Doxycycline hydrochloride and potassium clavulanate powder for aquaculture, their preparation methods and applications

Abstract

This invention discloses a powder containing doxycycline hydrochloride and potassium clavulanate for aquaculture, its preparation method, and its application, belonging to the field of biotechnology. This invention combines doxycycline hydrochloride and potassium clavulanate to enhance the antibacterial ability of aquatic animals against drug-resistant bacteria, thereby better treating various diseases caused by drug-resistant bacteria in aquatic animals. Further experiments demonstrate that potassium clavulanate can significantly reduce bacterial resistance and enhance the antibacterial activity of doxycycline hydrochloride. The preparation of a composite powder using the combination of doxycycline hydrochloride and potassium clavulanate is simple, uses readily available raw materials, and is suitable for large-scale industrial production. Its application in the treatment of aquatic animal diseases, verified through challenge experiments, demonstrates the effectiveness of this composite powder in treating bacterial infections in aquatic animals. It has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to doxycycline hydrochloride and potassium clavulanate powder for aquaculture, their preparation methods, and applications. Background Technology

[0002] Doxycycline is a semi-synthetic tetracycline antibiotic obtained by deoxygenating oxytetracycline at the 6α-position. Its antibacterial mechanism involves interfering with bacterial protein synthesis. It specifically binds to the A-position of the 30S subunit of the bacterial ribosome, thereby inhibiting the binding of aminoacyl-tRNA at this position and hindering peptide chain elongation. Furthermore, doxycycline increases the permeability of the pathogenic bacterial cell membrane, causing leakage of intracellular nucleotides and their important components, thus inhibiting bacterial DNA replication. Doxycycline is a broad-spectrum antibiotic with strong antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Escherichia coli, Aerobacterium aerogenes, and Shigella spp. It also has some effect against Rickettsia, Mycoplasma, Chlamydia, and Actinomyces. In aquaculture, it is mainly used to combat infections caused by Aeromonas hydrophila, Aeromonas vesiculosus, and Citrobacter freundii. Doxycycline hydrochloride is the hydrochloride salt of doxycycline, which has better water solubility, is more easily absorbed, and has high bioavailability, making it widely used in aquaculture. Doxycycline is an antibiotic developed in the 1960s and has a long history of use, beginning its application in aquaculture in 2002. However, with the continuous extension of its use and the irrational use of the drug, resistance to doxycycline in aquatic pathogens has become increasingly serious. Therefore, how to effectively reduce bacterial resistance and better exert the antibacterial activity of doxycycline through scientific and rational drug use strategies, innovative drug combination regimens, or novel drug formulation improvements is a pressing technical challenge in this field. Summary of the Invention

[0003] The purpose of this invention is to provide a powder of doxycycline hydrochloride and potassium clavulanate for aquaculture, its preparation method, and its application, in order to solve the problems existing in the prior art. This invention combines doxycycline hydrochloride with potassium clavulanate, which can enhance the antibacterial ability of aquatic animals against drug-resistant bacteria, thereby better treating various diseases in aquatic animals caused by drug-resistant bacteria.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the use of a composition of doxycycline hydrochloride and potassium clavulanate in the preparation of products that enhance the antimicrobial ability of aquatic animals against drug-resistant bacteria.

[0006] Optionally, the drug-resistant bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, and drug-resistant Citrobacter freundii.

[0007] Optionally, the drug-resistant bacteria are drug-resistant Aeromonas verrucosa.

[0008] Optionally, the composition comprises, by weight, 1-70 parts of doxycycline hydrochloride and 0.25-17.5 parts of potassium clavulanate.

[0009] Optionally, the composition comprises, by weight, 10-40 parts of doxycycline hydrochloride and 2.5-10 parts of potassium clavulanate.

[0010] Optionally, the product may include pharmaceutical formulations.

[0011] Optionally, the dosage form of the pharmaceutical preparation includes powder.

[0012] The present invention also provides a compound powder of doxycycline hydrochloride and potassium clavulanate for aquaculture, wherein the raw materials include 1-70 parts of doxycycline hydrochloride, 0.25-17.5 parts of potassium clavulanate and 12.50-98.75 parts of starch by weight.

[0013] Optionally, the preparation method includes the following steps:

[0014] The sieved doxycycline hydrochloride and potassium clavulanate were thoroughly mixed, and then the sieved starch was added and mixed again. The final mixture was then sieved again to obtain the aquatic doxycycline hydrochloride and potassium clavulanate compound powder.

[0015] This invention also provides the use of potassium clavulanate in reducing bacterial resistance to doxycycline hydrochloride.

[0016] The present invention discloses the following technical effects:

[0017] This invention combines doxycycline hydrochloride with potassium clavulanate to enhance the antibacterial ability of aquatic animals against drug-resistant bacteria, thereby improving the treatment of various diseases caused by drug-resistant bacteria in aquatic animals. Further experimental evidence demonstrates that potassium clavulanate significantly reduces bacterial resistance and enhances the antibacterial activity of doxycycline hydrochloride.

[0018] This invention utilizes a combination of doxycycline hydrochloride and potassium clavulanate to prepare a composite powder. The preparation process is simple, the raw materials are readily available, and it is suitable for large-scale industrial production. Its application in the treatment of aquatic animal diseases has been verified through challenge experiments, demonstrating the efficacy of this composite powder in treating bacterial infections in aquatic animals. It has broad application prospects. Attached Figure Description

[0019] 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.

[0020] Figure 1 This is a diagram of the chessboard method. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] This invention provides the application of potassium clavulanate in reducing bacterial resistance to doxycycline hydrochloride. Combined antibacterial studies have demonstrated a synergistic effect between doxycycline hydrochloride and potassium clavulanate in antibacterial activity.

[0027] In one specific embodiment, the drug-resistant bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, and drug-resistant Citrobacter freundii.

[0028] The present invention also uses doxycycline hydrochloride and potassium clavulanate to prepare a compound powder for aquaculture.

[0029] Optionally, the raw materials of the composite powder, by weight, include 1-70 parts of doxycycline hydrochloride and 0.25-17.5 parts of potassium clavulanate.

[0030] In a specific embodiment of the present invention, the raw materials of the composite powder, by weight, include 10-40 parts of doxycycline hydrochloride and 2.5-10 parts of potassium clavulanate.

[0031] The method for preparing the composite powder includes the following steps:

[0032] The sieved doxycycline hydrochloride and potassium clavulanate were thoroughly mixed, and then the sieved starch was added and mixed again. The final mixture was then sieved again to obtain the aquatic doxycycline hydrochloride and potassium clavulanate compound powder.

[0033] Optionally, the starch is 12.50-98.75 parts by weight.

[0034] In a specific embodiment of the present invention, the starch is 50-87.5 parts by weight.

[0035] This invention also provides the use of potassium clavulanate in reducing bacterial resistance to doxycycline hydrochloride.

[0036] In one specific embodiment, using yellow catfish as the experimental subject, the efficacy of the compound powder in treating bacterial infections in aquatic animals was verified through a challenge experiment.

[0037] Reagents and biomaterials involved in the embodiments of this invention:

[0038] Potassium clavulanate (CAS No.: 61177-45-5) was purchased from Jiangxi Ruiwei Biotechnology Co., Ltd., with a purity of 99%; doxycycline hydrochloride (CAS No.: 564-25-0) was purchased from Shandong Guobang Pharmaceutical Co., Ltd., as an active pharmaceutical ingredient, with a purity of 98%.

[0039] In this invention, drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, and drug-resistant Citrobacter freundii were all isolated from yellow catfish, and their species were confirmed through molecular biological verification.

[0040] The 16S sequence (SEQ ID NO.1) of the isolated drug-resistant Aeromonas verrucosa:

[0041]

[0042] The 16S sequence (SEQ ID NO.2) of the isolated drug-resistant Aeromonas hydrophila:

[0043]

[0044] The 16S sequence (SEQ ID NO.3) of the isolated drug-resistant Citrobacter freundii:

[0045]

[0046] Unless otherwise specified, all other experimental materials are standard materials in this field and can be purchased through conventional channels.

[0047] Example 1: Potassium clavulanate enhances the bactericidal activity of doxycycline hydrochloride against drug-resistant Aeromonas hydrophila.

[0048] 1. The minimum inhibitory concentrations (MICs) of potassium clavulanate and doxycycline hydrochloride against drug-resistant Aeromonas hydrophila were determined using the microdilution method.

[0049] 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.

[0050] 2. Determination of the combined effect of potassium clavulanate and doxycycline hydrochloride using the checkerboard method

[0051] Drug A: potassium clavulanate; Drug B: doxycycline.

[0052] See the diagram of the chessboard method. Figure 1 Take a 96-well bacterial culture plate, with row Y1 and column X1 as the rows and columns for drug A and drug B alone, respectively. Add 100 μL of standard drug A solution at concentrations of 1 / 8, 1 / 4, 1 / 2, 1, 2, and 4 MIC to wells 2-7 of row Y1, respectively; add 100 μL of standard drug B solution at concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC to wells 2-7 of column X1, respectively.

[0053] Add 50 μL of standard solution of drug A with concentrations of 1 / 8, 1 / 4, 1 / 2, 1, 2, and 4 MIC to wells 2-7 of row Y2-7, respectively; add 50 μL of standard solution of drug B with concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC to wells 2-7 of column X2-7, respectively.

[0054] Except for well X1Y1, add 100 μL of bacterial suspension to each of the other wells, and add 200 μL of bacterial suspension to well X1Y1.

[0055] At this time, the total volume of the liquid in the 96-well microplate is 200 μL. The 96-well microplate with the added samples is placed in an incubator at a constant temperature (28 °C) and cultured for 24 - 48 h. The lowest concentration at which the wells are observed to be clear is the MIC when the drugs are used in combination. The results are judged according to the equivalent midpoint method: all the tubes along the angular bisector of the 0 point are equivalent mid-tubes. For the combined use result, the lowest concentration without bacterial growth is read as 1 point. The point corresponding to this point on the X-axis is MIC A, and the point corresponding to it on the Y-axis is MIC B. The fractional inhibitory concentration (FIC) is used as the judgment basis for the combined drug sensitivity test, and the calculation formula is as follows:

[0056] FIC = ,

[0057] Among them, an FIC index ≤ 0.5 indicates a synergistic effect; 0.5 < FIC index ≤ 1 indicates an additive effect; 1 < FIC index ≤ 2 indicates an indifferent effect; and FIC index > 2 indicates an antagonistic effect.

[0058] Results: The MIC of clavulanic acid potassium against drug-resistant Aeromonas hydrophila is 16 μg / mL, and the MIC of doxycycline hydrochloride against drug-resistant Aeromonas hydrophila is 32 μg / mL. After the combined use of doxycycline hydrochloride and clavulanic acid potassium, the lowest concentrations at which the two drugs achieve no bacterial growth are: 2 μg / mL for clavulanic acid potassium and 16 μg / mL for doxycycline hydrochloride. The combined inhibitory index is 0.63, showing an additive effect.

[0059] Example 2: Enhancement of the bactericidal ability of doxycycline hydrochloride against drug-resistant Aeromonas veronii by clavulanic acid potassium

[0060] The measurement method is the same as that in Example 1.

[0061] Results: The MIC of clavulanic acid potassium against drug-resistant Aeromonas veronii is 128 μg / mL, and the MIC of doxycycline hydrochloride against drug-resistant Aeromonas veronii is 4 μg / mL. After the combined use of clavulanic acid potassium and doxycycline hydrochloride, the lowest concentrations at which the two drugs achieve no bacterial growth are: 32 μg / mL for clavulanic acid potassium and 0.5 μg / mL for doxycycline hydrochloride. The combined inhibitory index is 0.38, showing a synergistic effect.

[0062] Example 3: Enhancement of the bactericidal ability of doxycycline hydrochloride against drug-resistant Citrobacter freundii by clavulanic acid potassium

[0063] The measurement method is the same as that in Example 1.

[0064] Results: The MIC of potassium clavulanate against drug-resistant Aeromonas villi was 64 µg / mL, and the MIC of doxycycline hydrochloride against drug-resistant Aeromonas villi was 32 µg / mL. The minimum concentrations at which potassium clavulanate and doxycycline hydrochloride achieved sterile growth after combined use were: potassium clavulanate 32 µg / mL and doxycycline hydrochloride 4 µg / mL, with a combined inhibition index of 0.63, indicating an additive effect.

[0065] Example 4: Preparation of compound powder of doxycycline hydrochloride and potassium clavulanate

[0066] The compound powder of doxycycline hydrochloride and potassium clavulanate is prepared by mixing doxycycline hydrochloride, potassium clavulanate and starch in a certain proportion. The specific steps are as follows:

[0067] (1) Sift doxycycline hydrochloride, potassium clavulanate and starch separately and set aside.

[0068] (2) Weigh out doxycycline hydrochloride and potassium clavulanate separately according to a fixed ratio, and put them into a mixer to mix thoroughly;

[0069] (3) After the compounds in step (2) are mixed, sieved starch is added to the mixer in proportion and the mixture is continued. The mixed mixture is then sieved again to obtain the compound powder of doxycycline hydrochloride and potassium clavulanate of the present invention.

[0070] In the preparation of the above-mentioned compound powder of doxycycline hydrochloride and potassium clavulanate, the amount of doxycycline hydrochloride is 10-40 parts by weight, the amount of potassium clavulanate is 2.5-10 parts, and the amount of starch is 50-87.5 parts.

[0071] Example 5 Clinical Trial

[0072] 150 yellow catfish were randomly divided into four groups of 20 each.

[0073] The first group served as a blank control, receiving no viral load or treatment.

[0074] The second group served as a negative control, and was challenged with drug-resistant Aeromonas verrucosa derived from yellow catfish without treatment.

[0075] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish and treated with doxycycline hydrochloride at a dose of 20 mg / kg once a day for three consecutive days.

[0076] The fourth group, or treatment group 2, was challenged with drug-resistant Aeromonas villi from yellow catfish and treated with potassium clavulanate at a dose of 20 mg / kg, once a day for three consecutive days.

[0077] The fifth group, or treatment group 3, was challenged with drug-resistant Aeromonas villi from yellow catfish and treated with the compound powder of doxycycline hydrochloride and clavulanate potassium prepared in Example 4 (20 parts by weight of doxycycline hydrochloride, 5 parts of clavulanate potassium, and 75 parts of starch), at a dose of 20 mg / kg (calculated as doxycycline hydrochloride), once a day for three consecutive days.

[0078] After treatment, the yellow catfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 1.

[0079] Table 1. Test Results

[0080]

[0081] Based on the mortality results of each group in Table 1, the compound powder prepared from doxycycline hydrochloride and potassium clavulanate can be effectively used to treat diseases and infections in aquatic animals.

[0082] 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. The application of a composition of doxycycline hydrochloride and potassium clavulanate in the preparation of products that enhance the antibacterial ability of aquatic animals against drug-resistant bacteria; characterized in that, The drug-resistant bacteria are drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas verrucosa, and drug-resistant Citrobacter freundii; the composition comprises, by weight, 10-40 parts of doxycycline hydrochloride and 2.5-10 parts of potassium clavulanate.

2. The application according to claim 1, characterized in that, The drug-resistant bacteria are drug-resistant Aeromonas verrucosa.

3. The application according to claim 1, characterized in that, The products include pharmaceutical preparations.

4. The application according to claim 3, characterized in that, The dosage form of the pharmaceutical preparation includes powder.

5. A compound powder of doxycycline hydrochloride and potassium clavulanate for aquaculture, characterized in that, The raw materials, by weight, include 10-40 parts of doxycycline hydrochloride, 2.5-10 parts of potassium clavulanate, and 50-87.5 parts of starch.

6. The aquatic compound powder of doxycycline hydrochloride and potassium clavulanate according to claim 5, characterized in that, The preparation method includes the following steps: The sieved doxycycline hydrochloride and potassium clavulanate were thoroughly mixed, and then the sieved starch was added and mixed again. The final mixture was then sieved again to obtain the aquatic doxycycline hydrochloride and potassium clavulanate compound powder.

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