A traditional Chinese medicine composition for treating colibacillosis of chicken and a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for treating chicken colibacillosis and a preparation method and application thereof, and belongs to the technical field of traditional Chinese veterinary medicine. The application discloses a traditional Chinese medicine composition for treating chicken colibacillosis, which is composed of dandelion, radix drimydis, radix astragali, radix atractylodis macrocephalae, poria cocos and liquorice. 6 ) orthogonal test, and the best weight ratio is obtained by optimizing the diameter of the bacteriostatic circle as an index. In-vivo efficacy test shows that the 7-day survival rate of the composition reaches 85% for the O2:K1 infection of chicken colibacillosis, which is significantly higher than 55% of the model group (P<0.05). The composition can reduce pericarditis and perihepatitis, reduce the indexes of heart, liver and lung, increase the indexes of thymus and bursa of fabricius, reduce the bacterial load in blood and liver, reduce the levels of TNF-alpha, IL-1beta, IL-6, LPS and PCT in serum, increase the level of IL-10, increase the activities of SOD, CAT and GSH-Px, and reduce the content of MDA. The application has scientific proportion and definite curative effect, and can be used for preparing medicines for treating chicken colibacillosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese veterinary medicine technology, specifically to a traditional Chinese medicine composition for treating chicken colibacillosis, its preparation method, and its application. Background Technology

[0002] Avian colibacillosis is an acute or chronic bacterial infectious disease caused by avian pathogenic Escherichia coli (APEC). Clinically, it mainly manifests as septicemia, pericarditis, perihepatitis, and air sacculitis, and is one of the most serious bacterial diseases threatening poultry farming. Currently, prevention and control mainly rely on antibiotics. However, due to the numerous APEC serotypes and the long-term irrational use of antibiotics leading to the emergence of multidrug-resistant strains, the efficacy of traditional antibiotics has significantly decreased. Vaccine immunization, due to large serotype differences and limited cross-protection, is insufficient to meet clinical needs. Therefore, developing safe, effective, and drug-resistant traditional Chinese veterinary medicine preparations is of significant clinical importance.

[0003] Traditional Chinese medicine (TCM) compound formulas are characterized by multiple components and multiple targets, showing advantages in antibacterial, anti-inflammatory, and immunomodulatory effects. Dandelion, Mahonia japonica, Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis are all commonly used Chinese veterinary medicines, and modern pharmacological studies have confirmed their antibacterial, anti-inflammatory, antioxidant, and immune-enhancing activities. This invention's compound formula is modified from the classic formula "Si Jun Zi Tang" (Four Gentlemen Decoction), targeting the pathogenesis of chicken colibacillosis characterized by "damp-heat accumulation and intense heat toxicity." Dandelion and Mahonia japonica are used as the principal herbs for clearing heat and detoxifying; Astragalus membranaceus is used as the assistant herb to tonify qi and consolidate the exterior; Atractylodes macrocephala and Poria cocos are used as adjuvant herbs to strengthen the spleen and eliminate dampness; and Glycyrrhiza uralensis is used as the guiding herb to harmonize the other herbs. However, existing TCM compound formulas are mostly based on empirical compatibility, lacking systematic ratio optimization and rigorous efficacy verification, leading to unstable product quality and uncertain efficacy. Therefore, developing a scientifically formulated TCM composition with clear efficacy is a pressing technical problem to be solved in this field.

[0004] The above background information is provided only to aid in understanding the inventive concept and technical solution of this invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above information was disclosed on the filing date of this patent application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine composition for treating chicken colibacillosis, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A traditional Chinese medicine composition for treating chicken colibacillosis is made from the following raw materials in parts by weight: 14-26 parts of dandelion, 7-13 parts of Mahonia fortunei, 9-15 parts of Astragalus membranaceus, 7-13 parts of Atractylodes macrocephala, 4-8 parts of Poria cocos, and 6-10 parts of Glycyrrhiza uralensis.

[0008] Preferably, the traditional Chinese medicine composition for treating chicken colibacillosis is made from the following raw materials in parts by weight: 20-26 parts of dandelion, 10-13 parts of Mahonia fortunei, 12-15 parts of Astragalus membranaceus, 7-10 parts of Atractylodes macrocephala, 6-8 parts of Poria cocos, and 6-8 parts of Glycyrrhiza uralensis.

[0009] Preferably, the traditional Chinese medicine composition for treating chicken colibacillosis is made from the following raw materials in parts by weight: 26 parts dandelion, 13 parts Mahonia fortunei, 15 parts Astragalus membranaceus, 7 parts Atractylodes macrocephala, 6 parts Poria cocos, and 6 parts Glycyrrhiza uralensis.

[0010] Preferably, the dosage form of the traditional Chinese medicine composition includes one or more of the following: decoction, powder, granules, oral liquid, capsule, and tablet.

[0011] The present invention also provides a method for preparing a traditional Chinese medicine composition for treating chicken colibacillosis, comprising the following steps: soaking the raw material in 8-15 times the amount of water for 1-3 hours, boiling and then simmering for 30-60 minutes, and filtering; adding 6-12 times the amount of water to the residue and simmering for another 30-60 minutes, and filtering; adding 4-10 times the amount of water to the residue and simmering for another 20-50 minutes, and filtering; combining the three filtrates and concentrating under reduced pressure to a raw drug concentration of 0.2-1.0 g / mL.

[0012] Preferably, the preparation method of the traditional Chinese medicine composition for treating chicken colibacillosis includes the following steps: soaking the raw material in 12 times the amount of water for 2 hours, boiling and then simmering for 45 minutes, and filtering; adding 10 times the amount of water to the residue and simmering for another 45 minutes, and filtering; adding 8 times the amount of water to the residue and simmering for another 45 minutes, and filtering; combining the three filtrates and concentrating under reduced pressure to a raw drug concentration of 0.25-1.0 g / mL.

[0013] Preferably, the optimal dosage is 0.5 g / mL of crude drug.

[0014] The present invention also provides an application of a traditional Chinese medicine composition for treating chicken colibacillosis.

[0015] Preferably, the chicken colibacillosis is caused by infection with avian pathogenic Escherichia coli.

[0016] Preferably, the traditional Chinese medicine composition exerts its therapeutic effect through one or more of the following mechanisms: improving survival rate, reducing heart / liver / lung index, restoring thymus / bursa of Fabricius index, reducing blood and liver bacterial load, reducing serum TNF-α / IL-1β / IL-6 / LPS / PCT levels, increasing IL-10 levels, increasing SOD / CAT / GSH-Px activity, reducing MDA content, and alleviating pericarditis or perihepatitis.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The proportions have been optimized by the system. L18(3) is used. 6 An orthogonal experiment was conducted, using the diameter of the inhibition zone as an indicator, to investigate the antibacterial effects of different dosage combinations of six medicinal materials. Range analysis was used to determine the order of importance of each factor and the optimal ratio, overcoming the blindness of traditional empirical compatibility.

[0019] 2. Significant in vivo efficacy. In the APEC O2:K1-infected chick model, the 7-day survival rate of the treatment group treated with the medium dose (500 mg crude drug / mL) of the composition of the present invention reached 85%, which was significantly higher than the 55% of the model group (P<0.05). Moreover, the medium dose was more effective than the high and low doses, showing a non-linear dose-response relationship.

[0020] 3. Multi-target comprehensive regulation. The composition of this invention can reduce the indices of the heart, liver, and lungs, and increase the indices of the thymus and bursa of Fabricius; reduce the bacterial load in the blood and liver; reduce serum TNF-α, IL-1β, IL-6, LPS, and PCT levels, and increase IL-10 levels; increase the activity of SOD, CAT, and GSH-Px, and reduce MDA content; and alleviate the pathological damage of pericarditis and perihepatitis.

[0021] 4. Good safety profile. All raw materials used are commonly used traditional Chinese veterinary medicines listed in the Chinese Veterinary Pharmacopoeia, and no obvious toxic side effects were observed during the trial. Attached Figure Description

[0022] Figure 1 This image shows the therapeutic effect of the herbal composition of this invention on APEC-infected chicks. A is a flowchart of the animal experiment; B is a 7-day survival rate curve of chicks in each group; C is a gross lesion photograph of the heart and liver; D is an HE-stained image of heart tissue (200×); E is an HE-stained image of liver tissue (200×).

[0023] Figure 2 This is a graph showing the effect of the herbal composition of the present invention on serum indicators of APEC-infected chicks. In the graph, A represents blood bacterial load; B represents liver bacterial load; C represents serum TNF-α level; D represents serum IL-1β level; E represents serum IL-6 level; F represents serum IL-10 level; G represents serum LPS level; and H represents serum PCT level.

[0024] Figure 3 The figure shows the effect of the traditional Chinese medicine composition of this invention on immunoglobulins and antioxidant indicators in APEC-infected chicks. In the figure, A represents serum IgY level; B represents serum IgM level; C represents serum MDA content; D represents serum SOD activity; E represents serum CAT activity; and F represents serum GSH-Px activity. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments and the accompanying drawings. It should be emphasized that the following description is merely exemplary and is not intended to limit the scope or application of the present invention.

[0026] Screening Experiment 1

[0027] 1.1 Materials

[0028] APEC O2:K1 (TW-XM strain), provided by Professor Zhu Guoqiang of Yangzhou University. Dandelion, Mahonia japonica, Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, and Glycyrrhiza uralensis were purchased from Xiangjun Pharmacy in Nanning, Guangxi, and were identified as meeting the pharmacopoeia standards.

[0029] 1.2 Factor Levels

[0030] Dandelion (A), Mahonia (B), Astragalus (C), Atractylodes macrocephala (D), Poria cocos (E), and Glycyrrhiza uralensis (F) were used as the factors to be investigated, with each factor having three levels. The level design is shown in Table 1.

[0031] Table 1. Factor levels in orthogonal experiments (unit: g)

[0032]

[0033] 1.3 Orthogonal Experimental Design

[0034] L18(3) 6 The orthogonal array contains 18 matching combinations, as shown in Table 2.

[0035] Table 2 L18(3) 6 Orthogonal experimental design

[0036]

[0037] 1.4 Sample preparation and inhibition zone determination

[0038] Weigh the medicinal materials according to the combinations in Table 2, soak them in 12 times the amount of water for 2 hours, boil and then simmer for 45 minutes, then filter. Add 10 times the amount of water to the dregs and simmer for another 45 minutes, then filter. Add 8 times the amount of water to the dregs and simmer for another 45 minutes, then filter. Combine the three filtrates and concentrate under reduced pressure at 60℃ to a crude drug concentration of 1 g / mL. The diameter of the inhibition zone against APEC O2:K1 for each combination was determined using the Oxford cup method: the bacterial solution was evenly spread on MHA plates, Oxford cups were placed on them, 200 μL of sample was added to each cup, and the plates were incubated at 37℃ for 18–24 hours. The diameter of the inhibition zone was then measured. Each combination was repeated 3 times. The results are shown in Table 3.

[0039] Table 3. Diameter of inhibition zone in orthogonal experiment (mm, mean±SD, n=3)

[0040]

[0041] 1.5 Range Analysis and Optimal Proportion

[0042] The average value (K1, K2, K3) and range R of the inhibition zones at different levels of each factor were calculated, and the results are shown in Table 4.

[0043] Table 4 Range Analysis of Orthogonal Experiments

[0044]

[0045] Table 4 shows that the order of influence of each factor on the antibacterial effect is: B > A > C > D > E > F. The optimal combination is A3B3C3D1E2F1, namely 26g of dandelion, 13g of Mahonia fortunei, 15g of Astragalus membranaceus, 7g of Atractylodes macrocephala, 6g of Poria cocos, and 6g of Glycyrrhiza uralensis. A verification experiment was conducted using this ratio, and the diameter of the inhibition zone was measured to be 12.42±0.35mm (n=3), which is similar to the result of combination number 17 in the orthogonal table, indicating that this ratio has a stable antibacterial effect.

[0046] Clinical efficacy trial 1

[0047] 1.1 Animal Model and Grouping

[0048] One-day-old Guangxi Jinling Flower Chickens were raised to 14 days of age. The LD50 of APEC O2:K1 on 14-day-old chicks was determined using the modified Kohl's method, and was found to be 2.92 × 10⁻⁶. 7 CFU / chick. Select healthy chicks and inject them intraperitoneally with 100 μL of bacterial solution (containing 2.92 × 10⁻⁶ CFU / chick). 7 An infection model was established using CFU, and the control group was injected with an equal volume of physiological saline.

[0049] Four hundred and eighty 14-day-old chicks were randomly divided into six groups, with four replicates per group and twenty chicks per replicate: blank control group (C, physiological saline + drinking water), model group (M, bacterial solution + drinking water), low-dose compound group (CDD-L, bacterial solution + 250 mg crude drug / mL), medium-dose compound group (CDD-M, bacterial solution + 500 mg crude drug / mL), high-dose compound group (CDD-H, bacterial solution + 1000 mg crude drug / mL), and positive control group (PC, bacterial solution + 10% enrofloxacin soluble powder, calculated as 100 mg / kg of enrofloxacin active ingredient). Administration of the medication via gavage began one hour after challenge, once daily for three consecutive days.

[0050] 1.2 Detection Indicators

[0051] On day 7 post-challenge, 10 chicks from each group were harvested, and blood, heart, liver, lungs, spleen, thymus, and bursa of Fabricius were collected to calculate organ indices (organ weight / body weight, mg / g). Bacterial load was determined from blood and liver tissue. Serum was used to detect TNF-α, IL-1β, IL-6, IL-10, LPS, PCT, IgY, IgM, SOD, CAT, GSH-Px, and MDA. Heart and liver tissues were subjected to HE staining for pathological observation.

[0052] 1.3 Results

[0053] 1.3.1 Survival rate

[0054] See results Figure 1 B. The 7-day survival rate was 55% in the model group and 85% in the medium-dose compound group, showing a significant difference compared to the model group (P=0.0389, Log-rank test). The low-dose compound group had a survival rate of 70%, the high-dose group had a survival rate of 75%, and the positive control group had a survival rate of 80%.

[0055] 1.3.2 Gross lesions and histopathology

[0056] General observations Figure 1 C. In the model group, the surface of the heart was covered with a large amount of yellowish-white fibrinous exudate, and the pericardium was thickened; the surface of the liver was covered with a grayish-white fibrinous pseudomembrane. In the compound medium-dose group, the appearance of the heart and liver basically returned to normal.

[0057] HE staining of the heart ( Figure 1 D) The model group showed widening, edema, and rupture of myocardial fiber interstices, with a large number of inflammatory cells infiltrating; the compound medium-dose group showed that the myocardial structure had basically returned to normal, with only a small number of scattered inflammatory cells.

[0058] Liver HE staining ( Figure 1 E) The results showed that in the model group, hepatocytes were edematous, vacuolar degeneration was present, hepatic sinusoids were dilated, and a large number of inflammatory cells infiltrated; in the compound medium-dose group, the liver lobule structure was basically normal, and the inflammatory infiltration was significantly reduced.

[0059] 1.3.3 Organ Index

[0060] The organ indices of chicks in each group on day 7 are shown in Table 5. Compared with the control group, the indices of heart, liver, lung, and spleen in the model group were significantly increased (P<0.05), while the indices of thymus and bursa of Fabricius were significantly decreased (P<0.05). Compared with the model group, the indices of heart, liver, and lung in the compound medium-dose group were significantly decreased (P<0.05), while the indices of thymus and bursa of Fabricius were significantly increased (P<0.05).

[0061] Table 5. Organ index of chicks in each group on day 7 (mg / g, mean±SD, n=10)

[0062]

[0063] Note: Different superscript letters in the same row indicate significant differences between groups (P<0.05).

[0064] 1.3.4 Bacterial load

[0065] See results Figure 2 A, 2B. No bacteria were detected in the blood and liver of the control group. The bacterial load in the blood of the model group was (9.66±3.37)×10⁻⁶. 2 CFU / mL, liver bacterial load was (3.04±2.36)×10 5 CFU / g. Compared with the model group, the bacterial load in blood and liver of each treatment group was significantly reduced (P<0.05), with the bacterial load in blood and liver of the medium-dose compound group decreasing to (2.58±1.23)×10⁻⁶. 2 CFU / mL and (1.17±1.87)×10 4 CFU / g.

[0066] 1.3.5 Serum inflammatory factors and infection markers

[0067] See results Figure 2 C~2H. In the model group, the levels of TNF-α, IL-1β, IL-6, LPS, and PCT were all higher than in the blank group (P<0.05), while IL-10 was lower than in the blank group (P<0.05). In the medium-dose compound preparation group, all the above indicators were significantly reversed (P<0.05), approaching the levels of the blank group.

[0068] 1.3.6 Immunoglobulins and Antioxidant Indicators

[0069] See results Figure 3 A–3F. In the model group, IgY and IgM levels were higher than in the control group (P<0.05), while SOD, CAT, and GSH-Px activities were lower than in the control group (P<0.05), and MDA content was higher than in the control group (P<0.05). In the medium-dose compound group, IgY and IgM levels were significantly decreased (P<0.05), SOD, CAT, and GSH-Px activities were significantly increased (P<0.05), and MDA levels were significantly decreased (P<0.05), recovering to levels close to the control group.

[0070] The above results indicate that the traditional Chinese medicine composition provided by the present invention has a significant therapeutic effect on chicken colibacillosis caused by APEC O2:K1 in the dosage range of 250-1000 mg crude drug / mL, with 500 mg crude drug / mL being the optimal dose. Its effects involve direct antibacterial, anti-inflammatory, antioxidant and immunomodulatory effects.

[0071] Those skilled in the art will recognize that numerous variations are possible with respect to the above description, and therefore the embodiments are merely illustrative of one or more specific implementations.

[0072] Although exemplary embodiments of the invention have been described and illustrated, those skilled in the art will understand that various changes and substitutions can be made thereto without departing from the spirit of the invention. Furthermore, many modifications can be made to adapt specific situations to the doctrine of the invention without departing from the central concepts of the invention described herein. Therefore, the invention is not limited to the specific embodiments disclosed herein, but may include all embodiments and equivalents that fall within the scope of the invention.

Claims

1. A traditional Chinese medicine composition for treating chicken colibacillosis, characterized in that, It is made from the following raw materials in parts by weight: 14-26 parts dandelion, 7-13 parts Mahonia fortunei, 9-15 parts Astragalus membranaceus, 7-13 parts Atractylodes macrocephala, 4-8 parts Poria cocos, and 6-10 parts Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition for treating chicken colibacillosis according to claim 1, characterized in that, It is made from the following raw materials in parts by weight: 20-26 parts dandelion, 10-13 parts Mahonia fortunei, 12-15 parts Astragalus membranaceus, 7-10 parts Atractylodes macrocephala, 6-8 parts Poria cocos, and 6-8 parts Glycyrrhiza uralensis.

3. The traditional Chinese medicine composition for treating chicken colibacillosis according to claim 2, characterized in that, It is made from the following raw materials in parts by weight: 26 parts dandelion, 13 parts Mahonia fortunei, 15 parts Astragalus membranaceus, 7 parts Atractylodes macrocephala, 6 parts Poria cocos, and 6 parts Glycyrrhiza uralensis.

4. A traditional Chinese medicine composition for treating chicken colibacillosis according to any one of claims 1 to 3, characterized in that, The dosage forms of the traditional Chinese medicine composition include one or more of the following: decoction, powder, granules, oral liquid, capsule, and tablet.

5. A method for preparing a traditional Chinese medicine composition for treating chicken colibacillosis according to any one of claims 1-4, characterized in that, The process includes the following steps: Soak the raw medicinal material in 8-15 times the amount of water for 1-3 hours, boil and then simmer for 30-60 minutes, then filter; add 6-12 times the amount of water to the dregs and simmer for another 30-60 minutes, then filter; add 4-10 times the amount of water to the dregs and simmer for another 20-50 minutes, then filter; combine the three filtrates and concentrate under reduced pressure to a raw drug concentration of 0.2-1.0 g / mL.

6. The method for preparing a traditional Chinese medicine composition for treating chicken colibacillosis according to claim 5, characterized in that, Includes the following steps: Soak the raw material in 12 times the amount of water for 2 hours, boil and simmer for 45 minutes, then filter. Add 10 times the amount of water to the dregs and simmer for another 45 minutes, then filter. Add 8 times the amount of water to the dregs and simmer for another 45 minutes, then filter. Combine the three filtrates and concentrate under reduced pressure to a raw drug concentration of 0.25-1.0 g / mL.

7. The method for preparing a traditional Chinese medicine composition for treating chicken colibacillosis according to claim 6, characterized in that, The concentration of the crude drug is 0.5 g / mL.

8. The application of a traditional Chinese medicine composition for treating chicken colibacillosis prepared by the method according to any one of claims 5 to 7.

9. The application of the traditional Chinese medicine composition for treating chicken colibacillosis according to claim 8, characterized in that, The chicken colibacillosis is caused by infection with pathogenic avian Escherichia coli.

10. The application of the traditional Chinese medicine composition for treating chicken colibacillosis according to claim 8, characterized in that, The traditional Chinese medicine composition exerts its therapeutic effect through one or more of the following mechanisms: improving survival rate, reducing heart / liver / lung index, restoring thymus / bursa of Fabricius index, reducing blood and liver bacterial load, reducing serum TNF-α / IL-1β / IL-6 / LPS / PCT levels, increasing IL-10 levels, increasing SOD / CAT / GSH-Px activity, reducing MDA content, and alleviating pericarditis or perihepatitis.

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