A method for targeted fishing and screening of alpha-glucosidase inhibitors from dioscorea opposita

Abstract

This invention discloses a method for targeted screening and alpha-glucosidase inhibitors from yam, belonging to the field of bioanalytical technology. The method uses carboxyl-functionalized magnetic nanobeads to immobilize alpha-glucosidase. Using yam extract as a sample, co-incubation allows the inhibitor to specifically bind to the immobilized enzyme. After magnetic separation and elution, potential inhibitors are obtained, identified using UPLC-QTOF-MS / MS, and their activity is verified through in vitro enzyme inhibition experiments. Molecular docking technology is then used to analyze the site of action and the interaction forces. This invention preferably uses 80% ethanol as the extraction solvent. Six alpha-glucosidase inhibitors were screened from yam, among which diosgenin IV and diosgenin V showed superior activity compared to acarbose. This method is simple to operate, has high throughput, and high specificity, providing a new technical means for rapid screening of alpha-glucosidase inhibitors from natural products and offering important lead compounds for new drug development.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a method for targeted fishing and screening of α-glucosidase inhibitors from yam. Background Technology

[0002] Diabetes mellitus is a global chronic metabolic disease, with type 2 diabetes accounting for over 90% of cases. Postprandial hyperglycemia control is a key aspect of its management. Alpha-glucosidase inhibitors (AGIs) are oral hypoglycemic agents that competitively inhibit the activity of alpha-glucosidase on the brush border of the small intestine, thereby lowering postprandial blood glucose by delaying carbohydrate breakdown and glucose absorption. While currently used synthetic AGIs such as acarbose and voglibose are effective, they often cause gastrointestinal side effects such as bloating, flatulence, and diarrhea, which limits their clinical application. Therefore, developing novel AGIs with higher efficacy and fewer side effects has become an important direction in drug development.

[0003] Natural products, with their diverse structures and high safety profile, are a valuable resource for discovering novel lead compounds. Traditional medicinal plants have been shown to contain potential α-glucosidase inhibitory components. Yam (Dioscorea opposite Thunb.), a traditional Chinese medicine used in both food and medicine, possesses unique nutritional value and remarkable medicinal efficacy. Modern pharmacological studies have shown that yam extract has good hypoglycemic activity, suggesting that it may contain potential α-glucosidase inhibitors.

[0004] Currently, the screening of α-glucosidase inhibitors from traditional natural products mainly relies on methods such as crude extract preparation, in vitro inhibition model construction, and activity evaluation. For example, the traditional microplate in vitro inhibition method is cumbersome and time-consuming, not only inefficient but also difficult to miniaturize and automate, limiting its application in high-throughput screening. Furthermore, traditional methods cannot rapidly enrich and deeply analyze the binding characteristics of inhibitors to enzymes. Therefore, developing a new method for the rapid and effective discovery of α-glucosidase inhibitors directly from yam extracts has significant scientific value and industrialization prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a method for targeted fishing and screening of α-glucosidase inhibitors from yam, in order to solve the technical problems faced by existing α-glucosidase inhibitor fishing and screening methods, such as strong matrix interference, difficulty in capturing target inhibitors, low throughput, and single site of action.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] A method for targeted fishing and screening of α-glucosidase inhibitors from yam includes the following steps:

[0008] Step S1: Prepare immobilized enzyme, that is, use carboxyl-functionalized magnetic nanobeads to immobilize α-glucosidase through covalent bonds to obtain magnetic nanobead immobilized α-glucosidase.

[0009] Step S2: Extract yam samples to obtain yam extract;

[0010] Step S3: The immobilized enzyme obtained in step S1 is co-incubated with the yam extract obtained in step S2. The complex bound to the enzyme is collected by magnetic separation, and then elution is performed by adding elution buffer to obtain an elution buffer containing potential inhibitors.

[0011] Step S4: The compounds in the eluent obtained in step S3 were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS).

[0012] Step S5: Verify the in vitro α-glucosidase inhibitory activity of the compounds identified in Step S4 to determine the target compounds with inhibitory activity.

[0013] Furthermore, the specific operation of step S1 is as follows:

[0014] S11 carboxyl-functionalized magnetic nanobeads were washed with MES buffer and then dispersed in MES buffer;

[0015] S12 was added to EDC and Sulfo-NHS, and the reaction was carried out at room temperature in the dark with stirring to activate the carboxyl groups on the surface of the magnetic beads.

[0016] After washing away unreacted activating reagents in S13, the magnetic beads are redispersed in MES buffer.

[0017] Add α-glucosidase to S14, stir at room temperature in the dark to react, and fix the enzyme on the magnetic beads.

[0018] S15 was added with BSA and glycine, and the reaction was carried out in the dark with stirring to block unreacted active sites.

[0019] Immobilized enzyme was obtained after magnetic separation and purification of S16 and stored for later use.

[0020] Furthermore, the amount of carboxyl-functionalized magnetic nanobeads used was 50 mg, the pH of the MES buffer was 6.0, and the nanobeads were dispersed in 2 mL of MES buffer after each wash; the amount of EDC and Sulfo-NHS added was 50 mg each; the amount of α-glucosidase added was 2.5 mg; 200 µL of 10% BSA and 200 µL of 1 M glycine were added in the blocking step; the immobilized enzyme was stored in PBS containing 1 mg / mL BSA at 4 °C.

[0021] Furthermore, the extraction solvent for the yam samples was 80% ethanol.

[0022] Further, the specific operation of step S2 is as follows: after the yam sample is washed, dried, and sliced, it is ground into powder in liquid nitrogen; 10 g of yam powder is weighed, 200 mL of extraction solvent is added, and after shaking, ultrasonic extraction is performed, and the supernatant is collected by centrifugation; the ultrasonic extraction conditions are 20 kHz ultrasonication for 40 min, and the centrifugation conditions are 12000 r / min centrifugation for 10 min.

[0023] Further, the specific operation of step S3 is as follows: filter the yam extract, evaporate it to dryness, and reconstitute it with 80% ethanol; mix the buffer solution, reconstituted solution, and immobilized enzyme, and incubate them together at 37 °C with shaking for 2 h; after magnetic separation, discard the supernatant and wash the magnetic beads with pure water. Repeat the enrichment process 3 times, then elute with acetonitrile, and combine the eluents.

[0024] Further, in step S4, the detection conditions for UPLC-QTOF-MS / MS are as follows: chromatographic column: ACQUITY UPLC HSST3 column, 1.8 μm, 2.1 × 100 mm; mobile phase: water (A) - acetonitrile (B); elution program: 0-3 min 20% B, 3-5 min 50% B, 5-10 min 60% B, 10-15 min 70% B; flow rate: 0.2 mL / min; column temperature: 30 ℃; injection volume: 5 μL; detection wavelength: 274 nm; mass spectrometry uses electrospray ionization (ESI) positive and negative ion mode, with a scan range of m / z 50–1500.

[0025] Furthermore, in step S5, the in vitro α-glucosidase inhibitory activity was verified using the PNPG substrate method, with acarbose as a positive control, and the half-maximal inhibitory concentration (IC50) was calculated. 50 .

[0026] Furthermore, it also includes step S6: performing molecular docking of the active compound determined in step S5 with α-glucosidase, and analyzing its binding site and interaction force.

[0027] Furthermore, the α-glucosidase inhibitors screened by the method include yamin I, yamin III, yamin IV, yamin V, 2,4-dimethoxy-6,7-dihydroxyphenanthrene, and 3′-O-methylyamin III.

[0028] The present invention has the following beneficial effects:

[0029] This invention discloses a method for targeted fishing and screening of α-glucosidase inhibitors from yam. By comparing different extraction solvents, suitable extraction conditions were determined. α-glucosidase was immobilized using magnetic nanobeads as the target. Utilizing a strategy of specific binding of inhibitors to the enzyme's active site, combined with UPLC-QTOF-MS / MS identification and in vitro enzyme inhibition verification, six compounds with α-glucosidase inhibitory activity were successfully screened from yam. Among them, diosgenin IV and diosgenin V showed superior inhibitory activity compared to the clinically commonly used drug acarbose. Further molecular simulation studies showed that the binding pockets of these highly active compounds to α-glucosidase differed from those of acarbose, suggesting a potentially novel mechanism of action. This method is simple to operate, highly specific, and has high throughput, effectively overcoming the technical difficulties of traditional screening methods, such as strong matrix interference, difficulty in capturing target inhibitors, and single site of action. It provides efficient technical support for the discovery of novel hypoglycemic drugs from natural sources. Attached Figure Description

[0030] Figure 1 : Schematic diagram of the preparation process of immobilized enzymes. Figure 2 Transmission electron microscopy characterization of α-glucosidase labeled with magnetic nanoparticles. Figure 3 Comparison of extraction effects of different pretreatment solvents. Figure 4 Flowchart of targeted fishing and screening of α-glucosidase inhibitors. Figure 5 Chromatograms and MS of potential inhibitors 2 picture. Figure 6 : A molecular docking model diagram of the interaction between acarbose and α-glucosidase.

[0031] Figure 7 Molecular docking model diagram of yamin IV (A), yamin V (B) and α-glucosidase. Detailed Implementation

[0032] To better understand the present invention, specific embodiments are described in further detail below, but the scope of protection of the present invention is not limited to the following embodiments. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] Example 1: Preparation and characterization of immobilized α-glucosidase

[0034] This embodiment provides a method for preparing α-glucosidase immobilized with magnetic nanobeads, the specific steps of which are as follows:

[0035] (1) Weigh 50 mg of carboxyl-functionalized magnetic nanobeads (average particle size of about 50 nm, purchased from Beijing Zhongke Keyou Technology Co., Ltd.), place them on a magnetic separation rack, add 2 mL of MES buffer (0.1 M, pH 6.0), shake and wash 3 times, 1 min each time. After the last wash, discard the supernatant and redisperse the magnetic beads in 2 mL of MES buffer.

[0036] (2) Add 50 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mg of N-hydroxythiosuccinimide (Sulfo-NHS) to the above dispersion in sequence, and stir the mixture at room temperature in the dark for 1 h to activate the carboxyl groups on the surface of the magnetic nanobeads.

[0037] (3) After the reaction is complete, place the activated magnetic nanobeads on a magnetic separation rack and wash them three times with 2 mL of MES buffer each time to remove unreacted EDC and Sulfo-NHS. After the last wash, discard the supernatant and redisperse the magnetic beads in 2 mL of MES buffer.

[0038] (4) Add 2.5 mg of α-glucosidase (derived from Saccharomyces cerevisiae, enzyme activity ≥10 U / mg, purchased from Sigma-Aldrich) to the activated magnetic bead dispersion, and stir the reaction in the dark at room temperature for 1 h to fix the α-glucosidase on the surface of the nano magnetic beads through covalent bonds.

[0039] (5) To seal the unreacted active carboxyl sites on the surface of the magnetic beads, 200 µL of 10% (w / v) bovine serum albumin (BSA) and 200 µL of 1 M glycine solution were added to the reaction system, and the reaction was continued to be stirred in the dark for 1 h.

[0040] (6) After the reaction, the immobilized enzyme was separated and purified using a magnetic separator. The supernatant was discarded, and the enzyme was washed three times with PBS buffer (pH 7.4, containing 0.1% Tween-20). Finally, the immobilized enzyme was resuspended in 10 mL of storage buffer (PBS solution containing 1 mg / mL BSA, pH 7.4) and stored at 4 °C for later use.

[0041] (7) The morphology of the obtained immobilized enzyme was characterized using transmission electron microscopy (TEM). For example... Figure 2 As shown, the immobilized α-glucosidase nanoparticles exhibit a uniform spherical structure with good dispersibility and an average particle size of 52 ± 5 nm, indicating that α-glucosidase was successfully immobilized on the surface of the magnetic beads without causing significant aggregation.

[0042] Example 2: Screening and optimization of extraction solvents for yam samples

[0043] This embodiment optimizes the extraction solvent for active ingredients in yam, using total polyphenol content as the evaluation index. The specific steps are as follows:

[0044] (1) Take a fresh sample of iron yam, wash it with deionized water, let it air dry naturally, slice it, accurately weigh 10 g of slices and place them in a mortar, add liquid nitrogen to freeze quickly and grind them into fine powder;

[0045] (2) Take equal amounts of yam powder and add 200 mL of different extraction solvents at a material-to-liquid ratio of 1:20 (w / v). The extraction solvents include: water, methanol, 50% methanol (v / v), 80% methanol (v / v), ethanol, 50% ethanol (v / v), and 80% ethanol (v / v).

[0046] (3) After shaking the above mixture for 5 min, place it in an ultrasonic cleaner (20 kHz) for ultrasonic extraction for 40 min, and control the extraction temperature at 25 ± 2 ℃.

[0047] (4) After extraction, centrifuge at 12000 r / min for 10 min and collect the supernatant;

[0048] (5) The content of total polyphenols in each extract was determined by the Folin-Ciocalteu method: Take 0.5 mL of the appropriately diluted extract, add 2.5 mL of 10% Folin-Ciocalteu reagent, mix well and let stand for 5 min, then add 2.0 mL of 7.5% Na2CO3 solution, mix well and react in the dark for 30 min, measure the absorbance at a wavelength of 765 nm, and calculate the total polyphenol content by plotting a standard curve with gallic acid as the standard.

[0049] (6) For example Figure 3 As shown, the total polyphenol content in the extracts obtained from different extraction solvents varied significantly. The 80% ethanol extract had the highest total polyphenol content, followed by 80% methanol and water. Considering extraction efficiency, solvent safety, and compatibility with subsequent experiments, this invention selected 80% ethanol as the optimal extraction solvent.

[0050] Example 3: Targeted fishing of α-glucosidase inhibitors in yam extract

[0051] This embodiment uses immobilized α-glucosidase to target potential inhibitors in 80% ethanol extract of yam. The specific process is as follows: Figure 4 As shown:

[0052] (1) Take the supernatant extracted with 80% ethanol in Example 2, filter it through a 0.22 µm microporous membrane to remove particulate impurities, and collect the filtrate in a round-bottom flask;

[0053] (2) The filtrate was dried by low-pressure vacuum rotary evaporation at 45 °C. The residue was redissolved with 5 mL of 80% ethanol to obtain a yam extract solution.

[0054] (3) Add 400 µL of PBS buffer (pH 7.4), 300 µL of the above yam extract solution and 300 µL of the immobilized α-glucosidase suspension prepared in Example 1 to a 2 mL centrifuge tube in sequence. After mixing, place the tube in a constant temperature shaker and incubate it at 37°C in the dark for 2 h at a shaking speed of 200 r / min.

[0055] (4) After incubation, place the centrifuge tube on the magnetic separator rack and let it stand for 1 min to separate the immobilized enzyme from the solution. Carefully discard the supernatant (containing unbound sample components).

[0056] (5) Add 1 mL of pure water to the centrifuge tube, gently blow and wash the magnetic beads, discard the washing liquid after magnetic separation, and repeat the washing 3 times to remove the non-specific binding components of physical adsorption.

[0057] (6) Repeat the above operations (3) to (5) to enrich the yam extract three times;

[0058] (7) Add 500 µL of acetonitrile to the washed magnetic beads as the eluent, vortex for 2 min to dissociate the inhibitor that specifically binds to α-glucosidase and dissolve it in acetonitrile, and collect the eluent in a new centrifuge tube after magnetic separation.

[0059] Example 4: UPLC-QTOF-MS / MS identification of potential inhibitors

[0060] In this embodiment, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to identify compounds in the eluent collected in Example 3:

[0061] (1) The eluent obtained in Example 3 was filtered through a 0.22 µm microporous membrane and transferred to a vial for UPLC-QTOF-MS / MS analysis;

[0062] (2) UPLC conditions:

[0063] Chromatographic column: ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm, Waters).

[0064] Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile (containing 0.1% formic acid); Flow rate: 0.2 mL / min;

[0065] Column temperature: 30 ℃; injection volume: 5 μL; detection wavelength: 274 nm;

[0066] Elution gradient program: 0-3 min, 20% B; 3-5 min, 50% B; 5-10 min, 60% B; 10-15 min, 70% B;

[0067] (3) Mass spectrometry conditions:

[0068] Ion source: Electrospray ionization (ESI); Scanning mode: Full scan in both positive and negative ion modes; Nebulizer gas: High-purity nitrogen (purity ≥99.999%); Drying gas temperature: 320 ℃; Drying gas flow rate: 8 L / min; Sheath gas temperature: 350 ℃; Sheath gas flow rate: 11 L / min; Nebulizer pressure: 35 psi; Nozzle voltage: 3.5 kV (positive ion mode) / 3.0 kV (negative ion mode); Capillary voltage: 1000 V; Scanning range: m / z 50~1500; Secondary mass spectrometry collision energy: 20-40 eV (dynamically adjustable);

[0069] (4) Data acquisition and processing: SCIEX OS software was used for data processing. By comparing retention time, precise molecular weight and secondary mass spectrometry fragment information, the data was matched with a self-built natural product database and public databases such as PubChem and ChemSpider to preliminarily identify the structure of compounds.

[0070] (5) Result of the identification: such as Figure 5 As shown, six compounds were identified from the eluent, including yamin I, yamin III, yamin IV, yamin V, 2,4-dimethoxy-6,7-dihydroxyphenanthrene, and 3′-O-methylyamin III. These compounds are speculated to be potential α-glucosidase inhibitors.

[0071] Example 5: Verification of in vitro α-glucosidase inhibitory activity

[0072] In this embodiment, the PNPG substrate colorimetric method was used to evaluate the in vitro enzyme inhibitory activity of the compounds identified in Example 4, in order to confirm their α-glucosidase inhibitory effect:

[0073] (1) Take the reference standard of the compound identified in Example 4 (purity ≥98%, purchased from Chengdu EFA Biotechnology Co., Ltd.), dissolve it in methanol and prepare a series of concentration gradients (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM);

[0074] (2) Add 20 µL of control solutions of different concentrations and 80 µL of α-glucosidase solution (0.125 U / mL, prepared with PBS buffer) to a 96-well plate in sequence, and mix gently.

[0075] (3) Place the 96-well plate in a 37 ℃ constant temperature incubator for 10 min to allow the inhibitor to fully bind with the enzyme;

[0076] (4) Add 20 µL of 4-nitrophenyl-α-D-glucopyranoside (PNPG) substrate solution (final concentration 1 mM) to each well, mix well and continue to incubate at 37 °C in the dark for 10 min;

[0077] (5) After the reaction is complete, add 40 µL of 1 M Na2CO3 solution to each well to terminate the reaction, and immediately measure the absorbance (OD) at 405 nm using a microplate reader. 405 );

[0078] (6) The experiment set up a positive control group (acarbose, prepared in the same way with a series of concentrations), a negative control group (equal volume of PBS buffer) and a blank control group (without enzyme, containing equal volume of PBS buffer).

[0079] (7) The formula for calculating the inhibition rate is:

[0080]

[0081] In the formula: A0, A1, and A2 are the absorbance values ​​of the blank control group, the test sample group, and the positive control group, respectively.

[0082] (8) Using GraphPad Prism 8.0 software, with the logarithm of inhibitor concentration as the x-axis and the inhibition rate as the y-axis, a nonlinear regression curve (four-parameter logistic model) was fitted, and the half-maximal inhibitory concentration (IC50) was calculated. 50 );

[0083] (9) The results are shown in Table 1. Among the 6 compounds identified, diosgenin IV (IC) 50 = 0.15 µM), yam extract V (IC50) 50 The inhibitory activity of acarbose (IC50 = 0.22 µM) was superior to that of the positive control acarbose (IC50 = 0.22 µM). 50 = 0.26 µM), which has the potential to be a lead compound for novel α-glucosidase inhibitors.

[0084] Table 1. Half-maximal inhibitory concentrations (IC50) of six compounds against α-glucosidase 50 )

[0085] Acarbose (positive control) 0.26 Yam extract I 2.58 Yam Extract III 0.32 Yam extract IV 0.15 Yam Extract V 0.22 2,4-Dimethoxy-6,7-Dihydroxyphenanthrene 0.53 3′-O-methyl yamin III 0.46

[0086] Example 6: Molecular docking analysis of potential inhibition mechanism

[0087] This embodiment employs molecular docking technology to perform computer simulation analysis on the binding mode of the highly active compounds screened in Example 5 with α-glucosidase, in order to reveal their potential inhibitory mechanism:

[0088] (1) Protein and ligand preparation:

[0089] From the RCSB PDB database ( https: / / www.rcsb.org / Download the crystal structure of α-glucosidase (PDBID: 3WY1, derived from Saccharomyces cerevisiae, resolution 2.0 Å);

[0090] Use PyMol software to perform ligand removal and structural optimization, and save it in pdbqt format;

[0091] The structures of the ligand small molecules (acarbose, yamin IV, yamin V) were downloaded from the PubChem database, and after hydrogenation and charge adjustment, they were saved in pdbqt format.

[0092] (2) Molecular docking experiment

[0093] Integration software: AutoDock Vina 1.1.2;

[0094] Center coordinates of the grid box: x=23.5, y=11.2, z=14.8;

[0095] Box dimensions: x=30 Å, y=30 Å, z=30 Å, sufficient to cover the entire active site region;

[0096] Docking accuracy: Exhaust rate = 16, maximum output conformation number = 10;

[0097] The target compounds were attached to the active pockets of α-glucosidase to determine the optimal binding conformation;

[0098] (4) Interaction analysis:

[0099] The molecular docking results were visualized using PyMOL 2.5 software to show the three-dimensional spatial relationship between small molecules and amino acid residues in the active pocket.

[0100] Two-dimensional interaction diagrams were generated using Discovery Studio Visualizer software to analyze intermolecular forces such as hydrogen bonds and hydrophobic interactions.

[0101] (5) Acarbose docking results:

[0102] like Figure 6As shown, after acarbose enters the active pocket of α-glucosidase, it forms a stable hydrogen bond network with the following amino acid residues: Gly-94 (bond length 2.3 Å), Asp-48 (1.8 Å), Arg-457 (2.2 Å), Arg-456 (2.0 Å), and His-459 (2.7 Å). A total of 15 amino acid residues are involved in the interaction with acarbose, mainly through hydrogen bonds and van der Waals forces.

[0103] (6) Results of docking with highly active compounds:

[0104] like Figure 7 As shown, the molecular docking results of yamin IV (A), yamin V (B) with α-glucosidase indicate that:

[0105] Both enter a different binding pocket than acarbose, which is mainly composed of hydrophobic amino acid residues, making it more conducive to the binding of lipophilic small molecules;

[0106] Yam extract IV forms three hydrogen bonds with the active site (with Tyr-158, Gln-182 and Arg-315), while also exhibiting multiple hydrophobic interactions;

[0107] Yam extract V formed two hydrogen bonds with the active site (Thr-226 (2.0 Å) and Asn-301 (2.1 Å)).

[0108] Mechanism summary:

[0109] These results indicate that yamin IV and yamin V do not competitively bind to the classic binding site of acarbose, but rather exert their inhibitory activity by acting on an alternative hydrophobic cavity of α-glucosidase. This novel binding mode not only explains their higher inhibitory activity but also provides a structural basis for circumventing the gastrointestinal side effects of acarbose (which may be related to specific binding sites), and has significant medicinal chemical implications.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for targeted fishing and screening of α-glucosidase inhibitors from Dioscorea alata, characterized by, Includes the following steps: Step S1: Prepare immobilized enzyme, that is, use carboxyl-functionalized magnetic nanobeads to immobilize α-glucosidase through covalent bonds to obtain magnetic nanobead immobilized α-glucosidase. Step S2: Extract yam samples to obtain yam extract; Step S3: The immobilized enzyme obtained in step S1 is co-incubated with the yam extract obtained in step S2. The complex bound to the enzyme is collected by magnetic separation, and then elution is performed by adding elution buffer to obtain an elution buffer containing potential inhibitors. Step S4: The compounds in the eluent obtained in step S3 were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Step S5: Verify the in vitro α-glucosidase inhibitory activity of the compounds identified in Step S4 to determine the target compounds with inhibitory activity.

2. The method of claim 1, wherein, The specific operation of step S1 is as follows: S11. The carboxyl-functionalized magnetic nanobeads were washed with MES buffer and then dispersed in MES buffer. S12, add EDC and Sulfo-NHS, stir at room temperature in the dark to activate the carboxyl groups on the surface of the magnetic beads; S13. After washing away unreacted activating reagents, redisperse the magnetic beads in MES buffer. S14. Add α-glucosidase and stir at room temperature in the dark to fix the enzyme on the magnetic beads. S15. Add BSA and glycine, stir the mixture in the dark, and block the unreacted active sites. S16. After magnetic separation and purification, the immobilized enzyme is obtained and stored for later use.

3. The method of claim 2, wherein, In step S1: the amount of carboxyl-functionalized magnetic nanobeads used is 50 mg, the pH of the MES buffer is 6.0, and it is dispersed into 2 mL of MES buffer after each wash; the amount of EDC and Sulfo-NHS added is 50 mg each; the amount of α-glucosidase added is 2.5 mg; 200 µL of 10% BSA and 200 µL of 1 M glycine are added in the blocking step; the immobilized enzyme is stored in PBS containing 1 mg / mL BSA at 4 °C.

4. The method according to claim 1, characterized in that, In step S2, the extraction solvent for the yam sample is 80% ethanol.

5. The method according to claim 4, characterized in that, The specific operation of step S2 is as follows: After the yam sample is washed, dried, and sliced, it is ground into powder in liquid nitrogen; 10 g of yam powder is weighed, 200 mL of extraction solvent is added, and after shaking, ultrasonic extraction is performed. The supernatant is collected by centrifugation; the ultrasonic extraction conditions are 20 kHz ultrasonication for 40 min and centrifugation conditions are 12000 r / min centrifugation for 10 min.

6. The method according to claim 1, characterized in that, The specific operation of step S3 is as follows: filter the yam extract, evaporate it to dryness, and reconstitute it with 80% ethanol; mix the buffer solution, reconstituted solution and immobilized enzyme, and incubate them together at 37 °C with shaking for 2 h; after magnetic separation, discard the supernatant, wash the magnetic beads with pure water, repeat the enrichment 3 times, and then elute with acetonitrile, and combine the eluents.

7. The method of claim 1, wherein, In step S4, the detection conditions for UPLC-QTOF-MS / MS are as follows: chromatographic column: ACQUITY UPLC HSS T3 column, 1.8 μm, 2.1×100 mm; Mobile phase: Water (A) - Acetonitrile (B); Elution program: 0-3 min 20% B, 3-5 min 50% B, 5-10 min 60% B, 10-15 min 70% B; flow rate 0.2 mL / min, column temperature 30 ℃, injection volume 5 μL, detection wavelength 274 nm; mass spectrometry was performed in electrospray ionization positive and negative ion mode, with a scanning range of m / z 50–1500.

8. The method of claim 1, wherein, In step S5, the in vitro a-glucosidase inhibitory activity is verified using the p-nitrophenyl-a-D-glucopyranoside (PNPG) substrate method, with acarbose as the positive control, and the half-inhibitory concentration IC50is calculated. 50 .

9. The method of claim 1, wherein, It also includes step S6: performing molecular docking of the active compound determined in step S5 with α-glucosidase, and analyzing its binding site and interaction force.

10. The method of claim 1, wherein, The α-glucosidase inhibitors screened by the method include yamin I, yamin III, yamin IV, yamin V, 2,4-dimethoxy-6,7-dihydroxyphenanthrene, and 3′-O-methylyamin III.

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