A method for screening alpha-glucosidase inhibitors from pericarpium citri reticulatae and application thereof

Abstract

The application belongs to the technical field of medicine and chemical analysis, and particularly relates to a method for screening alpha-glucosidase inhibitors from pericarpium citri reticulatae and application. The method for screening alpha-glucosidase inhibitors from pericarpium citri reticulatae extract in the application uses 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 pericarpium citri reticulatae extract for incubation, then dissociates the obtained receptor-ligand complex, collects a dissociation solution for LC-MS analysis, and through data statistical analysis of compounds contained in the analysis sample and the blank sample, active ingredients capable of specifically binding to the alpha-glucosidase are obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine and chemical analysis, and particularly relates to a method for screening alpha-glucosidase inhibitors from pericarpium citri reticulatae and application thereof. BACKGROUND

[0002] Diabetes is a multi-factorial endocrine metabolic disorder characterized by high blood sugar, which is caused by insufficient insulin secretion, insulin resistance or both. Diabetes is widespread worldwide. According to the latest data released by the International Diabetes Federation (IDF), as of 2021, there are 537 million diabetes patients (20-79 years old) worldwide, which will reach 643 million in 2030 and 783 million in 2045. Therefore, the prevention and treatment of diabetes has become a global public health problem. Among diabetes patients, more than 90% of them are type 2 diabetes patients, which is the main type of diabetes.

[0003] At present, diabetes patients pay more attention to fasting blood sugar during the treatment process, but the control of postprandial blood sugar is obviously not enough. Modern research has found that diabetes often occurs after high blood sugar. In addition, postprandial hyperglycemia is easy to cause cardiovascular and nervous system diseases, and even may cause ketoacidosis to cause coma, and even threaten life. Therefore, controlling postprandial hyperglycemia is one of the important measures to prevent diabetes, reduce complications and reduce mortality.

[0004] Alpha-glucosidase is an enzyme that plays a key role in the metabolic process of the body and is closely related to many diseases caused by metabolic disorders, especially a key enzyme for the digestion of carbohydrates in the body. Research shows that alpha-glucosidase is one of the most important targets for controlling postprandial hyperglycemia. Alpha-glucosidase is mainly located on the brush border of small intestinal mucosal cells and is distributed in all segments of the small intestine, and plays an important role in the decomposition and metabolism of carbohydrates in the body. By promoting the decomposition of carbohydrates such as starch, sucrose and maltose in the intestinal tract into monosaccharides, the monosaccharides are absorbed by the epithelial cells of the upper small intestine and then enter the blood circulation, causing postprandial hyperglycemia.

[0005] Alpha-glucosidase inhibitors can competitively inhibit or reversibly inhibit the activity of alpha-glucosidase on the brush border of the small intestine, thereby delaying the conversion of polysaccharides into monosaccharides, thereby delaying the postprandial increase in blood glucose. In addition, food components are broken down and absorbed in the upper part of the small intestine, and after the use of alpha-glucosidase inhibitors, chyme can enter the distal ileum, where there is a large amount of small intestinal glucagon-like peptide-1 (GLP-1), and food can stimulate the increased secretion of GLP-1, thereby stimulating the secretion of insulin, thereby reducing the postprandial blood glucose concentration. At present, the commonly used alpha-glucosidase inhibitor drugs in clinical practice mainly include miglitol, acarbose and voglibose, etc. However, the above-mentioned drugs all have more or less certain side effects, such as gastrointestinal distension, etc., while reducing blood glucose. Therefore, it is expected in the art to develop new alpha-glucosidase inhibitors with less side effects.

[0006] Traditional Chinese medicine resources are a huge treasure trove of natural active ingredients, with the advantages of structural diversity, obvious effect and small side effects, so it is an important way to develop new alpha-glucosidase inhibitors to find compounds with inhibitory activity against alpha-glucosidase from traditional Chinese medicine resources with hypoglycemic effect. Pericarpium Citri Reticulatae is the mature dried pericarp of Citrus reticulata Blanco and its cultivated varieties of Rutaceae, which has a long history of use in China, and is not only a traditional Chinese medicine for medical treatment, but also a traditional food material for health care and diet therapy. Modern research has found that Pericarpium Citri Reticulatae has the effects of anti-inflammatory, anti-Alzheimer's disease (AD) and Parkinson's disease (PD), anticancer, bone protection, anti-obesity, skin protection and vascular regulation. In addition to the above-mentioned effects, modern research has also found that Pericarpium Citri Reticulatae extract can reduce the fasting blood glucose level of diabetic rats, suggesting that Pericarpium Citri Reticulatae has the effect of lowering blood glucose. Further exploration by researchers has found that Pericarpium Citri Reticulatae extract has a certain inhibitory effect on alpha-glucosidase, but there are few reports on the specific alpha-glucosidase inhibition. SUMMARY

[0007] Therefore, the technical problem to be solved by the present application is to provide a method for screening alpha-glucosidase inhibitors from Pericarpium Citri Reticulatae, which can quickly screen alpha-glucosidase inhibitors from Pericarpium Citri Reticulatae and its extracts by using affinity ultrafiltration screening combined with mass spectrometry technology;

[0008] The second technical problem to be solved by the present application is to provide the use of compounds in Pericarpium Citri Reticulatae for preparing alpha-glucosidase inhibitors.

[0009] To solve the above technical problems, the method for screening alpha-glucosidase inhibitors from Pericarpium Citri Reticulatae according to the present application comprises the following steps:

[0010] (1) incubation: taking the extract of pericarpium citri reticulatae to incubate with α-glucosidase, obtaining a sample solution containing α-glucosidase-ligand complex; taking the extract of pericarpium citri reticulatae to incubate with inactivated α-glucosidase solution, obtaining a blank solution;

[0011] (2) ultrafiltration-dissociation: respectively performing ultrafiltration treatment on the sample solution and the blank solution, and collecting the ultrafiltration to perform dissociation treatment by adding dissociation agent, collecting dissociation solution, and respectively obtaining dissociation solution of the sample solution and the blank solution;

[0012] (3) LC-MS analysis: respectively blowing dry the dissociation solution, collecting the residue to dissolve in constant volume solvent, respectively obtaining analysis sample of the sample solution and blank sample of the blank solution; and respectively performing LC-MS scanning and analysis on the analysis sample and the blank sample, obtaining LC-MS data;

[0013] (4) data statistical analysis: analyzing the LC-MS data, respectively obtaining information of each compound in the analysis sample and the blank sample, and preliminarily screening the required α-glucosidase inhibitor.

[0014] Specifically, in the step (1) of the method for screening α-glucosidase inhibitor from pericarpium citri reticulatae, the temperature of the incubation step is 35-40℃, and the incubation time is 20-40 min.

[0015] Preferably, the extract of pericarpium citri reticulatae and / or the α-glucosidase is prepared into a solution.

[0016] Preferably, the solution of the extract of pericarpium citri reticulatae and / or the α-glucosidase comprises ammonium acetate buffer solution.

[0017] Preferably, the solution of the extract of pericarpium citri reticulatae is prepared at a concentration of 5-15 mg / mL.

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

[0019] Specifically, in the step (1) of the method for screening α-glucosidase inhibitor from pericarpium citri reticulatae, the extract of pericarpium citri reticulatae comprises alcohol extract of pericarpium citri reticulatae or water extract of pericarpium citri reticulatae.

[0020] Preferably, the extract of pericarpium citri reticulatae is methanol extract of pericarpium citri reticulatae.

[0021] Specifically, in the step (2) of the method for screening α-glucosidase inhibitor from pericarpium citri reticulatae, the dissociation agent comprises 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 a step of rinsing the ultrafiltrate.

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

[0025] Specifically, in the method for screening α-glucosidase inhibitors from Pericarpium Citri Reticulatae, the step (3) comprises a step of LC-MS analysis.

[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 Pericarpium Citri Reticulatae, the step (3) comprises a step of LC-MS analysis.

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

[0029] Elution program comprises: 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 tray temperature: 10°C;

[0033] Column temperature: 35°C.

[0034] Specifically, in the method for screening α-glucosidase inhibitors from Pericarpium Citri Reticulatae, the step (3) comprises a step of LC-MS analysis. TM Pre-Column (2.1×5 mm, 1.7 μm) pre-column and Waters ACQUITY UPLC BEH C18 column (2.1 μ 100 mm, 1.7 μm) chromatographic column.

[0035] Specifically, in the method for screening α-glucosidase inhibitors from Pericarpium Citri Reticulatae, the step (3) comprises a step of LC-MS analysis.

[0036] Ion source voltage: 3400 V;

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

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

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

[0040] Nebulizer gas temperature: 350 °C;

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

[0042] m / z scan range 150-1000;

[0043] Internal calibration source Thermo Scientific EASY-IC TM Calibration of mass number was performed.

[0044] Specifically, in the method for screening α-glucosidase inhibitors from pericarpium citri reticulatae, the data statistical analysis step in step (4) comprises the following steps.

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

[0046] (b) calculating the peak area ratio of each compound in the analysis sample and the blank sample respectively, denoted as PAR value, and calculating the average PAR value of each compound, and performing t test on the peak areas of each compound in the analysis sample and the blank sample;

[0047] (c) the compounds with average PAR value > 1 and p < 0.05 (n = 4) after t test are preliminarily considered as active ingredients capable of specifically binding to α-glucosidase.

[0048] Preferably, the data statistical analysis step adopts Compound Discoverer TM software to process the collected LC-MS data. TM

[0049] Specifically, in the method for screening α-glucosidase inhibitors from pericarpium citri reticulatae, the data statistical analysis step in step (4) further comprises the steps of structure identification and activity verification of the preliminarily screened α-glucosidase inhibitors.

[0050] ​Specifically, the application further discloses a use of a flavonoid compound for preparing an alpha-glucosidase inhibitor, wherein the flavonoid compound comprises at least one of 5-hydroxy-3,7,3',4'-tetramethoxyflavone, norcitrusin, vitexin-3, naringin, 8-hydroxy-3,5,6,7,3',4'-hexamethoxyflavone, 4',5,,6,7-tetramethoxyflavone, tangeritin, neohesperidin, citrusin, 3,5,6,7,3',4'-hexamethoxyflavone, sweet orange flavone, iso-sweet orange flavone, hesperidin, naringenin, naringenin-7-O-glucoside, rutinoside, vitexin-2, heparoside, hesperetin or isoquercitrin.

[0051] The method for screening alpha-glucosidase inhibitors from the extract of dried tangerine or orange peel according to the application uses alpha-glucosidase as a target, and uses affinity ultrafiltration screening technology combined with liquid chromatography-mass spectrometry technology to screen alpha-glucosidase inhibitors.

[0052] The method for screening alpha-glucosidase inhibitors from the extract of dried tangerine or orange peel according to the application uses LC-MS analysis on the analysis sample and the blank sample, and uses statistical software to extract, align and calculate the PAR value and the average PAR value of each compound, and performs t test on the peak area of each compound in the sample group and the blank group, so that the compound with an average PAR value greater than 1 and a p value less than 0.05 (n=4) after t test is the alpha-glucosidase inhibitor active ingredient.

[0053] The application screens alpha-glucosidase inhibitors from pericarpium citri reticulatae with hypoglycemic efficacy, and 20 alpha-glucosidase inhibitors are screened from pericarpium citri reticulatae by using affinity ultrafiltration mass spectrometry, which are all identified as flavonoids. The application further carries out molecular docking on the screened active ingredients, tests the affinity of the screened active ingredients with alpha-glucosidase, and tests the inhibition degree of the screened active ingredients on the activity of alpha-glucosidase by using in-vitro enzyme activity inhibition verification. It is predicted by the molecular docking method that they all have certain affinity with alpha-glucosidase; it is found by the in-vitro enzyme activity inhibition experiment that they all have inhibitory activity on alpha-glucosidase, and even 6 components have stronger inhibitory activity on alpha-glucosidase than the positive control drug acarbose. The flavonoids involved in the application have positive significance for developing new alpha-glucosidase inhibitor drugs, and pericarpium citri reticulatae is a medicine and food dual-purpose traditional Chinese medicine, so the screened flavonoids provide a reference for the development of pericarpium citri reticulatae functional food.

[0054] The method for screening alpha-glucosidase inhibitors from pericarpium citri reticulatae extract in the application is targeted at the characteristics of pericarpium citri reticulatae, and can realize rapid and accurate screening of alpha-glucosidase inhibitors from pericarpium citri reticulatae by optimizing the entire affinity ultrafiltration screening process, especially by optimizing the detection and analysis program of LC-MS technology, thereby effectively overcoming the drawbacks of the traditional screening mode of separating first and then detecting activity in the screening and identification of natural active substances from complex traditional Chinese medicine extracts, and having the advantages of short experimental period and small workload, and can realize large-scale and high-throughput screening, which has positive significance for drug development. BRIEF DESCRIPTION OF DRAWINGS

[0055] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which,

[0056] Figure 1 The high-resolution mass spectrum total ion chromatogram of pericarpium citri reticulatae extract (Extract), pericarpium citri reticulatae blank sample (Control) and analysis sample (Sample) before incubation. DETAILED DESCRIPTION

[0057] In the following examples of the application, the information of instruments, reagents, control samples, materials and the like involved in the application is as follows:

[0058] Alpha-glucosidase (derived from Saccharomyces cerevisiae, EC number: 3.2.1.20, 100 units);

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

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

[0061] 0.1M Phosphate buffer salt (pH 6.8, Prolabo);

[0062] 0.1M Na2CO3 (Sigma);

[0063] Deionized water was purchased from Watsons;

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

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

[0066] Dried tangerine peel was provided by Beijing Tong Ren Tang Co., Ltd;

[0067] 96-well plates (Corning);

[0068] The control sample acarbose was purchased from Shanghai Yuye Co., Ltd, with purity >98%.

[0069] Example 1

[0070] This example is directed to affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried tangerine peel.

[0071] Preparation of samples

[0072] The dried tangerine peel was ground into powder (through an 80-mesh sieve), 100.0 g of the powder was weighed, 1000 mL of 70% methanol was added and mixed thoroughly, and ultrasonic extraction was performed at 25°C for 30 min. After ultrasonic extraction, the extract was filtered and collected, and the separated residue was repeatedly extracted for 3 times. The collected extract was recovered by a rotary evaporator at 40°C to obtain a dried extract sample of dried tangerine peel, 44 g.

[0073] 10 mg of the dried tangerine peel extract sample was weighed and dissolved in 1 mL of 10 mM ammonium acetate buffer solution (pH 6.86, Prolabo) to prepare a dried tangerine peel extract solution with a concentration of 10 mg / mL, which was ready for use.

[0074] Preparation of α-glucosidase solution

[0075] 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 40 U / mL, which was ready for use.

[0076] Another appropriate amount of α-glucosidase solution with a concentration of 40 U / mL was boiled in boiling water for 10 min to obtain an inactivated α-glucosidase solution, which was ready for use.

[0077] Screening of α-glucosidase inhibitors

[0078] Take 100 μL of the above prepared 10 mg / mL extract solution of Pericarpium Citri Reticulatae and 100 μL of 40 U / mL α-glucosidase solution, mix and incubate at 37°C for 30 min to obtain an incubation solution containing α-glucosidase-ligand complex and some unbound small molecule compounds. Transfer the above incubation solution to an ultrafiltration centrifuge tube and centrifuge 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] Continue to rinse the α-glucosidase-ligand complex with 250 μL of ammonium acetate buffer for 5 times to remove as much as possible unbound small molecule compounds, and centrifuge at 14000 r / min for 10 min after each rinse. Dissociate the collected α-glucosidase-ligand complex with 100 μL of methanol-water solution (50:50, pH 3.30) for 3 times to release small molecule ligands, and centrifuge at 14000 r / min for 15 min after each dissociation. Collect and combine all dissociation solutions, dry under nitrogen, and re-dissolve with 50 μL of methanol-water solution (50:50) to obtain an analysis sample.

[0080] Take another 100 μL of the same Pericarpium Citri Reticulatae extract sample solution (10 mg / mL) as a blank solution, add an equal amount of inactivated α-glucosidase solution instead of α-glucosidase solution for incubation, and perform the same parallel operation to obtain a blank sample.

[0081] The above analysis sample and blank sample are each operated in parallel four times, and subjected to LC-MS primary scan analysis.

[0082] Example 2

[0083] This example is based on the analysis sample and blank sample obtained in the preceding Example 1, which are subjected to LC-MS primary scan analysis.

[0084] The LC-MS analysis detection conditions are as follows: each chemical component in the above analysis sample and blank sample is obtained on a Waters ACQUITY UPLC BEH C18 column (2.1 x 100 mm, 1.7 um) equipped with a binary pump, a heated column oven, and a Waters ACQUITY UPLC BEH C18 VanGuard Pre-Column (2.1 x 5 mm, 1.7 um) pre-column. TM Flex UPLC (Thermo Scientific, USA) system. TM Pre-Column (2.1 x 5 mm, 1.7 um) pre-column.

[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 tray temperature: 10 °C;

[0091] Column temperature: 35 °C.

[0092] In this embodiment, the Orbitrap Exploris 240 (Thermo Scientific, USA) high-resolution mass spectrometer equipped with a heated ESI source was used 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 transmission tube temperature: 320 °C; nebulizing gas temperature: 350 °C; collision energy (%): 30, 45, 60; m / z scan range 150-1000. Internal calibration source Thermo Scientific EASY-IC TM Calibration of mass number was performed.

[0093] In this embodiment, the high-resolution mass spectra of the Chenpi extract (Extract) before incubation, the analysis sample (Sample) obtained by adding α-glucosidase and screening by affinity ultrafiltration, and the blank sample (Control) obtained by adding inactivated α-glucosidase and screening by affinity ultrafiltration are shown in Figure 1 .

[0094] Example 3

[0095] In this embodiment, Compound Discoverer TM (Thermo Scientific TM , version 3.2.0.421) software was used for data processing based on statistical analysis of the LC-MS data obtained in the above-mentioned Example 2. It should be noted that other software capable of analyzing compound structure data in the art can also be used for characterization, and the software selected in this embodiment is only an exemplary embodiment.

[0096] The first-order full scan mass spectrometry data of the analysis sample and the blank sample obtained in Example 2 were respectively imported into Compound Discoverer TM software (Thermo Scientific TM , version 3.2.0.421) for data processing.

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

[0098] According to the above method and parameters, the extraction process of the compounds was performed, and the molecular weight, retention time and peak area of each compound in each analysis sample and blank sample were obtained. 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 the blank group was subjected to t-test inspection. Finally, the compounds with an 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 compounds was determined by the obtained fragment ion information.

[0099] Based on the data obtained in Example 2 and through the above data statistical analysis, the 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 the reference substance.

[0100] Table 1. 20 alpha-glucosidase inhibitors screened from pericarpium citri reticulatae extract.

[0101]

[0102]

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

[0104]

[0105] Example 4

[0106] This example is based on the bioactivity evaluation of the compounds screened as described above, and molecular docking based on alpha-glucosidase and the active ingredients screened.

[0107] The molecular docking method can predict the affinity of a compound to a target, so the molecular docking method can be one of the methods for verifying whether the compound is a ligand of 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 for two-dimensional (2D) structure, and the 2D structure was converted to three-dimensional (3D) structure by ChemBio 3D Ultra 14.0 software. After the MM2 Minimize Energy force field optimization of the small molecule compound, it was saved in PDB format for future use. The alpha-glucosidase crystal (PDB ID: 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 removed by PyMol (version 2.4.0) software. Hydrogenation treatment was performed on the receptor using AutodockTools software, and converted to PDBQT format. The Ligand subprogram package processed the small molecule compound, calculated the number of rotatable bonds of the ligand, and saved it in pdbqt format. Energy grid calculation was performed using Auto Grid, with grid box setting as 40nm x 40nm x 40nm, grid spacing 1nm, and grid center coordinates as the default active center of the receptor. The compounds stored in the file were docked with the protein molecules using AutodockVina software, and the energy distribution results were viewed through the docking results to analyze the binding energy of the small molecule and alpha-glucosidase. The binding site with the lowest binding energy was taken as the optimal binding site of the compound and alpha-glucosidase, and finally the PYMOL software was used for drawing.

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

[0110] Before performing molecular docking of the screened small molecule compounds with α-glucosidase, the positive control drug acarbose was first subjected to molecular docking with α-glucosidase. The results showed that the Affinity value of acarbose with α-glucosidase was -7.1 kcal / mol, indicating that acarbose has moderate affinity with α-glucosidase, thereby verifying the effectiveness of the molecular docking process. Subsequently, 27 known compounds were subjected to molecular docking with α-glucosidase, and the results are shown in Table 2 below.

[0111] Table 2 Affinity of screened compounds with α-glucosidase and inhibition effect on α-glucosidase

[0112] No. Compound name Affinity (kcal / mol) C1 5-hydroxy-3,7,3',4'-tetramethoxyflavone -6.2 C2 Nordamnacanthal -6.1 C3 Vicenin-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 Tangeritin -5.9 C8 Neohesperidin -7.3 C9 Damnacanthal -6.8 C10 3,5,6,7,3',4'-hexamethoxyflavone -5.6 C11 Sweet orange flavone -6.1 C12 Isohesperetin -6.1 C13 Hesperidin -8.2 C14 Naringenin -6.8 C15 Naringenin-7-O-glucoside -7.3 C16 Eriodictyol -8.3 C17 Vicenin-2 -6.8 C18 Katsuteroside -8.8 C19 Hesperetin -6.8 C20 Isoquercitrin -6.9 Acarbose -7.1

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

[0114] Example 5

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

[0116] In this example, the inhibitory activity of the above-mentioned 20 screened compounds on α-glucosidase was tested.

[0117] 40 μL of sample with different concentrations was taken, 40 μL of 0.2 U / mL α-glucosidase was added, and incubation was performed at 37°C for 5 min, 20 μL of 2 mM pNPG was then added, and incubation was performed at 37°C for 30 min, 100 μL of 0.1 M Na2CO3 was then added to terminate the reaction, and the absorption value of the solution was immediately measured at 405 nm, the inhibition rate was calculated, and the IC 50 value was calculated. The blank group was replaced with an equal amount of phosphate buffer instead of the sample solution.

[0118] Inhibition rate (%) = (A a -A b ) / A a x 100%; wherein, A a represents the absorption value of the blank group, and Ab Absorbance values of the sample groups are shown. The specific test results are shown in Table 3, in which, 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 Inhibition of α-glucosidase by the screened compounds.

[0120]

[0121]

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

[0123] Example 6

[0124] This example is directed to affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in Pericarpium Citri Reticulatae Viride.

[0125] Preparation of samples

[0126] The Pericarpium Citri Reticulatae Viride raw material was ground into powder (through an 80-mesh sieve), 100.0 g of the powder was weighed, 1000 mL of 60% methanol was added to mix thoroughly, and ultrasonic extraction was performed at 30°C for 40 min. After ultrasonic extraction, the extract was filtered and collected, and the separated residue was repeatedly extracted 3 times by the above process. The collected extract was recovered by a rotary evaporator at 40°C to obtain a Pericarpium Citri Reticulatae Viride extract sample.

[0127] 10 mg of the Pericarpium Citri Reticulatae Viride extract sample was weighed, and 1 mL of 8 mM ammonium acetate buffer solution (pH 6.86, Pluronic) was added to dissolve the sample to prepare a Pericarpium Citri Reticulatae Viride extract solution with a concentration of 10 mg / mL for standby use.

[0128] Preparation of α-glucosidase solution

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

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

[0131] Screening of α-glucosidase inhibitors

[0132] Take 100 μL of the above prepared 10 mg / mL extract solution of dried orange peel and 100 μL of 30 U / mL α-glucosidase solution, mix and incubate at 35°C for 40 min to obtain an incubation solution containing α-glucosidase-ligand complex and some unbound small molecule compounds. Transfer the above incubation solution to an ultrafiltration centrifuge tube, centrifuge at 12000 r / min for 10 min, retain the α-glucosidase-ligand complex, and remove most of the buffer and unbound small molecule compounds.

[0133] Continue to rinse the α-glucosidase-ligand complex with 250 μL of ammonium acetate buffer for 5 times to remove as much unbound small molecule compounds as possible, centrifuge at 12000 r / min for 10 min after each rinse. Dissociate the collected α-glucosidase-ligand complex with 100 μL of methanol-water solution (40:60) for 3 times to release small molecule ligands, centrifuge at 12000 r / min for 15 min after each dissociation. Collect and combine all dissociation solutions, blow dry with nitrogen, and re-dissolve with 50 μL of methanol-water solution (40:60) to obtain an analysis sample.

[0134] Take another 100 μL of the same extract solution (10 mg / mL) of dried orange peel as a blank solution, add an equal amount of inactivated α-glucosidase solution instead of α-glucosidase solution for incubation, and perform the same parallel operation to obtain a blank sample.

[0135] The above analysis sample and blank sample are each operated in parallel four times, and subjected to LC-MS primary scan analysis.

[0136] Example 7

[0137] This example is directed to affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried orange peel.

[0138] Preparation of samples

[0139] The pericarpium citri reticulatae raw material was ground into powder (through an 80-mesh sieve), 100.0 g of the powder was weighed out, 1000 mL of 80% methanol was added and mixed thoroughly, and ultrasonic extraction was performed at 20°C for 20 min. After ultrasonic extraction, the extract was filtered and collected, and the separated residue was subjected to the above extraction process three times. The collected extracts were combined, and the reagent was recovered in a rotary evaporator at 40°C. The obtained extract was dried under reduced pressure to obtain a pericarpium citri reticulatae extract sample.

[0140] 10 mg of the pericarpium citri reticulatae extract sample was weighed out, 1 mL of 12 mM ammonium acetate buffer solution (pH 6.86, Plurilase) was added to dissolve the sample, and a pericarpium citri reticulatae extract solution with a concentration of 10 mg / mL was prepared for use.

[0141] Preparation of α-glucosidase solution

[0142] α-glucosidase was taken and dissolved in 12 mM ammonium acetate buffer solution (pH 6.86) to prepare an α-glucosidase solution with a concentration of 50 U / mL for use.

[0143] Another appropriate amount of α-glucosidase solution with a concentration of 50 U / mL was boiled in boiling water for 10 min to obtain an inactivated α-glucosidase solution for use.

[0144] Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-glucosidase inhibitors Preparation of samples Preparation of α-glucosidase solution Screening of α-gluc

[0145] 100 μL of the prepared 10 mg / mL pericarpium citri reticulatae extract solution and 100 μL of the 50 U / mL α-glucosidase solution were mixed and co-incubated at 40°C for 20 min to obtain an incubation solution containing α-glucosidase-ligand complexes and some unbound small molecule compounds. The incubation solution was transferred to an ultrafiltration centrifuge tube and centrifuged at 10000 r / min for 10 min. The α-glucosidase-ligand complexes were retained, and most of the buffer and unbound small molecule compounds were removed.

[0146] The α-glucosidase-ligand complexes were further rinsed with 250 μL of ammonium acetate buffer solution for 5 times to remove as much unbound small molecule compounds as possible. After each rinse, the solution was centrifuged at 10000 r / min for 10 min. The collected α-glucosidase-ligand complexes were dissociated with 100 μL of methanol-water solution (60:40) for 3 times to release small molecule ligands. After each dissociation, the solution was centrifuged at 10000 r / min for 15 min. The dissociation solutions were collected and combined, blown dry with nitrogen, and redissolved with 50 μL of methanol-water solution (60:40) to obtain an analysis sample.

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

[0148] The analysis sample and the blank sample were each operated in parallel four times, and subjected to LC-MS primary scanning analysis.

[0149] Example 8

[0150] This example is identical to Example 1 in the method of affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried orange peel, except that the dried orange peel was extracted based on the same concentration and amount of ethanol.

[0151] Example 9

[0152] This example is identical to Example 1 in the method of affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried orange peel, except that the dried orange peel was decocted and extracted based on the same amount of water.

[0153] Example 10

[0154] This example is identical to Example 1 in the method of affinity ultrafiltration mass spectrometry screening of α-glucosidase inhibitors in dried orange peel, except that in the rinsing step, the α-glucosidase-ligand complex was rinsed with 100 μL of ammonium acetate buffer three times to remove as much as possible the unbound small molecular compounds. Obviously, the above examples are only examples for the purpose of clarity, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. It is not necessary or possible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method of screening α-glucosidase inhibitors from pericarpium citri reticulatae, characterized by, The method comprises the following steps: (1) Incubation: Take the alcohol extract of dried tangerine or peel water extract of dried tangerine peel and add α-glucosidase to incubate at 35-40℃ for 20-40min to obtain a sample solution containing α-glucosidase-ligand complex; another alcohol extract of dried tangerine peel or water extract of dried tangerine peel is taken and added to the inactivated α-glucosidase solution to incubate to obtain a blank solution; (2) Ultrafiltration-dissociation: The sample solution and the blank solution are respectively subjected to ultrafiltration treatment, and the ultrafiltrate is collected, rinsed with ammonium acetate buffer solution, and then dissociated by adding methanol-water solution to collect the dissociation solution, thereby obtaining the dissociation solution of the sample solution and the blank solution respectively; the volume ratio of methanol-water solution is 40-60:60-40; (3) LC-MS analysis: The dissociation solution is dried, and the residue is collected and dissolved in a constant volume solvent to obtain an analysis sample of the sample solution and a blank sample of the blank solution respectively; and the analysis sample and the blank sample are respectively subjected to LC-MS scanning and analysis to obtain LC-MS data; The chromatographic conditions in the LC-MS analysis include: Mobile phase: A: water-0.1% formic acid; B: acetonitrile; Elution program includes: 0-7min, 2% B-20% B; 7-10min, 20% B-25% B; 10-20min, 25% B-40% B; 20-25min, 40% B-65% B; 25-30min, 65% B-95% B; Flow rate: 0.3mL / min; Injection volume: 2μL; Sample disc temperature: 10℃; Column temperature: 35℃; After chromatography, a high-resolution mass spectrometer with a heated ESI source is used for data collection, using positive ion mode, parameters as follows: Ion source voltage: 3400V; Sheath gas flow rate: 5.08 L / min; Auxiliary gas flow rate: 9.37L / min; Ion transmission tube temperature: 320℃; Atomization gas temperature: 350℃; HCD collision energy: 30%, 45%, 60%; m / z Scan range is 150-1000; Internal correction source Thermo Scientific EASY-IC TM Calibration of mass numbers was performed; (4) Data statistical analysis: The LC-MS data are analyzed to obtain the information of each compound in the analysis sample and the blank sample respectively, calculate the peak area ratio of each compound in the analysis sample and the blank sample, and perform t-test on the peak area of each compound to preliminarily screen the required α-glucosidase inhibitors.

2. The method for screening α-glucosidase inhibitors from pericarpium citri reticulatae according to claim 1, wherein, In the step (1), The alcohol extract of dried tangerine peel or the water extract of dried tangerine peel and / or the α-glucosidase are prepared into a solution; The solution of the alcohol extract of dried tangerine peel or the water extract of dried tangerine peel and / or the α-glucosidase includes an ammonium acetate buffer solution; The solution of the alcohol extract of dried tangerine peel or the water extract of dried tangerine peel is prepared at a concentration of 5-15mg / mL; The solution of the α-glucosidase is prepared at a concentration of 30-50U / mL.

3. The method for screening α-glucosidase inhibitors from pericarpium citri reticulatae according to claim 1 or 2, characterized in that, The alcohol extract of dried tangerine peel is a methanol extract of dried tangerine peel.

4. The method of claim 1, wherein the α-glucosidase inhibitor is selected from pericarpium citri reticulatae. In the step (3), the constant volume solvent includes a methanol-water solution; the volume ratio of the methanol-water solution is 40-60:60-40.

5. The method of claim 1, wherein the α-glucosidase inhibitor is selected from pericarpium citri reticulatae. In the step (4), the data statistical analysis step adopts Compound Discoverer TM software for data processing; Specifically, the data statistical analysis step includes: (a) importing the collected LC-MS data into Compound Discoverer software, extracting and aligning all compounds in the analysis sample and the blank sample respectively, obtaining the molecular weight, retention time and peak area information of each compound in the analysis sample and the blank sample respectively; TM software, extracting and aligning all compounds in the analysis sample and the blank sample respectively, obtaining the molecular weight, retention time and peak area information of each compound in the analysis sample and the blank sample respectively; (b) calculating the peak area ratio of each compound in the analysis sample and the blank sample respectively, denoted as PAR value, and calculating the average PAR value of each compound, and performing t-test on the peak area of each compound in the analysis sample and the blank sample; (c) the compound with average PAR value > 1 and p < 0.05 after t-test is preliminarily considered as an active ingredient capable of specifically binding with α-glucosidase.

6. The method of claim 1, wherein the α-glucosidase inhibitor is selected from pericarpium citri reticulatae. The step (4) further comprises the steps of structure identification and activity verification of the α-glucosidase inhibitor preliminarily screened.

7. The method of claim 1, wherein the α-glucosidase inhibitor is selected from pericarpium citri reticulatae. The α-glucosidase inhibitor is a flavonoid compound, and the flavonoid compound comprises at least one of 5-hydroxy-3,7,3',4'-tetramethoxyflavone, norobtusin, vitexin-3, naringin, 8-hydroxy-3,5,6,7,3',4'-hexamethoxyflavone, 4',5,6,7-tetramethoxyflavone, tangeritin, neohesperidin, obtusin, 3,5,6,7,3',4'-hexamethoxyflavone, sweet orange flavone, iso-sweet orange flavone, hesperidin, naringenin, naringenin-7-O-glucoside, rutinoside, vitexin-2, toxicodendrin, hesperetin or isoquercitrin.

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