Method for screening traditional Chinese medicine hypoglycemic active ingredients based on GLP-1 receptor

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for screening traditional Chinese medicine hypoglycemic active ingredients based on a GLP-1 receptor. According to the method for screening the traditional Chinese medicine hypoglycemic active ingredients based on the GLP-1 receptor, the GLP-1 receptor is added into a traditional Chinese medicine extract for incubation, then an obtained receptor-ligand compound is dissociated, and dissociation liquid is collected for LC-MS analysis; and performing data statistical analysis on compounds contained in the analysis sample and the blank sample to obtain the active component capable of being specifically bound with the GLP-1 receptor. The method for screening the traditional Chinese medicine hypoglycemic active ingredients based on the GLP-1 receptor can be used for screening GLP-1 receptor agonists in traditional Chinese medicines, is short in experimental period and small in workload, can realize large-scale and high-throughput screening, and has positive significance on development of diabetes medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and in particular relates to a method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor. Background Art

[0002] Diabetes is prevalent worldwide. According to the latest data released by the International Diabetes Federation (IDF), as of 2021, there were 537 million people with diabetes worldwide (aged 20-79 years old). This number is projected to reach 643 million by 2030 and 783 million by 2045. Diabetes has become the third most threatening non-communicable disease to human health and life, after cardiovascular disease and cancer, and the market potential for its medication is enormous.

[0003] According to research, natural GLP-1 is an incretin hormone transcribed from the body's own proglucagon gene. It is secreted by intestinal L cells, primarily located in the ileum, colon, and rectum. Its synthesis and secretion are induced by food intake, which can promote insulin secretion in hyperglycemic states, thereby lowering blood sugar. GLP-1 exerts its biological effects in the body primarily through interaction with the GLP-1 receptor (GLP-1R). Compared to other diabetes treatments, GLP-1 has two significant advantages: first, GLP-1 is secreted in response to high blood sugar levels and ceases secretion once blood sugar levels return to normal, thus preventing hypoglycemia. Second, GLP-1 can stimulate the proliferation of pancreatic beta cells and inhibit their apoptosis, which can even reverse type 2 diabetes. Due to these two advantages, GLP-1 holds great potential as a novel insulin secretagogue for the treatment of type 2 diabetes.

[0004] Since natural GLP-1 is extremely unstable in the body and has a serum half-life of only 2 minutes, there is an urgent need to find GLP-1 analogs, i.e., GLP-1 receptor agonists, including peptides and small molecule compounds, to replace GLP-1 to play a role. Currently, peptide GLP-1 agonists mainly include short-acting receptor agonists, which provide short-term receptor activation (such as exenatide and lixisenatide) or long-acting receptor agonists (such as albiglutide, semaglutide, dulaglutide, exenatide sustained-release microspheres and liraglutide), which continuously activate the GLP-1 receptor at the recommended dose. However, they all belong to injectable drugs and have the disadvantages of difficult operation, weak compliance and pain. Therefore, the development of new small molecule drugs suitable for oral administration is also a major research hotspot.

[0005] Traditional Chinese medicine (TCM) is a vast repository of naturally occurring active ingredients. Screening for active ingredients that specifically bind to the GLP-1 receptor from TCM could provide new insights and approaches for the development of GLP-1 receptor agonists. However, not only are there few reports on methods for screening GLP-1 receptor agonists from TCM extracts, but the traditional approach to screening and identifying naturally occurring active substances from complex TCM extracts typically requires isolation followed by activity testing, resulting in lengthy experimental cycles and a high workload.

[0006] Therefore, the field expects to establish a screening method for hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor, which can be used to screen GLP-1 receptor agonists in traditional Chinese medicine and has positive significance for the development of diabetes drugs. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on the GLP-1 receptor, which has the advantages of short experimental cycle and small workload, and can realize large-scale and high-throughput screening work;

[0008] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned method for screening hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor in the field of drug screening and analysis.

[0009] To solve the above technical problems, the present invention provides a method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor, comprising the following steps:

[0010] (1) taking the Chinese herbal medicine extract to be tested and adding it to a buffer solution to dissolve it, thereby obtaining a drug stock solution; taking the drug stock solution and adding it to a GLP-1 receptor solution for incubation, thereby obtaining a GLP-1 receptor-ligand conjugate; taking another drug stock solution and adding it to an amount of the buffer solution equal to that of the GLP-1 receptor solution for incubation, thereby obtaining a blank sample solution;

[0011] (2) adding the GLP-1 receptor-ligand conjugate to a dissociation solution for dissociation treatment to release the small molecule ligand that can bind to the GLP-1 receptor, and collecting the dissociation solution; the blank sample solution is subjected to a parallel operation;

[0012] (3) drying the dissociation solution, collecting the residue, adding a constant volume solvent to dissolve it, and obtaining an analytical sample. The blank sample solution is subjected to a parallel operation to obtain a blank sample; performing LC-MS analysis on the analytical sample and the blank sample respectively;

[0013] (4) performing statistical analysis on the compounds contained in the analysis sample and the blank sample to obtain the active ingredient that can specifically bind to the GLP-1 receptor.

[0014] Specifically, in the method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on the GLP-1 receptor, in step (1), the buffer comprises a HEPES buffer;

[0015] Preferably, the buffer comprises 30-80 mM HEPES buffer, pH 7.5±0.2; preferably, the buffer comprises 50 mM HEPES buffer, pH 7.5;

[0016] Preferably, the concentration of the GLP-1 receptor solution is 200-400 μg / mL, preferably 300 μg / mL;

[0017] Preferably, the incubation temperature of the incubation step is 20-30° C., and the incubation time is 40-80 min.

[0018] Specifically, the method for screening hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor, in step (1), further comprises the step of ultrafiltration and / or rinsing the reactants after incubation to obtain the desired GLP-1 receptor-ligand conjugate;

[0019] Preferably, the ultrafiltration treatment step includes the step of ultrafiltration membrane filtration;

[0020] Preferably, the rinsing step includes adding ammonium acetate buffer for rinsing.

[0021] Specifically, in the method for screening hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor, in step (2), the dissociation solution includes methanol.

[0022] Specifically, in the method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on the GLP-1 receptor, in the step (3), the constant volume solvent includes a methanol-water solution;

[0023] Preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v).

[0024] Specifically, in the method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor, in step (4), the data statistical analysis step adopts Compound Discoverer TM Software for data processing.

[0025] Specifically, the method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on the GLP-1 receptor, the data statistical analysis step includes:

[0026] (a) Import the data of the analytical sample and blank sample collected in step (3) into Compound Discoverer TMSoftware that performs peak alignment and peak extraction for all compounds in each sample by establishing an extraction process for any compound;

[0027] (b) calculating the peak area ratio of each compound in the analytical sample and the blank sample, respectively, and calculating the average peak area ratio of each compound (n=4);

[0028] (c) A t-test was performed on the peak areas of the analytical samples and blank samples for each compound. Compounds with an average peak area ratio between the analytical samples and blank samples greater than 1 (n=4) and p<0.05 (n=4) after the t-test were preliminarily considered to be active ingredients that can specifically bind to the GLP-1 receptor.

[0029] Specifically, in the method for screening hypoglycemic active ingredients of traditional Chinese medicine based on GLP-1 receptor, in step (b), the designed compound extraction process is "input files→select spectra→align retention time→detect compounds→group compounds."; wherein,

[0030] In the input files step, the graph to be processed is selected;

[0031] In the select spectra step, spectral characteristics such as retention time range and positive and negative ion modes are selected;

[0032] In the align retention time step, all compounds in all samples are peak aligned according to retention time;

[0033] In the detect compounds step, all compounds in the data are extracted by setting a series of parameters; preferably, the parameters include mass deviation range, intensity range, S / N threshold, minimum peak intensity, extracted ion settings, minimum and maximum elemental composition, etc.;

[0034] In the group compounds step, all the same compounds with different adduction modes are grouped.

[0035] Specifically, the method for screening the hypoglycemic active ingredients of traditional Chinese medicine based on the GLP-1 receptor, in step (4), further includes the step of structural identification of the screened active ingredients that can specifically bind to the GLP-1 receptor.

[0036] The present invention also discloses the application of the method for screening the glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor in the field of drug screening and analysis.

[0037] The present invention also discloses a method for screening the hypoglycemic active ingredients of traditional Chinese medicine from ginseng, which comprises the steps of using ginseng extract as raw material and screening the hypoglycemic active ingredients of traditional Chinese medicine according to the method;

[0038] Preferably, the ginseng extract is an alcohol extract of ginseng;

[0039] Preferably, the ginseng extract is a methanol extract of ginseng.

[0040] The GLP-1 receptor-based screening method for hypoglycemic active ingredients in traditional Chinese medicines described herein comprises adding a GLP-1 receptor to a traditional Chinese medicine extract, incubating the mixture, dissociating the resulting receptor-ligand complex, collecting the dissociation solution for LC-MS analysis, and statistically analyzing the compounds contained in the analytical sample and the blank sample to obtain active ingredients that specifically bind to the GLP-1 receptor. The GLP-1 receptor-based screening method for hypoglycemic active ingredients in traditional Chinese medicines described herein can be used to screen for GLP-1 receptor agonists in traditional Chinese medicines, effectively overcoming the drawbacks of the traditional screening model of first isolating and then testing the activity of natural active substances from complex traditional Chinese medicine extracts. It has the advantages of a short experimental cycle and a low workload, enabling large-scale, high-throughput screening, and has positive implications for the development of diabetes drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0042] Figure 1 They are respectively the mass spectra of the ginseng extract before co-incubation (Extract), the sample group with added GLP-1 receptor (Sample), and the blank group without added GLP-1 receptor (Control) in Example 1. DETAILED DESCRIPTION

[0043] In the following examples of the present invention, the present invention provides a method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor, which specifically includes the following steps.

[0044] (1) The Chinese herbal medicine extract to be tested is dissolved in a buffer solution to obtain a drug stock solution; a portion of the drug stock solution of the same state is taken as a sample group, and a GLP-1 receptor solution is added thereto for incubation to obtain a GLP-1 receptor-ligand conjugate; the other portion is taken as a blank group, and an equal amount of buffer solution is added thereto to replace the GLP-1 receptor solution, and the operation is carried out in parallel with the sample group to obtain a blank sample solution.

[0045] Preferably, in the following embodiments of the present invention, the incubated reactants further include a step of ultrafiltration treatment, preferably ultrafiltration centrifugation treatment, and the ultrafiltration centrifuge tube uses an ultrafiltration membrane AMICON ULTRA 0.5mL (Millipore) with a low protein binding rate. More preferably, the molecular retention capacity of the ultrafiltration centrifuge tube is 10KDa.

[0046] Preferably, in the following embodiments of the present invention, the reactants after incubation further include a rinsing step to wash away other unbound small molecule compounds; in the rinsing step, the buffer used to rinse the GLP-1 receptor-ligand conjugate is ammonium acetate buffer (150 mM, pH 7.5), the volume is 250 μL, and the number of rinsing is five times. After each rinsing, the centrifuge speed is 14000 r / min and the centrifugation is 10 min.

[0047] Preferably, in the following examples of the present invention, the buffer used for dissolving the Chinese herbal extracts, GLP-1 receptors and incubation is 50 mM HEPES buffer (pH 7.5).

[0048] Preferably, in the following examples of the present invention, the concentration of GLP-1 receptor is 300 μg / mL.

[0049] Preferably, in the following examples of the present invention, the incubation time with the Chinese herbal medicine extract solution is 60 min and the incubation temperature is 25°C.

[0050] (2) The obtained GLP-1 receptor-ligand conjugate is added to a dissociation solution for dissociation treatment to release the small molecule ligand that can bind to the GLP-1 receptor, and the dissociation solution is collected; the blank sample solution is subjected to the same parallel operation.

[0051] Preferably, in the dissociation step, the dissociation solution is preferably methanol, and the dissociation is preferably performed three times, with 100 μL of methanol used each time. After the dissociation, the centrifuge speed is 14,000 r / min, and the centrifugation is performed for 15 minutes.

[0052] (3) The dissociation solution was blown dry with nitrogen, and the residue was dissolved with an appropriate solvent and fixed to volume to obtain analytical samples of the sample group and blank samples of the blank group; the analytical samples of the sample group and the blank samples of the blank group were analyzed by liquid chromatography-mass spectrometry using the same analytical conditions.

[0053] Preferably, after the dissociation solution is dried with nitrogen, the solvent used to dissolve the residue and adjust the volume is methanol:water (v / v, 50:50), and the adjusted volume is 50 μL.

[0054] When the liquid chromatography-mass spectrometry technique is used for analysis, detection instruments, detection means and analysis conditions known in the art may be selected. Depending on the extract, the detection parameters may be adjusted and optimized within a reasonable range.

[0055] (4) Using the data statistical analysis method, all compounds in the analytical samples of the sample group and the blank samples of the blank group were statistically analyzed to obtain the active ingredients that can specifically bind to the GLP-1 receptor.

[0056] Preferably, the present invention uses Compound Discoverer TM (Thermo Scientific TM , version 3.2.0.421) software for data processing, the specific methods include:

[0057] (a) Importing data from eight samples collected by UHPLC-Orbitrap Exploris 240 into Compound Discoverer TM Software that performs peak alignment and peak extraction for all compounds in each sample by establishing a compound extraction process;

[0058] The designed compound extraction process is "input files→select spectra→align retention time→detect compounds→group compounds."; wherein, in the input files step, the spectrum to be processed is selected; in the select spectra step, the spectrum characteristics such as retention time range and positive and negative ion modes are selected; in the align retention time step, all compounds in all samples are peak aligned according to the retention time; in the detect compounds step, all compounds in the data are extracted by setting a series of parameters, such as; mass deviation range, intensity range, S / N threshold, minimum peak intensity, extracted ion settings, minimum and maximum elemental composition, etc.; in the group compounds step, all the same compounds with different adduction methods are classified. After running the extraction process, the information required for data statistical analysis is obtained, such as the molecular weight, retention time and peak area of ​​each compound in each sample, etc. These data will be used for subsequent analysis;

[0059] (b) Calculate the peak area ratio of each compound in the sample group and the blank group; In this step, calculate the peak area ratio of each compound in each pair of sample group and blank group (sample group / blank group), and calculate the average peak area ratio of each compound (n=4);

[0060] (c) The peak areas of each compound in the sample group and the blank group were subjected to a t-test. Compounds with an average peak area ratio between the sample group and the blank group greater than 1 (n=4) and p<0.05 (n=4) after the t-test were preliminarily considered to be active ingredients that can specifically bind to the GLP-1 receptor.

[0061] The method of the present invention further comprises the step of performing targeted MS / MS analysis on the identified active ingredient using mass spectrometry to identify the structure of the active ingredient.

[0062] Example 1

[0063] In this embodiment, the screening of hypoglycemic active ingredients based on the GLP-1 receptor in the traditional Chinese medicine ginseng is taken as an example to further illustrate the method and process of the present application.

[0064] Ginseng medicinal materials were crushed into fine powder, 15 g was weighed, and 150 mL of 70% methanol was added for ultrasonic extraction. The mixture was filtered and extracted three times. The extracts were combined and evaporated to dryness on a rotary evaporator at 40 ° C. The reagents were recovered and the obtained extract was dried under reduced pressure to obtain 3.5 g of ginseng extract sample.

[0065] Dissolve 2.5 mg of the ginseng extract in 1 mL of 50 mM HEPES buffer (pH 7.5) and filter through a 0.45 μm microporous filter to obtain a 2.5 mg / mL stock solution of ginseng extract. Separately, dissolve 300 μg of GLP-1 receptor (purchased from Sino Biological Technologies Co., Ltd., Cat. 13944-H02H) in 1 mL of 50 mM HEPES buffer (pH 7.5) to prepare a 300 μg / mL GLP-1 receptor solution.

[0066] 100 μL of the above-mentioned ginseng sample solution (2.5 mg / mL) was taken and placed in a 1.5 mL EP tube. Then 100 μL of 300 μg / mL GLP-1 receptor solution was added, incubated at 25°C for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 14000 r / min for 10 min; 250 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and centrifuged at 14000 r / min for 10 min, for a total of 5 rinses; then 100 μL of methanol was added for dissociation, and centrifuged at 14000 r / min for 15 min, for a total of 3 dissociations.

[0067] The dissociation solution was collected and dried with nitrogen, and then reconstituted with 50 μL of methanol: water (v / v, 50:50) for liquid chromatography-mass spectrometry analysis.

[0068] During the above operation, an equal amount of HEPES buffer was added to the blank group sample instead of the GLP-1 receptor solution and the operation was carried out in parallel with the sample group. The sample group and the blank group were respectively operated in parallel in quadruplicate.

[0069] The analytical samples of the sample group obtained above and the blank samples of the blank group were analyzed by liquid chromatography-mass spectrometry using the same analytical conditions.

[0070] In this example, UHPLC-Orbitrap Exploris 240 (Thermo Scientific, USA) was used for sample data acquisition, and a Vanquish equipped with a binary pump and a heated column oven was used. TM A Flex UHPLC system (ThermoScientific, USA) was used for the separation of multiple components.

[0071] In this embodiment, considering the case of ginseng extract, the optimized analytical chromatographic conditions in the LC-MS analysis step are selected as follows:

[0072] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm);

[0073] Precolumn ACQUITY UPLC BEH C18 VanGuard TM Pre-Column (2.1×5mm, 1.7μm);

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

[0075] Elution program: (v / v): 0-7min, 98%A: 2%B-80%A: 20%B; 7-10min, 80%A: 20%B-75%A: 25%B; 10-20min, 75%A: 25%B-60%A: 40%B; 20-25min, 60%A: 40%B-35%A: 65%B; 25-30min, 35%A: 65%B-5%A: 95%B; 30-31min, 5%A: 95%B-5%A: 95%B;

[0076] Flow rate: 0.3 mL / min;

[0077] Column temperature: 35°C;

[0078] Sample plate temperature: 10°C;

[0079] Injection volume: 2 μL.

[0080] In this embodiment, the optimized mass spectrometry conditions in the LC-MS analysis step are as follows: an Orbitrap Exploris 240 (Thermo Scientific, USA) high-resolution mass spectrometer equipped with a heated ESI source was used for data detection, in negative ion mode, with an ion source voltage of 2500 V; sheath gas: 5.08 L / min; auxiliary gas: 9.37 L / min; ion transfer tube temperature: 320° C.; nebulizer gas temperature: 350° C., a mass-to-charge ratio (m / z) scan range of 150-2000; and an internal calibration source, Thermo Scientific EASY-IC TM Perform mass calibration.

[0081] In this example, the mass spectra of the ginseng extract before incubation (Extract), the analysis sample group with the addition of GLP-1 receptor (Sample), and the blank sample group without the addition of GLP-1 receptor (Control) are shown in FIG. Figure 1 shown.

[0082] In this example, Compound Discoverer TM (Thermo Scientific TM , version 3.2.0.421) software was used for data processing.

[0083] Specific method: Import the above four analysis sample data and four blank sample data into CompoundDiscoverer TM Before data processing, the software established an extraction process for all compounds in the sample: "input files→select spectra→align retention time→detect compounds→group compounds." In the input files step, four sample group samples and four blank group samples were selected for subsequent data processing. In the select spectra step, the entire run time (0-30 min) and negative ion mode were selected. In the align retention time step, the mass deviation was set to 5 ppm. In the detect compounds step, the following parameters were set: mass deviation 5 ppm; S / N threshold: 3; minimum peak intensity 100,000; extracted ions: [MH] - , [M-H+HAc] - , [2M-H] - , [2M-H+HAc] - ; Minimum element composition: CHO; Maximum element composition: C 90 H 190 O90 ; In the group compounds step, the mass deviation was set to 5 ppm.

[0084] The compound extraction process was run to obtain the molecular weight, retention time, and peak area of ​​each compound in each sample. The peak area ratio of each compound in the sample group to the blank group was calculated, and the average peak area ratio for each compound was calculated (n=4). A t-test was performed on the peak areas of each compound in the sample group and the blank group. Compounds with an average peak area ratio greater than 1 (n=4) and a p<0.05 after the t-test (n=4) were preliminarily considered to be active ingredients that specifically bind to the GLP-1 receptor. The results are shown in Table 1 below.

[0085] Table 1 Active ingredients in ginseng extract that can specifically bind to GLP-1 receptors

[0086]

[0087]

[0088] Furthermore, in the experiment to verify the agonist activity of the five potential compounds listed in Table 1 on the GLP-1 receptor, the agonist activity of the above compounds on the GLP-1 receptor of HEK293 / CRE-Luc / GLP1R cells was tested.

[0089] The results showed that in the experiment of cAMP release in HEK293 / CRE-Luc / GLP1R cells, the EC50 value of the positive drug PF-06882961 was 0.1955nM, the EC50 value of the test sample 20(R)-ginsenoside Rh2 was 8.68μM, the EC50 value of the test sample ginsenoside Rk1 was 24.00μM, the EC50 value of the test sample 20(S)-ginsenoside Rh2 was 6.60μM, the EC50 value of the test sample ginsenoside Rg5 was 20.71μM, and the EC50 value of the test sample ginsenoside Compound K was 9.89μM.

[0090] The above research results show that the five compounds screened above have agonist activity on the GLP-1 receptor, and also demonstrate the effectiveness of the method of the present invention for screening GLP-1 receptor agonists from traditional Chinese medicine. The present invention is of great significance to the development of small molecule GLP-1R agonists.

[0091] Example 2

[0092] In this embodiment, the screening of hypoglycemic active ingredients based on GLP-1 receptor in the traditional Chinese medicine Gardenia jasminoides is taken as an example to further illustrate the method and process of the present application.

[0093] Dissolve 2 mg of Gardenia jasminoides extract in 1 mL of 50 mM HEPES buffer (pH 7.5) and filter through a 0.45 μm microporous filter to obtain a 2 mg / mL stock solution. Separately, dissolve 200 μg of GLP-1 receptor (purchased from Sino Biological Technologies, Inc., Cat. 13944-H02H) in 1 mL of 50 mM HEPES buffer (pH 7.5) to prepare a 200 μg / mL GLP-1 receptor solution.

[0094] 100 μL of the above sample solution was placed in a 1.5 mL EP tube, and then 100 μL of GLP-1 receptor solution was added. The tube was incubated at 25°C for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 14,000 r / min for 10 min. 250 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and the tube was centrifuged at 14,000 r / min for 10 min, for a total of 5 rinses. Subsequently, 100 μL of methanol was added for dissociation, and the tube was centrifuged at 14,000 r / min for 15 min, for a total of 3 dissociations.

[0095] The dissociation solution was collected and dried with nitrogen, then reconstituted with 50 μL of methanol:water (v / v, 40:60) for liquid chromatography-mass spectrometry analysis.

[0096] During the above operation, an equal amount of HEPES buffer was added to the blank group sample instead of the GLP-1 receptor solution and the operation was carried out in parallel with the sample group. The sample group and the blank group were respectively operated in parallel in quadruplicate.

[0097] The analytical samples of the sample group obtained above and the blank samples of the blank group were subjected to liquid chromatography-mass spectrometry analysis and data analysis under the same analytical conditions to screen and obtain the desired hypoglycemic active ingredients of traditional Chinese medicine.

[0098] This example can complete the screening and verification of hypoglycemic active ingredients in Gardenia jasminoides based on the above method.

[0099] Example 3

[0100] In this embodiment, the screening of hypoglycemic active ingredients based on GLP-1 receptor in the traditional Chinese medicine Puerariae Radix is ​​taken as an example to further illustrate the method and process of the present application.

[0101] 3 mg of Pueraria root extract was dissolved in 1 mL of HEPES buffer (50 mM, pH 7.5) and filtered through a 0.45 μm microporous filter to obtain a stock solution of 3 mg / mL. Separately, 400 μg of GLP-1 receptor (purchased from Sino Biological Technologies Co., Ltd., Cat. 13944-H02H) was dissolved in 1 mL of HEPES buffer (50 mM, pH 7.5) to prepare a 400 μg / mL GLP-1 receptor solution.

[0102] 100 μL of the above sample solution was placed in a 1.5 mL EP tube, and then 100 μL of GLP-1 receptor solution was added. The tube was incubated at 25°C for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 14,000 r / min for 10 min. 250 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and the tube was centrifuged at 14,000 r / min for 10 min, for a total of 5 rinses. Subsequently, 100 μL of methanol was added for dissociation, and the tube was centrifuged at 14,000 r / min for 15 min, for a total of 3 dissociations.

[0103] The dissociation solution was collected and dried with nitrogen, and then reconstituted with 50 μL of methanol: water (v / v, 60:40) for liquid chromatography-mass spectrometry analysis.

[0104] During the above operation, an equal amount of HEPES buffer was added to the blank group sample instead of the GLP-1 receptor solution and the operation was carried out in parallel with the sample group. The sample group and the blank group were respectively operated in parallel in quadruplicate.

[0105] The analytical samples of the sample group obtained above and the blank samples of the blank group were subjected to liquid chromatography-mass spectrometry analysis and data analysis under the same analytical conditions to screen and obtain the desired hypoglycemic active ingredients of traditional Chinese medicine.

[0106] This example can complete the screening and verification of hypoglycemic active ingredients in Pueraria lobata based on the above method.

[0107] Example 4

[0108] The method for screening hypoglycemic active ingredients described in this example is the same as that in Example 1, except that: 100 μL of the above-mentioned ginseng sample solution (2.5 mg / mL) was taken and placed in a 1.5 mL EP tube, and then 100 μL of 100 μg / mL GLP-1 receptor solution was added, incubated at 25° C. for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 14,000 r / min for 10 min; 250 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and centrifuged at 14,000 r / min for 10 min, for a total of 5 rinses; then 100 μL of methanol was added for dissociation, and centrifuged at 14,000 r / min for 15 min, for a total of 3 dissociations.

[0109] In this embodiment, the concentration of the GLP-1 receptor solution was 100 μg / mL, while the concentration of the ginseng sample solution was 2.5 mg / mL. The final screening result was not ideal, which may be due to the low concentration of the GLP-1 receptor solution.

[0110] Example 5

[0111] The method for screening hypoglycemic active ingredients described in this example is the same as that in Example 1, except that: 100 μL of the above-mentioned ginseng sample solution (2.5 mg / mL) was taken and placed in a 1.5 mL EP tube, and then 100 μL of 300 μg / mL GLP-1 receptor solution was added, incubated at 25° C. for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 10,000 r / min for 10 min; 250 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and centrifuged at 10,000 r / min for 10 min, for a total of 5 rinses; then 100 μL of methanol was added for dissociation, and centrifuged at 10,000 r / min for 15 min, for a total of 3 dissociations.

[0112] In this embodiment, the centrifuge speed was set to 10,000 rpm during the rinsing and dissociation steps. During the rinsing step, the purpose of post-rinsing centrifugation was to remove unbound small molecule compounds and the solution. Therefore, the less residual liquid in the centrifuge tube, the better the removal of small molecule compounds. During the dissociation step, the purpose of centrifugation was to collect the dissociated small molecule compounds. Therefore, the less residual liquid in the centrifuge tube, the better the collection of small molecule ligands.

[0113] Therefore, when the centrifuge speed is 10,000 r / min compared to 14,000 r / min, the amount of liquid remaining in the centrifuge tube during the rinsing and dissociation stages is less at 14,000 r / min, so the centrifuge speed was finally determined to be 14,000 r / min.

[0114] Example 6

[0115] The method for screening hypoglycemic active ingredients described in this example is the same as that in Example 1, except that: 100 μL of the above-mentioned ginseng sample solution (2.5 mg / mL) was taken and placed in a 1.5 mL EP tube, and then 100 μL of 300 μg / mL GLP-1 receptor solution was added, incubated at 25°C for 60 min, transferred to an ultrafiltration centrifuge tube AMICON ULTRA 0.5 mL (10 KDa, Millipore), and centrifuged at 14000 r / min for 10 min; 100 μL of ammonium acetate buffer (150 mM, pH 7.5) was added to rinse unbound small molecule compounds, and centrifuged at 14000 r / min for 10 min, for a total of 3 rinses; then 100 μL of methanol was added for dissociation, and centrifuged at 14000 r / min for 15 min, for a total of 3 dissociations.

[0116] In this embodiment, in the rinsing step, when the volume of the rinsing solution was 100 μL and the number of rinsing times was 3, it was found that the screening results were not ideal. This may be because the volume of the rinsing solution was insufficient and the number of rinsing times was too few, resulting in the incomplete removal of unbound small molecule compounds, causing interference with the results.

[0117] Therefore, in theory, increasing the volume of the rinsing solution and the number of rinsing times can more easily remove unbound small molecule compounds. Therefore, in the final implementation case, the volume of the rinsing solution was 250 μL and the number of rinsing times was 5.

[0118] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor, characterized in that: The steps include: (1) adding a buffer solution to dissolve the Chinese herbal extract to obtain a drug stock solution; adding the drug stock solution to a GLP-1 receptor solution for incubation to obtain a GLP-1 receptor-ligand conjugate; and adding another amount of the drug stock solution to an equal amount of the buffer solution to the GLP-1 receptor solution for incubation to obtain a blank sample solution; (2) adding the GLP-1 receptor-ligand conjugate to a dissociation solution for dissociation treatment to release the small molecule ligand that can bind to the GLP-1 receptor, and collecting the dissociation solution; the blank sample solution is operated in parallel; (3) drying the dissociation solution, collecting the residue, adding a constant volume solvent to dissolve, and obtaining an analytical sample. The blank sample solution is subjected to a parallel operation to obtain a blank sample; and the analytical sample and the blank sample are subjected to LC-MS analysis respectively; (4) performing statistical analysis on the compounds contained in the analysis sample and the blank sample to obtain the active ingredients that can specifically bind to the GLP-1 receptor.

2. The method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to claim 1, characterized in that: In the step (1), the buffer comprises HEPES buffer; Preferably, the buffer comprises 30-80 mM HEPES buffer, pH 7.5±0.2; Preferably, the concentration of the GLP-1 receptor solution is 200-400 μg / mL; Preferably, the incubation temperature of the incubation step is 20-30° C., and the incubation time is 40-80 min.

3. The method for screening the glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to claim 1 or 2, characterized in that: The step (1) further comprises the step of ultrafiltration and / or rinsing the incubated reactants to obtain the desired GLP-1 receptor-ligand conjugate; Preferably, the ultrafiltration treatment step includes the step of ultrafiltration membrane filtration; Preferably, the rinsing step includes adding ammonium acetate buffer for rinsing.

4. The method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to any one of claims 1 to 3, characterized in that: In the step (2), the dissociation solution comprises methanol; In the step (3), the constant volume solvent includes a methanol-water solution; Preferably, the volume ratio of the methanol-water solution is 40-60:60-40 (v / v).

5. The method for screening the glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to any one of claims 1 to 4, characterized in that: In step (4), the data statistical analysis step uses Compound Discoverer TM Software for data processing.

6. The method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to claim 5, characterized in that: The data statistical analysis step comprises: (a) Import the data of the analytical sample and blank sample collected in step (3) into Compound Discoverer TM Software that performs peak alignment and peak extraction for all compounds in each sample by establishing an extraction process for any compound; (b) calculating the peak area ratio of each compound in the analytical sample and the blank sample, respectively, and calculating the average peak area ratio of each compound; (c) A t-test was performed on the peak areas of the analytical samples and blank samples of each compound, and the compounds whose average ratio of the peak areas in the analytical samples and blank samples was greater than 1 and whose p value was less than 0.05 after the t-test were preliminarily regarded as active ingredients that could specifically bind to the GLP-1 receptor.

7. The method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to claim 6, characterized in that: In the step (b), the designed compound extraction process is "input files→select spectra→alignretention time→detect compounds→group compounds."; wherein, In the input files step, the graphs to be processed are selected; In the select spectra step, spectral characteristics such as retention time range and positive and negative ion modes are selected; In the align retention time step, all compounds in all samples are peak aligned according to retention time; In the detect compounds step, a series of parameters are set; preferably, the parameters include mass deviation range, intensity range, S / N threshold, minimum peak intensity, extraction ion setting, minimum and maximum elemental composition, etc. to extract all compounds in the data; In the group compounds step, all the same compounds with different adducting modes are grouped.

8. The method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to any one of claims 1 to 7, characterized in that: The step (4) also includes a step of structurally identifying the screened active ingredient that can specifically bind to the GLP-1 receptor.

9. Application of the method for screening glucose-lowering active ingredients of traditional Chinese medicine based on GLP-1 receptor according to any one of claims 1 to 8 in the field of drug screening and analysis.

10. A method for screening a Chinese medicine hypoglycemic active ingredient from ginseng, characterized in that: The method comprises the steps of using ginseng extract as raw material and screening the glucose-lowering active ingredients of traditional Chinese medicine according to the method described in any one of claims 1 to 8; Preferably, the ginseng extract is an alcohol extract of ginseng; Preferably, the ginseng extract is a methanol extract of ginseng.

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