A method for analyzing YZH treatment mechanism for liver injury based on network pharmacology

Abstract

The application discloses a method for analyzing the mechanism of YZH in treating liver injury based on network pharmacology, wherein magnetic solid-phase extraction microspheres prepared by modifying magnetic Fe3O4 with oleic acid, adding 4-vinyl ethyl benzoate and 2-methyl propionic acid-3-heptenyl ester are used to sufficiently extract main components in YZH, component identification is performed in combination with LC-MS / MS and a traditional Chinese medicine metabolite database, components and disease targets are predicted, a protein-protein interaction network is constructed, and core targets are screened out. Further confirmation is performed through GO function and KEGG pathway enrichment analysis, molecular docking and immunoblotting experiments, and it is found that YZH plays a liver protection role through regulating multiple pathways such as oxidative stress response and inflammatory signaling pathway. The method integrates analysis of multiple components, multiple targets and multiple pathways, analyzes the action mechanism of YZH in treating liver injury, and provides a standardized technical scheme for mechanism research of traditional Chinese medicine compounds.

Description

Technical Field

[0001] This invention belongs to the field of pharmacology, specifically relating to a method for analyzing the mechanism of YZH in treating liver injury based on network pharmacology. Background Technology

[0002] Liver injury is a common pathological condition of the liver, which can be caused by various factors such as drugs, alcohol, toxins, and infections. Among these, drug-induced liver injury accounts for an increasing proportion year by year, becoming a significant safety hazard in drug development and clinical use worldwide. Furthermore, current clinical treatments are still mainly symptomatic and supportive, lacking highly effective and low-toxicity specific drugs. Traditional Chinese medicine (TCM) compound formulas, due to their synergistic effects across multiple components and targets, have shown unique advantages in the prevention and treatment of liver injury. The classic compound Yin Zhi Huang oral liquid (YZH), composed of Scutellaria baicalensis, Gardenia jasminoides, and Lonicera japonica, has been clinically proven to have significant efficacy against liver injury; however, its mechanism of action remains unclear, hindering the modernization and international promotion of TCM.

[0003] Traditional Chinese medicine (TCM) research methods are often limited to single-component or single-target analysis, making it difficult to comprehensively reveal the overall pharmacodynamic mechanism of compound formulas. With the development of systems biology and bioinformatics, network pharmacology, as an interdisciplinary approach integrating multi-omics data, offers new insights into the mechanism of TCM compound formula research. However, current TCM research based on network pharmacology still faces the following technical bottlenecks: First, the complexity of TCM components makes it difficult for traditional extraction and identification methods to comprehensively capture active ingredients, especially for low-abundance and trace components; second, target prediction lacks accuracy, and existing database prediction algorithms are not fully adapted to the characteristics of TCM components, leading to a high false positive rate; third, pathway enrichment analysis often relies on single databases, lacking multi-source data integration, making it difficult to construct a complete action network; fourth, in the molecular docking verification process, the standardization of receptor protein pretreatment and active pocket identification is insufficient, affecting the reliability of docking results. Furthermore, the pathological mechanisms of liver injury involve multiple biological processes such as oxidative stress, inflammatory response, and apoptosis, making single-target treatment difficult to achieve ideal results. Meanwhile, although the application of magnetic solid phase extraction technology in the separation of Chinese medicine components can improve the extraction efficiency, existing magnetic bead materials have problems such as small specific surface area and low adsorption selectivity, resulting in insufficient integrity of component identification.

[0004] Therefore, there is an urgent need to establish a network pharmacology research method that integrates efficient component extraction, precise target prediction, multi-pathway enrichment analysis, and experimental verification to comprehensively reveal the mechanism of action of YZH in treating liver injury and provide a standardized technical path for the modernization of traditional Chinese medicine compound research. Based on the above background, this invention, through improved sample preparation processes, optimized database screening strategies, construction of a multi-dimensional network model, and combined with molecular docking and immunoblotting experiments for verification, has achieved a systematic analysis of the mechanism of YZH in treating liver injury, filling the gap in existing technologies for multi-target mechanism research of traditional Chinese medicine compound formulas. Summary of the Invention

[0005] The purpose of this invention is to provide an efficient method for analyzing the mechanism of YZH in treating liver injury based on network pharmacology.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for analyzing the mechanism of YZH treatment for liver injury includes: The active ingredients of YZH were extracted by ultrasonic extraction using an extraction solution. After centrifugation, the supernatant was blown with nitrogen and then extracted by ultrasonic extraction using a reconstituted solution to obtain the sample solution. The sample solution was identified by LC-MS / MS combined with a database of traditional Chinese medicine metabolites, and the component targets and disease targets were predicted. By constructing a protein-protein interaction network through the intersection of component targets and disease targets, core targets can be screened. We performed GO function and KEGG pathway enrichment analysis on the core target and verified the mechanism of action through molecular docking.

[0007] Preferably, the extract comprises methanol and acetonitrile.

[0008] Preferably, the volume ratio of methanol to acetonitrile is 160-1600:40-400.

[0009] Preferably, the reconstituted solution comprises acetonitrile and deionized water.

[0010] Preferably, the volume ratio of acetonitrile to deionized water is 30-300:30-300.

[0011] Preferably, the ultrasonic temperature is 4-6℃ and the power is 35-45kHz.

[0012] Preferably, ultrasonic extraction includes magnetic solid-phase extraction.

[0013] Preferably, the adsorbent for magnetic solid-phase extraction is magnetic solid-phase extraction microspheres.

[0014] Preferably, the eluent for magnetic solid-phase extraction includes sodium carbonate, deionized water, and ethanol.

[0015] Preferably, the mass-to-volume ratio of sodium carbonate to deionized water is 2.65-26.5 mg: 250-2500 μL.

[0016] Preferably, the volume ratio of ethanol to deionized water is 250-2500:250-2500.

[0017] Preferably, the magnetic solid-phase extraction microspheres comprise a modified magnetic Fe3O4 core and a polymer shell.

[0018] Preferably, the modified magnetic Fe3O4 core includes oleic acid.

[0019] Preferably, the polymeric monomers of the polymeric shell include ethyl 4-vinylbenzoate and 3-heptenyl 2-methylpropionic acid.

[0020] Preferably, the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate is 1.5-15:20-200.

[0021] Preferably, the mass ratio of modified magnetic Fe3O4 to 2-methylpropionic acid-3-heptenyl ester is 1.5-15:10-100.

[0022] Oleic acid inhibits the aggregation of Fe3O4 particles through steric hindrance. In the subsequent polymerization of modified magnetic Fe3O4 surface, ethyl 4-vinylbenzoate and 2-methylpropionic acid-3-heptenyl ester form a porous network, which increases the specific surface area of ​​magnetic microspheres, improves adsorption selectivity and recovery rate, and thus improves the extraction efficiency of YZH effective components.

[0023] Preferably, the core targets include TLR4, TNF, MMP2, MMP9, PPARA, and PTGS2.

[0024] More preferably, the polymeric monomers of the polymer shell include 1,6-hexanediol diacrylate. The mass ratio of 1,6-hexanediol diacrylate to ethyl 4-vinylbenzoate is 10-100:20-200. During the polymerization reaction, 1,6-hexanediol diacrylate undergoes free radical copolymerization with the double bonds of ethyl 4-vinylbenzoate, enhancing the mechanical stability and pore rigidity of the magnetic microspheres, further forming a dense cross-linked network, increasing the specific surface area of ​​the magnetic microspheres, improving the adsorption selectivity for active ingredients, reducing non-specific adsorption, and increasing the extraction and recovery rates.

[0025] The present invention also provides a method for preparing a sample solution, comprising: Preparation of sample solution: Methanol and acetonitrile were mixed, and L-2-chlorophenylalanine was added and mixed evenly to obtain the extract. Acetonitrile and deionized water were mixed evenly to obtain the reconstituted solution. YZH was mixed with the extract and vortexed for 25-35 s. The mixture was then ultrasonically extracted for 25-35 min at 4-6℃ and 35-45 kHz. The mixture was allowed to stand for 25-35 min at -22--18℃ and centrifuged for 10-20 min at 3-5℃ and 12000-14000 rpm. The supernatant was collected, dried under nitrogen, and the reconstituted solution was added. The mixture was vortexed for 25-35 s and ultrasonically extracted for 4-6 min at 4-6℃ and 35-45 kHz. The mixture was then centrifuged for 5-15 min at 3-5℃ and 12000-14000 rpm. The supernatant was collected to obtain the sample solution.

[0026] Preferably, the volume ratio of methanol to acetonitrile in the extract is 160-1600:40-400.

[0027] Preferably, the mass-to-volume ratio of L-2-chlorophenylalanine to methanol in the extract is 40-400 μg: 160-1600 μL.

[0028] Preferably, in the reconstituted solution, the volume ratio of acetonitrile to deionized water is 30-300:30-300.

[0029] Preferably, the volume ratio of YZH to the extract is 50-500:200-2000.

[0030] Preferably, the volume ratio of the reconstituted solution to the extract is 60-600:200-2000.

[0031] This invention also provides a method for collecting analytical data, comprising: Data acquisition: The sample solution was analyzed by LC-MS / MS to obtain raw data; the raw data was imported into preprocessing software for baseline filtering, peak identification, retention time correction and peak alignment to obtain a data matrix containing information such as retention time, mass-to-charge ratio and peak intensity. Characteristic peaks were identified by searching the library. The MS and MS / MS mass spectrometry information was matched with a database of metabolites specific to traditional Chinese medicine. The MS mass error was set to be less than 9-11 ppm. Metabolites were identified based on the secondary mass spectrometry matching score.

[0032] Preferably, the chromatographic column is an ACQUITY UPLC BEH C18.

[0033] Preferably, the mobile phase A is an aqueous solution of 1.5-2.5% acetonitrile containing 0.05-0.15% formic acid.

[0034] Preferably, the mobile phase B is an acetonitrile solution containing 0.05-0.15% formic acid.

[0035] Preferably, the injection volume is 2-4 μL.

[0036] Preferably, the column temperature is 39-41℃.

[0037] Preferably, the mass spectrometry spray voltage is 3400-3600V.

[0038] Preferably, the scanning range is 70-1050 m / z.

[0039] Preferably, the sheath gas velocity is 48-52 arb.

[0040] Preferably, the auxiliary airflow velocity is 12-14 arb.

[0041] Preferably, the heating temperature is 445-455℃.

[0042] Preferably, the capillary temperature is 315-325℃.

[0043] Preferably, the collision energy is 20-60 V.

[0044] Preferably, the first-order mass spectrometry resolution is 60,000-80,000.

[0045] Preferably, the resolution of the secondary mass spectrometer is 17000-18000.

[0046] Preferably, the pretreatment software includes Progenesis QI v3.0 software.

[0047] This invention also provides a method for predicting components and disease targets, comprising: Prediction of components and disease targets: The target targets of the target components in YZH are screened and predicted through the component database. The predicted targets of YZH are screened with a probability greater than 0.12. The names of the target proteins are standardized through the protein database to obtain the component targets. Using drug-induced liver injury as the keyword, the disease targets are predicted in the disease database. The protein names are standardized through the protein database to obtain the disease targets.

[0048] Preferably, the ingredient database includes TCMSP and the SwissTargetPrediction database.

[0049] Preferably, the protein database includes the UniProt database.

[0050] Preferably, the disease database includes the DisGeNET database.

[0051] This invention also provides a method for constructing a protein-protein interaction network, comprising: Construction of protein-protein interaction networks: The intersection of component targets and disease targets is obtained, and duplicates are removed. The intersection target data is imported into a protein-protein interaction database. "Human / mouse" is selected as the research species to construct a protein-protein interaction network. The core targets for treating diseases are screened out based on the average value greater than the calculated degree value.

[0052] Preferably, the protein-protein interaction database includes the STRING11.5 database.

[0053] This invention also provides a method for GO functional enrichment and KEGG pathway enrichment analysis, including: GO functional enrichment and KEGG pathway enrichment analysis: GO functional enrichment and KEGG pathway enrichment analysis were performed on the screened core targets. P < 0.05 was used as the screening criterion. Biological functions and signaling pathways were selected and represented by bar charts and bubble charts, respectively. Based on the relationship between protein targets and signaling pathways, a "component-target-pathway-disease" network was drawn.

[0054] This invention also provides a method for verifying molecular docking, comprising: Validation of molecular docking: Download the 3D structures of macromolecular receptors and small molecule ligands from molecular databases, with macromolecular receptors as the core target and small molecule ligands as the active ingredients; dehydrate and deligatinate the receptor protein, perform hydrogenation, calculate the charge and determine the rigid structure to obtain a pretreated receptor protein, determine the location and size of the active pocket, perform molecular docking, and select the conformation with the lowest binding energy for analysis.

[0055] Preferably, the molecular database includes the PDB database and the PubChem database.

[0056] Preferably, the software used for dehydration and deligation of the receptor protein is Pymol software.

[0057] Preferably, the software used for hydrogenation of the receptor protein, charge calculation, and determination of rigid structure is Autodock Tools.

[0058] Preferably, the molecular docking software is Autodock 4 software.

[0059] This invention also provides a method for preparing modified magnetic Fe3O4, comprising: Preparation of modified magnetic Fe3O4: Oleic acid was dissolved in acetone to obtain an acetone solution; FeCl3·6H2O and FeCl2·4H2O were dispersed and dissolved in deionized water and reacted at 75-85℃ with stirring for 25-35 min; acetone solution and 20-30wt% ammonia solution were added and reacted for 5-15 min; oleic acid was added and the reaction was stirred for 25-35 min; after magnetic separation, the mixture was washed with methanol and dried under vacuum at 55-65℃ for 10-15 h to obtain modified magnetic Fe3O4.

[0060] Preferably, in the acetone solution, the mass-to-volume ratio of oleic acid to acetone is 1.5-15g:10-100mL.

[0061] Preferably, the mass-to-volume ratio of FeCl3·6H2O to deionized water is 3.5-35g:150-1500mL.

[0062] Preferably, the mass-to-volume ratio of FeCl2·4H2O to deionized water is 1.2-14 g: 150-1500 mL.

[0063] Preferably, the volume-to-mass ratio of acetone solution to FeCl3·6H2O is 10-100 mL: 3.5-35 g.

[0064] Preferably, the volume ratio of acetone solution to ammonia solution is 10-100:15-150.

[0065] Preferably, the mass-to-volume ratio of oleic acid to acetone solution is 1.5-15g:10-100mL.

[0066] This invention also provides a method for preparing a magnetic emulsion, comprising: Preparation of magnetic emulsion: Polyvinyl alcohol, anhydrous sodium sulfate and hydroquinone were dissolved in deionized water to obtain an aqueous phase; ethyl 4-vinylbenzoate, 3-heptenyl 2-methylpropionic acid, toluene, benzoyl peroxide and modified magnetic Fe3O4 were uniformly mixed to obtain an oil phase; the oil phase was introduced into the circulating aqueous phase through an SPG membrane to obtain a magnetic emulsion.

[0067] Preferably, the mass-to-volume ratio of polyvinyl alcohol to deionized water is 1-10g: 250-2500mL.

[0068] Preferably, the mass ratio of anhydrous sodium sulfate to polyvinyl alcohol is 50-500 mg: 1-10 g.

[0069] Preferably, the mass ratio of hydroquinone to polyvinyl alcohol is 50-500 mg: 1-10 g.

[0070] Preferably, the mass ratio of ethyl 4-vinylbenzoate to toluene is 20-200:40-400.

[0071] Preferably, the mass ratio of 2-methylpropionic acid-3-heptenyl ester to toluene is 10-100:40-400.

[0072] Preferably, the mass ratio of benzoyl peroxide to ethyl 4-vinylbenzoate is 0.4-4:20-200.

[0073] Preferably, the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate is 1.5-15:20-200.

[0074] Preferably, the mass of the aqueous phase is measured by the mass of polyvinyl alcohol therein, the mass of the oil phase is measured by the mass of ethyl 4-vinylbenzoate therein, and the mass ratio of polyvinyl alcohol to ethyl 4-vinylbenzoate is 1-10:20-200.

[0075] More preferably, the oil phase comprises 1,6-hexanediol diacrylate.

[0076] More preferably, the mass ratio of 1,6-hexanediol diacrylate to ethyl 4-vinylbenzoate is 10-100:20-200.

[0077] This invention also provides a method for preparing magnetic solid-phase extraction microspheres, comprising: Preparation of magnetic solid-phase extraction microspheres: Under nitrogen protection, the magnetic emulsion was polymerized at 75-85℃ for 1-3 h, at 85-95℃ for 1-3 h, and at 90-100℃ for 5-7 h. After cooling, the microspheres were washed alternately with deionized water and methanol 3-5 times and then vacuum dried at 75-85℃ for 10-15 h to obtain magnetic solid-phase extraction microspheres.

[0078] The present invention also provides a method for preparing a sample solution, comprising: Preparation of sample solution: Methanol and acetonitrile were mixed, and L-2-chlorophenylalanine was added and mixed evenly to obtain the extract. Acetonitrile and deionized water were mixed evenly to obtain the reconstituted solution. Sodium carbonate was dissolved in deionized water, and ethanol was added and mixed evenly to obtain the eluent. YZH was mixed with the extract and vortexed for 25-35 s. The mixture was then ultrasonically extracted for 25-35 min at 4-6℃ and 35-45 kHz. The mixture was allowed to stand for 25-35 min at -22--18℃ and centrifuged for 10-20 min at 3-5℃ and 12000-14000 rpm. The supernatant was dried under nitrogen, and the reconstituted solution was added. The mixture was vortexed for 25-35 s and then mixed with magnetic solid-phase extraction microspheres. The mixture was kept at 25-35℃ and shaken for 15-25 min. After magnetic separation, the eluent was added to the magnetic solid-phase extraction microspheres and kept at 25-35℃ and shaken for 10-20 min. The mixture was then filtered to obtain the sample solution.

[0079] Preferably, the volume ratio of methanol to acetonitrile in the extract is 160-1600:40-400.

[0080] Preferably, the mass-to-volume ratio of L-2-chlorophenylalanine to methanol in the extract is 40-400 μg: 160-1600 μL.

[0081] Preferably, in the reconstituted solution, the volume ratio of acetonitrile to deionized water is 30-300:30-300.

[0082] Preferably, the mass-to-volume ratio of sodium carbonate to deionized water in the eluent is 2.65-26.5 mg: 250-2500 μL.

[0083] Preferably, the volume ratio of ethanol to deionized water in the eluent is 250-2500:250-2500.

[0084] Preferably, the volume ratio of YZH to the extract is 50-500:200-2000.

[0085] Preferably, the volume ratio of the reconstituted solution to the extract is 60-600:200-2000.

[0086] Preferably, the mass-to-volume ratio of magnetic solid-phase extraction microspheres to the complex solution is 0.6-60 g: 60-600 μL.

[0087] Preferably, the volume-to-mass ratio of the eluent to the magnetic solid-phase extraction microspheres is 0.5-5 mL: 0.6-60 g.

[0088] This invention utilizes magnetic solid-phase extraction microspheres prepared by modifying magnetic Fe3O4 with oleic acid and adding ethyl 4-vinylbenzoate and 3-heptenyl 2-methylpropionic acid to fully extract the main components of YZH. This is combined with LC-MS / MS and network pharmacology analysis to determine the mechanism of YZH in treating liver injury. Therefore, it has the following advantages: high extraction rate of effective components of YZH, high accuracy of target prediction, and successful elucidation of the multi-component, multi-target, and multi-pathway mechanism of action of YZH in treating liver injury. Thus, this invention is a highly efficient method for analyzing the mechanism of YZH in treating liver injury based on network pharmacology. Attached Figure Description

[0089] Figure 1 This is a schematic diagram of the total ion chromatogram of YZH.

[0090] Figure 2 A pie chart illustrating the content of YZH components by category.

[0091] Figure 3 This is a schematic diagram of the structural formula of the active ingredient in YZH.

[0092] Figure 4 This is a schematic diagram of the intersection of YZH components and disease targets.

[0093] Figure 5 This is a schematic diagram of a protein-protein interaction network.

[0094] Figure 6 A diagram illustrating the GO function of YZH.

[0095] Figure 7 A schematic diagram of the top 10 pathways enriched for the YZH core target.

[0096] Figure 8 This is a network diagram of components, targets, pathways, and diseases.

[0097] Figure 9 This is a schematic diagram of the molecular docking between the key target TLR4 and the active ingredient.

[0098] Figure 10 This is a schematic diagram illustrating the effect of YZH on the NF-κB signaling pathway in mouse liver. Detailed Implementation

[0099] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0100] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0101] Example 1: Preparation of sample solution: Methanol and acetonitrile were mixed, and L-2-chlorophenylalanine was added and mixed evenly to obtain the extract. Acetonitrile and deionized water were mixed evenly to obtain the reconstituted solution. YZH was mixed with the extract, vortexed for 30 s, and ultrasonically extracted for 30 min at 5 ℃ and 40 kHz. The mixture was then allowed to stand at -20 ℃ for 30 min, centrifuged at 4 ℃ and 13000 rpm for 15 min, and the supernatant was collected. The supernatant was dried under nitrogen, and the reconstituted solution was added. The mixture was vortexed for 30 s, ultrasonically extracted for 5 min at 5 ℃ and 40 kHz, and centrifuged at 4 ℃ and 13000 rpm for 10 min. The supernatant was collected to obtain the sample solution. In the extract, the volume ratio of methanol to acetonitrile was 320:80, and the mass-volume ratio of L-2-chlorophenylalanine to methanol was 80 μg:320 μL; in the reconstituted solution, the volume ratio of acetonitrile to deionized water was 60:60; the volume ratio of YZH to the extract was 100:400, and the volume ratio of the reconstituted solution to the extract was 120:400.

[0102] Data acquisition: The sample solution was analyzed by LC-MS / MS to obtain raw data. The raw data was then imported into Progenesis QI v3.0 software for baseline filtering, peak identification, retention time correction, and peak alignment to obtain a data matrix containing information such as retention time, mass-to-charge ratio, and peak intensity. Characteristic peaks were identified by searching the library. The MS and MS / MS mass spectrometry information was matched with a database of metabolites specific to traditional Chinese medicine. The MS mass error was set to less than 10 ppm. Metabolites were also identified based on the secondary mass spectrometry matching score. Chromatographic conditions: The column was an ACQUITY UPLC BEH C18, 100 mm × 2.1 mm id, 1.7 µm; mobile phase A was a 2% acetonitrile aqueous solution containing 0.1% formic acid; mobile phase B was an acetonitrile solution containing 0.1% formic acid; the injection volume was 3 μL; and the column temperature was 40 °C. Mass spectrometry conditions: the spray voltage was 3500 V; the scan range was 70–1050 m / z; the sheath gas flow rate was 50 arb; the auxiliary gas flow rate was 13 arb; the heating temperature was 450 °C; the capillary temperature was 320 °C; the collision energies were 20, 40, and 60 V; the primary mass spectrometry resolution was 70,000; and the secondary mass spectrometry resolution was 17,500.

[0103] Component and disease target prediction: Targets of the target components in YZH were screened and predicted using the TCMSP and SwissTargetPrediction databases. Predictable targets of YZH were screened based on a probability greater than 0.12. The names of the target proteins were normalized using the UniProt database to obtain the component targets. Disease targets were predicted using the DisGeNET database with "Drug Induced Liver Injury" as the keyword. The protein names were normalized using the UniProt database to obtain the disease targets.

[0104] Construction of protein-protein interaction networks: The intersection of component targets and disease targets was obtained, duplicates were removed, and Venn diagrams were drawn. The intersection target data were imported into the STRING11.5 database. "Human / Mouse" was selected as the research species to construct protein-protein interaction networks. The core targets for disease treatment were screened out based on the average value greater than the calculated degree value.

[0105] GO functional enrichment and KEGG pathway enrichment analysis: The core targets selected were analyzed for GO functional enrichment and KEGG pathway enrichment using the R language clusterProfiler. The top 10 biological functions and signaling pathways were selected with P < 0.05 as the screening criterion, and represented by bar charts and bubble charts, respectively. Based on the relationship between protein targets and signaling pathways, a "component-target-pathway-disease" network was drawn.

[0106] Validation of molecular docking: 3D structures of macromolecular receptors and small molecule ligands were downloaded from the PDB and PubChem databases, respectively, with the macromolecular receptor as the core target and the small molecule ligand as the active ingredient. The receptor protein was dehydrated and deligated using Pymol software, and hydrogenated, charged, and determined to have a rigid structure using Autodock Tools software to obtain a pretreated receptor protein. The core target protein and active ingredient were saved as "pdbqt" format files, the location and size of the active pocket were determined, and molecular docking was performed using Autodock 4 software. The conformation with the lowest binding energy was selected for analysis.

[0107] Example 2: The only difference between this example and Example 1 is the preparation of the sample solution.

[0108] Preparation of modified magnetic Fe3O4: Oleic acid was dissolved in acetone to obtain an acetone solution; FeCl3·6H2O and FeCl2·4H2O were dispersed and dissolved in deionized water and reacted at 80℃ with stirring for 30 min; acetone solution and 25wt% ammonia solution were added and reacted for 10 min; oleic acid was added and the reaction was stirred for 30 min; after magnetic separation, the mixture was washed with methanol and dried under vacuum at 60℃ for 12 h to obtain modified magnetic Fe3O4. In the acetone solution, the mass-to-volume ratio of oleic acid to acetone is 3 g: 20 mL; the mass-to-volume ratio of FeCl3·6H2O to deionized water is 7 g: 300 mL; the mass-to-volume ratio of FeCl2·4H2O to deionized water is 2.58 g: 300 mL; the volume-to-mass ratio of acetone solution to FeCl3·6H2O is 20 mL: 7 g; the volume ratio of acetone solution to ammonia solution is 20:30; and the mass-to-volume ratio of oleic acid to acetone solution is 3 g: 20 mL.

[0109] Preparation of magnetic emulsion: Polyvinyl alcohol, anhydrous sodium sulfate and hydroquinone were dissolved in deionized water to obtain an aqueous phase; ethyl 4-vinylbenzoate, 3-heptenyl 2-methylpropionic acid, toluene, benzoyl peroxide and modified magnetic Fe3O4 were uniformly mixed to obtain an oil phase; the oil phase was introduced into the circulating aqueous phase through an SPG membrane to obtain a magnetic emulsion. The mass-to-volume ratio of polyvinyl alcohol (PVA) to deionized water was 2 g:500 mL; the mass ratio of anhydrous sodium sulfate to PVA was 100 mg:2 g; the mass ratio of hydroquinone to PVA was 100 mg:2 g; the mass ratio of ethyl 4-vinylbenzoate to toluene was 40:80; the mass ratio of 2-methylpropionic acid-3-heptenyl ester to toluene was 20:80; the mass ratio of benzoyl peroxide to ethyl 4-vinylbenzoate was 0.8:40; and the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate was 3:40. The mass of the aqueous phase was measured by the mass of PVA contained therein, and the mass of the oil phase was measured by the mass of ethyl 4-vinylbenzoate contained therein. The mass ratio of PVA to ethyl 4-vinylbenzoate was 2:40.

[0110] Preparation of magnetic solid-phase extraction microspheres: Under nitrogen protection, the magnetic emulsion was polymerized at 80℃ for 2 h, at 90℃ for 2 h, and at 95℃ for 6 h. After cooling, it was washed 4 times alternately with deionized water and methanol, and then vacuum dried at 80℃ for 12 h to obtain magnetic solid-phase extraction microspheres.

[0111] Preparation of sample solution: Methanol and acetonitrile were mixed, and L-2-chlorophenylalanine was added and mixed evenly to obtain the extract. Acetonitrile and deionized water were mixed evenly to obtain the reconstituted solution. Sodium carbonate was dissolved in deionized water, and ethanol was added and mixed evenly to obtain the eluent. YZH was mixed with the extract, vortexed for 30 s, and ultrasonically extracted at 5℃ and 40 kHz for 30 min. The mixture was then allowed to stand at -20℃ for 30 min, centrifuged at 4℃ and 13000 rpm for 15 min, and the supernatant was dried under nitrogen. The reconstituted solution was added, vortexed for 30 s, and mixed with magnetic solid-phase extraction microspheres. The mixture was kept at 30℃ and shaken for 20 min. After magnetic separation, the eluent was added to the magnetic solid-phase extraction microspheres, and the mixture was kept at 30℃ and shaken for 15 min. The sample solution was obtained by filtration. In the extract, the volume ratio of methanol to acetonitrile was 320:80, and the mass-to-volume ratio of L-2-chlorophenylalanine to methanol was 80 μg:320 μL; in the reconstitution solution, the volume ratio of acetonitrile to deionized water was 60:60; in the eluent, the mass-to-volume ratio of sodium carbonate to deionized water was 5.3 mg:500 μL, and the volume ratio of ethanol to deionized water was 500:500; the volume ratio of YZH to the extract was 100:400, and the volume ratio of the reconstitution solution to the extract was 120:400; the mass-to-volume ratio of magnetic solid-phase extraction microspheres to the reconstitution solution was 1.2 g:120 μL, and the volume-to-mass ratio of the eluent to the magnetic solid-phase extraction microspheres was 1 mL:1.2 g.

[0112] Example 3: The only difference between this example and Example 2 is the preparation of the magnetic emulsion.

[0113] Preparation of magnetic emulsion: Polyvinyl alcohol, anhydrous sodium sulfate and hydroquinone were dissolved in deionized water to obtain an aqueous phase; ethyl 4-vinylbenzoate, 3-heptenyl 2-methylpropionic acid, toluene, benzoyl peroxide and modified magnetic Fe3O4 were uniformly mixed to obtain an oil phase; the oil phase was introduced into the circulating aqueous phase through an SPG membrane to obtain a magnetic emulsion. The mass-to-volume ratio of polyvinyl alcohol (PVA) to deionized water was 2 g:500 mL; the mass ratio of anhydrous sodium sulfate to PVA was 100 mg:2 g; the mass ratio of hydroquinone to PVA was 100 mg:2 g; the mass ratio of ethyl 4-vinylbenzoate to toluene was 60:80; the mass ratio of 2-methylpropionic acid-3-heptenyl ester to toluene was 20:80; the mass ratio of benzoyl peroxide to ethyl 4-vinylbenzoate was 0.8:40; and the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate was 3:40. The mass of the aqueous phase was measured by the mass of PVA contained therein, and the mass of the oil phase was measured by the mass of ethyl 4-vinylbenzoate contained therein. The mass ratio of PVA to ethyl 4-vinylbenzoate was 2:40.

[0114] Example 4: The only difference between this example and Example 2 is the preparation of the magnetic emulsion.

[0115] Preparation of magnetic emulsion: Polyvinyl alcohol, anhydrous sodium sulfate and hydroquinone were dissolved in deionized water to obtain an aqueous phase; ethyl 4-vinylbenzoate, 3-heptenyl 2-methylpropionic acid, toluene, benzoyl peroxide, 1,6-hexanediol diacrylate and modified magnetic Fe3O4 were uniformly mixed to obtain an oil phase; the oil phase was introduced into the circulating aqueous phase through an SPG membrane to obtain a magnetic emulsion. The mass-to-volume ratio of polyvinyl alcohol (PVA) to deionized water was 2 g:500 mL; the mass ratio of anhydrous sodium sulfate to PVA was 100 mg:2 g; the mass ratio of hydroquinone to PVA was 100 mg:2 g; the mass ratio of ethyl 4-vinylbenzoate to toluene was 40:80; the mass ratio of 2-methylpropionic acid-3-heptenyl ester to toluene was 20:80; the mass ratio of benzoyl peroxide to ethyl 4-vinylbenzoate was 0.8:40; the mass ratio of 1,6-hexanediol diacrylate to ethyl 4-vinylbenzoate was 20:40; and the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate was 3:40. The mass of the aqueous phase was measured by the mass of PVA contained therein, and the mass of the oil phase was measured by the mass of ethyl 4-vinylbenzoate contained therein. The mass ratio of PVA to ethyl 4-vinylbenzoate was 2:40.

[0116] Example 5: The only difference between this example and Example 2 is the preparation of the magnetic emulsion.

[0117] Preparation of magnetic emulsion: Polyvinyl alcohol, anhydrous sodium sulfate and hydroquinone were dissolved in deionized water to obtain an aqueous phase; ethyl 4-vinylbenzoate, 3-heptenyl 2-methylpropionic acid, toluene, benzoyl peroxide, 1,6-hexanediol diacrylate and modified magnetic Fe3O4 were uniformly mixed to obtain an oil phase; the oil phase was introduced into the circulating aqueous phase through an SPG membrane to obtain a magnetic emulsion. The mass-to-volume ratio of polyvinyl alcohol (PVA) to deionized water was 2 g:500 mL; the mass ratio of anhydrous sodium sulfate to PVA was 100 mg:2 g; the mass ratio of hydroquinone to PVA was 100 mg:2 g; the mass ratio of ethyl 4-vinylbenzoate to toluene was 40:80; the mass ratio of 2-methylpropionic acid-3-heptenyl ester to toluene was 20:80; the mass ratio of benzoyl peroxide to ethyl 4-vinylbenzoate was 0.8:40; the mass ratio of 1,6-hexanediol diacrylate to ethyl 4-vinylbenzoate was 40:40; and the mass ratio of modified magnetic Fe3O4 to ethyl 4-vinylbenzoate was 3:40. The mass of the aqueous phase was measured by the mass of PVA contained therein, and the mass of the oil phase was measured by the mass of ethyl 4-vinylbenzoate contained therein. The mass ratio of PVA to ethyl 4-vinylbenzoate was 2:40.

[0118] Comparative Example 1: The only difference between this comparative example and Example 2 is that ethyl 4-vinylbenzoate was not used in the preparation of the magnetic emulsion.

[0119] Comparative Example 2: The only difference between this comparative example and Example 2 is that 2-methylpropionic acid-3-heptenyl ester was not used in the preparation of the magnetic emulsion.

[0120] Comparative Example 3: This comparative example differs from Example 2 in that ethyl 4-vinylbenzoate and 3-heptenyl 2-methylpropionic acid were not used in the preparation of the magnetic emulsion.

[0121] Experimental Example 1: Specific surface area test of magnetic solid phase extraction microspheres.

[0122] Test samples: Magnetic solid microspheres prepared in Examples 2-5 and Comparative Examples 1-3.

[0123] Test method: Using high-purity nitrogen as the adsorbate, adsorption-desorption experiments were conducted at liquid nitrogen temperature of -196℃. Adsorption-desorption isotherms were plotted based on the amount of nitrogen adsorbed, and the specific surface area of ​​the magnetic solid-phase extraction microspheres was calculated according to the multi-point BET equation.

[0124] The specific surface area test results of the magnetic solid-phase extraction microspheres prepared in this invention are shown in Table 1.

[0125]

[0126] Example 2 used oleic acid to modify magnetic Fe3O4 and introduced ethyl 4-vinylbenzoate and 2-methylpropionic acid-3-heptenyl ester, which increased the hydrophilicity and roughness of the microsphere surface and increased the specific surface area of ​​the magnetic solid phase extraction microspheres. Example 3 increased the amount of ethyl 4-vinylbenzoate, which further improved the specific surface area of ​​the magnetic solid phase extraction microspheres. Example 4 introduced 1,6-hexanediol diacrylate, and Example 5 increased its amount, which promoted the formation and stability of the pore structure, increased the effective adsorption sites, and achieved the highest specific surface area. However, Comparative Example 1 did not use ethyl 4-vinylbenzoate, Comparative Example 2 did not use 2-methylpropionic acid-3-heptenyl ester, and Comparative Example 3 lacked both ethyl 4-vinylbenzoate and 2-methylpropionic acid-3-heptenyl ester, resulting in incomplete functionalization of the microsphere surface, poor pore development, and a significantly lower specific surface area than Examples 2-5.

[0127] Experimental Example 2: Test on the spiked recovery rate of baicalin by magnetic solid phase extraction microspheres.

[0128] Test samples: Magnetic solid-phase extraction microspheres prepared in Examples 2-5 and Comparative Examples 1-3.

[0129] Test method: Add 50 μg baicalein to 10 mL of YZH solution to obtain a spiked YZH solution with a concentration of C0; mix 320 μL of methanol with 80 μL of acetonitrile, add 80 μg of L-2-chlorophenylalanine, and mix thoroughly to obtain the extract; mix 60 μL of acetonitrile with 60 μL of deionized water to obtain the reconstituted solution; mix 100 μL of YZH solution and the spiked YZH solution with 400 μL of the extract, vortex for 30 s, sonicate at 5℃ and 40 kHz for 30 min, let stand at -20℃ for 30 min, and centrifuge at 4℃ and 13000 rpm for 15 minutes. After 30 min, the supernatant was dried under nitrogen, and 120 μL of the reconstitution solution was added. The mixture was vortexed for 30 s and then mixed with magnetic solid-phase extraction microspheres. The mixture was kept at 30 °C and shaken for 20 min. After magnetic separation, 1 mL of eluent was added to 1.2 g of magnetic solid-phase extraction microspheres and the mixture was kept at 30 °C and shaken for 15 min. The mixture was filtered to obtain the sample solution and the spiked sample solution. The sample solution and the spiked sample solution were analyzed by LC-MS / MS to obtain the concentrations of baicalein C1 and C2 in the YZH solution. The spiked recovery rate of baicalein was calculated according to the formula R (%) = (C2-C1) / C0 × 100%. Chromatographic conditions: The column was an ACQUITY UPLC BEH C18, 100 mm × 2.1 mm id, 1.7 µm; mobile phase A was a 2% acetonitrile aqueous solution containing 0.1% formic acid; mobile phase B was an acetonitrile solution containing 0.1% formic acid; the injection volume was 3 μL; and the column temperature was 40 °C. Mass spectrometry conditions: the spray voltage was 3500 V; the scan range was 70–1050 m / z; the sheath gas flow rate was 50 arb; the auxiliary gas flow rate was 13 arb; the heating temperature was 450 °C; the capillary temperature was 320 °C; the collision energies were 20, 40, and 60 V; the primary mass spectrometry resolution was 70,000; and the secondary mass spectrometry resolution was 17,500.

[0130] The spiked recovery rate of baicalein by the magnetic solid-phase extraction microspheres prepared in this invention is shown in Table 2.

[0131]

[0132] Example 2: The large specific surface area of ​​the magnetic solid-phase extraction microspheres provided ample adsorption sites, enabling baicalin to effectively bind to the microsphere surface and improving the adsorption-desorption efficiency of baicalin. Example 3: Increasing the amount of ethyl 4-vinylbenzoate optimized the pore structure, reducing the loss of baicalin during adsorption, and further improving the spiked recovery rate compared to Example 2. Example 4: Introducing 1,6-hexanediol diacrylate enhanced the affinity of the microspheres for polar molecules, improving the spiked recovery rate of baicalin. Example 5: The highly developed porous structure and stable surface functional groups enabled the microspheres to achieve optimal specific adsorption capacity for baicalin. Comparative Example 1 (without ethyl 4-vinylbenzoate), Comparative Example 2 (without 2-methylpropionic acid-3-heptenyl ester), and Comparative Example 3 (both ethyl 4-vinylbenzoate and 2-methylpropionic acid-3-heptenyl ester were missing), resulting in low specific surface area, insufficient and unevenly distributed surface active sites, and the inability to effectively capture some baicalin, leading to a decrease in recovery rate.

[0133] Experimental Case 3: Mechanism analysis of YZH treatment for drug-induced liver injury.

[0134] The total ion chromatogram of YZH is as follows: Figure 1 As shown in the pie chart of YZH component classification and content, Figure 2 As shown, a total of 230 compounds were identified. Among them, phenylpropanoids were the main components, accounting for 78.66%, organic oxides accounted for 9.24%, benzene rings accounted for 3.98%, lipids and lipid-like compounds accounted for 3.49%, organic acids accounted for 2.46%, organic heterocyclic compounds accounted for 1.8%, and the remaining components accounted for 0.37%.

[0135] The structural formula of the active ingredient in YZH is as follows: Figure 3 As shown, further identification of the components contained in the liver tissue revealed seven components that entered the target tissue, including aloin, apigenin-7-O-β-D-glucuronic acid, baicalin, luteolin, baicalin, irisin, and wogonin-7-O-glucuronide.

[0136] The intersection diagram of YZH components and disease targets is shown below. Figure 4 As shown, the intersection yields 32 common target sites; the protein-protein interaction network is as follows: Figure 5 As shown, Figure 5 (A) is a protein-protein interaction network diagram of potential targets for YZH treatment of liver injury. Figure 5 (B) is the core target map. Based on the calculated degree value, 6 core targets were selected, namely TLR4, TNF, MMP2, MMP9, PPARA and PTGS2.

[0137] YZH's GO function annotation is as follows: Figure 6As shown, biological processes mainly involve responses to oxidative stress, cellular responses to chemical stimuli, positive regulation of smooth muscle cell proliferation, regulation of inflammatory responses, responses to exogenous stimuli, responses to amyloid-β, responses to ethanol and reactive oxygen species metabolism, and alcohol responses. Cellular components are mainly enriched in cytoplasmic vesicle cavities, vesicle cavities, secretory granule cavities, the outer plasma membrane, vacuolar cavities, Ficolin-1-rich granules, azurophilic granule cavities, collagen-containing extracellular matrix, phagocytic cuvettes, and Ficolin-1-rich granule cavities. Molecular functions mainly include electron transfer activity, heat shock protein binding, heme binding, nuclear receptor activity, ligand-activated transcription factor activity, tetrapyrrole binding, glucose binding, hydrolase activity, serine endopeptidase activity, and oxidoreductase activity.

[0138] The top 10 pathways enriched by the YZH core target are as follows: Figure 7 As shown, the diseases involved include lipid and atherosclerosis, alcoholic liver disease, diabetic cardiomyopathy, insulin resistance, non-alcoholic fatty liver disease, Legionnaires' disease, and bladder cancer; the biological pathways involved include the AGE-RAGE signaling pathway, IL-17 signaling pathway, and C-type lectin receptor signaling pathway in diabetic complications; indicating that YZH treatment of liver injury is a synergistic effect of multiple targets, multiple pathways, and multiple pathways.

[0139] Component-target-pathway-disease network diagram as follows Figure 8 As shown, there are 34 nodes. The Chinese medicinal herbs Scutellaria baicalensis, Gardenia jasminoides, and Lonicera japonica are interconnected with their components baicalein and kaempferol, as well as their targets NOX4, TLR4, MMP2, MMP9, and TNF. This reflects the mechanism by which Chinese medicines act on different targets through the same active ingredient, and the same target is regulated by multiple active ingredients.

[0140] Table 3 shows the docking results between the key target TLR4 and the active ingredient molecules. Figure 9 As shown.

[0141] Table 3. Docking results between key target TLR4 and active ingredient molecules

[0142] Besides baicalin and wogonin-7-O-glucuronide, TLR4 can bind tightly to five other active ingredients to form stable conformations. Among them, TLR4 has the strongest binding affinity to aloin, followed by apigenin-7-O-β-D-glucuronic acid. The tight docking of these active ingredients with the target protein mainly occurs through hydrogen bonds and active pockets. Aloin interacts with ASN-44, TYR-46, LYS-47, and ASP-50 residues of TLR4 to form six hydrogen bonds; apigenin-7-O-β-D-glucuronic acid interacts with ASN-137, THR-136, and LEU-117 residues to form four hydrogen bonds; irisin interacts with HIS-199 and GLU-225 residues to form three hydrogen bonds; and baicalin interacts with ASN-624... The LYS-615 residues interact to form 4 hydrogen bonds; luteolin interacts with PHE-533, GLN-510, LYS560 and ASP536 residues to form 5 hydrogen bonds; baicalin interacts with HIS-458, LEU-434, ARG-460 and GLU-439 residues to form 6 hydrogen bonds; baicalin-7-O-glucuronide interacts with ASN-329, THR-308, ASN-309 and GLN-333 residues to form 5 hydrogen bonds.

[0143] Experiment 4: Immunoblot analysis to verify the effect of YZH on proteins related to the liver NF-κB signaling pathway.

[0144] Test methods: Six- to eight-week-old SPF-grade male C57BL / 6 mice were randomly divided into three groups: control group, model group, and YZH intervention group. Mice in the model group and YZH group were intraperitoneally injected with 0.1 mL of cyclophosphamide (80 mg / kg body weight) once daily for 5 consecutive days. The control group was injected with an equal volume of physiological saline. The YZH group was administered YZH oral solution (180 mg / kg) by gavage during model establishment, once daily for 12 consecutive days. The control group and model group were administered an equal volume of physiological saline by gavage. Mice were fed... Mice were housed in a constant temperature environment of 22±2℃ and a constant humidity of 55±5%, with a 12-hour light / dark cycle and free access to food and water. They were fasted for 12 hours before the experiment. Twenty-four hours after the last drug administration, mice were anesthetized intraperitoneally with sodium pentobarbital, and blood was collected from the abdominal aorta. Serum was separated. The left lobe of the liver was quickly removed, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and the expression levels of related proteins in the NF-κB signaling pathway in mouse liver tissue, including TLR4, p-IκBα, IκBα, p-P65, and P65, were detected by Western blotting.

[0145] The effects of YZH on the mouse liver NF-κB signaling pathway, such as Figure 10As shown, NF-κB is a downstream signaling molecule of TLR4. Once activated, it can induce an inflammatory cascade response, and the inflammatory response is closely related to the development of CTX-induced hepatotoxicity. The expression levels of TLR4, p-IKBα / IKBα, and p-P65 / P65 in the model group were significantly increased compared with those in the control group. After YZH intervention, the expression of inflammation-related proteins was significantly reduced. This indicates that YZH can alleviate liver injury by inhibiting the activation of the inflammation-related TLR4 / NF-κB signaling pathway.

[0146] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0147] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for analyzing the mechanism of YZH treatment for liver injury, comprising: The active ingredients of YZH were extracted by ultrasonic extraction using an extraction solution. After centrifugation, the supernatant was blown with nitrogen and then a reconstitution solution was added for ultrasonic extraction to obtain a sample solution. YZH is Compound Yin Zhi Huang Oral Solution. The sample solution was identified by LC-MS / MS combined with a database of traditional Chinese medicine metabolites, and the component targets and disease targets were predicted. By constructing a protein-protein interaction network through the intersection of component targets and disease targets, core targets can be screened. We performed GO function and KEGG pathway enrichment analysis on the core target and verified the mechanism of action through molecular docking. The ultrasonic extraction includes magnetic solid-phase extraction, wherein the adsorbent for the magnetic solid-phase extraction is magnetic solid-phase extraction microspheres, and the magnetic solid-phase extraction microspheres include a modified magnetic Fe3O4 core and a polymer shell. The modified magnetic Fe3O4 core includes oleic acid, and the polymer shell's monomers include ethyl 4-vinylbenzoate and 3-heptenyl 2-methylpropionic acid. The mass ratio of the modified magnetic Fe3O4 to ethyl 4-vinylbenzoate is 1.5-15:20-200, and the mass ratio of the modified magnetic Fe3O4 to 3-heptenyl 2-methylpropionic acid is 1.5-15:10-100.

2. The method for analyzing the mechanism of YZH treatment for liver injury according to claim 1, characterized in that, The extract comprises methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 160-1600:40-400.

3. The method for analyzing the mechanism of YZH treatment for liver injury according to claim 1, characterized in that, The reconstitution solution comprises acetonitrile and deionized water, wherein the volume ratio of acetonitrile to deionized water is 30-300:30-300.

4. The method for analyzing the mechanism of YZH treatment for liver injury according to claim 1, characterized in that, The ultrasonic temperature is 4-6℃ and the power is 35-45kHz.

5. The method for analyzing the mechanism of YZH treatment for liver injury according to claim 1, characterized in that, The eluent for the magnetic solid-phase extraction comprises sodium carbonate, deionized water, and ethanol, wherein the mass-to-volume ratio of sodium carbonate to deionized water is 2.65-26.5 mg: 250-2500 μL, and the volume ratio of ethanol to deionized water is 250-2500: 250-2500.

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