A traditional Chinese medicine composition for treating broiler ascites syndrome

Abstract

The application discloses a traditional Chinese medicine composition for treating broiler ascites syndrome, which is composed of poria cocos, tangeret peel, radix scutellariae, radix astragali, plantago seed, ginger, saffron, and Terminalia chebula, and the weight ratio is (15-20):(10-15):(5-10):(30-35):(15-20):(9-15):(3-9):(9-15). The traditional Chinese medicine composition is obtained by screening traditional Chinese medicine compound based on saffron and Terminalia chebula, and is effective in treating broiler ascites syndrome, safe and effective, and can treat the disease without recurrence, and further excavates the medicinal value of traditional Tibetan medicine in treating diseases of poultry and livestock.

Description

Technical Field

[0001] This invention relates to the field of animal medicine, specifically to a traditional Chinese medicine composition for treating ascites syndrome in broilers. Background Technology

[0002] Broiler ascites syndrome (BAS), also known as broiler pulmonary hypertension syndrome, is a non-infectious, outbreak-prone disease caused by pulmonary hypertension in broilers under relatively hypoxic conditions, accompanied by varying degrees of pathological changes in parenchymal organs such as the heart, liver, lungs, and kidneys. This disease is a nutritional and metabolic disorder affecting rapidly growing broilers, primarily occurring in 4-7 week old broilers during the winter and spring seasons. Clinically, broilers develop the disease around 28 days of age due to various causes of hypoxia, and mortality increases before slaughter. In recent years, the incidence of this disease has been steadily rising, with epidemic reports from more than ten provinces and cities in my country. Globally, approximately seven billion broilers are affected by BAS annually, resulting in economic losses of up to tens of billions of dollars. Therefore, the development of effective drugs for the prevention and treatment of BAS has become an urgent global issue.

[0003] my country is a large, multi-ethnic country with a long history, and has developed a unique system for the application of natural medicines over thousands of years. Tibetan medicine is an important ethnic resource in my country, with extremely high medicinal research value. It is mainly produced in the Qinghai-Tibet Plateau and areas inhabited by Tibetans. It is rich in variety and is another great treasure trove of medicinal resources in my country. Representative drugs include saffron, Terminalia chebula, and Terminalia chebula.

[0004] Saffron, the dried stigma of *Crocus sativus* L. (family Iridaceae), is also known as Tibetan saffron. It has a sweet taste and slightly cold nature, and enters the heart and liver meridians. It possesses the effects of promoting blood circulation and removing blood stasis, clearing heat and detoxifying, promoting diuresis and dampness elimination, relieving depression and calming the mind, and has strong physiological activity. Studies have shown that saffron can reduce blood pressure elevation and aortic media thickness in hypertensive rats. Crocin, one of the extracts of saffron, also has antihypertensive and normalizing effects. Crocin has a conjugated polyene structure and exhibits good antioxidant activity. Other in vivo and in vitro studies have also demonstrated that crocin possesses antioxidant, anti-inflammatory, and vascular protective properties.

[0005] Terminalia chebula, the dried, ripe fruit of Terminalia chebula (family Cynodontaceae), is mainly distributed in Tibet and Yunnan provinces of my country. It has a sweet and astringent taste, is neutral in nature, and possesses properties such as clearing heat and dampness, astringing and nourishing blood, and harmonizing other medicinal herbs. It is a common medicinal and edible herb. Studies have shown that the tannins in Terminalia chebula have a protective effect against high-altitude pulmonary hypertension. The tannins in Terminalia chebula, such as ellagic acid, chebulic tannic acid, and corilagin, have various pharmacological activities including anti-inflammatory and antioxidant effects.

[0006] Traditional screening of traditional Chinese medicine (TCM) compound formulas requires numerous animal models, resulting in long experimental cycles, high costs, and difficulty in meeting the needs of TCM mechanism research. With the rapid development of emerging disciplines such as data mining and network pharmacology, big data and fundamental theoretical support have been provided for TCM veterinary medicine research. Applying data mining and network pharmacology techniques to BAS research can help us quickly and accurately discover new TCM compound formulas, providing new research ideas for disease treatment.

[0007] Patent application CN113559230A discloses a traditional Chinese medicine composition for the prevention and treatment of broiler ascites syndrome, which uses herbs such as Astragalus membranaceus, dried ginger, tangerine peel, and Poria cocos. Patent application CN108576473A discloses a chicken feed suitable for the prevention and treatment of broiler ascites syndrome, which includes herbs such as Astragalus membranaceus, tangerine peel, Plantago asiatica, and Poria cocos. However, there are no reports on the research into saffron and Terminalia chebula, or on the application of combining saffron and Terminalia chebula with other traditional Chinese medicines in a specific order to treat broiler ascites syndrome. Summary of the Invention

[0008] In view of the above-mentioned prior art, the purpose of this invention is to provide a traditional Chinese medicine composition for treating broiler ascites syndrome. Based on the antioxidant, anti-inflammatory, vascular protective, and pulmonary hypertension therapeutic effects of saffron and Terminalia chebula, this invention explores the therapeutic effect of modified Tibetan medicine formulas on broiler ascites syndrome. First, a basic traditional Chinese medicine formula was selected, and then this basic formula was combined with saffron and Terminalia chebula to obtain a Tibetan saffron diuretic formula for preventing and treating BAS.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a traditional Chinese medicine composition comprising Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica seed, Zingiber officinale, Saffron saffron, and Terminalia chebula in a weight ratio of (15-20):(10-15):(5-10):(30-35):(15-20):(9-15):(3-9):(9-15).

[0011] Preferably, the traditional Chinese medicine composition is composed of Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica seed, fresh ginger, saffron, and Terminalia chebula in a weight ratio of 15:10:5:30:15:9:3:9.

[0012] A second aspect of the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps:

[0013] Weigh each component according to the weight ratio, then add 8-12 times the weight of water and soak for 10-15 hours. Finally, decoct twice, 1-2 hours each time, combine the two decoctions, concentrate and dry to obtain the final product.

[0014] Preferably, the decocting temperature is 90-100℃.

[0015] A third aspect of the present invention provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for treating chicken ascites syndrome.

[0016] In a fourth aspect, the present invention provides a medicament for treating chicken ascites syndrome, wherein the above-mentioned traditional Chinese medicine composition is the active ingredient.

[0017] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0018] Preferably, the excipients include one or more of starch, dextrin, and citric acid.

[0019] Preferably, the drug is a tablet, powder, premix, granule, or oral liquid.

[0020] The beneficial effects of this invention are:

[0021] (1) Based on the antioxidant, anti-inflammatory, vascular protective and pulmonary hypertension effects of saffron and Terminalia chebula, this invention explores the therapeutic effect of Tibetan medicine modified formula on broiler ascites syndrome. The final Chinese medicine composition is reasonably matched, safe and effective, and treats both the symptoms and the root cause without recurrence.

[0022] (2) Based on saffron and Terminalia chebula, this invention obtains a Chinese medicine composition that can effectively treat ascites syndrome in broilers by screening Chinese medicine compound prescriptions, and further explores the medicinal value of traditional Tibetan medicine in the treatment of poultry and livestock diseases.

[0023] (3) This invention uses data mining and network pharmacology and other technologies to select new Chinese medicine compound prescriptions for the treatment of BAS in a highly efficient and accurate manner and explore their therapeutic effects, based on big data-based Chinese medicine compound prescriptions and BAS trial models. This provides information data support and theoretical basis for the rational use of drugs and the development of new prescriptions in the clinical treatment of BAS. Attached Figure Description

[0024] Figure 1Distribution of medicinal properties of Chinese herbs in an effective prescription for treating ascites syndrome in broilers;

[0025] Figure 2 Distribution diagram of Chinese medicinal ingredients in an effective prescription for treating ascites syndrome in broilers;

[0026] Figure 3 Distribution of Chinese herbal medicines by meridian tropism in an effective prescription for treating ascites syndrome in broilers;

[0027] Figure 4 Cluster diagram of variables in traditional Chinese medicine;

[0028] Figure 5 Diagram of traditional Chinese medicine composition formulas;

[0029] Figure 6 Venny diagram of active ingredient targets and disease targets in basic traditional Chinese medicine formulas;

[0030] Figure 7 A "drug-active ingredient-target" network for treating ascites syndrome in broiler chickens using a basic traditional Chinese medicine formula.

[0031] Among them: the purple part is the Chinese medicine monomer, the circular part is the effective component of the Chinese medicine monomer, and the rectangle is the target point for treating ascites syndrome in broilers;

[0032] Figure 8 : A PPI network of potential targets for the treatment of ascites syndrome in broiler chickens using traditional Chinese medicine basic formulations;

[0033] Among them: the redder the color, the higher the target ranking, and the more important the target;

[0034] Figure 9 Key targets of traditional Chinese medicine basic formula for treating ascites syndrome in broilers;

[0035] Figure 10 :GO Function Analysis Diagram;

[0036] Wherein: green represents biological processes, orange represents cellular components, and purple represents molecular functions;

[0037] Figure 11 KEGG enrichment analysis plot;

[0038] Among them: the larger the bubble area, the more genes are enriched; the redder the bubble color, the more significant the enrichment.

[0039] Figure 12 Molecular docking diagram;

[0040] Wherein: the red part is the small molecule ligand, and the purple part is the large molecule receptor residue;

[0041] Figure 13 Molecular docking binding energy;

[0042] Among them: the redder the color, the greater the binding energy and the stronger the docking activity;

[0043] Figure 14 Symptoms of broiler chicken necropsy;

[0044] Figure 15 : A diagram showing the morphology of broiler lungs;

[0045] Figure 16 Comparison of ascites and cardiac index in broiler chickens; different letters indicate significant differences between groups, P < 0.05;

[0046] Figure 17 Diagram of broiler lung tissue; red arrows indicate respiratory capillaries; black arrows indicate blood vessel walls; blue arrows indicate edema fluid; green arrows indicate red blood cells; yellow arrows indicate inflammatory cell infiltration.

[0047] Figure 18 Comparison chart of oxidative stress indicators in broiler chickens;

[0048] Wherein: (A) is the detection of malondialdehyde (MDA) content in broilers; (B) is the detection of superoxide dismutase (SOD) activity in broilers; (C) is the detection of glutathione peroxidase (GPX) activity in broilers; (D) is the detection of catalase (CAT) activity in broilers; different letters indicate significant differences between groups, P < 0.05;

[0049] Figure 19 Comparison of NO content in broiler chickens; different letters indicate significant differences between groups, P < 0.05;

[0050] Figure 20 : Expression level of IL-6 protein in broiler lungs; different letters indicate significant differences between groups, P < 0.05;

[0051] Figure 21 : Comparison of broiler weights; different letters indicate significant differences between groups, P < 0.05. Detailed Implementation

[0052] 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.

[0053] As mentioned earlier, the incidence of broiler ascites syndrome (BAS) is on the rise, with epidemic reports from more than ten provinces and cities in my country. Globally, approximately seven billion broilers are affected by BAS annually, resulting in economic losses of up to tens of billions of US dollars. Therefore, developing effective drugs to prevent and treat BAS has become an urgent global issue.

[0054] Based on this, the present invention uses Tibetan medicines saffron and Terminalia chebula as the basis, and through scientific formulation, obtains a traditional Chinese medicine composition that can effectively treat ascites syndrome in broilers.

[0055] The inventors first learned through literature review that saffron can lower blood pressure and aortic membrane thickness in hypertensive rats, and Terminalia chebula also has a protective effect against high-altitude pulmonary hypertension. Therefore, the inventors first selected saffron and Terminalia chebula as components of the traditional Chinese medicine composition. Next, using keywords such as "broiler ascites syndrome" and "traditional Chinese medicine compound," the inventors searched and organized academic journals, dissertations, and other literature from the past ten years, selecting 128 traditional Chinese medicine prescriptions and obtaining 164 traditional Chinese medicines. Finally, the inventors analyzed the frequency, properties, and flavors of the 164 traditional Chinese medicines, and combined association analysis and cluster analysis methods, according to the principle of "principal, assistant, adjuvant, and guide," efficiently and accurately screened out the basic traditional Chinese medicine formula for symptomatic treatment of BAS, namely Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, Zingiber officinale, saffron, and Terminalia chebula, where the principal herbs are Poria cocos and saffron, the assistant herbs are Citrus reticulata peel and Terminalia chebula, the adjuvant herbs are Scutellaria baicalensis, Astragalus membranaceus, and Plantago asiatica, and the guide herb is Zingiber officinale.

[0056] 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.

[0057] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0058] The L-arginine used in this invention was purchased from Shandong Hongxin Biotechnology Co., Ltd., the MDA, SOD, GPX, NO and CAT combination kits were purchased from Jiangsu Beyotime Biotechnology Co., Ltd., China, the ELISA combination kits were purchased from Shanghai Jinma Biotechnology Co., Ltd., and the hematoxylin-eosin staining solution was purchased from Jiangsu Adison Biotechnology Co., Ltd.

[0059] Example 1: Screening of basic components of traditional Chinese medicine composition.

[0060] 1. Data Mining

[0061] (1) Searching on domestic and international websites such as CNKI, Wanfang Data Knowledge Service Platform, VIP Chinese Journal Service Platform, PubMed, NCBI and Sci-hub using keywords such as "broiler ascites syndrome", "Chinese herbal compound", "Ascites Syndrome" and "Plumonary Hypertension syndrome", we sorted out the academic journals, dissertations and other literature published from 2010 to 2022, and screened the relevant prescriptions to obtain 128 Chinese herbal prescriptions, which contain 164 Chinese herbs. According to the 2020 edition of "Pharmacopoeia of the People's Republic of China for Veterinary Medicine" and "Chinese Materia Medica", we standardized the names of the 164 Chinese herbs in the prescriptions and established a Chinese herbal compound database.

[0062] (2) The names of the 164 Chinese herbal medicines contained in the prescription were standardized according to the 2020 edition of the "Pharmacopoeia of Veterinary Medicine of the People's Republic of China" and "Chinese Materia Medica", and a database of Chinese herbal medicine compound prescriptions was established.

[0063] (3) Data Analysis

[0064] The frequency of drugs in 128 prescriptions was statistically analyzed using an Excel spreadsheet, and the results are shown in Table 1.

[0065] Table 1. Distribution of Chinese herbal medicines used more than 10 times in effective prescriptions for treating ascites syndrome in broilers.

[0066]

[0067]

[0068] Next, based on the 2020 edition of the *Veterinary Pharmacopoeia of the People's Republic of China*, the medicinal properties, flavors, meridian tropism, and efficacy of each herb were statistically analyzed. Association rule analysis and cluster analysis were then performed based on drug frequency. The results are shown below. Figures 1-3 .

[0069] exist Figures 1-3 Based on this, and combined with association analysis and cluster analysis methods, the data in Tables 2 and 3 were obtained.

[0070] Table 2. Prescription Combination Patterns of Two Traditional Chinese Medicine Herbs

[0071]

[0072] Table 3. Prescription Combination Patterns of Three Chinese Herbal Medicines

[0073]

[0074] The results were analyzed and a new prescription was developed, resulting in the basic formula: Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, and Zingiber officinale.

[0075] 2. Network pharmacology

[0076] (1) Screening of effective drug components

[0077] The chemical components of "Poria cocos", "Citrus reticulata peel", "Scutellaria baicalensis", "Astragalus membranaceus", "Plantago asiatica", and "Zingiber officinale" were obtained by searching the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and the Encyclopedia of Traditional Chinese Medicine (ETCM) databases, respectively. Then, the TCMSP database was used to screen for components based on oral bioavailability (OB) ≥ 30% and drug-likeness ratio (DL) ≥ 0.18. Components collected from the ETCM database were screened based on their drug-likeness ratio, categorized as medium or good. After removing duplicate components, the finally qualified active ingredients were obtained. The results are shown in Table 4.

[0078] Table 4. Active ingredients of Chinese herbs in the basic formula

[0079]

[0080]

[0081] (2) Collection of active ingredients and disease-related targets

[0082] Active ingredients were standardized using the PubChem database to obtain their PubChem ID and SMILEs (Simplified Molecular Input Line Entry System). These SMILEs were then imported into the SwissTargetPrediction database for target prediction. Searches were conducted in the Human Genome Database (GeneCard, https: / / www.genecards.org / ) and the Online Mendelian Inheritance Database (OMIM, http: / / omim.org / ) using the keywords "Ascites Syndrome" and "Broiler Ascites Syndrome," respectively, to collect relevant disease targets.

[0083] (3) Acquisition of potential targets

[0084] Using VENNY 2.1, the intersection of the targets of the above-mentioned active pharmaceutical ingredients and the targets of the disease was obtained and visualized, and a Venn diagram was drawn. Figure 6 ), and identified 166 potential drug targets for treating diseases.

[0085] (4) Construction of the “Drug-Active Ingredient-Potential Target” Network

[0086] Create Network and Tape files to map the active ingredients and traditional Chinese medicines corresponding to potential targets, construct a "drug-active ingredient-potential target" network, and import it into Cytoscape 3.9.1 software for visualization. Figure 7 Meanwhile, the CytoHubba plugin was used to analyze its centrality using the MCC algorithm (Maximum Clique Centrality) to obtain the main active ingredients. Finally, the main active ingredients of the basic traditional Chinese medicine formula were obtained, including scutellarin, scutellarin, gingerol, epiberberine, baicalin, and isootropin.

[0087] (5) PPI network construction

[0088] Potential targets were imported into the STRING database to construct a protein-protein interaction network. The species "Gallus gallus" was selected, and a minimum combined score > 0.4 was used for screening. Other parameters were left at their default values, and free targets were excluded. The TSV format files were then downloaded and imported into Cystscope 3.9.1 for visualization. The results are shown below. Figures 8-9 The core targets were selected based on the maximum clique centrality of the nodes, resulting in core targets such as IL-6, STAT3, SRC, and BCL2.

[0089] (6) GO enrichment analysis and KEGG pathway enrichment analysis of target sites

[0090] Intersecting targets of drugs and diseases were imported into the DAVID database, and relevant files were downloaded. Enrichment results were obtained by screening with P < 0.01 and FDR < 0.05, and the results were visualized using online bioinformatics tools. (See attached image). Figures 10-11 We obtained 68 GO enriched entries and 27 KEGG pathways, among which the MAPK signaling pathway had the most enriched targets, followed by the FOXO signaling pathway.

[0091] Based on the analysis of the above core targets, it is predicted that the basic formulation of traditional Chinese medicine mainly plays a key role in BAS by acting on the three core targets of IL-6, STAT3 and FOXO3a in the FOXO signaling pathway through components such as scutellaria baicalensis flavonoids, gingerol flavonoids and scutellaria baicalensis flavonoids.

[0092] 3. Molecular docking verification

[0093] To verify the accuracy of the core targets identified by network pharmacology screening, molecular docking was used for validation. Predicted core targets were paired with active ingredients in the basic formulation (molecular docking was performed; hydrogen bonds were formed between the main active ingredients and the targets, with binding energies less than -2 kcal·mol⁻¹ (except for 5-Hydroxy-3,7,8,2'-tetramethoxyflavone and FOXO3a) to obtain the action fraction. The 3D structures of the active ingredients were downloaded from PubChem, and the target protein structures were obtained from the PDB protein structure database. The protein structures were dehydrated using Pymol to remove other linking molecules, and then docking between the molecules and targets was performed using Autodock software. The molecular docking results were visualized using Pymol. The results are shown in [Figure number missing]. Figures 12-13 .

[0094] Figures 12-13 This indicates that there is good binding activity between the predicted core targets and active ingredients, suggesting that the basic formulation obtained from data mining may play a key role in BAS by acting on the three core targets of IL-6, STAT3 and FOXO3a through the main active ingredients such as baicalein, gingerol, and baicalein.

[0095] Example 2: Investigation of the prescription ratio

[0096] To investigate the optimal ratio between the drugs, a uniform design experiment was conducted in this embodiment.

[0097] Thirty 1-day-old Ross broiler chickens (male or female) were raised to 14 days old and randomly divided into 6 groups (AF group, 5 chickens per group). The specific grouping and medication ratios are shown in Table 5.

[0098] Table 5 Grouping and Drug Ratio

[0099]

[0100] Based on the single-factor screening, six factors—Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, and Zingiber officinale—were selected as the test factors for uniform design. Each factor was set with six levels. The experimental design was carried out using a 6-factor, 6-level U66 uniform design table (Table 6), and the uniform design experimental groups were obtained. The drugs were then prepared according to the proportions in Table 5.

[0101] Table 6. Uniform Experiment: 6 Factors and 6 Levels

[0102]

[0103] A 6-factor, 6-level U66 uniform design was used to establish a BAS (Breakfast Ascites Syndrome) experimental model through high-salt drinking water and a high-energy diet. The ascites-cardiac index (AHI) was measured at 35 days. The method for measuring the broiler ascites-cardiac index (AHI) was as follows: the broiler was dissected and the heart was separated. The left and right atria were removed along the coronary sulcus, and excess fat and blood clots were removed. The weight of the entire ventricle was recorded as TV. The right ventricle was removed along the anterior and posterior longitudinal grooves and weighed, recorded as RV. The ascites-cardiac index was calculated to determine the occurrence of broiler ascites syndrome. An AHI > 0.25, with ascites in the abdominal cavity, indicated that the broiler had broiler ascites syndrome. The AHI = Right Ventricular Weight (RV) / Total Ventricular Weight (TV).

[0104] The optimal compound ratio was analyzed using the quadratic polynomial stepwise regression method, and the results are shown in Table 7-10.

[0105] Table 7. The therapeutic effects of different proportions of the new traditional Chinese medicine compound on ascites syndrome in broiler chickens.

[0106]

[0107] Table 8. Regression coefficient tests for each regression term.

[0108]

[0109] Note: The P-value represents the significance of the experimental result. P < 0.05 indicates that the result is significant, and P < 0.01 indicates that the result is highly significant.

[0110] Table 9. Observed values, fitted values, and fitting errors of the formulation.

[0111]

[0112] Table 10 Results of Uniform Design Experiment

[0113]

[0114] Note: Correlation coefficient R = 0.999993; F-value = 17176.795208; Df = (4,1); P-value = 0.0057; residual standard deviation S = 0.000082; adjusted correlation coefficient Ra = 0.999964

[0115] The results showed that the optimal therapeutic effect was achieved when the basic formula ratio was Poria cocos: Citrus reticulata peel: Scutellaria baicalensis: Astragalus membranaceus: Plantago asiatica seed: Ginger = 15:10:5:30:15:9.

[0116] Based on the basic formulation, following the dosage ranges in Part II of the *Pharmacopoeia of the People's Republic of China for Veterinary Medicine (2020 Edition)*, and adhering to the principle of "principal, assistant, adjuvant, and guide" in drug compatibility, the ratio of Poria cocos: Citrus reticulata peel: Scutellaria baicalensis: Astragalus membranaceus: Plantago asiatica seed: Zingiber officinale: Saffron: Terminalia chebula was determined to be 15:10:5:30:15:9:3:9, resulting in a traditional Chinese medicine composition capable of treating ascites syndrome in broiler chickens. The formula diagram of the traditional Chinese medicine composition is shown below. Figure 5 .

[0117] Example 3: Preparation of traditional Chinese medicine composition.

[0118] Poria cocos, tangerine peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, ginger, saffron, and Terminalia chebula were mixed in a weight ratio of 15:10:5:30:15:9:3:9. Then, 8 times the weight of water was added and the mixture was soaked for 10 hours. Finally, the mixture was decocted twice at 100°C for 1 hour each time. The two decoctions were combined, concentrated, and dried to obtain the traditional Chinese medicine composition.

[0119] Example 4: Preparation of traditional Chinese medicine composition.

[0120] Poria cocos, tangerine peel, scutellaria baicalensis, astragalus membranaceus, plantain seed, ginger, saffron, and chebula were mixed in a weight ratio of 18:12:8:32:18:10:6:12. Then, 12 times the weight of water was added and the mixture was soaked for 15 hours. Finally, the mixture was decocted twice at 95°C for 2 hours each time. The two decoctions were combined, concentrated, and dried to obtain the traditional Chinese medicine composition.

[0121] Example 5: Preparation of traditional Chinese medicine composition.

[0122] Poria cocos, tangerine peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, ginger, saffron, and Terminalia chebula were mixed in a weight ratio of 20:15:10:35:20:15:9:15. Then, 10 times the weight of water was added and the mixture was soaked for 12 hours. Finally, the mixture was decocted twice at 100°C for 1.5 hours each time. The two decoctions were combined, concentrated, and dried to obtain the traditional Chinese medicine composition.

[0123] Example 6: Preparation of the basic formulation.

[0124] Poria cocos, tangerine peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, and ginger were mixed in a weight ratio of 15:10:5:30:15:9. Then, 8 times their weight of water were added and the mixture was soaked for 10 hours. Finally, the mixture was decocted twice at 100°C for 1 hour each time. The two decoctions were combined, concentrated, and dried to obtain the basic formula of traditional Chinese medicine.

[0125] Comparative Example 1: Preparation of Traditional Chinese Medicine Composition.

[0126] Poria cocos and saffron were mixed in a weight ratio of 15:3, then soaked in 8 times their weight of water for 10 hours. Finally, the mixture was decocted twice at 100°C for 1 hour each time. The two decoctions were combined, concentrated, and dried to obtain the traditional Chinese medicine composition.

[0127] Comparative Example 2: Preparation of Traditional Chinese Medicine Composition.

[0128] The dried tangerine peel and Terminalia chebula were mixed in a weight ratio of 10:9, then soaked in 8 times their weight of water for 10 hours. Finally, the mixture was decocted twice at 100°C for 1 hour each time. The two decoctions were combined, concentrated and dried to obtain the traditional Chinese medicine composition.

[0129] Comparative Example 3: Preparation of Traditional Chinese Medicine Composition.

[0130] Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica and ginger were mixed in a weight ratio of 5:30:15:9, then soaked in 8 times their weight of water for 10 hours. Finally, the mixture was decocted twice at 100°C for 1 hour each time. The two decoctions were combined, concentrated and dried to obtain the traditional Chinese medicine composition.

[0131] Experimental Example 1: Animal Experiment Verification.

[0132] 1. Experimental Grouping

[0133] Seventy 14-day-old Ross broiler chickens of similar weight were selected for the experiment. A blank control group, a model group, a positive control group, and experimental groups 1-4 were established. Ten chickens were randomly assigned to each group for the feeding experiment. The grouping and feeding methods are as follows:

[0134] Control group: Normal drinking water, basal feed;

[0135] Model group: Drinks saline solution with sodium ions of 0.12% by mass, and lard and fish oil are added to the basic feed; the amount of lard added is 3% of the weight of the basic feed, and the amount of fish oil added is 4% of the weight of the basic feed.

[0136] Experimental group 1 (low): Drinking saline solution with sodium ions of 0.12% by mass, with lard, fish oil and the basic formula prepared in Example 6 added to the basic feed, with 1.25g of the basic formula added per kg of basic feed;

[0137] Experimental Group 2 (medium): Drinking saline solution with sodium ions of 0.12% by mass, with lard, fish oil and the basic formula prepared in Example 6 added to the basic feed. The amount of lard added was 3% of the weight of the basic feed, and the amount of fish oil added was 4% of the weight of the basic feed; 2.5g of the basic formula was added per kg of basic feed.

[0138] Experimental group 3 (high): Drink saline solution with sodium ions of 0.12% by mass. The basic feed is supplemented with lard, fish oil and the basic formula prepared in Example 6. The amount of lard added is 3% of the weight of the basic feed and the amount of fish oil added is 4% of the weight of the basic feed. 5g of the basic formula is added per kg of basic feed.

[0139] Experimental group 4 (medium control): The diet consisted of drinking saline solution with a sodium ion content of 0.12% by mass. The basic feed was supplemented with lard, fish oil and the traditional Chinese medicine composition prepared in Example 3. The amount of lard added was 3% of the weight of the basic feed and the amount of fish oil added was 4% of the weight of the basic feed. 2.5g of the traditional Chinese medicine composition was added per kg of basic feed.

[0140] Positive control group (L-Arg): Drink saline solution with sodium ions of 0.12% by mass. Lard, fish oil and L-arginine were added to the feed. The amount of lard added was 3% of the weight of the basic feed, the amount of fish oil added was 4% of the weight of the basic feed, and 10g of L-arginine was added per kg of basic feed.

[0141] Free access to feed and water. The basic feed consists of the following components by weight: 15 parts corn, 10 parts wheat bran, 10 parts soybean meal, 5 parts grass meal, 5 parts wheat middlings, 3 parts oil, 3 parts limestone powder, 2 parts dicalcium phosphate, 0.5 parts sodium chloride, 0.5 parts lysine, and 0.5 parts methionine.

[0142] 2. Sample Collection

[0143] At 35 days and 42 days, five broiler chickens were randomly selected from each group, and blood and tissue samples were collected, as follows:

[0144] Blood sample collection: Collect 5 mL of blood from the wing vein, centrifuge at 4℃ and 3000 rpm for 10 min, collect serum, and store at -80℃ for later use.

[0145] Tissue specimen collection: After blood collection, the broiler chickens were euthanized by exsanguination through the carotid artery. The heart and lungs were separated and rinsed thoroughly with pre-cooled PBS. The right ventricle and the entire ventricle were isolated and weighed separately. A portion of lung tissue was fixed in 4% paraformaldehyde solution for section preparation and observation of pathological changes.

[0146] During the dissection, the symptoms of the blank group and the model group were compared (see...). Figure 14 The results show that the model group of broilers had a large amount of ascites, while the blank group did not have ascites, indicating that the experimental modeling was successful.

[0147] Compare the morphological changes of the lungs in each component (see Figure 15 It can be seen that the lung tissue structure of the model group broilers is abnormal, the blood vessel walls are significantly thickened, and there is inflammatory cell infiltration, while the lungs of the other group broilers are normal in size and shape.

[0148] 3. Determination of Ascites-Heart Index (AHI) in Broilers

[0149] After separating the heart, the left and right atria are cut off along the coronary sulcus, and excess fat and blood clots are removed. The weight of the entire ventricle is recorded as TV. The right ventricle is cut off along the anterior and posterior longitudinal grooves and weighed, recorded as RV. The ascites cardiac index is calculated to determine the occurrence of broiler ascites syndrome. An AHI > 0.25, with ascites in the abdominal cavity, indicates that the broiler has broiler ascites syndrome.

[0150] Ascites-related cardiac index (AHI) = Right ventricular weight (RV) / Total ventricular weight (TV)

[0151] See results Figure 16 Based on the anatomical observation, it was found that the broiler chickens in the model group suffered from ascites syndrome, while the other experimental groups did not suffer from ascites syndrome.

[0152] 4. Observation of lung morphological changes by HE staining

[0153] Fresh lung tissue was transversely sectioned along the left hilum and fixed with 4% paraformaldehyde. Following standard paraffin sectioning methods, lung sections approximately 4 μm thick were prepared and stained with hematoxylin and eosin. Pathological changes in the lungs were observed, and the results are shown below. Figure 17 .

[0154] Depend on Figure 17 It can be seen that the lung tissue of the model group showed structural abnormalities, significant thickening of the blood vessel walls, and infiltration of inflammatory cells; compared with the model group, the blood vessel wall thickness of the other broiler groups was reduced, and the infiltration of inflammatory cells was alleviated.

[0155] 5. Detection of oxidative stress indicators in broilers

[0156] (1) Take out the serum and tissue homogenate supernatant from -80℃ and place them in a refrigerator at 4℃ until the samples are gradually thawed for use.

[0157] (2) Test the tissue samples according to the instructions of the MDA, SOD, GPX and CAT combination kits.

[0158] See results Figure 18 Compared with the control group, the MDA level in the model group of broilers was significantly increased, while the SOD, CAT and GPX levels were significantly decreased. Compared with the model group, the MDA level in the positive control group, the medium-dose control group and the low, medium and high dose groups of broilers was significantly decreased, while the SOD, CAT and GPX levels were increased, with the medium-dose group showing the most significant effect.

[0159] 6. Detection of NO content in broiler chickens

[0160] (1) Take the serum sample out of -80℃ and place it in the refrigerator at 4℃ until the sample is gradually thawed for use.

[0161] (2) Serum samples were tested according to the instructions of the NO combination kit.

[0162] See results Figure 19 Compared with the control group, the NO level in the model group of broilers was significantly increased, while the NO level in the other experimental groups of broilers was significantly decreased.

[0163] 7. Enzyme-linked immunosorbent assay (ELISA)

[0164] (1) Take the tissue homogenate supernatant out of -80℃ and place it in a refrigerator at 4℃ until the sample is gradually thawed for later use.

[0165] (2) Test the tissue sample according to the instructions of the ELISA kit.

[0166] See results Figure 20 Compared with the control group, the expression level of IL-6 protein in the model group of broilers was significantly increased, while the expression level of IL-6 protein in the other experimental groups of broilers was decreased.

[0167] 8. Broiler weight measurement

[0168] The health status of the broilers was observed and recorded. Every 7 days, the weight of the broilers was measured and recorded in groups. Statistical analysis was then performed on the broiler weight at each stage. The results are shown in [Table missing]. Figure 21 It can be seen that the weight of the broilers in the model group was significantly lower than that of the broilers in other experimental groups.

[0169] The above experiments show that the basic herbal formula selected in this invention can effectively treat ascites syndrome in broilers, and the optimal dosage of the basic formula, 2.5 g / kg, was obtained. Furthermore, based on the data from the above experiments, it is evident that, compared to the basic formula, the addition of saffron and Terminalia chebula according to the principles of monarch-minister-assistant-guide compatibility results in a more effective treatment of ascites syndrome in broilers.

[0170] Experimental Example 2:

[0171] Based on Experiment 1, in order to verify the relationship between the principal, assistant, adjuvant, and guide components of the obtained traditional Chinese medicine composition, the inventors conducted further experiments.

[0172] (1) Test methods

[0173] Fifty 14-day-old Ross broiler chickens of similar weight were randomly divided into 5 groups. The grouping method is as follows:

[0174] Control group: Drink salt water, and add lard and fish oil to feed;

[0175] Experimental group: Drinking saline solution, and fed with lard, fish oil and the traditional Chinese medicine composition prepared in Example 3;

[0176] Control group 1: Drink saline solution, and feed containing lard, fish oil and the traditional Chinese medicine composition prepared in Comparative Example 1;

[0177] Control group 2: Drink saline solution, and feed containing lard, fish oil and the traditional Chinese medicine composition prepared in Comparative Example 2;

[0178] Control group 3: Drink saline solution, and add lard, fish oil and the traditional Chinese medicine composition prepared in control group 3 to feed.

[0179] Free access to feed and water was provided. At 35 days of age, 5 broilers from each group were randomly selected, and their AHI was calculated using the same method as in Experiment 1. The average value of the results from each group was taken.

[0180] (2) Test Results

[0181] Table 11 AHI values ​​of broilers in each group

[0182] Blank group 0.28 Experimental group 1 0.15 Control group 1 0.23 Control group 2 0.24 Control group 3 0.23

[0183] The data above shows that the AHI index of the blank group is greater than 0.25, while the AHI of the experimental group and experimental group 2 are both less than 0.25. This indicates that the traditional Chinese medicine composition provided by the present invention can effectively treat ascites syndrome in broilers, and that the components of the "principal, assistant, adjuvant and guide" have a synergistic effect.

[0184] 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 ascites syndrome in broiler chickens, characterized in that, The traditional Chinese medicine composition for treating ascites syndrome in broilers is made from Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, Zingiber officinale, Saffron saffron, and Terminalia chebula in a weight ratio of (15-20):(10-15):(5-10):(30-35):(15-20):(9-15):(3-9):(9-15).

2. The traditional Chinese medicine composition for treating ascites syndrome in broilers according to claim 1, characterized in that, The traditional Chinese medicine composition for treating ascites syndrome in broilers is made from Poria cocos, Citrus reticulata peel, Scutellaria baicalensis, Astragalus membranaceus, Plantago asiatica, Zingiber officinale, Saffron saffron, and Terminalia chebula in a mass ratio of 15:10:5:30:15:9:3:

9.

3. The method for preparing the traditional Chinese medicine composition for treating ascites syndrome in broilers according to claim 1, characterized in that, Includes the following steps: Weigh each component according to the weight ratio, then add 8-12 times the weight of water and soak for 10-15 hours. Finally, decoct twice, 1-2 hours each time, combine the two decoctions, concentrate and dry to obtain the final product.

4. The method for preparing the traditional Chinese medicine composition for treating ascites syndrome in broilers according to claim 3, characterized in that, The temperature for simmering is 90-100℃.

5. The use of the traditional Chinese medicine composition for treating ascites syndrome in chickens according to claim 1 in the preparation of a medicament for treating ascites syndrome in chickens.

6. A drug for treating ascites syndrome in chickens, characterized in that, The traditional Chinese medicine composition for treating ascites syndrome in broilers, as described in claim 1, is the sole active ingredient.

7. The medicament for treating chicken ascites syndrome according to claim 6, characterized in that, The drug is made from the traditional Chinese medicine composition for treating ascites syndrome in broilers as described in claim 1 and pharmaceutically acceptable excipients.

8. The medicament for treating chicken ascites syndrome according to claim 6, characterized in that, The drug is in the form of tablets, powders, premixes, granules, or oral liquids.

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