Application of isoliquiritigenin as polymyxin synergist

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of isoliquiritigenin as a polymyxin synergist. Through combination of isoliquiritigenin and polymyxin, it is found that isoliquiritigenin and polymyxin have a good effect on in-vivo and in-vitro salmonella inhibition. The isoliquiritigenin is safe in component, can achieve a good synergistic effect with the polymyxin, can significantly reduce the dosage of the polymyxin, is not easy to induce the generation of bacterial drug resistance, and has the characteristics of low price and good curative effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and discloses the application of isoliquiritigenin as a polymyxin synergist. Background Art

[0002] Salmonella is a common Gram-negative bacterium that is widely distributed in nature. When humans or animals eat food contaminated with Salmonella, they will experience symptoms such as vomiting and diarrhea, which can be life-threatening in severe cases. The gradual increase in Salmonella resistance began in the 1970s and has now become an important global problem. Due to the lack of new anti-infective drugs to combat infections caused by drug-resistant bacteria, polymyxin has been reintroduced for treatment and has become the last line of defense for antibiotic treatment of multidrug-resistant Salmonella.

[0003] Polymyxin was discovered in 1947 and has since been used as an antibiotic to combat infections caused by Gram-negative bacteria in humans and veterinary settings. However, due to its severe nephrotoxicity and neurotoxicity, its use rate declined significantly in the 1980s and it was only developed as a veterinary drug. However, with the increasing use of polymyxin in animal husbandry and agriculture, its therapeutic effect on Gram-negative bacteria has declined relatively, and Gram-negative bacteria resistance has become a major problem. The problem of polymyxin resistance based on Enterobacteriaceae is quite important and prominent, so it is urgent to screen effective polymyxin enhancers to treat polymyxin-resistant Enterobacteriaceae infections. Extracting natural compounds that have synergistic effects with polymyxin from natural compound libraries can effectively restore the sensitivity of polymyxin-resistant Enterobacteriaceae to polymyxin, which is also one of the strategies to effectively explore alternative methods for antibiotics and solve the problem of bacterial resistance.

[0004] Isoliquiritigenin is a flavonoid compound isolated from licorice, which has anti-tumor activity and is also an effective inhibitor of influenza virus replication. At present, there is no report on the use of isoliquiritigenin as a polymyxin synergist at home and abroad. Summary of the invention

[0005] The technical problem to be solved by the present invention is to make up for the deficiencies in the prior art. Firstly, the application of isoliquiritigenin as a polymyxin synergist is provided.

[0006] Another object of the present invention is to provide a composition comprising isoliquiritigenin and polymyxin.

[0007] Another object of the present invention is to provide use of the above composition in the preparation of drugs for resisting Salmonella infection.

[0008] The above technical objectives are achieved through the following solutions:

[0009] The invention discloses an application of isoliquiritigenin as a polymyxin synergist, wherein the isoliquiritigenin is combined with polymyxin.

[0010] The invention uses Salmonella as a target for screening a polymyxin enhancer, screens out isoliquiritigenin, and finds that isoliquiritigenin has no antibacterial ability within an effective concentration range. The combination of isoliquiritigenin and polymyxin can significantly enhance the sensitivity of polymyxin-resistant Salmonella to polymyxin.

[0011] Preferably, the polymyxin is polymyxin E.

[0012] Preferably, the concentration ratio of isoliquiritigenin to polymyxin is 25:8.

[0013] The invention also provides a composition consisting of isoliquiritigenin and polymyxin.

[0014] The use of the above composition in the preparation of drugs for resisting Salmonella infection is also within the protection scope of the present invention.

[0015] Furthermore, the Salmonella is Salmonella ATCC14028 or polymyxin-resistant Salmonella.

[0016] Furthermore, the dosage form of the drug against Salmonella infection is a drug dosage form acceptable to humans and / or animals; the above dosage forms include: powder, granules, tablets, capsules, aerosols, films, oral solutions, injections or suppositories.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention discloses the application of isoliquiritigenin as a polymyxin synergist. After isoliquiritigenin is combined with polymyxin, the minimum inhibitory concentration of polymyxin to Gram-negative bacteria can be significantly reduced. The plant-derived isoliquiritigenin is not only low in price and safe to use, but also avoids the generation of polymyxin drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Showing the checkerboard heat map and in vitro killing curve of isoliquiritigenin combined with polymyxin against Salmonella DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0022] The isoliquiritigenin of the present invention has a CAS registration number of I811912 and a molecular formula of C 15 H 12 O 4 , molecular mass 256.25.

[0023] Structural formula:

[0024]

[0025] The polymyxins used in the following examples are all polymyxin E.

[0026] Example 1 Isoliquiritigenin enhances the sensitivity of Salmonella to polymyxin in vitro

[0027] 1. Preparation before the test:

[0028] (1) Weigh an appropriate amount of isoliquiritigenin and dimethyl sulfoxide as solvent to make the drug concentration 50 mg / mL. Vortex mix and filter with a filter membrane. Prepare a stock solution of polymyxin with a concentration of 5120 mg / L according to CLSI for later use.

[0029] 2. The LPM broth with a pH of 5.8 after high-pressure sterilization was cooled for later use. The Salmonella standard strain ATCC14028 and the polymyxin-resistant Salmonella (17ES) used were both stored in the laboratory.

[0030] 3. MIC and FIC of isoliquiritigenin and polymyxin against Salmonella.

[0031] (1) The day before, inoculate the standard strain of Salmonella ATCC14028 and polymyxin-resistant Salmonella (17ES) onto LB agar plates and culture to an appropriate size.

[0032] (2) On the second day, the standard strain of Salmonella ATCC14028 and the polymyxin-resistant Salmonella (17ES) were inoculated into 4 mL of LB broth and shaken at 37°C and 180 rpm until the logarithmic growth phase;

[0033] (3) Use LPM broth with a pH of 5.8 to dilute the incubated bacteria to 100 times, about 10 6 CFU / mL, reserve;

[0034] (4) Take a sterile 96-well plate, add 180 μL of LPM broth medium to column 1, and add 100 μL of LPM broth medium to columns 2-10 respectively;

[0035] (5) Then add 20 μL of drug to the first column, mix by pipetting, and then pipette 100 μL into the second well, and so on, and pipette 100 μL into the tenth well and discard;

[0036] (6) Add 100 μL of diluted bacterial solution to wells 1 to 11 and 200 μL of LPM broth to well 12;

[0037] (6) Repeat steps (4) to (6) three times in parallel;

[0038] (7) Place the 96-well plate in a 37°C incubator for 16 h and read the results;

[0039] (8) Based on the MIC results, the checkerboard method was performed again. The concentration range of isoliquiritigenin was 1-100 mg / L, and the effective concentration range of polymyxin was 16-32 mg / L.

[0040] The MIC results are shown in Table 1. The MIC values ​​of isoliquiritigenin against the standard Salmonella bacteria and the polymyxin-resistant Salmonella are all >100 mg / L. When isoliquiritigenin is used in combination with polymyxin, the minimum inhibitory concentration of polymyxin against Salmonella can be significantly reduced. The results of the chessboard method are shown in Figure 1 As shown, the results of the chessboard method show that the FICI is less than 0.5, indicating that isoliquiritigenin and polymyxin have a good synergistic effect in the treatment of Salmonella.

[0041] Table 1

[0042]

[0043] 4. In vitro bactericidal curves of isoliquiritigenin and polymyxin against Salmonella

[0044] (1) The standard strain of Salmonella ATCC14028 and the polymyxin-resistant Salmonella (17ES) were inoculated into 4 mL of LB broth and shaken at 37°C and 180 rpm until the logarithmic growth phase;

[0045] (2) Use LPM broth with a pH of 5.8 to dilute the shaken bacterial solution 10 times, about 10 7 CFU / mL, reserve;

[0046] (3) Add 25 mg / L isoliquiritigenin or polymyxin with an inhibitory concentration to a LPM broth centrifuge tube with a pH value of 5.8, then add 0.4 mL of the bacterial solution diluted in step (2) to each tube, vortex thoroughly to mix, and set up a blank group as a quality control. Except that no drug was added to the control group, the other conditions were the same as the experimental group. The bacterial solution system was 4 mL, and then placed in a shaker and cultured at 37°C and 180 rpm.

[0047] (4) At 0, 3, 6, 9 and 24 h of culture, 100 μL of bacterial solution was respectively taken and added to a 2 mL centrifuge tube containing 0.85% saline for 10-fold gradient dilution. After dilution, 25 μL was dropped on LB agar medium and then cultured in a 37°C incubator for about 18 h before counting. The experimental data were statistically analyzed after three biological replicates. The number of colonies at each time point was counted and a sterilization curve was drawn.

[0048] Results: By Figure 1 The bactericidal curve shows that after 24 hours of combined use of isoliquiritigenin and polymyxin, the bacterial load decreased by more than 100 times compared with the use of polymyxin alone, indicating that the combination of isoliquiritigenin and polymyxin has a good effect of killing Salmonella in vitro.

[0049] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. The use of isoliquiritigenin as a polymyxin synergist, characterized in that: The isoliquiritigenin is combined with polymyxin.

2. The use according to claim 1, characterized in that: The polymyxin is polymyxin E.

3. The use according to claim 1, characterized in that: The concentration ratio of isoliquiritigenin to polymyxin is 25:

8.

4. A composition, characterized in that It is composed of isoliquiritigenin and polymyxin.

5. Use of the composition according to claim 4 in the preparation of a drug for resisting Salmonella infection.

6. The use according to claim 5, characterized in that: The Salmonella is Salmonella ATCC14028 or polymyxin-resistant Salmonella.

7. The use according to claim 5, characterized in that: The dosage form of the drug against Salmonella infection is a drug dosage form acceptable to humans and / or animals; the above dosage forms include: powder, granules, tablets, capsules, aerosols, films, oral solutions, injections or suppositories.

Images