Doxycycline hydrochloride and potassium clavulanate powder for aquatic products as well as preparation method and application of doxycycline hydrochloride and potassium clavulanate powder

Abstract

The invention discloses doxycycline hydrochloride and potassium clavulanate powder for aquatic products as well as a preparation method and application of the doxycycline hydrochloride and potassium clavulanate powder, and belongs to the technical field of biology. The doxycycline hydrochloride and the potassium clavulanate are combined for use, so that the antibacterial ability of the aquatic animals on drug-resistant bacteria can be enhanced, and various diseases of the aquatic animals caused by the drug-resistant bacteria can be better treated. It is further proved through experiments that potassium clavulanate can greatly reduce the drug resistance of bacteria and enhance the antibacterial activity of doxycycline hydrochloride. The doxycycline hydrochloride and the potassium clavulanate are combined to prepare the compound powder, the preparation process is simple, the raw materials are easy to obtain, and the compound powder is suitable for industrial large-scale production. When the compound powder is applied to treatment of aquatic animal diseases, the effect of the compound powder in treatment of aquatic animal bacterial infection diseases is verified through a challenge experiment. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to doxycycline hydrochloride and potassium clavulanate powder for aquatic products, and a preparation method and application thereof. Background Art

[0002] Doxycycline is a semi-synthetic tetracycline antibiotic obtained by deoxygenation of oxytetracycline at the 6α-position. Its antibacterial mechanism is to interfere with bacterial protein synthesis. It can specifically bind to the bacterial ribosome 30S subunit at the A position, thereby inhibiting the binding of aminoacyl-tRNA at this position and hindering the extension of the peptide chain. In addition, doxycycline can also increase the permeability of the cell membrane of pathogens, allowing nucleotides and their important components in the cell to leak out, thereby inhibiting the replication of bacterial DNA. Doxycycline is a broad-spectrum antibiotic with strong antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, gonococci, meningococci, Escherichia coli, Aeromonas, Shigella, etc. It also has certain effects on Rickettsia, Mycoplasma, Chlamydia, Actinomycetes, etc. In aquaculture, it is mainly used to fight against infections of pathogens such as Aeromonas hydrophila, Aeromonas veronii, and Citrobacter freundii. Doxycycline hydrochloride is the hydrochloride of doxycycline, which has better water solubility, easier absorption, high bioavailability, and is widely used in aquaculture. Doxycycline is an antibiotic developed in the 1960s and has a long history of use. It has been used in aquaculture since 2002. With the continuous extension of the use time and the irrational use of drugs, the resistance of aquatic pathogens to doxycycline is becoming more and more serious. Therefore, how to effectively reduce bacterial resistance through scientific and reasonable drug use strategies, innovative drug combination schemes, or new drug dosage form improvements, so as to better exert the antibacterial activity of doxycycline, is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0003] The purpose of the present invention is to provide a doxycycline hydrochloride and potassium clavulanate powder for aquaculture and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention combines doxycycline hydrochloride with 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.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides application of a composition of doxycycline hydrochloride and potassium clavulanate in preparing a product for enhancing the antibacterial ability of aquatic animals against drug-resistant bacteria.

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

[0007] Optionally, the drug-resistant bacteria is drug-resistant Aeromonas veronii.

[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 includes a pharmaceutical preparation and a feed additive.

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

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

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

[0014] The sieved doxycycline hydrochloride and potassium clavulanate are fully mixed, and then the sieved starch is added and continued to be mixed. The final mixture is further sieved to obtain the doxycycline hydrochloride and potassium clavulanate compound powder for aquaculture.

[0015] The invention also provides the use of potassium clavulanate in reducing the drug resistance of bacteria to doxycycline hydrochloride.

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

[0017] The present invention combines doxycycline hydrochloride with 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. The present invention further confirms through experiments that potassium clavulanate can greatly reduce the drug resistance of bacteria and enhance the antibacterial activity of doxycycline hydrochloride.

[0018] The present invention combines doxycycline hydrochloride with potassium clavulanate to prepare a composite powder, which has a simple preparation process, readily available raw materials, and is suitable for industrial large-scale production. The composite powder is used in the treatment of aquatic animal diseases, and the effect of the composite powder in treating bacterial infection diseases of aquatic animals is verified by a challenge experiment. The composite powder has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is a chessboard pattern diagram. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The present invention provides an application of potassium clavulanate in reducing bacterial resistance to doxycycline hydrochloride. The combined antibacterial effect proves that the combination of doxycycline hydrochloride and potassium clavulanate can produce a synergistic effect in antibacterial effect.

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

[0028] The present invention also combines doxycycline hydrochloride with potassium clavulanate to prepare a composite powder for aquaculture.

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

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

[0031] The composite powder preparation method comprises the following steps:

[0032] The sieved doxycycline hydrochloride and potassium clavulanate are fully mixed, and then the sieved starch is added and continued to be mixed. The final mixture is further sieved to obtain the doxycycline hydrochloride and potassium clavulanate compound powder for aquaculture.

[0033] Optionally, the starch is present in an amount of 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] The invention also provides the use of potassium clavulanate in reducing the drug resistance of bacteria to doxycycline hydrochloride.

[0036] In a specific embodiment, yellow catfish was used as the experimental subject, and the effect of the composite powder in treating bacterial infection diseases of aquatic animals was verified through a toxicity challenge experiment.

[0037] Reagents and biological materials involved in the embodiments of the present invention:

[0038] Potassium clavulanate (CAS No.: 61177-45-5) was purchased from Jiangxi Ruiweier Biotechnology Co., Ltd., with a content of 99%, and doxycycline hydrochloride (CAS No.: 564-25-0) was purchased from Shandong Guobang Pharmaceutical Co., Ltd., raw material, with a content of 98%;

[0039] In the present invention, the drug-resistant Aeromonas hydrophila, the drug-resistant Aeromonas veronii and the drug-resistant Citrobacter freundii are all separated from yellow catfish, and their species are confirmed through molecular biological verification.

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

[0041]

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

[0043]

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

[0045]

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

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

[0048] 1. The minimum inhibitory concentration of potassium clavulanate and doxycycline hydrochloride against drug-resistant Aeromonas hydrophila was determined by microdilution method

[0049] 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.

[0050] 2. Chessboard method for determining the effect of combined use of clavulanate potassium and doxycycline hydrochloride

[0051] Drug A: Potassium clavulanate, Drug B: Doxycycline.

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

[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 in row Y2-7, and 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 in column X2-7.

[0054] Except for well X1Y1, 100 μL of bacterial suspension was added to the remaining wells, and 200 μL of bacterial suspension was added 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 of the wells observed to be clear is the MIC during combined drug use. The results are determined by reading 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 results, the lowest concentration without bacterial growth is read as point 1. The point corresponding to this point on the X-axis is MIC A, and the point corresponding to 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]

[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; FIC index > 2 indicates an antagonistic effect.

[0058] Results: The MIC of clavulanate 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 clavulanate potassium, the lowest concentrations of the two drugs to achieve no bacterial growth are: clavulanate potassium is 2 μg / mL, and doxycycline hydrochloride is 16 μg / mL. The combined inhibitory index is 0.63, showing an additive effect.

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

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

[0061] Results: The MIC of clavulanate 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 clavulanate potassium and doxycycline hydrochloride, the lowest concentrations of the two drugs to achieve no bacterial growth are: clavulanate potassium is 32 μg / mL, and doxycycline hydrochloride is 0.5 μg / mL. The combined inhibitory index is 0.38, showing a synergistic effect.

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

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

[0064] Results: The MIC of potassium clavulanate against drug-resistant Aeromonas veronii was 64 μg / mL, and the MIC of doxycycline hydrochloride against drug-resistant Aeromonas veronii was 32 μg / mL. After potassium clavulanate and doxycycline hydrochloride were used together, the minimum concentration of the two drugs to achieve sterile growth was: potassium clavulanate was 32 μg / mL, doxycycline hydrochloride was 4 μg / mL, and the combined antibacterial index was 0.63, which was an additive effect.

[0065] Example 4 Preparation of Doxycycline Hydrochloride and Clavulanate Potassium Compound Powder

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

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

[0068] (2) weighing doxycycline hydrochloride and potassium clavulanate in a fixed ratio, placing them in a mixer in sequence and mixing them thoroughly;

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

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

[0071] Example 5 Clinical Trial

[0072] A total of 150 yellow catfish were randomly divided into four groups, with 20 fish in each group.

[0073] The first group was a blank control, without virus attack or treatment;

[0074] The second group was a negative control, which was challenged with drug-resistant Aeromonas veronii from yellow catfish and received no treatment;

[0075] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas veronii 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 was treatment group 2, which was challenged with drug-resistant Aeromonas veronii 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 was treatment group 3, which was challenged with drug-resistant Aeromonas veronii from yellow catfish, and treated with the compound powder of doxycycline hydrochloride and potassium clavulanate prepared in Example 4 (in parts by weight, 20 parts of doxycycline hydrochloride, 5 parts of potassium clavulanate, and 75 parts of starch), with a dose of 20 mg / kg (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] Group Number of dead animals mortality rate Blank control group 0 0.00％ Negative control group 30 100％ Treatment Group 1 (doxycycline hydrochloride) 21 70.0％ Treatment Group 2 (potassium clavulanate) 20 66.67％ Treatment group 3 (doxycycline hydrochloride and clavulanate potassium combination powder) 1 3.33％

[0081] According to the mortality results of each group in Table 1, the compound powder prepared by doxycycline hydrochloride and potassium clavulanate can be well used to treat disease infections of aquatic animals.

[0082] 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. Use of a combination of doxycycline hydrochloride and potassium clavulanate in the preparation of a product for enhancing the antibacterial ability of aquatic animals against drug-resistant bacteria.

2. The use according to claim 1, characterized in that: The drug-resistant bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii.

3. The use according to claim 2, characterized in that: The drug-resistant bacteria are drug-resistant Aeromonas veronii.

4. The use according to claim 1, characterized in that: The composition comprises, by weight, 1-70 parts of doxycycline hydrochloride and 0.25-17.5 parts of potassium clavulanate.

5. The use according to claim 4, characterized in that: The composition comprises, by weight, 10-40 parts of doxycycline hydrochloride and 2.5-10 parts of potassium clavulanate.

6. The use according to claim 1, characterized in that: The products include pharmaceutical preparations and feed additives.

7. The use according to claim 6, characterized in that: The dosage form of the pharmaceutical preparation includes powder.

8. A compound powder of doxycycline hydrochloride and clavulanate potassium for aquaculture, characterized in that: 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 in parts by weight.

9. The doxycycline hydrochloride and clavulanate potassium compound powder for aquaculture according to claim 8, characterized in that: The preparation method comprises the following steps: The sieved doxycycline hydrochloride and potassium clavulanate are fully mixed, and then the sieved starch is added and continued to be mixed. The final mixture is further sieved to obtain the doxycycline hydrochloride and potassium clavulanate compound powder for aquaculture.

10. Application of potassium clavulanate in reducing bacterial resistance to doxycycline hydrochloride.

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