Preparation method of enrofloxacin compound powder for preventing drug resistance
By preparing a compound powder containing enrofloxacin hydrochloride and gallic acid, the problem of enrofloxacin hydrochloride resistance in aquaculture has been solved, achieving a highly effective antibacterial treatment effect, which is suitable for industrial production.
Patent Information
- Application Number
- CN202510050992.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2045-01-13
AI Technical Summary
In aquaculture, the problem of enrofloxacin hydrochloride resistance leads to poor treatment effects. Existing new antibiotics have long development cycles and high costs, and biological methods are not ideal.
A compound powder for preventing drug-resistant enrofloxacin hydrochloride was prepared, containing enrofloxacin hydrochloride, gallic acid, solubilizer, and starch. Gallic acid is used to reduce drug resistance and enhance the antibacterial ability of enrofloxacin hydrochloride.
It effectively reduces the resistance of pathogens to enrofloxacin hydrochloride, enhances antibacterial activity, improves treatment efficacy, and is suitable for industrial production.
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Figure CN119837875B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to a method for preparing a compound powder of enrofloxacin hydrochloride for preventing drug resistance. 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 hydrochloride 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, the long duration of its use, and the improper and excessive use of drugs, resistance to enrofloxacin hydrochloride in aquaculture pathogens has become increasingly serious. Minimum inhibitory concentrations (MICs) have reached tens of PPM, rendering conventional doses ineffective in treating diseases, leading to numerous treatment failures and economic losses.
[0003] Currently, the main methods for controlling drug-resistant pathogens include: exploring new antibiotics; and using biological methods such as microorganisms and plant extracts to control diseases. However, the development cycle for new antibiotics is long and the cost is high; and the effectiveness of using biological methods such as microorganisms and plant extracts to control diseases is not ideal. Therefore, there is an urgent need to develop new, low-cost, and efficient control methods. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing enrofloxacin hydrochloride compound powder for preventing drug resistance, so as to solve the problems existing in the prior art.
[0005] To achieve the above objectives, the present invention provides the following solution:
[0006] One of the technical solutions of the present invention is a compound powder for preventing drug-resistant enrofloxacin hydrochloride, which, by mass parts, comprises the following raw materials: 1-30 parts of enrofloxacin hydrochloride, 2-46 parts of gallic acid, 0.5-9 parts of solubilizer, 0.5-9 parts of absorption promoter, and 0-92 parts of starch.
[0007] Furthermore, the compound powder for preventing drug-resistant enrofloxacin hydrochloride comprises, by weight, the following raw materials: 10-20 parts enrofloxacin hydrochloride, 20-40 parts gallic acid, 3-9 parts solubilizer, 3-9 parts absorption promoter, and 16-60 parts starch.
[0008] Furthermore, the raw materials of the anti-drug-resistant enrofloxacin hydrochloride compound powder also include 4-6 parts of baicalin.
[0009] Furthermore, the solubilizer comprises sodium dodecyl sulfonate;
[0010] The absorption enhancer includes mannose.
[0011] Gallic acid can drastically reduce the drug resistance of resistant bacteria and enhance the antibacterial ability of enrofloxacin hydrochloride, thereby restoring the therapeutic effect.
[0012] The second technical solution of the present invention: a method for preparing the above-mentioned compound powder for preventing drug-resistant enrofloxacin hydrochloride, comprising the following steps:
[0013] After mixing all raw materials except starch evenly, starch is added and mixed evenly to obtain the compound powder for preventing drug-resistant enrofloxacin hydrochloride.
[0014] The third technical solution of the present invention: the application of the above-mentioned anti-drug-resistant enrofloxacin hydrochloride compound powder in the preparation of drugs for preventing and treating pathogens.
[0015] The fourth technical solution of the present invention: the application of the above-mentioned anti-drug-resistant enrofloxacin hydrochloride compound powder in the preparation of a medicine for preventing and treating diseases caused by drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, or drug-resistant Citrobacter freundii.
[0016] Furthermore, the drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, or drug-resistant Citrobacter freundii are enrofloxacin-resistant Aeromonas hydrophila, Aeromonas vernix, or Citrobacter freundii.
[0017] The present invention discloses the following technical effects:
[0018] The compound powder of enrofloxacin hydrochloride of the present invention can effectively treat various diseases and infections in aquatic animals. Gallic acid in the powder can greatly reduce the resistance of pathogens to enrofloxacin hydrochloride and enhance the antibacterial activity of enrofloxacin hydrochloride.
[0019] The enrofloxacin hydrochloride compound powder of the present invention can increase the solubility of gallic acid, promote absorption, and enhance the antibacterial therapeutic effect.
[0020] The preparation of the anti-drug-resistant enrofloxacin hydrochloride compound powder of the present 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
[0021] 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.
[0022] Figure 1 This is a diagram of the chessboard method. Detailed Implementation
[0023] 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.
[0024] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.
[0025] 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.
[0026] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0027] 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.
[0028] In the following examples, "parts" refers to "parts by weight".
[0029] The identification results of drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, and drug-resistant Citrobacter freundii are as follows:
[0030] (1) 16S rDNA sequence of drug-resistant Aeromonas hydrophila from yellow catfish (SEQ ID NO.1):
[0031]
[0032]
[0033] (2) 16S rDNA sequence of drug-resistant Aeromonas verrucosa from yellow catfish (SEQ ID NO.2):
[0034]
[0035]
[0036] (3) 16S rDNA sequence of drug-resistant Citrobacter freundii from Procambarus clarkii (SEQ ID NO.3):
[0037]
[0038]
[0039] Example 1
[0040] Gallic acid enhances the bactericidal activity of enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila:
[0041] (1) The minimum inhibitory concentrations (minimum concentrations for sterile growth) of gallic acid and enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila were determined using the microdilution method, specifically:
[0042] First, add 200 μL of the drug working solution (the concentration of gallic acid drug working solution is 1024 μg / mL, which is prepared by diluting gallic acid standard stock solution (concentration 5120 μg / mL) with sterile MH broth; the concentration of enrofloxacin hydrochloride drug working solution is 256 μg / mL, which is prepared by diluting enrofloxacin hydrochloride standard stock solution (concentration 2560 μg / mL) with sterile MH broth) to the preset maximum drug concentration well. 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. After mixing by repeatedly pipetting 5 times, add 100 μL to the third well. Perform serial dilutions sequentially, and discard the 100 μL of liquid drawn from the last well. At this point, each well contains 100 μL of drug-containing broth, with gallic acid concentrations ranging from 512 μg / mL to 0.125 μg / mL and enrofloxacin hydrochloride concentrations ranging from 128 μg / mL to 0.0313 μg / mL. Then, 100 μL of a suspension of drug-resistant Aeromonas hydrophila (containing 10...) is added to each well containing the drug-containing broth. 7 The drug concentration (cfu / mL) is placed in a constant temperature incubator (28℃) and incubated for at least 24 hours. After that, observe whether there are clear wells. The drug concentration in the clear wells is the minimum concentration for sterile growth, which is the MIC.
[0043] (2) Determination of the combined effect of gallic acid and enrofloxacin hydrochloride using the checkerboard method
[0044] Take a 96-well bacterial culture plate, Y1 row and X1 column are the rows and columns for drug A (gallic acid) and drug B (enrofloxacin hydrochloride) alone, respectively, and the drug addition method is shown in Table 1;
[0045] Add drug A to wells 2-7 of row Y1. 100 μL of standard drug solutions with concentrations of 1 / 32, 1 / 16, 1 / 8, 1 / 4, 1 / 2, and 1 times the MIC (128 μg / mL) of drug A are respectively referred to as drug A 1 / 32, drug A 1 / 16, drug A 1 / 8, drug A 1 / 4, drug A 1 / 2, and drug A 1.
[0046] Add drug B to wells 2-7 of column X1. The concentrations of drug B are 1 / 8, 1 / 4, 1 / 2, 1, 2, and 4 times the MIC (16 μg / mL) of the standard drug solution, respectively, and 100 μL each (abbreviated as drug B 1 / 8, drug B 1 / 4, drug B 1 / 2, drug B 1, drug B 2, and drug B 4).
[0047] Add drug A to wells 2-7 of rows Y2-7. Add 50 μL of standard drug solution at concentrations of 1 / 16, 1 / 8, 1 / 4, 1 / 2, 1, and 2 times the MIC (128 μg / mL).
[0048] Add drug B to wells 2-7 in columns X2-7. The concentrations of drug B are 50 μL of standard drug solutions that are 1 / 4, 1 / 2, 1, 2, 4, and 8 times the MIC (16 μg / mL).
[0049] Add 200 μL of a bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10⁻⁶ bacteria) to well X1Y1. 7 Add 100 μL of a suspension of drug-resistant Aeromonas hydrophila (containing 10 cfu / mL) to each of the remaining wells. 7 The total volume of liquid in the 96-well plate is 200 μL (cfu / mL). Place the 96-well plate with the added sample in a constant temperature incubator (28℃) for 24–48 h and observe whether there are clear wells. The drug concentration in the clear wells is the lowest concentration at which sterile growth is achieved when the drugs are used in combination, which is the MIC.
[0050] Results were determined using the equivalent midpoint method: each tube along the angle bisector of the zero point was considered an equivalent midpoint tube. The lowest concentration at which sterile growth was observed was recorded as point 1. The point corresponding to this point on the X-axis was MICA (MIC for drug A in combination therapy), and the corresponding point on the Y-axis was MICB (MIC for drug B in combination therapy). The partial concentration inhibition index (FIC) was used as the basis for determining the effectiveness of the combined drug susceptibility test. The calculation formula is as follows:
[0051]
[0052] 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.
[0053] Table 1. Dosing Methods
[0054]
[0055] The results showed that the MIC of gallic acid against drug-resistant Aeromonas hydrophila was 128 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila was 16 μg / mL.
[0056] When gallic acid and enrofloxacin hydrochloride are used in combination, the minimum concentration for sterile growth is 4 μg / mL for gallic acid and 2 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.156, indicating a synergistic effect.
[0057] Example 2
[0058] Gallic acid enhances the bactericidal activity of enrofloxacin hydrochloride against drug-resistant Aeromonas verrucosa:
[0059] Same as Example 1, except that the bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10...) was used. 7 Replace (cfu / mL) with a bacterial suspension of drug-resistant Aeromonas verrucosa (containing 10) 7 (cfu / mL).
[0060] The results showed that the MIC of gallic acid against drug-resistant Aeromonas villus was 256 μg / mL, while the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas villus was 8 μg / mL. The minimum concentration for sterility after the combined use of gallic acid and enrofloxacin hydrochloride was 8 μg / mL for gallic acid and 4 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.531, indicating an additive effect.
[0061] Example 3
[0062] Gallic acid enhances the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii:
[0063] Same as Example 1, except that the bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10...) was used. 7 Replace the cfu / mL with a suspension of drug-resistant Citrobacter freundii (containing 10 CFU / mL). 7 (cfu / mL).
[0064] The results showed that the MIC of gallic acid against drug-resistant Citrobacter freundii was 512 μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii was 16 μg / mL. The minimum concentration for sterile growth after the combined use of gallic acid and enrofloxacin hydrochloride was 8 μg / mL for gallic acid and 4 μg / mL for enrofloxacin hydrochloride, with a combined inhibition index of 0.266, indicating a synergistic effect.
[0065] Example 4
[0066] Clinical trials:
[0067] (1) Preparation method of compound enrofloxacin hydrochloride and gallic acid powder for preventing drug resistance:
[0068] Enrofloxacin hydrochloride, gallic acid, solubilizer (sodium dodecyl sulfonate), absorption enhancer (mannose), and starch were sieved (70 mesh) separately and set aside.
[0069] Weigh out 10 parts of enrofloxacin hydrochloride, 20 parts of gallic acid, 3 parts of solubilizer, and 3 parts of absorption promoter, and place them in a mixer to mix thoroughly. Then add 64 parts of starch to the mixer, mix thoroughly, and sieve again (24 mesh) to obtain the compound enrofloxacin hydrochloride and gallic acid powder for preventing drug resistance.
[0070] (2) Preparation method of compound enrofloxacin hydrochloride, gallic acid and baicalein powder for preventing drug resistance:
[0071] Enrofloxacin hydrochloride, gallic acid, baicalin, solubilizer (sodium dodecyl sulfonate), absorption enhancer (mannose), and starch were sieved (70 mesh) separately and set aside.
[0072] Weigh out 10 parts of enrofloxacin hydrochloride, 20 parts of gallic acid, 5 parts of baicalein, 3 parts of solubilizer, and 3 parts of absorption promoter, and place them in a mixer to mix thoroughly. Then add 59 parts of starch to the mixer, mix thoroughly, and sieve again (24 mesh) to obtain a compound powder containing enrofloxacin hydrochloride, gallic acid, and baicalein to prevent drug resistance.
[0073] (3) Take 180 yellow catfish (average weight 128.1±12.6g / fish) and randomly divide them into six groups of 30 fish each.
[0074] The first group served as a blank control group, receiving no viral load or treatment.
[0075] The second group served as the negative control group, which was challenged with drug-resistant Aeromonas verrucosa derived from yellow catfish (challenge time was 2-3 days). The challenge method was to inject 0.1 mL of a 1×10⁻⁶ solution into each fish. 8 A successful challenge with Aeromonas vesiculosus culture at cfu / mL results in inflammation and protrusion of the cloaca, abdominal distension, and ascites upon autopsy, without treatment.
[0076] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish (the challenge method was the same as above). After successful challenge, enrofloxacin hydrochloride was administered at a dose of 15 mg / kg, twice a day for three consecutive days.
[0077] The fourth group was treatment group 2, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish (the challenge method was the same as above). After successful challenge, gallic acid was administered at a dose of 20 mg / kg, twice a day for three consecutive days.
[0078] The fifth group was treatment group 3, which was challenged with drug-resistant Aeromonas verrucosa from yellow catfish (the challenge method was the same as above). After successful challenge, the patient was treated with compound enrofloxacin hydrochloride and gallic acid powder to prevent drug resistance. The dosage was 15 mg / kg (calculated as enrofloxacin hydrochloride), twice a day for three consecutive days.
[0079] Group 6, or treatment group 4, was challenged with drug-resistant Aeromonas verrucosa from yellow catfish (challenge method as above). After successful challenge, patients were treated with a compound preparation of enrofloxacin hydrochloride, gallic acid and baicalein powder to prevent drug resistance. The dosage was 10 mg / kg (calculated as enrofloxacin hydrochloride), twice a day for three consecutive days.
[0080] After treatment, the yellow catfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 2.
[0081] Table 2 Experimental Results
[0082]
[0083]
[0084] Example 5
[0085] 150 yellow catfish (average weight 130.2±18.1g / fish) were randomly divided into five groups of 30 fish each.
[0086] The first group served as a blank control group, receiving no viral load or treatment.
[0087] The second group served as the negative control group, which was challenged with drug-resistant Aeromonas hydrophila derived from yellow catfish (challenge time was 2-3 days). The challenge method was to inject 0.1 mL of a 1×10⁻⁶ solution into each fish. 8 A cfu / mL drug-resistant Aeromonas hydrophila culture, when successfully challenged, showed abdominal distension, and autopsy revealed intestinal congestion and swelling, without treatment;
[0088] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas hydrophila from yellow catfish (the challenge method was the same as above). After successful challenge, enrofloxacin was administered at a dose of 15 mg / kg, twice a day for three consecutive days.
[0089] The fourth group was treatment group 2, which was challenged with drug-resistant Aeromonas hydrophila from yellow catfish (the challenge method was the same as above). After successful challenge, gallic acid was administered at a dose of 20 mg / kg, twice a day for three consecutive days.
[0090] The fifth group was treatment group 3, which was challenged with drug-resistant Aeromonas hydrophila from yellow catfish (the challenge method was the same as above). After successful challenge, the patient was treated with compound enrofloxacin and gallic acid powder to prevent drug resistance, at a dose of 15 mg / kg (calculated as enrofloxacin), twice a day for three consecutive days.
[0091] After treatment, the yellow catfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 3.
[0092] Table 3. Experimental Results
[0093]
[0094]
[0095] Example 6
[0096] 150 red swamp crayfish (average weight 19.7±4.8g / crayfish) were randomly divided into five groups of 30 crayfish each.
[0097] The first group served as a blank control group, receiving no viral load or treatment.
[0098] The second group served as the negative control group, which was challenged with drug-resistant Citrobacter freundii from Procambarus clarkii (challenge time was 2-3 days). The challenge method was to inject 0.1 mL of a 1×10⁻⁶ CFU / mL solution into each shrimp. 8 When challenged with a cfu / mL resistant Citrobacter freundii solution, the crawfish exhibited signs of slow movement, red and swollen tails, or even ulceration, without treatment.
[0099] The third group was treatment group 1, which was challenged with drug-resistant Citrobacter freundii from Procambarus clarkii (challenge method as above). After successful challenge, enrofloxacin was administered at a dose of 15 mg / kg, twice a day for three consecutive days.
[0100] The fourth group was treatment group 2, which was challenged with drug-resistant Citrobacter freundii from Procambarus clarkii (challenge method as above). After successful challenge, gallic acid was administered at a dose of 20 mg / kg twice a day for three consecutive days.
[0101] The fifth group was the treatment group 3, which was challenged with drug-resistant Citrobacter freundii from Procambarus clarkii (challenge method as above). After successful challenge, the patient was treated with compound enrofloxacin and gallic acid powder to prevent drug resistance, at a dose of 15 mg / kg (calculated as enrofloxacin), twice a day for three consecutive days.
[0102] After treatment, the red swamp crayfish were observed for 7 days, and the mortality rate was calculated. The results are shown in Table 4.
[0103] Table 4. Test Results
[0104]
[0105]
[0106] 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 use of a compound powder containing antidrug-resistant enrofloxacin hydrochloride in the preparation of a pharmaceutical agent for the prevention and treatment of diseases caused by drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas vernix, or drug-resistant Citrobacter freundii, characterized in that, The compound powder for preventing drug-resistant enrofloxacin hydrochloride comprises, by weight, the following raw materials: 1-30 parts enrofloxacin hydrochloride, 2-46 parts gallic acid, 0.5-9 parts solubilizer, 0.5-9 parts absorption promoter, and 0-92 parts starch; The raw materials also include 4 to 6 parts of baicalin.
2. The application according to claim 1, characterized in that, The solubilizer includes sodium dodecyl sulfonate; And / or, the absorption enhancer includes mannose.
3. The application according to claim 1, characterized in that, The preparation method of the enrofloxacin hydrochloride compound powder for preventing drug resistance includes the following steps: After mixing all raw materials except starch evenly, starch is added and mixed evenly to obtain the compound powder for preventing drug-resistant enrofloxacin hydrochloride.
4. The application according to claim 1, characterized in that, The drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas versicolor, or drug-resistant Citrobacter freundii are enrofloxacin-resistant Aeromonas hydrophila, Aeromonas versicolor, or Citrobacter freundii.