A method for analyzing and applying the mechanism of action of cinnamon extract in lowering uric acid based on network pharmacology.

Abstract

This application discloses a method and application for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology, belonging to the field of traditional Chinese medicine network pharmacology technology. The method includes: screening active components of cinnamon and establishing an active component database; obtaining the target genes corresponding to the active components of cinnamon; searching for pathogenic genes related to hyperuricemia using Genecards, OMIM, and TTD databases; taking the intersection of the target genes of cinnamon active components and hyperuricemia disease genes to screen common targets for cinnamon intervention in hyperuricemia; constructing a PPI protein interaction network of common targets based on the String database and Cytoscape software and screening core targets; performing GO functional enrichment and KEGG pathway enrichment analysis on the common targets to predict the molecular mechanism of cinnamon's uric acid-lowering effect; and verifying the uric acid-lowering activity and pathway of cinnamon extract through in vitro experiments. This technical solution can elucidate the mechanism of action of cinnamon in intervening in hyperuricemia and provide a scientific theoretical basis for cinnamon-based uric acid-lowering treatments.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine network pharmacology technology, specifically involving an analytical method and application method for the mechanism of action of cinnamon extract in lowering uric acid based on network pharmacology. Background Technology

[0002] With the improvement of living standards, the proportion of high-purine, high-sugar, high-fat, and high-protein diets has continued to rise, leading to a year-on-year increase in the incidence of hyperuricemia. It has now become the fourth largest metabolic chronic disease after the "three highs" (high blood pressure, high blood sugar, and high cholesterol), and can induce various complications such as gouty arthritis and urate nephropathy, seriously endangering human health. Currently, mainstream uric acid-lowering chemical drugs generally have adverse reactions such as liver and kidney toxicity and allergic reactions, making them unsuitable for long-term use. Therefore, discovering safe and effective uric acid-lowering active ingredients from natural plants that are both food and medicine has become a key research direction in the food and pharmaceutical fields.

[0003] Cinnamon, the dried bark of a tree in the Lauraceae family, is a typical medicinal and edible herb in traditional Chinese medicine. Traditional Chinese medicine records its multiple pharmacological effects. Current research has only preliminarily observed cinnamon's potential to lower uric acid, but its molecular mechanism of action has not yet been systematically elucidated. Existing techniques for analyzing the uric acid-lowering activity of traditional Chinese medicine have significant shortcomings. On the one hand, relying solely on in vitro enzymatic experiments can only verify the strength of the drug's efficacy, but cannot identify molecular targets and regulatory pathways. On the other hand, conventional network pharmacology analysis processes are fragmented, lacking unified and standardized screening criteria, and mostly only at the level of target prediction, without combining in vivo and in vitro experiments to verify the results, resulting in insufficient reliability of the analytical conclusions. Current technologies lack a complete analytical scheme integrating active ingredient screening, target prediction intersection, protein interaction network analysis, pathway enrichment, and in vivo and in vitro efficacy verification, making it impossible to accurately locate the core targets and key signaling pathways of cinnamon in intervening in hyperuricemia. This severely restricts the formulation optimization and industrialization of cinnamon-related uric acid-lowering health foods and drugs. Summary of the Invention

[0004] This application provides a method for analyzing and applying the mechanism of action of cinnamon extract in lowering uric acid based on network pharmacology. The purpose is to solve the technical problems of existing technologies lacking an integrated and complete analytical process and being unable to accurately elucidate the molecular mechanism of cinnamon in lowering uric acid. It constructs a standardized analytical method that integrates screening, target matching, network analysis, pathway prediction, and in vivo and in vitro validation, so as to clarify the target and pathway of cinnamon action and provide theoretical support for the development of related products.

[0005] In a first aspect, embodiments of this application provide a method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology, the method comprising: Step 1: Screening cinnamon active ingredients and establishing an active ingredient database; Step 2: Obtain the target genes corresponding to the active ingredients of cinnamon; Step 3: Use the Genecards, OMIM, and TTD databases to search for pathogenic genes related to hyperuricemia; Step 4: Intersection of the target genes of cinnamon active ingredients and genes of hyperuricemia disease is obtained to screen common targets of cinnamon intervention in hyperuricemia. Step 5: Construct a PPI protein interaction network with common targets based on the String database and Cytoscape software, and screen core targets; Step 6: Perform GO functional enrichment and KEGG pathway enrichment analysis on common targets to predict the molecular mechanism of cinnamon in lowering uric acid. Step 7: Combining in vitro xanthine oxidase inhibition experiments and in vivo zebrafish model experiments, the uric acid-lowering activity and mechanism of action of cinnamon extract were verified.

[0006] Furthermore, step 1 uses standardized database screening rules to limit the screening range of cinnamon active ingredients.

[0007] Furthermore, in step 1, cinnamon was searched in the TCMSP database, and oral bioavailability OB≥30% and drug-likeness DL>0.1 were set as screening criteria to obtain 6 core active ingredients, including: quercetin-3-methyl ether, N-[6-(9-acridylamino)hexyl]benzamide, kaempferol, β-sitosterol, isorhamnetin and quercetin.

[0008] Furthermore, in step 2, target genes are obtained by combining structural target prediction with the original target in the database through two paths, and then all target genes are summarized and integrated.

[0009] Furthermore, step 2 specifically includes: The active ingredients obtained in step 1 were queried for SMILES numbers in PubChem, imported into Swiss Target Prediction to screen for targets with a predicted probability > 0, and merged with the targets provided by TCMSP to remove duplicates, resulting in 242 cinnamon-related genes.

[0010] Further: In step 3, using Hyperuricemia as the search term, 1461 genes related to hyperuricemia were screened and obtained. In step 4, the Venn diagram was drawn using the MicroBioinformatics online platform to complete gene intersection, and 56 common target sites for cinnamon and hyperuricemia were found. In step 5, the human species is selected in the String database, the medium confidence level is set to 0.400, the interaction data is exported and imported into Cytoscape software for topological analysis, and the core targets INS, IL6, PPARG, CRP and PPARA are selected based on the Degree value.

[0011] Furthermore, step 6 uses the David database to perform KEGG pathway enrichment analysis, which is visualized through the MicroBio platform. A total of 91 signaling pathways were enriched, including the core pathways of AGE-RAGE, lipid and atherosclerosis pathway, TNF pathway, PPAR pathway, and PI3K-Akt pathway.

[0012] Furthermore, the GO functional enrichment in step 6 is divided into three categories: biological process BP, cellular component CC, and molecular function MF. Biological processes mainly involve responses to exogenous stimuli, negative regulation of apoptosis, lipid metabolism, and regulation of nitric oxide synthesis. Cellular components mainly include extracellular regions, protein complexes, and cell membranes. Molecular functions mainly include enzyme binding, oxidoreductase activity, and nuclear receptor binding.

[0013] Furthermore, the in vitro xanthine oxidase inhibition experimental system described in step 7 includes pH 7.5 sodium phosphate buffer, cinnamon extract, 0.1 U / mL xanthine oxidase, nitrotetrazole blue chromogenic agent, and 2 mmol / L hypoxanthine substrate. The enzyme inhibition rate is calculated by continuous detection at 560 nm wavelength for 90 min. The IC50 of cinnamon extract on xanthine oxidase inhibition is also determined. 50 It is 0.251 mg / mL.

[0014] Secondly, this application provides a method for developing and applying cinnamon-based uric acid-lowering products. This method uses the network pharmacology-based cinnamon extract uric acid-lowering mechanism analysis method described above to screen active ingredients of cinnamon for uric acid-lowering, analyze targets and pathways, and obtain uric acid-lowering health foods or uric acid-lowering drugs.

[0015] Thirdly, embodiments of this application provide a readable storage medium storing a program or instructions that, when executed by a processor, implement the steps of the method described above.

[0016] Fourthly, embodiments of this application provide a chip, the chip including a processor and a communication interface, the communication interface being coupled to the processor, the processor being used to run programs or instructions to implement the method described above.

[0017] The technical solution provided in this application provides an integrated analysis method that integrates multi-database screening, network pharmacology analysis, and in vitro and in vivo efficacy verification. It can quickly identify the core active ingredients, common targets, and key regulatory pathways of cinnamon in lowering uric acid. By standardizing parameters, it improves the accuracy of target screening and network analysis results. In vitro XOD inhibition experiments are supplemented by zebrafish models to corroborate the prediction conclusions, which significantly shortens the traditional mechanism research cycle and reduces R&D costs. It clearly elucidates the multi-target and multi-pathway synergistic mechanism of cinnamon in intervening in hyperuricemia, providing a complete and scientific theoretical basis and evaluation scheme for the development of cinnamon-based uric acid-lowering health foods and drugs. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology, as provided in Embodiment 1 of this application. Figure 2 This is a schematic diagram of the Venn diagram of cinnamon components and disease targets in hyperuricemia; Figure 3 This is a schematic diagram of the PPI network, a potential target for String cinnamon in the treatment of hyperuricemia. Figure 4 This is a network diagram of cinnamon active ingredients, targets, and hyperuricemia. Figure 5 This is a schematic diagram of GO function analysis; Figure 6 This is a schematic diagram of KEGG pathway enrichment analysis; Figure 7 This is a schematic diagram showing the XOD inhibition activity results of cinnamon extracts at different concentrations. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, specific embodiments of this application will be described in further detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining this application and not for limiting it. It should also be noted that, for ease of description, only the parts relevant to this application are shown in the drawings, not all of them. Before discussing exemplary embodiments in more detail, it should be mentioned that some exemplary embodiments are described as processes or methods depicted as flowcharts. Although the flowcharts describe operations (or steps) as sequential processes, many of these operations can be performed in parallel, concurrently, or simultaneously. Furthermore, the order of the operations can be rearranged. The process can be terminated when its operation is completed, but may also have additional steps not included in the drawings. The process can correspond to a method, function, procedure, subroutine, subroutine, etc.

[0020] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0021] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0022] The following, in conjunction with the accompanying drawings, provides a detailed explanation of the network pharmacology-based cinnamon extract uric acid-lowering mechanism analysis method provided in this application, through specific embodiments and application scenarios.

[0023] Example 1 Figure 1 This is a flowchart illustrating the method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology, as provided in Embodiment 1 of this application. Figure 1 As shown, the method specifically includes: Step 1: Screening cinnamon active ingredients and establishing an active ingredient database; Cinnamon active ingredients refer to biologically active chemical substances in cinnamon that can participate in the regulation of metabolism in the body, such as kaempferol and quercetin.

[0024] An active ingredient database refers to an electronic data sheet that centrally stores the molecular structure, physicochemical parameters, and pharmacokinetics of various active ingredients in cinnamon. Specifically, it can be a local Excel database or a dataset exported from an online search database.

[0025] Specifically, operators or the system can automatically set screening thresholds based on a dedicated database of chemical components of traditional Chinese medicine, extract information on active molecules that meet the standards, and compile and summarize them into a standardized dedicated database.

[0026] Step 2: Obtain the target genes corresponding to the active ingredients of cinnamon; Target genes refer to gene fragments that can be bound to and regulated by active ingredients after entering the human body. The gene expression products can participate in physiological processes related to uric acid metabolism, such as IL6 and PPARG.

[0027] This method allows for the retrieval of all corresponding gene sequence information through two channels: calling target prediction tools and using the target information built into the traditional Chinese medicine database.

[0028] Step 3: Use the Genecards, OMIM, and TTD databases to search for pathogenic genes related to hyperuricemia; Among these, Genecards refers to the Human Genome Comprehensive Database, which includes all human genes associated with diseases. OMIM refers to the Online Human Mendelian Inheritance Database, which specifically records pathogenic genes for hereditary metabolic diseases. TTD refers to the Therapeutic Target Database, which stores the genes that are drug targets for diseases.

[0029] Hyperuricemia-related pathogenic genes refer to genes whose abnormal expression can lead to abnormally high levels of uric acid in the human body, such as INS and CRP.

[0030] This solution allows users to batch export all associated pathogenic gene raw data from three types of databases by inputting standard English keywords for the disease.

[0031] Step 4: Intersection of the target genes of cinnamon active ingredients and genes of hyperuricemia disease is obtained to screen common targets of cinnamon intervention in hyperuricemia. Gene intersection refers to the presence of identical gene entries in two sets of gene data, representing pathogenic genes that can be directly regulated by cinnamon components.

[0032] Common targets refer to genes that are simultaneously targets of cinnamon active ingredients and genes causing high uric acid, and can serve as the core gene for cinnamon's intervention in the disease.

[0033] Specifically, two gene lists can be imported using an online bioinformatics analysis platform, which can automatically match overlapping genes and extract and save them separately.

[0034] Step 5: Construct a PPI protein interaction network with common targets based on the String database and Cytoscape software, and screen core targets; The String database refers to a protein-protein interaction retrieval database, used for querying the association between genes and their corresponding proteins.

[0035] Cytoscape is a professional software for visualizing and analyzing biological networks. Versions can be 3.8.0 or earlier and later. It can perform network topology calculations.

[0036] The PPI protein interaction network is a visual network diagram that shows the binding and regulatory relationships between proteins encoding common target sites.

[0037] Core targets refer to key genes with the highest correlation and connectivity in protein networks, which have the strongest regulatory effect on uric acid metabolism, such as IL6 and PPARA.

[0038] Specifically, the original protein association file can be exported from String, imported into Cytoscape to generate a visualized network, and key targets can be screened based on the network topology and Degree values.

[0039] Step 6: Perform GO functional enrichment and KEGG pathway enrichment analysis on common targets to predict the molecular mechanism of cinnamon in lowering uric acid. GO functional enrichment refers to gene ontology enrichment analysis, which interprets the physiological functions of genes from three dimensions: biological processes, cellular components, and molecular functions.

[0040] KEGG pathway enrichment refers to Kyoto Gene and Genomic Pathway Enrichment Analysis, used for the analysis of gene-mediated cell signaling and metabolic pathways.

[0041] The molecular mechanism refers to the intrinsic molecular regulatory logic by which the active ingredients of cinnamon reduce uric acid in the human body by regulating genes and pathways.

[0042] This approach can import common target gene sets into an enrichment analysis database, batch enrich corresponding functions and pathways, and combine the significance of enrichment to predict drug action pathways.

[0043] Step 7: Combining in vitro xanthine oxidase inhibition experiments and in vivo zebrafish model experiments, the uric acid-lowering activity and mechanism of action of cinnamon extract were verified.

[0044] The in vitro xanthine oxidase inhibition assay is an extracellular biochemical assay used to evaluate the inhibitory effect of cinnamon on key enzymes in uric acid production.

[0045] In vivo zebrafish model experiments refer to live animal model experiments, which use juvenile zebrafish to construct a high uric acid model to verify the in vivo efficacy of drugs.

[0046] Cinnamon extract refers to a mixture of active substances obtained from cinnamon medicinal materials through processes such as water extraction and alcohol extraction.

[0047] This technical solution can simultaneously conduct in vitro enzyme activity detection and in vivo zebrafish modeling and drug administration experiments, verifying the efficacy of the pathways predicted by network pharmacology at the molecular and in vivo levels.

[0048] The technical solution provided in this embodiment first mines active molecules, targets, and disease genes of traditional Chinese medicine based on multiple databases. It then predicts the potential molecular pathways of cinnamon in lowering uric acid through protein interaction and enrichment analysis. Finally, it verifies the results with a two-layer experiment using in vitro enzymology and in vivo animal models. This completes the analytical process from bioinformatics prediction to physical experimental evidence, solving the shortcomings of traditional single in vivo or in vitro experiments that can only evaluate efficacy in a one-sided way and cannot systematically analyze the synergistic mechanism of multiple targets. It comprehensively and systematically elucidates the multi-level molecular regulatory logic of cinnamon extract in intervening in high uric acid.

[0049] In one embodiment, step 1 may optionally employ standardized database screening rules to limit the screening range of cinnamon active ingredients.

[0050] Standardized database screening rules refer to pre-defined and reproducible molecular screening criteria used to eliminate chemical molecules with no bioavailability potential, such as oral bioavailability and drug-likeness threshold.

[0051] The screening scope can refer to the boundary of the set of cinnamon chemical components to be included in subsequent target prediction.

[0052] This approach allows for the setting of fixed physicochemical screening thresholds within a database of traditional Chinese medicine chemical components, ensuring that only qualified molecules are retained for downstream analysis.

[0053] This technical solution constrains the screening boundaries of active ingredients through unified and standardized screening rules, avoids the problem of experimental non-reproducibility caused by arbitrary manual screening, uniformly limits the range of molecules included in the analysis, ensures the reproducibility of the entire network pharmacology analysis process and the stability of data results, and improves the data credibility and scientific research standardization of mechanism analysis experiments.

[0054] In one embodiment, optionally, step 1 involves searching for cinnamon in the TCMSP database, setting oral bioavailability (OB) ≥ 30% and drug-likeness (DL) > 0.1 as screening criteria, to obtain six core active ingredients, including: quercetin-3-methyl ether, N-[6-(9-acridylamino)hexyl]benzamide, kaempferol, β-sitosterol, isorhamnetin, and quercetin.

[0055] Among them, the TCMSP database refers to the Traditional Chinese Medicine Systems Pharmacology Database, which specifically records the chemical components and pharmacokinetic parameters of Chinese medicinal materials.

[0056] Oral bioavailability (OB) refers to the percentage of molecules that can be absorbed into the bloodstream and exert their effects after a drug is taken orally.

[0057] Drug class DL refers to a quantitative score that indicates that the molecule conforms to the physicochemical characteristics of an orally administered drug.

[0058] The core active ingredient is a characteristic cinnamon molecule that meets the pharmacokinetics screening criteria and has potential for in vivo efficacy.

[0059] Specifically, you can enter the medicinal herb name "cinnamon" into the TCMSP database, manually input the OB and DL value thresholds for filtering, and export a list of all qualified active molecules.

[0060] This technical solution provides specific TCMSP database screening criteria, identifies six core effective molecules in cinnamon, and forms a fixed and completely replicable active ingredient extraction scheme. This eliminates data bias caused by inconsistent screening criteria among different operators, provides fixed molecular research objects for bioinformatics analysis, and ensures that the analysis results of different batches have cross-comparison value.

[0061] In one embodiment, optionally, step 2 uses two paths—structural target prediction and database native target—to obtain target points and then summarizes and integrates all target genes.

[0062] Structural target prediction refers to an analytical method that uses online prediction tools to match potential binding genes based on the chemical structure of active molecules.

[0063] Database native targets refer to the target information corresponding to medicinal material components that are pre-included in the Chinese medicine database and supported by existing literature.

[0064] The two pathways refer to two independent gene acquisition channels constructed respectively: molecular structure prediction and database-built target retrieval.

[0065] The summary and integration process involves merging the target genes obtained from the two channels and removing duplicate entries.

[0066] This approach extracts target points through two channels: a molecular structure prediction website and a traditional Chinese medicine database. The two gene lists are then merged, and duplicate gene entries are removed.

[0067] This technical solution employs a dual-channel parallel search for target genes, which, compared to searching a single database, avoids missing a large number of potential targets. The combination of these two pathways comprehensively captures genes regulated by cinnamon's active molecules, significantly reducing the probability of missed target detection, fully covering genes related to cinnamon's intervention in high uric acid, and enhancing the comprehensiveness of subsequent mechanism analysis.

[0068] In one embodiment, optionally, step 2 specifically includes: querying the SMILES number of the active ingredient obtained in step 1 in PubChem, importing it into Swiss Target Prediction to screen for targets with a predicted probability > 0, merging it with the targets provided by TCMSP to remove duplicates, and obtaining 242 cinnamon-related genes.

[0069] PubChem is a global public database of chemical molecules that stores the molecular structure codes of various compounds.

[0070] SMILES is a simplified linear input canonical code used to standardize the description of the chemical structure of compounds.

[0071] Swiss Target Prediction is an online tool for predicting small molecule targets, based on matching molecular structure with potential targets for human action.

[0072] Predictive probability refers to the numerical value by which a tool determines that a molecule has a binding interaction with its corresponding gene protein.

[0073] This method allows users to search for active molecule structure codes on PubChem, import the codes into a target prediction website to screen for effective targets, and then merge them with built-in targets in TCMSP and remove duplicate genes to obtain the total gene set.

[0074] This technical solution clarifies the standardized operation process of the dual-target retrieval path, and uses PubChem and SwissTarget Prediction with TCMSP to complete target mining. The operation steps are clear and replicable, and the total range of cinnamon target genes is precisely defined, avoiding the interference of target dataset size fluctuations on the stability of subsequent enrichment analysis results.

[0075] In one embodiment, optionally, step 3 uses "Hyperuricemia" as the search term to screen and obtain 1461 genes related to hyperuricemia; In step 4, the Venn diagram was drawn using the MicroBioinformatics online platform to complete gene intersection, and 56 common target sites for cinnamon and hyperuricemia were found. In step 5, human species are selected from the String database, the medium confidence level is set to 0.400, the interaction data is exported and imported into Cytoscape software for topological analysis, and the core targets INS, IL6, PPARG, CRP and PPARA are selected based on the Degree value.

[0076] Hyperuricemia is a standard English disease search keyword for hyperuricemia.

[0077] Microbioinformatics online platform refers to online bioinformatics visualization and analysis tools that can perform gene intersection and plotting operations.

[0078] Venn diagrams are charts used to visually represent the number of overlapping entries between two gene groups.

[0079] Medium confidence refers to the equal confidence level filtering parameter in the String database, used to limit the reliability of protein-protein interactions.

[0080] Topology analysis refers to a quantitative calculation method for the correlation between nodes in a protein-protein interaction network.

[0081] Degree value refers to the connectivity of network nodes; the higher the value, the more central the gene's regulatory role.

[0082] Specifically, the process involves inputting standard disease keywords to search for pathogenic genes, using online tools to find the intersection of two sets of genes, setting a fixed confidence level to export protein interaction data, and importing it into analysis software to calculate node connectivity and screen core regulatory genes.

[0083] This technical solution uniformly specifies disease gene retrieval keywords, intersection plotting tools, protein network analysis parameters, and core target ranges. The parameters throughout the entire process are fixed and uniform, which greatly reduces the interference of human manipulation variables on the analysis results and accurately identifies the key genes that regulate high uric acid in cinnamon, thus identifying key research targets for subsequent pathway enrichment.

[0084] In one embodiment, optionally, step 6 uses the David database to perform KEGG pathway enrichment analysis, visualizes it through the MicroBio platform, and obtains a total of 91 signaling pathways, including the AGE-RAGE pathway, lipid and atherosclerosis pathway, TNF pathway, PPAR pathway, and PI3K-Akt pathway.

[0085] The David database is a dedicated online database for gene function and pathway enrichment analysis.

[0086] The KEGG signaling pathway refers to a set of physiological pathways involving metabolism, inflammation, and lipid metabolism that are synergistically regulated by intracellular genes.

[0087] Visualization refers to converting enriched pathway data into intuitive charts such as bar charts and bubble charts.

[0088] The core pathway refers to the cell signaling pathways with the highest enrichment significance that are directly related to uric acid metabolism and inflammation regulation.

[0089] This approach involves uploading the common target gene set to the David database to perform pathway enrichment, exporting the enrichment results, and then using online bioinformatics tools to create visualization charts to screen for key regulatory pathways with high significance.

[0090] This technical solution utilizes a dedicated enrichment database and visualization tools to identify the five core regulatory pathways of cinnamon intervention in hyperuricemia, fixes the enrichment range of these pathways, standardizes the prediction of molecular pathway mechanisms, accurately identifies key signaling pathways in cinnamon regulation of uric acid metabolism and inflammatory responses, and simplifies the scope of subsequent experimental verification.

[0091] In one embodiment, optionally, the GO functional enrichment in step 6 is divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The biological process mainly involves response to exogenous stimuli, negative regulation of apoptosis, lipid metabolism, and regulation of nitric oxide synthesis. The cellular component mainly includes the extracellular region, protein complex, and cell membrane. The molecular function mainly includes enzyme binding, oxidoreductase activity, and nuclear receptor binding.

[0092] Biological processes (BP) refer to the overall physiological activities of cells involving genes, such as metabolism, stress, and apoptosis.

[0093] Cellular components (CC) refer to the location regions within a cell where gene-expressed proteins are distributed.

[0094] Molecular function (MF) refers to the biochemical activities possessed by a single protein molecule, such as enzyme binding and receptor binding.

[0095] Exogenous stimulus response refers to the regulatory response process of cells to exogenous drugs and metabolic toxins.

[0096] Oxidoreductase activity refers to the ability of a protein to catalyze redox biochemical reactions, and uric acid production depends on this type of enzyme.

[0097] After performing GO enrichment, this scheme categorizes the enriched items into three classes: BP, CC, and MF. It then selects and summarizes the functional items that are significantly enriched and associated with the pathogenesis of hyperuricemia.

[0098] This technical solution standardizes the three major classification systems of GO enrichment results, clarifies the physiological activities, cellular locations, and molecular biochemical functions regulated by the cinnamon target, analyzes the characteristics of cinnamon's effects from multiple levels of microscopic molecules, cells, and macroscopic physiology, and fully explains the underlying molecular functional logic of cinnamon in lowering uric acid by regulating inflammation, lipid metabolism, and oxidase activity.

[0099] In one embodiment, optionally, the in vitro xanthine oxidase inhibition experimental system in step 7 includes pH 7.5 sodium phosphate buffer, cinnamon extract, 0.1 U / mL xanthine oxidase, nitrotetrazolium chloride blue chromogenic agent, and 2 mmol / L hypoxanthine substrate. The enzyme inhibition rate is calculated by continuous detection at a wavelength of 560 nm for 90 min. The IC50 of cinnamon extract inhibiting xanthine oxidase is 0.251 mg / mL.

[0100] Xanthine oxidase is a key rate-limiting enzyme in the human body that catalyzes the production of uric acid from hypoxanthine; the higher the enzyme activity, the more uric acid is produced in the blood.

[0101] Sodium phosphate buffer is a solvent system that maintains a stable pH value in an in vitro reaction system.

[0102] Hypoxanthine substrates refer to the raw materials for enzyme-catalyzed reactions, which are catalyzed to produce uric acid as a colorimetric product.

[0103] IC50 refers to the half-inhibitory concentration, which represents the concentration of drug required to inhibit 50% of enzyme activity. The lower the value, the stronger the enzyme inhibitory activity.

[0104] This method can build an in vitro enzyme reaction system with a fixed concentration ratio, set a fixed absorption wavelength for continuous spectrophotometric detection, calculate the enzyme inhibition rate based on the absorbance change, and obtain the half-inhibition concentration value by fitting the curve.

[0105] This technical solution fixes all reagent ratios, detection parameters, and efficacy evaluation indicators for in vitro enzyme activity experiments, provides clear in vitro enzyme inhibition activity values ​​for cinnamon extract, and uses standardized biochemical experiments to corroborate the prediction conclusions of network pharmacology. It provides intuitive in vitro efficacy data support for bioinformatics analysis and achieves mutual verification between prediction and in vitro experimental data.

[0106] To enable those skilled in the art to better understand this solution, this application also provides a preferred embodiment.

[0107] This invention, for the first time, employs network pharmacology to reveal that the core targets of cinnamon in improving hyperuricemia are TNF, IL6, TP53, CASP3, and PPARG. The functional classifications (GOs) involved are: Biological Process (BP), mainly involving responses to external chemical stimuli, positive regulation of gene expression, positive regulation of miRNA transcription, negative regulation of apoptosis, responses to ethanol, and positive regulation of DNA template transcription; Cellular Component (CC), mainly involving protein complexes, extracellular regions, cell membrane, cytoplasm, membrane rafts, chromatin, and secretory granules; and Molecular Function (MF), mainly involving the binding of identical proteins, enzymes, protein complexes, proteases, proteins, ubiquitin-protein ligases, and transcriptional co-regulatory factors. KEGG pathway enrichment analysis yielded 133 signaling pathways, primarily involving the AGE-RAGE signaling pathway in diabetic complications, lipids and atherosclerosis, hepatitis B, cancer pathways, fluid shear stress and atherosclerosis, the IL-17 signaling pathway, the TNF signaling pathway, and the NF-κB signaling pathway. The main components of cinnamon involved in hyperuricemia include kaempferol, β-sitosterol, isorhamnetin, and quercetin. In vitro XOD activity inhibition experiments showed that the IC50 of cinnamon extract for XOD inhibition was 0.251 mg / mL.

[0108] The network pharmacology method described in this invention, combined with experimental verification, further clarifies the function of cinnamon in improving hyperuricemia, and analyzes its target and mechanism of action. This provides a reference for the preliminary screening of whether single Chinese medicines have the effect of improving hyperuricemia, and reduces the cycle and cost of traditional experiments.

[0109] The implementation process of this invention includes the following core steps: (1) Screening the active ingredients of cinnamon and establishing an active ingredient database.

[0110] Using the TCMSP database, a database and analysis platform for systems pharmacology of traditional Chinese medicine, with cinnamon as the keyword and oral bioavailability (OB) ≥30% and drug-likeness (DL) >0.1 as screening criteria, six core components were screened out, as detailed in Table 1.

[0111] Table 1: Main active ingredients of cinnamon

[0112] (2) Input the screened components into the PubChem website, query the SMILES number of each component, input it into Swiss Target Prediction, perform target prediction, select targets with a probability greater than 0 for sorting, integrate with the targets sorted in TCMSP and remove duplicates, and obtain 242 cinnamon-related genes.

[0113] (3) In the Genecards, OMIM and TTD databases, we searched for genes related to hyperuricemia using "Alcoholic brain injury" as the keyword and screened out 1461 genes related to hyperuricemia.

[0114] (4) Intersection screening of the relevant genes from steps (2) and (3) was performed to identify genes related to the effects of cinnamon on hyperuricemia. A Venn diagram was created online using MicroBio, and 56 cross-linked genes related to hyperuricemia associated with cinnamon were identified. (See [link to relevant documentation]). Figure 2 , Figure 2 This is a schematic diagram of the Venn diagram of cinnamon components and disease targets in hyperuricemia.

[0115] (5) The PPI network diagram of key genes in cinnamon was constructed using the String database and Cytoscape 3.8.0 software. In the String database (https: / / cn.string-db.org / ), the species "Homo sapiens" was selected, and the "medium comfidence" was set to 0.400 to obtain protein-protein interaction networks (PPIs). The results were then imported into Cytoscape 3.8.0 software for data analysis and PPI network diagram construction. (See [link to documentation]). Figure 3 , Figure 3 This is a schematic diagram of the PPI network, a potential target for String cinnamon in the treatment of hyperuricemia.

[0116] Import the intersection target points into the String online software, select "Multiple proteins," set the species parameter to "Homo Sapiens," and set the minimum interaction score to medium confidence (0.400) to obtain the protein-protein interaction (PPI). See [link to relevant documentation]. Figure 4 , Figure 4 This is a network diagram of cinnamon active ingredients, targets, and hyperuricemia. The TSV table was downloaded and imported into Cytoscape 3.8.0 for visualization and topology analysis to obtain the degree value of each target. Targets with the highest degree values ​​were identified as core targets. The PPI network diagram contains 556 nodes and 334 edges, representing 334 pairs of interactions between 56 proteins. The average node degree is 11.9, meaning that on average, each protein in the network directly interacts with approximately 12 other proteins. The average local clustering coefficient is 0.65. A value of 1 indicates that all neighbors are paired, forming a maximally connected cluster, while a value of 0 indicates no connections between neighbors. This suggests that proteins in the PPI network tend to form functional modules or complexes. Based on the degree values, the top 5 core targets were identified as INS, IL6, PPARG, CRP, and PPARA, indicating that these targets are likely the core targets for cinnamon's uric acid-lowering effects.

[0117] (6) Perform GO and KEGG enrichment analysis on the relevant genes from step (4).

[0118] Potential targets for cinnamon in lowering uric acid were uploaded to the DAVID database for GO analysis and KEGG pathway enrichment analysis, such as... Figure 5 and Figure 6 As shown, Figure 5 This is a diagram illustrating the functional analysis of GO. Figure 6 This is a schematic diagram of KEGG pathway enrichment analysis. The potential therapeutic mechanism of cinnamon for hyperuricemia was predicted and visualized using a microbioinformatics platform.

[0119] Fifty-six common intersection targets between the active components of cinnamon and those of hyperuricemia were identified, and GO gene function and KEGG enrichment analyses were performed. The top 10 entries were used for visualization analysis. Biological Process (BP) mainly involves responses to exogenous stimuli, positive regulation of cell population proliferation, negative regulation of apoptosis, lipid metabolism, ethanol response, and positive regulation of nitric oxide biosynthesis. Cellular Component (CC) mainly involves extracellular regions, cytoplasmic cell membrane pits, extracellular spaces, extracellular exosomes, receptor complexes, protein-containing complexes, and the plasma membrane. Molecular Function (MF) mainly involves enzyme binding, homologous protein binding, oxidoreductase activity, nuclear receptor activity, signal receptor binding, and protein homodimerization activity. KEGG pathway enrichment analysis yielded 91 signaling pathways, mainly involving the AGE-RAGE signaling pathway in diabetic complications, lipids and atherosclerosis, HIF-1 signaling pathway, cancer pathway, PI3K-Akt signaling pathway, hepatitis B, TNF signaling pathway, NOD-like receptor signaling pathway, FoxO signaling pathway, PPAR signaling pathway, and cAMP signaling pathway.

[0120] (7) In vitro XODH activity inhibition experiment verified the effect of cinnamon extract in lowering uric acid.

[0121] Add 100 μL of 50 mmol / L sodium phosphate buffer (pH 7.5), 30 μL of sample solution, 30 μL of 0.1 U / mL xanthine oxidase, and 30 μL of nitrotetrazole blue chromogenic reagent to a clear 96-well plate. Then, quickly add 30 μL of 2 mmol / L hypoxanthine solution. The reaction can be monitored at 560 nm using a microplate reader for 90 min, and the absorbance at 560 nm is measured. The data show that this detection method has good precision, repeatability, and stability. Therefore, this method can be used as a high-throughput model for the inhibitory capacity of xanthine oxidase. The formula for calculating the inhibitory capacity is as follows: The inhibition rate (%) of the sample is calculated as follows: (Net area under the blank curve - Net area under the sample curve) / Net area under the blank curve. 100% Figure 7 This is a schematic diagram showing the XOD inhibition activity of cinnamon extract at different concentrations. The XOD inhibition rate of cinnamon extract at different concentrations was measured, and the results are as follows. Figure 7As shown in the figure, the IC50 of XOD inhibition rate of cinnamon extract was 0.251 mg / mL, indicating excellent uric acid-lowering effect.

[0122] Example 2 Embodiment 2 of this application provides a method for the research and development of cinnamon-based uric acid-lowering products. The method uses the network pharmacology-based cinnamon extract mechanism analysis method described in the above embodiment to screen the active ingredients of cinnamon for lowering uric acid, and to analyze the target and pathway, so as to obtain uric acid-lowering health foods or uric acid-lowering drugs.

[0123] The method for developing and applying cinnamon-based uric acid-lowering products provided in this embodiment has the same execution process and beneficial effects as the above-described method embodiments. To avoid repetition, it will not be described again here.

[0124] Example 3 This application also provides a readable storage medium storing a program or instructions. When the program or instructions are executed by a processor, they implement the various processes of the above-described method for analyzing the mechanism of action of cinnamon extract in lowering uric acid based on network pharmacology, and achieve the same technical effect. To avoid repetition, these will not be described again here.

[0125] The processor is the processor in the electronic device described in the above embodiments. The readable storage medium includes computer-readable storage media, such as computer read-only memory (ROM), random access memory (RAM), magnetic disk, or optical disk.

[0126] Example 4 This application also provides a chip, which includes a processor and a communication interface. The communication interface is coupled to the processor. The processor is used to run programs or instructions to implement the various processes of the above-described method for analyzing the mechanism of action of cinnamon extract in lowering uric acid based on network pharmacology, and can achieve the same technical effect. To avoid repetition, it will not be described again here.

[0127] It should be understood that the chip mentioned in the embodiments of this application may also be referred to as a system-on-a-chip, device chip, chip system, or system-on-a-chip, etc.

[0128] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or system that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or system. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or system that includes that element. Furthermore, it should be noted that the scope of the methods and systems in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0129] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of this application, in essence, or the part that contributes to the prior art, can be embodied in the form of a computer software product. This computer software product is stored in a storage medium (such as ROM / RAM, disk, optical disk) and includes several instructions to cause a terminal (which may be a mobile phone, computer, server, or network device, etc.) to execute the methods described in the various embodiments of this application.

[0130] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms fall within the scope of protection of this application.

[0131] The above description is merely a preferred embodiment and the technical principles employed in this application. This application is not limited to the specific embodiments described herein. Various obvious changes, readjustments, and substitutions that can be made by those skilled in the art will not depart from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the concept of this application, and the scope of this application is determined by the scope of the claims.

Claims

1. A method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology, characterized in that, Includes the following steps: Step 1: Screening cinnamon active ingredients and establishing an active ingredient database; Step 2: Obtain the target genes corresponding to the active ingredients of cinnamon; Step 3: Use the Genecards, OMIM, and TTD databases to search for pathogenic genes related to hyperuricemia; Step 4: Intersection of the target genes of cinnamon active ingredients and genes of hyperuricemia disease is obtained to screen common targets of cinnamon intervention in hyperuricemia. Step 5: Construct a PPI protein interaction network with common targets based on the String database and Cytoscape software, and screen core targets; Step 6: Perform GO functional enrichment and KEGG pathway enrichment analysis on common targets to predict the molecular mechanism of cinnamon in lowering uric acid. Step 7: Combining in vitro xanthine oxidase inhibition experiments and in vivo zebrafish model experiments, the uric acid-lowering activity and mechanism of action of cinnamon extract were verified.

2. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that, Step 1 uses standardized database screening rules to limit the screening range of cinnamon active ingredients.

3. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 2, characterized in that, In step 1, cinnamon was searched in the TCMSP database. Oral bioavailability (OB) ≥ 30% and drug-likeness (DL) > 0.1 were set as screening criteria to obtain 6 core active ingredients, including: quercetin-3-methyl ether, N-[6-(9-acridylamino)hexyl]benzamide, kaempferol, β-sitosterol, isorhamnetin, and quercetin.

4. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that, Step 2 involves obtaining target points through two paths: structural target prediction and native target points from the database. All target genes are then aggregated and integrated.

5. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 4, characterized in that, Step 2 specifically includes: The active ingredients obtained in step 1 were queried for SMILES numbers in PubChem, imported into Swiss Target Prediction to screen for targets with a predicted probability > 0, and merged with the targets provided by TCMSP to remove duplicates, resulting in 242 cinnamon-related genes.

6. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that: In step 3, using Hyperuricemia as the search term, 1461 genes related to hyperuricemia were screened and obtained. In step 4, the Venn diagram was drawn using the MicroBioinformatics online platform to complete gene intersection, and 56 common target sites for cinnamon and hyperuricemia were found. In step 5, the human species is selected in the String database, the medium confidence level is set to 0.400, the interaction data is exported and imported into Cytoscape software for topological analysis, and the core targets INS, IL6, PPARG, CRP and PPARA are selected based on the Degree value.

7. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that, Step 6 uses the David database to perform KEGG pathway enrichment analysis, which is visualized on the MicroBio platform. A total of 91 signaling pathways were enriched, including the core pathways such as the AGE-RAGE pathway, lipid and atherosclerosis pathway, TNF pathway, PPAR pathway, and PI3K-Akt pathway.

8. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that, The GO functional enrichment in step 6 is divided into three categories: biological process BP, cellular component CC, and molecular function MF. Biological processes mainly involve responses to exogenous stimuli, negative regulation of apoptosis, lipid metabolism, and regulation of nitric oxide synthesis. Cellular components mainly include extracellular regions, protein complexes, and cell membranes. Molecular functions mainly include enzyme binding, oxidoreductase activity, and nuclear receptor binding.

9. The method for analyzing the uric acid-lowering mechanism of cinnamon extract based on network pharmacology according to claim 1, characterized in that, The in vitro xanthine oxidase inhibition experimental system described in step 7 includes pH 7.5 sodium phosphate buffer, cinnamon extract, 0.1 U / mL xanthine oxidase, nitrotetrazole blue chromogenic reagent, and 2 mmol / L hypoxanthine substrate. The enzyme inhibition rate is calculated by continuous detection at 560 nm wavelength for 90 min. The IC50 of cinnamon extract on xanthine oxidase inhibition is determined. 50 The concentration was 0.251 mg / mL.

10. A method for developing and applying a cinnamon-based uric acid-lowering product, characterized in that, The network pharmacology-based cinnamon extract mechanism analysis method described in any of claims 1-9 is used to screen the active ingredients of cinnamon for lowering uric acid, and to analyze the target and pathway of action, so as to obtain uric acid-lowering health food or uric acid-lowering drug.

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