Targeted screening method and application of xanthine oxidase inhibitor in radix puerariae

Abstract

The invention discloses a targeted screening method and application of a xanthine oxidase inhibitor in radix puerariae, and belongs to the technical field of medicinal chemistry. A surface chemical exchange method is carried out on xanthine oxidase through a ligand fishing technology, ligands of active groups of xanthine oxidase are modified to the surfaces of magnetic metal nanoparticles, the magnetic metal nanoparticles and a to-be-separated solution are incubated together, and then specific components with the xanthine oxidase inhibiting effect are successfully screened out through a UPLC-Q-TOF-MS / MS technology. The combination of the active component and the XOD active site is verified through molecular docking simulation to confirm that the active component is an effective inhibitor. The invention provides a reliable basis for further clinical research and development of drugs for treating hyperuricemia, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly relates to a method for targeted screening and application of xanthine oxidase inhibitors in Pueraria lobata Background Art

[0002] Xanthine oxidase (XOD) is a key enzyme that regulates uric acid production. Its function is to convert hypoxanthine into xanthine, convert xanthine into uric acid, and simultaneously generate reactive oxygen species (ROS). Abnormal elevation of the enzyme activity will lead to hyperuricemia, causing gout, uric acid nephropathy and cardiovascular diseases. By inhibiting the activity of XOD, the production of uric acid can be inhibited, and hyperuricemia (HUA) can be alleviated.

[0003] Currently, the clinically applied xanthine oxidase inhibitors mainly include two categories: chemically synthesized drugs (such as allopurinol, febuxostat) and natural products. Although the chemically synthesized drugs have definite curative effects, they have serious adverse reactions (such as allergic reactions, cardiovascular risks), and drug resistance will occur after long-term use. Natural products (such as flavonoids, polyphenolic compounds) have attracted much attention due to their structural diversity and low toxicity, but the screening rate of their active ingredients is low and the action mechanism is unclear. In addition, most of the existing inhibitors target the molybdenum active center of XOD, lacking the exploration of other regulatory sites of the enzyme, resulting in serious homogenization of inhibitor structures. Developing new type of highly efficient and low-toxic XOD inhibitors, especially mining multi-target inhibitors from traditional Chinese medicines, has become a current research hotspot.

[0004] Puerariae Lobatae Radix is a traditional Chinese medicinal material, and its main active ingredients are isoflavone compounds and a small amount of flavonoid substances. Research shows that the content of isoflavone components in Pueraria lobata is relatively high. Isoflavones can be divided into glycosides and aglycones. The former includes puerarin, daidzin, etc., and the latter mainly refers to daidzin, genistein, coumarin, isoglycyrrhizin glycoside, etc. A large number of studies have shown that flavonoid components can effectively inhibit the activity of XOD, reduce uric acid production and have potential kidney protection effects. Based on the active ingredients in total flavonoids of Pueraria lobata, finding specific compounds with effective inhibitory effects on XOD is of great significance for the treatment of hyperuricemia.

[0005] The existing XOD inhibitor screening technologies mainly include biochemical detection methods (such as ultraviolet spectrophotometry, HPLC method), high-throughput screening, immobilized enzyme affinity screening, virtual screening, etc. The traditional technologies have three common defects: they cannot quickly locate active ingredients from complex natural extracts; the screening and structure identification links are disjointed, resulting in a long research cycle; and there is a lack of in-depth verification of the interaction mechanism between active ingredients and target enzymes. Therefore, there is an urgent need to develop a screening method that integrates capture, high-efficiency identification and mechanism verification. Summary of the Invention

[0006] The present invention provides a targeted screening method for xanthine oxidase inhibitors in Pueraria lobata, based on a screening strategy integrating ligand fishing technology - liquid chromatography - mass spectrometry - molecular docking to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions: A targeted screening method for xanthine oxidase inhibitors in Pueraria lobata, comprising: co-incubating a purified extract of Pueraria lobata with Fe3O4@SiO2-XOD magnetic nanoparticles, separating the ligand-XOD inhibitor complex from the unbound complex by magnetic separation, then performing chromatographic analysis on the dissociated ligand-XOD inhibitor complex, subsequently performing mass spectrometry analysis by the UPLC-Q-TOF-MS / MS method to identify the ligand compounds released by the -XOD inhibitor complex, and verifying the binding mode and interaction of the ligand compounds with the XOD active site through molecular docking simulation to confirm effective XOD inhibitors.

[0008] Verifying the binding mode and interaction of the ligand compounds with the XOD active site through molecular docking simulation includes the following steps: a. Obtaining the crystal structure of XOD from the Protein Data Bank, removing water molecules and cofactors, and retaining the active site; b. Constructing and optimizing the three-dimensional structure of the screened ligand compounds; c. Using molecular docking software to calculate the binding energy and hydrogen bond and hydrophobic interaction sites between the ligand and XOD; d. Compounds with a binding energy ≤ -9.0 kcal / mol are confirmed as effective XOD inhibitors.

[0009] The specific steps of the co-incubation are as follows: incubating a 0.5 mg / ml Pueraria lobata extract with 5 mg of Fe3O4@SiO2-XOD magnetic nanoparticles at a volume ratio of 1:2 in a thermostatic shaker at 180 rpm and 30 °C for 30 minutes.

[0010] After co-incubation, the specific steps of magnetic separation are as follows: washing three times with PBS buffer to elute the compounds that did not specifically bind to XOD, dissociating with 99.9% methanol, and then using magnetic separation to aspirate the supernatant to obtain the final eluate.

[0011] The chromatographic analysis is specifically performed by using an HSS T3 chromatographic column, with mobile phase A being a water / acetonitrile (95 / 5, volume / volume) solution containing 0.1% formic acid, and mobile phase B being an acetonitrile / isopropanol / water (47.5 / 47.5 / 5, volume / volume / volume) solution containing 0.1% formic acid; the flow rate is 0.40 mL / min, the column temperature is 40 °C, and the injection volume is 3 μL.

[0012] The mass spectrometry is specifically performed in the multiple reaction monitoring (MRM) mode, and the ion source parameters are as follows: mass scanning range m / z: 70 - 1050. The ion spray voltage in the positive mode is 3400 V, the ion spray voltage in the negative mode is -2800 V, the declustering voltage is 50 V, the nebulizing gas is 50 psi, the auxiliary heating gas is 50 psi, the curtain gas is 30 psi, the ion source heating temperature is 400 °C, and the collision energy is cycled from 20 - 60 V.

[0013] The purified Pueraria lobata extract is prepared by the following method: The fresh Pueraria lobata is dried in the sun and then crushed into a powder passing through a 40 - 80 mesh sieve. The Pueraria lobata powder is fully mixed with 40% ethanol, heated under reflux for extraction, and the extraction operation is repeated twice. The two extraction solutions are combined, concentrated by a rotary evaporator, and then subjected to vacuum freeze-drying to obtain the purified extract.

[0014] In the molecular docking simulation, the crystal structure of XOD is selected from PDB ID: 1FIQ, and the ligand compounds include one or more of puerarin, daidzein, formononetin, and β-sitosterol.

[0015] The active pocket of the molecular docking covers the molybdenum cofactor binding region of XOD, and the hydrogen bond and hydrophobic interaction residues include GLU A:23, ARG A:32, PHE C:798, LEU A:27, and TRP B:283.

[0016] Application of the xanthine oxidase inhibitor obtained by the above screening method in the preparation of anti-hyperuricemia drugs.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. High-efficiency targeted screening and multi-component synchronous capture The present invention specifically captures potential inhibitors from Pueraria lobata extract through XOD-immobilized magnetic beads (Fe3O4@SiO2-XOD), and combines the ligand fishing technology to achieve synchronous fishing of multi-target active components (such as puerarin, daidzein, formononetin, and β-sitosterol), breaking through the limitation of the traditional method that cannot quickly locate active ingredients from complex natural products, and significantly improving the screening efficiency and accuracy.

[0018] 2. Combination of high-precision separation and identification techniques By using the UPLC-Q-TOF-MS / MS method, high-efficiency separation and accurate identification of active ingredients are achieved, the mass scanning range covers m / z 70 - 1050, and the detection rate of low-abundance compounds is ensured through the multiple reaction monitoring (MRM) mode, solving the problem of the disconnection between the screening and structure identification links in the prior art and shortening the research cycle.

[0019] 3. Molecular docking reveals the mechanism of action For the first time, the binding mode of the active ingredient to the active site of XOD was clarified through molecular docking simulation (AutoDock Vina), and the binding energy was as low as -9.9 kcal / mol (such as formononetin), revealing the key residues of hydrogen bonds (such as GLU A:23, ARG A:32) and hydrophobic interactions (such as TRP B:283, ILE A:66), providing a theoretical basis at the molecular level for the structural optimization and targeted design of inhibitors.

[0020] 4. Multi-dimensional technology integration and innovative application By deeply integrating ligand fishing, liquid chromatography-mass spectrometry, metabolomics and molecular docking techniques, not only can the component identification be achieved, but also the pharmacokinetic behavior of the active ingredient can be traced, and the active mechanism of inhibiting XOD can be comprehensively analyzed, providing an integrated solution from screening to mechanism verification for the preclinical research of hyperuricemia drugs.

[0021] 5. Promote the development and clinical transformation of natural drugs The screened XOD inhibitors (such as puerarin, β-sitosterol) have the characteristics of low toxicity and multi-target synergistic effects. Compared with traditional chemically synthesized drugs (such as allopurinol), they can significantly reduce the risks of allergy and cardiovascular diseases, laying a scientific foundation for the development of new, highly efficient and low-toxic anti-hyperuricemia drugs and having broad clinical application prospects.

[0022] In summary, the present invention solves the core problems such as low screening efficiency and unclear mechanism of natural product active ingredients through technological innovation, and provides an efficient and reliable method system for the targeted discovery, drug development and clinical transformation of traditional Chinese medicine active ingredients. Brief description of the drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 is the standard curve of rutin; Figure 2 is the graph of the in vitro inhibition rate of XOD; Figure 3 is the infrared spectrum of Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2-XOD; Figure 4For the recovery effect of magnetic nanoparticles, where A is the state diagram of Fe3O4@SiO2-XOD magnetic nanoparticles being ultrasonically and completely dispersed in PBS buffer solution, and B is the state diagram of Fe3O4@SiO2-XOD magnetic nanoparticles being rapidly separated from the suspension after being adsorbed by a magnet; Figure 5 It is the UHPLC-Q-TOF-MS / MSTIC diagram of the methanol eluate of XOD-targeted fishing for Pueraria lobata extract.

[0025] Figure 6 It is the statistical bar chart of KEGG compound classification.

[0026] Figure 7 It is the statistical bordered column chart of KEGG pathways.

[0027] Figure 8 It is the pie chart of HMDB compound classification.

[0028] Figure 9 It is the simulated molecular docking results of puerarin, daidzein, formononetin, and β-sitosterol in the examples of the present invention. Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application.

[0030] The embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

[0031] Example 1 Extraction of active ingredients from Pueraria lobata 1. Pretreatment of medicinal materials Wash, dry, crush into powder, and pass through a 40-60 mesh sieve the Chinese medicinal material of Pueraria lobata to obtain the crude powder of Pueraria lobata; weigh the crude powder, add a 40% ethanol solution with a solid-liquid ratio of 1:20 (g / ml), and extract twice at a constant water bath temperature of 80°C, and combine the filtrates.

[0032] Furthermore, concentrate the filtrate in a vacuum rotary evaporator to obtain the Pueraria lobata extract; then freeze-dry the extract after evaporation and concentration to obtain the Pueraria lobata extract powder.

[0033] Preferably, the freeze-drying is carried out in a vacuum freeze dryer.

[0034] 2. Determination of total flavonoid content in Pueraria lobata In this invention, rutin was used as the standard substance, and the UV method was adopted to determine the total flavonoid content in Pueraria lobata; the rutin standard curve at a wavelength of 510 nm is shown in Figure 1 , and the regression equation is Y = 0.1071X + 0.0054, R 2 = 0.9991, indicating that there is a linear relationship between the solution concentration and the absorbance in the range of 0.01 - 0.05 mg / ml.

[0035] After detection, the average content of total flavonoids in Pueraria lobata was 5.21 mg / g, and the RSD value was 0.69%, and the results are shown in Table 1.

[0036] Table 1 Determination results of total flavonoids in Pueraria lobata 3. Effect of Pueraria lobata extract on xanthine oxidase, a key target for hyperuricemia: Xanthine oxidase can decompose xanthine to generate uric acid, so the activity of xanthine oxidase can be judged according to the generation of uric acid. At present, the main research method for the inhibition experiment of xanthine oxidase is the spectrophotometry method, and the test basis is that uric acid has a characteristic absorption peak at 295 nm.

[0037] The specific method is as follows: (1) Prepare reagents Xanthine (XA, purity: 98%) and allopurinol (purity: 98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; xanthine oxidase (XOD, 50 u / mg) was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0038] (2) Preparation of test reagents Preparation of xanthine solution: Dissolve 2.28 mg XO with 1 ml of 1 mol / L NaOH solution, and then dilute it to 100 ml with PBS (0.1 mmol / L, pH: 7.2 - 7.4) solution.

[0039] Preparation of xanthine oxidase (XOD): Accurately weigh 0.5 mg XOD and dissolve it in 10 ml of PBS. At this time, the enzyme concentration is about 5 U / ml.

[0040] Preparation of test reagents to be measured: Weigh 32.7 mg of allopurinol and dissolve it in 40 ml of PBS. Respectively absorb 2.5 ml, 4.17 ml, 5.84 ml, 7.5 ml, 9.2 ml of xanthine solution and make up the volume to 10 ml; respectively weigh 15 mg, 25 mg, 35 mg, 45 mg, 55 mg of Pueraria lobata extract and dissolve them in 10 ml of PBS. At this time, the final concentrations of each test solution in the system are 1.5 mg / ml, 2.5 mg / ml, 3.5 mg / ml, 4.5 mg / ml, 5.5 mg / ml respectively.

[0041] Sample group: Mix 1 ml of samples with different concentrations, 2 ml of PBS solution and 0.5 ml of XOD, and wait for 15 minutes at 30 °C; then add 1 ml of xanthine solution and wait for 10 minutes at 30 °C, and measure the spectrophotometer at 295 nm.

[0042] Sample control group: As described in the sample group, replace 0.5 ml of XOD solution with 0.5 ml of PBS solution, and the other steps are the same.

[0043] Use allopurinol as the positive group and positive control group, and its operation steps are the same as those of the sample group and sample control group.

[0044] Blank group: Add 3 ml of PBS solution and 0.5 ml of XOD to the colorimetric tube, and wait for 15 minutes at 30 °C; then add 1 ml of xanthine solution and wait for 10 minutes at 30 °C, and measure the spectrophotometer at 295 nm.

[0045] Blank control group: As described in the blank group, replace 0.5 ml of XOD solution with 0.5 ml of PBS solution, and the other steps are the same.

[0046] Use the final concentration of the test solution in the system as the abscissa and the inhibition rate of xanthine oxidase as the ordinate to draw a curve, and analyze the inhibitory effects of allopurinol and pueraria extract on xanthine oxidase. The obtained results are as Figure 2 shown.

[0047] From Figure 2 it can be seen that the pueraria extract of the present invention has an obvious inhibitory effect on xanthine oxidase. Under the comparison of the same concentration of pueraria extract and allopurinol, the inhibitory effects are the same under high-concentration conditions, and its IC50 value is 3.18 mg / ml.

[0048] Example 2 Preparation and Characterization of Magnetic Particle-Fixed Xanthine Oxidase 1. Preparation of Magnetic Nanoparticle-Fixed XOD Measure 1 ml of Fe3O4 dispersion into 952 ml of ethanol and 278 ml of water. After ultrasonic homogenization, add 30 ml of ammonia water, and then add 12 ml of tetraethyl orthosilicate. After stirring at room temperature for 8 hours, magnetically separate the product, wash the product several times with ultrapure water, and freeze-dry to obtain the sample Fe3O4@SiO2.

[0049] Weigh 139 mg of Fe3O4@SiO2 magnetic nanoparticles, sonicate for 10 minutes to completely disperse Fe3O4@SiO2 in 280 ml of absolute ethanol, then add 2.8 ml of 3-aminopropyltriethoxysilane, stir at room temperature for 6 hours, magnetically separate the product, wash it several times with ultrapure water, and freeze-dry to obtain amino-functionalized Fe3O4@SiO2 magnetic nanoparticles.

[0050] Suspend 130 mg of amino-functionalized Fe3O4@SiO2 magnetic nanoparticles in 130 ml of glutaraldehyde solution for 1 h of activation, magnetically attract, discard the supernatant; wash three times with PBS buffer, magnetically separate, discard the supernatant; add 56 ml of XOD solution (50 μg / ml) to the magnetic beads, after 2 h of activation, magnetically separate, wash three times with PBS buffer, and magnetically separate to obtain the sample Fe3O4@SiO2-XOD, and store it in PBS buffer at 4 °C.

[0051] 2. Characterize the magnetic nanoparticles by infrared spectroscopy and magnetic responsiveness analysis Fourier transform infrared spectroscopy analysis: Use a Fourier transform infrared spectrometer to scan unmodified Fe3O4 magnetic nanoparticles, Fe3O4@SiO2 magnetic nanoparticles, and Fe3O4@SiO2-XOD nanoparticles respectively, and the scanning wavelength is 400 - 4000 cm -1 .

[0052] Magnetic responsiveness analysis: Fe3O4 magnetic nanomaterials have superparamagnetism. Use a magnet outside the container to conduct a recovery experiment on Fe3O4@SiO2-XOD magnetic nanoparticles uniformly dispersed in the container, and observe the magnetic responsiveness performance and recovery performance.

[0053] Use FT-IR analysis technology to deeply study the functional groups and chemical bonds on the surface of the synthetic materials. As Figure 3 shown: 580 cm -1 is caused by the stretching vibration of the Fe-O-Fe bond in Fe3O4 and is the characteristic absorption peak of Fe3O4; after surface modification of Fe3O4 magnetic nanoparticles with tetraethyl orthosilicate, a new absorption peak appears at 1091.3 cm -1 , which is caused by the asymmetric stretching vibration of Si-O-Si in the silica layer, and 756.3 cm -1 and 848.1 cm -1 are the symmetric contraction vibrations of Si-O-Si, indicating that the silica layer has been successfully coated on the surface of the magnetic particles; after adding 3-aminopropyltriethoxysilane to modify -NH2 on the Fe3O4@SiO2 magnetic nanoparticles and immobilize xanthine oxidase, the absorption peak becomes broader at 3100 cm -1 , and at 1592.1 cm -1The absorption peak at this position is enhanced, which is caused by the C-N stretching vibration and N-H bending vibration, indicating that the functional amino group has been attached to the magnetic nanoparticles. At the same time, the stretching vibration peak at 1738.1 cm -1 is the stretching vibration peak of C=O, indicating that XOD is coated on the surface of the material.

[0054] The recovery performance of the magnetic particles was verified by rapid recovery through the adsorption of an external magnet, as Figure 4 shown: Fe3O4@SiO2-XOD magnetic nanoparticles were ultrasonically and completely dispersed in PBS ( Figure 4 A), and the Fe3O4@SiO2-XOD magnetic nanoparticles were rapidly separated from the homogeneous suspension by magnet adsorption ( Figure 4 B), achieving the effect of rapid recovery; the results show that Fe3O4@SiO2-XOD has excellent water dispersibility and magnetic responsiveness, which is very beneficial for subsequent ligand screening.

[0055] Example 3. Screening of active ingredients Prepare a 1 ml solution of Pueraria lobata extract (0.5 mg / ml) with PBS buffer for ligand capture experiments. Add 5 mg of Fe3O4@SiO2-XOD magnetic nanoparticles to the prepared Pueraria lobata extract solution, incubate at 180 rmp and 30 °C in a constant temperature shaker for 30 min, perform magnetic separation, and wash the magnetic nanoparticles 3 times with PBS buffer; then add 3 ml of methanol (99.9%) to the magnetic nanomaterial ligand complex, incubate under the same conditions as above for 1 h, perform magnetic separation, and aspirate the supernatant into a centrifuge tube; filter the Pueraria lobata extract solution and the methanol eluate after targeted fishing with a 0.22 μm microporous filter membrane and inject it into a UHPLC-Q Exactive HF-X instrument for analysis.

[0056] UPLC-Q-TOF-MS / MS detection conditions: Chromatographic conditions: 3 μL of the sample was separated by an HSS T3 chromatographic column (100 mm × 2.1 mm i.d., 1.8 μm) and then entered the mass spectrometry detection. Mobile phase A was a solution of water / acetonitrile (95 / 5, volume / volume) containing 0.1% formic acid, and mobile phase B was a solution of acetonitrile / isopropanol / water (47.5 / 47.5 / 5, volume / volume / volume) containing 0.1% formic acid. The flow rate was 0.40 mL / min and the column temperature was 40 °C.

[0057] Mass spectrometry conditions: The mass spectrometry signals of the samples were collected in positive and negative ion scanning modes, and the mass scanning range was m / z: 70 - 1050. The positive mode ion spray voltage was 3400 V, the negative mode ion spray voltage was -2800 V, the declustering voltage was 50 V, the nebulizing gas was 50 psi, the auxiliary heating gas was 50 psi, the curtain gas was 30 psi, the ion source heating temperature was 400 °C, and the collision energy was cycled at 20 - 60 V.

[0058] Under the above chromatographic conditions, the Pueraria lobata extract solution and the methanol eluate after targeted fishing were identified and analyzed by UPLC-Q-TOF-MS / MS.

[0059] The methanol eluate sample of XOD targeted fishing extraction was analyzed, and the total ion (TIC) chromatogram of the mass spectrometry in the negative ion mode was obtained, as Figure 5 shown.

[0060] According to the retention time, exact molecular weight and MS / MS mass spectrometry information of each component, combined with the established compound database, a total of 727 components were identified in the methanol eluate after targeted fishing, as shown in Table 2, including phenylpropanoids, alkaloids, quinones, long-chain unsaturated fatty acids, and 44 of them belong to flavonoids.

[0061] Table 2 723 components identified by UPLC-Q-TOF-MS / MS in the methanol eluate of XOD targeted fishing extraction.

[0062] Focus on the study of these 44 components in the XOD-targeted fishing Pueraria extract. According to the secondary mass spectrometry diagrams of the compounds and their main fragmentation processes, these 44 flavonoid components were identified as puerarin, coumarin, baicalein, apigenin, emodin, anthocyanin, phloretin, lutein, dihydroquercetin, proanthocyanidin, isorhodeanone, ononin, formononetin, calycosin, sanggenon C, isovitexin, genistein, apiole, pterocarpin, alloimperatorin, cirsimaritin, daidzein, scutellarin, cytisine, catechol, kaempferol, naringenin, pinobanksin, isoliquiritigenin, limettin, chlorocimifugin, 2-hydroxyflavanone, 4-hydroxycoumarin, mosla chinensis maxim flavone, pisum sativum linn chalcone B, vitex trifolia l glucoside, sophoricoside, carthamin, genkwanin, dioscin I, kaempferol-7-O-glucoside, 6-hydroxydaidzein, 6,2'-dihydroxyflavanone, ω-hydroxyemodin. The mass spectrometry information related to these compounds by UPLC-Q-TOF-MS / MS is shown in Table 3.

[0063] Table 3 Mass spectrometry information of 44 flavonoid components in the Pueraria extract after XOD-targeted fishing The identified metabolites were compared with the KEGG Compound database to obtain a summary of metabolite classification and merged statistical mapping; KEGG Compound is a collection of small molecules, biopolymers, and other chemical substances related to biological systems. Through KEGG compound classification, metabolites are divided into ten categories: antibiotics, carbohydrates, hormones and neurotransmitters, lipids, nucleic acids, peptides, organic acids, steroids, vitamins and cofactors, and others. Among them, lipids, carbohydrates, and organic acids account for a relatively high proportion, as Figure 6 shown.

[0064] By comparing the information of the identified metabolites with the KEGG Compound ID, the metabolic pathway information participated by the metabolites can be obtained and mapped. It can be seen that the pathways participated by the Pueraria metabolites after fishing are divided into four categories: metabolic pathways, organic system pathways, human disease, and environmental information processing pathways. Among them, in the metabolic pathways, lipid metabolism and the biosynthesis metabolic pathway of other secondary metabolites are the most important; in the organic system pathways, the digestive system and the nervous system are the most important, as Figure 7 shown.

[0065] The identified compounds were classified and plotted in the Human Metabolome Database (HMDB). It can be seen that the metabolites were divided into 18 major categories, among which phenylpropanoids and polyketides, lipids and lipid-like molecules, organic heterocyclic compounds, organic acids and their derivatives, oxygen-containing organic compounds, benzene-ring compounds, nucleic acids and nucleotide compounds, alkaloids and their derivatives, nitrogen-containing heterocyclic compounds, lignans and neolignans were the major ones, as Figure 8 shown.

[0066] Example 4 Molecular Docking Molecular docking simulations were performed between the screened compounds puerarin, daidzein, formononetin, and β-sitosterol and xanthine oxidase (XOD). The specific method is as follows: First, access the Uniprot database (http: / / www.uniprot.org / ) to retrieve the Uniprot ID (such as P47989) of the target protein "xanthine oxidase (XOD)"; then, through the RCSB PDB database (http: / / www.rcsb.org / ), input the Uniprot ID to screen the resolved crystal structures, and preferentially select the complex structure containing molybdenum cofactor (Mo-co) (such as PDB ID: 1FIQ); download the receptor protein file in PDB format, remove water molecules, coenzyme molecules, and original ligands from the structure, and retain the complete enzyme structure; use Pymol software to dehydrate and remove impurities from the saved PDB file, and then perform hydrogenation with adt and other processes, and save it as a PDB format file for later use.

[0067] Access the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ), retrieve the active ingredients of Pueraria lobata (such as formononetin, CID: 5280378), download the 2D SDF format file, construct and optimize the 2D structure through Chem3D software, and then save it as a Mol2 file for later use.

[0068] Use Auto Dock TOOL and Vina software to determine the active pocket and calculate the binding energy, etc., to obtain the complex file; import the complex file into Pymol software for visualization to obtain the docking result diagram; import the complex into Discovery Studio software to obtain the 2D display diagram; as Figure 9 shown in Table 4, the binding energies between puerarin, daidzein, formononetin, and β-sitosterol and xanthine oxidase are -9.1, -9.0, -9.9, and -9.4 kcal / mol respectively, indicating that the binding between these four compounds and the XOD active site is stable.

[0069] Puerarin formed 5 hydrogen bonds in the active site of XOD. Among them, GLU A:23 and ARG A:32 are the core residues for puerarin to inhibit XOD, while ARG B:233, LYS B:269 and ARG C:680 enhance the overall inhibitory efficacy through assisting in stabilization; meanwhile, 4 hydrophobic interactions were formed. Among them, LEU A:27 and LEU A:41 directly stabilize the puerarin parent nucleus through hydrophobic interaction, while the conformational adaptability of ALA A:28 and the allosteric regulation of GLU C:676 jointly improve the binding efficiency, and it realizes the efficient inhibition of XOD activity through the synergy of hydrophobic core anchoring and distal conformational regulation.

[0070] Daidzein formed 5 hydrogen bonds in the active site of XOD. Among them, PHE C:798 and GLU C:1261 are the core residues for daidzein to inhibit XOD, and they dominate the binding through π-π stacking and strong polar interaction respectively; GLN C:1194 and MET C:1038 assist in stabilization through hydrogen bonds and weak polar interaction, while the conformational adaptability of ALA C:1079 further improves the binding rate; meanwhile, PHE C:798, ALA C:1078 and GLU C:1261 form a multi-level binding network through hydrophobic interaction, which is the core structural basis for daidzein to inhibit XOD. Among them, the π-π stacking of PHE C:798 dominates the hydrophobic anchoring, ALA C:1078 provides conformational adaptability, and the hydrophobic-polar synergistic effect of GLU C:1261 enhances the inhibitory efficiency.

[0071] Formononetin formed 4 hydrogen bonds in the active site of XOD. Among them, the strong hydrogen bonds of LYS C:1045 and SER C:1080 are the core driving forces for formononetin to inhibit XOD, while the auxiliary hydrogen bonds of ALA C:1078 and THR C:1083 further optimize the binding conformation; meanwhile, ALA C:910, ARG C:912, GLN C:1040 and ALA C:1078 form a multi-level binding network through hydrophobic interaction. Among them, the ALA residue stabilizes the parent nucleus through van der Waals force, and ARG and GLN expand the action range through hydrophobic-polar synergistic effect.

[0072] β-sitosterol mainly plays a hydrophobic role in inhibiting XOD activity, where the π-π stacking of TRP B:283 and the π-alkyl interaction of ILE A:66 jointly anchor the steroid nucleus, and residues such as LYS A:57 and HIS A:82 enhance stability through local hydrophobic contacts; the rigid ring of PRO B:224 and the flexible side chain of ASN B:288 synergistically regulate the shape of the active pocket to adapt to the three-dimensional structure of β-sitosterol; in short, residues such as LYS A:57, ILE A:66, and TRP B:283 dominate the binding of β-sitosterol to XOD through multi-level hydrophobic interactions, and residues such as PRO B:224 and HIS A:82 provide conformational support.

[0073] Table 4 Interaction sites of puerarin, daidzein, formononetin, and β-sitosterol with the active site of xanthine oxidase

[0074] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for targeted screening of xanthine oxidase inhibitors in Pueraria lobata, comprising: The purified extract of Pueraria lobata was incubated with Fe3O4@SiO2-XOD magnetic nanoparticles, and the ligand-XOD inhibitor complex and the unbound complex were separated by magnetism. The dissociated ligand-XOD inhibitor complex was then subjected to chromatographic analysis and then mass spectrometry analysis by UPLC-Q-TOF-MS / MS method to identify the ligand compound released from the XOD inhibitor complex. The binding mode and interaction of the ligand compound with the XOD active site were verified by molecular docking simulation to confirm that it was an effective XOD inhibitor.

2. The screening method according to claim 1, characterized in that The binding mode and interaction between the ligand compound and the XOD active site are verified by molecular docking simulation, comprising the following steps: a. Obtain the crystal structure of XOD from the protein database, remove water molecules and cofactors, and retain the active site; b. constructing and optimizing the three-dimensional structure of the ligand compound obtained by screening; c. Use molecular docking software to calculate the binding energy, hydrogen bond and hydrophobic interaction sites between the ligand and XOD; d. Compounds with binding energies ≤ -9.0 kcal / mol were identified as potent XOD inhibitors.

3. The screening method according to claim 1, wherein The specific steps of the co-incubation are: incubate 0.5 mg / ml Pueraria extract and 5 mg Fe3O4@SiO2-XOD magnetic nanoparticles at a volume ratio of 1:2 in a constant temperature oscillator at 180 rpm and 30°C for 30 minutes.

4. The screening method according to claim 1, characterized in that After co-incubation, the specific steps of magnetic separation are: washing three times with PBS buffer to elute the compounds that are not specifically bound to XOD, dissociating with 99.9% methanol, and then using magnetic separation to absorb the supernatant to obtain the final eluate.

5. The screening method according to claim 1, characterized in that The chromatographic separation is specifically performed by using an HSS T3 chromatographic column, wherein mobile phase A is a water / acetonitrile (95 / 5, volume / volume) solution containing 0.1% formic acid, and mobile phase B is an acetonitrile / isopropanol / water (47.5 / 47.5 / 5, volume / volume / volume) solution containing 0.1% formic acid; the flow rate is 0.40 mL / min, the column temperature is 40°C, and the injection volume is 3 μL.

6. The screening method according to claim 1, wherein the mass spectrometry analysis is performed in multiple reaction detection mode, and the ion source parameters are as follows: mass scanning range m / z: 70-1050; positive mode ion spray voltage is 3400 V, negative mode ion spray voltage is -2800 V, declustering voltage is 50 V, spray gas is 50 psi, auxiliary heating gas is 50 psi, curtain gas is 30 psi, ion source heating temperature is 400°C, and 20-60 V cycle collision energy.

7. The screening method according to claim 1, wherein The kudzu root purified extract is prepared by the following method: fresh kudzu root is dried in the sun, then crushed into powder that passes through a 40-80 mesh sieve, the kudzu root powder is fully mixed with 40% ethanol, heated under reflux for extraction, the extraction operation is repeated twice, the two extracts are combined and concentrated by a rotary evaporator, and then vacuum freeze-dried to obtain a purified extract.

8. The screening method according to claim 1, characterized in that, In the molecular docking simulation, the crystal structure of XOD is selected from PDB ID: 1FIQ, and the ligand compound includes one or more of puerarin, daidzein, formononetin, and β-sitosterol.

9. The screening method according to claim 1, wherein The active pocket of the molecular docking covers the molybdenum cofactor binding region of XOD, and the hydrogen bond and hydrophobic interaction residues include GLU A:23, ARG A:32, PHE C:798, LEU A:27, and TRP B:

283.

10. Use of the xanthine oxidase inhibitor obtained by the screening method according to any one of claims 1 to 9 in the preparation of a drug for reducing uric acid.

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