Method for rapidly screening and analyzing alpha-glucosidase inhibitor in traditional Chinese medicine

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction and analysis, and particularly relates to a method for rapidly screening and analyzing an alpha-glucosidase inhibitor in traditional Chinese medicine. According to the method for rapidly screening and analyzing the alpha-glucosidase inhibitor in the traditional Chinese medicine, affinity ultrafiltration screening of the alpha-glucosidase inhibitor in the traditional Chinese medicine and extracts of the traditional Chinese medicine is carried out by combining an affinity ultrafiltration screening technology with a liquid chromatography-mass spectrometry technology; the method comprises the following steps: adding alpha-glucosidase into a traditional Chinese medicine and an extract thereof, incubating, dissociating an obtained receptor-ligand compound, collecting dissociation liquid, carrying out LC-MS analysis, and carrying out data statistical analysis on compounds contained in an analysis sample and a blank sample, the active component capable of being specifically combined with the alpha-glucosidase is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and analysis of active ingredients of traditional Chinese medicines, and specifically relates to a method for rapid screening and analysis of alpha-glucosidase inhibitors in traditional Chinese medicines, in particular to a method for rapid screening and analysis of alpha-glucosidase inhibitors in astragalus. Background Art

[0002] Diabetes is an endocrine metabolic disorder caused by multiple causes and characterized by high blood sugar. It is caused by insufficient insulin secretion, insulin resistance, or both. Diabetes is widely prevalent worldwide. According to the latest data released by the International Diabetes Federation (IDF), as of 2021, there are as many as 537 million diabetic patients (20-79 years old) worldwide, which will reach 643 million by 2030 and 783 million by 2045. Therefore, the prevention and treatment of diabetes has become a global public health issue. Among diabetic patients, type 2 diabetes accounts for more than 90% of the total diabetic population, becoming the main type of diabetes.

[0003] α-Glucosidase is an enzyme that plays a key role in the body's metabolic process and is closely related to many diseases caused by metabolic disorders. At present, α-glucosidase inhibitors are one of the important drugs for the treatment of type 2 diabetes. Their target in the body is the α-glucosidase located on the brush border of the small intestinal mucosal cells. They can competitively inhibit or reversibly inhibit the activity of α-glucosidase on the brush border of the small intestine, thereby delaying the conversion of polysaccharides into monosaccharides, thereby delaying the increase in blood sugar after a meal. At present, the α-glucosidase inhibitor drugs that have been listed mainly include Miglitol, Acarbose, and Voglibose. While lowering blood sugar, they all produce certain side effects to a greater or lesser extent, such as gastrointestinal flatulence. Therefore, it is necessary to develop new α-glucosidase inhibitors with fewer side effects.

[0004] Traditional Chinese medicine and natural drug resources are a huge treasure trove of natural active ingredients, with the advantages of numerous active ingredients and few side effects. Screening for antidiabetic active ingredients targeting α-glucosidase from traditional Chinese medicine or natural drug resources with hypoglycemic efficacy (such as Astragalus) is conducive to the discovery of α-glucosidase inhibitors and the development of new hypoglycemic drugs. The traditional screening mode of active ingredients is mainly based on multiple extractions and separations. One is activity-oriented separation, that is, using in vitro efficacy evaluation to evaluate the activity of each stage of extraction and separation, tracking its active components, and then continuing to track the active components until the active monomer components are obtained; the other is to directly extract, separate, and identify the structure of the chemical components of traditional Chinese medicine, and then perform biological activity determination to determine the effective ingredients. The above two research strategies often have the problems of long experimental cycles and large workloads, and cannot achieve large-scale, high-throughput screening, and the trace active ingredients of this screening mode are easily lost during the separation process. It can be seen that screening α-glucosidase inhibitors from traditional Chinese medicine or natural drug resources with hypoglycemic efficacy requires certain research strategies.

[0005] At present, based on the affinity ultrafiltration screening technology of the biomacromolecule related to the disease as the target, the affinity ultrafiltration technology is coupled with liquid chromatography-mass spectrometry (LC-MS), and the small molecule active ingredients that can be combined with the target can be obtained by the affinity ultrafiltration technology, and then the rapid identification of the active ingredients is carried out by the LC-MS technology, which plays an important role in the early discovery of the active ingredients. However, in the traditional identification process, the conventional data processing method is to directly compare the difference of the chromatographic peak intensity of different compounds on the liquid chromatogram to determine which chromatographic peak is α-glucosidase inhibition, and then determine the structure with mass spectrum. This data processing method, especially when the chromatographic peaks overlap or some chromatographic peaks with little intensity are covered by the chromatographic peaks with high intensity, will produce the appearance of false positive / false negative results, affecting the result of screening. Therefore, how to quickly realize the screening of target substances, especially for the rapid and accurate analysis and identification of screening compounds, is still a problem to be solved in this area. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide a method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine and its extracts, wherein the method can rapidly screen α-glucosidase inhibitors from traditional Chinese medicine and its extracts by using affinity ultrafiltration screening combined with LC-MS technology;

[0007] The second technical problem to be solved by the present invention is to provide a method for rapidly screening and analyzing α-glucosidase inhibitors in Astragalus and its extracts.

[0008] In order to solve the above technical problems, the method of the present invention for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine comprises the following steps:

[0009] (1) Incubation: adding α-glucosidase to the Chinese herbal medicine and / or Chinese herbal medicine extract for incubation to obtain a sample solution containing α-glucosidase-ligand conjugates, while unbound small molecule compounds are released; and adding the Chinese herbal medicine and / or Chinese herbal medicine extract to an inactivated α-glucosidase solution for incubation to obtain a blank solution;

[0010] (2) Ultrafiltration-dissociation: The sample solution and the blank solution are subjected to ultrafiltration treatment respectively, the α-glucosidase-ligand conjugate is retained, and most of the incubation buffer and unbound small molecule compounds are removed; and the ultrafiltration product is collected and added with a dissociation agent for dissociation treatment, the α-glucosidase-ligand conjugate is dissociated to release the small molecule ligand compound, and the dissociation solution is collected to obtain the dissociation solution of the sample solution and the blank solution respectively;

[0011] (3) LC-MS analysis: drying the dissociation solution, collecting the residue and adding a constant volume solvent to dissolve, respectively obtaining an analytical sample of the sample solution and a blank sample of the blank solution; and performing LC-MS scanning and analysis on the analytical sample and the blank sample to obtain LC-MS data;

[0012] (4) Data statistical analysis: The LC-MS data were analyzed to obtain information about each compound in the analytical sample and the blank sample, respectively, to preliminarily screen the desired α-glucosidase inhibitors, and to perform structural identification and activity verification.

[0013] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in step (1), the temperature of the incubation step is 35-40° C., and the incubation time is 20-40 min;

[0014] Preferably, the Chinese medicine, Chinese medicine extract and / or the α-glucosidase are formulated into a solution;

[0015] Preferably, the dissolving solution of the traditional Chinese medicine, the traditional Chinese medicine extract and / or the α-glucosidase comprises an ammonium acetate buffer solution; preferably, the concentration of the ammonium acetate buffer solution is 5-15 mM, more preferably 10 mM;

[0016] Preferably, the solution concentration of the Chinese medicine and / or Chinese medicine extract is 3-8 mg / mL;

[0017] Preferably, the concentration of the α-glucosidase solution is 30-50 U / mL.

[0018] Preferably, in the incubation step, 100 μL of 5 mg / mL sample is selected and incubated with 100 μL of 40 U / mL α-glucosidase.

[0019] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in step (2):

[0020] The ultrafiltration step uses an ultrafiltration membrane with a low protein binding rate; preferably, the molecular retention capacity of the ultrafiltration membrane is 8-15Kda; and / or,

[0021] The dissociation agent comprises a methanol-water solution; preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v); and / or,

[0022] Preferably, the step (2) further comprises the step of rinsing the ultrafiltrate to remove as many unbound compounds as possible;

[0023] Preferably, the rinsing step uses an ammonium acetate buffer solution; preferably, the concentration of the ammonium acetate buffer solution is 5-15mM, more preferably 10mM;

[0024] Preferably, after the rinsing step, a centrifugal treatment step is also included, and the centrifugal speed is preferably 12000-14000r / min.

[0025] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in the step (3), the constant volume solvent includes a methanol-water solution;

[0026] Preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v).

[0027] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in step (4), the data statistical analysis step adopts Compound Discoverer TM Software for data processing;

[0028] Specifically, the data statistical analysis step includes:

[0029] (a) Importing collected LC-MS data into Compound Discoverer TM Software, aligning and extracting all compounds in the analytical sample and blank sample, respectively, to obtain the molecular weight, retention time and peak area information of each compound in the analytical sample and blank sample;

[0030] (b) calculating the peak area ratio of each compound in the analytical sample and the blank sample as the PAR value, and calculating the average PAR value of each compound, and performing a t-test on the peak areas of each compound in the analytical sample and the blank sample;

[0031] (c) Compounds with an average PAR value > 1 and p < 0.05 (n = 4) after t-test were preliminarily considered as active ingredients that can specifically bind to α-glucosidase.

[0032] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in step (a), the procedure of the extraction step is "input files→select spectra→align retention time→detect compounds→group compounds."; wherein,

[0033] In the input files step, four sample groups and a blank group of samples are imported;

[0034] In the select spectra step, the running time of the data to be processed and the positive and negative ion modes are selected for the next step of processing;

[0035] In the align retention time step, the maximum time drift is set to 0.2 min and the mass deviation is 5 ppm;

[0036] In the detect compounds step, all compounds in the raw data are extracted using the component elucidator algorithm, with the following parameters set: mass deviation: 5 ppm, signal-to-noise ratio threshold: 3, minimum peak intensity: 100,000, extracted ions: [MH] - and [M-H+HAc] - / [M+H] + and [M+Na] + , minimum element composition: CHO, maximum element composition: C 90 H 190 O 90 N 10 S 10 ;

[0037] In the group compounds step, the mass deviation is set to 5 ppm, and the preferred ion is [MH] - / [M+H] + .

[0038] Specifically, the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, in step (4), further comprises the step of performing MS / MS analysis on the initially screened active ingredients to identify and confirm their structures.

[0039] The present invention also discloses a method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus, the method comprising the steps of using Astragalus extract as a raw material and performing screening and analysis according to the method;

[0040] Preferably, the astragalus extract is an alcohol extract of astragalus, preferably a methanol extract; preferably, the methanol extract is an extract with 60-80% (v / v) methanol.

[0041] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus, in the LC-MS analysis step, the chromatographic conditions include:

[0042] Chromatographic column: Waters ACQUITY UPLC BEH C18 column, pre-column ACQUITY UPLC BEH C18VanGuard TM Pre-Column;

[0043] Mobile phase: A: water-0.1% formic acid, B: acetonitrile;

[0044] The elution procedures included: 0-7 min, 2% B-20% B; 7-10 min, 20% B-25% B; 10-20 min, 25% B-40% B; 20-25 min, 40% B-65% B; 25-30 min, 65% B-95% B;

[0045] Flow rate: 0.3 mL / min;

[0046] Column temperature: 35°C:

[0047] Sample pan temperature: 10°C;

[0048] Injection volume: 2 μL.

[0049] Specifically, in the method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus, in the LC-MS analysis step, a high-resolution mass spectrometer equipped with a heated ESI source is used for data detection, and a UHPLC-OrbitrapExploris 240 is used for sample data acquisition in negative ion mode, and the parameters are as follows:

[0050] Ion source voltage: 2500 V; sheath gas flow rate: 5.08 L / min; auxiliary gas flow rate: 9.37 L / min; ion transfer tube temperature: 320°C; nebulizer gas temperature: 350°C, m / z scanning range: 150-2000; internal calibration source Thermo Scientific EASY-ICTM was used for mass number calibration.

[0051] The invention also discloses the method for rapid screening and analysis of alpha-glucosidase inhibitors in traditional Chinese medicine or the application of the method for rapid screening and analysis of alpha-glucosidase inhibitors in astragalus in the field of drug screening and analysis.

[0052] The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine and extracts thereof of the present invention utilizes affinity ultrafiltration screening technology in combination with liquid chromatography-mass spectrometry technology to perform affinity ultrafiltration screening of α-glucosidase inhibitors, adds α-glucosidase to the traditional Chinese medicine and / or extracts thereof for incubation, dissociates the obtained receptor-ligand complex, collects the dissociation solution for LC-MS analysis, and performs data statistical analysis on the compounds contained in the analysis sample and the blank sample to obtain the active component that can specifically bind to the α-glucosidase.

[0053] The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine and its extracts of the present invention is to perform LC-MS analysis on the analysis samples and blank samples, and use Compound Discoverer TM The software extracts and aligns compounds, calculates the PAR value of each compound and the average PAR value, and performs a t-test on the peak area of ​​each compound in the sample group and the blank group. Compounds with an average PAR value > 1 and p < 0.05 (n = 4) after the t-test are screened as active ingredients of α-glucosidase inhibitors, and can quickly and accurately screen out active ingredients of α-glucosidase inhibitors.

[0054] The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine and its extracts of the present invention is particularly suitable for rapid screening of α-glucosidase inhibitors in astragalus extracts, effectively overcoming the drawbacks of the traditional screening mode of first separating and then detecting the activity for screening and identifying natural active substances from complex traditional Chinese medicine extracts, has the advantages of short experimental cycle and small workload, can realize large-scale and high-throughput screening, and has positive significance for drug development.

[0055] The method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus membranaceus of the present invention can realize rapid and accurate screening of α-glucosidase inhibitors from Astragalus membranaceus by optimizing the entire affinity ultrafiltration screening process, especially by optimizing the detection and analysis procedures of LC-MS technology, according to the characteristics of Astragalus membranaceus. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0057] Figure 1The total ion current graphs of the astragalus extract (Extract) before incubation in Example 1, the analysis sample (Sample) obtained by affinity ultrafiltration screening with the addition of α-glucosidase, and the blank sample (Control) obtained by affinity ultrafiltration screening with the addition of inactivated α-glucosidase;

[0058] Figure 2 The peak intensity differences and MS / MS spectra of the representative ligand compounds in Example 1 in the analysis sample (Sample) and the blank sample (Control) are shown. DETAILED DESCRIPTION

[0059] In the following embodiments of the present invention, the instruments, reagents, reference substances and materials involved include:

[0060] Vanquish with binary pump and heated column oven TM Flex UHPLC (Thermo Scientific, USA) system, Orbitrap Exploris 240 (Thermo Scientific, USA) high-resolution mass spectrometer equipped with a heated ESI source;

[0061] LC-MS grade formic acid was purchased from Fisher-Scientific (Fair Lawn, USA), LC-MS grade acetonitrile and methanol were purchased from Merck (Germany), and deionized water was purchased from Watsons; Astragalus root was provided by Beijing Tong Ren Tang Co., Ltd.; ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore); 10 mM ammonium acetate buffer solution (pH 6.86, Prilep); α-glucosidase (from Saccharomyces cerevisiae, EC number: 3.2.1.20, 100 units); centrifuge: Multifuge X Pro Series (Thermo Scientific).

[0062] Example 1

[0063] This example uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to screen α-glucosidase inhibitors from Astragalus.

[0064] Grind the Astragalus membranaceus into fine powder, weigh 15 g, add 150 mL 70% methanol for ultrasonic extraction, filter, and repeat the extraction twice. Combine the extracts and evaporate to dryness on a rotary evaporator at 40°C to obtain 2.3 g of Astragalus membranaceus sample.

[0065] Weigh 20 mg of the above-mentioned astragalus sample, add 4 mL of 10 mM ammonium acetate buffer solution (pH 6.86) to dissolve the sample, and filter through a 0.45 μm microporous filter membrane to obtain an astragalus extract stock solution with a concentration of 5 mg / mL.

[0066] Take α-glucosidase, dissolve it in 10 mM ammonium acetate buffer solution (pH 6.86), and prepare an α-glucosidase solution with a concentration of 40 U / mL.

[0067] The α-glucosidase solution with a concentration of 40 U / mL was boiled in boiling water for 10 minutes to obtain an inactivated α-glucosidase solution.

[0068] Take 100 μL of the above-mentioned Astragalus extract stock solution sample (5 mg / mL), place it in a 1.5 mL EP tube as the sample solution, then add 100 μL of 40 U / mL α-glucosidase, incubate at 37 ° C for 30 min, obtain α-glucosidase-ligand conjugate, and the unbound small molecule compounds are freed. The above-mentioned incubation product is transferred to an ultrafiltration centrifuge tube, centrifuged at 14000 r / min for 10 min, retain the α-glucosidase-ligand conjugate, and remove most of the incubation buffer and unbound small molecule compounds. Continue to add 250 μL of ammonium acetate buffer, centrifuge at 14000 r / min for 10 min to remove the rinse solution, and rinse 5 times in total to remove as many unbound compounds as possible. Subsequently, 100 μL of methanol: water (50:50, v / v, pH 3.30) was added for dissociation treatment to dissociate the α-glucosidase-ligand complex and release the small molecule ligand compound. The solution was centrifuged at 14000 r / min for 15 min and the dissociation was repeated 3 times to collect the dissociation solution of the sample solution.

[0069] Another 100 μL sample of the above-mentioned Astragalus extract stock solution (5 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace α-glucosidase, and the same parallel operation was performed to obtain a dissociation solution of the blank solution. The sample solution and the blank solution were respectively operated in parallel in quadruplicate.

[0070] The dissociation solutions of the sample solution and blank solution were blown dry with nitrogen, and the residue was redissolved with 50 μL of methanol: water (50:50, v / v) to obtain analytical samples and blank samples, respectively, and then subjected to LC-MS primary scanning and analysis.

[0071] In this embodiment, the LC-MS analysis conditions of the complex solution containing the ligand are as follows:

[0072] Chromatographic conditions:

[0073] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1×100mm, 1.7μm), pre-column ACQUITY UPLC BEH C18 VanGuard TM Pre-Column (2.1×5mm, 1.7μm);

[0074] Mobile phase: A: water (0.1% formic acid), B: acetonitrile;

[0075] Elution program: 0-7 min, 2% B-20% B; 7-10 min, 20% B-25% B; 10-20 min, 25% B-40% B; 20-25 min, 40% B-65% B; 25-30 min, 65% B-95% B;

[0076] Flow rate: 0.3 mL / min;

[0077] Column temperature: 35 °C;

[0078] Sample pan temperature 10°C;

[0079] Injection volume: 2 μL.

[0080] In this embodiment, the LC-MS analysis and detection step uses an OrbitrapExploris 240 (Thermo Scientific, USA) high-resolution mass spectrometer equipped with a heated ESI source for data detection, and uses UHPLC-Orbitrap Exploris 240 (Thermo Scientific, USA) for sample data acquisition, negative ion mode, ion source voltage: 2500V; sheath gas flow rate: 5.08L / min; auxiliary gas flow rate: 9.37L / min; ion transfer tube temperature: 320°C; nebulizer gas temperature: 350°C, m / z scanning range of 150-2000, internal calibration source Thermo Scientific EASY-IC TM Perform mass calibration.

[0081] In this example, the total ion currents of the astragalus extract before incubation (Extract), the analysis sample obtained by affinity ultrafiltration screening by adding α-glucosidase (Sample), and the blank sample obtained by affinity ultrafiltration screening by adding inactivated α-glucosidase (Control) are shown in Figure 1 shown.

[0082] In this embodiment, the above LC-MS data statistical analysis step was performed using Compound Discoverer TM (Thermo ScientificTM , version 3.2.0.421) software was used for data processing.

[0083] Import the four sample group data and four blank group data into Compound Discoverer TM Software was used to align and extract all compounds in the sample group and blank group samples.

[0084] The specific extraction process is "input files→select spectra→align retention time→detect compounds→group compounds." In the input files step, four sample group samples and four blank group sample data are imported; in the select spectra step, the entire run time (0-30min) and negative ion mode are selected; in the align retention time step, the mass deviation is set to 5ppm; in the detect compounds step, a series of parameters are set as follows: mass deviation 5ppm; S / N threshold: 3; minimum peak intensity 100000; extracted ions: [MH]-, [M-H+HAc]-; minimum element composition: CHO; maximum element composition: C 90 H 190 O 90 In the group compounds step, the mass tolerance was set to 5 ppm and the preferred ion was [MH] - .

[0085] In this embodiment, according to the above method and parameters, the compound extraction process is run to obtain the molecular weight, retention time and peak area of ​​each compound in each analytical sample and blank sample. The peak area ratio (PAR value) of each compound in the analytical sample and the blank sample is calculated, that is, the PAR value of each compound = the peak area of ​​the compound in the analytical sample / the peak area of ​​the compound in the blank sample, and the average PAR value of each compound is calculated (n = 4); the peak area of ​​each compound in the analytical sample and the blank sample is subjected to a t test. The compounds with an average peak area ratio of greater than 1 (n = 4) in the sample group and in the blank group and p < 0.05 (n = 4) after the t test are preliminarily regarded as active ingredients that can specifically bind to α-glucosidase.

[0086] In this example, compounds with average PAR>1 and p<0.05 (n=4) were selected as α-glucosidase inhibitors. Results A total of 8 α-glucosidase inhibitors were screened from the Astragalus extract, and the results are shown in Table 1 below.

[0087] Table 1 Screening results of α-glucosidase inhibitors from Astragalus extract

[0088]

[0089] a Average of four tests

[0090] In order to determine whether there is indeed a difference in the peak intensity of the above-screened compounds between the analytical samples and the blank samples, the ion chromatograms of the above-mentioned compounds in the analytical samples and the blank samples were extracted respectively. The results showed that the peak intensity of the above-mentioned compounds in the sample group was increased compared with that in the blank group, indicating the reliability of the data statistical processing method in this experiment. Figure 2 The peak intensity differences of some representative compounds in the sample group and the blank group are shown. The screened compounds were subjected to targeted MS / MS analysis to obtain their MS / MS spectra, and compared with the reference substances to determine their structures.

[0091] Example 2 Molecular docking experiment

[0092] This example verifies the activity of the components screened out in Example 1.

[0093] 1. Small molecule ligands

[0094] Based on the results of affinity mass spectrometry screening experiments, the above 8 chemical components were obtained, and their two-dimensional (2D) structures in SDF format were downloaded from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. The 2D structures of the compounds were converted to three-dimensional (3D) structures using ChemBio 3DUltra 14.0 software, and the small molecule compounds were optimized by MM2Minimize Energy force field. The converted molecules were saved in PDB format for future use.

[0095] 2. Preparation of receptor protein

[0096] The α-glucosidase crystal (PDBID: 5ZCB) was downloaded from the PDB database (https: / / www.rcsb.org / ) as the receptor model for computer molecular docking, and the water molecules and small ligand molecules were deleted using PyMol software.

[0097] 3. Molecular docking preparation

[0098] The receptor was hydrogenated using Autodock Tools software and converted into PDBQT format; the Ligand subroutine package was used to process small molecule compounds, calculate the number of rotatable bonds of the ligand, and save it in pdbqt format. AutoGrid was used to calculate the energy grid, and the grid box (gridbox) was set to 40nm×40nm×40nm, the grid spacing was 1nm, and the grid center coordinates were the default receptor active center. The compounds in the stored file were docked with protein molecules using Autodock Vina software, and the energy distribution results were viewed through the docking results to analyze the binding energy of the small molecule and the receptor. The binding site with the lowest binding energy was used as the optimal binding site for the compound and α-glucosidase, and finally the PYMOL software was used to draw the map.

[0099] In this example, the molecular docking results of each compound are shown in Table 2 below.

[0100] Table 2 Molecular docking results of the screened active ingredients and α-glucosidase

[0101] No. Compound Name Affinity activity (kcal / mol) 1 Formononetin -6.6 2 Soyasaponin I -8.0 3 Calycosin -6.3 4 Biochanin A -6.7 5 Astragaloside III -8.7 6 Isoastragaloside IV -8.2 7 Astragaloside II -7.5 8 Astragaloside IV -7.9 Acarbose -7.1

[0102] In the above molecular docking results, Affitnity (kcal / mol) < -5 is usually considered to have affinity activity. The Affitnity (kcal / mol) of the above compounds are all < -5. It can be seen that the active ingredients screened in this experiment all have the ability to inhibit α-glucosidase activity.

[0103] Example 3

[0104] This example verifies the α-glucosidase inhibitory activity of the above-screened active ingredients.

[0105] 1. Instruments, reagents, reference substances and materials

[0106] Microplate reader: Epoch 2 microplate reader (BioTek); 0.1 M phosphate buffer (pH 6.8, Prilep); p-Nitrophenyl-α-D-glucoside (pNPG, purity >99%, MacLean); α-glucosidase (derived from Saccharomyces cerevisiae, EC number: 3.2.1.20, 100 units); 0.1 M Na2CO3 (Sigma); Formononetin, Calycosin, Soyasaponin I, Biochanin A, Astragaloside III, Isoastragaloside IV, Astragaloside II, Astragaloside IV were all purchased from Shanghai Yuanye Co., Ltd., with a purity of >98%; 96-well plate (Corning).

[0107] 2. Solution preparation

[0108] The reference substance was prepared into solutions of different concentrations using 0.1 M phosphate buffer (pH 6.8), α-glucosidase was prepared into a solution with a concentration of 0.2 U / mL using 0.1 M phosphate buffer (pH 6.8), and pNPG was prepared into a solution with a concentration of 2 mM using 0.1 M phosphate buffer (pH 6.8).

[0109] 3. Activity verification experiment

[0110] In this example, the in vitro inhibitory activity verification experiment of α-glucosidase was performed according to the method in the references with appropriate modifications.

[0111] Take 40 μL of samples of different concentrations, add 40 μL of 0.2 U / mL α-glucosidase, incubate at 37 °C for 5 min, add 20 μL of 2 mM pNPG, incubate at 37 °C for 30 min, add 100 μL of 0.1 M Na2CO3 to terminate the reaction, immediately measure the absorbance of the solution at 405 nm, calculate the inhibition rate, and calculate the IC 50 The blank group was replaced with an equal amount of phosphate buffer instead of sample solution.

[0112] Inhibition rate (%) = (A a -A b ) / A a x 100%; where A a represents the absorbance value of the blank group, A b represents the absorption value of the sample group. The experimental results are shown in Table 3 below.

[0113] Table 3 Results of in vitro enzyme activity inhibition experiments of active ingredients screened from Astragalus:

[0114] No. Compound Name IC50(mM) 1 Formononetin 0.47 2 Soyasaponin I 0.86 3 Calycosin 1.05 4 Biochanin A 0.043 5 Astragaloside III UT 6 Isoastragaloside IV UT 7 Astragaloside II UT 8 Astragaloside IV UT Acarbose 5.25

[0115] UT has poor water solubility and its activity has not been tested.

[0116] As shown in Table 3 above, Formononetin (formononetin, IC 50 value is 0.47mM), Soyasaponin I (IC 50 value is 0.86mM), Calycosin (calycosin, IC 50 value is 1.05mM), Biochanin A (Biochanin A, IC 50 The inhibitory activity of α-glucosidase was stronger than that of acarbose (IC 50 value is 5.25mM); AstragalosideIII (astragaloside III), Isoastragaloside IV (isoastragaloside IV), Astragaloside II (astragaloside II), and Astragaloside IV (astragaloside I) are difficult to dissolve in water and cannot be verified for their α-glucosidase inhibitory activity in vitro. Instead, the molecular docking method was used to verify their activity. In addition, the molecular docking method was also used to verify the activity of other compounds.

[0117] Example 4

[0118] This example uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to screen α-glucosidase inhibitors from Astragalus.

[0119] Grind the Astragalus membranaceus into fine powder, weigh 15 g, add 150 mL of 60% methanol for ultrasonic extraction, filter, repeat the extraction twice, combine the extracts, and evaporate to dryness on a rotary evaporator at 40°C to obtain an Astragalus membranaceus sample.

[0120] Weigh 20 mg of the above-mentioned astragalus sample, add 4 mL of 10 mM ammonium acetate buffer solution (pH 6.86) to dissolve the sample, and filter through a 0.45 μm microporous filter to obtain an astragalus extract stock solution with a concentration of 5 mg / mL.

[0121] α-glucosidase was taken and dissolved in 10 mM ammonium acetate buffer solution (pH 6.86) to prepare an α-glucosidase solution with a concentration of 30 U / mL.

[0122] The α-glucosidase solution with a concentration of 30 U / mL was boiled in boiling water for 10 min to obtain an inactivated α-glucosidase solution.

[0123] Take 100 μL of the above-mentioned Astragalus extract stock solution sample (5 mg / mL), place it in a 1.5 mL EP tube as the sample solution, then add 100 μL of 30 U / mL α-glucosidase, incubate at 35 ° C for 40 min, obtain α-glucosidase-ligand conjugate, and the unbound small molecule compounds are freed. The above-mentioned incubation product is transferred to an ultrafiltration centrifuge tube, centrifuged at 14000 r / min for 10 min, retain the α-glucosidase-ligand conjugate, and remove most of the incubation buffer and unbound small molecule compounds. Continue to add 250 μL of ammonium acetate buffer, centrifuge at 14000 r / min for 10 min to remove the rinse solution, and rinse 5 times in total to remove as many unbound compounds as possible. Then, 100 μL of methanol: water (40:60, v / v) was added for dissociation treatment to dissociate the α-glucosidase-ligand complex and release the small molecule ligand compound. The sample was centrifuged at 14000 r / min for 15 min and the dissociation was repeated 3 times to collect the dissociation solution of the sample solution.

[0124] Another 100 μL sample of the above-mentioned Astragalus extract stock solution (5 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace α-glucosidase, and the same parallel operation was performed to obtain a dissociation solution of the blank solution. The sample solution and the blank solution were respectively operated in parallel in quadruplicate.

[0125] The dissociation solutions of the sample solution and blank solution were blown dry with nitrogen, and the residue was redissolved with 50 μL of methanol: water (40:60, v / v) to obtain analytical samples and blank samples, respectively, and then subjected to LC-MS primary scanning and analysis.

[0126] In this embodiment, the steps of LC-MS detection, analysis and data analysis and processing are the same as those in Embodiment 1.

[0127] Example 5

[0128] This example uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to screen α-glucosidase inhibitors from Astragalus.

[0129] Grind the Astragalus membranaceus into fine powder, weigh 15 g, add 150 mL 80% methanol for ultrasonic extraction, filter, repeat the extraction twice, combine the extracts, and evaporate to dryness on a rotary evaporator at 40°C to obtain an Astragalus membranaceus sample.

[0130] Weigh 20 mg of the above-mentioned astragalus sample, add 4 mL of 10 mM ammonium acetate buffer solution (pH 6.86) to dissolve the sample, and filter through a 0.45 um microporous filter membrane to obtain an astragalus extract stock solution with a concentration of 5 mg / mL.

[0131] α-glucosidase was taken and dissolved in 10 mM ammonium acetate buffer solution (pH 6.86) to prepare an α-glucosidase solution with a concentration of 50 U / mL.

[0132] The α-glucosidase solution with a concentration of 50 U / mL was boiled in boiling water for 10 minutes to obtain an inactivated α-glucosidase solution.

[0133] Take 100 μL of the above-mentioned Astragalus extract stock solution sample (5 mg / mL), place it in a 1.5 mL EP tube as the sample solution, then add 100 μL of 50 U / mL α-glucosidase, incubate at 40 ° C for 20 min, obtain α-glucosidase-ligand conjugate, and the unbound small molecule compounds are freed. The above-mentioned incubation product is transferred to an ultrafiltration centrifuge tube, centrifuged at 14000 r / min for 10 min, retain the α-glucosidase-ligand conjugate, and remove most of the incubation buffer and unbound small molecule compounds. Continue to add 250 μL of ammonium acetate buffer, centrifuge at 14000 r / min for 10 min to remove the rinse solution, and rinse 5 times in total to remove as many unbound compounds as possible. Then, 100 μL of methanol: water (60:40, v / v) was added for dissociation treatment to dissociate the α-glucosidase-ligand complex and release the small molecule ligand compound. The sample was centrifuged at 14000 r / min for 15 min and the dissociation was repeated 3 times to collect the dissociation solution of the sample solution.

[0134] Another 100 μL sample of the above-mentioned Astragalus extract stock solution (5 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace α-glucosidase, and the same parallel operation was performed to obtain a dissociation solution of the blank solution. The sample solution and the blank solution were respectively operated in parallel in quadruplicate.

[0135] The dissociation solutions of the sample solution and blank solution were blown dry with nitrogen, and the residue was redissolved with 50 μL of methanol: water (60:40, v / v) to obtain analytical samples and blank samples, respectively, and then subjected to LC-MS primary scanning and analysis.

[0136] In this embodiment, the steps of LC-MS detection, analysis and data analysis and processing are the same as those in Embodiment 1.

[0137] Example 6

[0138] This example uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to screen α-glucosidase inhibitors from Cornus officinalis.

[0139] The medicinal material of Cornus officinalis was crushed into fine powder, 150 g was weighed, and 1500 mL of 70% methanol was added for ultrasonic extraction. The extraction was repeated 3 times, and the extracts were combined and evaporated to dryness in a rotary evaporator at 40°C. The reagents were recovered and the obtained extract was dried under reduced pressure to obtain 74.94 g of Cornus officinalis sample.

[0140] Weigh 12 mg of Cornus officinalis sample, add 4 mL of ammonium acetate buffer solution (pH 6.86) to dissolve the sample, and filter through a 0.45 um microporous membrane to obtain a stock solution of Cornus officinalis extract with a concentration of 3 mg / mL.

[0141] Take α-glucosidase, dissolve it in ammonium acetate buffer solution (pH 6.86), and prepare an α-glucosidase solution with a concentration of 40 U / mL.

[0142] Preparation of inactivated α-glucosidase solution: Boil α-glucosidase solution with a concentration of 40 U / mL in boiling water for 10 minutes.

[0143] Take 100 μL of the above-mentioned Cornus officinalis extract stock solution sample (3 mg / mL), place it in a 1.5 mL EP tube as the sample solution, then add 100 μL of 40 U / mL α-glucosidase, incubate at 37 ° C for 30 min, obtain α-glucosidase-ligand conjugate, and the unbound small molecule compounds are freed. The above-mentioned incubation product is transferred to an ultrafiltration centrifuge tube, centrifuged at 14000 r / min for 10 min, retain the α-glucosidase-ligand conjugate, and remove most of the incubation buffer and unbound small molecule compounds. Continue to add 250 μL of ammonium acetate buffer, centrifuge at 14000 r / min for 10 min to remove the rinse solution, and rinse 5 times in total to remove as many unbound compounds as possible. Subsequently, 100 μL of methanol: water (50:50, v / v, pH 3.30) was added for dissociation treatment to dissociate the α-glucosidase-ligand complex and release the small molecule ligand compound. The solution was centrifuged at 14000 r / min for 15 min and the dissociation was repeated 3 times to collect the dissociation solution of the sample solution.

[0144] Another 100 μL sample of the above-mentioned Cornus officinalis extract stock solution (3 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace α-glucosidase, and the same parallel operation was performed to obtain a dissociation solution of the blank solution. The sample solution and the blank solution were respectively operated in parallel four times.

[0145] The dissociation solutions of the sample solution and blank solution were blown dry with nitrogen, and the residue was redissolved with 50 μL of methanol: water (50:50, v / v) to obtain analytical samples and blank samples, respectively, and then subjected to LC-MS primary scanning and analysis.

[0146] In this embodiment, the steps of LC-MS detection, analysis and data analysis and processing are the same as those in Embodiment 1.

[0147] Example 7

[0148] The screening method for α-glucosidase described in this example is the same as that in Example 1, the only difference is that the Astragalus extracts used in the sample solution and the blank solution are obtained in different ways.

[0149] In this embodiment, the astragalus extract raw material is extracted with n-hexane, chloroform and 70% ethanol in turn by hot reflux for 2.5 hours, the temperature is controlled at 75°C, and then the 70% ethanol extract is extracted with n-butanol (water-saturated solution), the organic phase solution is concentrated under reduced pressure to dryness, and finally, methanol is added for redissolution to obtain a 10 μg / mL sample solution.

[0150] In this embodiment, due to the different processing methods of the affinity screening sample solution, in the affinity screening, any small molecule compound that can react with α-glucosidase by affinity can be screened out. In this embodiment, compared with the affinity screening directly performed after dissolving the methanol extract of Astragalus membranaceus with a buffer in Example 1, the screening method in this embodiment requires multiple treatments, and the process operation is slightly complicated.

[0151] Example 8

[0152] The screening method of α-glucosidase described in this example is the same as that in Example 1, the only difference is that, during the affinity screening, the mixed solution is added to a 0.5 mL ultrafiltration centrifuge tube (Ultracel-PL regenerated cellulose ultrafiltration membrane, 10000 MW) in the ultrafiltration step, and centrifuged at room temperature at a speed of 10000 r / min for 10 min, and the filtrate is discarded.

[0153] In the scheme of this embodiment, in order to remove the liquid in the centrifuge tube as much as possible during centrifugation after the incubation product is transferred to the centrifuge tube, the speed of the centrifuge is controlled to 10000 r / min compared to the speed of 14000 r / min used in Example 1. The liquid residue in the centrifuge tube is slightly more, and the effect of removing the unbound compounds in the centrifuge tube is inferior. However, the scheme in Example 1 can completely remove the unbound small molecule compounds.

[0154] Example 9

[0155] The screening method for α-glucosidase described in this example is the same as that in Example 1, the only difference is that in the dissociation step, 200 μL CH3OH-H2O (1:1, v / v) (pH 3.30) is added, and centrifugation is performed at 10000 r / min for 15 min. The steps are repeated three times to dissociate the components bound to the α-glucosidase and collect the filtrate.

[0156] In this embodiment, the effect of the dissociation step is not as good as that in Example 1. In Example 1, a centrifugal speed of 14000 r / min is used, and the dissociated small molecule compounds are easier to be collected.

[0157] Comparative Example 1

[0158] The screening method of α-glucosidase in this comparative example is the same as that in Example 1, except that inactivated α-glucosidase is not added to the blank solution, and an equal amount of 10 mM ammonium acetate buffer solution (pH 6.86) is used to dissolve the inactivated α-glucosidase instead.

[0159] In this comparative example, more nonspecific adsorption components were screened out, and since inactivated α-glucosidase was added to replace α-glucosidase in Example 1, the influence of nonspecific adsorption on the experiment can be eliminated, ensuring the smooth progress of the experimental process.

[0160] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine, characterized in that: The steps include: (1) Incubation: adding α-glucosidase to the Chinese herbal medicine and / or Chinese herbal medicine extract for incubation to obtain a sample solution containing α-glucosidase-ligand conjugate; adding the Chinese herbal medicine and / or Chinese herbal medicine extract to an inactivated α-glucosidase solution for incubation to obtain a blank solution; (2) Ultrafiltration-dissociation: The sample solution and the blank solution are subjected to ultrafiltration treatment respectively, and the ultrafiltrate is collected and added with a dissociation agent for dissociation treatment, and the dissociation solution is collected to obtain the dissociation solution of the sample solution and the blank solution respectively; (3) LC-MS analysis: drying the dissociation solution, collecting the residue and adding a constant volume solvent to dissolve, respectively obtaining an analytical sample of the sample solution and a blank sample of the blank solution; and performing LC-MS scanning and analysis on the analytical sample and the blank sample to obtain LC-MS data; (4) Data statistical analysis: The LC-MS data were analyzed to obtain information about each compound in the analytical sample and the blank sample, respectively, to preliminarily screen the desired α-glucosidase inhibitors, and to perform structural identification and activity verification.

2. The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step (1), the temperature of the incubation step is 35-40° C., and the incubation time is 20-40 min; Preferably, the Chinese medicine, Chinese medicine extract and / or the α-glucosidase are formulated into a solution; Preferably, the dissolving solution of the Chinese medicine, Chinese medicine extract and / or the α-glucosidase comprises an ammonium acetate buffer solution; Preferably, the solution concentration of the Chinese medicine and / or Chinese medicine extract is 3-8 mg / mL; Preferably, the concentration of the α-glucosidase solution is 30-50 U / mL.

3. The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine according to claim 1 or 2, characterized in that: In the step (2): The ultrafiltration step uses an ultrafiltration membrane with a low protein binding rate; preferably, the molecular retention capacity of the ultrafiltration membrane is 8-15Kda; and / or, The dissociation agent comprises a methanol-water solution; preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v); and / or, Preferably, the step (2) further comprises the step of rinsing the ultrafiltrate; Preferably, the rinsing step uses an ammonium acetate buffer solution.

4. The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine according to any one of claims 1 to 3, characterized in that: In the step (3), the constant volume solvent includes a methanol-water solution; Preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v).

5. The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine according to any one of claims 1 to 4, characterized in that: In step (4), the data statistical analysis step uses Compound Discoverer TM Software for data processing; Specifically, the data statistical analysis step includes: (a) Importing collected LC-MS data into Compound Discoverer TM Software, extracting and aligning all compounds in the analytical sample and blank sample, respectively, to obtain the molecular weight, retention time and peak area information of each compound in the analytical sample and blank sample; (b) calculating the peak area ratio of each compound in the analytical sample and the blank sample as the PAR value, and calculating the average PAR value of each compound, and performing a t-test on the peak areas of each compound in the analytical sample and the blank sample; (c) Compounds with an average PAR value > 1 and p < 0.05 (n = 4) after t-test were preliminarily considered as active ingredients that can specifically bind to α-glucosidase.

6. The method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicine according to claim 5, characterized in that: In the step (a), the procedure of the extraction step is "input files→select spectra→alignretention time→detect compounds→group compounds."; wherein, In the input files step, four sample groups and a blank group of samples are imported; In the select spectra step, the running time of the data to be processed and the positive and negative ion modes are selected for the next step of processing; In the align retention time step, the maximum time drift is set to 0.2 min and the mass deviation is 5 ppm; In the detect compounds step, all compounds in the raw data are extracted using the component elucidator algorithm, with the following parameters set: mass deviation: 5 ppm, signal-to-noise ratio threshold: 3, minimum peak intensity: 100,000, extracted ions: [MH] - and [M-H+HAc] - / [M+H] + and [M+Na] + , minimum element composition: CHO, maximum element composition: C 90 H 190 O 90 N 10 S 10 ; In the group compounds step, the mass deviation is set to 5 ppm, and the preferred ion is [MH] - / [M+H] + .

7. A method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus, characterized in that: The method comprises the steps of using astragalus extract as raw material and performing screening and analysis according to the method of any one of claims 1 to 6; Preferably, the astragalus extract is an alcohol extract of astragalus; Preferably, the astragalus extract is a methanol extract of astragalus.

8. The method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus according to claim 7, characterized in that: In the LC-MS analysis step, the chromatographic conditions include: Chromatographic column: Waters ACQUITY UPLC BEH C18 column, pre-column ACQUITY UPLC BEH C18VanGuard TM Pre-Column; Mobile phase: A: water-0.1% formic acid, B: acetonitrile; The elution procedures included: 0-7 min, 2% B-20% B; 7-10 min, 20% B-25% B; 10-20 min, 25% B-40% B; 20-25 min, 40% B-65% B; 25-30 min, 65% B-95% B; Flow rate: 0.3 mL / min; Column temperature: 35°C: Sample pan temperature: 10°C; Injection volume: 2 μL.

9. The method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus according to claim 7 or 8, characterized in that: In the LC-MS analysis step, a high-resolution mass spectrometer equipped with a heated ESI source is used for data detection, using a negative ion mode with the following parameters: Ion source voltage: 2500 V; sheath gas flow rate: 5.08 L / min; auxiliary gas flow rate: 9.37 L / min; ion transfer tube temperature: 320°C; nebulizer gas temperature: 350°C, m / z scanning range: 150-2000; internal calibration source Thermo Scientific EASY-ICTM was used for mass number calibration.

10. Use of the method for rapid screening and analysis of α-glucosidase inhibitors in traditional Chinese medicines according to any one of claims 1 to 6 or the method for rapid screening and analysis of α-glucosidase inhibitors in Astragalus membranaceus according to any one of claims 7 to 9 in the field of drug screening and analysis.

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