CN112159443A - Alpha-glucosidase inhibitor extracted from flos Rosae Davuricae and its preparation method - Google Patents
Alpha-glucosidase inhibitor extracted from flos Rosae Davuricae and its preparation method Download PDFInfo
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Abstract
The invention provides an alpha-glucosidase inhibitor, which comprises quercetin-7-O-beta-D-glucuronic acid, gallic acid, 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 alpha-glucosidase inhibitor of the composition with the mass ratio of 3:1:3 has the highest inhibition rate. The invention relates to a medicine for reducing postprandial blood sugar of diabetics.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to an alpha-glucosidase inhibitor extracted from gynura segetum, and an extraction method and application thereof.
Background
The golden dew plum (pollen. L) is a shrub, up to 0.51 m, with multiple branches. The twigs are reddish brown and become long and soft when young. Feathery compound leaf, 5(3) small leaf, long, inverted egg-shaped long round or egg-shaped needle, length of 0.72 cm, flat or slightly reverse-rolled edge, single edge, sharp or blunt tip, yellow wedge-shaped base, wide inverted egg shape. The cleome fruit is nearly oval, the cooked product is brown and brown, has a length of about 15 mm, is born on grasslands, gravel slopes, irrigated areas and the like, is produced in Qinghai, Tibet Sichuan and the like, is a Tibetan medicine commonly used medicinal material, namely Tibet medicine Banna, Banna recorded in Jingzhu herbal, is mainly used for treating dyspepsia and encephalopathy, can dry yellow water due to carbon, and is found to mainly contain chemical components such as flavonoids, tannins, organic acids, phenolic acids and the like in modern research. The fact that the Jinlumei monomeric compound has higher alpha-glucosidase inhibitory activity is found for the first time. Alpha-glucosidase (AG) is a type of disaccharide hydrolase distributed on the surface of the intestinal epithelial mucosa, and includes maltase, isomaltase, sucrase, trehalase, and the like. The enzyme can convert disaccharide taken into human body into monosaccharide which is easy to be absorbed by human body, and has important effect on sugar catabolism. The alpha-glucosidase inhibitor is a medicine for reducing postprandial blood sugar and treating diabetes, and achieves the purpose of reducing the postprandial blood sugar by competitive inhibition with AG.
The prior art has the problems that: at present, acarbose, which is the most commonly used alpha-glucosidase inhibitor blood sugar-reducing drug, can effectively control diabetes and reduce blood sugar, but is limited in applicable population, is not suitable for the population taking high-protein and high-fat foods such as meat, eggs and the like as staple food, and is also suitable for the population with low income because of slow decomposition and absorption in the small intestine, prolonged retention time and increased gas production caused by glycolysis of intestinal bacteria, abdominal distension, bellyache, diarrhea and the like. Other hypoglycemic drugs also have side effects of varying degrees and social problems.
Disclosure of Invention
The key technical problem to be solved by the invention is to provide an alpha-glucosidase inhibitor extracted from the Jinlu plum and verify the inhibitory activity of the alpha-glucosidase inhibitor.
In order to solve the technical problems, the invention adopts the following technical scheme:
1. an α -glucosidase inhibitor, comprising:
(1) quercetin-7-O- β -D-glucuronic acid;
(2) gallic acid;
(3)3, 4, 6-tri-O-galloyl- β -D-glucose;
(4) two or three of quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose.
2. An α -glucosidase inhibitor, comprising: quercetin-7-O- β -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 preparation method of an alpha-glucosidase inhibitor comprises the following steps:
(1) extraction: weighing 500g of shade-dried leaf of Dendranthema indicum, sieving with a 40-mesh sieve, performing reflux extraction with 70% ethanol at 80 deg.C for 3 times (each time for 2 hr), mixing extractive solutions, and concentrating to obtain 0.6L of crude extract.
(2) Separation: loading the crude extract onto macroporous adsorbent resin (AB-8), sequentially eluting with 10L methanol with mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, concentrating the collected eluate, and rotary steaming and drying. Using a high performance liquid chromatograph, the column model: welch Ultimate MB-C18, column temperature 30 ℃, mobile phase: 99% ultrapure water, methanol, detection conditions: 5-30% methanol, 60min, and detecting wavelength of 254nm, 280nm and 360 nm. The eluate eluted by macroporous adsorbent resin is filtered through 0.25 μm organic filter membrane to obtain sample with sample amount of 10 μ L, and the sample amount of each of the mother liquor of quercetin-7-O- β -D-glucuronic acid, gallic acid, and 3,4, 6-tri-O-galloyl- β -D-glucose reference substance is 5 μ L. Determining 20% methanol elution part as eluate containing target components by the above detection, and concentrating the eluate containing target components in 80 deg.C water to constant weight to obtain enriched product containing target components.
(3) Preparation: passing the enrichment containing the target component through a preparative liquid chromatograph, wherein a chromatographic column is DAC-HB50, mobile phase methanol is adopted, the column temperature is 30 ℃, the mobile phase methanol (A) -2% glacial acetic acid (B) has the mass concentration of 45%, isocratic elution is carried out, the flow rate of the mobile phase is 50mL/min, the detection wavelengths are 254nm and 280nm, the sample injection amount is 3mL, a repeated sample injection mode is adopted, the sample injection interval is 50min, and the prepared solution is collected, wherein the detection method is the same as the detection method (2), so that the compound 1 quercetin-7-O-beta-D-glucuronic acid monomer is 1.6877g in mass and 97.6516% in purity.
Removing quercetin-7-O-beta-D-glucuronic acid component from the concentrate of the collected target component, and passing through a rapid purification system by adopting a preparative chromatographic column: dubhe C18, hanbang, yin, column temperature 30 ℃, methanol, 1% glacial acetic acid as mobile phase, 20% methanol isocratic elution, mobile phase flow rate of 50mL/min, detection wavelength of 254nm and 280nm, sample injection amount of 1.5mL, repeated sample injection mode, sample injection interval of 30min, concentration of collected peak preparation solution, detection by high performance liquid chromatography, column model: platisil ODS, C18 column 4.6X 250mm, 5 μm, column temperature 30 ℃, mobile phase: 1% glacial acetic acid, acetonitrile, a mobile phase flow rate of 1mL/min, and detection conditions of: 20-35% acetonitrile for 30min, and detection wavelengths of 254nm, 280nm and 360 nm. The eluent eluted by the macroporous absorption resin passes through an organic filter membrane with the thickness of 0.25 μm for sample preparation, and the sample injection amount is 10 μ L. The obtained compound 2, gallic acid (18.6 mg) and purity (95.7021%), respectively, and the compound 33, 4, 6-tri-O-galloyl-beta-D-glucose (253.8 mg) and purity (96.1672%).
4. A method for verifying the inhibitory activity of an alpha-glucosidase inhibitor, comprising: the enzyme inhibitory activity was measured by an alpha-glucosidase in vitro activity measurement method. The specific determination method is as follows:
AG inhibitor screenModel selection principle: and (3) carrying out AG catalytic hydrolysis on PNPG to generate p-nitrophenol (PNP), wherein the PNP has an absorption peak at the wavelength of 400nm, measuring the output of PNG, and calculating the inhibition activity of the chrysosporium odoratum monomer compound on AG. The enzyme inhibitory activity was measured using a fully automatic enzyme calibrator and the reaction was performed in non-detachable 96-well plates. The reaction system is 200 mu L: in 125. mu.L of phosphate buffer and 8.92X 10-3Adding PNPG 25 μ L with concentration of 1.953 × 10-2After 25 mu L of mg/mL sample and 25 mu L of AG 0.05U/mL, the sample is incubated at 37 ℃ for 20min, absorbance is measured at 400nm wavelength by using a full-automatic enzyme calibration instrument, and distilled water is used as a blank control instead of enzyme solution. Each sample was subjected to 3 replicates and averaged. AG activity unit definition: the amount of PNG released by enzymatic hydrolysis of PNPG (OD value) per minute was found to be within the range of pH 6.8 at 37 ℃. Definition of inhibitor activity units: the amount of inhibitor required to reduce 1 enzyme activity unit under the same conditions. To eliminate the effect of the sample and substrate PNPG on the assay results, it is necessary to determine background absorbance values for the sample and substrate. Calibration was performed using 0.05mol/mL phosphate buffer instead of sample and substrate.
The inhibition ratio (%) - (A1-A3) - (A2-A4) ]/(A1-A3) × 100%
A1: the original enzyme activity; a2: enzyme activity after adding inhibitor; a3: a PNPG background; a4: sample background
The results show that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin-7-O-beta-D-glucuronic acid is 94.67%, IC500.259 mg/mL; the alpha-glucosidase inhibition rate of 3,4, 6-tri-O-galloyl-beta-D-glucose is 96.34%, IC500.01 mg/mL; the alpha-glucosidase inhibition rate of gallic acid is 67.19%, IC50It was 0.831 mg/mL.
5. A film coated tablet for lowering postprandial blood glucose comprising: common film-coated tablet adjuvant (100mg)
6. A capsule for lowering postprandial blood glucose comprising: capsule adjuvant (100mg)
Has the advantages that:
in the application, the chrysolepsis 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. So far, few documents report the isolation and preparation of active compounds from the leaf of Dendrocalamus hamiltonii. The invention adopts macroporous adsorption resin and a liquid chromatograph to prepare and separate the hypoglycemic active compounds quercetin-7-O-beta-D-glucuronic acid, 3,4, 6-tri-O-galloyl-beta-D-glucose and gallic acid in the Jinlumei, obtains the compound with higher purity, and further performs glycosidase activity experiments on the compound, and the result shows that the alpha-glucosidase inhibitor has high inhibition rate.
Drawings
FIG. 1 is a flow chart of the process for extracting the compound of the Jinlumei.
FIG. 2 is a high performance liquid chromatography chromatogram of quercetin-7-O-beta-D-glucuronic acid; wherein, peak 1 in the figure is compound 1 (quercetin-7-O-beta-D-glucuronic acid), and X axis: time (min), Y-axis: the peak on the left side of the compound content (mV) is the peak containing compounds 2 and 3.
FIG. 3 is a chromatogram obtained by rapidly optimizing the system for preparing gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose; wherein, the peak 2 is compound 2 (gallic acid), the peak 3 is compound 3(3,4, 6-tri-O-galloyl-beta-D-glucose) X axis: time (min), Y-axis: compound content (mV), BC pump, B pump water, C pump methanol (chromatographically pure).
FIG. 4 is a high performance liquid chromatography assay of quercetin-7-O- β -D-glucuronic acid; wherein the peak is compound 1 (quercetin-7-O-beta-D-glucuronic acid), and the X axis: time (min), Y-axis: compound content (mV).
FIG. 5 is a high performance liquid chromatography assay of gallic acid; wherein the peak is representative of compound 2 (gallic acid), X-axis: time (min), Y-axis: compound content (mV).
FIG. 6 is a liquid chromatographic assay of 3,4, 6-tri-O-galloyl- β -D-glucose solution; wherein, the peak is represented by compound 3(3,4, 6-tri-O-galloyl-beta-D-glucose), X axis: time (min), Y-axis: compound content (mV).
FIG. 7 is a schematic diagram of the structure of a 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 invention
The methods and devices used in the following examples of the present invention are conventional methods and devices unless otherwise specified; the equipment and the reagent are all conventional equipment and reagents purchased by a reagent company. In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention is provided in connection with the specific embodiments. 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 invention with unnecessary details, only the technical solutions and/or processing steps closely related to the technical solutions of the present invention are shown in the embodiments, and other details that are not relevant are omitted.
Example 1
This example provides a group of α -glucosidase inhibitors, comprising:
(1) quercetin-7-O- β -D-glucuronic acid;
(2) gallic acid;
(3)3, 4, 6-tri-O-galloyl-beta-D-glucose.
(4) Two or three of quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose.
Example 2
This example provides a method for extracting α -glucosidase inhibitor of example 1 from ludew plum, comprising:
(1) extraction: weighing dried leaf of herba Dendrobii 500g in shade at a ratio of 1:20g/mL, extracting with 70% ethanol under reflux at 80 deg.C for 3 times, each for 2 hr, mixing extractive solutions, and concentrating to obtain 0.6L crude extract.
(2) Separation: loading the crude extract onto macroporous adsorbent resin (AB-8), sequentially eluting with 10L methanol (with mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), and performing high performance liquid chromatography (LC3000 Anhui Instrument science and technology, Inc.), column model: welch Ultimate MB-C18 (4.6X 25mm, 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 360 nm. The eluate eluted by macroporous adsorbent resin is filtered through 0.25 μm organic filter membrane to obtain sample with sample amount of 10 μ L, and the sample amount of each of the mother liquor of quercetin-7-O- β -D-glucuronic acid, gallic acid, and 3,4, 6-tri-O-galloyl- β -D-glucose reference substance is 5 μ L. Determining 20% methanol elution part as eluate containing target components by the above detection, and concentrating the eluate containing target components in 80 deg.C water to constant weight to obtain enriched product containing target components.
(3) Preparation: the enrichment containing the target component is processed by a preparative liquid chromatograph (DAC-50), the adopted chromatographic column is DAC-HB50, the mobile phase is methanol, the liquid phase is methanol (chromatographic purity), the mass concentration of the methanol is 45%, isocratic elution is carried out, 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 solution is collected, the detection method is the same as the step (2), and the quercetin-7-O-beta-D-glucuronic acid (compound 1) monomer with the mass of 1.6877g and the purity of 97.6516% is obtained.
Removing quercetin-7-O-beta-D-glucuronic acid component from the concentrate of the collected target component, and purifying by a rapid purification system (BUCHI Peveleries Prep) by using a preparative chromatographic column: dubhe C18(10mm multiplied by 250mm 5um) of Jiangyin Hanbang, the column temperature is 30 ℃, the mobile phase is methanol (chromatographic pure), 1% glacial acetic acid and 20% methanol for isocratic elution, the flow rate of the mobile phase is 50mL/min, the detection wavelength is 254nm and 280nm, the sample injection amount is 1.5mL, a repeated sample injection mode is adopted, the sample injection interval is 30min, and the collected preparation solution of each peak is concentrated and is to be detected. Using high performance liquid chromatography (Agilent 1260, Agilent, usa, Agilent), column model: platisil ODS, C18 column 4.6X 250mm, 5 μm), column temperature 30 ℃, mobile phase: 1% glacial acetic acid, Acetonitrile (AR), a mobile phase flow rate of 1mL/min, and detection conditions of: 20-35% Acetonitrile (AR), 30min, detection wavelength 254nm, 280nm and 360 nm. The eluent eluted by the macroporous absorption resin passes through an organic filter membrane with the thickness of 0.25 μm for sample preparation, and the sample injection amount is 10 μ L.
Example 3
This example provides a method for verifying the inhibitory activity of an α -glucosidase inhibitor, comprising: the enzyme inhibitory activity was measured by an alpha-glucosidase in vitro activity measurement method. The specific determination method is as follows:
principle of AG inhibitor screening model: and (3) carrying out AG catalytic hydrolysis on PNPG to generate p-nitrophenol (PNP), wherein the PNP has an absorption peak at the wavelength of 400nm, measuring the output of PNG, and calculating the inhibition activity of the chrysosporium odoratum monomer compound on AG. The enzyme inhibitory activity was measured using a fully automatic enzyme calibrator and the reaction was performed in non-detachable 96-well plates. The reaction system is 200 mu L: in 125. mu.L of phosphate buffer and 8.92X 10-3Adding PNPG 25 μ L with concentration of 1.953 × 10-2After 25 mu L of mg/mL sample and 25 mu L of AG 0.05U/mL, the sample is incubated at 37 ℃ for 20min, absorbance is measured at 400nm wavelength by using a full-automatic enzyme calibration instrument, and distilled water is used as a blank control instead of enzyme solution. Each sample was subjected to 3 replicates and averaged. AG activity unit definition: the amount of PNG released by enzymatic hydrolysis of PNPG (OD value) per minute was found to be within the range of pH 6.8 at 37 ℃. Definition of inhibitor activity units: the amount of inhibitor required to reduce 1 enzyme activity unit under the same conditions. To eliminate the effect of the sample and substrate PNPG on the assay results, it is necessary to determine background absorbance values for the sample and substrate. Calibration was performed using 0.05mol/mL phosphate buffer instead of sample and substrate.
The inhibition ratio (%) - (A1-A3) - (A2-A4) ]/(A1-A3) × 100%
A1: the original enzyme activity; a2: enzyme activity after adding inhibitor; a3: a PNPG background; a4: sample background
The results show that: when the sample concentration is 1.25mg/mL, the alpha-glucosidase inhibition rate of quercetin-7-O-beta-D-glucuronic acid is 94.67%, IC500.259 mg/mL; the alpha-glucosidase inhibition rate of 3,4, 6-tri-O-galloyl-beta-D-glucose is 96.34%, IC500.01 mg/mL; the alpha-glucosidase inhibition rate of gallic acid is 67.19%, IC50It was 0.831 mg/mL.
Example 4
The embodiment provides a method for verifying the inhibitory activity of an alpha-glucosidase inhibitor composition, which comprises the following steps: using the method described in example 3, the activity of α -glucosidase inhibitors from different proportions of compound A, B and C was determined as follows at a sample concentration of 1.25 mg/mL:
TABLE 1 Activity of alpha-glucosidase inhibitors for compounds A, B and C formulated in varying proportions
| Numbering | Mass ratio of compound (A: B: C) | Inhibition ratio (%) | |
| 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, and the common film-coated tablet preparation consists of the following mass components (100 mg):
the preparation method comprises the following steps: adopts wet granulation tabletting and film coating process. Firstly, the three medicinal powders of quercetin-7-O-beta-D-glucuronic acid, 3,4, 6-tri-O-galloyl-beta-D-glucose and gallic acid pass through a 40-mesh sieve, then starch, a proper amount of talcum powder and ethanol are added for mixing, the ethanol is added in portions, and the mixture is granulated in a wet mixing granulator. The resulting granules were then dried in an oven (40 ℃,4 h). And (3) finishing, sieving with a 20-mesh sieve, adding the low-substituted hydroxypropyl cellulose, the micropowder silica gel and the rest of talcum powder, and tabletting with a single-punch tablet machine to obtain the tablet core. Rotating the tablet core in a coating pan, spraying hydroxypropyl methylcellulose solution uniformly, blowing 40 ° hot air to evaporate the solution, standing at room temperature for 6-8 hr to solidify completely, and drying at 50 deg.C for 12-24 hr to remove residual organic solvent.
Example 6
The embodiment provides a formula and a preparation method of a capsule preparation for reducing postprandial blood sugar, which consists of the following components in percentage by mass (100 mg):
| composition (I) | Raw materials/auxiliary materials | 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 | 10％ |
| Low-substituted hydroxypropyl cellulose | Disintegrating agent | 6.5％ |
| Ethanol | Wetting agent | 75.0％ |
| Silica gel micropowder | Glidants | 0.5％ |
| Talcum powder | Anti-sticking agent and lubricant | 1.0％ |
The preparation method comprises the following steps: the granulation process was the same as the preparation process in example 5, and the granules were filled into capsule shell No. 4 using a hard capsule filling machine.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (9)
1. An alpha-glucosidase inhibitor, wherein the inhibitor is quercetin-7-O-beta-D-glucuronic acid.
2. An alpha-glucosidase inhibitor, wherein the inhibitor is gallic acid.
3. An alpha-glucosidase inhibitor, wherein the inhibitor is 3,4, 6-tri-O-galloyl-beta-D-glucose.
4. An α -glucosidase inhibitor, wherein the inhibitor comprises: quercetin-7-O-beta-D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl-beta-D-glucose.
5. The α -glucosidase inhibitor of claim 4, wherein the mass ratio of quercetin-7-O- β -D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl- β -D-glucose is 3:1: 3.
6. The α -glucosidase inhibitor of claim 4, wherein the mass ratio of quercetin-7-O- β -D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl- β -D-glucose is 3:2: 2.
7. The α -glucosidase inhibitor of claim 4, wherein the mass ratio of quercetin-7-O- β -D-glucuronic acid, gallic acid and 3,4, 6-tri-O-galloyl- β -D-glucose is 2:1: 2.
8. A preparation method of an alpha-glucosidase inhibitor comprises the following steps:
(1) extraction: weighing 500g of shade-dried leaf of Chrysanthemum indicum, sieving with a 40-mesh sieve, performing reflux extraction with 70% ethanol at 80 deg.C for 3 times (each time for 2 hr), mixing extractive solutions, and concentrating to obtain 0.6L of crude extract;
(2) separation: loading the crude extract onto macroporous adsorbent resin (AB-8), sequentially eluting with 10L methanol with mass concentration of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, concentrating the collected eluate, and rotary steaming and drying. Using a high performance liquid chromatograph, the column model: welch Ultimate MB-C18, column temperature 30 ℃, mobile phase: 99% ultrapure water, methanol, detection conditions: 5-30% methanol, 60min, and detecting wavelength of 254nm, 280nm and 360 nm. The eluate eluted by macroporous adsorbent resin is filtered through 0.25 μm organic filter membrane to obtain sample with sample amount of 10 μ L, and the sample amount of each of the mother liquor of quercetin-7-O- β -D-glucuronic acid, gallic acid, and 3,4, 6-tri-O-galloyl- β -D-glucose reference substance is 5 μ L. Determining 20% of the methanol eluate as eluate containing target components, and concentrating the eluate containing target components in 80 deg.C water to constant weight to obtain enriched substance containing target components;
(3) preparation: passing the enrichment containing the target component through a preparative liquid chromatograph, wherein a chromatographic column is DAC-HB50, mobile phase methanol is adopted, the column temperature is 30 ℃, the mobile phase methanol (A) -2% glacial acetic acid (B) has the mass concentration of 45%, isocratic elution is carried out, the flow rate of the mobile phase is 50mL/min, the detection wavelengths are 254nm and 280nm, the sample injection amount is 3mL, a repeated sample injection mode is adopted, the sample injection interval is 50min, and the prepared solution is collected, wherein the quality of the quercetin-7-O-beta-D-glucuronic acid monomer is 1.6877g, and the purity is 97.6516% as the detection method in the step (2);
removing quercetin-7-O-beta-D-glucuronic acid component from the concentrate of the collected target component, and passing through a rapid purification system by adopting a preparative chromatographic column: dubhe C18, hanbang, yin, column temperature 30 ℃, methanol, 1% glacial acetic acid as mobile phase, 20% methanol isocratic elution, mobile phase flow rate of 50mL/min, detection wavelength of 254nm and 280nm, sample injection amount of 1.5mL, repeated sample injection mode, sample injection interval of 30min, concentration of collected peak preparation solution, detection by high performance liquid chromatography, column model: platisil ODS, C18 column 4.6X 250mm, 5 μm, column temperature 30 ℃, mobile phase: 1% glacial acetic acid, acetonitrile, a mobile phase flow rate of 1mL/min, and detection conditions of: 20-35% acetonitrile for 30min, and detection wavelengths of 254nm, 280nm and 360 nm. The eluent eluted by the macroporous absorption resin passes through an organic filter membrane with the thickness of 0.25 μm for sample preparation, and the sample injection amount is 10 μ L. The obtained gallic acid has a mass of 18.6mg and a purity of 95.7021%, respectively, and 3,4, 6-tri-O-galloyl-beta-D-glucose has a mass of 253.8mg and a purity of 96.1672%.
9. Use of an α -glucosidase inhibitor as defined in any of claims 1-4 for lowering postprandial blood glucose in a diabetic patient.
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