Application of kaempferol and shikonin as antibacterial synergists against drug-resistant Riemerella anatipestifer

Abstract

The present invention provides the use of kaempferol and shikonin as antibacterial synergists in combating drug-resistant Riemerella anatipestifer, that is, as synergists for enrofloxacin. The present invention studies kaempferol and shikonin and, for the first time, uses them in combination with enrofloxacin to combat drug-resistant Riemerella anatipestifer infection. The present invention has found through research that kaempferol and shikonin can effectively enhance the sensitivity of drug-resistant Riemerella anatipestifer to enrofloxacin, effectively improve the efficacy of antibiotics against drug-resistant Riemerella anatipestifer, and reduce the dosage of antibiotics, which is of great significance for resolving clinical drug-resistant Riemerella anatipestifer infections. The present invention provides technical support for providing safe and effective auxiliary drugs for the treatment of important drug-resistant Riemerella anatipestifer infections in veterinary clinics, as well as for controlling bacterial resistance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, specifically to antibacterial synergist technology, and relates to the application of natural compounds kaempferol and shikonin in combination with enrofloxacin to combat drug-resistant Riemerella anatipestifer. Background Art

[0002] Riemerella anatipestifer is a contagious disease caused by the bacterium Riemerella anatipestifer, also known as duck infectious serositis, new duck disease, duck septicemia, and duck plague syndrome. Riemerella anatipestifer is a Gram-negative pathogen that primarily infects ducklings aged 2-3 weeks. Infection in ducklings can cause encephalitis, fibrosing pericarditis, and hepatitis. The bacterium has a high infection rate and mortality rate, and once spread, it can cause significant economic losses to duck farms. Currently, the main clinical treatments for Riemerella anatipestifer are antibiotics such as enrofloxacin, gentamicin, kanamycin, and cephalosporins. Enrofloxacin is widely used in veterinary practice, but due to its widespread irregular use, enrofloxacin resistance is increasing. There is an urgent need to find new treatments to combat enrofloxacin-resistant Riemerella anatipestifer and achieve the goal of reducing or replacing antibiotics. However, methods to reduce enrofloxacin resistance are currently lacking in the field.

[0003] In recent years, studies have shown that Chinese herbal monomers, when used in combination with antimicrobial drugs, can increase antibiotic sensitivity and enhance the antibacterial effect of antibiotics. These monomers can act as antimicrobial synergists to address bacterial resistance, becoming a green additive to replace antibiotics or hormones.

[0004] The present invention aims to explore pure natural ingredients that can be used to treat Riemerella anatipestifer, reduce drug resistance, and act as antibacterial synergists. Kaempferol is a flavonoid compound with antioxidant, anti-inflammatory, anticancer, and antibacterial properties. It has inhibitory effects against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, and Shigella dysenteriae. Pure kaempferol is a yellow crystalline powder that is slightly soluble in water and soluble in dimethyl sulfoxide, hot ethanol, ether, and alkali. Kaempferol is primarily found in the rhizomes of Kaempferia galanga (Zingiberaceae) and is widely found in various vegetables and fruits. Shikonin is a crystalline powder or purple flaky crystals that is soluble in ethanol, vegetable oils, and organic solvents but insoluble in water. Shikonin can be extracted from the roots of Lithospermum erythrorhizon (Boraginaceae). Shikonin has anti-inflammatory, antibacterial, and anticancer properties and is currently used clinically to treat skin diseases, acute and chronic hepatitis, flat warts, cervicitis, and hepatic ascites. Topical application can treat burns and promote wound healing. Currently, the clinical use of shikonin and kaempferol is still in the exploratory stage, and there has been no research on their use in the clinical treatment and prevention of Riemerella anatipestifer. Therefore, the present invention's exploration of kaempferol and shikonin is of great significance for the future clinical treatment and prevention of enrofloxacin-resistant Riemerella anatipestifer infection. Summary of the Invention

[0005] The present invention has discovered two new synergists, namely kaempferol and shikonin, which can be used in combination with enrofloxacin to effectively reduce the minimum inhibitory concentration of enrofloxacin and enhance the sensitivity of drug-resistant Riemerella anatipestifer to enrofloxacin, thereby reducing the dosage of antibiotics, solving the serious problem of drug resistance of Riemerella anatipestifer, and achieving the purpose of reducing and replacing antibiotics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In one aspect, the present invention provides the use of kaempferol in combating drug-resistant Riemerella anatipestifer, that is, kaempferol is used in combination with enrofloxacin to combat drug-resistant Riemerella anatipestifer.

[0008] Furthermore, the dosage ratio of enrofloxacin and kaempferol used in combination is 1:0.25 to 1:4.

[0009] Another aspect of the present invention provides the use of shikonin in combating drug-resistant Riemerella anatipestifer, that is, shikonin is used in combination with enrofloxacin to combat drug-resistant Riemerella anatipestifer.

[0010] Furthermore, the enrofloxacin and shikonin are used in combination at a dosage ratio of 1:0.5 to 1:2.

[0011] On the other hand, the present invention provides the use of a combination of kaempferol and shikonin in combating drug-resistant Riemerella anatipestifer, that is, the combination of kaempferol and shikonin is used in combination with enrofloxacin to combat drug-resistant Riemerella anatipestifer.

[0012] Furthermore, the dosage ratio of enrofloxacin, kaempferol and shikonin can be any one of the following ratios: 4:1:1; 10:1:4; 5:8:2; 8:1:1; 40:1:4; 10:4:1.

[0013] In another aspect of the present invention, an enrofloxacin synergist is provided for combating drug-resistant Riemerella anatipestifer. The synergist comprises any one of the following: kaempferol, shikonin, or a combination of kaempferol and shikonin.

[0014] In another aspect of the present invention, a pharmaceutical composition for treating drug-resistant Riemerella anatipestifer is provided, wherein the composition comprises any one of the following: kaempferol and enrofloxacin, shikonin and enrofloxacin, or kaempferol, shikonin and enrofloxacin.

[0015] In another aspect of the present invention, a pharmaceutical preparation for use against drug-resistant Riemerella anatipestifer is provided, comprising any one of the following combinations: a preparation comprising kaempferol and enrofloxacin, shikonin and enrofloxacin, or kaempferol, shikonin, and enrofloxacin as active ingredients.

[0016] In another aspect of the present invention, an emulsion is provided, wherein the main components of the emulsion include 0.25% by weight of kaempferol, 1.0% by weight of shikonin, and 10% by weight of enrofloxacin.

[0017] The present invention has the following outstanding beneficial effects:

[0018] 1. This invention explores a new use of kaempferol: as a synergist for enrofloxacin. Kaempferol has been little studied in veterinary clinical practice. This invention investigates kaempferol and, for the first time, uses it to combat drug-resistant Riemerella anatipestifer infections. This invention provides early technical support for the development of safe and effective adjuvant medications for the treatment of important drug-resistant Riemerella anatipestifer infections in veterinary clinical practice, as well as for the control of bacterial resistance.

[0019] 2. This invention explores a new use of shikonin as an enrofloxacin synergist. Shikonin has been little studied in veterinary clinical practice. This invention is the first to use shikonin to combat drug-resistant Riemerella anatipestifer infections. This provides early technical support for the development of safe and effective adjuvant drugs for the treatment of important drug-resistant Riemerella anatipestifer infections in veterinary clinical practice, as well as for the control of bacterial resistance.

[0020] 3. The present invention has found through research that kaempferol and shikonin can effectively enhance the sensitivity of drug-resistant Riemerella anatipestifer to enrofloxacin, effectively improving the efficacy of the above antibiotics against drug-resistant Riemerella anatipestifer, which is of great significance for solving clinical drug-resistant Riemerella anatipestifer infection.

[0021] 4. The present invention has been tested and shown that the combination of kaempferol and shikonin with enrofloxacin significantly reduces the minimum inhibitory concentration of enrofloxacin and reduces the amount of antibiotics used. It was found that the combination of kaempferol and enrofloxacin, and shikonin and enrofloxacin, has a synergistic antibacterial effect.

[0022] 5. Experiments of the present invention have shown that mixing kaempferol and shikonin in different proportions and using them in combination with enrofloxacin can reduce the minimum inhibitory concentration of enrofloxacin, and has a more significant effect than the combination of kaempferol and shikonin with enrofloxacin separately, while also reducing the usage of the three drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results show the FIC of enrofloxacin combined with kaempferol against 15 strains of Riemerella anatipestifer and the multiple reduction of enrofloxacin MIC.

[0024] Figure 2 The results show the FIC of enrofloxacin combined with shikonin against 15 strains of Riemerella anatipestifer and the multiple reduction of enrofloxacin MIC. DETAILED DESCRIPTION

[0025] To more intuitively illustrate the embodiments or technical solutions of this invention, the following describes the experiments involved in the embodiments of this invention to illustrate the beneficial effects of the present invention. The effects of the present invention are not limited to the description of the embodiments of the present invention. For those skilled in the art, other experimental results can be obtained based on these without inventive efforts.

[0026] The present invention has no special limitation on kaempferol, shikonin, enrofloxacin, etc., and those conventionally used in the art can be used. Enrofloxacin is a chemically synthesized antibacterial agent belonging to the fluoroquinolone class. It is insoluble in water and easily soluble in sodium hydroxide solution. It is a slightly yellow or light yellow crystalline powder and tastes bitter. Enrofloxacin is relatively completely absorbed by oral administration or intramuscular injection. When enrofloxacin is used as an animal drug, the drug has a long half-life in the animal body and good tissue distribution. It is a broad-spectrum antibacterial agent and has antibacterial effects on Gram-positive bacteria, Gram-negative bacteria and mycoplasmas. Example 1. Combined drug sensitivity test of kaempferol and enrofloxacin against Riemerella anatipestifer

[0027] 1. Test consumables

[0028] 1. Reagents and drugs

[0029] Kaempferol: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0030] Enrofloxacin: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0031] Kaempferol was dissolved in DMSO to prepare a stock solution of 10240 μg / mL and stored in a refrigerator at -20°C.

[0032] Enrofloxacin was dissolved in 0.1 mol / L sodium hydroxide to prepare 5120 μg / mL as a stock solution and stored in a -20°C refrigerator.

[0033] 2. Strains

[0034] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The Riemerella anatipestifer used in this invention was stored in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine, Qingdao Agricultural University. E. coli was activated by streaking onto MacConkey medium, cultured overnight in a 37°C incubator, and stored in a refrigerator at 4°C until use. Riemerella anatipestifer was activated by streaking onto blood agar plates, cultured in a 37.5°C 5% CO2 incubator for 24 hours, and stored in a refrigerator at 4°C until use.

[0035] 3. Culture medium

[0036] LB broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0037] MH broth medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0038] MacConkey agar medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual.

[0039] 5% sheep blood agar medium: sterile defibrinated sheep blood: Shanghai Yuanye Biotechnology Co., Ltd.; Columbia blood agar base: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instructions, cooled to about 55°C after autoclaving, and then added 5% sterile defibrinated sheep blood according to the amount.

[0040] 5% calf serum TSB liquid medium: Newborn calf serum: Solebao Biotechnology Co., Ltd.; TSB liquid medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual, and after autoclaving, 5% calf serum was added according to the amount after cooling.

[0041] 4. Instruments

[0042] Steam sterilizer (Tomy Digital Biology, Japan); single-person single-sided vertical clean bench (Shanghai Longyue Instrument Equipment Co., Ltd.); micro-adjustable pipette (Transferpette); electronic analytical balance (Ohaus Instrument (Changzhou) Co., Ltd.); desktop full-temperature oscillating incubator (Tianjin Labterui Instrument Equipment Co., Ltd.), electric constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.); ultraviolet-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.), and carbon dioxide incubator (Shanghai Lishen Scientific Instrument Co., Ltd.).

[0043] 2. Test methods

[0044] 1. Preparation of test bacterial solution

[0045] Take out the E. coli standard strain ATCC 25922 stored in a -80℃ refrigerator and dissolve it at room temperature. In a clean bench, use the three-zone streak method to inoculate the bacterial solution on MacConkey agar for recovery. Place the plate upside down in a 37℃ constant temperature incubator and incubate for 18-24 hours. Use an inoculation loop to pick a single colony and inoculate it into 3mL MH broth. Incubate it in a 37℃ constant temperature incubator overnight. Take the bacterial solution into MH broth medium and adjust the bacterial solution to 0.5 McFarland turbidity standard (concentration of about 10 8 CFU / mL), and the bacterial solution was diluted 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL was used as the test bacterial solution. 15 strains of Riemerella anatipestifer stored in a -80°C refrigerator were taken out and dissolved at room temperature. The bacterial solution was inoculated on a blood agar plate using the three-zone streak method in a clean bench for recovery. The plate was inverted and placed in a 5% CO2 carbon dioxide incubator for 24 to 48 hours. A single colony was picked for purification again and cultured for 24 to 48 hours. A single colony was picked using an inoculation loop and inoculated into 3 mL of TSB broth containing 5% calf serum. The plate was placed in a shaker at 37.5°C and 220 rpm / min for 6 to 8 hours. The bacterial solution was adjusted to a concentration of about 10 using an ultraviolet spectrophotometer. 8 CFU / mL, the bacterial solution was diluted 100 times with TSB broth to a concentration of about 10 6 CFU / mL, as the test bacterial solution.

[0046] 2. Determination of the Minimum Inhibitory Concentration (MIC) of a Single Drug

[0047] Using the microbroth dilution method specified by CLSI and taking ATCC 25922 as the quality control strain, susceptibility tests were performed on the prepared drugs. At the same time, the MIC of enrofloxacin and kaempferol was determined for 15 strains of Riemerella anatipestifer. A 96-well sterile microplate was used, and MIC determinations were designed at concentration gradients of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, 1 / 1280, 1 / 2560, and 1 / 5120 times the stock solution. An eight-channel pipette was used to aspirate TSB broth containing 5% calf serum, and 80 μL of broth was added to the first column, and 50 μL of broth was added to the remaining columns. 20 μL of enrofloxacin / kaempferol was added to the first column and serially diluted to the tenth column, with 50 μL of broth and drug fixed in each well. After the test bacterial suspension was mixed evenly, 50 μL was aspirated with an eight-channel pipette and added to each well of the 96-well plate, with a total volume of 100 μL. At the same time, positive wells (without adding drugs) were set in the 11th column and negative wells (without adding bacteria) were set in the 12th column for control. It was then cultured in a carbon dioxide incubator at 37.5°C with 5% CO2 for 24 h, and the results were observed and recorded. Each test was repeated twice. (The DMSO content in each well was less than or equal to 5% to eliminate the effect of DMSO on bacteria).

[0048] 3. Combined susceptibility test

[0049] Taking ATCC 25922 as the quality control strain and using the MIC results as the reference basis, 15 strains of Riemerella anatipestifer were tested by the microbroth dilution checkerboard method using a 96-well sterile microplate. The combination was designed at 1-fold, 1 / 2-fold, 1 / 4-fold, 1 / 8-fold, 1 / 16-fold, 1 / 32-fold, and 1 / 64-fold MIC of the two drugs. 80 μL of broth was added to the first column, and 50 μL of broth was added to the remaining columns. 20 μL of kaempferol was added to the first vertical column and serially diluted horizontally to the seventh column. Enrofloxacin was added to the first eight wells of the first row and serially diluted vertically to the seventh row, with 50 μL of broth and drug fixed in each well. After the test bacterial suspension was mixed evenly, 50 μL was aspirated with an eight-channel pipette and added to each well of the 96-well plate, with a total volume of 100 μL. At the same time, the individual MIC of kaempferol, the individual MIC of enrofloxacin, negative wells (without adding bacteria), and positive wells (without adding drugs) were set as controls. It was then cultured in a carbon dioxide incubator at 37.5°C with 5% CO2 for 24 h, and the results were observed and recorded. Each test was repeated twice.

[0050] Calculation and result judgment of the FICI index (fractional inhibitory concentration index FICI):

[0051] FIC index = (MIC of drug combination A / MIC of drug A alone) + (MIC of drug combination B / MIC of drug B alone)

[0052] FIC index standard: When FIC ≤ 0.5, it is a synergistic effect; when 0.5 < FIC ≤ 1, it is an additive effect;

[0053] When 1 < FIC ≤ 2, it has no relevant effect; when FIC > 2, it has an antagonistic effect.

[0054] III. Test Results

[0055] The MIC value of the drug sensitivity test is determined by the minimum concentration at which no bacterial growth is observed macroscopically as the MIC of this drug.

[0056] Taking strains No. 37-2, 43-2, 44-2, 47, and 53 as examples, the combined drug sensitivity results are shown in Table 1

[0057] Table 1 MIC determination results of kaempferol and enrofloxacin against Riemerella anatipestifer strains

[0058]

[0059]

[0060] As can be seen from Table 1, the MIC of enrofloxacin against Riemerella anatipestifer is 8 μg / mL - 64 μg / mL, and the MIC of kaempferol against Riemerella anatipestifer is from 32 μg / mL to 64 μg / mL. The FIC index shows that the interaction between the two drugs is a synergistic effect.

[0061] Among the 15 selected Riemerella anatipestifer strains, as Figure 1 shown, the FIC values are all ≤ 0.5, and kaempferol and enrofloxacin show a synergistic effect. Analysis of the drug sensitivity results of kaempferol combined with enrofloxacin shows that after combination, the MIC of enrofloxacin decreased to 1 / 4 of the MIC of enrofloxacin used alone (the MIC value changed by 4 times) in 4 strains, decreased to 1 / 8 MIC (the MIC value changed by 8 times) in 6 strains, decreased to 1 / 16 MIC in 3 strains, and decreased to 1 / 32 MIC in 2 strains. The highest MIC value of kaempferol alone in the drug sensitivity test was 512 μg / mL in 1 strain, and the lowest MIC value was 16 μg / mL in 1 strain. Kaempferol combined with enrofloxacin can reduce the MIC of kaempferol by 4 - 128 times, up to 128 times in 1 strain; at least 4 times in 2 strains; the MIC value of kaempferol decreased by 8 times in most strains, in 6 strains.

[0062] Note: When enrofloxacin is combined with kaempferol

[0063] 37-2: FIC = [8÷64 + 2÷32] = 0.19;

[0064] 43-2: FIC = [2÷16 + 8÷64] = 0.25;

[0065] 44-2: FIC = [2÷8 + 8÷64] = 0.38;

[0066] 47: FIC=[2÷16+8÷64]=0.25;

[0067] 53: FIC=[2÷16+8÷64]=0.25;

[0068] The unit of drug dosage is μg / mL.

[0069] The enrofloxacin to kaempferol dosage ratio is the calculated enrofloxacin to kaempferol dosage ratio that achieves the synergistic FIC value. Because enrofloxacin and kaempferol are both present in the same 100 μL system, this ratio also represents the concentration ratio of enrofloxacin to kaempferol that achieves the synergistic FIC value.

[0070] Example 2: Combined drug susceptibility test of shikonin and enrofloxacin against Riemerella anatipestifer

[0071] 1. Test consumables

[0072] 1. Reagents and drugs

[0073] Shikonin: produced by Chengdu Pusi Biotechnology Co., Ltd., content: 98%.

[0074] Enrofloxacin: produced by Shanghai McLean Biochemical Technology Co., Ltd., content: 98%.

[0075] Shikonin was dissolved in DMSO to prepare a stock solution of 10240 μg / mL and stored in a refrigerator at -20°C.

[0076] Enrofloxacin was prepared with 0.1 mol / L sodium hydroxide to a concentration of 5120 μg / mL as a stock solution and stored frozen at -20°C.

[0077] 2. Strains

[0078] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The Riemerella anatipestifer used in this invention was stored in the Veterinary Pharmacology Laboratory of the College of Veterinary Medicine, Qingdao Agricultural University. E. coli was activated by streaking onto MacConkey medium, cultured overnight in a 37°C incubator, and stored in a refrigerator at 4°C until use. Riemerella anatipestifer was activated by streaking onto blood agar plates, cultured in a 37.5°C 5% CO2 incubator for 24 hours, and stored in a refrigerator at 4°C until use.

[0079] 3. Culture medium

[0080] Same as Example 1.

[0081] 4. Instruments

[0082] Same as Example 1.

[0083] 2. Test methods

[0084] 1. Preparation of test bacterial solution

[0085] Dissolve the E. coli standard strain ATCC 25922 stored in -80℃ glycerol at room temperature, resuscitate the bacterial solution and inoculate it on MacConkey agar using the three-zone streak method in a clean bench, incubate it inverted at 37℃ in an incubator for 18-24 hours, pick a typical single colony and inoculate it into LB broth, and incubate it in a 37℃ incubator overnight. Take the bacterial solution and add it to MH broth medium to adjust the bacterial solution to a concentration of about 10 8 CFU / mL, dilute the bacterial solution 100 times with MH broth to make the bacterial solution concentration 10 6 CFU / mL was used as the test bacterial solution. 15 strains of Riemerella anatipestifer stored in a -80°C refrigerator were taken out and dissolved at room temperature. The bacterial solution was inoculated on a blood agar plate using the three-zone streak method in a clean bench for recovery. The plate was inverted and placed in a 5% CO2 incubator for 24 to 48 hours. A single colony was picked for purification again and cultured for 24 to 48 hours. A single colony was picked using an inoculation loop and inoculated into 3 mL of TSB broth containing 5% calf serum. The plate was placed in a shaker at 37.5°C and 220 rpm / min for 6 to 8 hours. The bacterial solution was adjusted to a concentration of about 10 using a UV spectrophotometer. 8 CFU / mL, the bacterial solution was diluted 100 times with TSB broth to a concentration of about 10 6 CFU / mL, as the test bacterial solution.

[0086] 3. Determination of single-drug susceptibility MIC

[0087] ATCC 25922 was used as a quality control strain, and 15 drug-resistant Riemerella anatipestifer strains were used as test strains. MICs for enrofloxacin and shikonin were determined using the CLSI-specified broth microdilution method. A sterile 96-well microplate was used, with a concentration gradient of 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, 1 / 1280, 1 / 2560, and 1 / 5120 times the stock solution. TSB broth was pipetted using an eight-channel pipette, with 80 μL added to the first column and 50 μL added to the remaining columns. A 20 μL dilution series of enrofloxacin / shikonin was added to the first column, and the dilution series continued until the tenth column, with a fixed 50 μL broth and drug content in each well. After the test bacterial solution was mixed, 50 μL was pipetted into each well of the 96-well plate using an eight-channel pipette, for a total volume of 100 μL. For control, place the positive wells (without drug) in column 11 and the negative wells (without bacteria) in column 12. Incubate the wells in a 37.5°C, 5% CO2 incubator for 24 hours. Observe and record the results. Repeat each test twice. The lowest concentration gradient in the wells with no visible turbidity is used as the MIC.

[0088] 3. Combined drug sensitivity test

[0089] Using ATCC 25922 as a quality control strain and MIC results as a reference, a microbroth dilution checkerboard assay was performed in a 96-well sterile microplate. Combinations of two drugs were tested at 1x, 1 / 2x, 1 / 4x, 1 / 8x, 1 / 16x, 1 / 32x, and 1 / 64x the MIC. 80 μL of broth was added to the first column, and 50 μL of broth was added to the remaining columns. Shikonin (20 μL) was added to the first vertical column, and a gradient dilution was performed horizontally to the seventh column. Enrofloxacin was added to the first eight wells of the first horizontal row, and a gradient dilution was performed vertically to the seventh row, with a fixed 50 μL broth and drug content in each well. After mixing the test bacterial solution, 50 μL was pipetted using an eight-channel pipette and added to each well of the 96-well plate, for a total volume of 100 μL. Control wells were also set with the MIC of shikonin alone, the MIC of enrofloxacin alone, and negative and positive wells (without bacteria). The cells were placed in a 37.5°C 5% CO2 incubator for 24 hours, and the results were observed and recorded. Each test was repeated twice.

[0090] The calculation of the FICI index (graded inhibitory concentration index FICI) and the result judgment are the same as those in Example 1.

[0091] 3. Test results

[0092] The MIC value of the drug sensitivity test was determined by taking the minimum concentration at which no bacterial growth was observed with the naked eye as the MIC of the drug.

[0093] Taking strains 41-2, 53, 10, 18, and 19 as examples, the combined drug sensitivity results are shown in Table 2

[0094] Table 2 MIC determination results of shikonin and enrofloxacin against drug-resistant Riemerella anatipestifer strains

[0095]

[0096]

[0097] As shown in Table 2, the MIC of enrofloxacin against Riemerella anatipestifer is 16 μg / mL to 128 μg / mL, and the MIC of shikonin against Riemerella anatipestifer is 8 μg / mL to 32 μg / mL. The FIC index indicates that the interaction between the two drugs is synergistic.

[0098] Among the 15 strains of Riemerella anatipestifer selected, Figure 2As shown, the FIC values of 8 strains were ≤0.5, indicating a synergistic effect between enrofloxacin and shikonin; the FIC values of 7 strains were 0.5 < FIC ≤ 1, indicating an additive effect between the two drugs at this time. Analysis of the results showed that under the action of shikonin, the MIC of enrofloxacin decreased to 1 / 2 MIC (MIC value changed by 2 times) in 1 strain, decreased to 1 / 4 MIC in 1 strain, decreased to 1 / 8 MIC (MIC value changed by 8 times) in 3 strains, decreased to 1 / 16 MIC in 3 strains, decreased to 1 / 32 MIC in 6 strains, and decreased to 1 / 64 MIC in 1 strain. The highest MIC value of shikonin alone was 64 μg / mL in 1 strain, and the lowest MIC value was 2 μg / mL in 1 strain. When shikonin was combined with enrofloxacin, the MIC of shikonin decreased by 2 - 64 times, up to 64 times in 1 strain; the lowest decrease was 2 times in 6 strains.

[0099] Note: When enrofloxacin and shikonin are combined

[0100] 41 - 2: FIC = [8÷64 + 4÷32] = 0.25;

[0101] 53: FIC = [2÷16 + 2÷8] = 0.38; [[ID=X]]

[0102] 10: FIC = [2÷64 + 2÷32] = 0.09;

[0103] 18: FIC = [2÷64 + 4÷16] = 0.28;

[0104] 19: FIC = [8÷128 + 4÷16] = 0.31;

[0105] The unit of drug dosage is μg / mL.

[0106] The drug dosage ratio of enrofloxacin and shikonin is the calculated dosage ratio of enrofloxacin and shikonin when reaching the synergistic FIC value. Enrofloxacin and shikonin are in the same 100 - μL system, and this ratio is also the concentration ratio of enrofloxacin and shikonin when reaching the synergistic FIC value.

[0107] Experimental Example 3. Effects of the combined use of kaempferol, shikonin and enrofloxacin on Riemerella anatipestifer I. Test method

[0108] In order to determine whether kaempferol, shikonin and enrofloxacin have a synergistic antibacterial effect and their optimal ratio, this study selected a representative strain (strain 53) in which the FIC values of kaempferol and shikonin combined with enrofloxacin were both <0.5, and a representative strain (strain 47) in which the FIC value of enrofloxacin and shikonin combined was 1 (with additive effect), and determined the effects of different ratios of kaempferol and shikonin on the MIC of enrofloxacin. Taking strains 53 and 47 from the above experiment as an example, ATCC25922 was used as a quality control strain. Kaempferol and shikonin were mixed at weight ratios (or concentration ratios) of 1:1, 1:4, and 4:1. The MIC of the mixture of the two drugs was first determined. Based on the MIC results, a combined drug susceptibility test with enrofloxacin was performed. A 96-well sterile microplate was used using the microbroth dilution checkerboard method. Combinations of the two drugs at 1x, 1 / 2x, 1 / 4x, 1 / 8x, 1 / 16x, 1 / 32x, and 1 / 64x the MIC were designed. 80 μL of broth was added to the first column, and 50 μL of broth was added to the remaining columns. 20 μL of the mixed drug was added to the first vertical column, and the mixture was diluted horizontally to the seventh column. Enrofloxacin was added to the first eight wells of the first horizontal row, and the mixture was diluted vertically to the seventh row. 50 μL of broth and drug were fixed in each well. After the test bacterial solution is mixed, 50 μL is pipetted using an eight-channel pipette and added to each well of a 96-well plate for a total volume of 100 μL. Control wells are also set up, including the MIC of the drug mixture alone, the MIC of enrofloxacin alone, and negative and positive wells (without the addition of bacteria). The plates are incubated in a 37.5°C, 5% CO2 incubator for 24 hours. The results are observed and recorded. Each test is repeated twice.

[0109] The calculation of the FICI index (graded inhibitory concentration index FICI) and the result judgment are the same as those in Example 1.

[0110] 2. Test results

[0111] The MIC value of the drug sensitivity test was determined by taking the minimum concentration at which no bacterial growth was observed with the naked eye as the MIC of the drug.

[0112] The combined drug sensitivity results are shown in Tables 3 and 4:

[0113] Table 3 MIC test results of mixtures of kaempferol and shikonin in different ratios combined with enrofloxacin against drug-resistant Riemerella anatipestifer strain 53

[0114]

[0115]

[0116] Table 4 MIC determination results of mixtures of kaempferol and shikonin in different ratios combined with enrofloxacin against drug-resistant Riemerella anatipestifer strain 47

[0117]

[0118] As shown in Tables 3 and 4, the MIC of enrofloxacin against both strains of Riemerella anatipestifer was 16 μg / mL. After combined susceptibility testing, the MIC of enrofloxacin decreased to 2 μg / mL. When kaempferol and shikonin were mixed in different ratios, the MIC values of the mixtures varied significantly compared to those of either kaempferol or shikonin alone. Combined susceptibility testing of enrofloxacin with the mixed drugs was performed, and the results were analyzed.

[0119] In the two selected strains of Riemerella anatipestifer, as shown in Table 3, when the three drugs were combined, the MIC of enrofloxacin could be reduced to 1 / 8 MIC. When the ratio of kaempferol to shikonin was 1:1, the MIC value of the mixture decreased from 8 μg / mL to 1 μg / mL, a decrease of 8 times. The mixture showed a synergistic effect with enrofloxacin (FIC=0.25). At this time, the dosage ratio of enrofloxacin, kaempferol, and shikonin was 4:1:1; when the ratio of kaempferol to shikonin was 1:4, the MIC value of the mixture decreased from 4 μg / mL to 1 μg / mL. / mL decreased to 1 μg / mL, a decrease of 4 times, and the mixture showed synergistic effect with enrofloxacin (FIC=0.38). At this time, the dosage ratio of enrofloxacin: kaempferol: shikonin was 10:1:4; when the kaempferol: shikonin ratio was 4:1, the MIC value of the mixture with this ratio decreased from 16 μg / mL to 4 μg / mL, also a decrease of 4 times. The mixture showed synergistic effect with enrofloxacin (FIC=0.38). At this time, the dosage ratio of enrofloxacin: kaempferol: shikonin was 5:8:2. Comparing Table 3 with Tables 1 and 2, the FIC value of the mixture of kaempferol and shikonin combined with enrofloxacin was ≤ the FIC value of kaempferol alone or the FIC value of shikonin combined with enrofloxacin. Although there was no significant change in the FIC values of the two drugs, a comparison of the MIC values of the drugs revealed that the MIC value of the mixture of kaempferol and shikonin could decrease by up to 16 times compared with that before the mixture, from 64 μg / mL to 4 μg / mL.

[0120] As shown in Table 4, the MIC of enrofloxacin can be reduced to 1 / 8 MIC. When the ratio of kaempferol to shikonin is 1:1, the MIC value of the mixture decreases from 8 μg / mL to 0.5 μg / mL, a decrease of 16 times. The mixture has a synergistic effect with enrofloxacin (FIC=0.19). At this time, the drug dosage ratio of enrofloxacin: kaempferol: shikonin is 8:1:1; when the ratio of kaempferol to shikonin is 1:4, the MIC value of the mixture decreases from 4 μg / mL to 0.25 μg / mL. The MIC value of the mixture decreased 32-fold from 32 μg / mL to 1 μg / mL at a drug ratio of 40:1:4 for enrofloxacin:kaempferol:shikonin. Table 4 shows a synergistic effect with enrofloxacin (FIC = 0.19) at a drug ratio of 40:1:4 for enrofloxacin:kaempferol:shikonin. At a 4:1 ratio, the MIC value of the mixture decreased 32-fold from 32 μg / mL to 1 μg / mL. The mixture also showed a synergistic effect with enrofloxacin (FIC = 0.16) at a drug ratio of 10:4:1 for enrofloxacin:kaempferol:shikonin. Comparing Table 4 with Table 1, the FIC value of the combination of kaempferol and enrofloxacin was 0.25, while the FIC value of the combination of shikonin and enrofloxacin was 1. When kaempferol and shikonin were mixed at different ratios and used in combination with enrofloxacin, the FIC values of the three combinations were all lower than those of the combination of the two.

[0121] The results showed that the synergistic effect of kaempferol and shikonin when mixed in appropriate proportions and used in combination with enrofloxacin was more pronounced, with a more significant effect compared to enrofloxacin. This can reduce the dosage of the three drugs and has significant implications for subsequent clinical use. Since the MIC value of the mixture against drug-resistant Riemerella anatipestifer was the lowest when the kaempferol:shikonin ratio was 1:4, and this mixture exhibited a synergistic effect when combined with enrofloxacin, the synergistic effect of the three agents was optimal when the ratio of enrofloxacin:kaempferol:shikonin was (10-40):1:4, and the antibacterial effect was best when the ratio of enrofloxacin:kaempferol:shikonin was 40:1:4.

[0122] Example 4: Preparation of a mixed emulsion of kaempferol, shikonin and enrofloxacin

[0123] Soy lecithin, Span 80, and Tween 80 were selected as emulsifiers, sodium methylcellulose as the aqueous phase stabilizer, aluminum stearate as the oil phase stabilizer, and injectable soybean oil as the oil phase. Injectable soybean oil is prone to rancidity, necessitating the addition of antioxidants during preparation. Vitamin E (VE) has excellent antioxidant properties and is harmless to the body, so it was chosen as an excipient to maintain drug stability. An orthogonal design was used to determine the optimal emulsion ratio. The final ratios were 10% emulsifier, 65% injectable soybean oil, and 1% stabilizer. Experimental results showed that the optimal effect was achieved when the kaempferol-shikonin mixture ratio was 1:4 and the enrofloxacin content was 8 times that of the kaempferol-shikonin mixture (i.e., a ratio of 40:1:4 for enrofloxacin:kaempferol:shikonin). Therefore, the emulsion contained 0.25% kaempferol, 1.0% shikonin, and 10% enrofloxacin. Soybean oil for injection is taken in appropriate proportions, and aluminum stearate is added. Heat to dissolve, then mix thoroughly with the remaining soybean oil and Span 80. After autoclaving at 121°C, remove from the mixture, cool to room temperature, and then add kaempferol, shikonin, and vitamin E to prepare the oil phase. Sodium methylcellulose is added in appropriate proportions to sterile water for injection and dissolved by heating until transparent. Tween 80 is then added. After the Tween 80 dissolves, enrofloxacin is added and stirred thoroughly to prepare the aqueous phase. The aqueous phase is slowly added to the oil phase with continuous stirring to produce colostrum. Finally, mechanical shearing is performed in a colloid mill to obtain an emulsion.

Claims

1. The application of kaempferol in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer is characterized by: Kaempferol combined with enrofloxacin against drug-resistant Riemerella anatipestifer.

2. The use of kaempferol as claimed in claim 1 in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer, characterized in that: The dosage ratio of enrofloxacin and kaempferol used in combination is 1:0.25 to 1:

4.

3. The application of shikonin in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer is characterized by: Combination of shikonin and enrofloxacin against drug-resistant Riemerella anatipestifer.

4. The use of shikonin as claimed in claim 3 in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer, characterized in that: The dosage ratio of enrofloxacin to shikonin is 1:0.5 to 1:

2.

5. The use of kaempferol and shikonin in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer, characterized by: Kaempferol and shikonin combination with enrofloxacin against drug-resistant Riemerella anatipestifer.

6. The use of kaempferol and shikonin as claimed in claim 5 in the preparation of a medicament for combating drug-resistant Riemerella anatipestifer, characterized in that: The dosage ratio of enrofloxacin, kaempferol and shikonin can be any one of the following ratios: 4:1:1; 10:1:4; 5:8:2; 8:1:1; 40:1:4; or 10:4:

1.

7. A pharmaceutical composition for treating drug-resistant Riemerella anatipestifer, characterized by: The composition comprises any one of the following: kaempferol and enrofloxacin, shikonin and enrofloxacin, and kaempferol, shikonin and enrofloxacin.

8. A pharmaceutical preparation for use against drug-resistant Riemerella anatipestifer, characterized in that: The pharmaceutical preparation is prepared using the pharmaceutical composition according to claim 7 as an active ingredient.

9. An emulsion, characterized in that: The main components of the emulsion include 0.25% by weight of kaempferol, 1.0% by weight of shikonin, and 10% by weight of enrofloxacin.

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