Method for detecting bloody component of Xiaoyu tablets and method for constructing xerophthalmia target network
Through liquid chromatography-mass spectrometry detection and network pharmacology technology, a network of targets for dysophthalmic tablets into blood and dry eye targets was constructed, which solved the detection and target prediction problems of dysophthalmic tablets in the treatment of dry eye, revealed the efficacy characteristics of multi-component and multi-targets, and provided a theoretical basis for the treatment of dry eye.
Patent Information
- Application Number
- CN202510172815.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art lacks systematic detection methods for the blood-injected components of illustrative tablets and the construction of target networks for dry eye targets, and cannot effectively predict its core drug-effective ingredients and dry eye-related targets, which limits the application of illustrative tablets in the treatment of dry eye.
The blood components of the illustrative tablets were detected by liquid chromatography-mass spectrometry detection technology, combined with network pharmacology technology to build a dry eye target network, chemical components were identified through UFLC-Triple Q-TOF-MS/MS detection technology, and biological targets of dry eye were screened using databases such as DrugBank and PharmGkb. Visual processing was used to establish a 'active ingredient-target-disease' interaction network.
The full chemical component identification of the blood-injected components of the illustrative tablets and the construction of the target network for dry eye disease were realized, and the efficacy characteristics of multi-component and multi-target points were revealed, providing theoretical basis and data support for the treatment of dry eye disease.
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Figure CN120254093A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of traditional Chinese medicine, and specifically relates to a method for detecting the blood components of Zhangyanming tablets and a method for constructing a dry eye target network. Background Art
[0002] The prescription of Zhangyanming has forty years of experience in finished medicine. Its tablet prescription comes from Professor Li Zaoyun, a national ophthalmology authority and a famous traditional Chinese medicine expert. On the basis of the traditional famous prescriptions Yiqi Congming Decoction and Zhujing Pills, it is developed by combining its own medication experience and is recorded in the "Practical Basic Traditional Chinese Medicine Preparation Handbook" and is currently included in the first volume of the 2020 edition of the Chinese Pharmacopoeia.
[0003] Zhangyanming tablets are a compound traditional Chinese patent medicine composed of twenty-two Chinese herbal medicines, mainly including Chinese dodder seeds, prepared rehmannia root, codonopsis pilosula, wolfberry fruit, astragalus membranaceus, buddleja officinalis, chrysanthemum, chuanxiong rhizome, kudzu root and other medicinal materials, and have the effects of tonifying the liver and kidney, removing nebula and improving eyesight, and are mainly used to treat symptoms such as dryness and discomfort caused by deficiency of the liver and kidney.
[0004] On the basis of having detected its whole chemical composition and the blood components in Beagle dogs, the present invention aims to screen the blood components of Zhangyanming extract and their action targets through network pharmacology technology, predict its core pharmacodynamic components and dry eye-related action targets, and lay a theoretical foundation and data support for the development and utilization of Zhangyanming tablets for dry eye. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for detecting the blood components of Zhangyanming tablets and a method for constructing a dry eye target network.
[0006] The method for detecting the blood components of Zhangyanming tablets of the present invention includes the following steps:
[0007] A. Administer Zhangyanming tablets to animals, collect serum after a predetermined time as the experimental group, and use the serum of animals that have not been administered Zhangyanming tablets as the control group;
[0008] B. Detect the experimental group and the control group by liquid chromatography-mass spectrometry;
[0009] For the chromatography, the conditions are: use a Kinetex C18 chromatographic column 100×2.1mm 2.6μm, Waters, USA, the column temperature is 40°C, the mobile phase system consists of water-A phase and acetonitrile-B phase, both containing 0.1% formic acid v / v, the flow rate is 0.3mL / min, the total elution time is: 45min, and the gradient elution method is: 5% to 95% acetonitrile-B phase from 0 to 30min, 95% acetonitrile-B phase from 30 to 35%, 95% to 5% acetonitrile-B phase from 35 to 37min, and 5% acetonitrile-B phase from 37 to 45min.
[0010] For the mass spectrometry, the conditions are:
[0011] Electrospray ionization source ESI, detection in positive and negative ion modes. In the positive ion mode, the spray voltage is set to: 5500 V; in the negative ion mode, it is set to: -4500 V. The spray gas pressure is set to 55 psi, the auxiliary heating gas pressure is 50 psi, the ion source temperature is maintained at 550 °C, the collision voltage is 30 V, the declustering voltage is 80 V, the curtain gas pressure is 35 psi, the collision cell pressure is 10 psi, and the scanning mass-to-charge ratio m / z range is: 50 - 1500. Nitrogen is used as the spray gas and auxiliary gas, and The software collects data in the information-dependent acquisition (IDA) mode.
[0012] Preferably, step A is as follows:
[0013] One day before the experiment, beagle dogs are fasted for 12 h and allowed free access to water. After collecting blank blood samples, the beagle dogs are given a single oral administration of Zhangyanming extract at 499.5 mg / kg. Blood samples (2 mL) are collected at 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h after administration. The blood samples are centrifuged at 2500 g for 15 min at 4 °C, and the supernatant is collected.
[0014] Preferably, the method for preparing the injection specimen for liquid chromatography in step B is as follows: Take 100 μL of beagle dog serum sample, add 1 mL of ethyl acetate, vortex for 5 min, and then centrifuge at 10000 g for 15 min at 4 °C. Transfer 700 μL of the supernatant, dry it under nitrogen, and immediately reconstitute it for detection or store it at -80 °C. Before detection, the sample is reconstituted with 100 μL of methanol: distilled water solution (4:1, v / v), vortexed for 5 min, ultrasonically treated for 10 min until completely dissolved, and then centrifuged at 10000 g for 20 min at 4 °C to obtain the sample solution for injection.
[0015] Preferably, the components entering the blood are shown in Table 1.
[0016] Preferably, for the components entering the blood, the key active components are: luteolin, apigenin, obacunone, methyl myristate, albiflorin, paeonolide D, angelicin, dibutyl sebacate, puerarin, formononetin, sclareolide, oleic acid, adenosine, benzyl-β-primeveroside, naringenin, odoratin, senkyunolide O, ethyl oleate.
[0017] The second object of the present invention is to provide a method for constructing a dry eye target network, which includes the following steps:
[0018] A. Collection of dry eye disease-related targets
[0019] Based on drug target and pathway databases such as DrugBank, PharmGkb, MetaCore, Ingenuity Pathway Analysis, TherapeuticTargets Database, and Potential Drug Target Database, a dry eye biological target database and a disease-related target set were screened and constructed. After sorting and merging, a disease target list corresponding to dry eye was obtained;
[0020] B. Construction of the "drug ingredient - target - disease" network
[0021] The above-mentioned blood components of Zhangyanming tablets and the intersection targets of dry eye were imported into Cytoscape 3.9.0 for visualization processing to construct a network diagram of "Zhangyanming extract - blood components - intersection targets - dry eye disease". Among them, the green nodes represent the blood-active components of Zhangyanming extract, and the red nodes represent the intersection targets of the active components and dry eye disease. The color is positively correlated with the number of related targets;
[0022] C. Construction of the PPI network
[0023] The String database was used to analyze the intersection targets to obtain a protein-protein interaction (PPI) network diagram, revealing the interaction relationships between the targets;
[0024] D. Signal pathway enrichment analysis
[0025] The DAVID database was used to perform gene function classification system and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the targets, revealing the biological functions and signal pathways of Zhangyanming extract in the treatment of dry eye.
[0026] Based on the UFLC-Triple Q-TOF-MS / MS detection technology, after completing the structural identification and component characterization of all chemical components and blood components of Zhangyanming extract and establishing its chemomics database, the blood components are used as ligands for molecular docking simulation. Based on drug target and pathway databases such as DrugBank, PharmGkb, MetaCore, Ingenuity Pathway Analysis, Therapeutic Targets Database, Potential Drug Target Database, etc., a biological target database for dry eye and a disease-related target set are screened and constructed. Cytoscape is used to construct a component-action target network for the screened active ingredient (group) action targets (group), where the nodes represent chemical components or potential targets, and the connecting lines represent the interaction between components and targets; further, the Network Analysis plugin and CentiScaPe 1.2 plugin are used to statistically analyze the parameters of the action network (including the position and size of each node, etc.), establish the "active ingredient-target-pathway-disease" interaction network and topological characteristics, measure the pharmacodynamic characteristics of multi-level drug interactions, and predict the core interaction targets. Description of the Drawings
[0027] Figure 1 is the number of common targets between the blood components of Zhangyanming tablets and dry eye disease;
[0028] Figure 2 is the network diagram of "Zhangyanming tablets-blood components-main targets-disease";
[0029] Figure 3 is the protein PPI network diagram of the main action targets of the blood components of Zhangyanming tablets;
[0030] Figure 4 is the GO pathway enrichment analysis;
[0031] Figure 5 is the KEGG pathway enrichment analysis. Detailed Embodiments
[0032] The following examples are further illustrations of the present invention, rather than limitations thereof.
[0033] Example 1:
[0034] I. Experimental Methods
[0035] 1.1 Experimental Instruments
[0036] Table 1 Experimental Instruments
[0037]
[0038] 1.2 Experimental reagents and drugs
[0039] Table 2 Experimental reagents and drugs
[0040]
[0041] 1.3 Experimental animals
[0042] Experimental Beagle dogs were purchased from Fuzhou Zhenhe Experimental Animal Technology Development Co., Ltd. (License number for experimental animal production: SCXK (Min) 2018-0001). There were equal numbers of males and females, and their body weights were controlled within the range of 10-18 kg. They were housed in Guangdong Ryan Institute of Pharmaceutical Research Co., Ltd. (License number for experimental animal use: SYXK (Yue) 2021-0246). A constant temperature and humidity environment was maintained, with the temperature controlled within the range of 21-23 °C and the humidity within the range of 40-70%. A 12 h dark / light cycle was maintained. All animal experiment protocols and procedures were approved, guided, and supervised by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and the Animal Ethics Committee of Guangdong Ryan Institute of Pharmaceutical Research Co., Ltd. The ethical approval number was: IA-TA20230602, Guangzhou, China.
[0043] 1.4 Collection of serum samples from Beagle dogs after drug administration
[0044] One day before the start of the experiment, the Beagle dogs were fasted for 12 h and allowed free access to water. After collecting blank blood samples, the Beagle dogs were given a single oral dose of Zhangyanming extract at 499.5 mg / kg. Blood samples (2 mL) were collected at 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h after drug administration. The blood samples were centrifuged at 2500 g for 15 min at 4 °C. The supernatant was collected and stored at -80 °C until taken out before analysis.
[0045] 1.5 Preparation of serum samples after drug administration and injection conditions
[0046] Take 100 μL of Beagle dog serum sample, add 1 mL of ethyl acetate, vortex for 5 min, and then centrifuge at 10000 g for 15 min at 4 °C. Transfer 700 μL of the supernatant, dry it under nitrogen, and immediately reconstitute it for detection or store it at -80 °C. Before detection, the sample was reconstituted with 100 μL of methanol: distilled water solution (4:1, v / v), vortexed for 5 min, sonicated for 10 min until completely dissolved, and then centrifuged at 10000 g for 20 min at 4 °C. Take 10 μL of the supernatant and perform detection and analysis using ultra-fast liquid chromatography quadrupole time-of-flight tandem mass spectrometry and an electrospray ionization source.
[0047] Chromatographic conditions: A Kinetex C18 column (100×2.1 mm, 2.6 μm, Waters, USA) was used, and the column temperature was 40 °C. The mobile phase system consisted of water (A) and acetonitrile (B), both containing 0.1% formic acid (v / v). The flow rate was 0.3 mL / min, and the total elution time was 45 min. The gradient elution method was set as shown in Table 3:
[0048] Table 3 Gradient elution conditions
[0049]
[0050] UFLC-Q-TOF-MS / MS mass spectrometry parameters: Electrospray ionization source (ESI), detection in positive and negative ion modes. In the positive ion mode, the spray voltage was set to 5500 V; in the negative ion mode, it was set to -4500 V. The spray gas pressure was set to 55 psi, the auxiliary heating gas pressure was 50 psi, the ion source temperature was maintained at 550 °C, the collision voltage was 30 V, the declustering voltage was 80 V, the curtain gas pressure was 35 psi, and the collision cell pressure was 10 psi. The scanning mass-to-charge ratio (m / z) range was 50 - 1500. Nitrogen was used as the spray gas and auxiliary gas, and data were collected using software in information-dependent acquisition (IDA) mode.
[0051] 1.6 Analysis of the components entering the blood of Zhangyanming Extract
[0052] When analyzing LC-MS / MS data, software 2.2 and Markerview 1.2 (AB Sciex, USA) were used to perform noise reduction, peak alignment, and identification processing on the mass spectrometry data. The identification methods included chromatographic retention time, literature reference, ion fragmentation analysis, comparison with a mass spectrometry library (Natural Products HR-MS / MS Spectral Library, Version 1.0, ABSciex, USA), MetabolitePilot software (Version 1.0, AB Sciex, USA), and comparison with some available reference standards. To ensure the reproducibility of the experimental results, the prototype and metabolite components shared by at least 2 - 3 beagle dogs were screened out and included in further analysis, with a response value above 2000.
[0053] 1.7 Network pharmacology analysis
[0054] Collection of targets corresponding to the components entering the blood:
[0055] The TCMSP database and the Swiss Target Prediction website were used to retrieve the targets of the components of Zhangyanming entering the blood, and their targets were collected. The detailed information of the components entering the blood is shown in Table 1 in the appendix.
[0056] Collection of targets related to dry eye disease
[0057] Based on drug target and pathway databases such as DrugBank, PharmGkb, MetaCore, Ingenuity Pathway Analysis, TherapeuticTargets Database, Potential Drug Target Database, etc., a dry eye biological target database and a disease-related target set were screened and constructed. After sorting and merging, a list of disease targets corresponding to dry eye was obtained.
[0058] Construction of "drug ingredient - target - disease" network
[0059] The above-mentioned blood components of Zhangyanming tablets and the intersection targets of dry eye were imported into Cytoscape 3.9.0 for visualization processing to construct a network diagram of "Zhangyanming extract - blood components - intersection targets - dry eye disease". Among them, the green nodes represent the blood-active components of Zhangyanming extract, and the red nodes represent the intersection targets of the active components and dry eye disease. The color is positively correlated with the number of related targets.
[0060] Construction of PPI network
[0061] The String database (https: / / string-db.org / ) was used to analyze the intersection targets to obtain a protein-protein interaction (PPI) network diagram, revealing the interaction relationships between the targets.
[0062] Signal pathway enrichment analysis
[0063] The DAVID database (https: / / david.ncifcrf.gov / ) was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the targets. To reveal the biological functions and signal pathways of Zhangyanming extract in treating dry eye, p < 0.05 indicates that the results have statistical differences.
[0064] II. Experimental results
[0065] By retrieving and predicting the targets corresponding to the blood components of Zhangyanming tablets through TCMSP and Swiss Target Prediction, a total of 303 component-related targets were obtained. Through the Genecards database, a total of 1647 dry eye-related targets were retrieved. Taking the intersection with the targets corresponding to the blood components of Zhangyanming tablets through the Venny 2.1.0 website, as Figure 1As shown, 335 intersection action targets were obtained.
[0066] The above-mentioned blood components of Zhangyanming Tablets and the intersection targets of dry eye were imported into Cytoscape 3.9.0 for visualization processing to construct a "Zhangyanming Tablets - blood components - targets - diseases" network diagram, as Figure 2 shown. The green nodes represent the blood-active components of Zhangyanming Tablets, and the red nodes represent the related targets corresponding to the components. The color is positively correlated with the number of related targets. Analysis of this network shows that there are 327 nodes and 1119 edges in this network diagram. Among them, the same active component may correspond to different targets, and different active components may also correspond to the same target, indicating that Zhangyanming Tablets act on diseases through multiple components and multiple targets.
[0067] The key blood-active components of Zhangyanming Tablets include: luteolin, apigenin, obacunone, methyl myristate, albiflorin, moutanpioside D, angelicin, dibutyl sebacate, puerarin, formononetin, sclareolide, oleic acid, adenosine, benzyl-β-primeveroside, naringenin, odoratin, senkyunolide O, ethyl oleate.
[0068] Analysis of the PPI blood component - disease target interaction network shows that there are 31 core targets in total, including: TNF, IL6, BCL2, CASP3, etc., as Figure 3 shown.
[0069] Using the DAVID database to perform enrichment analysis on the action targets, a total of 1432 GO enrichment entries were obtained, among which 1078 belong to Biological Process (BP), 137 belong to Cellular Component (CC), and 216 belong to Molecular Function (MF). The target genes are mainly involved in the positive regulation of IL-10 production, the positive regulation of T cell proliferation, immune cell adhesion, the negative regulation of apoptosis, etc. ( Figure 4 )
[0070] 183 KEGG pathways were enriched, and the related pathways include cancer pathways, neuroactive ligand - receptor interactions, PI3K - Akt signaling pathways, lipid and atherosclerosis, calcium signaling pathways, etc., providing ideas and directions for the pharmacodynamic evaluation and mechanism of action research on exploring the new indications of Zhangyanming Tablets in regulating dry eye in the next chapter ( Figure 5 )
[0071] Table 1 Analysis of Blood Components of Zhangyanming Extract
[0072]
[0073]
[0074]
[0075]
[0076]
Claims
1. A method for detecting the blood components of Zhangyanming tablets, characterized in that, Including the following steps: A. Administer Zhangyanming tablets to animals. After a predetermined time, collect the serum as the experimental group, and use the serum of animals that have not been administered Zhangyanming tablets as the control group; B. Detect the experimental group and the control group by liquid chromatography-mass spectrometry; For the chromatography mentioned above, the conditions are as follows: Use a Kinetex C18 chromatographic column 100×2.1mm 2.6μm, Waters, USA, the column temperature is 40°C, the mobile phase system consists of water-A phase and acetonitrile-B phase, both containing 0.1% formic acid v / v, the flow rate is 0.3 mL / min, the total elution time is: 45 min, and the gradient elution method is: 5% to 95% acetonitrile-B phase from 0 to 30 min, 95% acetonitrile-B phase from 30 to 35%, 95% to 5% acetonitrile-B phase from 35 to 37 min, and 5% acetonitrile-B phase from 37 to 45 min. For the mass spectrometry mentioned above, the conditions are as follows: Electrospray ionization source ESI, detection in positive and negative ion modes. In the positive ion mode, the spray voltage is set to: 5500 V; in the negative ion mode, it is set to: -4500 V. The spray gas pressure is set to 55 psi, the auxiliary heating gas pressure is 50 psi, the ion source temperature is maintained at 550 °C, the collision voltage is 30 V, the declustering voltage is 80 V, the curtain gas pressure is 35 psi, the collision cell pressure is 10 psi, the scanning mass-to-charge ratio m / z range is: 50 - 1500, nitrogen is used as the spray gas and auxiliary gas, using The software collects data in information-dependent acquisition mode.
2. The detection method according to claim 1, wherein The step A is as follows: One day before the start of the experiment, Beagle dogs are fasted for 12 h, allowed to drink water freely. After collecting blank blood samples, Beagle dogs are given a single oral administration of Zhangyanming extract, 499.5 mg / kg. Blood samples (2 mL) are collected at 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h after administration. The blood samples are centrifuged at 2500 g for 15 min at 4°C, and the supernatant is collected.
3. The detection method according to claim 1, wherein The method for preparing the injection specimen for the liquid chromatography in step B is as follows: Take 100 μL of Beagle dog serum sample, add 1 mL of ethyl acetate, vortex for 5 min, and then centrifuge at 10000 g for 15 min at 4°C. Transfer 700 μL of the supernatant, dry it with nitrogen, immediately reconstitute it for detection or store it at -80°C. Before detection, the sample is reconstituted with 100 μL of methanol: distilled water solution 4:1, v / v, vortex for 5 min, ultrasonicate for 10 min until completely dissolved, and then centrifuge at 10000 g for 20 min at 4°C to obtain the sample solution for injection.
4. The detection method according to claim 1, wherein The blood components are as follows; 5. The detection method according to claim 1, characterized in that For the blood components mentioned above, the key active components are: luteolin, apigenin, obacunone, methyl myristate, albiflorin, paeonolide D, angelicin, dibutyl sebacate, puerarin, formononetin, sclareolide, oleic acid, adenosine, benzyl-β-primeveroside, naringenin, odoratin, senkyunolide O, ethyl oleate.
6. A method for constructing a dry eye target network, characterized in that, Including the following steps: A. Collection of dry eye disease-related targets Based on drug target and pathway databases such as DrugBank, PharmGkb, MetaCore, Ingenuity Pathway Analysis, TherapeuticTargets Database, Potential Drug Target Database, etc., screen and construct a dry eye biological target database and a disease-related target set. After sorting and merging, obtain the disease target list corresponding to dry eye; B. Construction of the "drug ingredient-target-disease" network The blood-entry components of Zhangyanming tablets and the intersection targets of dry eye disease in Claim 4 or 5 were imported into Cytoscape 3.9.0 for visualization, and a network diagram of "Zhangyanming extract - blood-entry components - intersection targets - dry eye disease" was constructed. Among them, the green nodes represent the blood-entry active components of Zhangyanming extract, and the red nodes represent the intersection targets of the active components and dry eye disease. The color is positively correlated with the number of related targets; C. Construction of PPI network The String database was used to analyze the intersection targets to obtain a protein-protein interaction (PPI) network diagram, revealing the interaction relationships between the targets; D. Signal pathway enrichment analysis The DAVID database was used to perform gene function classification system and Kyoto Encyclopedia of Genes and Genomes enrichment analysis on the targets, revealing the biological functions and signal pathways of Zhangyanming extract in the treatment of dry eye disease.