A synergistic antibacterial composition containing carvacrol, forsythoside and hesperetin and its use

Abstract

The application discloses a synergistic antibacterial composition containing carvacol, forsythoside and hesperetin and application thereof, and belongs to the technical field of antibacterial drugs for aquaculture. Three kinds of carvacol, forsythoside and hesperetin with synergistic effects on neomycin sulfate are screened from Chinese herbal medicines; further, the carvacol, forsythoside and hesperetin are combined with neomycin sulfate, and it is found that the composition has a significant inhibiting effect on Aeromonas hydrophila, and the highest bacteriostatic rate can reach 94.63%; the bacteriostatic effect is persistent, the risk of antibiotic residue and drug resistance transmission can be significantly reduced, the composition can be used for preparing veterinary drugs or aquatic feed additives, and has important application value for preventing and treating hemorrhagic septicemia caused by Aeromonas hydrophila in aquaculture.

Description

Technical Field

[0001] This invention relates to a synergistic antibacterial composition containing carvacrol, forsythoside and hesperidin and its application, belonging to the technical field of antibacterial drugs for aquaculture. Background Technology

[0002] Aeromonas hydrophila ( Aeromonas hydrophila This bacterium is one of the main pathogens in freshwater farmed fish, shrimp, and crabs, capable of causing explosive hemorrhagic septicemia with a mortality rate as high as 95%, resulting in severe economic losses to the aquaculture industry. Simultaneously, this bacterium is a zoonotic pathogen, threatening public health through the consumption of contaminated aquatic products. Therefore, developing safe and efficient antimicrobial strategies for aquaculture has significant economic value and public health implications.

[0003] Currently, the control of Aeromonas hydrophila in aquaculture mainly relies on antibiotics (such as neomycin sulfate), but long-term overuse has led to serious drug resistance problems. Aeromonas hydrophila possesses multiple drug resistance mechanisms, including outer membrane permeability barriers, plasmid-mediated resistance genes, and biofilm protection. Therefore, there is an urgent need to develop alternative or adjunctive antimicrobial strategies that can reduce antibiotic dosage and delay the development of drug resistance.

[0004] In order to reduce the use of antibiotics and avoid the risk of drug resistance in current production, a variety of antibiotic reduction and control measures have been gradually promoted. Common methods include compound antibacterial Chinese herbal medicines, reduced use of antibiotics alone, probiotic biological antagonism, water microecological regulation, and physical disinfection as an auxiliary control measure.

[0005] Traditional Chinese medicine, with its advantages of being natural, low in toxicity, and less likely to induce drug resistance, is considered a potential alternative or synergist to antibiotics. Probiotics inhibit the proliferation of pathogens by competing for colonization sites and secreting antibacterial substances, thereby reducing the bacterial population in aquaculture water. Microecological regulation improves the aquaculture environment by optimizing the balance of bacterial and algal communities in the water, thus reducing the incidence of diseases. Reduced antibiotic use alleviates the problem of drug resistance accumulation caused by long-term antibiotic abuse by controlling the dosage.

[0006] However, existing antibiotic reduction methods still have many significant shortcomings, greatly limiting their practical application effectiveness. For instance, the antibacterial effects of single-herb Chinese medicines are relatively weak, making it difficult to address bacterial outbreaks in high-density aquaculture. Probiotics and microecological regulation technologies have long onset periods and are ineffective against acute outbreaks caused by Aeromonas hydrophila, only suitable for routine prevention and unable to address sudden epidemics. While low-dose single antibiotics can reduce dosage in the short term, long-term low-concentration drug stress accelerates bacterial resistance gene mutations and spread, inducing stronger multidrug resistance in strains. Furthermore, various single antibiotic reduction technologies generally suffer from weak antibacterial targeting and insufficient ability to disrupt pathogen biofilms, failing to completely eliminate Aeromonas hydrophila from water and farmed animals, resulting in high disease recurrence rates and remaining a safety hazard in aquaculture.

[0007] Therefore, how to leverage the natural antibacterial advantages of traditional Chinese medicine, and achieve low-dose synergistic antibacterial effects through scientific formulation and synergistic optimization, can effectively solve the core problems of drug-resistant Aeromonas hydrophila, persistent biofilm infection, recurrent disease outbreaks, and antibiotic reduction and quality improvement in aquaculture with less drug dosage and lower control costs. This research also aims to address the industry pain points of existing antibiotic reduction technologies, such as slow onset, weak effect, and poor stability. It has significant research value and application significance for building a green, safe, and sustainable disease control system for aquaculture, delaying the evolution of drug resistance in aquatic pathogens, and ensuring stable production and increased income in the aquaculture industry. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a synergistic antibacterial composition containing carvacrol, forsythoside, and hesperidin, and its applications. This invention screened three compounds—carvacrol, forsythoside, and hesperidin—that exhibit synergistic effects with neomycin sulfate from traditional Chinese medicine. Further, by combining carvacrol, forsythoside, and hesperidin with neomycin sulfate, it was found that this composition significantly inhibits Aeromonas hydrophila, achieving an inhibition rate of up to 94.63%, with a long-lasting antibacterial effect. It significantly reduces antibiotic residues and the risk of antibiotic resistance transmission, and can be used to prepare veterinary drugs or aquatic feed additives. It has significant application value in preventing and treating hemorrhagic septicemia caused by Aeromonas hydrophila in aquaculture.

[0009] The first objective of this invention is to provide an antibacterial composition comprising carvacrol, forsythoside, hesperidin, and neomycin sulfate; wherein the mass ratio of carvacrol, forsythoside, hesperidin, and neomycin sulfate is 1~64:1~128:1~64:0.25.

[0010] In one embodiment, the mass ratio of carvacrol, forsythoside, hesperidin, and neomycin sulfate is 11.35~39.1:36.35~78.2:8.1~36.35:0.25.

[0011] In one embodiment, the mass ratio of carvacrol, forsythoside, hesperidin, and neomycin sulfate is 10~20:60~80:6~10:0.25.

[0012] In one embodiment, the mass ratio of carvacrol, forsythoside, hesperidin, and neomycin sulfate is 13.6:64.6:8.1:0.25.

[0013] In one embodiment, the antibacterial composition is used to inhibit Aeromonas hydrophila.

[0014] In one embodiment, the antibacterial composition is in the form of a liquid or a solid.

[0015] In one embodiment, the liquid dosage form is any one of a solution, suspension, emulsion, spray, lotion, or drops.

[0016] In one embodiment, the solid dosage form is any one of powder, granules, tablets, and capsules.

[0017] A second object of the present invention is to provide a method for inhibiting Aeromonas hydrophila by adding the antibacterial composition described in any one of the above claims to a system containing Aeromonas hydrophila.

[0018] In one embodiment, the system is an aquaculture system or a natural aquatic environment system.

[0019] A third object of the present invention is to provide a product comprising the antibacterial composition described in any one of the preceding claims.

[0020] In one embodiment, the product is a veterinary drug or an aquatic feed additive.

[0021] In one embodiment, the veterinary drug further contains pharmaceutically acceptable pharmaceutical excipients.

[0022] In one embodiment, the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field; the pharmaceutical excipient includes one or more of the following: binders, diluents, fillers, humectants, disintegrants, absorption enhancers, surfactants, colorants, lubricants, and coating materials.

[0023] In one embodiment, the veterinary drug is in the form of a liquid or a solid.

[0024] In one embodiment, the liquid dosage form is any one of a solution, suspension, emulsion, spray, lotion, or drops.

[0025] In one embodiment, the solid dosage form is any one of powder, granules, tablets, and capsules.

[0026] A fourth object of the present invention is to provide the use of the antibacterial composition described in any of the above claims in the preparation of anti-Aeromonas hydrophila drugs and feed additives for aquaculture.

[0027] Beneficial effects (1) Significant synergistic effect. The present invention verified by checkerboard method that the three Chinese herbal monomers carvacrol, forsythoside and hesperidin all have significant synergistic antibacterial effect with neomycin sulfate, with FICI values ​​of 0.375, 0.5 and 0.5 respectively (FICI ≤0.5 is synergistic), which confirms that the antibacterial effect of the three combined with neomycin sulfate is significantly better than that of each component used alone.

[0028] (2) Significantly reduced antibiotic dosage. This invention found that when forsythoside, hesperidin, carvacrol and neomycin sulfate were in a mass ratio of 64.6:8.1:13.6:0.25, the inhibition rate against Aeromonas hydrophila could reach 94.63%; the effective concentration of neomycin sulfate was only 0.25 mg / L, which is 1 / 32 of the MIC (8 mg / L) of neomycin sulfate alone, and the antibiotic dosage was reduced by 96.9%, which greatly reduced the risk of antibiotic residues and the spread of drug resistance.

[0029] (3) Long-lasting bactericidal effect. Time-growth curve experiments show that the composition of the present invention can continuously inhibit the proliferation of Aeromonas hydrophila, and there is no rebound regrowth within 24 hours.

[0030] (4) The preparation method is simple. All components of the composition described in this invention are commercially available herbal monomers and antibiotics, which can be used by mixing them in proportion, making it suitable for industrial production. Attached Figure Description

[0031] Figure 1 This study aims to determine the bactericidal curve of the antibacterial composition against Aeromonas hydrophila. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0033] In the examples, solutions without a specified solvent are water; operations without a specified temperature refer to room temperature, which is 15-35°C; and percentages without a specified meaning refer to mass percentages.

[0034] Raw materials involved in the examples: All the key monomers involved in the examples are commercially available, including carvacrol purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., forsythoside purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and hesperidin purchased from Sinopharm Chemical Reagent Co., Ltd. Neomycin sulfate was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0035] Aeromonas hydrophila was purchased from the China Agricultural Microbial Culture Collection Center, with accession number ACCC 01748.

[0036] Culture media involved in the examples: LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride.

[0037] Example 1: Screening of herbal monomers with synergistic effects with neomycin sulfate 1. Activation and culture of Aeromonas hydrophila Frozen Aeromonas hydrophila were removed from a -80°C freezer, streaked onto LB agar, and incubated at 28°C for 24 h. Subsequently, one colony was picked and inoculated into 5 mL of LB agar, and incubated at 28°C and 150 rpm for 12 h to obtain an Aeromonas hydrophila seed culture. This seed culture was then inoculated into 50 mL of LB agar at a 2% v / v inoculation rate, and incubated at 28°C and 150 rpm for 6 h to reach the logarithmic growth phase. The culture was then diluted with PBS to a concentration of 1×10⁻⁶. 5 CFU / mL available for use.

[0038] 2. MIC determination of herbal monomers against Aeromonas hydrophila The MICs of 37 traditional Chinese medicine monomers against Aeromonas hydrophila were determined using the micro-broth dilution method. The specific method is as follows: Each herbal medicine monomer was serially diluted twice with LB liquid medium to prepare solutions with concentrations of 2048, 1024, 512, 256, 128, 64, 32, and 16 mg / L, respectively. Add 100 μL of the above-mentioned concentration of drug solution and 100 μL of a 1×10⁻⁶ concentration to each well of a 96-well plate. 5 Bacterial suspensions of CFU / mL were used to achieve final concentrations of 1024, 512, 256, 128, 64, 32, 16, and 8 mg / L for the herbal medicines, respectively. The solutions were then incubated at 28°C and 150 rpm for 24 h. A bacterial suspension without added drugs served as a positive control, and a culture medium without bacteria served as a negative control. OD values ​​were measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 The minimum concentration at which bacterial growth is completely inhibited is defined as the MIC. Each experiment was repeated three times.

[0039] The results are shown in Table 1. The results indicate that among the 37 Chinese herbal monomers, sanguinarine has the lowest MIC of 64 mg / L; 6 compounds including carvacrol and hesperidin have an MIC of 128 mg / L; 21 compounds including forsythin have an MIC of 256~512 mg / L; the remaining 9 compounds have an MIC of 1024 mg / L. The MIC of the positive control neomycin sulfate is 8 mg / L.

[0040] Table 1 MIC of Chinese herbal monomers against Aeromonas hydrophila

[0041] 3. Screening of Chinese herbal monomers with synergistic effect with neomycin sulfate Based on the MIC results in step 2, 28 Chinese herbal monomers with MIC ≤ 512 mg / L were selected, and were combined with neomycin sulfate respectively by the checkerboard microdilution method to determine the combined antibacterial effect; The specific method is as follows: Each Chinese herbal monomer and neomycin sulfate were serially two-fold diluted in a 96-well plate respectively, covering a concentration range from 1 / 16×MIC to 2×MIC. The Chinese herbal monomers were added along the horizontal direction, and neomycin sulfate was added along the vertical direction to form cross concentration combinations. 100 μL of bacterial suspension with a concentration of 1×10 5 CFU / mL was added to each well, and after incubation at 28°C and 150 rpm for 24 h, the OD 600 was measured.

[0042] Taking carvacrol as an example, the addition amount of each reagent is shown in Table 2 (the MIC of carvacrol is 128 mg / L, and the MIC of neomycin sulfate is 8 mg / L): Table 2

[0043] The minimum concentration that completely inhibits bacterial growth is defined as MIC, and the FICI is calculated according to the following formula to screen out Chinese herbal monomers with FICI ≤ 0.5, that is, with synergistic effect; FICI = MIC(Chinese herbal monomer in combination) / MIC(Chinese herbal monomer alone) + MIC(neomycin sulfate in combination) / MIC(neomycin sulfate alone).

[0044] Wherein, FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 1.0 indicates an additive effect, 1.0 < FICI ≤ 2.0 indicates an indifferent effect, and FICI > 2.0 indicates an antagonistic effect.

[0045] Table 3 Interaction of the combination of Chinese herbal monomers and neomycin sulfate against Aeromonas hydrophila

[0046] The results are shown in Table 3. The results indicate that the FICI values ​​of carvacrol, forsythoside, and hesperidin with neomycin sulfate were all ≤0.5, specifically 0.375, 0.5, and 0.5, respectively, indicating synergistic effects. Among the remaining combinations, ferulic acid, magnolol, succinic acid, paeoniflorin, andrographolide, and berberine showed additive effects (FICI 0.56–0.75), while the remaining 19 combinations showed no antagonistic effects (FICI > 1.0).

[0047] Example 2: Synergistic antibacterial effects of carvacrol, forsythoside, hesperidin and neomycin sulfate Aeromonas hydrophila was activated according to the method in Example 1, and a concentration of 1×10⁻⁶ was prepared. 5 A bacterial suspension of CFU / mL was prepared for further testing. The synergistic antibacterial effects of carvacrol, forsythoside, hesperidin, and neomycin sulfate were further determined, including the following treatment groups: Group 1 (Negative control group): 100 μL of a 1×10⁻⁶ concentration was added to a 96-well plate. 5 CFU / mL bacterial suspension and 100 μL LB medium; Group 2 (blank group): 200 μL of LB solution was added to the 96-well plate; Group 3 (64.6 mg / L forsythoside monotherapy): 100 μL of a 1×10⁻⁶ mg / L forsythoside monotherapy was added to a 96-well plate. 5 The bacterial suspension of CFU / mL, 12.9 μL of LB solution containing 1 mg / mL forsythoside, and 87.1 μL of LB solution resulted in a final concentration of forsythoside in the total system of 64.6 mg / L. Group 4 (8.1 mg / L hesperidin alone): 100 μL of a 1×10⁻⁶ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension of CFU / mL, 1.6 μL of LB solution containing 1 mg / mL hesperidin, and 98.4 μL of LB solution resulted in a final concentration of hesperidin of 8.1 mg / L in the total system. Group 5 (13.6 mg / L carvacrol alone): 100 μL of carvacrol at a concentration of 1×10⁻⁶ mg / L was added to a 96-well plate. 5 The bacterial suspension of CFU / mL, 2.7 μL of LB solution containing 1 mg / mL carvacrol, and 97.3 μL of LB solution resulted in a final concentration of carvacrol of 13.6 mg / L in the total system. Group 6 (0.25 mg / L neomycin sulfate monotherapy): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5A bacterial suspension of CFU / mL, 5 μL containing 10 mg / L neomycin sulfate, and 95 μL LB solution, resulted in a final concentration of neomycin sulfate of 0.25 mg / L in the system. Group 7 (64.6 mg / L forsythoside + 8.1 mg / L hesperidin + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension containing CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of 1 mg / mL hesperidin, 2.7 μL of 1 mg / mL carvacrol, and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.8 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, carvacrol, and neomycin sulfate in the total system 64.6, 8.1, 13.6, and 0.25 mg / L, respectively. Group 8 (64.6 mg / L ferulic acid + 8.1 mg / L hesperidin + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension containing CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL ferulic acid, 1.6 μL of 1 mg / mL hesperidin, 2.7 μL of 1 mg / mL carvacrol, and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.8 μL of LB solution was added to make the final concentrations of ferulic acid, hesperidin, carvacrol, and neomycin sulfate in the total system 64.6, 8.1, 13.6, and 0.25 mg / L, respectively. Group 9 (combination therapy of 64.6 mg / L forsythoside + 8.1 mg / L magnolol + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension containing CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of 1 mg / mL magnolol, 2.7 μL of 1 mg / mL carvacrol, and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.8 μL of LB solution was added to make the final concentrations of forsythoside, magnolol, carvacrol, and neomycin sulfate in the total system 64.6, 8.1, 13.6, and 0.25 mg / L, respectively. Group 10 (64.6 mg / L forsythoside + 8.1 mg / L hesperidin + 13.6 mg / L succinic acid + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension containing CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of 1 mg / mL hesperidin, 2.7 μL of 1 mg / mL succinic acid and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.8 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, succinic acid and neomycin sulfate in the total system 64.6, 8.1, 13.6 and 0.25 mg / L, respectively. Group 11 (72.2 mg / L hesperidin + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension was prepared at CFU / mL, and 14.5 μL of LB solution containing 1 mg / mL hesperidin, 2.7 μL of carvacrol containing 1 mg / mL carvacrol, and 5 μL of neomycin sulfate containing 10 mg / L were added. Then, 77.8 μL of LB solution was added to make the final concentrations of hesperidin, carvacrol, and neomycin sulfate in the total system 72.7, 13.6, and 0.25 mg / L, respectively. Group 12 (72.2 mg / L forsythoside + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension was prepared at CFU / mL, and 14.5 μL of LB solution containing 1 mg / mL forsythoside, 2.7 μL of carvacrol containing 1 mg / mL and 5 μL of neomycin sulfate containing 10 mg / L were added. Then, 77.8 μL of LB solution was added to make the final concentrations of forsythoside, carvacrol and neomycin sulfate in the total system 72.7, 13.6 and 0.25 mg / L, respectively. Group 13 (78.2 mg / L forsythoside + 8.1 mg / L hesperidin + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension containing CFU / mL was prepared, along with 15.6 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of hesperidin containing 1 mg / mL, and 5 μL of neomycin sulfate containing 10 mg / L. Then, 77.8 μL of LB solution was added to achieve final concentrations of forsythoside, hesperidin, and neomycin sulfate in the total system of 78.2, 8.1, and 0.25 mg / L, respectively. Group 14 (78.2 mg / L carvacrol + 8.1 mg / L hesperidin + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension of CFU / mL was prepared, along with 1.6 μL of LB solution containing 1 mg / mL hesperidin, 15.6 μL of carvacrol containing 1 mg / mL carvacrol, and 5 μL of neomycin sulfate containing 10 mg / L. Then, 77.8 μL of LB solution was added to achieve final concentrations of 8.1, 78.2, and 0.25 mg / L for hesperidin, carvacrol, and neomycin sulfate in the total system, respectively. Group 15 (64.6 mg / L forsythoside + 21.7 mg / L hesperidin + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension containing CFU / mL was prepared, along with 12.9 μL of LB solution containing 1 mg / mL forsythoside, 4.3 μL of hesperidin containing 1 mg / mL, and 5 μL of neomycin sulfate containing 10 mg / L. Then, 77.8 μL of LB solution was added to achieve final concentrations of forsythoside, hesperidin, and neomycin sulfate in the total system of 64.6, 21.7, and 0.25 mg / L, respectively. Group 16 (64.6 mg / L forsythoside + 21.7 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension of CFU / mL was prepared, along with 12.9 μL of LB solution containing 1 mg / mL forsythoside, 4.3 μL of carvacrol containing 1 mg / mL, and 5 μL of neomycin sulfate containing 10 mg / L. Then, 77.8 μL of LB solution was added to achieve final concentrations of forsythoside, carvacrol, and neomycin sulfate in the total system of 64.6, 21.7, and 0.25 mg / L, respectively. Group 17 (combination therapy of 36.35 mg / L forsythoside + 36.35 mg / L hesperidin + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension contained CFU / mL, and 7.27 μL of LB solution containing 1 mg / mL forsythoside, 7.27 μL of hesperidin containing 1 mg / mL, 2.7 μL of carvacrol containing 1 mg / mL, and 5 μL of neomycin sulfate containing 10 mg / L were added. Then, 77.76 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, carvacrol, and neomycin sulfate in the total system 36.35, 36.35, 13.6, and 0.25 mg / L, respectively. Group 18 (39.1 mg / L forsythoside + 8.1 mg / L hesperidin + 39.1 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension contained CFU / mL, and 7.82 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of 1 mg / mL hesperidin, 7.82 μL of 1 mg / mL carvacrol, and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.76 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, carvacrol, and neomycin sulfate in the total system 39.1, 8.1, 39.1, and 0.25 mg / L, respectively. Group 19 (64.6 mg / L forsythoside + 11.35 mg / L hesperidin + 11.35 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension contained CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL forsythoside, 2.27 μL of 1 mg / mL hesperidin, 2.27 μL of 1 mg / mL carvacrol, and 5 μL of 10 mg / L neomycin sulfate were added. Then, 77.56 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, carvacrol, and neomycin sulfate in the total system 64.6, 11.35, 11.35, and 0.25 mg / L, respectively. After culturing the above groups at 28℃ and 150 rpm for 24 h, the inhibition rate was calculated. Antibacterial rate (%) = [(Control group OD - Experimental group OD) / (Control group OD - Blank group OD)] × 100% The measurement results are shown in Table 4: Table 4 Determination of antibacterial rate

[0048] The results showed that when forsythoside, hesperidin, or carvacrol were replaced with ferulic acid, magnolol, or succinic acid (groups 8-10), which only have an additive effect in the checkerboard method, the antibacterial rate decreased significantly, indicating the irreplaceable nature of the three monomers in the formulation. Further elimination of any monomer to construct ternary formulations (groups 11-16) resulted in significantly lower antibacterial rates than the quaternary formulations, with reductions ranging from 8.17 to 32.55 percentage points. Among these, the formulation excluding carvacrol (group 14, 81.26%) showed the largest reduction, followed by the formulation excluding hesperidin (group 12, 74.38%), indicating that the contribution of each monomer to the synergistic effect was, in descending order: carvacrol > hesperidin > forsythoside. When the concentration ratio of monomers was adjusted (groups 17-19, the concentrations of forsythoside and hesperidin, forsythoside and carvacrol, and hesperidin and carvacrol were evenly distributed), the antibacterial rate was lower than that of the optimal formula (84.05%-90.23%), proving that the determined concentration ratio has reached or is close to the optimal balance point.

[0049] Example 3: Time-growth curve of the optimal formulation against Aeromonas hydrophila Aeromonas hydrophila was activated according to the method in Example 1, and a concentration of 1×10⁻⁶ was prepared. 5 A bacterial suspension of CFU / mL was prepared for further testing. Growth curves of Aeromonas hydrophila treated with carvacrol, forsythoside, hesperidin, and neomycin sulfate were determined, including the following treatment groups: Group 1 (Negative control group): 100 μL of a 1×10⁻⁶ concentration was added to a 96-well plate. 5 CFU / mL bacterial suspension and 100 μL LB medium; Group 2 (64.6 mg / L forsythoside monotherapy): 100 μL of a 1×10⁻⁶ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension was prepared at CFU / mL, and 12.9 μL of LB solution containing 1 mg / mL forsythoside was prepared. Then, 87.1 μL of LB solution was added to make the final concentration of forsythoside in the total system 64.6 mg / L. Group 3 (8.1 mg / L hesperidin alone): 100 μL of a 1×10⁻⁶ mg / L solution was added to each well of a 96-well plate. 5 The bacterial suspension was prepared at CFU / mL, and 1.6 μL of LB solution containing 1 mg / mL hesperidin was added. Then, 98.4 μL of LB solution was added to make the final concentration of hesperidin in the total system 8.1 mg / L. Group 4 (13.6 mg / L carvacrol alone): 100 μL of carvacrol at a concentration of 1×10⁻⁶ mg / L was added to a 96-well plate. 5A bacterial suspension of CFU / mL and 2.7 μL of LB solution containing 1 mg / mL carvacrol were added, followed by 97.3 μL of LB solution to bring the final concentration of carvacrol in the total system to 13.6 mg / L. Group 5 (0.25 mg / L neomycin sulfate monotherapy): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension of CFU / mL and 5 μL of neomycin sulfate at a concentration of 10 mg / L were added, followed by 95 μL of LB solution to bring the final concentration of neomycin sulfate in the system to 0.25 mg / L. Group 6 (64.6 mg / L forsythoside + 8.1 mg / L hesperidin + 13.6 mg / L carvacrol + 0.25 mg / L neomycin sulfate combination group): 100 μL of a 1×10⁻⁵ mg / L solution was added to each well of a 96-well plate. 5 A bacterial suspension of CFU / mL was prepared, and 12.9 μL of LB solution containing 1 mg / mL forsythoside, 1.6 μL of hesperidin containing 1 mg / mL, 2.7 μL of carvacrol containing 1 mg / mL, and 5 μL of neomycin sulfate containing 10 mg / L were added. Then, 77.8 μL of LB solution was added to make the final concentrations of forsythoside, hesperidin, carvacrol, and neomycin sulfate in the total system 64.6, 8.1, 13.6, and 0.25 mg / L, respectively. The above treatment groups were cultured at 28℃ and 180 rpm with shaking. Samples were taken at 0, 4, 8, 12, 16, 20, and 24 h to measure OD. 600 Plot the growth curve and measure the results as follows: Figure 1 As shown.

[0050] OD of the control group 600 The value continuously increased over time, indicating that the bacteria exhibited normal logarithmic growth under conditions without drug intervention. The growth curves of the four single-drug groups (forsythoside, hesperidin, carvacrol, and neomycin sulfate) largely overlapped with the control group, except for the OD value at the plateau phase. 600 The values ​​were slightly lower, indicating that the inhibitory effect of each individual component on bacterial proliferation was extremely limited at the tested concentrations. In stark contrast, the OD values ​​of the optimal combination group were much higher. 600 The concentration remained at the initial inoculation level throughout the 24-hour incubation period, without any upward trend. The results indicate that none of the four components, when used individually, could effectively inhibit the growth of Aeromonas hydrophila; however, the combined use achieved a complete and sustained antibacterial effect, and no recovery of resistance was observed, confirming that the formulation possesses irreversible bactericidal properties.

[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An antibacterial composition, characterized in that, The antibacterial composition comprises carvacrol, forsythoside, hesperidin, and neomycin sulfate; the mass ratio of carvacrol, forsythoside, hesperidin, and neomycin sulfate is 1~64:1~128:1~64:0.

25.

2. The antibacterial composition according to claim 1, characterized in that, The antibacterial composition is used to inhibit Aeromonas hydrophila.

3. The antibacterial composition according to claim 1, characterized in that, The antibacterial composition is in liquid or solid form.

4. A method for inhibiting Aeromonas hydrophila, characterized in that, The antibacterial composition according to any one of claims 1 to 3 is added to a system containing Aeromonas hydrophila.

5. A product characterized in that, The product contains the antibacterial composition according to any one of claims 1 to 3.

6. The product according to claim 5, characterized in that, The product is a veterinary drug or an aquatic feed additive.

7. The product according to claim 5, characterized in that, The veterinary drug also contains pharmaceutically acceptable excipients.

8. The product according to claim 5, characterized in that, The pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; the pharmaceutical excipients include one or more of the following: binders, diluents, fillers, humectants, disintegrants, absorption enhancers, surfactants, colorants, lubricants, and coating materials.

9. The product according to claim 5, characterized in that, The veterinary drug is in liquid or solid form.

10. The use of the antibacterial composition according to any one of claims 1 to 3 in the preparation of drugs against Aeromonas hydrophila and feed additives for aquaculture.

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