Application of curcumin and enrofloxacin hydrochloride in resisting drug-resistant bacterium infection of aquatic products

Abstract

The invention discloses application of curcumin and enrofloxacin hydrochloride in resisting drug-resistant bacterium infection of aquatic products, and belongs to the technical field of medicines. The invention provides an application of curcumin in any one of the following items: (1) an application in preparation of an enrofloxacin hydrochloride synergist for resisting aquatic product drug-resistant bacterium infection; (2) application in preparation of a product for enhancing the activity of enrofloxacin hydrochloride for resisting aquatic drug-resistant bacteria; the aquatic drug-resistant bacteria comprise drug-resistant citrobacter freundii, drug-resistant aeromonas veronii or drug-resistant aeromonas hydrophila. Experiments prove that curcumin can obviously reduce the drug resistance of bacteria and enhance the antibacterial activity of enrofloxacin hydrochloride, and especially has a synergistic effect on drug-resistant citrobacter freundii. The invention provides a new thought for treatment of drug-resistant bacteria. The curcumin and enrofloxacin hydrochloride compound powder provided by the invention is simple in preparation process, easily available in raw materials, suitable for industrial large-scale production and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of curcumin in combination with enrofloxacin hydrochloride in resisting aquatic drug-resistant bacteria infection. Background Art

[0002] Enrofloxacin hydrochloride is a light yellow crystalline powder with a slightly bitter taste. It is slightly soluble in water or ethanol. It is a broad-spectrum antibacterial drug that has a highly effective killing effect on various Gram-positive bacteria, Gram-negative bacteria, mycoplasmas, etc. Enrofloxacin is an animal-specific drug approved for use in the last century and has a long history of application in animal husbandry and aquaculture. In aquaculture, enrofloxacin hydrochloride is mainly used to fight against bacterial infections such as Aeromonas, Citrobacter, and Streptococcus. It has excellent antibacterial effects and is favored by the majority of farmers. However, due to the excessive singleness of effective antibiotics in aquaculture, long application time, and irregular use and abuse of drugs, the drug resistance of enrofloxacin to pathogens in aquaculture has become more and more serious, and the minimum inhibitory concentration has reached dozens of PPM. Conventional doses can no longer treat the disease, resulting in a large number of treatment failures and economic losses.

[0003] Turmeric is a perennial herb widely cultivated in tropical regions of Asia. Curcumin is a phenolic pigment extracted from the rhizome of turmeric and is an important active ingredient of turmeric. It has a wide range of pharmacological effects such as anti-tumor, anti-oxidation, anti-inflammatory, and hypolipidemic. The present invention has found that curcumin can reduce the drug resistance of aquatic drug-resistant bacteria and enhance the antibacterial ability of enrofloxacin hydrochloride, thereby restoring the therapeutic effect. Summary of the invention

[0004] The purpose of the present invention is to provide an application of curcumin in synergistic effect with enrofloxacin hydrochloride in preventing aquatic drug-resistant bacteria infection, so as to solve the problems existing in the above-mentioned prior art. The present invention finds 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 on drug-resistant Citrobacter freundii and drug-resistant Aeromonas veronii. The present invention provides a new idea for the treatment of drug-resistant bacteria.

[0005] To achieve the above object, the present invention provides the following solutions:

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

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

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

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

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

[0011] The aquatic drug-resistant bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas veronii 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 resisting aquatic drug-resistant bacteria infection, which comprises the composition and pharmaceutically acceptable excipients.

[0015] The present invention also provides the use of curcumin in combination with enrofloxacin hydrochloride in the preparation of a medicine for resisting infection by drug-resistant Citrobacter freundii.

[0016] The present invention also provides the use of curcumin in combination with enrofloxacin hydrochloride in the preparation of a drug for resisting drug-resistant Aeromonas veronii infection.

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

[0018] Through experimental verification, the present invention found 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 on drug-resistant Citrobacter freundii. The present invention provides a new idea for the treatment of drug-resistant bacteria.

[0019] The curcumin and enrofloxacin hydrochloride compound powder provided by the invention has a simple preparation process, readily available raw materials, is suitable for industrial large-scale production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 Schematic diagram of a 96-well bacterial culture plate. DETAILED DESCRIPTION

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

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0027] The curcumin used in the present invention was purchased from Ningbo 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 veronii, and drug-resistant Aeromonas hydrophila used in the present invention are obtained by self-isolation, wherein the 16S sequences of the drug-resistant Citrobacter freundii, drug-resistant Aeromonas veronii, and drug-resistant Aeromonas hydrophila are shown in SEQ ID NO.1-SEQ ID NO.3, and sequencing verification shows that the three bacteria are Citrobacter freundii, Aeromonas veronii, 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 increases the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii

[0036] 1. Multiple dilution

[0037] First, 200 μL of drug working solution was added to the preset maximum drug concentration well, and then 100 μL of sterilized MH broth was added to the remaining wells. Then, 100 μL of drug working solution was added to the second well from the maximum drug concentration well, and 100 μL was added to the third well after repeated pipetting 5 times to mix. The dilution was performed backward in turn, and the 100 μL liquid aspirated from the last well was discarded. At this time, each well contained 100 μL of drug-containing broth, and the drug concentration was 512 μg / mL to 0.002 μg / mL.

[0038] 2. Chessboard method to determine the combined effect of curcumin and enrofloxacin hydrochloride

[0039] Take a 96-well bacterial culture plate (see Figure 1 ), Y1 row and X1 column are the rows and columns for drug A and drug B to be used separately. 100μL of standard drug solution with drug A concentration of 1 / 4, 1 / 2, 1, 2, 4, 8MIC was added to wells 2-7 of Y1 row respectively; 100μL of standard drug solution with drug B concentration of 1 / 4, 1 / 2, 1, 2, 4, 8MIC was added to wells 2-7 of X1 column respectively. 50μL of standard solution with drug A concentration of 1 / 4, 1 / 2, 1, 2, 4, 8MIC was added to wells 2-7 of Y2-7 row respectively; 50μL of standard solution with drug B concentration of 1 / 4, 1 / 2, 1, 2, 4, 8MIC was added to wells 2-7 of X2-7 column respectively. 100μL of bacterial suspension was added to each well except well X1Y1, and 200μL of bacterial suspension was added to well X1Y1. At this time, the total volume of liquid in the 96-well microplate was 200μL. Place the 96-well microplate with the sample in a constant temperature incubator (28°C) and incubate for 24-48 hours. The lowest concentration of the clear well is the MIC for combined drug use. The results are determined by the equivalent midpoint method: each tube along the 0-point angle bisector is an equivalent midpoint tube. The lowest concentration of sterile growth is 1 point. The point on the X-axis corresponding to this point is MIC A, and the point on the Y-axis is MIC B. The partial concentration inhibition index (FIC) is used as the basis for the combined drug sensitivity test. The calculation formula is as follows:

[0040]

[0041] FIC index ≤ 0.5 indicates synergistic effect; FIC index 0.5-1 indicates additive effect; FIC index 1-2 indicates irrelevant effect; FIC index > 2 indicates antagonistic effect.

[0042] Results: The MIC of curcumin against drug-resistant Citrobacter freundii was 256μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii was 16μg / mL. After curcumin and enrofloxacin hydrochloride were used together, the minimum concentration of sterile growth was: curcumin was 64μg / mL, enrofloxacin hydrochloride was 2μg / mL, and the combined antibacterial index was 0.375, which was a synergistic effect.

[0043] Example 2 Curcumin increases the bactericidal ability of enrofloxacin against drug-resistant Aeromonas veronii

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

[0045] Results: The MIC of curcumin against drug-resistant Aeromonas veronii was 512 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas veronii was 8 μg / mL. After curcumin and enrofloxacin hydrochloride were used together, the minimum concentration for sterile growth was 64 μg / mL for curcumin and 1 μg / mL for enrofloxacin hydrochloride, and the combined antibacterial index was 0.25, which was a synergistic effect.

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

[0047] The determination 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. After the solid dose of curcumin was used in combination with enrofloxacin, the minimum concentration of sterile growth was: curcumin was 16 μg / mL, enrofloxacin hydrochloride was 8 μg / mL, and the combined antibacterial index was 0.531, which was an additive effect.

[0049] Example 4 Yellow catfish test

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

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

[0052] Put curcumin and enrofloxacin hydrochloride in a mixer in sequence and mix thoroughly;

[0053] After mixing, starch is added and the mixture is continued to be mixed. The mixed mixture is then sieved through a 24-mesh sieve to obtain a compound powder of curcumin and enrofloxacin hydrochloride.

[0054] 2. Grouping and Dosing

[0055] A total of 150 yellow catfish were randomly divided into five groups, with 30 fish in each group.

[0056] The first group was a blank control, without virus attack or treatment;

[0057] The second group was a negative control, which was challenged with drug-resistant Aeromonas veronii from yellow catfish and not treated;

[0058] The third group was the enrofloxacin hydrochloride treatment group, which was challenged with resistant Aeromonas veronii 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 veronii 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 used drug-resistant Aeromonas veronii from yellow catfish as the challenge agent and was treated with a compound powder of curcumin and enrofloxacin hydrochloride at a dose of 10 mg / kg (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 Test results of yellow catfish

[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％ Combination therapy group 1 3.33％

[0064] Example 5 Procambarus clarkii test

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

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

[0067] Put curcumin and enrofloxacin hydrochloride in a mixer in sequence and mix thoroughly;

[0068] After mixing, starch is added and the mixture is continued to be mixed. The mixed mixture is then sieved through a 24-mesh sieve to obtain a compound powder of curcumin and enrofloxacin hydrochloride.

[0069] 2. Grouping and Dosing

[0070] A total of 150 Procambarus clarkii were randomly divided into five groups, with 30 crayfish in each group.

[0071] The first group was a blank control, without virus attack or treatment;

[0072] The second group was a negative control, which was challenged with Citrobacter freundii from Procambarus clarkii and received no treatment;

[0073] The third group was the enrofloxacin hydrochloride treatment group, which was challenged with Citrobacter freundii from Procambarus clarkii 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 used Citrobacter freundii from Procambarus clarkii as the toxin challenge and was treated with a compound powder of curcumin and enrofloxacin hydrochloride at a dose of 10 mg / kg (as enrofloxacin hydrochloride), twice a day for three consecutive days.

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

[0077] Table 2 Test results of 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％ Combination therapy group 1 3.33％

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of curcumin in any of the following: (1) Application of enrofloxacin hydrochloride in the preparation of an anti-aquatic drug-resistant bacterial infection enhancer; (2) Application in the preparation of products that enhance the activity of enrofloxacin hydrochloride against aquatic drug-resistant bacteria; The aquatic drug-resistant bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas veronii or drug-resistant Aeromonas hydrophila.

2. A composition for resisting aquatic drug-resistant bacteria infection, characterized in that: The composition comprises curcumin and enrofloxacin hydrochloride; The aquatic drug-resistant bacteria include drug-resistant Citrobacter freundii, drug-resistant Aeromonas veronii or drug-resistant Aeromonas hydrophila.

3. The composition according to claim 2, characterized in that The weight ratio of curcumin to enrofloxacin hydrochloride is (4-55):(1-30).

4. The composition according to claim 3, characterized in that The weight ratio of the curcumin to enrofloxacin hydrochloride is (20-40):(10-20).

5. A product for preventing aquatic drug-resistant bacteria infection, characterized in that: The product comprises the composition according to any one of claims 2 to 4, and pharmaceutically acceptable excipients.

6. Application of curcumin synergistically with enrofloxacin hydrochloride in the preparation of drugs for treating drug-resistant Citrobacter freundii infection.

7. The application of curcumin synergistically with enrofloxacin hydrochloride in the preparation of drugs for treating drug-resistant Aeromonas vermifuge infection.

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