Traditional Chinese medicine composition for preventing and treating avian pathogenic escherichia coli as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and treating avian pathogenic escherichia coli as well as a preparation method and application thereof, and relates to the technical field of veterinary drugs. According to the invention, screening is carried out through subtraction genomics and reverse network pharmacology, and based on the compatibility theory of monarch, minister, assistant and guide and the traditional Chinese veterinary medicine and pharmacy differentiation theory, a traditional Chinese medicine composition, namely the radix angelicae and fructus forsythiae intestine health formula, for preventing and treating avian pathogenic escherichia coli is formed. According to the traditional Chinese medicine composition, fructus forsythiae and radix angelicae are used as monarch drugs, radix isatidis and clinopodium polycephalum are used as ministerial drugs, fructus aurantii immaturus, mint and plantain herb are used as adjuvant drugs, and liquorice is used as a conductant drug, so that the traditional Chinese medicine composition can inhibit reproduction of avian pathogenic escherichia coli, destroy formation of biological membranes of the avian pathogenic escherichia coli and relieve pathological injuries of diseased poultry caused by APEC; the traditional Chinese medicine composition has a good treatment effect on avian pathogenic escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of poultry medicine technology, and in particular to a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry, its preparation method, and its application. Background Technology

[0002] Pathogenic Escherichia coli (Avian pathogenic Escherichia coli) Avian pathogenic Escherichia coli Pathogenic Escherichia coli (E. coli), a type of extraintestinal pathogenic Escherichia coli, can cause localized or systemic infections in poultry such as broilers, turkeys, quails, pigeons, and waterfowl, mainly manifesting as acute septicemia, perihepatitis, pericarditis, and air sacculitis. This not only affects poultry health and production but also causes significant economic losses for farmers. Poultry farming, as a crucial pillar industry in 21st-century agricultural development, plays an irreplaceable role in ensuring the supply of animal-derived foods such as meat and eggs and promoting rural economic development. However, diseases caused by pathogenic E. coli in poultry seriously endanger the development of the poultry farming industry and may even transmit the danger to humans through the food chain.

[0003] Since the complete ban on antibiotics in feed in 2020, the poultry industry has faced a surge in pressure to control diseases such as peritonitis, perihepatitis, and air sacculitis caused by APEC. Finding safe, efficient, low-residue alternatives that are less likely to induce drug resistance has become a core issue for ensuring poultry health and the sustainable development of the poultry industry. Traditional Chinese medicine, as a unique medicinal resource and cultural treasure of my country, possesses unique advantages such as complex components, diverse mechanisms of action, low likelihood of inducing drug resistance, and minimal toxicity. It is considered one of the most promising "antibiotic alternatives," and its application in poultry disease prevention and control is gradually gaining attention.

[0004] Therefore, providing a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry has important theoretical value and practical significance for promoting the green and healthy development of poultry farming. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a traditional Chinese medicine composition for the prevention and treatment of avian pathogenic Escherichia coli, its preparation method, and its application. After screening using subtractive genomics and reverse network pharmacology, this invention, based on the theory of "principal, assistant, adjuvant, and guide" drug combinations and the dialectical theory of traditional Chinese veterinary medicine, has formed a traditional Chinese medicine composition for the prevention and treatment of avian pathogenic Escherichia coli. This composition uses Forsythia suspensa and Angelica dahurica as principal herbs, Isatis indigotica and Dioscorea opposita as assistant herbs, Citrus aurantium, Mentha haplocalyx, and Plantago asiatica as adjuvant herbs, and Glycyrrhiza uralensis as guide herb. It can inhibit the reproduction of avian pathogenic Escherichia coli, disrupt the formation of avian pathogenic Escherichia coli biofilms, alleviate pathological damage to poultry caused by APEC, and has a good therapeutic effect on avian pathogenic Escherichia coli.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a traditional Chinese medicine composition for preventing and treating pathogenic Escherichia coli in birds, comprising the following components in parts by weight: Forsythia suspensa 7-9 parts, Angelica dahurica 7-9 parts, Isatis indigotica 0.8-1.2 parts, Dioscorea opposita 7-7.5 parts, Citrus aurantium 0.8-1.2 parts, Mentha haplocalyx 3-3.5 parts, Plantago asiatica 7-9 parts, Glycyrrhiza uralensis 7-9 parts.

[0007] Preferably, the traditional Chinese medicine composition is prepared from the following components: Forsythia suspensa 8 parts, Angelica dahurica 8 parts, Isatis indigotica 1 part, Dioscorea opposita 7.15 parts, Citrus aurantium 1 part, Mentha haplocalyx 3.26 parts, Plantago asiatica 8 parts, Glycyrrhiza uralensis 8 parts.

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition for preventing and treating pathogenic Escherichia coli in birds, comprising the following steps: Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and Glycyrrhiza uralensis were mixed in proportion to mass to obtain a mixture. Water was added to the mixture to soak it, and it was decocted twice over low heat. After each decoction, the mixture was filtered and the decoction was collected. The two decoctions were combined and concentrated to a concentration of 0.5-1.5 g / mL to obtain a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in birds—Zhiqiao Changkang Formula (ZCF).

[0009] Preferably, the mass ratio of the mixture to water is 1 g: (8-12) mL.

[0010] Preferably, the soaking time is 20-30 hours.

[0011] Ideally, each simmering time should be 50-70 minutes.

[0012] In a third aspect, the present invention provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a product for treating pathogenic Escherichia coli in birds.

[0013] In a fourth aspect, the present invention provides a product for treating avian pathogenic Escherichia coli, the product comprising the above-mentioned traditional Chinese medicine composition for preventing and treating avian pathogenic Escherichia coli.

[0014] Preferably, the product for treating pathogenic Escherichia coli in birds also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutically acceptable excipients include disintegrants, lubricants, sweeteners, or binders.

[0016] Preferably, the dosage form of the product used to treat pathogenic Escherichia coli in birds is a decoction, a soluble powder, or an injection.

[0017] The beneficial effects of this invention are: 1. This invention uses subtractive genomics and reverse network pharmacology for screening, and based on the theory of "principal, assistant, adjuvant and guide" compatibility and the dialectical theory of traditional Chinese veterinary medicine, it has formed a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in birds - Zhiqiao Changkang Formula. This traditional Chinese medicine composition includes eight medicinal materials: Forsythia suspensa, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica and Glycyrrhiza uralensis.

[0018] The traditional Chinese medicine composition prepared by this invention can effectively inhibit the reproduction of pathogenic Escherichia coli in birds, disrupt the formation of biofilms of pathogenic Escherichia coli in birds, and reduce pathological damage caused by APEC, thus playing a therapeutic role in diseased birds. This invention also verifies through experiments that the therapeutic effect is optimal when the dosage of this traditional Chinese medicine composition is 2.5 g / kg.

[0019] 2. This invention is based on the theory of "principal, assistant, adjuvant, and guide" drug compatibility and the dialectical theory of traditional Chinese veterinary medicine. It utilizes Forsythia suspensa and Angelica dahurica as principal drugs, Isatis indigotica and Dioscorea opposita as assistant drugs, Citrus aurantium, Mentha haplocalyx, and Plantago asiatica as adjuvant drugs, and Glycyrrhiza uralensis as guide drug, to form a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in birds. Forsythia suspensa has the effects of clearing heat and detoxifying, and dispersing wind-heat; Angelica dahurica has the effects of relieving exterior syndromes and dispersing cold, and drying dampness and stopping leukorrhea; Isatis indigotica has the effects of clearing heat and detoxifying, cooling blood and relieving sore throat; Dioscorea opposita has the effects of astringing and stopping bleeding; Citrus aurantium has the effects of breaking up qi stagnation and eliminating stagnation, and resolving phlegm and dispersing masses; Mentha haplocalyx has the effects of dispersing wind-heat and soothing the liver and regulating qi; Plantago asiatica has the effects of clearing heat and detoxifying, and promoting diuresis and eliminating dampness; and Glycyrrhiza uralensis has the effects of tonifying the spleen and replenishing qi. Attached Figure Description

[0020] Figure 1 : Protein model structure diagram modeled using Swiss-Model and visualized using PyMOL molecular graphics system, where A is ArcB, B is emrY, and C is UGDH; Figure 2 DoGSiteScorer visualization of drug-derived regions; Figure 3 Network diagram of "target-active ingredient-traditional Chinese medicine"; Figure 4 Distribution of medicinal properties of Chinese medicinal herbs; Figure 5 The distribution of medicinal properties in Chinese medicinal herbs; Figure 6 The distribution of Chinese medicinal herbs according to their meridian tropism; Figure 7 : Molecular docking results diagram; Figure 8 Figure: Molecular dynamics simulation results; Figure 9 : Growth curve determination results of 8 Chinese herbal medicines corresponding to APEC strain JN1704; Figure 10 : Growth curve determination results of 8 Chinese herbal medicines corresponding to APEC strain PZ112; Figure 11 : Results of the determination of the effect of traditional Chinese medicine composition used to prevent and treat pathogenic Escherichia coli in birds on the physiological characteristics of Escherichia coli; Figure 12 : Visual observation of tissue changes and body composition in poultry; Figure 13 HE pathological section of poultry intestinal tissue; Figure 14 Comparison chart of intestinal bacterial load in poultry; Figure 15 : Effect of inhibiting the expression levels of Escherichia coli virulence factors and target proteins; Figure 16 Graph showing changes in inflammatory factors, immune proteins, and tight junction proteins detected by ELISA; Figure 17 Effects on the expression levels of proteins in the PI3K-AKT signaling pathway; Figure 18 Effects on the expression levels of proteins in the NF-κB signaling pathway. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0023] In existing technologies, pathogenic Escherichia coli in birds can cause local or systemic infections, affecting the development of the poultry industry. Traditional Chinese medicine (TCM) has advantages such as diverse mechanisms of action, low likelihood of drug resistance, and minimal toxicity. Therefore, developing a TCM composition to replace antibiotics for the prevention and treatment of pathogenic Escherichia coli in birds has significant theoretical and practical value for promoting the green and healthy development of the poultry farming industry.

[0024] Based on this, the present invention provides a traditional Chinese medicine composition for the prevention and treatment of avian Escherichia coli, comprising Forsythia suspensa, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and Glycyrrhiza uralensis. Forsythia suspensa and Angelica dahurica serve as principal herbs, Isatis indigotica and Dioscorea opposita as assistant herbs, Citrus aurantium, Mentha haplocalyx, and Plantago asiatica as adjuvant herbs, and Glycyrrhiza uralensis as guiding herb.

[0025] Compared with single Chinese medicines, this Chinese medicine composition has the following advantages in the prevention and treatment of avian Escherichia coli: (1) Comprehensive therapeutic effect with multiple targets. Through the synergistic effect of multiple components, the compound can not only inhibit bacteria, but also regulate the body's immunity, repair the intestinal mucosa, and relieve endotoxemia, achieving a combination of "eliminating pathogens" and "strengthening the body's resistance". (2) Reduced drug resistance. The compound acts on multiple targets, making it more difficult for bacteria to develop drug resistance through single gene mutations. (3) Improved in vivo microenvironment. Single medicines have excellent antibacterial effects in vitro, but may become ineffective in vivo due to metabolic and other factors; the compound can indirectly inhibit the growth of pathogens by improving the intestinal environment. (4) Holistic treatment based on syndrome differentiation. Traditional Chinese veterinary medicine treatment targets the overall state of the diseased body, rather than just the pathogen. The compound is more in line with its holistic concept and the principle of syndrome differentiation and treatment.

[0026] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0027] In this invention, Forsythia suspensa, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and Glycyrrhiza uralensis were all purchased from Hebei Baicao Fanghe Pharmaceutical Co., Ltd.; Colistin sulfate was purchased from Hefei Qianfang Animal Health Technology Co., Ltd.

[0028] Example 1: Screening of traditional Chinese medicine compositions for the prevention and treatment of pathogenic Escherichia coli in birds 1. Subtractive genomics: The whole-genome proteins of 5737 APEC O78 strains were obtained using the Uniprot website. These proteins were compared with the KEGG-specific metabolic pathway of GGA (chicken genome assembly). Through comparative analysis, common metabolic pathways between the pathogen and the host were excluded to preserve the APEC-specific metabolic pathway, resulting in 1125 proteins. Subsequently, paralogous proteins were removed from the obtained APEC proteome using CD-HIT, yielding 700 proteins. The FASTA files of these 700 protein sequences were then compared with the whole-genome proteins of the host chicken using BLASTp (with a truncation parameter E value of 10). -3 The screening process involved excluding homologous proteins and identifying 220 non-homologous proteins for drug target prioritization (only non-homologous proteins were selected to avoid drug cross-reactions and side effects). Then, the DEG database was used to perform BLASTp (E value ≤ 10) on the screened non-homologous proteins. -5 After processing (bit score > 100, identity > 30%), 88 proteins essential for APEC production were identified; these 88 essential proteins were then analyzed using BLASTp (E value ≤ 10) from the DrugBank database. -5 After processing, 63 drug-forming proteins that could serve as drug targets were identified; the complete toxicity information of these drug-forming proteins was analyzed using the VFDB database, and BLASTp (E value ≤ 10) was used to further analyze them. -5After processing, virulence factor prediction was performed, and proteins related to APEC virulence were selected, resulting in 20 virulence factor proteins. The Fasta sequences of these virulence factor proteins were analyzed using the CARD database, and BLASTp (E value ≤ 10) was then applied. -5 After processing, three APEC O78 resistance proteins were screened out. Finally, KASS was used to analyze the metabolic pathways of the obtained proteins. pSORTB v.3.0.3 and CELLP v.2.5 were used to perform subcellular localization of the shortlisted proteins, and proteins located in the cytoplasm and bacterial membrane were selected as the final screening proteins.

[0029] Ultimately, UGDH, AcrB, and emrT were selected as target proteins.

[0030] 2. Reverse network pharmacology: (1) Construction of target protein model: The amino acid sequence of the target protein was retrieved from the UniProt database. Using the Swiss-Model platform and a PDB database as a template (sequence identity threshold ≥30%, allowing automatic template selection), a three-dimensional protein structure model was constructed in "High Quality" mode. The model was then visualized and validated using PyMOL software to check the spatial conformation of key functional sites. The final model was saved in PDB format for subsequent molecular docking and kinetic simulation analysis.

[0031] Build a model using Swiss-Model and validate it using PyMOL visualization, as shown below. Figure 1 As shown. By Figure 1 As can be seen, the InPreferred Regions of the Laplace plot of the AcrB model in A is 91.95%, the InPreferred Regions of the Laplace plot of the UGDH model in C is 95.6%, and the InPreferred Regions of the Laplace plot of the emrY model in B is 97.45%, indicating that the model structure is reasonably constructed and can be used for subsequent simulation verification experiments.

[0032] (2) Drugability analysis of the model: The constructed PDB file was uploaded to the DoGSiteScorer online server for druggability analysis of the protein binding pocket, obtaining key parameters including pocket volume, score, and physicochemical characteristics. The druggable regions of the obtained file were visualized and validated using UCSF Chimera X for subsequent molecular docking analysis. The results are as follows: Figure 2 As shown.

[0033] (3) Screening of active ingredients: In the TCMSP database, a search was conducted using the target protein as a keyword, retaining compounds with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0. Given that database predictions may not match actual results, PubMed, CNKI, and other literature databases were also searched, retaining all compounds experimentally reported to bind to the target to ensure the reliability of the results. The results are shown in Table 1.

[0034] Table 1. Screened active ingredients and their target proteins As shown in Table 1, a total of 12 active ingredients were identified, namely: ursolic acid, bergamot lactone, theobromine, naringenin, glycyrrhizin A, thymol, eugenol, estradiol, (-)-epigallocatechin gallate, glycerol, coumarin, and sucrose. Among these, no possible compounds were found to bind to the target emrY, while compounds were found to bind to UGDH and AcrB, respectively.

[0035] (4) Reverse screening of Chinese medicinal materials: Active ingredients were obtained by inputting them into the HERB, TCMSP, and PubMed databases, and medicinal herbs containing these active ingredients were screened. The names of the screened medicinal herbs were standardized according to the 2020 edition of the *Pharmacopoeia of the People's Republic of China for Veterinary Medicine* and *Chinese Materia Medica*, and usable medicinal herbs were retained.

[0036] A total of 102 Chinese medicinal herbs were obtained targeting UGDH proteins. After standardizing their names and removing duplicates, 83 herbs were selected. A total of 158 Chinese medicinal herbs were obtained targeting AcrB proteins. After standardizing their names and removing duplicates, 130 herbs were selected. Through literature screening, a total of 126 Chinese medicinal herbs were identified that could be used for further research.

[0037] (5) Construction of the reverse network “target-active ingredient-traditional Chinese medicine”: Create Network and Tape files to map the active ingredients and traditional Chinese medicines corresponding to potential targets, construct a "target-active ingredient-traditional Chinese medicine" network, and import it into Cytoscape 3.9.1 software for visualization, such as... Figure 3 As shown, the centrality of the clique is analyzed using the CytoHubba plugin via the MCC (Maximum Clique Centrality) algorithm.

[0038] Depend on Figure 3It can be seen that there are 120 nodes and 138 edges in the "target-active ingredient-traditional Chinese medicine" network. The MCC algorithm was used to identify the central nodes in the cluster. The redder the color, the higher the score and the more central the node. The top-scoring traditional Chinese medicines are: Forsythia suspensa, Citrus aurantium, Citrus reticulata peel, Coriander, Plantago asiatica, Dioscorea opposita, Angelica dahurica, Ephedra sinica, Prunus armeniaca, Isatis indigotica, Kuding tea, Jujube, Trollius chinensis, Paederia scandens, Artemisia argyi, Actinidia chinensis root, and Mentha haplocalyx. It is speculated that these traditional Chinese medicines are the main traditional Chinese medicines in the new compound treatment for pathogenic Escherichia coli infection in birds.

[0039] (6) Construction of new traditional Chinese medicine compound prescriptions: After summarizing and removing duplicates from the medicinal materials collected for the two targets mentioned above, a total of 107 medicinal materials were obtained. The medicinal properties, flavors, and meridian tropism of these materials were analyzed, and the results are as follows: Figures 4-6 As shown.

[0040] Depend on Figure 4 It can be seen that cold, warm, and cool are the three most frequently used medicinal properties. Figure 5 It can be seen that bitter, sweet, and pungent are the three most frequently occurring medicinal flavors. Figure 6 It can be seen that the frequency of use of drugs related to the lung meridian is the highest, at 45 times (accounting for 45%); followed by drugs that belong to the lung meridian, at 37 times (accounting for 37%).

[0041] Finally, based on the online ranking of "target-active ingredient-traditional Chinese medicine", the top 17 herbs were selected for formulation, and licorice was chosen as the guiding herb from among 126 herbs for combined use. Based on the principles of "principal, assistant, adjuvant, and guide", a new traditional Chinese medicine compound containing 8 herbs was obtained: Forsythia suspensa, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and licorice.

[0042] 3. Verification: (1) Molecular docking: To verify the accuracy and binding affinity of the targets and active ingredients screened by subtractive genomics and network pharmacology, molecular docking was performed on the screened target proteins and the active ingredients of the core traditional Chinese medicine. The main active ingredients were docked separately with the core targets, and a binding energy ≤ -5.0 kcal / mol was used as the criterion for stable binding. The specific steps are as follows: The 3D structure (SDF format) of the active ingredient was downloaded from the PubChem database. It was then converted to PDBQT format using Open Babel 3.1.1 software. AutoDock Tools 1.5.6 was used to hydrogenate the small molecule ligands, calculate the Gasteiger charge, and remove redundant conformations using an energy minimization algorithm, retaining the thermodynamically stable lowest-energy conformation for subsequent docking. The 3D structure of the target protein predicted in Swiss-Mode was downloaded and pre-processed using PyMOL 2.5 software: crystal water, endogenous ligands (such as ATP and substrate analogs), and heteroatoms were precisely removed from the crystal structure. AutoDock Tools was used to supplement missing hydrogen bonds and assign atomic charges, finally saving the result as a PDBQT file. Semi-flexible docking was performed using AutoDock Vina 1.2.0 software. A docking grid was defined centered on the previously predicted spatial coordinates of the target protein's active pocket, with the grid center coordinates (x, y, z) set to ensure complete coverage of the active pocket and surrounding flexible region. During docking, the protein structure is kept rigid, allowing the rotatable bonds of small ligand molecules to rotate freely. The exhaustiveness=32 is the default setting to balance computational efficiency and result reliability. Other parameters follow the software's default standards. Each "active ingredient-core target" combination is run independently in three parallel docking experiments, and the result with the lowest binding energy and the highest conformational overlap is selected as the optimal docking conformation.

[0043] The optimal docking conformation was visualized using PyMOL 2.5 software, clearly revealing the spatial fit pattern between the active ingredient and the target protein's active pocket. The distribution characteristics of key binding forces, such as hydrogen bonds, hydrophobic interactions, and van der Waals forces, were observed, and high-resolution 3D structural images were saved. The PDBQT file of the optimal docking conformation was imported into Discovery Studio 2019 software, and the interaction data were quantitatively analyzed using the "Ligand Interactions" module. A 2D docking schematic diagram was generated, annotating key interacting residues, hydrogen bond lengths (≤3.5 Å for effective hydrogen bonds), and hydrophobic interaction regions. A binding energy ≤-5.0 kcal / mol was used as the criterion for stable binding, and combinations of "active ingredient-core target" meeting the criteria were statistically analyzed.

[0044] result Figure 7 As shown. By Figure 7It can be seen that the binding energies of the core active ingredients and target proteins all meet the stability criteria. Specifically, the binding energies of AcrB and Ursolic acid are -9.6 kcal / mol, AcrB and Majudin are -7 kcal / mol, AcrB and Naringenin are -8 kcal / mol, UGDH and eugenol and saccharose are both -5.8 kcal / mol, and UGDH and glycerin are -4 kcal / mol. Furthermore, the pyMOL visualization shows that all stably bound "ingredient-target" complexes form effective hydrogen bonds, and the amino acid residues linked by these hydrogen bonds are key functional sites of the target proteins. Specifically, in the AcrB-Ursolic acid complex, Ursolic acid... There is a stable hydrogen bond between acid and THR-C:748 (L-threonine); in the complex AcrB-Majudin, there are stable hydrogen bonds between Majudin and THR-C:222 (L-threonine) and GLY-A:51 (glycine); in the complex AcrB-Naringenin, there are stable hydrogen bonds between Naringenin and ALA-B:159 (alanine), Naringenin and LYS-B:770 (lysine), and Naringenin and TYR-B:758 (tyrosine); in the complex UGDH-Eugenol, there are stable hydrogen bonds between Eugenol and THR-A:83 (L-threonine), Eugenol and THR-A:118 (L-threonine), and Eugenol and LYS-A:256 (lysine). In the UGDH-Glycerin complex, stable hydrogen bonds are formed between Glycerin and LYS-A:116 (lysine), Glycerin and PRO-A:140 (proline), Glycerin and GLU-A:141 (glutamic acid), and Glycerin and VAL-A:159 (valine). Similarly, in the UGDH-Saccharose complex, stable hydrogen bonds are formed between Saccharose and ASN-A:312 (asparagine), UGDH and TYRA:10 (tyrosine), Saccharose and CYS-A:253 (cysteine), Saccharose and VAL-A:11 (valine), and Saccharose and LYS-A:116 (lysine). This indicates that compounds AcrB and UGDH have a relatively stable binding ability with traditional Chinese medicine components, forming stable complexes.

[0045] (2) Molecular dynamics simulation: Molecular dynamics (MD) simulations of protein-ligand complexes were performed using Discovery Studio 2019 software. The ligand-compound files of the active components and target proteins AcrB and UGDHD from the previous studies were analyzed using Protein Report to obtain basic information such as crystal resolution, amino acid chain composition, and residue integrity. Combined with the Isotropic Displacement (IDI) analysis results (blue indicates more stable atomic positions), the most stable structural chains and redundant structures were retained after comprehensive evaluation. Automatic structure correction and preprocessing were performed using the Prepare Protein tool in the Macromolecules module to generate a Prep file. Subsequently, the CHARMM36 force field was specified and parameters were assigned using the Change Forcefield function in the Simulation panel. The preprocessed protein structure was then placed in a solvent chamber for solvation using the Solvation workflow with default parameters. The Standard Dynamics Cascade kinetics workflow was then initiated, setting up five stages: Optimization I, Optimization II, Heating, Equilibrium, and Production Simulation. The equilibrium stage simulation time was set to 20 ps, ​​and the production stage simulation time to 200 ps. In the Advanced parameters, the number of processors was set to 8 to improve computational efficiency, while other parameters remained at their default values. After submitting the task, the running status was monitored using Jobs Explorer. After the simulation, the Analyze Trajectory tool was used for trajectory analysis. The simulated product was input as the target molecule, and RMSD and RMSF were selected as the analysis types. The RMSD reference molecule was set to the preprocessed Prep structure, and both the atomic groups and the fitted atomic groups were set to the main chain. RMSF analysis focused on the atomic fluctuations of each residue in the protein to reflect the flexibility characteristics of different regions (especially residues around the active pocket). By systematically analyzing the stability of the RMSD curve and the RMSF numerical distribution, the conformational stability and local residue flexibility of the complex during the simulation were comprehensively assessed.

[0046] The results are as follows Figure 8 As shown. By Figure 8It can be seen that the mean RMSD values ​​and standard deviations of AcrB and Ursolic acid are 1.33497414 ±0.254534482, AcrB and Majudin are 1.36219582 and ±0.267733299, AcrB and Naringenin are 1.3186418 and ±0.262251551, UGDH and eugenol are 0.973195 and ±0.169125594, UGDH and glycerin are 1.46213 and ±0.358385134, and UGDH and saccharose are 0.934253 and ±0.128491003. RMSD fluctuation analysis revealed that the UGDH-saccharose complex essentially reached equilibrium after a period of kinetic simulation, exhibiting only small fluctuations. In contrast, the AcrB-Majudinn complex did not reach equilibrium during molecular dynamics simulations and showed significantly larger fluctuations, indicating relatively poor structural stability during the simulation process. Further investigation of the molecular dynamic parameters of the ligand complexes revealed significant differences in the RMSD fluctuation characteristics between the AcrB and UGDH ligand complexes. Figure 8 GI indicates that the RMSF value of the AcrB ligand complex fluctuates around 1 nm after binding, with a local peak of up to 2 nm in the mid-term simulation. This is related to the function of AcrB as a drug efflux pump, where the residues in its binding pocket need to achieve substrate transport through dynamic movement, resulting in easy conformational changes in binding. Figure 8 JL results show that the RMSF value of the UGDH ligand complex fluctuates around 0.5 nm after binding, which is smaller than that of the AcrB ligand complex, only half that of the AcrB complex. In particular, the RMSF value of the UGDH active site residues is as low as 0.37 nm, indicating that the ligands intercalate into the binding pocket through complementary spatial structures, forming a multi-point anchoring effect of "multiple hydrogen bonds + hydrophobic stacking." This also induces conformational locking of the protein, enhancing secondary structure stability and significantly restricting disordered atomic movement. These results confirm that the active ingredients of the new traditional Chinese medicine compound bind to the UGDH target with high stability and specificity, are not easily affected by environmental interference, and can reduce the risk of off-target effects. The RMSF fluctuation of the AcrB ligand complex at around 1 nm is precisely matched to its drug efflux pump function, and the dynamic conformation facilitates substrate binding and release, providing key dynamic evidence for optimizing ligand interactions and enhancing targeted inhibitory activity.

[0047] Example 2: In vivo antibacterial test of Escherichia coli Test strains: Standard strain Escherichia coli JN1704 O78 and test strain Escherichia coli PZ1112 O78 were provided by the Institute of Poultry, Shandong Academy of Agricultural Sciences (Shandong Specific Pathogen-Free Chicken Research Center); Specific experimental steps: (1) Activation of strain: The glycerol storage tube containing Escherichia coli was slowly thawed until the mixture of glycerol and bacterial solution was completely melted. 100 μL of the thawed bacterial solution was inoculated into LB liquid medium and cultured at 37°C and 220 rpm for 12 h to allow the strain to enter the logarithmic growth phase. (2) Determination of growth curves: The experimental strain and the standard strain of Escherichia coli were inoculated into 5 mL of antibiotic-free LB liquid medium and cultured overnight at 37℃ and 220 rpm. The next day, 10 mL of antibiotic-free LB liquid medium was dispensed into 50 mL Erlenmeyer flasks and inoculated with the overnight cultured strains. The initial OD600 nm was adjusted to ≈0.05 and cultured at 37℃ and 220 rpm with shaking. The OD600 nm was measured every 1 h with a spectrophotometer until the growth plateau was reached. The growth curves of each strain were plotted. One-way ANOVA and LSD (Least Significant Difference) post-hoc test were used to analyze the differences. The results are shown in Figure 9.

[0048] (5) The MIC and MBC of single Chinese medicine against Escherichia coli were determined by serial dilution: The original solutions of each single Chinese medicine were diluted to 1000, 500, 250, 125, 62.5, 31.25 and 15.6 in LB liquid medium in 96-well plates. 100 μL of diluted bacterial solution was added to each well, mixed well and incubated at 37 ℃ for 24 h.

[0049] If the culture medium is transparent, the result can be directly determined; if the culture medium is dark in color and the result is difficult to determine, after the culture is completed, take an appropriate amount of culture medium and streak it on a solid culture medium. Observe the result after 24 h of incubation, and take the lowest dilution with no bacterial growth as the MIC of each tested extract. When measuring the minimum bactericidal concentration (MBC), select a culture with no bacterial growth, inoculate it into nutrient agar medium, and incubate at a constant temperature (37℃) for 18 h. The minimum drug concentration with ≤5 colonies growing in the medium is the MBC of the drug. The results are shown in Table 2.

[0050] Table 2. MIC and MBC values ​​of various traditional Chinese medicines against Escherichia coli standard strains and test strains. Depend on Figures 9-10It can be seen that, compared with the standard strain, the inhibition of *E. coli* growth curves by *Forsythia suspensa*, *Isatis indigotica*, *Citrus aurantium*, *Mentha haplocalyx*, *Plantago asiatica*, and *Glycyrrhiza uralensis* was dose-dependent. The inhibition of *E. coli* growth curves by *Angelica dahurica* showed a sudden decrease at 10 h, which was followed by a recovery after 2 h. However, the inhibition of blood flow interruption was complete at 2 MIC, and the *E. coli* growth curve became completely flat. These results indicate that the antibacterial effects of *Forsythia suspensa* and *Glycyrrhiza uralensis* in the herbal combination are dominant against *E. coli*.

[0051] As shown in Table 2, among the eight traditional Chinese medicines, licorice showed the best efficacy against the standard strain JN1704 at a MIC of 31.25 mg / mL. -1 The next most effective was forsythia, isatis root, and peppermint, at 62.5 mg / mL. -1 Finally, Angelica dahurica, Desmodium styracifolium, and Citrus aurantium were added, at a dose of 125 mg / mL. -1 Licorice was also the most effective form of MBC, at 31.25 mg / mL. -1 The second highest was forsythia, at 62.5 mg / mL. -1 The other strains showed poorer results. Compared to the standard strain JN1704, licorice and peppermint showed the best MIC and MBC for the test strain PZ1112, at 31.25 mg / mL. -1 The next most potent drugs were Angelica dahurica, Isatis indigotica, and cyclophosphamide, at 62.5 mg / mL. -1 Finally, forsythia, immature bitter orange, and plantain were added, at a dose of 125 mg / mL. -1 Combined with the growth curve, forsythia, licorice, and peppermint are the main active ingredients.

[0052] Both licorice and peppermint contain naringenin, a chemical component targeting AcrB, while naringenin is one of the natural flavonoids with good antibacterial activity against E. coli. Therefore, naringenin in licorice and peppermint exerts its antibacterial effect by disrupting the integrity of bacterial cell membranes. The main chemical components of the principal and assistant herbs target UGDH and are primarily used for anti-inflammatory treatment. Consequently, their antibacterial zone effect was found to be better than that of the selected principal and assistant herbs in antibacterial tests. Forsythia and Angelica dahurica were chosen as principal herbs based on their medicinal properties, meridian tropism, and the principles of principal, assistant, and adjuvant herbs. Chicken colibacillosis is a damp-heat disease, and forsythia, being slightly cold, can relieve damp-heat. Angelica dahurica enters the large intestine and stomach meridians and can promote diuresis and conduct heat, treating the target organ point-to-point. Isatis root and blood-activating herbs were chosen as assistant herbs because they coordinate with the principal herbs and can clear heat and cool the blood.

[0053] Example 3: A traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha, Plantago asiatica, and Glycyrrhiza uralensis were mixed in a mass ratio of 8:8:1:7.15:1:3.26:8:8 to obtain a mixture. The mixture was then mixed with water at a ratio of 1 g:10 mL and soaked for 24 hours. The mixture was then decocted twice over low heat, with each decoction lasting 1 hour. After each decoction, the mixture was filtered, and the decoctions were collected. The two decoctions were combined and concentrated to a concentration of 1.0 g / mL to obtain a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry, abbreviated as ZCF.

[0054] Example 4: A traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha, Plantago asiatica, and Glycyrrhiza uralensis were mixed in a mass ratio of 7:7:0.8:7:0.8:3:7:7 to obtain a mixture. The mixture was then mixed with water at a ratio of 1 g:8 mL and soaked for 20 hours. The mixture was then decocted twice over low heat, each time for 40 minutes. After each decoction, the mixture was filtered, and the decoction was collected. The two decoctions were combined and concentrated to a concentration of 0.5 g / mL. This is the traditional Chinese medicine composition used to prevent and treat pathogenic Escherichia coli in poultry, abbreviated as ZCF.

[0055] Example 5: A traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha, Plantago asiatica, and Glycyrrhiza uralensis were mixed in a mass ratio of 9:9:1.2:7.5:1.2:3.5:9:9 to obtain a mixture. The mixture was then mixed with water at a ratio of 1g:12mL and soaked for 24 hours. The mixture was then decocted twice over low heat, each time for 70 minutes. After each decoction, the mixture was filtered, and the decoction was collected. The two decoctions were combined and concentrated to a concentration of 1.5 g / mL. This is the traditional Chinese medicine composition used to prevent and treat pathogenic Escherichia coli in poultry, abbreviated as ZCF.

[0056] Comparative Example 1: The difference between this comparative example and Example 3 is that the traditional Chinese medicine composition only includes the principal herbs (Forsythia suspensa and Angelica dahurica). Specifically: Forsythia and Angelica dahurica were mixed at a mass ratio of 8:8 to obtain a mixture. The mixture was then mixed with water at a material-liquid ratio of 1g:10mL and soaked for 24 hours. The mixture was then decocted twice over low heat, with each decoction lasting 1 hour. After each decoction, the mixture was filtered, and the decoction was collected. The two decoctions were combined and concentrated to a concentration of 1.0g / mL to obtain the Chinese herbal composition.

[0057] Comparative Example 2: The difference between this comparative example and Example 3 is that the traditional Chinese medicine composition only includes the auxiliary herbs (Isatis root and blood-activating agent). Specifically: Mix Isatis root and Duanxueliu at a mass ratio of 1:7.15 to obtain a mixture; mix the mixture with water at a material-to-liquid ratio of 1g:10mL, soak for 24 hours, decoct twice over low heat, each time for 1 hour, filter after each decoction, collect the decoction, combine the two decoctions, and concentrate to a concentration of 1.0g / mL to obtain the traditional Chinese medicine composition.

[0058] Comparative Example 3: The difference between this comparative example and Example 3 is that the herbal composition only includes adjuvant herbs (Fructus Aurantii Immaturus, Herba Menthae, Herba Plantaginis, and Radix Glycyrrhizae). Specifically: The following mixtures were prepared: Citrus aurantium, Mentha haplocalyx, Plantago asiatica and Glycyrrhiza uralensis were mixed in a mass ratio of 1:3.26:8:8 to obtain a mixture; the mixture was then mixed with water in a material-liquid ratio of 1g:10mL and soaked for 24 hours. The mixture was then decocted twice over low heat, with each decoction lasting 1 hour. After each decoction, the mixture was filtered, and the decoctions were collected. The two decoctions were combined and concentrated to a concentration of 1.0g / mL to obtain the Chinese herbal composition.

[0059] Test Example 1: Antibacterial Test 1. MIC determination of the traditional Chinese medicine composition ZCF used for the prevention and treatment of avian pathogenic Escherichia coli against avian pathogenic Escherichia coli APEC O78: Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha, Plantago asiatica, and Glycyrrhiza uralensis were mixed in a mass ratio of 8.00:8.00:1.00:7.15:1.00:3.26:8.00:8.00 to obtain a ZCF mixture. 500 mg of ZCF was accurately weighed and ground, dissolved in sterile nutrient broth, and brought to a final volume of 1 mL in a centrifuge tube to prepare a ZCF solution with a concentration of 500 mg / mL for later use.

[0060] The concentration of avian pathogenic Escherichia coli APEC O78 bacterial solution was adjusted to 1×10 using nutritious meat broth. 5 CFU / mL. Take a 96-well cell culture plate. Add 100 μL of ZCF solution directly to the first well. Add 100 μL of nutrient broth to each well up to the eighth well. Add 100 μL of ZCF solution to the second well, mix well, and then serially dilute to the eighth well. Add 20 μL of bacterial suspension to each well. Simultaneously set up blank controls, ZCF solution controls, and bacterial suspension controls. Incubate at 37℃ for 18 h, then plate the plate and incubate at 37℃ for another 12 h. The MIC is the drug dilution corresponding to no bacterial growth or a colony count less than 3 on the plate. Each experiment is repeated 3 times. When determining the minimum bactericidal concentration (MBC), select cultures with no bacterial growth, inoculate them onto nutrient agar medium, and incubate at a constant temperature (37℃) for 18 h. The minimum drug concentration with ≤5 colonies growing in the medium is the MBC of the drug.

[0061] 2. Determination of the growth curve of a traditional Chinese medicine composition used for the prevention and treatment of pathogenic Escherichia coli in avian birds against APEC O78: Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha, Plantago asiatica, and Glycyrrhiza uralensis were mixed in a mass ratio of 8:8:1:7.15:1:3.26:8:8. 100g of the mixed Chinese herbal medicines were soaked in 10 times the amount of water overnight, then decocted twice over low heat for 1 hour each time. The two decoctions were mixed and brought to a final volume of 1 g / mL to obtain a ZCF solution, which was stored at -20℃.

[0062] The APEC O78 strain was inoculated into nutrient broth and cultured at 37 °C for 18 h to obtain bacterial suspension. The bacterial suspension was then inoculated into broth medium at an inoculation rate of 5% (v / v), and ZCF solution was added to make the final concentrations MIC, 1 / 2 MIC, and 1 / 4 MIC, respectively. A blank control group without ZCF was also set up.

[0063] Record the moment the drug is added as 0h, and immediately take a sample to measure the OD. 600 The nm value was measured after incubation at 37 ℃ and 120 r / min on a shaker, and the OD values ​​were determined at 2, 4, 6, 8, 10, 12, and 14 h. 600 nm value. And with time as the x-axis, OD 600 Using nm as the ordinate, a growth curve of ZCF inhibiting APEC O78 strain was plotted, and the results are as follows: Figure 11 As shown.

[0064] 3. The effect of a traditional Chinese medicine composition used for the prevention and treatment of pathogenic Escherichia coli in poultry on APECO78 biofilm was determined using a semi-quantitative crystal violet staining method: The overnight cultured APEC O78 bacterial suspension was adjusted to approximately 1×10⁻⁶ using Mueller-Hinton broth (MHB). 7The concentration was set at CFU / mL. Then, 100 μL of bacterial culture and 100 μL of MHB medium containing different concentrations of the traditional Chinese medicine composition were added to each well of a 96-well polystyrene plate, resulting in final concentrations of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC of the traditional Chinese medicine composition. Wells containing no drug were used as a positive control, and wells containing only the medium were used as a negative control. The plates were incubated at 37°C for 24 hours to allow biofilm formation. After incubation, airborne bacteria and medium were carefully discarded from the wells, and the plates were gently washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove unattached bacteria. Next, 200 μL of methanol was added to each well for fixation for 15 minutes, and the methanol was discarded before air drying. Then, 1% (w / v) crystal violet solution was added for staining for 20 minutes, followed by thorough rinsing with deionized water until no free dye remained. After the plates were completely dry, 200 μL of 33% glacial acetic acid solution was added to each well to elute the dye bound to the biofilm. Finally, 100 μL of the eluent was transferred to a new 96-well plate, and the absorbance (OD) was measured at 570 nm using a microplate reader. 570 Each concentration was tested in 6 replicates, and the experiment was independently repeated three times. The results are as follows: Figure 11 As shown.

[0065] Depend on Figure 11 As can be seen from A, ZCF has a certain inhibitory effect on the reproduction of pathogenic Escherichia coli in birds, with an inhibition zone diameter of 22±1 mm and an inhibitory effect of S. Figure 11 As indicated by B in the diagram, the MIC and MBC of ZCF against avian pathogenic Escherichia coli are 62.5 mg / mL. -1 and 125 mg•mL -1 This indicates that ZCF exhibits antibacterial effects even at low concentrations. Figure 11 The study found that at a concentration of 1 / 2 MIC, ZCF only alleviated the growth of *E. coli* during the lag phase, delaying the exponential phase by 2 hours, after which the growth returned to normal. 1 / 2 MIC showed no significant antibacterial effect, while the antibacterial effects of 1 MIC and 2 MIC were dose-dependent. They effectively reduced the total number of *E. coli* bacteria after delaying the exponential phase to 6 hours, and after a 2-hour plateau phase, the bacteria entered the decline phase. These results indicate that 1 MIC and 2 MIC have better antibacterial effects. Figure 11 As shown in Figure D, the study on Escherichia coli biofilm using ZCF revealed that ZCF significantly disrupted biofilm formation, reducing it by nearly 80% compared to normal E. coli biofilm growth. In conclusion, ZCF exhibits a strong antibacterial effect against APEC O78 strain.

[0066] Experimental Example 2: Animal Experiment 1. Laboratory animals and their treatment One-day-old Hy-Line White commercial chickens were used as experimental animals. These Hy-Line White chickens were purchased from Dongyue Poultry Breeding Co., Ltd. in Tai'an City, Shandong Province. After being fed normally for 7 days, they were examined and found to be normal. Each chicken was weighed and randomly divided into 6 groups (healthy control group, challenge control group, high-, medium-, and low-dose groups, and antibiotic control group), with 20 chickens in each group. Specific treatments for each group are shown in Table 3. Table 3 Specific treatments for each treatment group Among them, avian pathogenic Escherichia coli PZ1112 O78 bacterial suspension was used for challenge treatment via intraperitoneal injection, with a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, injection volume 0.5mL / animal 2. Sample collection and testing The clinical manifestations of the experimental chickens were observed and recorded. After the experiment, blood and tissue samples were collected and weighed. The details are as follows: 2.1 Blood samples: 5 mL of blood was collected from the heart of the poultry, centrifuged at 3000 rpm for 10 min at 4℃, and the serum was collected and stored at -80℃ for later use to detect the protein content of inflammatory factors and immune factors.

[0067] 2.2 Tissue Samples: After blood collection, broiler chickens were euthanized by exsanguination via the carotid artery and subjected to necropsy. The small intestine was then separated and rinsed thoroughly with pre-cooled PBS. A portion of the intestinal tissue was fixed in 4% paraformaldehyde solution for section preparation to observe pathological changes.

[0068] 2.3 Immune index: After the end of the experimental observation (7 days), the surviving diseased chickens were weighed and euthanized. The thymus, spleen and bursa of Fabricius were removed and weighed, and the corresponding immune organ indices were calculated.

[0069] 2.4 Bacterial load detection: Small intestinal homogenates were cultured and quantitatively analyzed on days 3 and 7 post-challenge. The intestinal contents were longitudinally dissected, washed with PBS, and 0.1 g of tissue homogenate was serially diluted. 100 μL of the homogenate was then evenly spread onto MacConkey agar and incubated at 37°C for 24 h. The bacterial load was calculated from plates with 30-300 colonies. Three replicates were set for each group before spreading onto MacConkey agar.

[0070] 2.5 Enzyme-linked immunosorbent assay (ELISA): Serum samples were removed from a -80°C freezer and allowed to stand at room temperature until serum precipitation. The serum was then separated and the ELISA kit was used to detect the expression levels of inflammatory factors (TNF-α, IL-1β, IL-6), tight junction proteins ZO-1 and Claudin-1, and immune factor IFN-γ in the serum of patients with Escherichia coli infection.

[0071] 2.6 Detection of relative expression levels of virulence gene mRNA using real-time fluorescence PCR: (1) Primers for real-time PCR were synthesized according to the sequences and methods provided in the literature, including UGDH, AcrB, iss, Fimc, iron, and GAPDH. The primer sequences were synthesized by Shanghai Shenggong Technology Co., Ltd.

[0072] (2) The experimental groups were a blank control group (APEC O78 without drug treatment) and a drug treatment group (the final concentration of the traditional Chinese medicine composition was the optimum effective concentration), with 3 biological replicates in each group. Single colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 180 r / min with shaking until OD. 600 ≈0.6, the corresponding concentration of compound was added to the drug treatment group, and after culturing for another 12 h, bacterial RNA was extracted from each group using a bacterial total RNA extraction kit.

[0073] (3) Quantify the extracted RNA to μg (20 μL) and place it in an EP tube. Add 2 μL of primer, then add DEPC-H2O to a total volume of 22 μL. Add 4 μL of dNTP, incubate at 70℃ for 5 min, and immediately place on ice. Then add 8 μL of 5×buffer, 4 μL of DTT, 1 μL of RNase inhibitor, and 1 μL of M-mLV to make the total reverse transcription volume 40 μL. Mix and centrifuge, and place the EP tube in a 37℃ water bath for 2 h to obtain the reverse transcription product. Take 6 μL of the reverse transcription product, 2 μL each of forward and reverse primers, 8 μL of dNTP Mix, 5 μL of 10×buffer, and 0.3 μL of rTaq enzyme, and add sterile double-distilled water to a total volume of 50 μL to construct the amplification reaction system. Reaction conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, followed by 35 cycles, and finally 72℃ extension for 10 min.

[0074] (4) Construct a quantitative PCR system by adding 1.2 μL of cDNA template, 0.8 μL each of upstream and downstream primers, 10 μL of FastStart DNA Master SYBR Green I, and ddH2O to a final volume of 20 μL. Under light-protected conditions, add the quantitative PCR system to the quantitative PCR plate and seal it for PCR. Set the reaction conditions as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, and repeat for 40 cycles, followed by a final extension at 72℃ for 10 min. After the reaction, use LightCycler® 96 SW 1.1 software to generate melting curves for each amplification product. Calculate the 2-ΔΔCT value based on the relative quantitative data, which is the fold increase in gene expression in each experimental group after internal control normalization compared to the control group, and statistically analyze the differences between the experimental groups. The calculation formula is as follows: 2-ΔΔCT = 2-[(CT test gene - CT internal reference gene) treatment group - (CT test gene - CT internal reference gene) untreated group].

[0075] 2.7 Detection of PI3K-AKT / NF-κB signaling pathway protein expression levels in small intestinal tissue by Western blot: (1) On days 35 and 42 of the experiment, three chicks were selected from each group. After being euthanized, their small intestines were immediately separated. After rinsing with pre-cooled PBS, 1 mL of lysis buffer (a mixture of protease and phosphatase inhibitors: lysis buffer = 1:100) was added. After homogenization, the mixture was centrifuged at 12,000 rpm / min and 4°C for 10 min, and then placed on ice for 20 min. The supernatant was then transferred to a new centrifuge tube and centrifuged at 12,000 rpm / min and 4°C for 10 min. The supernatant was then collected to obtain the total protein lysis buffer. (2) Protein concentration was determined by the BCA method.

[0076] (3) Electrophoresis: Prepare a clean glass plate, fix it in the slot of the electrophoresis tank, fill the middle of the glass plate with deionized water, and let it stand for 20 minutes. If the plate does not sink, the glass plate is considered to have good sealing and can be used to prepare the gel. Pour off the deionized water and absorb the residual moisture with filter paper. Add the separating gel to the glass plate, then add anhydrous ethanol, flatten the separating gel, and let it stand in an incubator at 37°C for 20 minutes to solidify. After the separating gel solidifies, pour off the anhydrous ethanol, add the stacking gel, and slowly insert the comb that matches the size of the gaps in the glass plate vertically into the separating gel to avoid air bubbles. Let it stand in an incubator at 37°C for 20 minutes to solidify. Place the glass plate and slot into the electrophoresis tank, add the electrophoresis solution, slowly pull the comb out vertically, and use a needle to straighten the gel between each well to keep it vertical. Add the protein pre-staining marker and protein sample to the well. Cover the electrode, connect the positive and negative terminals correctly (red for positive, black for negative), and set the parameters to 150 V, 220 mA, and electrophoresis for 90 min.

[0077] (4) Transfer: After electrophoresis, remove the glass plate, measure the required gel size according to the protein size, cut it out and place it in the transfer buffer for later use. Cut an appropriate size PVDF membrane, soak it in methanol for 20 s to activate it, then rinse it in deionized water for 10 s, and finally place it in the transfer buffer for later use. Arrange the transfer "sandwich" in the following order from negative electrode to positive electrode: sponge, gel, PVDF membrane, sponge. Note that when placing each layer, use a pipette to drip a small amount of transfer buffer and slowly lower it to avoid generating air bubbles. Place the "transfer sandwich" in the transfer tank and add transfer buffer until it can cover the entire gel. Set the parameters to 300 V, 220 mA, and 80 min. Note that the transfer process requires an ice bath environment to prevent protein degradation.

[0078] (5) Sealing: Place 5% skim milk powder or 5% BSA on a shaker at room temperature and shake well. After the transfer is complete, use tweezers to remove the PVDF membrane and quickly place it into 5% skim milk powder or 5% BSA, and seal it on a shaker at room temperature for 90-120 min.

[0079] (6) Antibody incubation: After blocking, wash the PVDF membrane three times with TBST for 10 min each time. Cut the PVDF membrane according to the size of the target protein, soak it in the corresponding primary antibody, and incubate overnight at 4°C. Remove the bands and wash the PVDF membrane three times with TBST for 10 min each time. Place the secondary antibody on a shaker at room temperature 30 min in advance, mix well, and cool to room temperature. Incubate in the secondary antibody at room temperature for 1 h. Remove the bands and wash the PVDF membrane three times with TBST for 10 min each time.

[0080] (7) Development: Mix solution A and solution B in a 1:1 ratio. Before development, absorb the excess water on the strip. Immerse the strip completely in the developing solution for about 2 minutes. Then take out the strip and develop it with a developing instrument.

[0081] 3. Experimental Conclusions 3.1 Visual and necropsy findings in poultry: Depend on Figure 12 As shown in Figure A, compared to the Control group, poultry in the APEC O78 group exhibited significant pericarditis, pericardial effusion, and pericardial thickening containing yellowish-white fibrinous exudate. Compared to the control group, poultry in the ZCF medium-dose group and the antibiotic control group showed less pericarditis, less pericardial effusion, and no pericardial thickening. Poultry in the ZCF low-dose and high-dose groups showed a small amount of pericardial effusion, and the low-dose group showed slight pericardial thickening.

[0082] like Figure 12 As shown in Figure B, compared with the Control group, the liver in the APEC O78 group was significantly congested and swollen; the liver tissue in the antibiotic control group and the medium-dose group showed no bleeding points, the liver tissue morphology was relatively normal, and there was no obvious enlargement or degeneration; the low-dose group had a few hemorrhages and the liver was slightly swollen, while the high-dose group had enlarged and congested liver. Compared with the Control group, the intestinal tissue in the APEC O78 group was significantly enlarged, with yellow-green watery stool in the intestinal lumen, and the intestinal mucosa was rough and the intestinal wall was thickened; no obvious bleeding was observed in the intestinal mucosa in the antibiotic control group and the medium-dose group; the intestinal wall was thickened and there were a few bleeding points in the intestinal mucosa in the low-dose ZCF group, while the intestinal wall was not significantly thickened in the high-dose group.

[0083] like Figure 12 As shown in Figure C, the heart / body weight ratios of poultry in the APEC O78 group and the low-dose group were significantly heavier than those in other groups, with heart / body weight ratios of 1.25 and 1.14, respectively, which were closely related to the occurrence of pericarditis. Figure 12 As shown in D, the liver / body weight ratio of poultry in each group showed significant enlargement in the APEC O78 group (value 5.1) and a significant decrease in the ZCF medium-dose group (value 3.9). Figure 12 As shown in E, there was no significant difference in gut-to-body weight ratio among the groups. Figure 12 As shown in F, poultry infected with APEC O78 strain experienced significant weight loss due to diarrhea and loss of appetite, while the weight changes in the other groups were not obvious.

[0084] 3.2 Effects on the immune organs of poultry infected with pathogenic Escherichia coli The immune organ index is closely related to the level of lymphocyte proliferation within the organ and can directly reflect the strength of the body's immune function. The results are shown in Table 4.

[0085] Table 4 Results of Immune Indices in Poultry Immune Organs As shown in Table 4, compared with the Control group (thymus index 3.571±0.11, bursa of Fabricius index 3.969±0.10, spleen index 1.889±0.11), the APEC O78 group had significantly lower thymus index (2.597±0.13, bursa of Fabricius index 2.873±0.17, spleen index 2.469±0.15) than the Control group (2.597±0.13, 2.873±0.17, 2.469±0.15). P <0.05 indicates that APEC O78 infection can significantly inhibit the development of poultry immune organs, thereby impairing the body's immune function. After intervention with a traditional Chinese medicine composition used to prevent and treat pathogenic Escherichia coli in poultry, the thymus, bursa of Fabricius, and spleen indices in both the Colistin group and the medium-dose group were significantly improved. P <0.05 indicates that the traditional Chinese medicine composition used to prevent and treat pathogenic Escherichia coli in birds can reduce the damage of pathogens to immune organs. However, there was no significant difference in the immune organ index between the low-dose group and the high-dose group. P >0.05).

[0086] 3.3 HE section observation of histopathological damage in poultry infected with pathogenic Escherichia coli The results are as follows Figure 13 As shown. By Figure 13 It can be seen that the duodenal tissue structure in the blank group was basically normal, with abundant and intact intestinal villi, and neatly and tightly arranged epithelial cells without obvious cell shedding; the crypt structure was tightly arranged without obvious crypt space expansion; and no obvious inflammatory cell infiltration was observed. Normal red blood cells were visible, as shown by the blue arrows. Compared with the blank group, no significant abnormal changes were observed in the antibiotic control group and the medium-dose treatment group. After challenge, the intestinal tissue structure in the E. coli infection group and the low-dose treatment group was moderately abnormal, with a large amount of epithelial layer shedding and the lamina propria exposed, as shown by the gray arrows; the crypt structure was tightly arranged, with a small amount of cell edema and cytoplasmic vacuolation, as shown by the green arrows; a small number of inflammatory cells appeared in the tissue, as shown by the red arrows, and a small number of cells were necrotic with fragmented, condensed, and deeply stained nuclei, as shown by the black arrows; the intestinal tissue structure in the high-dose group was slightly abnormal, with neatly and tightly arranged epithelial cells and a small amount of cell shedding, as shown by the gray arrows.

[0087] 3.4 Results of bacterial load detection in animal intestinal tissue after treatment The results are as follows Figure 14 As shown. Intestinal bacterial load reflects the level of pathogenic bacteria colonization and reproduction in the intestine, and its level reflects the therapeutic effect of traditional Chinese medicine. Compared with the Control group, the intestinal bacterial load in the APEC O78 group was significantly increased ( P<0.05); and compared with the APEC O78 group, after treatment with Colistin and the traditional Chinese medicine composition prepared in this invention, the intestinal bacterial content decreased significantly, and the intestinal bacterial load in the medium-dose treatment group was significantly reduced ( P <0.05).

[0088] 3.5 Effects on the expression levels of virulence factors in poultry infected with pathogenic Escherichia coli The results are as follows Figure 15 As shown. Compared with the APEC O78 group, the expression levels of virulence-related genes iss, FimC, and iron were significantly decreased after treatment with the traditional Chinese medicine composition prepared in this invention. P <0.05), and the expression levels of target genes AcrB and UGDH were also significantly decreased. Among them, the FimC gene is closely related to the adhesion and colonization ability of bacteria, and its downregulation can directly reduce the adhesion efficiency of the strain to intestinal mucosal epithelial cells; the iron gene is involved in the iron ion uptake process of pathogenic bacteria, and its suppressed expression will affect the survival and reproduction of the strain in the host intestine; the iss gene can enhance the serum resistance and pathogenicity of the strain, and its reduced expression can effectively weaken the pathogenic potential of pathogenic bacteria; the UGDH and AcrB genes are related to the metabolic regulation and invasion ability of the strain, and the decrease in the expression levels of the two helps to reduce damage to intestinal tissues.

[0089] 3.6 Effects on the expression levels of inflammatory factors, tight junction proteins, and immune factors The results are as follows Figure 16 As shown. By Figure 16 As can be seen from the AC results, compared with the APEC O78 group, all ZCF dosage groups effectively reduced the levels of three inflammatory factors, IL-6, TNF-α, and IL-1β, in infected poultry. The medium-dose group showed the most significant decrease in inflammatory factors, confirming its outstanding role in inhibiting the inflammatory response. The serum levels of IL-6, TNF-α, and IL-1β decreased by 73.8.3%, 47.4%, and 74.4%, respectively, compared with the APEC O78 group. This indicates that ZCF has a significant effect in inhibiting the intestinal inflammatory response induced by APEC O78 infection.

[0090] Depend on Figure 16 As can be seen from D and G, compared with the APEC O78 group, the medium-dose group showed a significant decrease in both IL-10 and IFN-γ, two key immune factors. P <0.05), indicating that ZCF can enhance the immunity of poultry by regulating the expression of immune factors and provide immune support for the repair of intestinal damage.

[0091] Depend on Figure 16 As can be seen from E and F, the ZO-1 and Claudin-1 genes in poultry from the APEC O78 group were significantly decreased.P <0.05), this change was reversed in the Colistin group and the medium-dose ZCF treatment group, with significant increases in ZO-1 and Claudin-1 genes ( P <0.05), indicating that ZCF may have protective and repair functions on the intestines of poultry infected with Escherichia coli.

[0092] 3.7 Effects on the expression levels of proteins in the PI3K-AKT and NF-κB signaling pathways The results are as follows Figures 17-18 As shown. The active ingredients of the new traditional Chinese medicine compound successfully activated the UGDH protease, thereby stimulating a significant increase in HAS2 and CD44 proteins. P <0.05), successfully activating the downstream PI3K-AKT pathway.

[0093] like Figure 17 As shown, compared with the Control group, the expression level of P-PI3K protein in the intestinal tissue of poultry in the APEC O78 group was significantly increased ( P <0.05), P-AKT protein expression level was significantly increased ( P <0.05); Compared with the APEC O78 group, the expression level of P-PI3K protein in the intestinal tissue of poultry in the Colistin group and the ZCF medium-dose treatment group was significantly decreased ( P <0.05), P-AKT protein expression level decreased significantly ( P <0.05). The above results indicate that after avian pathogenic Escherichia coli infection in poultry, the expression levels of P-PI3K and P-AKT proteins in the body are increased, the PI3K-AKT signaling pathway is activated, and ZCF can reduce the activity of the upstream UGDH enzyme in this pathway, thereby inhibiting the activation of this signaling pathway.

[0094] like Figure 18 As shown, compared with the Control group, the expression level of P-P65 protein in the intestinal tissue of poultry in the APEC O78 group was increased ( P <0.05), the expression level of P-IKBα protein was significantly increased ( P <0.05); Compared with the APEC O78 group, the expression level of P-P65 protein in the intestinal tissue of poultry in the Colistin group and the ZCF medium-dose treatment group was significantly decreased ( P <0.05), the expression level of P-IKBα protein decreased significantly ( P <0.05). The above results indicate that the expression levels of P-P65 and P-IKBα proteins are increased in poultry infected with pathogenic Escherichia coli, and the NF-κB signaling pathway is activated, while ZCF can inhibit the activation of this pathway.

[0095] In summary, after treatment with a medium dose of the traditional Chinese medicine composition, the levels of p-PI3K, p-AKT, and p-p65 were significantly downregulated compared to the model group. P <0.05), p-IκBα levels significantly rebounded ( P <0.05), and the levels of each phosphorylated protein were close to those of the normal control group. This result directly confirms that the traditional Chinese medicine composition prepared in this invention can effectively inhibit the overactivation of the PI3K-AKT and NF-κB pathways by reducing the expression level of UGDH protein.

[0096] Trial Example 3: Clinical Trial One-day-old Hy-Line White commercial chickens were used as experimental subjects. After being fed normally for 7 days, Hy-Line chicks with no abnormalities were selected for subsequent experiments.

[0097] Healthy Hy-Line chicks were injected intraperitoneally with a solution of avian pathogenic Escherichia coli PZ1112 O78, at a dose of 0.2 mL per chick. The chicks' mental state, feed intake, weight, and fecal condition were then observed. Infection was considered successful when chicks exhibited symptoms such as lethargy, drooping wings, standing alone and listless, drowsiness, yellowish-white watery droppings, fecal contamination of the vent and surrounding feathers, and a tendency to move after being driven away or become immobile when driven away.

[0098] This experiment consisted of six treatment groups: a blank control group, a challenge control group, the traditional Chinese medicine composition groups prepared in Example 3, and Comparative Examples 1-3. Except for the blank control group, all treatments used successfully infected chicks. Each treatment group contained 30 chickens, and the details of each treatment group are as follows: Blank control group: Healthy chicks were selected and administered physiological saline by gavage; Control group: Successfully infected chicks were selected and administered physiological saline by gavage; Example 3: Selected successfully infected chicks were administered the traditional Chinese medicine composition prepared in Example 1 for the prevention and treatment of pathogenic Escherichia coli in birds via gavage; Comparative Example 1: Selected successfully infected chicks were gavaged with the traditional Chinese medicine composition prepared in Comparative Example 1. Comparative Example 2: Selected successfully infected chicks were gavaged with the traditional Chinese medicine composition prepared in Comparative Example 2; Comparative Example 3: Selected successfully infected chicks were gavaged with the traditional Chinese medicine composition prepared in Comparative Example 3.

[0099] The dosage of both the traditional Chinese medicine composition and physiological saline was 25 mg per chick administered via gavage. All treatment groups underwent gavage for 7 consecutive days, once daily, ensuring consistent feeding and management across all groups. The chicks' condition was recorded daily, and the cure rate was calculated after 7 days. The results are shown in Table 5.

[0100] Cure rate (%) = (Number of cured chicks / Total number of chicks) × 100%.

[0101] When the clinical symptoms of chicks disappear, such as lethargy, drooping wings, standing alone and listless, drowsiness, yellowish-white watery droppings, cloaca and surrounding feathers being soiled with feces, walking when driven away, being unable to move when driven away, or even death, they are considered cured.

[0102] Table 5. Cure rate of chicks in each treatment group As shown in Table 5, the number of diseased chicks in the control group was 30. After treatment with only the principal herbs (Forsythia suspensa and Angelica dahurica), assistant herbs (Isatis indigotica and Desmodium styracifolium), and adjuvant herbs (Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and Glycyrrhiza uralensis), the number of diseased chicks decreased by 12, 7, and 4, respectively. However, after treatment with the traditional Chinese medicine composition prepared in this invention for the prevention and treatment of pathogenic Escherichia coli in birds, the number of diseased chicks decreased by 29, with a cure rate as high as 96.7%. Therefore, the principal herbs (Forsythia suspensa and Angelica dahurica), assistant herbs (Isatis indigotica and Desmodium styracifolium), and adjuvant herbs have a synergistic effect in improving the cure rate of pathogenic Escherichia coli in birds.

[0103] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in poultry, characterized in that, The components include the following parts by weight: Forsythia suspensa 7-9 parts, Angelica dahurica 7-9 parts, Isatis indigotica 0.8-1.2 parts, Dioscorea opposita 7-7.5 parts, Citrus aurantium 0.8-1.2 parts, Mentha haplocalyx 3-3.5 parts, Plantago asiatica 7-9 parts, Glycyrrhiza uralensis 7-9 parts.

2. The traditional Chinese medicine composition for preventing and treating pathogenic Escherichia coli in birds as described in claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following components: Forsythia suspensa 8 parts, Angelica dahurica 8 parts, Isatis indigotica 1 part, Dioscorea opposita 7.15 parts, Citrus aurantium 1 part, Mentha haplocalyx 3.26 parts, Plantago asiatica 8 parts, Glycyrrhiza uralensis 8 parts.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Includes the following steps: Forsythia, Angelica dahurica, Isatis indigotica, Dioscorea opposita, Citrus aurantium, Mentha haplocalyx, Plantago asiatica, and Glycyrrhiza uralensis were mixed to obtain a mixture. Water was added to the mixture to soak it, and it was decocted twice over low heat. After each decoction, the mixture was filtered and the decoction was collected. The two decoctions were combined and concentrated to a concentration of 0.5-1.5 g / mL to obtain a traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in birds.

4. The method for preparing the traditional Chinese medicine composition as described in claim 3, characterized in that, The mass ratio of the mixture to water is 1g:(8-12)mL; the soaking time is 20-30h, and the decoction time is 50-70min each time.

5. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a product for treating pathogenic Escherichia coli in birds.

6. A product for treating pathogenic Escherichia coli in birds, characterized in that, The product includes the traditional Chinese medicine composition for the prevention and treatment of pathogenic Escherichia coli in birds as described in claim 1 or 2.

7. The product for treating pathogenic Escherichia coli in birds as described in claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

8. The product for treating pathogenic Escherichia coli in birds as described in claim 7, characterized in that, Pharmaceutically acceptable excipients include disintegrants, lubricants, sweeteners, or binders.

9. The product for treating pathogenic Escherichia coli in birds as described in claim 6, characterized in that, Products used to treat pathogenic Escherichia coli in birds are available in the form of decoctions, soluble powders, or injections.

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