Application of liquiritigenin in preparation of bactericide or antibiotic synergist

Abstract

The invention discloses an application of liquiritigenin in preparation of a bactericide or an antibiotic synergist. The invention discloses the synergistic effect, the antibacterial property and the synergistic mechanism of liquiritigenin on colistin and the potential value of in-vivo combined application of liquiritigenin. On the basis, a feasible treatment scheme is provided to cope with the increasingly aggravated drug resistance problem, the use curative effect of the colistin is effectively enhanced, and the clinical service life of the colistin is prolonged. The possibility that liquiritigenin is used as a colistin synergist to resist infection of multi-drug-resistant pathogenic bacteria is reduced. The liquiritigenin can reduce the minimum inhibitory concentration of the colistin to drug-resistant gram-negative bacteria by 16 times, and the antibacterial activity of the colistin is greatly improved.

Description

Technical Field

[0001] This invention relates to the application of glycyrrhizin in the preparation of bactericides or antibiotic synergists, and belongs to the field of glycyrrhizin application. Background Technology

[0002] With the emergence and spread of multidrug-resistant bacteria (MDRs), bacterial resistance to antibiotics poses a serious threat to global public health. Recent reports indicate a high number of deaths worldwide related to bacterial MDRs, particularly in Africa where healthcare is relatively underdeveloped. Carbapenem-producing Enterobacteriaceae are among the most significant drug-resistant bacteria threatening human and animal health. Colistin (polymyxin B1), a cationic cyclic peptide antibiotic, is a last resort against carbapenem-producing Enterobacteriaceae. Colistin exerts its antibacterial effect by binding to the lipopolysaccharide of the outer membrane of Gram-negative bacteria, mediating cell membrane rupture, leakage of intracellular contents, and ultimately bacterial lysis. However, the transferable colistin resistance gene (mcr-1), encoding phosphoethanolamine transferase, reduces the negative charge of lipid A and mediates acquired colistin resistance. Subsequently, other mcr variants of the mcr-1 gene, including mcr-2-10, were discovered, and their prevalence has been greatly enhanced due to their plasmid-borne nature and horizontal spread. (The text also mentions animal-derived bacteria in my country in 2016, but this appears unrelated to the previous sentences and may be a separate, incomplete thought.) mcr-1 The isolation rate of positive Enterobacteriaceae exceeded 30%, and the carrier rate in healthy individuals exceeded 14%. mcr-1The positive pathogen infection rate was 1.7% (Wang Y, Xu C, Zhang R, Chen Y, Shen Y, Hu F, Liu D, LuJ, Guo Y, Xia X, Jiang J, Wang C, Xu S, Walsh TR, Shen J. Changes in colistinresistance and mcr-1 abundance in Escherichia coli of animal and humanorigins following the ban of colistin-positive additives in China: anepidemiological comparative study. Lancet Infect Dis. 2020 Oct;20(10):1161-1171. doi: 10.1016 / S1473-3099(20)30149-3.), which greatly challenges the effectiveness of colistin, leading to treatment failure in carbapenem-producing Enterobacteriaceae infections. Therefore, there is an urgent need for means to overcome MCR-mediated colistin resistance. Due to high costs, long cycles, and low profitability, the pipeline for developing new antibiotics has been exhausted since the late 1990s, and the development speed of new antibacterial drugs has lagged far behind the development speed of resistance, leading to the post-antibiotic era. In contrast, antibiotic adjuvants overcome antibiotic resistance by slowing the development of resistance and improving the efficacy of antibiotics, and are a cost-effective strategy to combat multidrug-resistant (MDR) bacteria. Clavulanic acid, a β-lactamase inhibitor, is a prime example of success, largely salvaging the efficacy of penicillin and cephalosporin antibiotics. Therefore, developing colistin adjuvants is an important means to combat colistin resistance.

[0003] Plants constitute the largest biomass on Earth and have evolved many drug-like secondary metabolites to combat infection. It has been reported that from 1981 to 2010, approximately 65% ​​of approved drugs were either natural compounds or their semi-synthetic derivatives. As of 2018, the FDA had received over 800 research applications or pre-conference applications for plant-based drugs and approved two new drug applications (tea polyphenols and Fulyzaq). This indicates that plant-derived small molecules are a promising source of antibacterial lead compounds. Furthermore, the proven efficacy, structural diversity, abundant sources, and safety profiles of natural compounds suggest that identifying small-molecule natural compounds with synergistic activities from plants and combining them with existing important antibacterial drugs to enhance their antibacterial efficacy and restore sensitivity to multidrug-resistant pathogens is currently a crucial strategy for addressing bacterial resistance.

[0004] Glycyrrhizin, chemically known as (2S)-7-hydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one, is a dihydroflavonoid compound isolated from the rhizome of licorice, possessing a C6-C3-C6 flavonoid skeleton. Glycyrrhizin is slightly soluble in water and readily soluble in organic solvents such as DMSO, methanol, and ethanol. It exhibits various pharmacological activities, including antitumor, antibacterial, anti-infective, and anti-inflammatory effects. Although glycyrrhizin has been found to possess multiple physiological activities, research on its role in controlling bacterial resistance and its antibacterial effects when used in combination with colistin remains relatively limited. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an application of glycyrrhizin in the preparation of bactericides or antibiotic synergists.

[0006] Technical solution: The present invention provides an antibacterial agent or antibiotic synergist containing glycyrrhizin.

[0007] The antibacterial agent also contains colistin.

[0008] The concentration of glycyrrhizin is 2-4 μg / mL.

[0009] The concentration of the colistin is 64~128 μg / mL.

[0010] The bacteria in question are either colistin-resistant bacteria mediated by chromosome mutations or drug-resistant bacteria mediated by the mcr gene.

[0011] The bacteria include multidrug-resistant bacteria. Salmonella D14-P2 , E. coli B2 ( mcr -1) E. coli G92 ( mcr -1) E. coli BL-21-pET28a- mcr -1. Salmonella ( Salmonella 15E343 ( mcr-3 ), Klebsiella pneumoniae ( K. pneumoniae )19-2-1 ( mcr-8 ) or E. coli ( Escherichia coli ATCC 25922.

[0012] The present invention also provides a reagent for restoring the sensitivity of mcr-positive colistin-resistant bacteria to colistin, which contains glycyrrhizin.

[0013] This invention also provides the use of glycyrrhizin in the preparation of bactericides and / or antibiotic synergists.

[0014] The bactericide and / or antibiotic synergist also contains colistin.

[0015] The concentration of glycyrrhizin is 2-4 μg / mL; the concentration of colistin is 64-128 μg / mL.

[0016] The present invention also provides the application of glycyrrhizin in restoring the sensitivity of mcr-positive colistin-resistant bacteria to colistin.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention provides the use of glycyrrhizin as an antibacterial drug, restoring the sensitivity of colistin-resistant bacteria, which is one of the strategies for solving the problem of bacterial resistance. 2. This invention provides the synergistic effect of glycyrrhizin on colistin, its antibacterial properties, synergistic mechanism, and the potential value of combined in vivo application. Based on this, it provides a feasible treatment plan to address the increasingly serious drug resistance problem, effectively enhancing the efficacy of colistin and extending its clinical use. 3. It provides the possibility of using glycyrrhizin as a colistin synergist against multidrug-resistant pathogens. Glycyrrhizin can reduce the minimum inhibitory concentration of colistin against drug-resistant Gram-negative bacteria by up to 16 times, greatly improving the antibacterial activity of colistin. Attached Figure Description

[0018] Figure 1 To verify the in vitro synergistic activity of glycyrrhizin and colistin through combined drug sensitivity testing; Figure 2 The killing activity of glycyrrhizin and colistin against drug-resistant bacteria at different stages: A: logarithmic growth phase drug-resistant bacteria; B: stationary phase drug-resistant bacteria; Figure 3 The antibacterial activity of glycyrrhizin compared to other antibiotics: A: Meropenem; B: Gentamicin; Figure 4The antibacterial activity of glycyrrhizin compared to other antibiotics: A: Tetracycline; B: Ciprofloxacin; Figure 5 The antibacterial activity of glycyrrhizin compared to other antibiotics: A: rifampin; B: colistin. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0020] Example 1: Synergistic antibacterial activity of glycyrrhizin and colistin The checkerboard dilution method was used to determine the effectiveness of glycyrrhizin combined with colistin against multidrug-resistant bacteria. Salmonella D14- P2 , E. coli B2 ( mcr -1) E. coli G92 ( mcr -1) E. coli BL-21-pET28a- mcr -1. Salmonella ( Salmonella 15E343 ( mcr-3 ), Klebsiella pneumoniae ( K. pneumoniae )19-2-1 ( mcr-8 ), Escherichia coli ( Escherichia coli The synergistic antibacterial activity of ATCC 25922. Among them, E. coli B2 ( mcr -1) Salmonella 15E343 ( mcr-3 ), K. pneumoniae 19-2-1 ( mcr-8 The strain was published in the article: Huang Y, Wang Z, Liu Z, Huan Q, Liu Y, Li R, Wang M, Xiao X. Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin. Phytomedicine. 2023Aug;117:154886. doi: 10.1016 / j.phymed.2023.154886. Salmonella D14-P2 , E. coli G92 ( mcr -1) E. coli BL-21-pET28a- mcr -1、 Salmonella 15E343 ( mcr-3 ), Escherichia coli ( Escherichia coli The ATCC 25922 strain was published in the article: Xu T, Fang D, Li F, Wang Z, Liu YA. Dietary Source of High Level of Fluoroquinolone Tolerance in mcr -CarryingGram-Negative Bacteria. Research (Wash DC). 2023 Oct 6;6:0245. doi:10.34133 / research.0245. The specific steps of the chessboard dilution method are as follows: (1) Dilute the bacterial suspension of the test strain with CAMHB broth medium to make the bacterial suspension concentration 1×10⁻⁶. 6 CFU / mL.

[0021] (2) Colistin was dissolved in sterile ultrapure water as recommended by CLSI guidelines and diluted with CAMHB broth medium to obtain a colistin solution with a concentration of 256 μg / mL.

[0022] (3) Glycyrrhizin was dissolved in 5% DMSO and diluted with CAMHB broth medium to obtain a glycyrrhizin solution with a concentration of 256 μg / mL.

[0023] (4) Take a 96-well plate and add 100 μL of MHB broth medium to each well. Add 100 μL of the colistin solution prepared in step (2) to each well in the last row and serially dilute from the eighth row to the second row. Add 100 μL of the glycyrrhizin solution prepared in step (3) to each well in the first column and serially dilute to the seventh column. That is, the actual concentration of glycyrrhizin is 0-256 μg / mL and the actual concentration of colistin is 0-4 μg / mL. Then add 100 μL of the bacterial suspension prepared in step (1) to each well and incubate at 37℃ for 16-20 h. Observe the lowest concentration combination of glycyrrhizin and colistin that inhibits bacterial growth.

[0024] The method for calculating fractionated inhibitory concentration (FICI) is as follows: FICI = MIC (Drug A in combination) / MIC (Drug A alone) + MIC (Drug B in combination) / MIC (Drug B alone) Experimental results are as follows Figure 1As shown, for both colistin-resistant bacteria mediated by chromosomal mutations and those mediated by the mcr gene, whether Escherichia coli or Salmonella, liquiritin can restore the sensitivity of resistant bacteria to colistin, and the FICI values are all less than 0.3. The interaction type between drugs is determined by the FICI value: FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 2 indicates an indifferent effect, and FICI > 2 indicates an antagonistic effect. Therefore, liquiritin and colistin have a synergistic effect. However, for bacteria sensitive to colistin, liquiritin does not enhance the antibacterial activity of colistin. Liquiritin can restore the sensitivity of mcr-positive colistin-resistant bacteria to colistin, reducing the MIC of colistin against resistant bacteria by 16 - 32 times.

[0025] Example 2. Time-kill curve of the combination of liquiritin and colistin The multidrug-resistant bacterium Escherichia coli B2 (mcr - 1) was cultured in CAMHB broth medium at 37 °C until the exponential and stationary phases, and the bacterial suspension was diluted to 10 6 or 10 8 CFU / mL concentrations with CAMHB broth. Then, bacteria were treated separately or in combination with different concentrations of liquiritin and colistin. At 0, 4, 8, 12, and 24 h, 50 μL of the bacterial suspension was taken, serially diluted 10-fold, and dropped on LB agar plates. After culturing at 37 °C for 24 h, the colony-forming units (CFU / mL) were calculated. All experiments were performed with 3 biological replicates.

[0026] The results showed ( Figure 2 A), that the combination of liquiritin (LQ, 64 μg / mL) and colistin (COL, 2 μg / mL) could kill bacteria in the logarithmic growth phase, reducing the bacterial load by more than 10,000 times. However, as shown in Figure 2 B, for bacteria in the stationary phase, the combination of liquiritin (64 μg / mL) and colistin (2 μg / mL) had almost no killing effect; when the concentration of liquiritin was further increased to 128 μg / mL, its combination with colistin (2 μg / mL) also had almost no killing effect. When the concentration of colistin was increased to 4 μg / mL, its combination with liquiritin (64 μg / mL) had a stronger killing effect, reducing the bacterial load by a factor of 100. These results further indicated that liquiritin could indeed significantly enhance the bactericidal activity of colistin against mcr-positive pathogens. It can be seen that for both logarithmic-phase and stationary-phase bacteria, the combination of liquiritin and colistin can reduce the bacterial count of the multidrug-resistant bacterium Escherichia coli B2 (mcr - 1) below the detection limit (10 2 CFU / mL), achieving bactericidal activity, while using colistin or liquiritin alone could not achieve bactericidal activity.

[0027] Comparative Example 1: Antibacterial Activity of Glycyrrhizin in Contrast with Different Types of Antibiotics Following the method in Example 1, the effects of glycyrrhizin on different antibiotics (meropenem, gentamicin, tetracycline, ciprofloxacin, rifampin, and colistin) were investigated. E. coli B2 ( mcr -1) antibacterial activity.

[0028] Table 1. Synergistic activity of different antibiotics with LQ against mcr-1 positive Escherichia coli B2

[0029] The results are shown in Figure 1 and... Figures 3-5 As shown, LQ did not exhibit synergistic effects against the other five different types of antibiotics (meropenem, gentamicin, tetracycline, ciprofloxacin, and rifampin).

Claims

1. An antibacterial agent or antibiotic potentiator, characterized in that, The glycyrrhizin.

2. The antimicrobial agent or antibiotic potentiator of claim 1, wherein, The concentration of the glycyrrhizin is 2-4 μg / mL.

3. The antimicrobial agent or antibiotic potentiator of claim 1, wherein, The antibacterial agent further contains colistin.

4. The antimicrobial agent or antibiotic potentiator of claim 3, wherein, The concentration of the colistin is 64-128 μg / mL.

5. The antimicrobial agent or antibiotic potentiator of claim 1, wherein, The bacteria are colistin-resistant bacteria mediated by chromosomal mutation or mcr gene.

6. The antimicrobial agent or antibiotic potentiator of claim 1, wherein, The bacteria include multidrug-resistant bacteria. Salmonella D14-P2 , E.coli B2 ( mcr -1) E.coli G92 ( mcr -1) E.coli BL-21-pET28a- mcr -1、 Salmonella 15E343( mcr-3 ), K. pneumoniae 19-2-1( mcr-8 )or Escherichia coli ATCC25922.

7. An agent for restoring the susceptibility of an mcr-positive colistin-resistant bacterium to colistin, characterized in that, The glycyrrhizin.

8. The use of glycyrrhizin in the preparation of a bactericide and / or an antibiotic synergist.

9. Use according to claim 8, characterized in that, The bactericide and / or the antibiotic synergist further contain colistin.

10. The use of glycyrrhizin in the restoration of the sensitivity of mcr-positive colistin-resistant bacteria to colistin.

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