Lidocaine-limonene compositions and their use in the preparation of salmonella inhibiting preparations

Abstract

This invention belongs to the field of antibacterial preparation technology, and discloses a lidocaine-limonene composition and its use in the preparation of Salmonella inhibitors. This invention discovers that lidocaine and limonene, when used alone, have weak antibacterial effects against Salmonella, but their combined application exhibits a significant synergistic bactericidal effect. Simultaneously, lidocaine, while enhancing the bactericidal activity of limonene, also possesses multiple pharmacological effects such as local anesthesia, analgesia, anti-inflammation, and wound healing promotion, giving this composition multi-target therapeutic advantages. Compared to traditional antibiotics, this invention has a more efficient antibacterial effect and can reduce antibiotic use, which is of great significance in addressing the problem of drug-resistant Salmonella infections.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial preparation technology, and specifically discloses the lidocaine-limonene composition and its use in the preparation of Salmonella inhibitors. Background Technology

[0002] Salmonella is a Gram-negative bacillus widely found in nature, animal intestines, and various foods. It is an important pathogen causing foodborne gastroenteritis, typhoid fever, and various systemic infections. In recent years, with the widespread use of antibiotics, multidrug-resistant strains of Salmonella and other multidrug-resistant strains have emerged, posing a serious challenge to clinical treatment and becoming a major threat to global public health.

[0003] Limonene is a monocyclic monoterpene natural compound widely found in the essential oils of citrus fruit peels, possessing broad-spectrum antibacterial, antioxidant, and anti-inflammatory activities. Studies have shown that limonene can disrupt the structural integrity of bacterial cell membranes and interfere with bacterial energy metabolism, thereby exerting a bactericidal effect. However, the concentration of limonene used alone against Salmonella is often high, limiting its clinical application. Therefore, finding synergists that can synergistically enhance the antibacterial activity of limonene is an important way to expand the application of natural antibacterial compounds.

[0004] Lidocaine is an amide-type local anesthetic that also possesses antiarrhythmic properties. Clinically, it is widely used for local anesthesia, postoperative analgesia, and the treatment of arrhythmias. Recent studies have found that lidocaine also exhibits significant anti-inflammatory activity, inhibiting the release of various inflammatory cytokines and regulating inflammatory responses. Furthermore, lidocaine has been shown to accelerate wound healing by regulating local blood flow and promoting tissue repair. In addition, some studies have reported that lidocaine has a certain interfering effect on bacterial cell membranes, but its individual antibacterial activity is limited. Summary of the Invention

[0005] In view of this, the present invention explores the antibacterial effect of lidocaine and limonene in combination against Salmonella, aiming to develop a novel combination formulation with both high bactericidal and multiple therapeutic effects to overcome the shortcomings of the prior art.

[0006] To achieve the above technical objectives, the technical solution adopted by this invention is as follows:

[0007] An antimicrobial composition for inhibiting Salmonella, comprising lidocaine and limonene.

[0008] Preferably, the concentration of limonene used is 1 / 8 MIC to 1 / 4 MIC; and the concentration of lidocaine used is 500 μg / mL to 1000 μg / mL.

[0009] Preferably, the Salmonella includes one or both of Salmonella ATCC 14028 or Salmonella 11S, a clinical isolate from broilers.

[0010] Preferably, the lidocaine is lidocaine hydrochloride, and the limonene has a purity of ≥98%.

[0011] The present invention also claims protection for the use of the said antimicrobial composition in the preparation of preparations for inhibiting Salmonella infection.

[0012] Furthermore, the formulation contains a pharmaceutically acceptable carrier selected from at least one of solvents, gel matrices, emulsifiers, penetration enhancers, stabilizers, and preservatives.

[0013] Furthermore, the formulation is any one of an injection, ointment, gel, spray, or lotion.

[0014] Furthermore, the preparation is used to treat at least one of foodborne Salmonella infection, open wound Salmonella infection, and postoperative wound Salmonella infection.

[0015] Furthermore, the formulation also has synergistic bactericidal, local anesthetic and analgesic, anti-inflammatory, and wound-healing effects.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] (1) Significant synergistic bactericidal effect: Lidocaine and limonene have weak bactericidal effect on Salmonella when used alone (the bactericidal amount is less than 1×log when used alone), but when used together, they have a significant synergistic bacteriostatic effect (the bactericidal amount is reduced by more than 4×log), achieving the effect of non-antibiotic combined bactericidal effect, avoiding the use of antibiotics and reducing the risk of drug resistance.

[0018] (2) Multiple pharmacological advantages: Lidocaine can enhance the bactericidal activity of limonene, while also playing analgesic, anti-inflammatory and wound healing roles. It is especially suitable for clinical scenarios that require both antibacterial and repair effects, such as open wound infection and postoperative infected wound.

[0019] (3) Reduce antibiotic dependence: This composition uses limonene of natural origin as the main antibacterial component, combined with lidocaine for synergistic effect, eliminating the need for traditional antibiotics and helping to alleviate the problem of drug-resistant bacteria. Attached Figure Description

[0020] Figure 1 The figure shows the statistical results of the bactericidal effect of lidocaine and limonene combined against Salmonella ATCC 14028.

[0021] Figure 2The figure shows the statistical results of the bactericidal effect of lidocaine and limonene combined on the clinical strain Salmonella 11S. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0024] The limonene used is from the [Maclean] brand, with a purity of ≥98%;

[0025] The lidocaine hydrochloride mentioned is from [Shanghai Zhaohui Pharmaceutical Co., Ltd.], with a content of 2%.

[0026] Test strains: Salmonella standard strain ATCC 14028 (laboratory collection or commercially available), and Salmonella clinical strain Salmonella 11S isolated from a broiler farm (both Salmonella 11S clinical strains were provided by this laboratory. The public may obtain the above biological materials with the applicant's consent; the above biological materials are only used to repeat the experiments related to this invention and not for other purposes). Drug resistance information is as follows:

[0027] Table 1. Antibiotic susceptibility of Salmonella

[0028] Note: R indicates drug resistance, S indicates drug sensitivity.

[0029] Judgment principle: If the bacterial count reduction in the experimental group is two log reductions on top of the bacterial count reductions in the limonene-only treatment and lidocaine-only treatment, it indicates that limonene and lidocaine have a synergistic bactericidal effect.

[0030] Example 1: MIC determination of limonene and lidocaine against Salmonella ATCC 14028

[0031] 1. Experimental materials:

[0032] (1) Cool the MH broth that has been autoclaved for later use.

[0033] (2) Prepare the experimental raw materials or materials: Limonene: [Maclean], purity ≥98%; lidocaine hydrochloride and test strains: for example, Salmonella standard strain ATCC 14028 (laboratory collection).

[0034] 2. Preparations before the experiment:

[0035] (1) Salmonella ATCC 14028 and Salmonella 11S were passaged on agar medium.

[0036] (2) Limonene was sterilized by filtration through a 0.22 μm filter membrane, and then packaged and stored at 4°C in the dark.

[0037] (3) 2% lidocaine hydrochloride was sterilized by filtration through a 0.22 μm filter membrane, aliquoted and stored at 4°C in the dark.

[0038] 3. MIC Measurement Experiment:

[0039] (1) Inoculate a single colony into a centrifuge tube containing 4 mL of MH broth, place it in a shaker at 37°C and shake at 180 rpm for 4 h, and then remove it;

[0040] (2) Dilute the incubated bacterial culture with MH broth to approximately 10. 6 CFU / mL, for later use;

[0041] (3) Take a sterile 96-well plate, add 180 μL of MH broth medium to well 1, and add 100 μL of MH broth medium to wells 2-12.

[0042] (4) Add 20 μL of limonene or lidocaine hydrochloride to well 1, mix well and then transfer 100 μL to well 2. Repeat this serial dilution process. Discard 100 μL from well 10.

[0043] (5) Add 100 μL of diluted bacterial solution to wells 1-11, and add 100 μL of MH broth to well 12 as a blank control;

[0044] (6) The final concentrations (V / V) of limonene were: 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078%, 0.039%, 0.0195%, and 0.0098%. Well 11 was the growth control and well 12 was the blank control.

[0045] (7) The final concentrations (μg / ml) of lidocaine hydrochloride were: 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.906, and 1.953. Well 11 was the growth control and well 12 was the blank control.

[0046] (8) Each drug is repeated in 3 rows;

[0047] (9) Place the 96-well plate in a 37°C incubator for 16–18 h and read the results.

[0048] The results are shown in Table 2. The MIC value of limonene against Salmonella ATCC 14028 was 0.625%, and the MIC value of lidocaine hydrochloride against Salmonella ATCC 14028 was >1000 μg / ml. The MIC value of limonene against Salmonella 11S was 1.25%, and the MIC value of lidocaine hydrochloride against Salmonella 11S was >1000 μg / ml. At concentrations below the MIC values, the bacteria were not significantly inhibited.

[0049] Table 2. MICs of limonene and lidocaine against Salmonella.

[0050] Example 2: The bactericidal effect of lidocaine and limonene combined on Salmonella standard strain ATCC 14028

[0051] 1. Experimental materials:

[0052] Same as Example 1.

[0053] 2. Sterilization curve experiment:

[0054] (1) Inoculate a single colony of Salmonella into MH broth and incubate at 37°C and 180 rpm for 4 h with shaking; dilute 10 times with MH broth to obtain a bacterial concentration of approximately 10. 7 CFU / mL, for later use.

[0055] (2) This experiment uses a 4 mL reaction system and is divided into 6 groups:

[0056] ① Blank control group: 3.6 mL MH broth + 0.4 mL bacterial culture;

[0057] ②Lidocaine alone group: lidocaine (1000μg / ml) + bacterial culture;

[0058] ③ Limonene alone group: 1 / 4 MIC limonene + bacterial culture;

[0059] ④ Limonene alone: ​​1 / 8 MIC limonene + bacterial culture;

[0060] ⑤ Combined group 1: 1 / 4 MIC limonene + lidocaine (1000 μg / ml) + bacterial culture;

[0061] ⑥ Combined group 2: 1 / 8 MIC limonene + lidocaine (1000μg / ml) + bacterial culture.

[0062] (3) After preparation, place it in a shaker at 37℃ and 180 rpm for 24 h.

[0063] (4) At 24 h, 100 μL of bacterial culture was taken from each tube and added to a centrifuge tube containing 900 μL of 0.85% physiological saline for a 10-fold serial dilution. 25 μL was dropped onto MH agar medium and incubated at 37℃ for 16-18 h for colony counting. The experiment was performed after three biological replicates.

[0064] The results are shown in Table 3 and Figure 1 As shown, the bacteria in the blank control group grew normally (approximately 7 log CFU / mL); there was no significant difference in bacterial count between the lidocaine-only group and the control group; the limonene-only group showed a decrease of <1 log CFU / mL compared to the blank control group, indicating that the inhibitory effect of each single drug on Salmonella ATCC 14028 was not significant at this concentration; however, the bacterial count in combination groups 1 and 2 decreased by 6.22 log values ​​and 3.4 log values, respectively, compared to the control group, demonstrating that the combined application of lidocaine and limonene has a significant synergistic bactericidal effect on Salmonella.

[0065] Table 3. Bactericidal effect of lidocaine and limonene combined against Salmonella ATCC 14028

[0066] Example 3: The bactericidal effect of lidocaine and limonene combined against Salmonella 11S

[0067] 1. Experimental materials:

[0068] Same as Example 1.

[0069] 2. Sterilization curve experiment:

[0070] Same as Example 2.

[0071] The results are shown in Table 4 and Figure 2 As shown, bacteria in the blank control group grew normally (approximately 7 log CFU / mL); there was no significant difference in bacterial count between the lidocaine-only group and the control group; the limonene-only group showed a decrease of <1 log CFU / mL compared to the blank control group, indicating that the inhibitory effect of each single drug on Salmonella 11S was not significant at this concentration; however, the bacterial count in combination group 1 and combination group 2 decreased by 6.59 log values ​​and 3.69 log values ​​respectively compared to the control group, demonstrating that the combined application of lidocaine and limonene has a significant synergistic bactericidal effect on Salmonella.

[0072] Table 4. Bactericidal effect of lidocaine and limonene combined against Salmonella 11S

[0073] The specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibacterial composition for inhibiting Salmonella, characterized in that, It contains lidocaine and limonene.

2. The antibacterial composition according to claim 1, characterized in that, The concentration of limonene used is 1 / 8 MIC to 1 / 4 MIC; the concentration of lidocaine used is 500 μg / mL to 1000 μg / mL.

3. The antibacterial composition according to claim 1 or 2, characterized in that, The Salmonella mentioned includes Salmonella ATCC 14028 or clinically resistant strains from broilers.

4. The antibacterial composition according to claim 1, characterized in that, The lidocaine is lidocaine hydrochloride, and the limonene has a purity of ≥98%.

5. Use of the antimicrobial composition according to any one of claims 1 to 4 in the preparation of an agent for inhibiting Salmonella infection.

6. The use according to claim 5, characterized in that, The formulation contains a pharmaceutically acceptable carrier selected from at least one of solvents, gel matrices, emulsifiers, penetration enhancers, stabilizers, and preservatives.

7. The use according to claim 6, characterized in that, The preparation is any one of an injection, ointment, gel, spray, or lotion.

8. The use according to claim 5, characterized in that, The preparation is used to treat at least one of foodborne Salmonella infection, open wound Salmonella infection, and postoperative wound Salmonella infection.

9. The use according to claim 5, characterized in that, The formulation has synergistic bactericidal, local anesthetic and analgesic, anti-inflammatory, and wound-healing effects.

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