Application of curcumin in combination with enrofloxacin hydrochloride in the treatment of drug-resistant bacterial infections in aquatic animals

Abstract

This invention discloses the application of curcumin in synergistic action with enrofloxacin hydrochloride in combating drug-resistant aquatic bacterial infections, belonging to the field of pharmaceutical technology. This invention provides the application of curcumin in any of the following: (1) in the preparation of an enrofloxacin hydrochloride synergist against drug-resistant aquatic bacterial infections; (2) in the preparation of a product that enhances the activity of enrofloxacin hydrochloride against drug-resistant aquatic bacteria; wherein the drug-resistant aquatic bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas vernix, or drug-resistant Aeromonas hydrophila. Experimental verification shows that this invention can significantly reduce bacterial resistance and enhance the antibacterial activity of enrofloxacin hydrochloride, especially showing a synergistic effect against drug-resistant Citrobacter freundii. This invention provides a new approach for the treatment of drug-resistant bacteria. The curcumin and enrofloxacin hydrochloride compound powder provided by this invention has a simple preparation process, readily available raw materials, is suitable for large-scale industrial production, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of curcumin in combination with enrofloxacin hydrochloride in the treatment of drug-resistant bacterial infections in aquatic organisms. Background Technology

[0002] Enrofloxacin hydrochloride is a pale yellow crystalline powder with a slightly bitter taste, and is slightly soluble in water or ethanol. It is a broad-spectrum antibiotic, highly effective against various Gram-positive bacteria, Gram-negative bacteria, and mycoplasma. Enrofloxacin was approved for use as an animal-specific drug in the last century and has a long history of application in livestock and aquaculture. In aquaculture, enrofloxacin hydrochloride is mainly used to combat infections caused by bacteria such as Aeromonas, Citrobacter, and Streptococcus, exhibiting excellent antibacterial effects and gaining popularity among farmers. However, due to the limited availability of effective antibiotics in aquaculture, prolonged use, and the improper and excessive use of drugs, resistance to enrofloxacin among aquaculture pathogens has become increasingly serious, with minimum inhibitory concentrations reaching tens of PPM. Conventional doses are no longer sufficient to treat diseases, leading to numerous treatment failures and economic losses.

[0003] Turmeric is a perennial herb widely cultivated in tropical Asia. Curcumin, a phenolic pigment extracted from the rhizome of turmeric, is an important active ingredient in turmeric and possesses a wide range of pharmacological effects, including anti-tumor, antioxidant, anti-inflammatory, and lipid-lowering properties. This invention has found that curcumin can reduce the drug resistance of aquatic antibiotic-resistant bacteria and enhance the antibacterial activity of enrofloxacin hydrochloride, thereby restoring therapeutic efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide the application of curcumin in synergistic action with enrofloxacin hydrochloride in combating drug-resistant bacterial infections in aquatic organisms, thereby addressing the problems existing in the prior art. This invention reveals that curcumin can significantly reduce the drug resistance of drug-resistant aquatic bacteria and enhance the antibacterial activity of enrofloxacin hydrochloride, particularly showing a synergistic effect against drug-resistant *Citrobacter freundii* and drug-resistant *Aeromonas vesiculosus*. This invention provides a new approach to the treatment of drug-resistant bacteria.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the use of curcumin in any of the following:

[0007] (1) Application in the preparation of enrofloxacin hydrochloride as an anti-drug-resistant aquatic bacteria infection enhancer;

[0008] (2) Application in the preparation of products that enhance the activity of enrofloxacin hydrochloride against drug-resistant aquatic bacteria;

[0009] The aquatic drug-resistant bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas vernix, or drug-resistant Aeromonas hydrophila.

[0010] The present invention also provides a composition for combating drug-resistant aquatic bacteria infection, the composition comprising curcumin and enrofloxacin hydrochloride;

[0011] The aquatic drug-resistant bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas vernix, or drug-resistant Aeromonas hydrophila.

[0012] Optionally, the weight ratio of curcumin to enrofloxacin hydrochloride is (4-55):(1-30).

[0013] Preferably, the weight ratio of curcumin to enrofloxacin hydrochloride is (20-40):(10-20).

[0014] The present invention also provides a product for combating drug-resistant bacterial infections in aquatic organisms, the product comprising the aforementioned composition and pharmaceutically acceptable excipients.

[0015] This invention also provides the application of curcumin in combination with enrofloxacin hydrochloride in the preparation of a drug for treating drug-resistant Citrobacter freundii infection.

[0016] This invention also provides the application of curcumin in combination with enrofloxacin hydrochloride in the preparation of drugs against drug-resistant Aeromonas verrucosa infections.

[0017] The present invention discloses the following technical effects:

[0018] Experimental verification revealed that curcumin can significantly reduce the drug resistance of aquatic drug-resistant bacteria and enhance the antibacterial activity of enrofloxacin hydrochloride, especially showing a synergistic effect against drug-resistant Citrobacter freundii. This invention provides a new approach for the treatment of drug-resistant bacteria.

[0019] The preparation process of the curcumin and enrofloxacin hydrochloride compound powder provided by this invention is simple, the raw materials are readily available, it is suitable for large-scale industrial production, and has broad application prospects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of a 96-well bacterial culture plate. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The curcumin used in this invention was purchased from Ningbo Traditional Chinese Medicine Pharmaceutical Factory (40%, CAS: 458-37-7), and enrofloxacin hydrochloride was purchased from Jiangsu Yiheng Pharmaceutical Co., Ltd. (98%, CAS: 112732-17-9).

[0028] The drug-resistant Citrobacter freundii, drug-resistant Aeromonas vernix, and drug-resistant Aeromonas hydrophila used in this invention were obtained by self-isolation. The 16S sequences of the drug-resistant Citrobacter freundii, drug-resistant Aeromonas vernix, and drug-resistant Aeromonas hydrophila are shown in SEQ ID NO.1-SEQ ID NO.3. Sequencing verification confirmed that the three bacteria are Citrobacter freundii, Aeromonas vernix, and Aeromonas hydrophila, respectively.

[0029] SEQ ID NO.1:

[0030]

[0031] SEQ ID NO.2:

[0032]

[0033] SEQ ID NO.3:

[0034]

[0035] Example 1: Curcumin enhances the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii.

[0036] 1. Serial dilution

[0037] First, add 200 μL of the drug working solution to the well with the preset maximum drug concentration. Then, add 100 μL of sterile MH broth to each of the remaining wells. Next, add 100 μL of the drug working solution from the maximum drug concentration well to the second well. Mix thoroughly by pipetting 5 times, then add 100 μL to the third well. Continue this serial dilution process, discarding the 100 μL of liquid drawn from the last well. At this point, each well contains 100 μL of drug-containing broth, with a drug concentration ranging from 512 μg / mL to 0.002 μg / mL.

[0038] 2. Determination of the combined effect of curcumin and enrofloxacin hydrochloride using the checkerboard method

[0039] Take a 96-well bacterial culture plate (see...) Figure 1 Row Y1 and column X1 are the rows and columns for individual administration of drug A and drug B, respectively. In row Y1, wells 2-7 are each added with 100 μL of standard drug A solution at concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC. In column X1, wells 2-7 are each added with 100 μL of standard drug B solution at concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC. In row Y2-7, wells 2-7 are each added with 50 μL of standard drug A solution at concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC. In column X2-7, wells 2-7 are each added with 50 μL of standard drug B solution at concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC. Except for well X1Y1, all other wells are added with 100 μL of bacterial suspension, and well X1Y1 is added with 200 μL of bacterial suspension. At this point, the total volume of liquid in the 96-well plate is 200 μL. Place the 96-well microplates containing the sample in a constant temperature incubator (28℃) for 24-48 hours. The lowest concentration observed in the clear wells is the MIC for combined drug use. Results are determined using the equivalent midpoint method: each tube along the bisector of the angle at 0 is considered an equivalent midpoint. The lowest concentration at which no sterile growth is observed is designated as point 1. The point corresponding to this point on the X-axis is MIC A, and the corresponding point on the Y-axis is MIC B. The partial inhibition index (FIC) is used as the basis for judging the combined drug susceptibility test. The calculation formula is as follows:

[0040]

[0041] A FIC index ≤ 0.5 indicates a synergistic effect; a FIC index of 0.5-1 indicates an additive effect; a FIC index of 1-2 indicates an unrelated effect; and a FIC index > 2 indicates an antagonistic effect.

[0042] Results: The MIC of curcumin against drug-resistant Citrobacter freundii was determined to be 256 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii was 16 μg / mL. The minimum concentration for sterility after combined use of curcumin and enrofloxacin hydrochloride was 64 μg / mL for curcumin and 2 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.375, indicating a synergistic effect.

[0043] Example 2: Curcumin enhances the bactericidal ability of enrofloxacin against drug-resistant Aeromonas verrucosa.

[0044] The measurement method is the same as in Example 1.

[0045] Results: The MIC of curcumin against drug-resistant Aeromonas verrucosa was 512 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas verrucosa was 8 μg / mL. The minimum concentration for sterility after combined use of curcumin and enrofloxacin hydrochloride was 64 μg / mL for curcumin and 1 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.25, indicating a synergistic effect.

[0046] Example 3: Curcumin enhances the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila.

[0047] The measurement method is the same as in Example 1.

[0048] Results: The MIC of curcumin against drug-resistant Aeromonas hydrophila was 512 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila was 16 μg / mL. The minimum concentration for sterility when curcumin solid dosage form was combined with enrofloxacin was 16 μg / mL for curcumin and 8 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.531, indicating an additive effect.

[0049] Example 4: Yellow catfish experiment

[0050] 1. Preparation of a compound powder of curcumin and enrofloxacin hydrochloride

[0051] Weigh out 20 parts curcumin, 10 parts enrofloxacin hydrochloride and 70 parts starch by weight, pass them through a 70-mesh sieve and set aside.

[0052] Place curcumin and enrofloxacin hydrochloride into a mixer and mix thoroughly.

[0053] After mixing, add starch and continue mixing. Then pass the mixture through a 24-mesh sieve to obtain a compound powder of curcumin and enrofloxacin hydrochloride.

[0054] 2. Grouping and drug administration

[0055] 150 yellow catfish were randomly divided into five groups of 30 each.

[0056] The first group served as a blank control, receiving no viral load or treatment.

[0057] The second group served as a negative control, and was challenged with drug-resistant Aeromonas verrucosa derived from yellow catfish without treatment.

[0058] The third group was the enrofloxacin hydrochloride treatment group, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish and treated with enrofloxacin hydrochloride at a dose of 10 mg / kg, twice a day for three consecutive days.

[0059] The fourth group was the curcumin treatment group, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish and treated with curcumin at a dose of 10 mg / kg, twice a day for three consecutive days.

[0060] The fifth group was the combined treatment group, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish and treated with a compound powder of curcumin and enrofloxacin hydrochloride at a dose of 10 mg / kg (calculated as enrofloxacin hydrochloride), twice a day for three consecutive days.

[0061] After treatment, the yellow catfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 1.

[0062] Table 1 Results of the Yellow Catfish Experiment

[0063] Group Number of dead animals mortality rate Blank control group 0 0.00％ negative control group 30 100％ Enrofloxacin hydrochloride treatment group 21 70.00％ Curcumin group 18 60％ Combined treatment group 1 3.33％

[0064] Example 5: Red swamp crayfish experiment

[0065] 1. Preparation of a compound powder of curcumin and enrofloxacin hydrochloride

[0066] Weigh out 40 parts of curcumin, 20 parts of enrofloxacin hydrochloride, and 40 parts of starch by weight, pass them through a 70-mesh sieve, and set aside.

[0067] Place curcumin and enrofloxacin hydrochloride into a mixer and mix thoroughly.

[0068] After mixing, add starch and continue mixing. Then pass the mixture through a 24-mesh sieve to obtain a compound powder of curcumin and enrofloxacin hydrochloride.

[0069] 2. Grouping and drug administration

[0070] 150 red swamp crayfish were randomly divided into five groups of 30 each.

[0071] The first group served as a blank control, receiving no viral load or treatment.

[0072] The second group served as a negative control, and was challenged with *Citrobacter freundii* from *Procambarus clarkii* without treatment.

[0073] The third group was the enrofloxacin hydrochloride treatment group, which was challenged with Citrobacter freundii from red swamp crayfish and treated with enrofloxacin hydrochloride at a dose of 10 mg / kg, twice a day for three consecutive days.

[0074] The fourth group was the curcumin treatment group, which was challenged with Citrobacter freundii from Procambarus clarkii and treated with curcumin at a dose of 10 mg / kg, twice a day for three consecutive days.

[0075] The fifth group was the combined treatment group, which was challenged with Citrobacter freundii from red swamp crayfish and treated with a compound powder of curcumin and enrofloxacin hydrochloride at a dose of 10 mg / kg (calculated as enrofloxacin hydrochloride), twice a day for three consecutive days.

[0076] After treatment, the red swamp crayfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 2.

[0077] Table 2. Results of the experiment with *Procambarus clarkii*

[0078] Group Number of dead animals mortality rate Blank control group 1 3.33％ negative control group 30 100％ Enrofloxacin hydrochloride treatment group 20 66.67％ Curcumin group 20 66.67％ Combined treatment group 1 3.33％

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of curcumin in any of the following: (1) Application in the preparation of enrofloxacin hydrochloride as an adjuvant against drug-resistant aquatic bacteria; (2) Application in the preparation of products that enhance the activity of enrofloxacin hydrochloride against drug-resistant aquatic bacteria; The aquatic drug-resistant bacteria are drug-resistant Citrobacter freundii or drug-resistant Aeromonas verrucosa.

2. Application of curcumin in combination with enrofloxacin hydrochloride in the preparation of drugs against drug-resistant Citrobacter freundii infection.

3. Application of curcumin in combination with enrofloxacin hydrochloride in the preparation of drugs against drug-resistant Aeromonas verrucosa infections.

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