Preparation method of drug-resistant florfenicol compound powder

Abstract

The application discloses a preparation method of a drug-resistant florfenicol compound powder, and belongs to the technical field of drug preparation. The drug-resistant florfenicol compound powder comprises the following raw materials in mass fractions: 2-40 parts of florfenicol, 1-20 parts of quercetin, 0.5-14 parts of a solubilizer and 20-93.5 parts of starch. The drug-resistant florfenicol compound powder can treat various diseases and infections of aquatic animals, and the quercetin in the powder can greatly reduce the drug resistance of pathogenic bacteria to florfenicol and enhance the antibacterial activity of florfenicol.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, in particular to a method for preparing a florfenicol compound powder for preventing drug resistance. Background Art

[0002] Florfenicol is a broad-spectrum antibiotic of the amide class that primarily exerts its antibacterial effects by inhibiting peptidyl transferase activity. Approved for marketing in my country since the 1990s, it is primarily used to treat diseases in livestock and aquatic animals. Florfenicol is an excellent antibiotic favored by aquaculture companies and farmers for its low toxicity, broad antimicrobial spectrum, rapid absorption and excretion, and short withdrawal period. However, with increasing years of use and widespread abuse, florfenicol resistance in aquaculture pathogens is becoming increasingly severe. The minimum inhibitory concentration (MIC) for certain bacteria reaches tens of parts per million (PPM), making conventional doses ineffective for treating the disease, leading to numerous treatment failures and economic losses.

[0003] Currently, the main approaches to combating drug-resistant pathogens include exploring new antibiotics and using biological methods such as microorganisms and plant extracts to combat diseases. However, the development of new antibiotics is time-consuming and expensive, and the effectiveness of biological methods such as microorganisms and plant extracts in combating diseases is not ideal. Therefore, there is an urgent need to develop new, low-cost, and effective control methods. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a florfenicol compound powder for preventing drug resistance, so as 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 is a florfenicol compound powder for preventing drug resistance, which comprises the following raw materials in parts by mass: 2 to 40 parts of florfenicol, 1 to 20 parts of quercetin, 0.5 to 14 parts of a solubilizer and 20 to 93.5 parts of starch.

[0007] Furthermore, the anti-resistance florfenicol compound powder comprises the following raw materials, calculated by mass: 10 to 30 parts of florfenicol, 5 to 15 parts of quercetin, 3 to 10 parts of solubilizer and 39 to 79 parts of starch.

[0008] Furthermore, the raw materials of the florfenicol compound powder for preventing drug resistance also include 3 to 6 parts of emodin.

[0009] Furthermore, the solubilizing agent includes sodium dodecyl sulfate.

[0010] Quercetin can dramatically reduce the drug resistance of drug-resistant bacteria and enhance the antibacterial ability of florfenicol, thereby restoring the therapeutic effect.

[0011] The second technical solution of the present invention is a method for preparing the above-mentioned florfenicol anti-resistance compound powder, comprising the following steps:

[0012] The raw materials other than starch are mixed evenly, and then starch is added and mixed evenly to obtain the florfenicol resistance preventing compound powder.

[0013] The third technical solution of the present invention: an application of the above-mentioned florfenicol resistance-preventing compound powder in the preparation of drugs for preventing and treating pathogens.

[0014] The fourth technical solution of the present invention: an application of the above-mentioned anti-resistance florfenicol compound powder in the preparation of a medicament for preventing and treating diseases caused by drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veseri or drug-resistant Citrobacter freundii.

[0015] Furthermore, the drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas welchii or drug-resistant Citrobacter freundii is florfenicol-resistant Aeromonas hydrophila, Aeromonas welchii or Citrobacter freundii.

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

[0017] The florfenicol resistance-preventing compound powder of the present invention can effectively treat various disease infections of aquatic animals. The quercetin in the powder can greatly reduce the resistance of pathogens to florfenicol and enhance the antibacterial activity of florfenicol.

[0018] The florfenicol resistance-preventing compound powder of the present invention can increase the solubility of quercetin, promote absorption, and enhance the antibacterial therapeutic effect.

[0019] The preparation process of the florfenicol resistance-preventing compound powder of the present invention is simple, the raw materials are readily available, the powder is suitable for industrial large-scale production, and the application prospect is broad. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0021] Figure 1 This is a chessboard pattern diagram. DETAILED DESCRIPTION

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

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

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

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

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

[0027] The “parts” described in the following examples are all “parts by weight”.

[0028] The identification results of drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veseri, and drug-resistant Citrobacter freundii are as follows:

[0029] (1) Drug-resistant Aeromonas hydrophila from yellow catfish

[0030]

[0031]

[0032] (2) Drug-resistant Aeromonas vermiformis from yellow catfish

[0033]

[0034]

[0035] (3) Drug-resistant Citrobacter freundii from Procambarus clarkii

[0036]

[0037]

[0038]

[0039] Example 1

[0040] Quercetin increases the bactericidal ability of florfenicol against drug-resistant Aeromonas hydrophila:

[0041] (1) The minimum inhibitory concentrations (minimum concentrations that result in sterile growth) of quercetin and florfenicol against drug-resistant Aeromonas hydrophila were determined by microdilution method, specifically:

[0042] First, draw 200 μL of drug working solution (the concentration of quercetin drug working solution is 1024 μg / mL, which is prepared by diluting the quercetin standard stock solution (concentration 5120 μg / mL) with sterilized MH broth; the concentration of florfenicol drug working solution is 256 μg / mL, which is prepared by diluting the florfenicol 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 mixing 5 times, draw 100 μL and add it to the third well. Perform doubling dilutions in sequence backwards, and discard the 100 μL liquid aspirated from the last well. At this time, each well was filled with 100 μL of drug-containing broth, with the concentration of quercetin ranging from 512 μg / mL to 0.125 μg / mL and the concentration of florfenicol ranging from 128 μg / mL to 0.0313 μg / mL. 100 μL of drug-resistant Aeromonas hydrophila bacterial suspension (containing 10 7 cfu / mL), and cultured in a constant temperature incubator (28°C) for at least 24 hours, and then observed to see if there were clear holes. The drug concentration in the clear holes was the minimum concentration for sterile growth, which was the MIC.

[0043] (2) Determination of the combined effect of quercetin and florfenicol using the checkerboard method

[0044] Take a 96-well bacterial culture plate, with row Y1 and column X1 being the rows and columns for drug A (quercetin) and drug B (florfenicol) 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. 100 μL of standard drug solution containing concentrations of 1 / 256, 1 / 64, 1 / 32, 1 / 16, 1 / 8, and 1 / 4 times the MIC (512 μg / mL) was used (referred to as drug A 1 / 256, drug A 1 / 64, drug A 1 / 32, drug A 1 / 16, drug A 1 / 8, and drug A 1 / 4).

[0046] Add drug B to wells 2 to 7 of column X1. 100 μL of standard drug solution with concentrations of 1 / 8, 1 / 4, 1 / 2, 1, 2, and 4 times the MIC (16 μg / mL) is added to each well (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 / 128, 1 / 32, 1 / 16, 1 / 8, 1 / 4, and 1 / 2 times the MIC (512 μg / mL), respectively, with 50 μL of each standard solution.

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

[0049] Add 200 μL of drug-resistant Aeromonas hydrophila bacterial suspension (containing 10 7 cfu / mL), and 100 μL of 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 200 μL, the 96-well plate with the sample added is placed in a constant temperature incubator (28°C) and cultured for at least 24 hours, and then the clear wells are observed. The drug concentration in the clear wells is the minimum concentration for sterile growth when the drug is used in combination, which is the MIC.

[0050] The results were determined using the equivalent midpoint method: all tubes along the 0-point angle bisector were equivalent midpoint tubes. The lowest concentration for sterile growth was defined as point 1. The point on the X-axis corresponding to this point was MICA (MIC for the combination of drug A), and the point on the Y-axis was MICB (MIC for the combination of drug B). The fractional inhibition index (FIC) was used as the basis for combined drug susceptibility testing, and the calculation formula was as follows:

[0051]

[0052] An FIC index of ≤0.5 indicates a synergistic effect; an FIC index of 0.5-1 indicates an additive effect; an FIC index of 1-2 indicates an irrelevant effect; and an FIC index of >2 indicates an antagonistic effect.

[0053] Table 1 Dosing method

[0054]

[0055]

[0056] The results showed that the MIC of quercetin against drug-resistant Aeromonas hydrophila was 512 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas hydrophila was 16 μg / mL.

[0057] After quercetin and florfenicol were used together, the minimum concentrations for sterile growth were 8 μg / mL for quercetin and 4 μg / mL for florfenicol, and the combined inhibition index was 0.266, indicating a synergistic effect.

[0058] Example 2

[0059] Quercetin increases the bactericidal ability of florfenicol against drug-resistant Aeromonas vermiformis:

[0060] 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 wiltshireii (containing 10 7 cfu / mL).

[0061] The results showed that the MIC of quercetin against drug-resistant Aeromonas vermiformis was 512 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas vermiformis was 16 μg / mL. When quercetin and florfenicol were used together, the minimum concentrations for sterile growth were 32 μg / mL for quercetin and 4 μg / mL for florfenicol, with a combined inhibition index of 0.313, indicating a synergistic effect.

[0062] Example 3

[0063] Quercetin increases the bactericidal ability of florfenicol against drug-resistant Citrobacter freundii:

[0064] 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).

[0065] The results showed that the MIC of quercetin against drug-resistant Citrobacter freundii was 512 μg / mL, and the MIC of florfenicol against drug-resistant Citrobacter freundii was 8 μg / mL. When quercetin and florfenicol were used together, the minimum concentrations for sterile growth were 8 μg / mL for quercetin and 2 μg / mL for florfenicol, with a combined inhibition index of 0.266, indicating a synergistic effect.

[0066] Example 4

[0067] Clinical trials:

[0068] (1) Preparation method of anti-drug resistance compound florfenicol and quercetin powder:

[0069] Florfenicol, quercetin, solubilizer (sodium lauryl sulfate), and starch were sieved (70 mesh) and set aside;

[0070] 20 parts of florfenicol, 10 parts of quercetin, and 6.5 parts of a solubilizer were weighed separately, and placed in a mixer in sequence and mixed thoroughly; then 63.5 parts of starch were added to the mixer, mixed thoroughly, and sieved again (24 mesh) to obtain a drug resistance-preventing compound florfenicol and quercetin powder.

[0071] (2) Preparation method of anti-drug resistance compound florfenicol, quercetin and emodin powder:

[0072] Florfenicol, quercetin, emodin, solubilizer (sodium lauryl sulfate), and starch were sieved (70 mesh) and set aside;

[0073] 20 parts of florfenicol, 10 parts of quercetin, 4 parts of emodin, and 6.5 parts of a solubilizer were weighed respectively, and placed in a mixer in sequence and mixed thoroughly; then 59.5 parts of starch were added to the mixer, mixed thoroughly, and sieved again (24 mesh) to obtain a drug resistance-preventing compound florfenicol, quercetin, and emodin powder.

[0074] (3) A total of 180 yellow catfish (average weight 127.3±12.7 g / fish) were randomly divided into six groups, with 30 fish in each group.

[0075] The first group was the blank control group, without challenge or treatment;

[0076] The second group was the negative control group, which was challenged with drug-resistant Aeromonas vermiformis 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 vermiformis liquid, successful infection is manifested by prominent inflammation of the cloaca, distended abdomen, and ascites on autopsy, without treatment;

[0077] The third group was treatment group 1, which was challenged with drug-resistant Aeromonas vermiformis from yellow catfish (the challenge method was the same as above). After successful challenge, florfenicol was given for treatment at a dose of 15 mg / kg, twice a day, for three consecutive days.

[0078] The fourth group was treatment group 2, which was challenged with drug-resistant Aeromonas vermiformis from yellow catfish (the challenge method was the same as above). After successful challenge, quercetin was given for treatment at a dose of 20 mg / kg, twice a day, for three consecutive days.

[0079] The fifth group was treatment group 3, which was challenged with drug-resistant Aeromonas vermiformis from yellow catfish (the challenge method was the same as above). After successful challenge, the anti-resistance compound florfenicol and quercetin powder was given for treatment at a dose of 15 mg / kg (calculated as florfenicol), twice a day for three consecutive days.

[0080] The sixth group was treatment group 4, which was challenged with drug-resistant Aeromonas vermiformis from yellow catfish (the challenge method was the same as above). After successful challenge, the anti-resistance compound florfenicol, quercetin and emodin powder were given for treatment at a dose of 10 mg / kg (calculated as florfenicol), twice a day for three consecutive days.

[0081] The yellow catfish were observed for 7 days after treatment, and the mortality rate was calculated. The results are shown in Table 2.

[0082] Table 2 Test results

[0083]

[0084] Example 5

[0085] A total of 150 yellow catfish (average weight 150.4±20.5g / fish) were randomly divided into five groups, with 30 fish in each group.

[0086] The first group was the blank control group, without 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 7 cfu / mL of resistant Aeromonas hydrophila solution. Successful infection is manifested by abdominal distension and autopsy reveals intestinal congestion and swelling, but no treatment is given.

[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, florfenicol 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, quercetin was given for treatment at a dose of 20 mg / kg, twice a day, for three consecutive days.

[0090] The fourth 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 florfenicol and quercetin powder was given for treatment at a dose of 15 mg / kg (calculated as florfenicol), 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 Test results

[0093] Group Death digit mortality rate(%) Blank control group 0 0.00 Negative control group 30 100 Treatment group 1 (florfenicol) 18 60.00 Treatment group 2 (quercetin) 22 73.33 Treatment group 3 (anti-resistance compound florfenicol and quercetin powder) 2 6.67

[0094] Example 6

[0095] A total of 150 Procambarus clarkii (average weight 20.3±5.2g / crawfish) were randomly divided into five groups, with 30 crawfish in each group.

[0096] The first group was the blank control group, without challenge or treatment;

[0097] The second group was the negative control group, and the drug-resistant Citrobacter freundii from Procambarus clarkii was used for the challenge. The challenge time was , and the challenge method was to inject 0.1 mL of 1×10 8 cfu / mL of drug-resistant Citrobacter freundii liquid, the manifestation of successful infection is slow movement of Procambarus clarkii, redness and swelling or even ulceration of the tail, without treatment;

[0098] 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, florfenicol was given for treatment at a dose of 15 mg / kg, twice a day, for three consecutive days.

[0099] 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, quercetin was given for treatment at a dose of 20 mg / kg, twice a day for three consecutive days.

[0100] 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 florfenicol and quercetin powder was given for treatment at a dose of 15 mg / kg (calculated as florfenicol), twice a day for three consecutive days.

[0101] The crayfish were observed for 7 days after treatment and the mortality rate was calculated. The results are shown in Table 4.

[0102] Table 4 Test results

[0103] Group Death digit mortality rate(%) Blank control group 1 3.33 Negative control group 30 100 Treatment group 1 (florfenicol) 20 66.67 Treatment group 2 (quercetin) 24 80.00 Treatment group 3 (anti-resistance compound florfenicol and quercetin powder) 1 3.33

[0104] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the 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 fall within the scope of protection determined by the claims of the present invention.

Claims

1. An application of a florfenicol-resistant compound powder in the preparation of a medicament for preventing and treating diseases caused by drug-resistant Aeromonas freundii or drug-resistant Citrobacter freundii, characterized in that: The florfenicol compound powder for preventing drug resistance comprises the following raw materials in parts by mass: 2-40 parts of florfenicol, 1-20 parts of quercetin, 0.5-14 parts of a solubilizer, and 20-93.5 parts of starch.

2. The use according to claim 1, characterized in that The raw materials also include 3 to 6 parts of emodin.

3. The use according to claim 1, characterized in that The solubilizing agent includes sodium lauryl sulfate.

4. The use according to claim 1, characterized in that The preparation method of the anti-resistance florfenicol compound powder comprises the following steps: The raw materials other than starch are mixed evenly, and then starch is added and mixed evenly to obtain the florfenicol resistance preventing compound powder.

5. The use according to claim 1, characterized in that The drug-resistant Aeromonas welchii or drug-resistant Citrobacter freundii is florfenicol-resistant Aeromonas welchii or Citrobacter freundii.

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