Method for screening alpha-glucosidase inhibitor from pericarpium citri reticulatae and application of alpha-glucosidase inhibitor

Abstract

The invention belongs to the technical field of medicine and chemical analysis, and particularly relates to a method for screening an alpha-glucosidase inhibitor from pericarpium citri reticulatae and application of the alpha-glucosidase inhibitor. According to the method for screening the alpha-glucosidase inhibitor from the pericarpium citri reticulatae extract, alpha-glucosidase is taken as a target, and affinity ultrafiltration screening of the alpha-glucosidase inhibitor 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 pericarpium citri reticulatae extract, incubating, then dissociating an obtained receptor-ligand compound, collecting dissociation liquid, carrying out LC-MS (Liquid Chromatography-Mass Spectrometer) 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 medicine and chemical analysis, and particularly relates to a method for screening alpha-glucosidase inhibitors from tangerine peel and application thereof. 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] At present, during the treatment of diabetes, people with diabetes pay more attention to fasting blood sugar, but obviously not enough attention is paid to the control of postprandial blood sugar. Modern research has found that diabetes often develops from postprandial hyperglycemia. In addition, postprandial hyperglycemia is easily accompanied by cardiovascular and cerebrovascular and nervous system diseases, and may even cause ketoacidosis, leading to coma, and even life-threatening. Therefore, controlling postprandial hyperglycemia is one of the important measures to prevent diabetes, reduce complications and reduce mortality.

[0004] α-glucosidase is an enzyme that plays a key role in the body's metabolism and is closely related to many diseases caused by metabolic disorders, especially a key enzyme in the digestion of carbohydrates in the body. Studies have shown that α-glucosidase is one of the most important targets for controlling postprandial hyperglycemia. α-glucosidase is mainly located on the brush border of the small intestinal mucosal cells and distributed in various sections of the small intestine. It plays an important role in the catabolism of carbohydrates in the body, by promoting the decomposition of carbohydrates such as starch, sucrose, and maltose in intestinal food into monosaccharides, which are absorbed by the epithelial cells in the upper small intestine and enter the blood circulation, causing postprandial blood sugar to rise.

[0005] α-glucosidase inhibitors 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. In addition, food ingredients are decomposed and absorbed in the middle and upper parts of the small intestine. After using α-glucosidase inhibitors, chyme can enter the distal ileum, where there is a large amount of intestinal glucagon-like peptide-1 (GLP-1). Food stimulates the secretion of GLP-1 to increase, thereby stimulating the secretion of insulin, thereby reducing blood sugar concentration after a meal. At present, the α-glucosidase inhibitors commonly used in clinical practice mainly include Miglitol, Acarbose, and Voglibose. However, the above-mentioned drugs all produce certain side effects more or less while lowering blood sugar, such as gastrointestinal flatulence, etc. Therefore, the art expects to develop new α-glucosidase inhibitors with small side effects.

[0006] Traditional Chinese medicine resources are a huge treasure trove of natural active ingredients, with the advantages of diverse structures, obvious effects and few side effects. Therefore, it is an important way to develop new α-glucosidase inhibitors to find compounds with inhibitory activity against α-glucosidase from traditional Chinese medicine resources with hypoglycemic effects. Tangerine peel is the mature dried peel of the Rutaceae plant Citrus reticulata Blanco and its cultivated varieties. It has a long history of use in China. It is not only a traditional Chinese medicine in medical clinics, but also a traditional food for health care and diet therapy. Modern research has found that tangerine peel has anti-inflammatory, anti-Alzheimer's disease (AD) and Parkinson's disease (PD), anti-cancer, bone protection, anti-obesity, skin protection, and blood vessel regulation. In addition to the above effects, modern research has also found that tangerine peel extract can reduce the fasting blood sugar value of diabetic rats, indicating that tangerine peel has the effect of lowering blood sugar. Some researchers have found through further exploration that tangerine peel extract has a certain inhibitory effect on α-glucosidase, but the specific α-glucosidase inhibition situation is rarely reported. Summary of the invention

[0007] To this end, the technical problem to be solved by the present invention is to provide a method for screening α-glucosidase inhibitors from tangerine peel, wherein the method can quickly screen α-glucosidase inhibitors from tangerine peel and its extracts by using affinity ultrafiltration screening combined with mass spectrometry technology;

[0008] The second technical problem to be solved by the present invention is to provide the use of compounds in tangerine peel for preparing α-glucosidase inhibitors.

[0009] In order to solve the above technical problems, the method for screening α-glucosidase inhibitors from tangerine peel of the present invention comprises the following steps:

[0010] (1) Incubation: taking a dried tangerine peel extract and adding α-glucosidase to incubate, thereby obtaining a sample solution containing an α-glucosidase-ligand complex; taking another dried tangerine peel extract and adding an inactivated α-glucosidase solution to incubate, thereby obtaining a blank solution;

[0011] (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;

[0012] (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;

[0013] (4) Data statistical analysis: The LC-MS data are analyzed to obtain information about each compound in the analytical sample and the blank sample, respectively, and the desired α-glucosidase inhibitors are preliminarily screened.

[0014] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (1), the temperature of the incubation step is 35-40° C., and the incubation time is 20-40 min;

[0015] Preferably, the tangerine peel extract and / or the α-glucosidase are formulated into a solution;

[0016] Preferably, the solution for dissolving the tangerine peel extract and / or the α-glucosidase comprises an ammonium acetate buffer solution;

[0017] Preferably, the solution concentration of the tangerine peel extract is 5-15 mg / mL;

[0018] Preferably, the concentration of the α-glucosidase solution is 30-50 U / mL.

[0019] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (1), the dried tangerine peel extract includes an alcohol extract of dried tangerine peel or a water extract of dried tangerine peel;

[0020] Preferably, the tangerine peel extract is a methanol extract of tangerine peel.

[0021] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (2), the dissociating agent comprises a methanol-water solution;

[0022] Preferably, the volume ratio of the methanol-water solution is 40-60%:60-40% (v / v);

[0023] Preferably, the step (2) further comprises the step of rinsing the ultrafiltrate;

[0024] Preferably, the rinsing step uses an ammonium acetate buffer solution.

[0025] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, 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 screening α-glucosidase inhibitors from dried tangerine peel, in the step (3), in the LC-MS analysis step, the chromatographic conditions include:

[0028] Mobile phase: A: water-0.1% formic acid (v / v); B: acetonitrile;

[0029] 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;

[0030] Flow rate: 0.3 mL / min;

[0031] Injection volume: 2 μL;

[0032] Sample pan temperature: 10°C;

[0033] Column temperature: 35°C.

[0034] Specifically, in the method for screening α-glucosidase inhibitors from dried orange peel, in the step (3), in the LC-MS analysis step, the chromatographic column comprises a column connected to an ACQUITY UPLC BEH C18 VanGuard TM Waters ACQUITY UPLC BEH C18 column (2.1μ100mm, 1.7μm) with Pre-Column (2.1×5mm, 1.7μm).

[0035] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (3), in the LC-MS analysis step, a high-resolution mass spectrometer equipped with a heated ESI source is used for data collection, and a positive ion mode is used, and the parameters are as follows:

[0036] Ion source voltage: 3400V;

[0037] Sheath gas flow rate: 5.08 L / min;

[0038] Auxiliary gas flow rate: 9.37L / min;

[0039] Ion transfer tube temperature: 320°C;

[0040] Atomizing gas temperature: 350℃;

[0041] HCD collision energy (%): 30, 45, 60;

[0042] The m / z scanning range was 150-1000;

[0043] Internal Calibration Source Thermo Scientific EASY-IC TM Perform mass calibration.

[0044] Specifically, in the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (4), the data statistical analysis step comprises:

[0045] (a) importing the collected LC-MS data into statistical software, extracting and aligning all compounds in the analytical sample and blank sample, respectively, and obtaining the molecular weight, retention time and peak area information of each compound in the analytical sample and blank sample;

[0046] (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;

[0047] (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.

[0048] Preferably, the data statistical analysis step uses Compound Discoverer TM The software performs data processing, that is, imports the collected LC-MS data into Compound Discoverer TM Software processing.

[0049] Specifically, the method for screening α-glucosidase inhibitors from dried tangerine peel, in the step (4), further comprises the steps of structural identification and activity verification of the α-glucosidase inhibitors required in the preliminary screening.

[0050] Specifically, the present invention also discloses a use of a flavonoid compound for preparing an α-glucosidase inhibitor, wherein the flavonoid compound includes at least one of 5-hydroxy-3,7,3',4'-tetramethoxyflavone, demethylnobiletin, vecinin-3, naringin, 8-hydroxy-3,5,6,7,3',4'-hexamethoxyflavone, 4',5,,6,7-tetramethoxyflavone, hesperidin, neohesperidin, nobiletin, 3,5,6,7,3',4'-hexamethoxyflavone, sweet orange flavonoid, isoquercetin, hesperidin, naringenin, naringenin-7-O-glucoside, eriocitrin, vecinin-2, wild sumac glycoside, hesperidin or isoquercitrin.

[0051] The method for screening alpha-glucosidase inhibitors from tangerine peel extract of the present invention takes alpha-glucosidase as a target, uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to perform affinity ultrafiltration screening of alpha-glucosidase inhibitors, adds alpha-glucosidase to the tangerine peel extract for incubation, then 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 alpha-glucosidase.

[0052] The method for screening alpha-glucosidase inhibitors from tangerine peel extracts of the present invention comprises the following steps: performing LC-MS analysis on analysis samples and blank samples, extracting and aligning compounds using statistical software on the obtained data, calculating the PAR value and the average PAR value of each compound, performing a t test on the peak areas of each compound in a sample group and a blank group, screening compounds with an average PAR value greater than 1 and p less than 0.05 (n=4) after the t test as active ingredients of the alpha-glucosidase inhibitors, and being able to quickly and accurately screen out active ingredients of the alpha-glucosidase inhibitors.

[0053] The present invention screens α-glucosidase inhibitors from dried tangerine peel with hypoglycemic efficacy, and uses affinity ultrafiltration mass spectrometry technology to screen out 20 α-glucosidase inhibitors from dried tangerine peel, which are all identified as flavonoid compounds. The present invention further performs molecular docking on the screened active ingredients, tests the affinity of the screened active ingredients with α-glucosidase, and uses in vitro enzyme activity inhibition to verify the degree of inhibition of the screened active ingredients on α-glucosidase activity. It is predicted by the molecular docking method that they all have a certain affinity with α-glucosidase; it is found through in vitro enzyme activity inhibition experiments that they all have inhibitory activity on α-glucosidase, and even 6 ingredients have stronger inhibitory activity on α-glucosidase than the positive control drug acarbose. The flavonoid compounds involved in the present invention have positive significance for the development of new α-glucosidase inhibitor drugs. At the same time, dried tangerine peel is a Chinese medicine for both medicinal and edible purposes, and the flavonoid compounds screened by the present invention provide a reference for the development of dried tangerine peel functional foods.

[0054] The method for screening alpha-glucosidase inhibitors from tangerine peel extracts of the present invention can realize rapid and accurate screening of alpha-glucosidase inhibitors from tangerine peel by optimizing the entire affinity ultrafiltration screening process, especially by optimizing the detection and analysis procedures of LC-MS technology, aiming at the characteristics of tangerine peel, effectively overcoming the drawbacks of the traditional screening mode of first separating and then detecting the activity of natural active substances in screening and identifying natural active substances from complex 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 the development of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] 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:

[0056] Figure 1 The high-resolution mass spectrometry total ion current graphs of tangerine peel extract (Extract), tangerine peel blank sample (Control) and analytical sample (Sample) before incubation. DETAILED DESCRIPTION

[0057] In the following examples of the present invention, the information of instruments, reagents, reference substances, materials, etc. involved are as follows:

[0058] α-Glucosidase (from Saccharomyces cerevisiae, EC number: 3.2.1.20, 100 units);

[0059] p-Nitrophenyl-α-D-glucoside (pNPG, purity >99%, MacLean);

[0060] 10 mM ammonium acetate buffer solution (pH 6.86, Priligy);

[0061] 0.1 M phosphate buffered saline (pH 6.8, Priligy);

[0062] 0.1 M Na2CO3 (Sigma);

[0063] Deionized water was purchased from Watsons;

[0064] LC-MS grade formic acid was purchased from Fisher-Scientific (Fair Lawn, USA);

[0065] LC-MS grade acetonitrile and methanol were purchased from Merck (Germany);

[0066] Tangerine peel was provided by Beijing Tong Ren Tang Co., Ltd.

[0067] 96-well plate (Corning);

[0068] The reference substance acarbose was purchased from Shanghai Yuanye Co., Ltd. with a purity of >98%.

[0069] Example 1

[0070] In this example, affinity ultrafiltration mass spectrometry screening was performed on α-glucosidase inhibitors in dried orange peel.

[0071] Sample preparation

[0072] Take the dried tangerine peel raw material and grind it into powder (pass through an 80-mesh sieve), weigh 100.0g of the powder, add 1000mL70% methanol and mix thoroughly, carry out ultrasonic extraction at 25°C for 30min, filter and collect the extract after ultrasonication, repeat the above-mentioned extraction process 3 times for the separated filter residue, merge the collected extracts, and recycle the reagents on a rotary evaporator at 40°C. The obtained extract is dried under reduced pressure to obtain 44g of dried tangerine peel extract sample.

[0073] Weigh 10 mg of the above tangerine peel extract sample, add 1 mL of 10 mM ammonium acetate buffer solution (pH 6.86, Prilep) to dissolve the sample, and the obtained tangerine peel extract solution has a concentration of 10 mg / mL and is set aside.

[0074] Preparation of α-glucosidase solution

[0075] Take α-glucosidase and add 10 mM ammonium acetate buffer solution (pH 6.86) to fully dissolve it, and prepare an α-glucosidase solution with a concentration of 40 U / mL for use.

[0076] Take another appropriate amount of α-glucosidase solution with a concentration of 40 U / mL, boil it in boiling water for 10 min to obtain an inactivated α-glucosidase solution for later use.

[0077] Screening for α-glucosidase inhibitors

[0078] 100 μL of the above-prepared 10 mg / mL tangerine peel extract solution sample was mixed with 100 μL of 40 U / mL α-glucosidase solution, and incubated at 37° C. for 30 min to obtain an incubation solution containing an α-glucosidase-ligand complex and some unbound small molecule compounds. The above incubation solution was transferred to an ultrafiltration centrifuge tube and centrifuged at 14000 r / min for 10 min to retain the α-glucosidase-ligand complex and remove most of the buffer and unbound small molecule compounds.

[0079] The α-glucosidase-ligand complex was rinsed 5 times with 250 μL ammonium acetate buffer to remove unbound small molecules as much as possible, and centrifuged at 14000 r / min for 10 min after each rinse. The collected α-glucosidase-ligand complex was dissociated 3 times with 100 μL methanol-water solution (50:50, pH 3.30) to release small molecule ligands, and centrifuged at 14000 r / min for 15 min after each dissociation. All dissociation solutions were collected and combined, dried with nitrogen, and re-dissolved with 50 μL methanol-water solution (50:50) to obtain analytical samples.

[0080] Another 100 μL of the same tangerine peel extract sample solution (10 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace the α-glucosidase solution for incubation, and the same parallel operation was performed to obtain a blank sample.

[0081] The above analytical samples and blank samples were respectively processed in quadruplicate and subjected to LC-MS primary scanning analysis.

[0082] Example 2

[0083] This example is based on the LC-MS primary scanning analysis of the analytical sample and blank sample obtained in the aforementioned Example 1.

[0084] The LC-MS analysis and detection conditions are as follows: The chemical components in the above analytical samples and blank samples were quantified in a Vanquish equipped with a binary pump and a heated column oven. TM Flex UPLC (Thermo Scientific, USA) system and connected to ACQUITY UPLC BEH C18 VanGuard TM Good separation was obtained on a Waters ACQUITY UPLCBEH C18 column (2.1×100mm, 1.7um) with a Pre-Column (2.1×5mm, 1.7um).

[0085] In this embodiment, the chromatographic separation conditions are as follows:

[0086] Mobile phase: A: water (containing 0.1 v / v% formic acid), B: acetonitrile;

[0087] 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;

[0088] Flow rate: 0.3 mL / min;

[0089] Injection volume: 2 μL;

[0090] Sample pan temperature: 10°C;

[0091] Column temperature: 35°C.

[0092] This example is based on an Orbitrap Exploris 240 (Thermo Scientific, USA) high-resolution mass spectrometer equipped with a heated ESI source for data acquisition, positive ion mode, ion source voltage: 3400 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; collision energy (%): 30, 45, 60; m / z scanning range: 150-1000. Internal calibration source Thermo Scientific EASY-IC TM Perform mass calibration.

[0093] In this embodiment, the total ion current of the high-resolution mass spectrometry of the dried tangerine peel extract (Extract) before incubation, 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 is shown in Figure 1 shown.

[0094] Example 3

[0095] In this example, Compound Discoverer was used to analyze the LC-MS data obtained in Example 2. TM (ThermoScientific TM , version 3.2.0.421) software for data processing. It should be noted that other software in the field that can perform compound structure data analysis can be used for characterization. This example selects this software for analysis only as an exemplary implementation.

[0096] The first-level full-scan mass spectrometry data of the analytical sample and blank sample obtained in Example 2 were imported into Compound Discoverer TM Software (ThermoScientific TM , version 3.2.0.421) for data processing.

[0097] First, all compounds in the first-level full-scan mass spectrometry data of the analytical samples and blank samples were extracted and aligned. The specific extraction process is "input files→select spectra→align retention time→detect compounds→group compounds." In the input files step, the data of four sample groups and four blank groups were imported; in the select spectra step, the entire run time (0-30min) and the positive ion mode were selected; in the align retention time step, the mass deviation was set to 5ppm; in the detect compounds step, a series of parameters were set as follows: mass deviation 5ppm; S / N threshold: 3; minimum peak intensity 100000; extracted ions: [M+H] + ; Minimum element composition: CHO; Maximum element composition: C 90 H 190 O 90 In the group compounds step, ions with different adducting modes were combined and the mass deviation was set to 5 ppm.

[0098] According to the above method and parameters, the compound extraction process was run to obtain the molecular weight, retention time and peak area of ​​each compound in each analytical sample and blank sample, and then the peak area ratio (PAR value, i.e., sample group peak area / blank group peak area) and average peak area ratio (average PAR value) of each compound were calculated, and the peak area of ​​each compound in the sample group and blank group was tested by t-test. Finally, the compounds with average PAR value>1 and p<0.05 (n=4) were selected as α-glucosidase inhibitors. On this basis, the parent ions of the screened compounds were subjected to targeted MS / MS analysis, and the structure of the compound was determined by the obtained fragment ion information.

[0099] Based on the data obtained in Example 2 and through the above data statistical analysis, compounds with an average PAR value >1 and p<0.05 (n=4) were selected as α-glucosidase inhibitors. As a result, 20 flavonoid compounds were screened out, as shown in Table 1 below, and their fragment information was further obtained by high-resolution mass spectrometry (see Table 1 below) and the structure was determined by comparison with reference substances.

[0100] Table 1 Twenty α-glucosidase inhibitors screened from tangerine peel extract.

[0101]

[0102]

[0103] The structures of 20 compounds screened in this example are as follows:

[0104]

[0105] Example 4

[0106] In this example, biological activity evaluation was performed based on the aforementioned screened compounds, and molecular docking was performed based on α-glucosidase and the screened active ingredients.

[0107] The molecular docking method can predict the affinity of a compound to a target, so the molecular docking method can be used as one of the methods to verify whether the compound is a ligand for the target.

[0108] Among the active ingredients screened by affinity mass spectrometry, the compounds with clear structures were downloaded from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database, and their 2D structures were converted to three-dimensional (3D) structures using ChemBio 3D Ultra 14.0 software. After the small molecule compounds were optimized by MM2 Minimize Energy force field, they were saved in PDB format for future use. α-glucosidase crystals (PDB ID: 5ZCB) were downloaded from the PDB database (https: / / www.rcsb.org / ) as the receptor model for computer molecular docking, and water molecules and small ligand molecules were deleted using PyMol (version 2.4.0) software. The receptor was hydrogenated using AutodockTools software and converted into PDBQT format; the small molecule compounds were processed by the Ligand subroutine package, the number of rotatable bonds of the ligand was calculated, and saved in pdbqt format. Auto Grid was used for energy grid calculation. The grid box 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 files were docked with protein molecules using AutodockVina software. The energy distribution results were viewed through the docking results to analyze the binding energy of small molecules and α-glucosidase. 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.

[0109] Generally, when using AutoDock (version 1.5.6) for molecular docking, when Affinity <= -5 kcal / mol, it is considered that the compound has a certain binding affinity with the target protein, and when Affinity <= -7 kcal / mol, it indicates that the compound has a moderate binding affinity with the target protein.

[0110] Before the screened small molecule compounds were molecularly docked with α-glucosidase, the positive control drug acarbose was first molecularly docked with α-glucosidase in this example. The results showed that its affinity value with α-glucosidase was -7.1kcal / mol, indicating that acarbose had a moderate affinity with α-glucosidase, verifying the effectiveness of this molecular docking process. Subsequently, 27 known compounds were molecularly docked with α-glucosidase, and the results are shown in Table 2 below.

[0111] Table 2 Affinity of the screened compounds to α-glucosidase and their inhibitory effects on α-glucosidase.

[0112] serial number Compound Name Affinity (kcal / mol) C1 5-Hydroxy-3,7,3',4'-tetramethoxyflavone -6.2 C2 Demethylnobiletin -6.1 C3 Vetsenin-3 -8.1 C4 Naringin -7.2 C5 8-Hydroxy-3,5,6,7,3′,4′-hexamethoxyflavone -6.2 C6 4′,5,,6,7-Tetramethoxyflavone -5.6 C7 Hesperidin -5.9 C8 Neohesperidin -7.3 C9 Nobiletin -6.8 C10 3,5,6,7,3′,4′-Hexamethoxyflavone -5.6 C11 Sweet orange flavonoids -6.1 C12 Isoflavone -6.1 C13 Hesperidin -8.2 C14 Naringenin -6.8 C15 Naringenin-7-O-glucoside -7.3 C16 Eriocitrin -8.3 C17 Vetsenin-2 -6.8 C18 Rhusin -8.8 C19 Hesperidin -6.8 C20 Isoquercetin -6.9 Acarbose -7.1

[0113] As can be seen from Table 2, the affinity of the above compounds to α-glucosidase is <-5 kcal / mol, indicating that they have affinity to α-glucosidase, and also verifying the reliability of the affinity screening results of this experiment.

[0114] Example 5

[0115] In this example, the in vitro inhibitory activity of the above-screened active ingredients on α-glucosidase was evaluated.

[0116] In this example, the inhibitory activities of the above 20 screened compounds on α-glucosidase were tested respectively.

[0117] 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, then add 20 μL of 2 mM pNPG, incubate at 37 °C for 30 min, then add 100 μL of 0.1 M Na2CO3 to terminate the reaction, and immediately measure the absorbance of the solution at 405 nm to calculate the inhibition rate and IC 50 The blank group was replaced with an equal amount of phosphate buffer instead of sample solution.

[0118] Inhibition rate (%) = (A a -A b ) / A a ×100%; where A a represents the absorbance value of the blank group, Ab Indicates the absorption value of the sample group. The specific test results are shown in Table 3, where: a Inhibition rate of the compound at 2.5 mM (%); b Inhibition rate of the compound at 5 mM (%); c Inhibition rate (%) of the compound at 10 mM.

[0119] Table 3 Inhibitory effects of the screened compounds on α-glucosidase.

[0120]

[0121]

[0122] As shown in Table 3, these 20 compounds all have inhibitory activity against α-glucosidase, among which 6 compounds, Vicenin-3 (C3, IC50: 0.41mM), Naringenin (C14, IC50: 0.40mM), Vicenin-2 (C17, IC50: 0.43mM), Hesperetin (C19, IC50: 0.32mM), Isoquercitroside (C20, IC50: 1.46mM) and Rhoifolin (C18, IC50: 5.28mM) have stronger inhibitory activity against α-glucosidase than the positive control drug acarbose (IC50: 6.41mM).

[0123] Example 6

[0124] In this example, affinity ultrafiltration mass spectrometry screening was performed on α-glucosidase inhibitors in dried orange peel.

[0125] Sample preparation

[0126] Take the dried tangerine peel raw material and crush it into powder (pass through an 80-mesh sieve), weigh 100.0g of the powder, add 1000mL of 60% methanol and mix thoroughly, perform ultrasonic extraction at 30°C for 40min, filter and collect the extract after ultrasonication, repeat the above extraction process 3 times for the separated filter residue, combine the collected extracts, and recover the reagents on a rotary evaporator at 40°C. The obtained extract is dried under reduced pressure to obtain a dried tangerine peel extract sample.

[0127] Weigh 10 mg of the above tangerine peel extract sample, add 1 mL of 8 mM ammonium acetate buffer solution (pH 6.86, Prilep) to dissolve the sample, and the prepared tangerine peel extract solution has a concentration of 10 mg / mL and is set aside.

[0128] Preparation of α-glucosidase solution

[0129] Take α-glucosidase and add 8 mM ammonium acetate buffer solution (pH 6.86) to fully dissolve it, and prepare an α-glucosidase solution with a concentration of 30 U / mL for use.

[0130] Take another appropriate amount of α-glucosidase solution with a concentration of 30 U / mL, boil it in boiling water for 10 min to obtain an inactivated α-glucosidase solution for later use.

[0131] Screening for α-glucosidase inhibitors

[0132] 100 μL of the above-prepared 10 mg / mL tangerine peel extract solution sample was mixed with 100 μL of 30 U / mL α-glucosidase solution, and incubated at 35° C. for 40 min to obtain an incubation solution containing an α-glucosidase-ligand complex and some unbound small molecule compounds. The above incubation solution was transferred to an ultrafiltration centrifuge tube and centrifuged at 12000 r / min for 10 min to retain the α-glucosidase-ligand complex and remove most of the buffer and unbound small molecule compounds.

[0133] The α-glucosidase-ligand complex was rinsed 5 times with 250 μL ammonium acetate buffer to remove unbound small molecules as much as possible, and centrifuged at 12000 r / min for 10 min after each rinse. The collected α-glucosidase-ligand complex was dissociated 3 times with 100 μL methanol-water solution (40:60) to release small molecule ligands, and centrifuged at 12000 r / min for 15 min after each dissociation. All dissociation solutions were collected and combined, dried with nitrogen, and re-dissolved with 50 μL methanol-water solution (40:60) to obtain analytical samples.

[0134] Another 100 μL of the same tangerine peel extract sample solution (10 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace the α-glucosidase solution for incubation, and the same parallel operation was performed to obtain a blank sample.

[0135] The above analytical samples and blank samples were respectively processed in quadruplicate and subjected to LC-MS primary scanning analysis.

[0136] Example 7

[0137] In this example, affinity ultrafiltration mass spectrometry screening was performed on α-glucosidase inhibitors in dried orange peel.

[0138] Sample preparation

[0139] Take the dried tangerine peel raw material and crush it into powder (pass through an 80-mesh sieve), weigh 100.0g of the powder, add 1000mL of 80% methanol and mix thoroughly, perform ultrasonic extraction at 20°C for 20min, filter and collect the extract after ultrasonication, repeat the above extraction process 3 times for the separated filter residue, combine the collected extracts, and recover the reagents on a rotary evaporator at 40°C. The obtained extract is dried under reduced pressure to obtain a dried tangerine peel extract sample.

[0140] Weigh 10 mg of the above tangerine peel extract sample, add 1 mL of 12 mM ammonium acetate buffer solution (pH 6.86, Prilep) to dissolve the sample, and the prepared tangerine peel extract solution has a concentration of 10 mg / mL and is set aside.

[0141] Preparation of α-glucosidase solution

[0142] Take α-glucosidase and add 12 mM ammonium acetate buffer solution (pH 6.86) to fully dissolve it, and prepare an α-glucosidase solution with a concentration of 50 U / mL for use.

[0143] Take another appropriate amount of α-glucosidase solution with a concentration of 50 U / mL, boil it in boiling water for 10 min to obtain an inactivated α-glucosidase solution for later use.

[0144] Screening for α-glucosidase inhibitors

[0145] 100 μL of the above-prepared 10 mg / mL tangerine peel extract solution sample was mixed with 100 μL of 50 U / mL α-glucosidase solution, and incubated at 40° C. for 20 min to obtain an incubation solution containing an α-glucosidase-ligand complex and some unbound small molecule compounds. The above incubation solution was transferred to an ultrafiltration centrifuge tube and centrifuged at 10000 r / min for 10 min to retain the α-glucosidase-ligand complex and remove most of the buffer and unbound small molecule compounds.

[0146] The α-glucosidase-ligand complex was rinsed 5 times with 250 μL ammonium acetate buffer to remove unbound small molecules as much as possible, and centrifuged at 10000 r / min for 10 min after each rinse. The collected α-glucosidase-ligand complex was dissociated 3 times with 100 μL methanol-water solution (60:40) to release small molecule ligands, and centrifuged at 10000 r / min for 15 min after each dissociation. All dissociation solutions were collected and combined, dried with nitrogen, and re-dissolved with 50 μL methanol-water solution (60:40) to obtain analytical samples.

[0147] Another 100 μL of the same tangerine peel extract sample solution (10 mg / mL) was taken as a blank solution, and an equal amount of inactivated α-glucosidase solution was added to replace the α-glucosidase solution for incubation, and the same parallel operation was performed to obtain a blank sample.

[0148] The above analytical samples and blank samples were respectively processed in quadruplicate and subjected to LC-MS primary scanning analysis.

[0149] Example 8

[0150] The method for affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried tangerine peel in this embodiment is the same as that in Embodiment 1, except that the dried tangerine peel is extracted based on ethanol with the same concentration and amount.

[0151] Example 9

[0152] The method for affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried tangerine peel in this embodiment is the same as that in Embodiment 1, except that the dried tangerine peel is decocted and extracted based on the same amount of water.

[0153] Example 10

[0154] The method for affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried orange peel in this embodiment is the same as that in Example 1, except that, in the rinsing step, the α-glucosidase-ligand complex is rinsed 3 times with 100 μL ammonium acetate buffer to remove unbound small molecules as much as possible. Obviously, the above embodiment is only an example for clear explanation, and is not a limitation of the embodiment. For those of ordinary skill in the art, other different forms of changes or variations can also be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the embodiments here. And the obvious changes or variations derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for screening α-glucosidase inhibitors from dried orange peel, characterized in that: The steps include: (1) Incubation: taking a dried tangerine peel extract and adding α-glucosidase to incubate, thereby obtaining a sample solution containing an α-glucosidase-ligand complex; taking another dried tangerine peel extract and adding an inactivated α-glucosidase solution to incubate, thereby obtaining 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 are analyzed to obtain information about each compound in the analytical sample and the blank sample, respectively, and the desired α-glucosidase inhibitors are preliminarily screened.

2. The method for screening α-glucosidase inhibitors from dried orange peel 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 tangerine peel extract and / or the α-glucosidase are formulated into a solution; Preferably, the solution for dissolving the tangerine peel extract and / or the α-glucosidase comprises an ammonium acetate buffer solution; Preferably, the solution concentration of the tangerine peel extract is 5-15 mg / mL; Preferably, the concentration of the α-glucosidase solution is 30-50 U / mL.

3. The method for screening α-glucosidase inhibitors from dried orange peel according to claim 1 or 2, characterized in that: In the step (1), the tangerine peel extract includes an alcohol extract of tangerine peel or a water extract of tangerine peel; Preferably, the tangerine peel extract is a methanol extract of tangerine peel.

4. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 3, characterized in that: In the step (2), the dissociation agent comprises a methanol-water solution; Preferably, the volume ratio of the methanol-water solution is 40-60 v / v%:60-40 v / v%; Preferably, the step (2) further comprises the step of rinsing the ultrafiltrate; Preferably, the rinsing step uses an ammonium acetate buffer solution.

5. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 4, 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 v / v%:60-40 v / v%.

6. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 5, characterized in that: In the step (3), in the LC-MS analysis step, the chromatographic conditions include: Mobile phase: A: water-0.1 v / v% 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; Injection volume: 2 μL; Sample pan temperature: 10°C; Column temperature: 35°C.

7. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 6, characterized in that: In the step (3), in the LC-MS analysis step, a high-resolution mass spectrometer equipped with a heated ESI source is used for data collection, and a positive ion mode is used, and the parameters are as follows: Ion source voltage: 3400V; Sheath gas flow rate: 5.08 L / min; Auxiliary gas flow rate: 9.37L / min; Ion transfer tube temperature: 320°C; Atomizing gas temperature: 350℃; HCD collision energy: 30%, 45%, 60%; The m / z scanning range was 150-1000; Internal Calibration Source Thermo Scientific EASY-IC TM Perform mass calibration.

8. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 7, characterized in that: In step (4), the data statistical analysis step includes: (a) importing the collected LC-MS data into statistical software, extracting and aligning all compounds in the analytical sample and blank sample, respectively, and obtaining 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 after t-test were preliminarily considered as active ingredients that can specifically bind to α-glucosidase.

9. The method for screening α-glucosidase inhibitors from dried orange peel according to any one of claims 1 to 8, characterized in that: The step (4) also includes the steps of structural identification and activity verification of the α-glucosidase inhibitors required in the preliminary screening.

10. A use of a flavonoid compound for preparing an α-glucosidase inhibitor, characterized in that: The flavonoid compounds include: at least one of 5-hydroxy-3,7,3',4'-tetramethoxyflavone, demethyl nobiletin, vicinin-3, naringin, 8-hydroxy-3,5,6,7,3',4'-hexamethoxyflavone, 4',5,,6,7-tetramethoxyflavone, hesperidin, neohesperidin, nobiletin, 3,5,6,7,3',4'-hexamethoxyflavone, sweet orange flavonoids, isocitronellal, hesperidin, naringenin, naringenin-7-O-glucoside, eriocitrin, vicinin-2, wild sumac glycoside, hesperidin or isoquercitrin.

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