A traditional Chinese medicine composition for treating chicken pullorum salmonella, a preparation method and application thereof

Abstract

This invention discloses a traditional Chinese medicine composition for treating Salmonella pullorum, its preparation method, and its application, belonging to the field of medical preparation technology. This invention utilizes network pharmacology screening and, based on the principles of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine formulation and the theory of syndrome differentiation in traditional veterinary medicine, has developed a traditional Chinese medicine composition for treating Salmonella pullorum. This composition uses Pulsatilla chinensis and Phellodendron chinense as principal herbs, Scutellaria baicalensis and Portulaca oleracea as assistant herbs, Pogostemon cablin and Atractylodes macrocephala as adjuvant herbs, and Glycyrrhiza uralensis as guiding herb; its main functions are clearing heat and detoxifying, drying dampness and stopping diarrhea, with the auxiliary function of strengthening the spleen and consolidating the body's foundation. It exhibits good therapeutic effects against Salmonella pullorum.

Description

Technical Field

[0001] This invention relates to the field of medical preparations technology, specifically to a traditional Chinese medicine composition for treating Salmonella pullorum disease, its preparation method, and its application. Background Technology

[0002] Pullorosis is caused by Salmonella pullorum (…). Salmonella pullorum This acute, septicemic infectious disease in chickens is clinically characterized by the excretion of white, pasty feces. It has high morbidity and mortality rates, seriously impacting the healthy development of the poultry farming industry. The disease primarily affects chicks aged 1-3 weeks, causing mass mortality. Adult chickens are often asymptomatic carriers. It can be transmitted vertically and horizontally. Currently, antibiotics are the main treatment method, but while antibiotic treatment can reduce mortality in poultry, it cannot eradicate the pathogen.

[0003] Traditional Chinese medicine (TCM), as a unique medical 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 alternatives to antibiotics, and its application in poultry disease prevention and control is gradually gaining attention. In TCM veterinary medicine theory, pullorum disease in chickens is classified as infectious dysentery, a condition characterized by damp-heat accumulation and spleen and stomach deficiency. TCM has a natural advantage in antibacterial applications, exhibiting resistance to drug resistance and minimal toxicity. While inhibiting bacteria, it can also improve the physical condition of poultry, achieving a "strengthening" effect while treating the disease. Finding safe, efficient, low-residue alternatives that are unlikely to induce drug resistance has become a core issue for ensuring poultry health and the sustainable development of the poultry industry.

[0004] Therefore, providing a traditional Chinese medicine composition for treating Salmonella pullorum in chickens has important theoretical value and practical significance for promoting the green and healthy development of the poultry farming industry. 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 treating Salmonella pullorum disease in chickens, its preparation method, and its application.

[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 treating Salmonella pullorum disease in chickens, comprising the following raw materials in parts by weight: 2-4 parts of Pulsatilla chinensis, 3-5 parts of Phellodendron chinense, 2-4 parts of Scutellaria baicalensis, 2-4 parts of Portulaca oleracea, 2-4 parts of Pogostemon cablin, 2-4 parts of Atractylodes macrocephala, and 2-4 parts of Glycyrrhiza uralensis.

[0007] Furthermore, it includes the following raw materials in parts by weight: 3 parts Pulsatilla chinensis, 4 parts Phellodendron chinense, 3 parts Scutellaria baicalensis, 3 parts Portulaca oleracea, 3 parts Pogostemon cablin, 3 parts Atractylodes macrocephala, and 3 parts Glycyrrhiza uralensis.

[0008] A second aspect of the present invention provides a method for preparing the aforementioned traditional Chinese medicine composition, comprising the following steps: Mix Pulsatilla chinensis, Phellodendron chinense, Scutellaria baicalensis, Portulaca oleracea, Pogostemon cablin, Atractylodes macrocephala, and Glycyrrhiza uralensis to obtain a mixture; add water to the mixture and soak it, then decoct it twice over low heat. Filter after each decoction and collect the decoction; combine the two decoctions and concentrate them to a concentration of 0.5-1.5 g / mL to obtain a traditional Chinese medicine composition for treating Salmonella pullorum in chickens.

[0009] Furthermore, the mass-to-volume 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.

[0010] A third aspect of the present invention provides the use of the aforementioned traditional Chinese medicine composition in the preparation of a product for treating Salmonella pullorum disease in chickens.

[0011] In a fourth aspect, the present invention provides a product for treating Salmonella pullorum, the product comprising the aforementioned traditional Chinese medicine composition for treating Salmonella pullorum.

[0012] Furthermore, this also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the pharmaceutically acceptable excipients include disintegrants, lubricants, sweeteners, or binders.

[0014] Furthermore, the product can be in the form of a decoction, a soluble powder, or an injection.

[0015] The beneficial effects of this invention are: This invention utilizes network pharmacology for screening and, based on the principles of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine and the theory of syndrome differentiation in veterinary medicine, has developed a traditional Chinese medicine composition for treating Salmonella pullorum disease. This composition uses Pulsatilla chinensis and Phellodendron chinense as principal herbs, Scutellaria baicalensis and Portulaca oleracea as assistant herbs, Pogostemon cablin and Atractylodes macrocephala as adjuvant herbs, and Glycyrrhiza uralensis as guiding herb. Its primary functions are clearing heat and detoxifying, drying dampness and stopping diarrhea, while its secondary functions are strengthening the spleen and consolidating the body's foundation. It exhibits good therapeutic effects against Salmonella pullorum disease. Attached Figure Description

[0016] Figure 1 A diagram showing the distribution of drug properties in an effective prescription for treating Salmonella pullorum disease in chickens.

[0017] Figure 2 A diagram showing the distribution of medicinal ingredients in an effective prescription for treating Salmonella pullorum disease in chickens.

[0018] Figure 3 A diagram showing the meridian distribution of drugs in an effective prescription for treating Salmonella pullorum disease in chickens.

[0019] Figure 4This is a cluster diagram of variables related to traditional Chinese medicine.

[0020] Figure 5 Venny diagram showing the active ingredient targets and disease targets of new traditional Chinese medicine compound preparations.

[0021] Figure 6 A "drug-active ingredient-target" network for a new traditional Chinese medicine compound for the treatment of Salmonella pullorum disease in chickens.

[0022] Figure 7 A PPI network for potential targets of new traditional Chinese medicine compound for treating Salmonella pullorum disease in chickens.

[0023] Figure 8 This is a key target for the treatment of Salmonella pullorum in chickens using a new traditional Chinese medicine compound.

[0024] Figure 9 Analysis of GO functionality.

[0025] Figure 10 This is a graph showing KEGG enrichment analysis.

[0026] Figure 11 This refers to the molecular docking binding energy. Detailed Implementation

[0027] 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.

[0028] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0029] In traditional Chinese veterinary medicine, chicken pullorum disease is classified as "epidemic dysentery," characterized by weak spleen and stomach, internal damp-heat, and excessive pathogenic factors. Therefore, the key to treatment lies in clearing heat and detoxifying, strengthening the spleen and drying dampness. In the novel traditional Chinese medicine compound provided by this invention, Pulsatilla chinensis has the effects of clearing heat and detoxifying, cooling blood and stopping dysentery, and has significant antibacterial effects. One of its main active ingredients is saponin compounds, which can significantly inhibit the function of inflammatory factors and have anti-inflammatory effects. Phellodendron chinense has the effects of clearing heat and dampness, stopping dysentery and consolidating the intestines. It contains alkaloids, limonene and other components, and has anti-inflammatory and antidiarrheal effects. Scutellaria baicalensis has the effects of clearing heat and drying dampness, purging fire and stopping dysentery. Its main components are flavonoids and flavonoid glycosides, which have antibacterial, anti-inflammatory and immune-enhancing effects. Portulaca oleracea has the effects of clearing heat and detoxifying, cooling blood and stopping dysentery. It contains flavonoids, alkaloids, coumarins and other components, and has anti-inflammatory, antifungal and antitumor effects. Patchouli has the effects of resolving dampness and harmonizing the middle jiao, reducing bitterness and coldness, and enters the spleen and stomach meridians. The polysaccharides in Atractylodes macrocephala have a certain effect on improving the symptoms of enteritis. Glycyrrhiza uralensis has a significant effect on harmonizing the various drugs and is a commonly used drug in the formulation. In the novel compound, it has the effect of regulating digestive and immune functions. Therefore, the new compound formula of this invention mainly focuses on clearing heat and detoxifying, drying dampness and stopping diarrhea, and secondarily on strengthening the spleen and consolidating the foundation. It can not only treat the symptoms of Salmonella pullorum infection in chickens, but also improve the state of the spleen and stomach damaged by the disease. It treats both the symptoms and the root cause and has a clear therapeutic effect on chicken pullorum.

[0030] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0031] Example 1: Screening of a traditional Chinese medicine composition for treating Salmonella pullorum disease in chickens Data mining and the development of new traditional Chinese medicine (TCM) formulas: Searches were conducted on websites such as CNKI, PubMed, and Wanfang Data Knowledge Service Platform using keywords such as "Salmonella pullorum," "traditional Chinese medicine," and "bacterial enteritis in chickens." Literature published since the database's inception was compiled and screened, resulting in 112 TCM formulas. After further screening, 123 TCM herbs were identified. The frequency of occurrence of the herbs in the 112 formulas was statistically analyzed, followed by association rule and cluster analysis. The properties, flavors, meridian tropism, and efficacy of the 123 herbs were statistically analyzed according to the 2020 edition of the *Pharmacopoeia of the People's Republic of China*. Based on the results, new formulas were developed.

[0032] 1.1 Analysis of Medication Frequency The frequency of use of 123 Chinese herbs in 112 prescriptions was statistically analyzed. Among them, 21 herbs were used more than 10 times. The top 10 herbs in terms of frequency of use were Coptis chinensis (51 times), Pulsatilla chinensis (43 times), Scutellaria baicalensis (42 times), Phellodendron chinense (38 times), Terminalia chebula (33 times), Lonicera japonica (23 times), Glycyrrhiza uralensis (21 times), Fraxinus chinensis (18 times), Portulaca oleracea (17 times), and Astragalus membranaceus (17 times).

[0033] 1.2 Drug Properties Analysis A statistical analysis of the medicinal properties of 123 herbs was performed, and a pie chart was created in an Excel file, such as... Figure 1 The results showed that cold, warm, and neutral drugs were used most frequently. Cold drugs were used 56.9% (66 times), warm drugs were used 25.9% (30 times), neutral drugs were used 15.5% (18 times), hot drugs were used 1.7% (2 times), and cooling drugs were used 0%.

[0034] 1.3 Analysis of Drug Flavors A statistical analysis of the 123 medicinal ingredients was conducted, and a pie chart was created in an Excel file, such as... Figure 2 The results showed that bitter, sweet, and pungent drugs were used most frequently, with bitter drugs accounting for 39.7% (46 times), sweet drugs for 25.9% (30 times), pungent drugs for 23.3% (27 times), sour drugs for 9.5% (11 times), and salty drugs for 5.2% (6 times).

[0035] 1.4 Analysis of Drug Meridian Tropism The meridian tropism of 123 Chinese medicinal herbs was statistically analyzed, and a bar chart was created in an Excel file. The results are as follows: Figure 3 The results showed that the drugs associated with the Large Intestine Meridian were used most frequently, 59 times (17%); followed by drugs associated with the Stomach Meridian, 52 times (16.7%); drugs associated with the Liver Meridian were used 48 times (16.3%), drugs associated with the Spleen Meridian were used 44 times (13.5%), drugs associated with the Lung Meridian were used 44 times (12.4%), drugs associated with the Heart Meridian were used 33 times (10.3%), drugs associated with the Kidney Meridian were used 29 times (4.6%), drugs associated with the Gallbladder Meridian were used 17 times (3.7%), drugs associated with the Bladder Meridian were used 14 times (3%), drugs associated with the Small Intestine Meridian were used 9 times (1.9%), and drugs associated with the Triple Energizer Meridian were used 2 times (0.6%).

[0036] 1.5 Association rule analysis of prescriptions Association rule analysis was performed on 112 prescriptions using SPSS Modeler software. The minimum conditional support was set to 10 and the confidence level to 0.8. The results of the strong association were obtained, as shown in Tables 1 and 2. There were 13 commonly used Chinese medicine combinations, of which 3 were combinations of two herbs and 10 were combinations of three herbs.

[0037] Table 1. Prescription Combination Patterns of Two Traditional Chinese Medicine Herbs Table 2. Prescription Combination Patterns of Three Chinese Herbs 1.6 Cluster Analysis of Drugs Cluster analysis was performed on 21 drugs that were used more than 10 times, resulting in a cluster analysis phylogenetic diagram, such as... Figure 4 Using a distance of 15 as a dividing line, the 21 medicinal herbs can be divided into five categories: Category 1 includes Isatis tinctoria leaf, Gardenia jasminoides fruit, Isatis indigotica root, and Pogostemon cablin; Category 2 includes Astragalus membranaceus and Atractylodes macrocephala; Category 3 includes Citrus reticulata peel, Punica granatum peel, Atractylodes lancea rhizome, Crataegus pinnatifida fruit, and Fraxinus chinensis bark; Category 4 includes Lonicera japonica flower and Forsythia suspensa fruit; Category 5 includes Portulaca oleracea fruit; Category 6 includes Sophora flavescens root, Glycyrrhiza uralensis root, and Terminalia chebula fruit; and Category 7 includes Pulsatilla chinensis root, Phellodendron chinense bark, Coptis chinensis root, and Scutellaria baicalensis root.

[0038] 1.7 New Traditional Chinese Medicine Compound Based on the results of association rules and cluster analysis, and combined with the TCM principle of "principal, assistant, adjuvant, and guide" in herbal formulation, a new compound prescription was formulated. The principal herbs are Pulsatilla chinensis and Phellodendron chinense; the assistant herbs are Scutellaria baicalensis and Portulaca oleracea; the adjuvant herbs are Pogostemon cablin and Atractylodes macrocephala; and the guiding herb is Glycyrrhiza uralensis. The prescription analysis is as follows: Chief herbs (Pulsatilla chinensis, Phellodendron chinense): Pulsatilla chinensis is cold in nature and bitter in taste. It enters the large intestine and stomach meridians and has the classic effects of clearing heat and detoxifying, cooling blood and stopping dysentery. It is a core medicine for treating dysentery caused by heat toxins.

[0039] Phellodendron bark is bitter and cold in nature, and enters the kidney, bladder, and large intestine meridians. It can clear damp-heat in the lower burner, purge fire and detoxify, stop dysentery and strengthen the intestines, precisely targeting the symptoms of internal damp-heat in the intestines.

[0040] Pulsatilla chinensis and Phellodendron chinense are the principal herbs. The two herbs work together to directly target the main disease, symptoms, and pathogenic factors of this disease. This aligns with the core pathogenesis of chicken dysentery caused by damp-heat stagnation in the intestines, and forms the core framework of the entire formula for eliminating pathogenic factors and stopping dysentery.

[0041] Assistant herbs (Scutellaria baicalensis, Portulaca oleracea): Scutellaria baicalensis is bitter and cold in nature, and is good at clearing heat and drying dampness, purging fire and detoxifying, cooling blood and stopping dysentery. It is especially good at clearing damp-heat and fire toxins in the intestines.

[0042] Purslane is sour and cold in nature, and enters the large intestine and liver meridians. It has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is a commonly used folk medicine for treating dysentery caused by heat toxins.

[0043] Scutellaria baicalensis and Portulaca oleracea are used as assistant herbs to enhance the effects of clearing heat and detoxifying, drying dampness and stopping dysentery, and strengthen the overall therapeutic effect of the compound.

[0044] Adjuvant herbs (patchouli, atractylodes): Patchouli enters the spleen, stomach, and lung meridians, and can resolve dampness and harmonize the middle jiao, invigorate the spleen and stimulate appetite, and dispel foulness and turbidity. It can effectively neutralize the bitter and cold nature of the formula, protect the spleen and stomach functions, and at the same time resolve dampness and turbidity in the body, improving the state of dampness and stagnation in the spleen and stomach of livestock and poultry and their poor digestion.

[0045] Atractylodes macrocephala has a sweet and bitter taste and is warm in nature. It enters the spleen and stomach meridians. Its core functions are to strengthen the spleen and replenish qi, dry dampness and promote diuresis, directly addressing the fundamental pathogenesis of chicken diarrhea, namely "weak spleen and stomach and internal dampness".

[0046] Patchouli and Atractylodes macrocephala are used as adjuvant herbs to strengthen the spleen, resolve dampness, and harmonize the properties of other herbs, addressing the root cause of the disease, namely "weakness of the spleen and stomach," while also mitigating the drawbacks of the bitter and cold herbs in the formula.

[0047] Guiding herb (licorice): Licorice is the main ingredient in this formula. It is sweet and neutral in nature, and enters the heart, lung, spleen, and stomach meridians. Licorice can harmonize the properties of all the herbs in the formula, moderate their bitter and cold nature, and balance their hot and cold properties, making the overall formula mild and preventing any imbalance that could harm the body. Licorice can also replenish qi and strengthen the spleen and stomach, regulate the digestive function and immune function of livestock and poultry, help repair spleen and stomach damage, enhance the body's resistance, and aid in post-illness recovery.

[0048] In summary, this novel traditional Chinese medicine compound formula is meticulously formulated, with clear primary and secondary components, addressing both the symptoms and the root cause. The core of the formula is clearing heat and detoxifying, drying dampness and stopping diarrhea, rapidly eliminating intestinal pathogens and damp heat, inhibiting pathogenic bacteria, and eliminating intestinal inflammation, thus resolving the external symptoms of pullorum disease in chickens. Simultaneously, it supplements this with spleen-strengthening and qi-tonifying, consolidating the body's foundation and nourishing the vital energy, repairing damaged spleen and stomach function, regulating bodily functions, enhancing immunity, and preventing recurrence. Compared to single symptomatic treatments, this compound formula achieves the dual effect of treating both the symptoms and the root cause, demonstrating a significant and stable therapeutic effect on various diseases caused by Salmonella pullorum infection in chickens.

[0049] Example 2: Pharmacological Analysis of a New Traditional Chinese Medicine Compound 2.1 Drug active ingredient target prediction and disease target acquisition After searching the TCMSP database for drugs and screening them according to standards, a total of 214 effective active ingredients were obtained, including 11 from Pulsatilla chinensis, 37 from Phellodendron chinense, 36 from Scutellaria baicalensis, 10 from Portulaca oleracea, 11 from Pogostemon cablin, 7 from Atractylodes macrocephala, and 92 from Glycyrrhiza uralensis. After summarizing and removing duplicates from all effective ingredients, 189 remained. The SMILEs of these 189 active ingredients were imported into the SwissTargetPrediction database for target prediction, and the results were summarized to obtain a total of 977 targets. A search for "avian salmonellosis" in the GeneCard database yielded 692 disease targets.

[0050] 2.2 Acquisition of potential targets The targets of the active ingredients and disease targets were imported into the VENNY platform, their intersection was calculated, and then visualized using Cytoscape software, ultimately yielding 132 potential targets, such as... Figure 5 .

[0051] 2.3 “Drug-Active Ingredient-Potential Target” Network 132 potential targets and 189 active ingredients were imported into Cytoscape 3.9.10 software to construct a network and visualize it, such as... Figure 6 The network has 184 nodes and 194 edges. The MCC algorithm can be used to identify the central nodes in the network. The compounds with higher rankings include ZINC01615307, isorhamnetin, aureusidin, glycyrrhizin B, and 8β-ethoxyatractylenolide III.

[0052] 2.4 PPI Network Data on 132 potential target proteins were imported into the STRING database. Protein-protein interaction networks were constructed according to standard procedures. The results were downloaded in TSV format and visualized using Cytoscape 3.9.10. The MCC algorithm was used to screen the top 20 core targets, including TP53, BCL2, and STAT3. Results are as follows: Figure 7 and Figure 8 .

[0053] 2.5 GO enrichment analysis Data from 132 potential targets were imported into the DAVID database for analysis. After downloading the results, statistical screening was performed. A total of 534 GO enrichment entries were obtained, including 411 entries in biological processes (BP), mainly including negative regulation of apoptosis, apoptosis, protein phosphorylation, positive regulation of transcription mediated by RNA polymerase II, positive regulation of gene expression, positive regulation of classical NF-κB signaling, positive regulation of MAPK cascade reactions, regulation of cell population proliferation, positive regulation of apoptosis, and response to lipopolysaccharide; 62 entries in cellular components (CC), mainly including protein complexes, cytoplasm, cytoplasm, cell membrane, membrane rafts, focal adhesion, glutamatergic synapses, perinuclear regions of the cytoplasm, postsynaptic structures, and cell surface; and 61 entries in molecular functions (MF), mainly including protein kinase activity, kinase activity, same-protein binding, protein tyrosine kinase activity, enzyme binding, serine / threonine kinase activity, transferase activity, ATP binding, and nucleotide binding. Rank the items according to their p-values ​​and create bar charts with the top 10 items, such as... Figure 9 .

[0054] GO enrichment analysis revealed that, in terms of biological processes, the targets were mainly concentrated on responses to lipopolysaccharide (LPS), positive regulation of the NF-κB signaling pathway, positive regulation of the MAPK cascade, and regulation of apoptosis. Following infection with *Salmonella pullorum*, its LPS activates the NF-κB and MAPK signaling pathways in the host, releasing a large number of inflammatory factors. Simultaneously, *Salmonella* injects effector proteins into host cells via the type III secretion system, thereby regulating the host's apoptosis process. The enrichment of these targets indicates that the targets screened in this invention are closely related to the pathological process of *Salmonella pullorum*. Regarding cellular components, the core targets were mainly distributed in the cell membrane and cytoplasm. When *Salmonella* invades host cells, it first enters the cell via endocytosis, forming *Salmonella*-containing vesicles in the cytoplasm and colonizing. The enrichment of these targets indicates that the subcellular distribution of the target proteins is consistent with the infection pathway of *Salmonella*. In terms of molecular function, the core targets were significantly enriched in functional items such as protein kinase activity and protein tyrosine kinase activity. Multiple effector proteins secreted by the Salmonella type III secretion system can interact with protein kinases in the host's MAPK pathway, thereby affecting the host's immune function. The enrichment of these molecular functions suggests that the active ingredients of traditional Chinese medicine may affect the pathogen's infection by competitively binding to host protein kinases and blocking Salmonella's manipulation of signaling pathways.

[0055] 2.6 KEGG pathway enrichment analysis 141 signal pathways were obtained, and the top 10 pathways were used to create a bubble chart, as shown below. Figure 10 This mainly includes signaling pathways such as apoptosis, Fc epsilon RI signaling pathway, and EGFR inhibitor resistance.

[0056] KEGG pathway enrichment analysis showed that the enzymes were mainly enriched in signaling pathways such as apoptosis and EGFR inhibitor resistance. The effector proteins secreted by Salmonella's type III secretion system can target and inhibit the MAPK signaling pathway, allowing bacteria to evade the immune system. Meanwhile, active ingredients in traditional Chinese medicine can activate the host's immune function by inhibiting the EGFR signaling pathway, thus achieving antibacterial effects. Therefore, the enrichment of these pathways suggests that important active ingredients may inhibit bacterial intracellular survival by suppressing Salmonella's interference with the MAPK pathway.

[0057] 2.7 Molecular docking verification To verify the accuracy and binding affinity of the screened targets and active ingredients, 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. Specifically, the top 5 effective active ingredients (ZINC01615307, isorhamnetin, aureusidin, glycyrrhizin B, 8β-ethoxy atractylenolide III) and the top 3 target proteins (TP53, BCL2, STAT3) were selected for molecular docking. The results were visualized, as shown below. Figure 11 And Table 3.

[0058] Table 3 Molecular docking table Molecular docking results showed that the screened core TCM components could effectively dock with their corresponding core targets, mainly through hydrogen bonds to form stable bonds, exhibiting strong overall affinity. Among them, isorhamnetin showed the best binding affinity to STAT3, suggesting a possible strong interaction between the two.

[0059] STAT3 is a signal transduction and transcription activator. Its family contains seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. They play crucial roles in cellular responses to cytokines and growth factors, regulating biological processes such as cell growth and programmed cell death. STAT3 is a redox regulatory protein whose function is strictly influenced by the intracellular redox environment. It promotes cell survival and anti-inflammatory responses. Under normal circumstances, STAT3 is located in the cytoplasm. When subjected to specific stimuli, such as bacterial infection, it migrates from the cytoplasm to the nucleus, regulating gene expression. When Salmonella enters the host, it activates STAT3, a signal transduction and transcription activator, in the later stages of infection. Phosphorylation of STAT3 induces the expression of nearly a hundred genes, participating in the proliferation of Salmonella in intestinal epithelial cells. This phenomenon is known as Salmonella infection-induced host cell transcriptional reprogramming. Therefore, after Salmonella infection, it invades the host's intestinal epithelial cells, stimulating STAT3 in the cytoplasm to enter the cell nucleus, regulating gene expression, and producing proteins that promote cell survival for Salmonella to use, thus promoting bacterial reproduction and ensuring its survival.

[0060] Isorhamnetin is a 3-O-methylated metabolite of quercetin and belongs to the flavonoid class of compounds. Isorhamnetin has therapeutic effects on a variety of inflammatory diseases. It can regulate the MAPK signaling pathway, and ERK1 / 2 within it can indirectly affect cell survival by influencing oxidative metabolism and energy balance.

[0061] Therefore, isorhamnetin can regulate the MAPK signaling pathway, and by utilizing ERK1 / 2, it can increase the intracellular SOD content, thereby affecting the intracellular redox environment, which in turn affects the activity of STAT3, weakens the regulation of gene expression by STAT3, and inhibits the reproduction of Salmonella in the host, thus playing a therapeutic role.

[0062] In conclusion, the new traditional Chinese medicine compound can treat fowl pullorum disease through multiple synergistic effects.

[0063] Example 3: A traditional Chinese medicine composition for treating Salmonella pullorum in chickens Take 3g of Pulsatilla chinensis, 4g of Phellodendron chinense, 3g of Scutellaria baicalensis, 3g of Portulaca oleracea, 3g of Pogostemon cablin, 3g of Atractylodes macrocephala, and 3g of Glycyrrhiza uralensis to obtain a mixture; mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours, then decoct twice over low heat, each time for 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate to a concentration of 1.0g / mL to obtain a traditional Chinese medicine composition for treating Salmonella pullorum in chickens.

[0064] Example 4: A traditional Chinese medicine composition for treating Salmonella pullorum in chickens Take 4g of Pulsatilla chinensis, 5g of Phellodendron chinense, 4g of Scutellaria baicalensis, 4g of Portulaca oleracea, 4g of Pogostemon cablin, 4g of Atractylodes macrocephala, and 4g of Glycyrrhiza uralensis to obtain a mixture; mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours, then decoct twice over low heat, each time for 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate to a concentration of 1.0g / mL to obtain a traditional Chinese medicine composition for treating Salmonella pullorum in chickens.

[0065] Example 5: A traditional Chinese medicine composition for treating Salmonella pullorum in chickens Take 2g of Pulsatilla chinensis, 3g of Phellodendron chinense, 2g of Scutellaria baicalensis, 2g of Portulaca oleracea, 2g of Pogostemon cablin, 2g of Atractylodes macrocephala, and 2g of Glycyrrhiza uralensis to obtain a mixture; mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours, then decoct twice over low heat, each time for 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate to a concentration of 1.0g / mL to obtain a traditional Chinese medicine composition for treating Salmonella pullorum in chickens.

[0066] Comparative Example 1 The difference between this comparative example and Example 3 is that only the principal herb is used in the preparation of the traditional Chinese medicine composition, as detailed below: Take 3g of Pulsatilla chinensis and 4g of Phellodendron chinense and mix them to obtain a mixture. Mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours. Decoction is carried out twice over low heat, with each decoction lasting 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate them to a concentration of 1.0g / mL to obtain the traditional Chinese medicine composition.

[0067] Comparative Example 2 The difference between this comparative example and Example 3 is that only the auxiliary medicine is used in the preparation process of the traditional Chinese medicine composition, as detailed below: Take 3g of Scutellaria baicalensis and 3g of Portulaca oleracea and mix them to obtain a mixture. Mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours. Decoction is carried out twice over low heat, each time for 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate to a concentration of 1.0g / mL to obtain the traditional Chinese medicine composition.

[0068] Comparative Example 3 The difference between this comparative example and Example 3 is that only adjuvant herbs are used in the preparation of the traditional Chinese medicine composition, as detailed below: Take 4g of agastache rugosa, 4g of atractylodes macrocephala and 4g of licorice and mix them to obtain a mixture. Mix the mixture with water at a ratio of 1g:10mL and soak for 24 hours. Decoction is carried out twice over low heat, each time for 1 hour. After each decoction, filter and collect the decoction. Combine the two decoctions and concentrate to a concentration of 1.0g / mL to obtain the traditional Chinese medicine composition.

[0069] Experimental Example 1: Animal Experiment 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. Healthy Hy-Line chicks were injected intraperitoneally with Salmonella pullorum solution, wherein the Salmonella pullorum strain was ATCC 10398, and the dosage was 10. 7 CFU / chick, then observe the chick's mental state, feed intake, weight, and feces. When chicks refuse to eat, are sensitive to cold, curl up, droop their wings, are depressed or lethargic, excrete white, sticky, pale yellow, or pale green watery feces, sometimes with the anus blocked by hardened fecal matter, and have difficulty breathing, the infection is considered successful.

[0070] 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 group: Selected successfully infected chicks were administered the traditional Chinese medicine composition prepared in Example 1 for the treatment of Salmonella pullorum disease by gavage; Comparative Example 1: Selected successfully infected chicks were gavaged with the traditional Chinese medicine composition prepared in Comparative Example 1. Comparative Example 2: Successfully infected chicks were selected and administered the traditional Chinese medicine composition prepared in Comparative Example 2 by gavage. Comparative Example 3: Selected successfully infected chicks were gavaged with the traditional Chinese medicine composition prepared in Comparative Example 3.

[0071] 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. Chicks were considered cured when the following clinical symptoms disappeared: refusal to eat, sensitivity to cold, huddled body, drooping wings, lethargy or drowsiness, excretion of white, viscous, pale yellow, or pale green loose feces, sometimes with the anus blocked by hardened fecal matter, and difficulty breathing. The cure rate is published below: Cure rate (%) = (Number of cured chicks / Total number of chicks) × 100%.

[0072] The results are shown in Table 4.

[0073] Table 4 Results of chick treatment in each group According to the results in Table 4, the number of diseased chicks in the challenge control group was 30. After treatment with only the principal herbs (Pulsatilla chinensis, Phellodendron chinense), assistant herbs (Scutellaria baicalensis, Portulaca oleracea), and adjuvant herbs (Pogostemon cablin, Atractylodes macrocephala, Glycyrrhiza uralensis), the number of diseased chicks decreased by 13, 8, and 3, respectively. However, after treatment with the traditional Chinese medicine composition for treating Salmonella pullorum prepared in this invention, the number of diseased chicks decreased by 28, and the cure rate was as high as 93.3%. Therefore, it can be seen that the principal herbs, assistant herbs, and adjuvant herbs of this invention have a synergistic effect in improving the cure rate of Salmonella pullorum.

[0074] 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 treating Salmonella pullorum disease in chickens, characterized in that, The raw materials include the following parts by weight: 2-4 parts of Pulsatilla chinensis, 3-5 parts of Phellodendron chinense, 2-4 parts of Scutellaria baicalensis, 2-4 parts of Portulaca oleracea, 2-4 parts of Pogostemon cablin, 2-4 parts of Atractylodes macrocephala, and 2-4 parts of Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials include the following parts by weight: 3 parts Pulsatilla chinensis, 4 parts Phellodendron chinense, 3 parts Scutellaria baicalensis, 3 parts Portulaca oleracea, 3 parts Pogostemon cablin, 3 parts Atractylodes macrocephala, and 3 parts Glycyrrhiza uralensis.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Includes the following steps: Mix Pulsatilla chinensis, Phellodendron chinense, Scutellaria baicalensis, Portulaca oleracea, Pogostemon cablin, Atractylodes macrocephala, and Glycyrrhiza uralensis to obtain a mixture; add water to the mixture and soak it, then decoct it twice over low heat. Filter after each decoction and collect the decoction; combine the two decoctions and concentrate them to a concentration of 0.5-1.5 g / mL to obtain a traditional Chinese medicine composition for treating Salmonella pullorum in chickens.

4. The preparation method according to claim 3, characterized in that, The mass-to-volume 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 Salmonella pullorum disease in chickens.

6. A product for treating Salmonella pullorum in chickens, characterized in that, The product includes the traditional Chinese medicine composition for treating Salmonella pullorum disease as described in claim 1 or 2.

7. The product for treating Salmonella pullorum disease in chickens according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

8. The product for treating Salmonella pullorum disease in chickens according to claim 7, characterized in that, Pharmaceutically acceptable excipients include disintegrants, lubricants, sweeteners, or binders.

9. The product for treating Salmonella pullorum disease in chickens according to claim 6, characterized in that, The product is available in decoction, soluble powder, or injection form.

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