Use of p-coumaric acid in the preparation of a medicament for treating colibacillosis in chickens

Abstract

This invention discloses the application of p-coumaric acid in the preparation of drugs for treating chicken colibacillosis. This invention is the first to discover that p-coumaric acid has significant antibacterial activity against pathogenic avian Escherichia coli O2:K1, with a minimum inhibitory concentration (MIC) of 3.125 mg / mL. P-coumaric acid can disrupt bacterial cell membrane integrity in a concentration-dependent manner, inhibiting biofilm formation and bacterial spread, and significantly downregulates the expression of multiple virulence genes at sub-inhibitory concentrations. In vivo experiments showed that oral administration of p-coumaric acid at 100 mg / kg significantly improved the survival rate of infected chicks (83.33% vs 45.83%), alleviated pericarditis and perihepatitis, reduced the indices of the heart, liver, lungs, and spleen, restored the thymus and bursa of Fabricius indices, significantly reduced blood and liver bacterial load, regulated inflammatory cytokine and immunoglobulin levels, and improved oxidative stress. P-coumaric acid possesses both direct antibacterial and host immunomodulatory effects, and can be used to prepare safe and effective drugs against chicken colibacillosis.

Description

Technical Field

[0001] This invention relates to the field of veterinary drug technology, specifically to the application of p-coumaric acid in the preparation of drugs for treating chicken colibacillosis. 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. Due to the numerous APEC serotypes and increasingly prominent drug resistance issues, the efficacy of traditional antibiotics is declining, making the development of novel anti-infective drugs of great significance.

[0003] p-Coumaric acid (also known as p-hydroxycinnamic acid) is a natural phenolic acid compound widely found in fruits, vegetables, and medicinal plants, possessing various biological activities such as antibacterial, anti-inflammatory, and antioxidant effects. However, there is currently a lack of relevant technical solutions and theoretical support for applying p-coumaric acid to the prevention and control of avian colibacillosis. The antibacterial activity, in vivo efficacy, and mechanism of action of p-coumaric acid against pathogenic Escherichia coli (especially the highly pathogenic serotype O2:K1) in birds are still unclear, and its regulatory potential on key APEC virulence genes has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of p-coumaric acid in the preparation of drugs for treating chicken colibacillosis.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] Application of coumaric acid in the preparation of drugs for treating chicken colibacillosis.

[0007] Furthermore, the chicken colibacillosis is caused by avian pathogenic Escherichia coli O2:K1.

[0008] Furthermore, the treatment includes a direct antibacterial effect, which is manifested by at least one of: disrupting bacterial cell membrane integrity, inhibiting biofilm formation, inhibiting bacterial spread, and downregulating virulence gene expression.

[0009] Furthermore, the virulence gene is selected from at least one of luxS, fimH, ompA, tsh, iss, iutA, ibeA, lpxC, and pgsA.

[0010] Furthermore, the treatment includes host immune modulation, which manifests as: reducing serum TNF-α, IL-1β, IL-6, LPS, and PCT levels, increasing IL-10 levels, increasing SOD, CAT, and GSH-Px activities, and reducing MDA content by at least one of these.

[0011] Preferably, the p-coumaric acid is prepared into a solution using 0.5% sodium carboxymethyl cellulose and then administered by gavage.

[0012] Preferably, the purity of the p-coumaric acid is ≥98%.

[0013] Preferably, the dosing regimen is once daily for 3 consecutive days.

[0014] Preferably, the dosage is 50-200 mg / kg per chick per day.

[0015] Preferably, the dosage is 100 mg / kg.

[0016] The beneficial effects of this invention compared to the prior art include:

[0017] 1. Clear in vitro antibacterial activity: The minimum inhibitory concentration (MIC) of p-coumaric acid against APEC O2:K1 is 3.125 mg / mL. It can inhibit bacterial growth in a concentration-dependent manner, disrupt the integrity of bacterial cell membranes leading to leakage of nucleic acids and proteins, inhibit biofilm formation (1 / 2MIC inhibition rate 71.4%) and bacterial spread (1 / 2MIC inhibition rate 47.6%).

[0018] 2. Downregulation of virulence genes: At sub-inhibitory concentrations, p-coumaric acid significantly downregulated the expression of several key virulence genes, including quorum sensing gene luxS, adhesion-related genes fimH and tsh, invasion-related genes ompA and ibeA, iron uptake gene iutA, immune escape gene iss, as well as lipopolysaccharide synthesis gene lpxC and phospholipid synthesis gene pgsA.

[0019] 3. Significant in vivo efficacy: In the APEC O2:K1-infected chick model, the 7-day survival rate of the medium-dose (100 mg / kg) coumaric acid treatment group reached 83.33%, significantly higher than the 45.83% in the model group (P<0.01). It can alleviate pericarditis and perihepatitis, reduce the indices of the heart, liver, lungs, and spleen, restore the indices of the thymus and bursa of Fabricius, and significantly reduce the bacterial load in the blood and liver.

[0020] 4. Regulation of inflammation and immunity: Coumaric acid can significantly reduce the levels of serum pro-inflammatory factors TNF-α, IL-1β, and IL-6, and increase the level of anti-inflammatory factor IL-10; reduce the levels of endotoxin LPS and infection marker PCT; and regulate immunoglobulin IgY and IgM to normal levels.

[0021] 5. Antioxidant effect: Coumaric acid can significantly increase the activity of SOD, CAT, and GSH-Px in serum and liver, reduce MDA content, and alleviate oxidative stress damage.

[0022] 6. Good safety profile: Coumaric acid is a naturally sourced phenolic acid compound, and no significant toxic side effects were observed at the experimental dose. Attached Figure Description

[0023] Figure 1 The effect of different concentrations of coumaric acid on the growth curves of APEC O2:K1 is shown in the figure.

[0024] Figure 2 This is a quantitative graph showing the inhibitory effect of different concentrations of p-coumaric acid on APEC O2:K1 biofilm formation.

[0025] Figure 3 The graph shows the inhibitory effect of coumaric acid on the diffusion of APEC O2:K1 on solid culture medium; where A is a representative image and B is a bar chart for quantitative analysis.

[0026] Figure 4 The diagram shows the effect of coumaric acid on the cell membrane integrity of APEC O2:K1 cells; where A represents nucleic acid leakage and B represents protein leakage.

[0027] Figure 5 Figure showing the effect of coumaric acid on the mRNA expression of the APEC O2:K1 virulence gene;

[0028] Figure 6 Survival curves of chicks in each group after infection with APEC O2:K1;

[0029] Figure 7 Gross lesion photos of the heart and liver of chicks in each group are shown. Among them, C is the blank control group, M is the model group, pCA-L, pCA-M, and pCA-H are the low, medium, and high dose groups of p-coumaric acid, respectively, and PC is the positive control group.

[0030] Figure 8 The bar charts show the bacterial load in the liver and blood of chicks in each group. Figure A shows the effect of p-coumaric acid on the bacterial load in the blood of infected chicks, and Figure B shows the effect of p-coumaric acid on the bacterial load in the liver of infected chicks.

[0031] Figure 9 A bar chart showing the serum inflammatory factors, infection markers, and immunoglobulin levels of chicks in each group;

[0032] Figure 10 Bar charts showing antioxidant indices in serum and liver of chicks in each group;

[0033] Figure 11 HE staining images of chick heart tissue from each group (200×), where: C is the blank control group, M is the model group, pCA-L, pCA-M, and pCA-H are the low, medium, and high dose groups of p-coumaric acid, respectively, and PC is the positive control group;

[0034] Figure 12 HE staining images of chick liver tissue from each group (200×), where: C is the blank control group, M is the model group, pCA-L, pCA-M, and pCA-H are the low, medium, and high dose groups of p-coumaric acid, respectively, and PC is the positive control group. Detailed Implementation

[0035] 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.

[0036] In vitro test 1: Determination of in vitro antibacterial activity against coumaric acid

[0037] 1.1 Materials and Methods

[0038] p-Coumaric acid (purity ≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. APEC O2:K1 (TW-XM strain) was provided by Professor Zhu Guoqiang of Yangzhou University. The minimum inhibitory concentration (MIC) of p-coumaric acid against APEC O2:K1 was determined using the micro-broth dilution method. p-Coumaric acid was dissolved in DMSO and then serially diluted with MHB broth to a final concentration of approximately 1×10⁻⁶. 6 CFU / mL bacterial suspension was incubated at 37°C for 16–18 h, with the lowest concentration at which no bacterial growth was observed to be visible to the naked eye defined as the MIC. Growth curves were determined using a 96-well plate method. 1% of the bacterial culture was inoculated overnight into LB medium containing different concentrations of p-coumaric acid (MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC), and incubated at 37°C with shaking for 18 h. OD was measured every 30 min. 600 value.

[0039] 1.2 Results

[0040] The MIC of p-coumaric acid for APEC O2:K1 is 3.125 mg / mL. Growth curve ( Figure 1 The results showed that the MIC group completely inhibited bacterial growth, the 1 / 2MIC group began to recover growth after 7 hours of culture, and the 1 / 4MIC and 1 / 8MIC groups had weaker inhibitory effects, indicating that coumaric acid can inhibit APEC proliferation in a concentration-dependent manner.

[0041] In vitro experiment 2: Inhibitory effect of coumaric acid on APEC biofilm formation

[0042] 2.1 Methods

[0043] The effect of coumaric acid on APEC biofilm formation was determined using the crystal violet staining method. A concentration of 1×10⁻⁶ was used. 6CFU / mL bacterial suspension was inoculated into 96-well plates, and p-coumaric acid was added at concentrations of 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC, respectively. The bacterial suspension without added drug served as a control, and a blank culture medium was also included. After incubation at 37°C for 48 h, the supernatant was discarded, and the samples were washed with PBS and fixed with methanol. The samples were stained with 0.1% crystal violet, dissolved in 33% glacial acetic acid, and the OD was measured. 570 The value is used to calculate the biofilm inhibition rate.

[0044] 2.2 Results

[0045] The results are as follows Figure 2 As shown, p-coumaric acid inhibited APEC biofilm formation in a concentration-dependent manner, with inhibition rates of 71.4%, 31.3%, and 6.6% at 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, respectively (P<0.01). These results indicate that p-coumaric acid can effectively inhibit APEC biofilm formation.

[0046] In vitro experiment 3: Inhibitory effect of coumaric acid on APEC diffusion ability

[0047] 3.1 Methods

[0048] A diffusion inhibition assay was performed using drug-containing plates. MHA plates containing different concentrations of p-coumaric acid (1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, and MIC) were prepared, with drug-free plates serving as controls. 2 μL of logarithmic-phase APEC bacterial suspension was inoculated into the center of each plate and incubated at 37°C for 16–18 h. Colony diameter was then measured, and the colony diameter inhibition rate was calculated.

[0049] 3.2 Results

[0050] The results are as follows Figure 3 As shown in the figure, APEC colonies spread significantly on drug-free plates; on drug-treated plates, the colony diffusion area decreased with increasing drug concentration, and the edges became clearer, with the MIC group completely inhibiting APEC growth. Quantitative analysis showed that the inhibition rates of 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC on colony diameter were 47.6% (P<0.01), 23.6% (P<0.05), and 6.2%, respectively. This indicates that p-coumaric acid can effectively inhibit the diffusion of APEC on the surface of solid culture media.

[0051] In vitro experiment 4: Effect of coumaric acid on APEC cell membrane integrity

[0052] 4.1 Methods

[0053] Log-phase APEC bacterial culture was taken, and 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC of p-coumaric acid were added respectively, with the untreated bacterial culture as a control. The cultures were incubated at 37°C with shaking for 2 hours. After centrifugation, the supernatant was collected, and the nucleic acid concentration (OD) was determined. 260) and protein concentration (OD) 280 ).

[0054] 4.2 Results

[0055] The results are as follows Figure 4 As shown in the figure, compared with the control group, the concentrations of nucleic acids and proteins in the supernatant of the coumaric acid-treated group were significantly increased (P<0.01 or P<0.001), showing a concentration-dependent increase, with the 1 / 2 MIC group showing the largest leakage. This indicates that coumaric acid directly disrupts the cell membrane integrity of APEC cells, leading to the leakage of intracellular macromolecules.

[0056] In vitro experiment 5: Effect of coumaric acid on the expression of APEC virulence genes

[0057] 5.1 Methods

[0058] Log-phase APEC bacterial cultures were treated with p-coumaric acid at concentrations of 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC for 2 hours, respectively. Total RNA was extracted, and after reverse transcription, the mRNA expression levels of virulence genes (luxS, pgsA, lpxC, fimH, ompA, ibeA, tsh, iss, iutA) were detected by qPCR, with 16S rRNA as an internal control.

[0059] 5.2 Results

[0060] The results are as follows Figure 5 As shown, coumarin significantly downregulated the expression of all the aforementioned virulence genes in a dose-dependent manner (P<0.05–P<0.001). Among them, the key quorum sensing gene luxS was significantly suppressed, while genes related to adhesion, invasion, iron uptake, and immune escape all showed a progressively downregulated trend. This indicates that subinhibitory concentrations of coumarin systematically interfere with the APEC virulence regulatory network.

[0061] In vivo efficacy test 1: The therapeutic effect of coumaric acid on APEC-infected chicks

[0062] 1.1 Animal Model and Grouping

[0063] APEC O2:K1 (2.92×10⁻⁶) was administered via intraperitoneal injection. 7 A chicken colibacillosis model was established by infecting 14-day-old Guangxi Jinling Flower chickens with CFU / chicken. 144 chicks were randomly divided into 6 groups (n=24), and each group underwent the following treatment:

[0064] Blank control group (C): Intraperitoneal injection of normal saline, followed by gavage administration of 1 mL of drinking water;

[0065] Model group (M): Intraperitoneal injection of bacterial solution, followed by gavage administration of 1 mL of 0.5% CMC-Na;

[0066] Low-dose pCA-L group: Intraperitoneal injection of bacterial solution, followed by gavage administration of 1 mL pCA solution (prepared with 0.5% CMC-Na, dose 50 mg / kg).

[0067] The medium-dose pCA-M group of coumaric acid was administered via intraperitoneal injection of bacterial solution and by gavage of 1 mL of pCA solution (prepared with 0.5% CMC-Na, dose 100 mg / kg).

[0068] High-dose coumaric acid group (pCA-H): Intraperitoneal injection of bacterial solution, followed by gavage administration of 1 mL of coumaric acid solution (prepared with 0.5% CMC-Na, dose 200 mg / kg).

[0069] Positive control group (PC): Intraperitoneal injection of bacterial solution, followed by oral gavage administration of 1 mL of 10% enrofloxacin soluble powder solution (prepared with 0.5% CMC-Na, based on 100 mg / kg of enrofloxacin active ingredient).

[0070] One hour after the viral challenge, the medication was administered via gavage once daily for three consecutive days.

[0071] 1.2 Detection Indicators

[0072] Mortality was recorded after 7 consecutive days of observation. On day 7, 10 chicks from each group were harvested, weighed, and their hearts, livers, lungs, spleens, thymus, and bursae were collected to calculate organ indices. Blood and liver samples were collected to determine bacterial load. Serum samples were collected 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 collected for HE staining and pathological observation.

[0073] 1.3 Results

[0074] 1.3.1 Survival rate

[0075] The results are as follows Figure 6 As shown, the 7-day survival rate was 45.83% in the model group, 83.33% in the medium-dose coumaric acid group (P<0.01), 70.83% in the low-dose group, 66.67% in the high-dose group, and 79.16% in the positive control group (P<0.05). This indicates that a medium dose of coumaric acid can significantly improve the survival rate of infected chicks.

[0076] 1.3.2 Gross lesions

[0077] General observations Figure 7 In the model group, the surface of the heart had a large amount of yellowish-white fibrinous exudate, and the liver was covered with a grayish-white pseudomembrane; in the coumaric acid medium-dose group, the appearance of the heart and liver basically returned to normal.

[0078] 1.3.3 Organ Index

[0079] The organ indices of chicks in each group on day 7 are shown in Table 1. Compared with the control group, the indices of heart, liver, lung, and spleen in the model group were significantly increased (P<0.001), while the indices of thymus and bursa of Fabricius were significantly decreased (P<0.05 or P<0.001). Compared with the model group, the indices of heart, liver, lung, and spleen in the medium-dose p-coumaric acid group were significantly decreased (P<0.001), while the indices of thymus and bursa of Fabricius were significantly increased (P<0.05 or P<0.01).

[0080] Table 1. Organ index of chicks in each group on day 7 (mg / g, mean±SD, n=10)

[0081]

[0082] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0083] 1.3.4 Bacterial load

[0084] See results Figure 8 Compared with the model group, the bacterial load in the blood and liver of each treatment group was significantly reduced (P<0.01 or P<0.001), with the medium dose group of coumaric acid showing the best effect.

[0085] 1.3.5 Inflammatory factors, infection markers and immunoglobulins

[0086] The results are as follows Figure 9 As shown in the figure. Compared with the model group, the serum levels of TNF-α, IL-1β, IL-6, LPS, and PCT in the coumaric acid medium-dose group were significantly decreased (P<0.05 to P<0.001), while the IL-10 level was significantly increased (P<0.05); IgY and IgM were significantly decreased, recovering to levels close to those in the blank group (P<0.05).

[0087] 1.3.6 Antioxidant Indicators

[0088] The results are as follows Figure 10 As shown, the activities of SOD, CAT, and GSH-Px in serum and liver were significantly increased (P<0.01) and the content of MDA was significantly decreased (P<0.01) in the medium-dose coumaric acid group, indicating that it can effectively improve oxidative stress caused by infection.

[0089] 1.3.7 Histopathological observation

[0090] HE staining of the heart ( Figure 11 The results showed that: in the blank group, myocardial fibers were neatly arranged with no inflammatory cell infiltration; in the model group, myocardial fibers were edematous and broken, with a large number of inflammatory cells infiltrating; in the coumaric acid medium-dose group, the myocardial structure basically returned to normal, with only a small number of scattered inflammatory cells.

[0091] Liver HE staining ( Figure 12 The results showed that the liver lobule structure was intact and the hepatic cords were neatly arranged in the blank group; the hepatocytes in the model group were edematous, vacuolar, dilated, and infiltrated with a large number of inflammatory cells; the liver lobule structure in the medium-dose coumaric acid group was basically normal and the inflammatory infiltration was significantly reduced.

[0092] The above results indicate that p-coumaric acid has a significant therapeutic effect on APEC O2:K1-induced Escherichia coli infection in chickens. Its mechanism of action includes directly disrupting bacterial cell membranes, inhibiting biofilms and virulence factors, and regulating host inflammation and oxidative stress responses.

[0093] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention. Furthermore, various different embodiments of the present invention can be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed by the present invention.

Claims

1. Application of coumaric acid in the preparation of drugs for treating chicken colibacillosis.

2. The use of p-coumaric acid according to claim 1 for the preparation of a medicament for the treatment of colibacillosis in chickens, characterized in that, The chicken colibacillosis is caused by avian pathogenic Escherichia coli O2:K1.

3. The use of p-coumaric acid according to claim 1 or 2 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The treatment includes direct antibacterial effects, which are manifested by at least one of the following: disrupting bacterial cell membrane integrity, inhibiting biofilm formation, inhibiting bacterial spread, and downregulating virulence gene expression.

4. The application of p-coumaric acid in the preparation of a drug for treating chicken colibacillosis according to claim 3, characterized in that, The virulence gene is selected from at least one of luxS, fimH, ompA, tsh, iss, iutA, ibeA, lpxC, and pgsA.

5. The use of p-coumaric acid according to claim 1 or 2 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The treatment includes host immune modulation, which manifests as: reducing serum TNF-α, IL-1β, IL-6, LPS, and PCT levels, increasing IL-10 levels, increasing SOD, CAT, and GSH-Px activities, and reducing MDA content by at least one of these.

6. The use of p-coumaric acid according to claim 1 or 2 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The p-coumaric acid was prepared into a solution using 0.5% sodium carboxymethyl cellulose and then administered by gavage.

7. The application of p-coumaric acid according to claim 6 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The purity of the p-coumaric acid is ≥98%.

8. The application of p-coumaric acid according to claim 6 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The dosing regimen is once daily for 3 consecutive days.

9. The application of p-coumaric acid according to claim 8 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The dosage is 50-200 mg / kg per chick per day.

10. The application of p-coumaric acid according to claim 9 in the preparation of a drug for treating chicken colibacillosis, characterized in that, The dosage is 100 mg / kg.

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