Preparation method of anti-drug resistance enrofloxacin hydrochloride compound powder

Abstract

The invention discloses a preparation method of anti-drug resistance enrofloxacin hydrochloride compound powder, and belongs to the technical field of medicine preparation. The anti-drug resistance enrofloxacin hydrochloride compound powder is prepared from the following raw materials in parts by mass: 1 to 30 parts of enrofloxacin hydrochloride, 2 to 46 parts of gallic acid, 0.5 to 9 parts of solubilizer, 0.5 to 9 parts of absorption enhancer and 0 to 92 parts of starch. The drug-resistant enrofloxacin hydrochloride compound powder can well treat various disease infections of aquatic animals, the gallic acid in the powder can greatly reduce the drug resistance of pathogenic bacteria to enrofloxacin hydrochloride, and the antibacterial activity of enrofloxacin hydrochloride is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, in particular to a method for preparing an anti-drug resistance enrofloxacin hydrochloride compound powder. 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 hydrochloride 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, the long application time, and the irregular use and abuse of drugs, the drug resistance of enrofloxacin hydrochloride to pathogens in aquaculture is becoming 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] At present, the main methods for preventing and controlling drug-resistant pathogens are: exploring new antibiotics; using biological methods such as microorganisms and plant extracts to prevent and control diseases. However, the development cycle of new antibiotics is long and the cost is high; the effect of using biological methods such as microorganisms and plant extracts to prevent and control diseases is not very ideal. Therefore, it is urgent to develop new low-cost and efficient prevention and control methods. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing an anti-resistance enrofloxacin hydrochloride compound powder to solve the problems existing in the above-mentioned prior art.

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

[0006] One of the technical solutions of the present invention: the anti-drug resistance enrofloxacin hydrochloride compound powder comprises the following raw materials, measured by weight: 1 to 30 parts of enrofloxacin hydrochloride, 2 to 46 parts of gallic acid, 0.5 to 9 parts of solubilizer, 0.5 to 9 parts of absorption promoter and 0 to 92 parts of starch.

[0007] Furthermore, the anti-resistance enrofloxacin hydrochloride compound powder comprises the following raw materials, measured by mass: 10 to 20 parts of enrofloxacin hydrochloride, 20 to 40 parts of gallic acid, 3 to 9 parts of solubilizer, 3 to 9 parts of absorption promoter and 16 to 60 parts of starch.

[0008] Furthermore, the raw materials of the anti-resistance enrofloxacin hydrochloride compound powder also include 4 to 6 parts of baicalin.

[0009] Further, the solubilizing agent includes sodium dodecyl sulfate;

[0010] The absorption enhancer includes mannose.

[0011] Gallic acid can drastically reduce the drug resistance of drug-resistant bacteria and enhance the antibacterial ability of enrofloxacin hydrochloride, thereby restoring the therapeutic effect.

[0012] The second technical solution of the present invention is a method for preparing the above-mentioned enrofloxacin hydrochloride compound powder for preventing drug resistance, comprising the following steps:

[0013] The raw materials other than starch are mixed evenly, and then starch is added and mixed evenly to obtain the anti-resistance enrofloxacin hydrochloride compound powder.

[0014] The third technical solution of the present invention: an application of the above-mentioned enrofloxacin hydrochloride compound powder for preventing and treating drug resistance in the preparation of drugs for preventing and treating pathogens.

[0015] The fourth technical solution of the present invention: an application of the above-mentioned enrofloxacin hydrochloride compound powder for preventing and treating diseases caused by drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii.

[0016] Furthermore, the drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii is Aeromonas hydrophila, Aeromonas veronii or Citrobacter freundii resistant to enrofloxacin hydrochloride.

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

[0018] The anti-resistance enrofloxacin hydrochloride compound powder of the present invention can well treat various disease infections of aquatic animals, and the 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 anti-resistance enrofloxacin hydrochloride compound powder of the present invention can increase the solubility of gallic acid, promote absorption, and enhance the antibacterial treatment effect.

[0020] The preparation of the anti-drug resistance enrofloxacin hydrochloride compound powder of the present invention is simple, the raw materials are easily available, the powder is suitable for industrial large-scale production, and the application prospect is broad. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 This is a chessboard pattern diagram. DETAILED DESCRIPTION

[0023] 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.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0025] 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.

[0026] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The “parts” described in the following examples are all “parts by mass”.

[0029] The identification results of drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii, 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 veronii 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 increases the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila:

[0041] (1) The minimum inhibitory concentration (minimum concentration for sterile growth) of gallic acid and enrofloxacin hydrochloride against drug-resistant Aeromonas hydrophila was determined by microdilution method, specifically:

[0042] First, draw 200 μL of 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 sterilized 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 sterilized MH broth) and add it to the preset maximum drug concentration well, then add 100 μL of sterile MH broth to the remaining wells, then draw 100 μL of drug working solution from the maximum drug concentration well and add it to the second well. After repeatedly pipetting and beating 5 times to mix, draw 100 μL and add it to the third well. Make multiple dilutions in sequence backwards, and discard the 100 μL of liquid aspirated from the last well. At this time, each well contains 100 μL of drug-containing broth, the concentration of gallic acid is 512 μg / mL to 0.125 μg / mL, and the concentration of enrofloxacin hydrochloride is 128 μg / mL to 0.0313 μg / mL. Add 100 μL of drug-resistant Aeromonas hydrophila bacterial suspension (containing 10 7 cfu / mL), and then cultured in a constant temperature incubator (28°C) for at least 24 hours, and then observed whether there were clear holes. The drug concentration in the clear holes was the minimum concentration for sterile growth, which was the MIC.

[0043] (2) Chessboard method to determine the combined effect of gallic acid and enrofloxacin hydrochloride

[0044] Take a 96-well bacterial culture plate, and the Y1 row and X1 column are the rows and columns for drug A (gallic acid) and drug B (enrofloxacin hydrochloride) to be used alone, respectively. The dosing method is shown in Table 1;

[0045] Drug A was added to wells 2 to 7 of row Y1. The concentrations of drug A were 1 / 32, 1 / 16, 1 / 8, 1 / 4, 1 / 2, and 1 times the MIC (128 μg / mL), 100 μL each of standard drug solutions (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 to 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), 100 μL of standard drug solution (referred to as drug B 1 / 8, drug B 1 / 4, drug B 1 / 2, drug B 1, drug B 2, and drug B 4);

[0047] Drug A was also added to wells 2 to 7 of rows Y2 to 7. The concentrations of drug A were 1 / 16, 1 / 8, 1 / 4, 1 / 2, 1, and 2 times the MIC (128 μg / mL), 50 μL each of standard drug solutions;

[0048] Drug B was also added to wells 2 to 7 of columns X2 to 7. The concentrations of drug B were 50 μL of standard drug solutions that were 1 / 4, 1 / 2, 1, 2, 4, and 8 times the MIC (16 μg / mL), respectively.

[0049] Add 200 μL of the bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10 7 cfu / mL), and 100 μL of the bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10 7 cfu / mL), at this time, the total volume of the liquid in the 96-well plate is 200μL, the 96-well plate with the sample is placed in a constant temperature incubator (28℃) for 24-48h, and the drug concentration in the clear well is the minimum concentration for aseptic growth when the drug is used in combination, which is the MIC.

[0050] The results are determined by reading the equivalent midpoint method: all tubes along the 0-point angle bisector are equivalent midpoint tubes, and the lowest concentration of sterile growth is read as point 1. The point on the X-axis corresponding to this point is MICA (MIC for drug A compound), and the point on the Y-axis is MICB (MIC for drug B compound). The partial concentration inhibition index (FIC) is used as the basis for the combined drug sensitivity test, and the calculation formula is as follows:

[0051]

[0052] 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.

[0053] Table 1 Dosing method

[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] After gallic acid was used in combination with enrofloxacin hydrochloride, the minimum concentrations for sterile growth were 4 μg / mL of gallic acid and 2 μg / mL of enrofloxacin hydrochloride, and the combined antibacterial index was 0.156, indicating a synergistic effect.

[0057] Example 2

[0058] Gallic acid increases the bactericidal ability of enrofloxacin hydrochloride against drug-resistant Aeromonas veronii:

[0059] Same as Example 1, except that the bacterial suspension of drug-resistant Aeromonas hydrophila (containing 10 7 cfu / mL) was replaced with a bacterial suspension of drug-resistant Aeromonas veronii (containing 10 7 cfu / mL).

[0060] The results showed that the MIC of gallic acid against drug-resistant Aeromonas veronii was 256μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Aeromonas veronii was 8μg / mL. After the combination of gallic acid and enrofloxacin hydrochloride, the minimum concentration for sterile growth was 8μg / mL of gallic acid and 4μg / mL of enrofloxacin hydrochloride, and the combined antibacterial index was 0.531, which was an additive effect.

[0061] Example 3

[0062] Gallic acid increases 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 7 cfu / mL) was replaced with a bacterial suspension of drug-resistant Citrobacter freundii (containing 10 7 cfu / mL).

[0064] The results showed that the MIC of gallic acid against drug-resistant Citrobacter freundii was 512μg / mL, and the MIC of enrofloxacin hydrochloride against drug-resistant Citrobacter freundii was 16μg / mL. After the combination of gallic acid and enrofloxacin hydrochloride, the minimum concentration for sterile growth was 8μg / mL of gallic acid and 4μg / mL of enrofloxacin hydrochloride, and the combined antibacterial index was 0.266, which was a synergistic effect.

[0065] Example 4

[0066] Clinical trials:

[0067] (1) Preparation method of anti-drug resistance compound enrofloxacin hydrochloride and gallic acid powder:

[0068] Enrofloxacin hydrochloride, gallic acid, solubilizer (sodium dodecyl sulfate), absorption promoter (mannose), and starch are sieved (70 meshes) respectively and set aside;

[0069] Weigh 10 parts of enrofloxacin hydrochloride, 20 parts of gallic acid, 3 parts of solubilizer, and 3 parts of absorption promoter respectively, put them into a mixer in sequence and mix them evenly; then add 64 parts of starch into the mixer, mix evenly, and sieve again (24 mesh) to obtain the anti-resistance compound enrofloxacin hydrochloride and gallic acid powder.

[0070] (2) Preparation method of anti-drug resistance compound enrofloxacin hydrochloride, gallic acid and baicalin powder:

[0071] Enrofloxacin hydrochloride, gallic acid, baicalein, solubilizer (sodium dodecyl sulfate), absorption enhancer (mannose), and starch were sieved (70 meshes) respectively and set aside;

[0072] Weigh 10 parts of enrofloxacin hydrochloride, 20 parts of gallic acid, 5 parts of baicalein, 3 parts of solubilizer, and 3 parts of absorption promoter respectively, put them into a mixer in sequence and mix them evenly; then add 59 parts of starch into the mixer, mix evenly, and sieve again (24 mesh) to obtain the anti-resistance compound enrofloxacin hydrochloride, gallic acid and baicalein powder.

[0073] (3) A total of 180 yellow catfish (average weight 128.1±12.6 g / fish) were randomly divided into six groups, with 30 fish in each group.

[0074] The first group was the blank control group, without any challenge or treatment;

[0075] The second group was the negative control group, which was challenged with drug-resistant Aeromonas veronii from yellow catfish (the challenge time was 2 to 3 days). The challenge method was to inject 0.1 mL of 1×10 8 cfu / mL of Aeromonas verticillioides liquid, successful attack is manifested by prominent inflammation of the cloaca, distended abdomen, ascites on autopsy, no treatment;

[0076] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas veronii from yellow catfish (the challenge method was the same as above). After successful challenge, enrofloxacin hydrochloride was given for treatment 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 veronii from yellow catfish (the challenge method was the same as above). After successful challenge, gallic acid was given for treatment 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 veronii from yellow catfish (the challenge method was the same as above). After successful challenge, the anti-resistance compound enrofloxacin hydrochloride and gallic acid powder was given for treatment at a dose of 15 mg / kg (as enrofloxacin hydrochloride), twice a day for three consecutive days.

[0079] The sixth group was treatment group 4, which was challenged with drug-resistant Aeromonas veronii from yellow catfish (the challenge method was the same as above). After successful challenge, the anti-resistance compound enrofloxacin hydrochloride, gallic acid and baicalin powder were given for treatment, with a dose of 10 mg / kg (as enrofloxacin hydrochloride), twice a day for three consecutive days.

[0080] The yellow catfish were observed for 7 days after treatment and the mortality rate was calculated. The results are shown in Table 2.

[0081] Table 2 Test results

[0082]

[0083]

[0084] Example 5

[0085] A total of 150 yellow catfish (average body weight of 130.2±18.1 g / fish) were randomly divided into five groups, with 30 fish in each group.

[0086] The first group was the blank control group, without any challenge or treatment;

[0087] The second group was the negative control group, which was challenged with drug-resistant Aeromonas hydrophila from yellow catfish (the challenge time was 2 to 3 days). The challenge method was to inject 0.1 mL of 1×10 8 cfu / mL of resistant Aeromonas hydrophila liquid, successful attack manifested as abdominal distension, autopsy revealed intestinal congestion and swelling, no 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 given for treatment 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 given for treatment 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 anti-resistance compound enrofloxacin and gallic acid powder was given for treatment at a dose of 15 mg / kg (as enrofloxacin), twice a day for three consecutive days.

[0091] The yellow catfish were observed for 7 days after treatment and the mortality rate was calculated. The results are shown in Table 3.

[0092] Table 3 Test results

[0093]

[0094]

[0095] Example 6

[0096] A total of 150 Procambarus clarkii (average weight 19.7±4.8 g / crawfish) were randomly divided into five groups, with 30 crawfish in each group.

[0097] The first group was the blank control group, without any challenge or treatment;

[0098] The second group was the negative control group, which was challenged with resistant Citrobacter freundii from Procambarus clarkii (the challenge time was 2 to 3 days). The challenge method was to inject 0.1 mL of 1×10 8 cfu / mL of resistant Citrobacter freundii liquid, successful infection is manifested by slow movement of Procambarus clarkii, redness and swelling of the tail, or even ulceration, and no treatment is given;

[0099] The third group was treatment group 1, which was challenged with resistant Citrobacter freundii from Procambarus clarkii (the challenge method was the same as above). After successful challenge, enrofloxacin was given for treatment 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 resistant Citrobacter freundii from Procambarus clarkii (the challenge method was the same as above). After successful challenge, gallic acid was given for treatment at a dose of 20 mg / kg, twice a day, for three consecutive days.

[0101] The fifth group was treatment group 3, which was challenged with resistant Citrobacter freundii from Procambarus clarkii (the challenge method was the same as above). After successful challenge, the anti-resistance compound enrofloxacin and gallic acid powder was given for treatment at a dose of 15 mg / kg (as enrofloxacin), twice a day for three consecutive days.

[0102] The crayfish were observed for 7 days after treatment 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 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. An anti-drug resistance enrofloxacin hydrochloride compound powder, characterized in that: The invention comprises the following raw materials in parts by mass: 1 to 30 parts of enrofloxacin hydrochloride, 2 to 46 parts of gallic acid, 0.5 to 9 parts of solubilizer, 0.5 to 9 parts of absorption promoter and 0 to 92 parts of starch.

2. The anti-resistance enrofloxacin hydrochloride compound powder according to claim 1, characterized in that: The raw materials also include 4 to 6 portions of baicalin.

3. The anti-resistance enrofloxacin hydrochloride compound powder according to claim 1, characterized in that: The solubilizing agent includes sodium dodecyl sulfate; And / or, the absorption enhancer includes mannose.

4. A method for preparing the anti-resistance enrofloxacin hydrochloride compound powder according to any one of claims 1 to 3, characterized in that: The following steps are involved: The raw materials other than starch are mixed evenly, and then starch is added and mixed evenly to obtain the anti-resistance enrofloxacin hydrochloride compound powder.

5. Use of the enrofloxacin hydrochloride compound powder for preventing drug resistance according to any one of claims 1 to 3 in the preparation of drugs for preventing and treating pathogens.

6. Use of the enrofloxacin hydrochloride compound powder for preventing drug resistance according to any one of claims 1 to 3 in the preparation of a medicament for preventing and treating diseases caused by drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii.

7. The use according to claim 6, characterized in that: The drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii are Aeromonas hydrophila, Aeromonas veronii or Citrobacter freundii that are resistant to enrofloxacin hydrochloride.

Images