detection wavelength 254nm, 280nm and 360nm. The eluent eluted by macroporous adsorption resin is filtered by an organic filter membrane with the thickness of 0.25 mu m to prepare samples, the sample injection amount is 10 mu L, and the sample injection amounts of quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose reference mother liquor are 5 mu L respectively. The detection proves that the 20% methanol elution part is the eluent containing the target component, and the eluent containing the target component is concentrated to constant weight in water at 80 ℃ to obtain the enrichment substance containing the target component.
(3) Preparation: the enrichment containing target components is processed by a preparative liquid chromatograph, a chromatographic column is DAC-HB50, mobile phase methanol is adopted, the column temperature is 30 ℃, the mass concentration of the mobile phase methanol (A) -2% glacial acetic acid (B) is 45%, the methanol is eluted at equal degree, the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 3mL, the repeated sample injection mode is adopted, the sample injection interval is 50min, the preparation liquid is collected, and the detection method is the same as (2), so that the compound 1 quercetin-7-O-beta-D-glucuronic acid monomer is 1.6877g, and the purity is 97.6516%.
Removing quercetin-7-O-beta-D-glucuronic acid component from the concentrate of the collected target components, and purifying by a rapid purification system, wherein a preparative chromatographic column is adopted: the method comprises the steps of (1) performing isocratic elution on a mobile phase of methanol, 1% glacial acetic acid and 20% methanol at a column temperature of 30 ℃, wherein the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 1.5mL, adopting a repeated sample injection mode, the sample injection interval is 30min, concentrating the collected peak preparation liquid, detecting by using a high performance liquid chromatograph, and the model of the column: platisil ODS, C18 column 4.6X250 mm,5 μm column temperature 30 ℃, mobile phase: 1% glacial acetic acid and acetonitrile, the flow rate of a mobile phase is 1mL/min, and the detection conditions are as follows: 20-35% acetonitrile, 30min, detection wavelength 254nm, 280nm and 360nm. The eluent eluted by the macroporous adsorption resin is filtered by an organic filter membrane with the thickness of 0.25 mu m to prepare samples, and the sample injection amount is 10 mu L. The mass of the obtained compound 2 gallic acid is 18.6mg, the purity is 95.7021%, and the mass of the compound 3,4, 6-tri-O-galloyl-beta-D-glucose is 253.8mg, and the purity is 96.1672%.
4. A method for verifying the inhibitory activity of an α -glucosidase inhibitor, comprising: the method for measuring the in-vitro activity of the alpha-glucosidase is adopted to measure the enzyme inhibition activity. The specific measurement method is as follows:
AG inhibitor screening model principle: the AG catalyzes and hydrolyzes PNPG to generate p-nitrophenol (PNP), PNP has an absorption peak at the wavelength of 400nm, the output of PNG is measured, and the inhibition activity of the monomer compound of the golden plum on AG is calculated. The inhibition activity of the enzyme was measured using a fully automatic microplate reader, and the reaction was performed in an undetachable 96-well plate. The reaction system was 200. Mu.L: in 125. Mu.L phosphate buffer and 8.92X 10 -3 The PNPG 25. Mu.L was added at a concentration of 1.953X 10 -2 After 25. Mu.L of mg/mL sample and 0.05U/mL AG 25. Mu.L, the mixture was incubated at 37℃for 20min, absorbance was measured at 400nm using a fully automatic microplate reader, and distilled water was used as a blank instead of the enzyme solution. Each sample was subjected to 3 replicates and the average was taken. AG viability unit definition: the amount of PNG released per minute by enzymatic hydrolysis of PNPG (OD value) at 37℃pH 6.8. Inhibitor activity unit definition: inhibition required to reduce 1 enzyme activity unit under the same conditionsAmount of formulation. In order to eliminate the influence of the sample and the substrate PNPG on the measurement result, the background absorption values of the sample and the substrate need to be measured. Calibration was performed with 0.05mol/mL phosphate buffer instead of sample and substrate.
Inhibition ratio (%) = [ (A1-A3) - (A2-A4) ]/(A1-A3) ×100%
A1: original enzyme activity; a2: enzyme activity after inhibitor addition; a3: PNPG background; a4: sample background
The results show that: at a sample concentration of 1.25mg/mL, the inhibition rate of alpha-glucosidase of quercetin-7-O-beta-D-glucuronic acid was 94.67%, IC 50 0.259mg/mL; the alpha-glucosidase inhibition rate of 3,4, 6-tri-O-galloyl-beta-D-glucose is 96.34%, IC 50 0.01mg/mL; the inhibition rate of alpha-glucosidase of gallic acid is 67.19%, IC 50 Is 0.831mg/mL.
5. A film coated tablet for reducing postprandial blood glucose comprising: common film-coated tablet auxiliary material (100 mg)
6. A capsule for reducing postprandial blood glucose comprising: capsule auxiliary material (100 mg)
The beneficial effects are that:
the golden dew plum compound quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose have the effect of reducing blood sugar. To date, there are few reports of the isolation of active compounds from the golden plum. The application adopts macroporous adsorption resin and a liquid chromatograph to prepare and separate hypoglycemic active compounds of quercetin-7-O-beta-D-glucuronic acid, 3,4, 6-tri-O-galloyl-beta-D-glucose and gallic acid in the golden plum, and obtains the compound with higher purity, and further carries out glycosidase activity experiments on the compound, so that the result shows that the alpha-glucosidase inhibitor has high inhibition rate.
Drawings
Fig. 1 is a flow chart of the extraction process of the golden plum compound.
FIG. 2 is a high performance liquid chromatography of quercetin-7-O-beta-D-glucuronic acid; wherein, peak 1 in the figure is compound 1 (quercetin-7-O-beta-D-glucuronic acid), X axis: time (min), Y axis: compound content (mV), left peak is peak containing compounds 2, 3.
FIG. 3 is a chromatogram of gallic acid, 3,4, 6-tri-O-galloyl-beta-D-glucose rapid optimization system preparation; wherein, the 2 peak is compound 2 (gallic acid), and the 3 peak is compound 3 (3, 4, 6-tri-O-galloyl-beta-D-glucose) X axis: time (min), Y axis: compound content (mV), BC is pump, B pump is water, C pump is methanol (chromatographic purity).
FIG. 4 high performance liquid chromatography detection chart of quercetin-7-O-beta-D-glucuronic acid; wherein the peak is compound 1 (quercetin-7-O-beta-D-glucuronic acid), X axis: time (min), Y axis: compound content (mV).
FIG. 5 is a high performance liquid chromatography detection chart of gallic acid; wherein the peak represents compound 2 (gallic acid), X-axis: time (min), Y axis: compound content (mV).
FIG. 6 is a liquid chromatography detection chart of 3,4, 6-tri-O-galloyl-beta-D-glucose; wherein the peak represents compound 3 (3, 4, 6-tri-O-galloyl- β -D-glucose), X-axis: time (min), Y axis: compound content (mV).
FIG. 7 is a schematic diagram of the structure of the compound; wherein A is quercetin-7-O-beta-D-glucuronic acid, B is gallic acid, and C is 3,4, 6-tri-O-galloyl-beta-D-glucose.
Detailed description of the preferred embodiments
The methods and apparatus used in the following examples of the present application are conventional methods and apparatus unless otherwise specified; the equipment and the reagent are conventional equipment and reagents purchased by reagent companies. In order to make the objects, technical solutions and advantages of the present patent more apparent, the following detailed description of the present patent refers to the field of 'electric digital data processing'. Examples of these preferred embodiments are illustrated in the specific examples.
It should be noted that, in order to avoid obscuring the technical solutions of the present application due to unnecessary details, only the technical solutions and/or processing steps closely related to the solutions according to the present application are shown in the embodiments, and other details having little relation are omitted.
Example 1
This example provides a set of α -glucosidase inhibitors comprising:
(1) quercetin-7-O-beta-D-glucuronic acid;
(2) Gallic acid;
(3) 3,4, 6-tri-O-galloyl- β -D-glucose.
(4) quercetin-7-O- β -D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl- β -D-glucose.
Example 2
This example provides a method for extracting an α -glucosidase inhibitor of example 1 from a golden plum, comprising:
(1) Extracting: weighing 500g of dried golden plum leaves in the shade, carrying out reflux extraction for 3 times at 80 ℃ by using 70% ethanol according to the feed liquid ratio of 1:20g/mL, extracting for 2 hours each time, combining the extracting solutions, and concentrating to obtain 0.6L of crude extract.
(2) Separating: the crude extract was subjected to macroporous adsorption resin (AB-8), and eluted with 10L of methanol having a mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% by volume in this order, and subjected to a high performance liquid chromatograph (LC 3000 Anhui Instrument technologies Co., ltd.) to obtain a column type: welch Ultimate MB-C18 (4.6X25 mm,5 μm), column temperature 30 ℃, mobile phase: 99% ultrapure water (UP), methanol (AR), detection conditions: 5-30% methanol (AR), 60min, detection wavelength 254nm, 280nm and 360nm. The eluent eluted by macroporous adsorption resin is filtered by an organic filter membrane with the thickness of 0.25 mu m to prepare samples, the sample injection amount is 10 mu L, and the sample injection amounts of quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose reference mother liquor are 5 mu L respectively. The detection proves that the 20% methanol elution part is the eluent containing the target component, and the eluent containing the target component is concentrated to constant weight in water at 80 ℃ to obtain the enrichment substance containing the target component.
(3) Preparation: the enrichment containing target components is processed by a preparative liquid chromatograph (DAC-50), a chromatographic column is DAC-HB50, a mobile phase methanol is methanol (chromatographic purity), the mass concentration of the methanol is 45%, the methanol is eluted at equal degree, the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 3mL, a repeated sample injection mode is adopted, the sample injection interval is 50min, the preparation liquid is collected, and the detection method is separated from (2), so that the quercetin-7-O-beta-D-glucuronic acid (compound 1) monomer has the mass of 1.6877g and the purity of 97.6516%.
The concentrate from which the target components were collected was freed from the components of quercetin-7-O-beta-D-glucuronic acid by a rapid purification system (BUCHI Peveleris Prep) using a preparative chromatographic column: the method comprises the steps of (1) (10 mm multiplied by 250mm 5 um) Dubhe C18 (Jiangyin Hanbang), wherein the column temperature is 30 ℃, the mobile phase is methanol (chromatographic purity), 1% glacial acetic acid and 20% methanol, the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 1.5mL, the repeated sample injection mode is adopted, the sample injection interval is 30min, and the collected peak preparation solutions are concentrated to be tested. Column model using high performance liquid chromatograph (Agilent 1260, agilent, america): platisil ODS, C18 column 4.6X250 mm,5 μm) column temperature 30 ℃, mobile phase: 1% glacial acetic acid, acetonitrile (AR), mobile phase flow rate 1mL/min, detection conditions were: 20-35% Acetonitrile (AR), 30min, detection wavelength 254nm, 280nm and 360nm. The eluent eluted by the macroporous adsorption resin is filtered by an organic filter membrane with the thickness of 0.25 mu m to prepare samples, and the sample injection amount is 10 mu L.
Example 3
This example provides a method for verifying the inhibitory activity of an α -glucosidase inhibitor, comprising: the method for measuring the in-vitro activity of the alpha-glucosidase is adopted to measure the enzyme inhibition activity. The specific measurement method is as follows:
AG inhibitor screening model principle: the AG catalyzes and hydrolyzes PNPG to generate p-nitrophenol (PNP), PNP has an absorption peak at the wavelength of 400nm, the output of PNG is measured, and the inhibition activity of the monomer compound of the golden plum on AG is calculated. The inhibition activity of the enzyme was measured using a fully automatic microplate reader, and the reaction was performed in an undetachable 96-well plate. The reaction system was 200. Mu.L: in 125. Mu.L phosphate buffer and 8.92X 10 -3 The PNPG 25. Mu.L was added at a concentration of 1.953X 10 -2 After 25. Mu.L of mg/mL sample and 0.05U/mL AG 25. Mu.L, the mixture was incubated at 37℃for 20min, absorbance was measured at 400nm using a fully automatic microplate reader, and distilled water was used as a blank instead of the enzyme solution. Each sample was subjected to 3 replicates and the average was taken. AG viability unit definition: the amount of PNG released per minute by enzymatic hydrolysis of PNPG (OD value) at 37℃pH 6.8. Inhibitor activity unit definition: the amount of inhibitor required to reduce 1 enzyme activity unit under the same conditions. In order to eliminate the influence of the sample and the substrate PNPG on the measurement result, the background absorption values of the sample and the substrate need to be measured. Calibration was performed with 0.05mol/mL phosphate buffer instead of sample and substrate.
Inhibition ratio (%) = [ (A1-A3) - (A2-A4) ]/(A1-A3) ×100%
A1: original enzyme activity; a2: enzyme activity after inhibitor addition; a3: PNPG background; a4: sample background
The results show that: at a sample concentration of 1.25mg/mL, the inhibition rate of alpha-glucosidase of quercetin-7-O-beta-D-glucuronic acid was 94.67%, IC 50 0.259mg/mL; the alpha-glucosidase inhibition rate of 3,4, 6-tri-O-galloyl-beta-D-glucose is 96.34%, IC 50 0.01mg/mL; the inhibition rate of alpha-glucosidase of gallic acid is 67.19%, IC 50 Is 0.831mg/mL.
Example 4
The present example provides a method for validating inhibition activity of an α -glucosidase inhibitor formulation, comprising: the α -glucosidase inhibitor activity of compounds A, B and C in different proportions was determined using the method described in example 3, with the following results at a sample concentration of 1.25 mg/mL:
table 1 alpha-glucosidase inhibitor Activity of Compounds A, B and C in different proportions
| Numbering device | Mass ratio of the compounds (A: B: C) | Inhibition ratio (%) | IC 50 |
| 1 | 3:2:2 | 86 | 0.264 |
| 2 | 2:1:2 | 88 | 0.0125 |
| 3 | 1:3:2 | 75 | 0.783 |
| 4 | 1:2:3 | 73 | 0.945 |
| 5 | 1:1:1 | 85 | 0.283 |
| 6 | 2:3:3 | 78 | 0.569 |
| 7 | 3:3:1 | 69 | 1.250 |
| 8 | 2:2:1 | 80 | 0.315 |
| 9 | 3:1:3 | 97 | 0.01 |
Example 5
The embodiment provides a formula and a preparation method of a common film-coated tablet preparation for reducing postprandial blood sugar, wherein the common film-coated tablet preparation comprises the following components in parts by mass (100 mg):
the preparation method comprises the following steps: wet granulation tabletting and film coating processes are adopted. Firstly, three kinds of medicinal powder of quercetin-7-O-beta-D-glucuronic acid, 3,4, 6-tri-O-galloyl-beta-D-glucose and gallic acid are sieved by a 40-mesh sieve, then starch, a proper amount of talcum powder and ethanol are added for mixing, the ethanol is added in a plurality of times, and the mixture is granulated in a wet mixing granulator. The resulting granules were then dried in an oven (40 ℃ C., 4 h). And (3) finishing, sieving with a 20-mesh sieve, adding low-substituted hydroxypropyl cellulose, micro powder silica gel and the rest talcum powder, and tabletting by a single-punch tablet machine to obtain tablet cores. The tablet core is put into a coating pot to rotate, meanwhile, the solution of hydroxypropyl methyl cellulose is evenly sprayed, 40 DEG hot air is blown in to evaporate the solution, the tablet core is naturally placed at room temperature for 6-8 hours to solidify completely, and the tablet core is dried at 50 ℃ for 12-24 hours to remove the residual organic solvent.
Example 6
The embodiment provides a capsule preparation formula for reducing postprandial blood sugar and a preparation method, wherein the capsule preparation formula comprises the following components in parts by mass (100 mg):
| composition of the components | Raw materials/adjuvants | Quality of |
| Quercetin-7-O-beta-D-glucuronic acid | Raw materials | 3.0％ |
| 3,4, 6-tri-O-galloyl-beta-D-glucose | Raw materials | 1.0％ |
| Gallic acid | Raw materials | 3.0％ |
| Modified starch | Diluent agent | 10％ |
| Low substituted hydroxypropyl cellulose | Disintegrating agent | 6.5％ |
| Ethanol | Wetting agent | 75.0％ |
| Micro powder silica gel | Glidant | 0.5％ |
| Talc powder | Anti-sticking agent, lubricant | 1.0％ |
The preparation method comprises the following steps: the granulation process was the same as in example 5, and the granules were then filled into capsule shells No. 4 using a hard capsule filling machine.
The foregoing is merely illustrative of the embodiments of this application and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of the application, and it is intended to cover all modifications and variations as fall within the scope of the application.
Claims (1)
1. A method for preparing an α -glucosidase inhibitor, characterized in that the α -glucosidase inhibitor comprises: quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose, wherein the mass ratio of the quercetin to the gallic acid is one of 3:1:3, 3:2:2 or 2:1:2; the preparation method of the quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose comprises the following steps:
(1) Extracting: weighing 500g of dried golden plum leaves, sieving with a 40-mesh sieve, carrying out reflux extraction with 70% ethanol for 3 times at 80 ℃ in a feed-liquid ratio of 1:20g/mL, extracting for 2 hours each time, combining the extracting solutions, and concentrating to obtain 0.6L of crude extract;
(2) Separating: the crude extract is put on macroporous adsorption resin AB-8, and is eluted by 10L volume of methanol with the mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, the collected eluent is concentrated, evaporated and dried in a rotary way, and the high performance liquid chromatograph is used for column model: welch Ultimate MB-C18, column temperature 30 ℃, mobile phase: 99% ultrapure water, methanol, detection conditions: 5-30% methanol, 60min, detecting 254nm, 280nm and 360nm of eluent eluted by macroporous adsorption resin, preparing sample by using 0.25 μm organic filter membrane, wherein the sample injection amount is 10 mu L, the sample injection amount of quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose reference mother liquor is 5 mu L respectively, determining that 20% of methanol elution part is eluent containing target components by the detection, concentrating the eluent containing the target components in water at 80 ℃ to constant weight, and obtaining enrichment substance containing the target components;
(3) Preparation: the enrichment containing target components is processed by a preparative liquid chromatograph, wherein a chromatographic column is DAC-HB50, mobile phase methanol is adopted, the column temperature is 30 ℃, the mass concentration of the mobile phase methanol A and-2% glacial acetic acid B is 45%, the methanol is eluted at equal degree, the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 3mL, a repeated sample injection mode is adopted, the sample injection interval is 50min, the preparation liquid is collected, and the quercetin-7-O-beta-D-glucuronic acid monomer is obtained by the detection method (2), wherein the mass of the quercetin-7-O-beta-D-glucuronic acid monomer is 1.6877g, and the purity is 97.6516%;
removing quercetin-7-O-beta-D-glucuronic acid component from the concentrate of the collected target components, and purifying by a rapid purification system, wherein a preparative chromatographic column is adopted: the method comprises the steps of (1) performing isocratic elution on a mobile phase of methanol, 1% glacial acetic acid and 20% methanol at a column temperature of 30 ℃, wherein the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 1.5mL, adopting a repeated sample injection mode, the sample injection interval is 30min, concentrating the collected peak preparation liquid, detecting by using a high performance liquid chromatograph, and the model of the column: platisil ODS, C18 column 4.6X250 mm,5 μm column temperature 30 ℃, mobile phase: 1% glacial acetic acid and acetonitrile, the flow rate of a mobile phase is 1mL/min, and the detection conditions are as follows: 20-35% acetonitrile, 30min, detection wavelengths 254nm, 280nm and 360nm, and eluting with macroporous adsorption resin, wherein the eluent is subjected to sample preparation by using a 0.25 μm organic filter membrane, the sample injection amount is 10 mu L, and the quality of gallic acid is 18.6mg, the purity is 95.7021%, the quality of 3,4, 6-tri-O-galloyl-beta-D-glucose is 253.8mg, and the purity is 96.1672%.
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