CN117427146A - Application of mulberry flavone and colistin in preparing medicine for inhibiting acinetobacter baumannii infection - Google Patents

Abstract

The application of mulberry flavone and colistin in preparing medicine for inhibiting Acinetobacter baumannii infection belongs to the field of biological medicine technology. The invention provides an application of combined use of mulberry flavone and colistin in preparing a medicament for inhibiting acinetobacter baumannii infection. The acinetobacter baumannii comprises colistin-resistant acinetobacter baumannii or sensitive acinetobacter baumannii. The invention provides a method for treating infection caused by colistin-resistant Acinetobacter baumannii by utilizing mulberry flavone to act in cooperation with colistin to play a role in resisting Acinetobacter baumannii. When the mulberry flavone and the colistin are combined at a certain concentration, all colistin-resistant Acinetobacter baumannii are killed within 2 hours, and the synergistic effect of the mulberry flavone and the colistin is proved.

Description

The use of molin and colistin in the preparation of drugs for inhibiting Acinetobacter baumannii infection application

Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of molin and colistin in preparing drugs for inhibiting Acinetobacter baumannii infection.

Background technique

Acinetobacter baumannii (Ab) is a non-fermenting Gram-negative opportunistic pathogen that easily causes infections in people with low immunity. It is one of the important pathogenic bacteria in hospital infection outbreaks and usually causes ventilator infections. associated pneumonia (VAP), bacteremia, meningitis, endocarditis, and urinary tract and skin infections.

The main antibiotics used clinically for Acinetobacter baumannii are carbapenems and lactamase inhibitors. However, with the increase in the detection rate of carbapenem-resistant Baumann in recent years and the lack of new drug development, clinicians are forced to use traditional The antibiotic polymyxins (polymyxin B and colistin) serve as the last line of defense in MDRAB treatment. Polymyxins were first discovered in the 1940s and are a family of natural polybasic cyclic lipopeptides. Polymyxin B and colistin were first introduced into clinical practice in the late 1950s. However, colistin has significant dose-limiting nephrotoxicity in clinical use, and its therapeutic window is very narrow. Secondly, acute toxicity severely limits the maximum dose of intravenous administration. Many cases of colistin-resistant Acinetobacter baumannii have emerged worldwide. Therefore, there is an urgent need to find new treatment strategies to improve efficacy and reduce toxicity.

Contents of the invention

In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide the application of molin and colistin in the preparation of drugs for inhibiting Acinetobacter baumannii infection. Morin can inhibit polymyxin-resistant A. baumannii. Infections caused by Acinetobacter.

In order to achieve the above objects, the present invention provides the following technical solutions:

In one aspect, the present invention provides the use of tannin and colistin in combination to prepare a medicament for inhibiting Acinetobacter baumannii infection.

Through research on Acinetobacter baumannii biofilm and intracellular ATP levels, it was found that linlin can significantly inhibit the formation of Acinetobacter baumannii biofilm and reduce intracellular ATP levels, thereby reducing the risk of Acinetobacter baumannii The aggregation reduces the obstacles to drug diffusion, disrupts bacterial homeostasis, and ultimately enhances the antibacterial effect of colistin. Morin flavone can significantly reduce the MIC value of colistin-resistant Acinetobacter baumannii against colistin, and cooperate with colistin to exert anti-Acinetobacter baumannii effects, thereby treating colistin-resistant Acinetobacter baumannii. of infection.

In the described application, the Acinetobacter baumannii is colistin-resistant Acinetobacter baumannii or sensitive Acinetobacter baumannii.

According to the application, the combined use of linsonin and colistin reduces the biofilm formation ability of Acinetobacter baumannii.

A pharmaceutical composition for inhibiting Acinetobacter baumannii infection, the components of the pharmaceutical composition include independently packaged molin and colistin.

In the pharmaceutical composition, the mass concentration of linsonin is 4-32 μg/mL, and the mass concentration of colistin is 0.5-16 μg/mL.

In the pharmaceutical composition, the mass concentration of colistin is 0.5-8 μg/mL.

The dosage form of the pharmaceutical composition is selected from the group consisting of injections, ointments or tablets.

The preparation method of the pharmaceutical composition for inhibiting Acinetobacter baumannii infection according to any one of the above is to prepare a linsonin solution and a colistin solution respectively, and then mix them evenly with pharmaceutically acceptable ingredients to obtain a pharmaceutical composition.

According to the preparation method, the solvent of the molin solution is dimethyl sulfoxide.

According to the preparation method, the solvent of the colistin solution is ddH ₂ O.

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

The invention provides the application of molin and colistin in the preparation of medicines for inhibiting Acinetobacter baumannii infection, and particularly has good effects in inhibiting colistin-resistant Acinetobacter baumannii. Morinone has a synergistic effect with colistin in inhibiting Acinetobacter baumannii, thereby reducing the concentration of colistin used in the treatment of A. baumannii infections. When linlin was combined with colistin at a concentration of 1/4 MIC, colistin-resistant Acinetobacter baumannii was completely killed within 2 hours, proving that there is a synergistic effect between linlin and colistin, which greatly reduces the The dosage of colistin improves the safety of treatment.

Description of the drawings

Figure 1 shows the checkerboard experiment of molin and colistin on colistin-resistant Acinetobacter baumannii ATCC19606R, AB13R, AB18R and colistin-sensitive Acinetobacter baumannii ATCC19606. FIC <0.5 indicates synergistic effect;

Figure 2 is the time-killing curve of molin and/or colistin against colistin-resistant Acinetobacter baumannii, where: A is the number of bacteria in strain ATCC19606R within 24 hours after treatment with different drug concentrations alone or in combination. Change trend; B is the change trend of the number of bacteria in strain AB13R within 24 hours after being treated with different drugs;

Figure 3 shows the inhibitory effect of morilin and/or colistin on the biofilm formation of ATCC19606R, which has a strong ability to form biofilms. A is the inhibitory effect of morilin alone on biofilm with increasing concentration gradient; B is the inhibitory effect of morilin on biofilm with increasing concentration gradient. Inhibitory effect of flavonoids combined with colistin on Acinetobacter baumannii biofilm;

Figure 4 shows the safety assessment of the combination of molin and/or colistin, where: A is the hemolysis test on sheep red blood cells, and B is the cytotoxicity measured by the CCK8 method.

Detailed ways

The technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Molinin of the present invention was purchased from MCE Company, and colistin was purchased from Selleck Company.

Example 1: Checkerboard test to detect the synergistic effect of molin and colistin

Acinetobacter baumannii clinical isolates AB13, AB18 and standard strain ATCC19606 from different sources were induced to colistin resistance to varying degrees, and the resulting resistant strains were named: AB13R, AB18R and ATCC19606R. Among them, AB13, AB18, ATCC19606, AB13R, AB18R and ATCC19606R are deposited in the Pathogenic Microbiology Laboratory of Yangzhou University. The solvent of molin is DMSO, and the solvent of colistin is ddH ₂ O. The stock solution is prepared and then diluted with MH broth culture medium to the required concentration (27g/1000mL distilled water).

Checkerboard test was used to detect the synergistic effect of molin and colistin: in a 96-well plate, double the concentration of molin (0 to 32 μg/mL) in each column and double the colistin (0 to 512 μg/mL) in each row. Each well was inoculated with 100 μL of test strain suspension (5 × 10 ⁵ CFU/mL), with a final volume of 200 μL, and then incubated at 37°C for 24 h. The results are shown in Figure 1. It can be seen that molin reduces the AB13R MIC value by 4 to 64 times, the AB18RMIC value by 64 times, the ATCC19606 RMIC value by 16 to 64 times, and the ATCC19606 MIC value by 2 to 4 times. It can be seen that different concentrations of molin significantly reduced the MIC value of colistin-resistant Acinetobacter baumannii against colistin, and also reduced the MIC value of colistin-sensitive Acinetobacter baumannii against colistin. . It is suggested that molin can cooperate with colistin to exert antibacterial effects, thereby treating infections caused by colistin-resistant Acinetobacter baumannii or reducing the dosage of colistin in treating colistin-sensitive Acinetobacter baumannii.

Example 2: Time-killing curve to detect the synergistic effect of molin and colistin

In order to further prove the synergistic effect of molin and colistin, a time killing curve test was conducted on the two strains of colistin-resistant Acinetobacter baumannii that were proven effective above. Different drugs were added to 5×10 ⁸ CFU/mL AB13R and 5×10 ⁶ CFU/mL ATCC19606R inoculum, and cultured on a shaking table at 37°C. Samples were taken every 2 hours for plate counting. After 12 hours, samples were taken every 4 hours for plate counting. count. The drug concentration selected in this experiment was set based on the MIC concentration in the previous results. MIC, 1/2MIC, and 1/4MIC concentrations were selected for separate and combined treatment. The results are shown in Figure 2. It can be seen that there is no significant difference in the bacterial growth between the medication alone group and the control group within 24 hours. However, when linlin was combined with colistin, all bacteria were killed within 2 hours, proving the synergistic effect of linlin and colistin.

Example 3: Combined use of molin and colistin reduces the biofilm formation ability of Acinetobacter baumannii

Biofilm formation is an important way for bacteria to resist antibiotics. Bacteria can grow in biofilms and escape the body's immune response, thereby hindering the effectiveness of antibacterial drugs. This experiment selected the ATCC19606R strain with strong biofilm formation ability. A 96-well plate was inoculated with 1 × 10 ⁶ CFU bacterial solution, treated with different concentrations of drugs, and cultured statically at 37°C for 48 hours. Biofilm formation was quantified by measuring the OD ₅₉₀ value through crystal violet staining. The results are shown in Figure 3. It can be seen that when linlin is used alone, it can inhibit the formation of biofilm in a concentration-dependent manner. When linlin is combined with colistin, it significantly reduces the biofilm formation of Acinetobacter baumannii. ability.

Example 4: Safety evaluation of the combined use of linsonin and colistin

The safety of a drug is one of the important factors that determine whether the drug has R&D and application value. The safety of the combined use of molin and colistin was initially evaluated through the sheep red blood cell hemolysis test and the CCK8 cytotoxicity test. First, take fresh 8% sheep red blood cells, wash them 3 times with PBS, and treat them with colistin and molin alone or in combination. An equal volume of 0.2% Triton X-100 is used as a positive control, and PBS is used as a negative control. 37 Incubate for 1 hour at ℃, and measure the absorbance of hemoglobin at 576 nm with a microplate reader. The results are shown in Figure 4A. The hemolysis rates in the drug combination concentrations used were all lower than 5%, indicating that significant hemolysis will not occur within the effective combined drug concentration of linsonin and colistin in vitro.

Human alveolar basal epithelial cells A549 cells were used to conduct CCK-8 cytotoxicity experiments to detect the cytotoxic effect of colistin combined with molin on mammalian cells. First, the cells were seeded in a 96-well plate at about 2×10 ³ /well and cultured for 16 to 24 hours. Then, the cells were treated with different concentrations of linsonin and colistin alone or in combination for 24 hours. Only drugs and culture medium were added to the wells. As a blank control. After 24 hours of culture, add 10 μL of CCK8 solution to each well, incubate at 37°C for 2 hours, and detect the absorbance at 450 nm. The results are shown in Figure 4B. When the concentration of colistin used was within 16 μg/mL, linsonin did not increase the cytotoxicity of colistin to epithelial cells A549. These results indicate that the highest concentrations of tannin and colistin that are effective as antibacterial agents in vitro are safe.

Claims (10)

1. Application of the combined use of molin and colistin in the preparation of drugs for inhibiting Acinetobacter baumannii infection. 2. The application according to claim 1, characterized in that the Acinetobacter baumannii is colistin-resistant Acinetobacter baumannii or sensitive Acinetobacter baumannii. 3. The application according to claim 1, characterized in that the combined use of molin and colistin reduces the biofilm formation ability of Acinetobacter baumannii. 4. A pharmaceutical composition for inhibiting Acinetobacter baumannii infection, characterized in that the components of the pharmaceutical composition include independently packaged molin and colistin. 5. The pharmaceutical composition according to claim 4, wherein the mass concentration of linsonin is 4-32 μg/mL, and the mass concentration of colistin is 0.5-16 μg/mL. 6. The pharmaceutical composition according to claim 4, wherein the mass concentration of colistin is 0.5-8 μg/mL. 7. The pharmaceutical composition according to claim 4, wherein the dosage form of the pharmaceutical composition is selected from the group consisting of injections, ointments or tablets. 8. The preparation method of the pharmaceutical composition for inhibiting Acinetobacter baumannii infection according to any one of claims 4 to 7, characterized in that: preparing linsonin solution and colistin solution respectively, and pharmaceutically acceptable ingredients After mixing evenly, a pharmaceutical composition is obtained. 9. The preparation method according to claim 8, wherein the solvent of the linsonin solution is dimethyl sulfoxide. 10. The preparation method according to claim 8, wherein the solvent of the colistin solution is _ddH2O .