Use of tannic acid in the preparation of a medicine for inhibiting actinobacillus pleuropneumoniae in pigs

Abstract

The application discloses application of tannic acid in preparation of a medicine for inhibiting porcine actinobacillus pleuropneumoniae. The tannic acid is applied in inhibition of the porcine actinobacillus pleuropneumoniae and preparation of the medicine for inhibiting the porcine actinobacillus pleuropneumoniae, and is specifically applied in inhibition of growth and / or proliferation of the porcine actinobacillus pleuropneumoniae and preparation of the medicine. The medicine is mainly a veterinary preparation, and also contains drugs and compositions thereof. Through in-vitro bacteriostatic experiments and growth curve analysis observation, it is found that the tannic acid has a clear inhibitory effect on the above important pathogenic bacteria of pigs, and also shows stable bacteriostatic activity on different serotype strains of the porcine actinobacillus pleuropneumoniae, thereby providing a new technical scheme for prevention and control of important bacterial diseases of pigs.

Description

Technical Field

[0001] This invention relates to the field of veterinary anti-infective drugs, specifically to the application of tannic acid in the preparation of drugs that inhibit Actinobacillus pleuropneumoniae. Background Technology

[0002] Porcine infectious pleuropneumonia ( porcine contagious pleuropneumonia PCP is caused by Actinobacillus pleuropneumoniae (PCP). Actinobacillus pleuropneumoniae Actinobacillus pleuropneumoniae (APP) is a highly contagious respiratory bacterial disease caused by contact infection. There are multiple serotypes of Actinobacillus pleuropneumoniae in my country, and the prevalent serotypes are diverse. Studies by Li Guo et al. (2022) and Nan Xiangzhu et al. (2023) have shown that serotypes 1, 2, 5, and 7 have a high detection rate in monitoring in many parts of China.

[0003] Current prevention and control mainly rely on vaccination and antimicrobial therapy. However, due to the numerous serotypes of Actinobacillus pleuropneumoniae (APP) and the low cross-immunity between different serotypes, vaccine protection is challenging, and the stability of prevention and control remains uncertain. Meanwhile, clinical treatment heavily relies on antibiotics; however, the excessive and irrational use of antibiotics has led to increasingly severe drug resistance in APP. Hennig-Pauka et al. (2022) reported that Actinobacillus pleuropneumoniae isolated in Germany showed significant resistance to tetracycline, penicillin, and gentamicin. Xu Minsheng (2023) found that 20 APP strains isolated and identified from Guangdong Province between 2019 and 2021 all exhibited multidrug resistance, with 8 or more drug-resistant strains, accounting for 50%. The resistance rates to tetracyclines, sulfonamides, and chloramphenicol were particularly high, at 77.5%, 65.0%, and 55%, respectively. 0.0%; Chen Pengxu et al. (2025) isolated an APP strain from a large-scale pig farm in Fuyang, Anhui Province. The strain was resistant to tetracycline and lincomycin and exhibited a multidrug resistance phenotype; Wang Jiafan et al. (2021) found that 11 APP isolates from Sichuan Province showed multidrug resistance, with a resistance rate of 90.90% to tetracycline and doxycycline; Peng Zhifeng et al. (2020) found that 5 APP isolates from Henan Province had a 100% resistance rate to tetracycline and a 60% resistance rate to penicillin; Wei-Fan Kwan et al. (2025) isolated 96 APP isolates from Taiwan, China, which showed high resistance to florfenicol, amoxicillin, and ampicillin (45.1%-68.4%). Therefore, under the industrial background of "limiting and reducing antibiotic use", developing alternative active ingredients that can effectively inhibit APP is of great significance for reducing the pressure of antibiotic use and improving the control effect.

[0004] Therefore, existing technologies lack new uses and applications for preparing drugs to inhibit important porcine pathogens such as Actinobacillus pleuropneumoniae. Summary of the Invention

[0005] To address the problems existing in the background technology, and considering the current situation where the serotypes of Actinobacillus pleuropneumoniae are complex, vaccine cross-protection is limited, and clinical prevention and control are heavily reliant on antibacterial drugs with increasingly prominent drug resistance issues, this invention provides a novel use of a natural active ingredient with a clear source, high safety, and clear antibacterial activity: the application of tannic acid in the preparation of drugs that inhibit Actinobacillus pleuropneumoniae, in order to reduce the pressure of traditional antibiotic use and improve the stability and effectiveness of prevention and control of bacterial diseases in pigs.

[0006] The technical solution adopted in this invention is as follows: I. Application of tannic acid in inhibiting Actinobacillus pleuropneumoniae and in the preparation of drugs for inhibiting Actinobacillus pleuropneumoniae.

[0007] The porcine Actinobacillus pleuropneumoniae mentioned therein includes serotypes 1, 5 and / or 7.

[0008] The application of tannic acid in altering the cell structure of Actinobacillus pleuropneumoniae and in pharmaceutical manufacturing.

[0009] When the cell culture medium containing Actinobacillus pleuropneumoniae was treated with the tannic acid, the cell structure of Actinobacillus pleuropneumoniae was altered.

[0010] The inhibitory effect was verified by minimum inhibitory concentration (MIC) determination and growth curve experiments. Different serotypes of *Actinomyces pleuropneumoniae* showed essentially the same inhibitory sensitivity to the tannic acid.

[0011] The drug is a veterinary preparation. It is used to prevent and treat porcine infectious pleuropneumonia.

[0012] II. A drug for inhibiting Actinobacillus pleuropneumoniae in pigs, wherein the main active ingredient of the drug comprises the tannic acid.

[0013] The only active ingredient in the drug is tannic acid.

[0014] The drug also includes pharmaceutically acceptable excipients, carriers, and / or diluents.

[0015] The drug is an oral tablet, capsule, oral liquid, granule or injection.

[0016] III. A pharmaceutical composition for inhibiting Actinobacillus pleuropneumoniae, comprising the above-mentioned drug and one or more adjuvant drugs available in veterinary clinical practice.

[0017] The tannic acid described in this application is derived from the active substances in Polygonatum odoratum.

[0018] The beneficial effects of this invention include: To address the aforementioned technical problems, this invention provides a novel use of tannic acid in the preparation of drugs that inhibit *Actinobacillus pleuropneumoniae*. Through in vitro antibacterial experiments, growth curve analysis, and morphological observation, the applicant discovered that tannic acid has a clear inhibitory effect on the aforementioned important porcine pathogens, exhibiting stable antibacterial activity against different serotypes of *Actinobacillus pleuropneumoniae*. This demonstrates that tannic acid can be used as a novel candidate active ingredient for anti-infective purposes in the development of related formulations.

[0019] Furthermore, in some embodiments, the tannic acid described in this invention can be used alone as the main active ingredient in the preparation of veterinary preparations, or it can be combined with one or more adjuvant drugs available in veterinary clinical practice to prepare a pharmaceutical composition for inhibiting or controlling Actinobacillus pleuropneumoniae infection and related diseases, thereby improving the overall prevention and control effect and reducing dependence on traditional antibiotics.

[0020] The adjuvant drugs can be commonly used veterinary drug components in the art, such as antibacterial drugs, anti-inflammatory drugs, antipyretic analgesics, expectorants and bronchodilators, and / or immune enhancers. For example, the antibacterial drugs can be selected from β-lactams, tetracyclines, macrolides, fluoroquinolones, amides, sulfonamides, etc.; the anti-inflammatory drugs can be selected from nonsteroidal anti-inflammatory drugs; the antipyretic analgesics can be selected from commonly used antipyretic analgesic components; the expectorants and bronchodilators can be selected from commonly used expectorants or bronchodilator-related components; and the immune enhancers can be selected from vitamins, trace elements, or other immunomodulatory components. The selection, compatibility, and dosage of the above adjuvant drugs can be routinely adjusted by those skilled in the art based on the animal's condition, degree of infection, and route of administration. Attached Figure Description

[0021] Figure 1 Growth curves for tannic acid-resistant APP serum type 1. Figure 2 Growth curves for tannic acid-resistant APP serum type 5. Figure 3 Growth curves for tannic acid-resistant APP serum type 7. Figure 4 This is a comparison of the electron microscopy morphology of APP bacteria before and after tannic acid treatment. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Those skilled in the art can make equivalent substitutions or appropriate changes to the conditions in the embodiments without departing from the spirit of the present invention, and all such substitutions should fall within the protection scope of the present invention. Unless otherwise stated, the reagents, methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in this technical field.

[0023] Unless otherwise stated, the culture media, serum, solvents, etc. used in the examples are all commercially available products. The following examples use *Actinomyces pleuropneumoniae* (Porcine Actinobacillus pleuropneumoniae). Actinobacillus pleuropneumoniae The invention uses serum serotypes 1, 5, and 7 of APP as examples for illustration. The activity verification and evaluation of tannic acid for other serum serotypes of APP can also be performed by referring to the method described in this specification.

[0024] Example 1: Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of tannic acid against APP 1. Materials (1) Strains: Serum types 1, 5 and 7 of Actinobacillus pleuropneumoniae (APP) were laboratory-preserved strains.

[0025] (2) Culture medium: Liquid / solid culture medium suitable for APP growth (TSB / TSA or MH broth / agar, etc.) were purchased from BD Company, and calf serum was purchased from Sijiqing Company.

[0026] (3) Drugs and solvents: Tannic acid; solvents may include DMSO or other soluble solvents that do not inhibit bacteria; sterile PBS or sterile distilled water.

[0027] (4) Consumables and instruments: sterile 96-well polystyrene microplates, 0.22μm filter membranes, constant temperature incubators, and microplate readers (OD500). 600 (Reading), etc.

[0028] 2. Test methods Refer to the broth micro-dilution method MIC plate preparation: Under aseptic conditions, tannic acid was dissolved and serially diluted 2-fold. Working solutions of different concentrations were added to wells 1 to 11 of a 96-well plate (50 μL per well). An equal volume of culture medium and the same final concentration of solvent were added to well 12 as a growth control. A blank control well (culture medium only) was set up for background correction. At least three replicates were set up for each concentration.

[0029] Inoculum preparation: Each serotype of APP strain was streaked onto TSA agar plates containing 5% fetal bovine serum (FBS) and incubated at 37°C for 12 h. Single colonies were picked and inoculated into TSB liquid medium containing 5% FBS and incubated overnight at 37°C. The cultured bacterial solution was then transferred 1:1000 to fresh TSB liquid medium containing 5% FBS and incubated at 37°C until the absorbance OD reached a certain level. 600 When the concentration was 0.6, the bacterial cells were collected by centrifugation (5000 rpm, 5 min), resuspended in the same liquid medium, and adjusted to approximately 1×10⁶. 8 CFU / mL; then dilute proportionally to achieve a final bacterial concentration of approximately 5 × 10⁻⁶ after adding to the well plate. 5 CFU / mL.

[0030] (5) Adding culture: Add an equal volume of bacterial suspension (50 μL) to each well, mix gently, and incubate in a 37°C incubator for 12–16 h.

[0031] (6) Result determination: Under the premise that the blank control well is uncontaminated and the bacteria in the growth control well grow well, the lowest drug concentration at which no bacteria grow is observed by the naked eye is determined as the MIC value of the test bacteria; the bacterial solution of each well is taken and spotted or spread on a TSA solid plate, and incubated at 37℃ for 18–24 h. The presence or absence of bacterial growth is observed, and the drug concentration corresponding to the number of colonies counted on the plate ≤ 5 is determined as the MBC value of the test bacteria.

[0032] 3. Results The MIC / MBC results of tannic acid against APP serotypes 1, 5, and 7 are shown in Table 1. The results indicate that tannic acid has antibacterial activity against all the above-mentioned APP serotypes, with a MIC of 32 μg / mL and an MBC of 64 μg / mL for all serotypes; however, the MIC and MBC against Streptococcus suis type 2 and Enterococcus faecalis were 1024 μg / mL and 2048 μg / mL, respectively, while it was not sensitive to Escherichia coli.

[0033] Table 1. MIC and MBC of tannic acid against bacteria Example 2: Effect of Tannic Acid on APP Growth Curve 1. Materials APP serum strains type 1, 5, and 7; tannic acid; culture medium as in Example 1.

[0034] 2. Test methods (1) Grouping and drug concentration: For each serotype, the following groups were set up: growth control group (no drug added, only an equal volume of solvent added) and tannic acid treatment group. The treatment concentration can be set to 0.25×MIC, 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC.

[0035] (2) Inoculation and culture: Take the logarithmic phase bacterial culture of each serotype and adjust the inoculation concentration so that the initial bacterial count of the system is about 1×10⁻⁶. 6 CFU / mL, incubated at 37℃.

[0036] (3) Viable cell count: Samples were taken at time points (0, 2, 4, 6, 8, 10, 12 h) starting from 0 h, with a sample volume of 100 μL. The samples were serially diluted 10-fold and then plated onto TSA solid medium. Each time point was replicated at least 3 plates per group. After incubation at 37℃ for 18–24 h, CFU were counted.

[0037] (4) Plotting: Plot the growth curve with time as the horizontal axis and log10 (CFU / mL) as the vertical axis.

[0038] 3. Results Growth curve results as follows Figure 1 – Figure 3 As shown (corresponding to serotypes 1, 5, and 7, respectively). Compared with the growth control group, the tannic acid treatment group showed significant inhibition of proliferation, manifested as prolonged lag phase and decreased logarithmic growth rate; significant inhibition of APP growth was observed under 1×MIC conditions, and the three serotypes showed similar inhibitory trends, suggesting that tannic acid has stable antibacterial activity against different serotypes of APP.

[0039] Example 3: Morphological observation of APP cells after tannic acid treatment 1. Materials APP (represented by serum type 5); tannic acid; fixative (2.5% glutaraldehyde); PBS; graded ethanol, etc.; scanning electron microscope equipment and routine consumables.

[0040] 2. Test methods (1) Bacterial treatment: Take the logarithmic phase bacterial culture of APP and divide it into a control group (add an equal amount of solvent) and a treatment group (add tannic acid to 1×MIC), and incubate at 37℃ for 4 h.

[0041] (2) Collection and washing: After the treatment, the cells were collected by centrifugation (5000 rpm, 5 min) and gently washed twice with PBS.

[0042] (3) Fixation: Add 2.5% glutaraldehyde and fix overnight at 4°C; after washing with PBS, proceed to the gradient dehydration step.

[0043] (4) Dehydration: Dehydration was carried out in sequence with 30%, 50%, 70%, 80%, 90% and 100% ethanol gradient, each step for 10-15 min, and 100% repeated twice.

[0044] (5) SEM sample preparation: After drying, spray gold, observe and photograph under a scanning electron microscope.

[0045] 3. Morphological results of the results, such as Figure 4 As shown, the control group of APP cells exhibited intact surface structures and regular morphology; the tannic acid-treated group showed varying degrees of shrinkage, depression, or rupture on the cell surface, with some cells having indistinct boundaries. These morphological changes suggest that tannic acid can disrupt the structure of APP cells, consistent with its in vitro antibacterial activity.

[0046] Example 4: Statistical analysis of the consistency of antibacterial effect of tannic acid against different serotypes of APP 1. Method The results of APP for different serotypes in Example 1 (MIC) and Example 2 (growth curve) were summarized, and the antibacterial trends and differences in MIC of serotypes 1, 5 and 7 under the same concentration of tannic acid treatment were compared.

[0047] 2. Results The results showed that tannic acid had a stable inhibitory effect on APP serotypes 1, 5, and 7; the MICs of the three serotypes were consistent; and the growth curves showed similar inhibitory trends, indicating that tannic acid has a stable antibacterial effect on APP across serotypes, providing experimental evidence for its potential as a candidate active ingredient for APP control.

[0048] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0049] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. Application of tannic acid in inhibiting Actinobacillus pleuropneumoniae in porcine pleuropneumoniae and in the preparation of drugs for inhibiting Actinobacillus pleuropneumoniae.

2. The application according to claim 1, wherein the porcine Actinobacillus pleuropneumoniae comprises serotypes 1, 5, and / or 7.

3. The application according to any one of claims 1-2, wherein the tannic acid is used in altering the cell structure of Actinobacillus pleuropneumoniae.

4. The application according to claim 1, wherein the drug is a veterinary preparation.

5. The application according to claim 1, wherein the drug is used to prevent and treat porcine infectious pleuropneumonia.

6. A drug for inhibiting Actinobacillus pleuropneumoniae in porcines, characterized in that: The main component of the drug includes tannic acid.

7. The drug according to claim 6, characterized in that: The only active ingredient in the drug is tannic acid.

8. The drug according to claim 6, characterized in that: The drug also includes pharmaceutically acceptable excipients, carriers, and / or diluents.

9. The drug according to claim 6, characterized in that: The drug is an oral tablet, capsule, oral liquid, granule or injection.

10. A pharmaceutical composition for inhibiting Actinobacillus pleuropneumoniae, characterized in that, It includes the drug as described in any one of claims 6-9, and one or more adjuvant drugs that are available in veterinary clinical practice.

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