Application of kaempferol and alkannin as antibacterial synergists in resisting drug-resistant riemerella anatipestifer

Abstract

The invention provides application of kaempferol and alkannin as antibacterial synergists in resisting drug-resistant riemerella anatipestifer, namely, the kaempferol and the alkannin are used as synergists of enrofloxacin. According to the invention, kaempferol and alkannin are researched, and kaempferol and alkannin are combined with enrofloxacin for resisting drug-resistant riemerella anatipestifer infection for the first time. Research finds that kaempferol and alkannin can effectively enhance the sensitivity of the drug-resistant riemerella anatipestifer to enrofloxacin, effectively improve the drug effect of antibiotics to the drug-resistant riemerella anatipestifer and reduce the dosage of the antibiotics, which is of great significance in solving clinical drug-resistant riemerella anatipestifer infection. And a technical support is provided for veterinary clinical treatment of important drug-resistant riemerella anatipestifer infection diseases and drug resistance control of bacteria.

Description

Technical Field

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

[0002] Riemerella anatipestifer disease is a contact infectious disease caused by Riemerella anatipestifer, also known as duck infectious serositis, new duck disease, duck septicemia, duck plague syndrome, etc. Riemerella anatipestifer is a Gram-negative pathogen that mainly infects ducklings aged 2-3 weeks. After the ducklings are infected with the bacteria, they will cause encephalitis, fibrosing pericarditis, and hepatitis. The bacteria have a high infection rate and high mortality rate. Once spread, it will cause huge economic losses to duck farms. At present, the drugs used to treat Riemerella anatipestifer in clinical practice are mainly antibiotics such as enrofloxacin, gentamicin, kanamycin, cephalosporins, etc. At present, enrofloxacin is widely used in veterinary clinics, but due to most of the irregular use, the resistance to enrofloxacin is getting higher and higher. It is urgent to find new treatment methods to combat enrofloxacin-resistant Riemerella anatipestifer and achieve the purpose of reducing resistance and replacing resistance, but there is still a lack of methods in this field that can reduce the resistance to enrofloxacin.

[0003] In recent years, studies have shown that when Chinese medicine monomers are used in combination with antibacterial drugs, they can increase the sensitivity of antibiotics and enhance the antibacterial effect of antibiotics. Chinese medicine monomers can be used as an antibacterial enhancer to solve the problem of bacterial resistance, thus becoming a green additive to replace antibiotics or hormone drugs.

[0004] The present invention aims to explore a pure natural ingredient that can be used for the treatment of Riemerella anatipestifer disease, reduce drug resistance, and can be used as an antibacterial synergist. Kaempferol is a flavonoid compound, which has antioxidant, anti-inflammatory, anti-cancer, antibacterial and other effects, and has inhibitory effects on Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, and Shigella dysenteriae. The pure product of kaempferol is a yellow crystalline powder, slightly soluble in water, soluble in dimethyl sulfoxide, hot ethanol, ether and alkali. Kaempferol is mainly found in the rhizome of Kaempferia galanga, a plant of the ginger family, and is widely found in various vegetables and fruits. Shikonin is a crystalline powder or purple flaky crystal, soluble in ethanol, vegetable oil, organic solvent, and insoluble in water. Shikonin can be extracted from the root of Lithospermum erythrorhizon, a plant of the Boraginaceae family. Shikonin has anti-inflammatory, antibacterial, anti-cancer and other effects. At present, it can be used clinically to treat skin diseases, acute and chronic hepatitis, flat warts, cervicitis, hepatic ascites and other diseases. Local application can treat burns and promote wound healing. At present, the clinical use of shikonin and kaempferol is still in the exploratory stage, and there is no relevant research on the use of kaempferol and shikonin in clinical treatment and prevention of Riemerella anatipestifer. Therefore, the exploration of kaempferol and shikonin in the present invention 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] Another aspect of 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, wherein 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 drug-resistant Riemerella anatipestifer is provided, the composition comprising any one of the following: kaempferol and enrofloxacin, shikonin and enrofloxacin, kaempferol, shikonin and enrofloxacin.

[0015] In another aspect of the present invention, a pharmaceutical preparation for resisting drug-resistant Riemerella anatipestifer is provided, wherein the pharmaceutical preparation comprises any one of the following combinations: a preparation prepared with 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. The present invention explores a new use of kaempferol, that is, as a synergist of enrofloxacin. Kaempferol has been less studied in veterinary clinics. The present invention studies kaempferol and uses it for the first time to combat drug-resistant Riemerella anatipestifer infection. It provides early technical support for providing safe and effective auxiliary drugs for the treatment of important drug-resistant Riemerella anatipestifer infection diseases in veterinary clinics and for controlling bacterial resistance.

[0019] 2. The present invention explores a new use of shikonin, namely, as a synergist of enrofloxacin. There are few studies on shikonin in veterinary clinics. The present invention is the first to use shikonin to combat drug-resistant Riemerella anatipestifer infection. It provides early technical support for providing safe and effective auxiliary drugs for the treatment of important drug-resistant Riemerella anatipestifer infection diseases in veterinary clinics and for controlling 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, and effectively improve the efficacy of the above antibiotics on drug-resistant Riemerella anatipestifer, which is of great significance for solving clinical drug-resistant Riemerella anatipestifer infection.

[0021] 4. The present invention has been shown through experiments 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 is found that the combination of kaempferol and enrofloxacin, and shikonin and enrofloxacin all have synergistic antibacterial effects.

[0022] 5. The present invention has been tested and shown that the combination of kaempferol and shikonin in different proportions and enrofloxacin can reduce the minimum inhibitory concentration of enrofloxacin, and has a more significant effect than the combination of kaempferol and shikonin with enrofloxacin respectively, and can also reduce the usage of the three drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] In order to more intuitively illustrate the embodiments or technical solutions of this invention, the experiments involved in the embodiments of this invention are described below 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 ordinary technicians in this field, other experimental results can be obtained based on these without creative work.

[0026] The present invention has no special limitation on kaempferol, shikonin, enrofloxacin, etc., and those conventionally used in the art may be used. Enrofloxacin is a chemically synthesized antibacterial agent belonging to the fluoroquinolone class. It is insoluble in water, easily soluble in sodium hydroxide solution, and is a slightly yellow or light yellow crystalline powder with a bitter taste. 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 -20°C refrigerator.

[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 the present invention is stored in the Veterinary Pharmacology Laboratory of the School of Veterinary Medicine of Qingdao Agricultural University. Escherichia coli is activated by streaking on a MacConkey medium, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator for use. Riemerella anatipestifer is activated by streaking on a blood plate medium, cultured in a 37.5°C 5% CO2 carbon dioxide incubator for 24 hours, and stored in a 4°C refrigerator for 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., prepare according to the instructions, cool to about 55°C after high pressure, and then add 5% sterile defibrinated sheep blood according to the amount.

[0040] 5% calf serum TSB liquid culture medium: Newborn calf serum: Solebao Biotechnology Co., Ltd.; TSB liquid culture medium: Qingdao Haibo Biotechnology Co., Ltd., prepared according to the instruction manual, after high pressure, wait for cooling and add 5% calf serum according to the amount.

[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 Labteri Instrument Equipment Co., Ltd.), electric constant temperature incubator (Shanghai Senxin Experimental Instrument Co., Ltd.); UV-visible spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.); electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.), 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. Inoculate the bacterial solution on MacConkey agar in a clean bench using the three-zone streak method for recovery. Place the plate upside down in a 37℃ constant temperature incubator for 18-24 hours. Use an inoculation loop to pick a single colony and inoculate it in 3mL MH broth. Incubate it in a 37℃ constant temperature incubator overnight. Take the bacterial solution to the 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, as the test bacterial solution. Take out 15 strains of Riemerella anatipestifer stored in a -80℃ refrigerator and dissolve them at room temperature. In the clean bench, use the three-zone streak method to inoculate the bacterial solution on the blood agar plate for recovery, and invert the plate in a carbon dioxide incubator with 5% CO2 for 24 to 48 hours. Pick a single colony for purification again, and incubate for 24 to 48 hours. Use an inoculation loop to pick a single colony and inoculate it in 3mL TSB broth containing 5% calf serum, and place it in a shaker at 37.5℃ and 220rpm / min for 6 to 8 hours. Use a UV spectrophotometer to adjust the bacterial solution to a concentration of about 10 8 CFU / mL, the bacterial solution was diluted 100 times with TSB broth, and the concentration was 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 against 15 strains of Riemerella anatipestifer was determined. A 96-well sterile microplate was used, and MIC was determined 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, 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 solution 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. The plate was incubated 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 influence of DMSO on bacteria).

[0048] 3. Combined susceptibility test

[0049] Using ATCC 25922 as the quality control strain and taking the MIC results as the reference basis, the microbroth dilution checkerboard method was used to test 15 strains of Riemerella anatipestifer with 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 solution 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. The plate was incubated 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 A in combination / MIC of drug A alone) + (MIC of drug B in combination / 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 with the naked eye as the MIC of the 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] It can be seen from Table 1 that the MIC of enrofloxacin against Riemerella anatipestifer is 8 μg / mL - 64 μg / mL, and the MIC of kaempferol against Riemerella anatipestifer is 32 μg / mL - 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 combined drug sensitivity results of kaempferol and 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. The combination of kaempferol and 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 dosage ratio of enrofloxacin to kaempferol is the calculated dosage ratio of enrofloxacin to kaempferol when the synergistic FIC value is reached. Because enrofloxacin and kaempferol are both in the 100 μL system, the ratio is also the concentration ratio of enrofloxacin to kaempferol when the synergistic FIC value is reached.

[0070] Example 2: Combined drug sensitivity 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 -20°C refrigerator.

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

[0077] 2. Strains

[0078] Escherichia coli standard strain ATCC 25922: purchased from Qingdao Haibo Biotechnology Co., Ltd. The Riemerella anatipestifer used in the present invention is stored in the Veterinary Pharmacology Laboratory of the School of Veterinary Medicine of Qingdao Agricultural University. Escherichia coli is activated by streaking on a MacConkey medium, cultured overnight in a 37°C incubator, and stored in a 4°C refrigerator for use. Riemerella anatipestifer is activated by streaking on a blood plate medium, cultured in a 37.5°C 5% CO2 carbon dioxide incubator for 24 hours, and stored in a 4°C refrigerator for 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 standard strain of E. coli 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 18-24h, pick a typical single colony and inoculate it in 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, as the test bacterial solution. Take out 15 strains of Riemerella anatipestifer stored in a -80℃ refrigerator and dissolve them at room temperature. In the clean bench, use the three-zone streak method to inoculate the bacterial solution on the blood agar plate for recovery, and invert the plate in a 5% CO2 incubator for 24 to 48 hours. Pick a single colony for purification again, and incubate for 24 to 48 hours. Use an inoculation loop to pick a single colony and inoculate it in 3mL TSB broth containing 5% calf serum, and place it in a shaker at 37.5℃ and 220rpm / min for 6 to 8 hours. Use a UV spectrophotometer to adjust the bacterial solution to a concentration of about 10 8 CFU / mL, the bacterial solution was diluted 100 times with TSB broth, and the concentration was about 10 6 CFU / mL, as the test bacterial solution.

[0086] 3. Determination of single drug susceptibility MIC

[0087] ATCC 25922 was used as the quality control strain, and 15 drug-resistant Riemerella anatipestifer were used as the test strains. The MIC of enrofloxacin and shikonin was determined by the microbroth dilution method specified by CLSI. A sterile 96-well microplate was used, and the MIC was determined at 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 mother solution. TSB broth was drawn using an eight-channel pipette, 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 / shikonin was added to the first column, and the gradient was diluted to the tenth column, with 50 μL of broth and drug fixed in each well. After the test bacterial solution was mixed, 50 μL was drawn into each well of the 96-well plate with an eight-channel pipette, and the total volume was 100 μL. At the same time, set the positive wells (without drug addition) in the 11th column and the negative wells (without bacteria addition) in the 12th column for control. Place them in a carbon dioxide incubator at 37.5℃ and 5% CO2 for 24 hours, observe and record the results. Repeat each test twice. When reading, take the lowest concentration gradient in the wells with no turbidity visible to the naked eye as the MIC.

[0088] 3. Combined drug sensitivity test

[0089] Using ATCC 25922 as the quality control strain and the MIC result as a reference, a 96-well sterile microplate was used for the microbroth dilution chessboard test, and the two drugs were designed to be combined at 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 16 times, 1 / 32 times and 1 / 64 times the MIC. 80 μL of broth was added to the first column, 50 μL of broth was added to the remaining columns, 20 μL of shikonin was added to the first vertical column, and the horizontal gradient was diluted to the seventh column. Enrofloxacin was added to the first eight wells of the first horizontal row, and the vertical gradient was diluted to the seventh row. Each well had 50 μL of broth and drug fixed. After the test bacterial solution was mixed, 50 μL was drawn 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 MIC of shikonin alone, the MIC of enrofloxacin alone, the negative wells (without bacteria) and the positive wells (without drug) were set as controls. The cells were placed in a carbon dioxide incubator at 37.5°C and 5% CO2 for 24 hours, and the results were observed and recorded. Each test was repeated twice.

[0090] The calculation of FICI index (Fractional 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 is determined by taking the minimum concentration at which no bacterial growth is 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. At this time, the FIC index shows 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, showing 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) for 1 strain, decreased to 1 / 4 MIC for 1 strain, decreased to 1 / 8 MIC (MIC value changed by 8 times) for 3 strains, decreased to 1 / 16 MIC for 3 strains, decreased to 1 / 32 MIC for 6 strains, and decreased to 1 / 64 MIC for 1 strain. The highest MIC value of shikonin alone for drug sensitivity was 64 μg / mL for 1 strain, and the lowest MIC value was 2 μg / mL for 1 strain. When shikonin was combined with enrofloxacin, the MIC of shikonin decreased by 2 - 64 times, up to 64 times for 1 strain; the lowest decrease was 2 times for 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;

[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] where the unit of drug dosage is μg / mL.

[0106] The drug dosage ratio of enrofloxacin to shikonin is the calculated dosage ratio of enrofloxacin to 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 to 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 methods

[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 effect of kaempferol and shikonin mixed in different ratios on the MIC of enrofloxacin. Taking strains 53 and 47 in the above experiment as examples, ATCC25922 was used as a quality control strain, kaempferol and shikonin were mixed at a weight ratio (or concentration ratio) of 1:1, 1:4, and 4:1, and the MIC of the mixture of the two drugs was first determined, and the MIC results were used as a reference for the combined drug sensitivity test with enrofloxacin. The 96-well sterile microplate was used for the microbroth dilution chessboard method, and the two drugs were designed to be combined at 1 times, 1 / 2 times, 1 / 4 times, 1 / 8 times, 1 / 16 times, 1 / 32 times, and 1 / 64 times the MIC. 80 μL of broth was added to the first column, 50 μL of broth was added to the remaining columns, 20 μL of mixed drugs was added to the first vertical column, and the horizontal gradient was diluted to the seventh column. Enrofloxacin was added to the first eight wells of the first horizontal row, and the vertical gradient was diluted to the seventh row, and 50 μL of broth and drugs were fixed in each well. After the test bacterial solution is mixed, 50 μL is drawn 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, set the mixed drug single MIC, enrofloxacin single MIC, (no bacteria) negative and (no drug) positive wells as controls. Place in a carbon dioxide incubator at 37.5℃ 5% CO2 for 24 hours, observe and record the results. Repeat each test twice.

[0109] The calculation of FICI index (Fractional 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 is determined by taking the minimum concentration at which no bacterial growth is 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 proportions combined with enrofloxacin against drug-resistant Riemerella anatipestifer strain 53

[0114]

[0115]

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

[0117]

[0118] As shown in Tables 3 and 4, the MIC of enrofloxacin against the two strains of Riemerella anatipestifer was 16 μg / mL, and the MIC value of enrofloxacin was reduced to 2 μg / mL after combined drug sensitivity test. After kaempferol and shikonin were mixed in different proportions, the MIC value of the mixture changed to varying degrees compared with kaempferol / shikonin alone. Enrofloxacin was subjected to combined drug sensitivity test with the mixed drug and the results were analyzed.

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

[0120] As shown in Table 4, the MIC of enrofloxacin can be reduced to 1 / 8MIC. 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. g / mL, also decreased by 16 times, the mixture and enrofloxacin showed synergistic effect (FIC = 0.19), at this time the drug dosage ratio was enrofloxacin: kaempferol: shikonin was 40:1:4; when kaempferol: shikonin was 4:1, the MIC value of the mixture decreased from 32μg / mL to 1μg / mL, decreased by 32 times, the mixture and enrofloxacin showed synergistic effect (FIC = 0.16), at this time the drug dosage ratio was enrofloxacin: kaempferol: shikonin was 10:4:1. Comparing Table 4 with Table 1, the FIC value of kaempferol combined with enrofloxacin was 0.25, and the FIC value of shikonin combined with enrofloxacin was 1. When kaempferol and shikonin were mixed in different proportions and used in combination with enrofloxacin, the FIC values ​​of the three combined were all lower than when the two were used in combination.

[0121] The results showed that the synergistic effect of kaempferol and shikonin mixed in proportion and used in combination with enrofloxacin was more obvious, and it had a more significant effect in comparison, which could reduce the dosage of the three drugs and was of great significance for subsequent clinical use. Since the MIC value of the mixture against drug-resistant Riemerella anatipestifer was the lowest when the ratio of kaempferol to shikonin was 1:4, and the mixture showed a synergistic effect when used in combination with enrofloxacin, therefore, when the ratio of enrofloxacin to kaempferol to shikonin was (10-40) to 1:4, the synergistic effect of the three was optimal, and when the ratio of enrofloxacin to kaempferol to shikonin was 40 to 1:4, its antibacterial effect was good.

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

[0123] Soybean lecithin, Span 80, and Tween 80 were selected as emulsifiers, sodium methylcellulose was used as a water phase stabilizer, aluminum stearate was used as an oil phase stabilizer, and soybean oil for injection was used as an oil phase. Soybean oil for injection is prone to rancidity, and antioxidants need to be added during preparation. VE has a good antioxidant effect and is harmless to the body, so VE is selected as an excipient to maintain drug stability. The optimal ratio of the emulsion was selected by orthogonal experimental design. Finally, the emulsifier was 10%, the soybean oil for injection was 65%, and the stabilizer was 1%. According to the test, the effect is better when the mixing ratio of kaempferol and shikonin is 1:4 and the drug content of enrofloxacin is 8 times that of the mixture of kaempferol and shikonin (that is, when the ratio of enrofloxacin: kaempferol: shikonin is 40:1:4). Therefore, the content of kaempferol in the emulsion is 0.25%, the content of shikonin is 1.0%, and the content of enrofloxacin is 10%. Take soybean oil for injection in proportion, add aluminum stearate, heat to dissolve, and then mix evenly with the remaining soybean oil and Span 80, sterilize at 121°C, take out, put it at room temperature, add kaempferol, shikonin and VE to prepare the oil phase. Add sodium methylcellulose to sterile water for injection in proportion, heat and dissolve until transparent, add Tween 80, wait for Tween 80 to dissolve, add enrofloxacin, stir evenly, and prepare the water phase. Slowly add the water phase to the oil phase and stir continuously to make colostrum. Finally, after mechanical shearing in a colloid mill, an emulsion is obtained.

Claims

1. The application of kaempferol in 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 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 combating drug-resistant Riemerella anatipestifer is characterized by: Shikonin combined with enrofloxacin against drug-resistant Riemerella anatipestifer.

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

2.

5. The application of kaempferol and shikonin in combating drug-resistant Riemerella anatipestifer is characterized by: Kaempferol and shikonin combination with enrofloxacin against drug-resistant Riemerella anatipestifer.

6. The use of kaempferol and shikonin in combating drug-resistant Riemerella anatipestifer as claimed in claim 5, 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; 10:4:

1.

7. An enrofloxacin synergist for combating drug-resistant Riemerella anatipestifer, characterized in that: The synergist includes any one of the following: kaempferol, shikonin, or a combination of kaempferol and shikonin.

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

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

10. 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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