Use of traditional Chinese medicine monomer in preparation of bacterial efflux pump inhibitor or antibiotic activity enhancing drug and combined bacteriostatic composition

Abstract

The present application relates to the technical field of bacteriostatic drugs, and particularly relates to application of traditional Chinese medicine monomers in preparation of bacterial efflux pump AcrB inhibitor or antibiotic activity improving drug and combined bacteriostatic composition. The traditional Chinese medicine monomers include red cedar flavone, stigmasterol, trichosanthin diol-3-benzoic acid ester, glycyrrhizic acid, alpha-bosanol, agastachin, acaciin, triptonylline C, rhein acid diglucoside, brassicasterol ferulate, cholesterin ferulate or calophyllolide G. The traditional Chinese medicine monomers can inhibit the efflux activity of AcrB, so that the drug molecules entering the bacteria continuously accumulate, thereby recovering the bactericidal efficiency of the existing antibacterial drugs and reducing the drug resistance of bacteria to the antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacteriostatic drugs, in particular to application of a traditional Chinese medicine monomer in preparation of a bacterial efflux pump inhibitor or an antibiotic activity improving drug and a combined bacteriostatic composition. BACKGROUND

[0002] At present, antibiotics have been widely used in the treatment of bacterial diseases, but due to non-standard use, bacterial drug resistance is becoming more and more serious, the drug resistance spectrum is continuously widening, and cross resistance and multiple drug resistance are also intensified. There are many mechanisms of bacterial drug resistance, mainly including: bacteria produce hydrolysis or inactivation enzymes, changes in the target of antibacterial drugs, changes in bacterial cell membrane permeability, formation of bacterial biofilm, and bacterial active efflux mechanism. The latter three mechanisms have poor specificity, that is, they will produce drug resistance to different types or antibacterial drugs with different mechanisms, which are closely related to multiple drug resistance of antibacterial drugs, and among them, the active efflux mechanism is the main one. Therefore, inhibiting or interfering with the bacterial active efflux mechanism is conducive to reducing bacterial drug resistance. SUMMARY

[0003] In view of the technical problem that non-standard use of antibiotics in the prior art causes increasingly serious drug resistance, the present application provides application of a traditional Chinese medicine monomer in preparation of a bacterial efflux pump AcrB inhibitor.

[0004] In addition, the present application also provides application of a traditional Chinese medicine monomer in preparation of an antibiotic activity improving drug.

[0005] In addition, the present application also provides a combined bacteriostatic composition.

[0006] In order to achieve the above application purposes, the embodiments of the present application adopt the following technical solutions:

[0007] The application of the traditional Chinese medicine monomer in preparation of the bacterial efflux pump AcrB inhibitor, wherein the traditional Chinese medicine monomer includes Sequoiaflavone, Stigmasterol, Karounidiol 3-benzoate, glycyrrhizin, Alpha spinasterol, Agastachin, Musennin, Wilfornine C, Rhein diglucoside, Campesteryl ferulate, Cholesteryl ferulate or Yadanzioside G.

[0008] AcrAB-TolC is a complex composed of three parts, a fusion protein (AcrA) existing in the cytoplasmic space of bacteria, an inner membrane transporter (AcrB) and an outer membrane channel protein (TolC). AcrB transporter uses proton power to squeeze the compound out of the TolC channel and outside, playing a major role in drug transport. The above-mentioned traditional Chinese medicine monomer can inhibit the efflux mechanism of AcrB, so that the drug molecules entering the bacterial body continuously accumulate, thereby restoring the bactericidal efficiency of the existing antibacterial drugs and reducing the drug resistance of bacteria to the antibacterial drugs.

[0009] Preferably, the bacteria are Enterobacteriaceae bacteria. AcrAB-TolC is the most important drug efflux pump of Enterobacteriaceae bacteria, and the above-mentioned traditional Chinese medicine monomer has an outstanding inhibitory effect on the efflux pump AcrB of Enterobacteriaceae bacteria. Enterobacteriaceae bacteria include Escherichia coli, Klebsiella pneumoniae, Salmonella, Enterobacter aerogenes, Shigella, etc.

[0010] Preferably, the traditional Chinese medicine monomer is glycyrrhizic acid, which has good solubility, low price, and is widely used in various foods as a sweetener of food, and has a wide safety range. Glycyrrhizic acid binds to efflux pump transporter AcrB through hydrogen bonds and hydrophobic forces, thereby inhibiting its efflux activity, and can accumulate antibiotics in the bacterial body to play an antibacterial role.

[0011] In addition, the embodiments of the present application also provide the use of the traditional Chinese medicine monomer in the preparation of a drug for improving the activity of antibiotics, wherein the traditional Chinese medicine monomer includes red cedar flavone, stigmasterol, trichosanthin diol-3-benzoate, glycyrrhizic acid, alpha-bosanol, agastachin, terihanin, triptolide ning base C, rhein acid diglucoside, linolenic acid ferulate, cholesterolic acid ferulate or brucea glycoside G, and the antibiotics include cefotaxime sodium, fosfomycin sodium or chloramphenicol. The above-mentioned antibiotics are substrates of AcrAB-TolC efflux pump, and the use of the above-mentioned traditional Chinese medicine monomer and the above-mentioned antibiotics together has a synergistic effect or an additive effect on killing bacteria, especially multi-drug resistant Enterobacteriaceae bacteria, and can significantly reduce the use amount of the above-mentioned antibiotics.

[0012] Preferably, the traditional Chinese medicine monomer is glycyrrhizic acid. Glycyrrhizic acid has the advantages of safety, wide source and low price, and can reduce the minimum inhibitory concentration of the above-mentioned antibiotics. For example, 3.125 mg / mL concentration of glycyrrhizic acid can reduce the MIC of cefotaxime sodium to 1 / 8 of the original, 3.125 mg / mL of glycyrrhizic acid can reduce the MIC of fosfomycin sodium to 1 / 8 of the original, and 12.5 mg / mL of glycyrrhizic acid can reduce the MIC of chloramphenicol to 1 / 2 of the original.

[0013] In addition, the present application also provides a combined bacteriostatic composition comprising a traditional Chinese medicine monomer and an antibiotic, wherein the traditional Chinese medicine monomer is at least one of redwood flavone, stigmasterol, trichosanthin diol-3-benzoate, glycyrrhizic acid, a-bosisto-rol, agastachin, acapulcoine, triptonyl C, rhein acid diglucoside, campesterol ferulate, cholesterin ferulate or bruceanol G, and the antibiotic comprises at least one of sodium cefotaxime, sodium fosfomycin or chloramphenicol. The traditional Chinese medicine monomer and the antibiotic can be used in combination to reduce the usage amount of the antibiotic.

[0014] Preferably, the traditional Chinese medicine monomer is glycyrrhizic acid.

[0015] Preferably, the antibiotic is sodium cefotaxime or sodium fosfomycin. The glycyrrhizic acid and the sodium cefotaxime or the sodium fosfomycin have a synergistic effect on inhibiting bacteria, especially multi-drug resistant Enterobacteriaceae, and can achieve excellent bacteriostatic effect at a lower usage amount. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A 2D schematic diagram of the binding of glycyrrhizic acid and AcrB protein in Example 1;

[0017] Figure 2 A Nile red efflux test result in Example 2. DETAILED DESCRIPTION

[0018] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0019] The software AutoDockTools 1.5.6, Autodock Vina, Raccoon used in the following examples are from The Scripps Institute, the crystal structure of E. coli AcrB protein is from PDB (Protein Data Bank), the structure of the traditional Chinese medicine monomer compound is from ZINC database, E. coli E320 is preserved in the laboratory of Hebei Agricultural University, MHB nutrient broth, 96-well sterile microplate, sodium cefotaxime, sodium fosfomycin and chloramphenicol are purchased from Beijing Coolab Technology Co., Ltd., glycyrrhizic acid, redwood flavone, stigmasterol, trichosanthin diol-3-benzoate, glycyrrhizic acid, a-bosisto-rol, agastachin, acapulcoine, triptonyl C, rhein acid diglucoside, campesterol ferulate, cholesterin ferulate and bruceanol G are purchased from Shanghai Mayreel Chemical Technology Co., Ltd., carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) and Nile red are purchased from Beijing Solabio Technology Co., Ltd.

[0020] Example 1

[0021] The application provides application of a traditional Chinese medicine monomer in preparation of a bacterial efflux pump AcrB inhibitor.

[0022] 1. Binding of the traditional Chinese medicine monomer to the AcrB protein receptor

[0023] The crystal structure of the E. coli AcrB protein is processed by AutoDockTools, water molecules are deleted, charges are added, hydrogen atoms are added, and the structure is converted into a pdbqt format. The structure of the traditional Chinese medicine monomer compound is converted into a pdbqt format by Raccoon.

[0024] The traditional Chinese medicine monomer and the AcrB protein are subjected to molecular docking simulation by AutoDock Vina software, and are analyzed by DSVisualizer software, and the results are shown in Table 1.

[0025] Table 1 Binding of each traditional Chinese medicine monomer to the AcrB protein receptor

[0026]

[0027] Molecular binding can cause energy reduction, so that the binding force is negative, and the greater the negative value, the more stable the binding.

[0028] As shown in Table 1, each traditional Chinese medicine monomer has good binding to the AcrB protein receptor.

[0029] Taking glycyrrhizin as an example, the 2D result of binding to the AcrB protein is shown in Figure 1 The glycyrrhizin ligand forms hydrogen bonds with Gly126, Gln125, Ser48, Tyr49, Thr85 and Thr87 of the AcrB protein (indicated by the words on the dotted line), and forms hydrophobic forces with Phe628, Phe615, Phe178, Val612 and Ile277. Figure 1

[0030] 2. Nile red efflux experiment

[0031] The E. coli E320 is cultured for 14 hours, centrifuged at 4400 rpm for 10 min, and the supernatant is discarded, and then the bacteria are washed three times in a 50 mM / L K3PO3 (pH = 7) buffer containing 1 mM MgCl2, and then the bacteria are suspended in the same buffer to OD 660 ​= 1.0, allowed to stand for 15 minutes, then a 3 ml aliquot was transferred to a 4 mL centrifuge tube and CCCP was added to a final concentration of 10 μM. After standing for 15 minutes, glycyrrhizic acid monomer was added to 6.25 mg / ml and 3.125 mg / ml, respectively. After mixing and standing for 15 minutes, the mixture was centrifuged at 4400 rpm for 5 minutes, the supernatant was discarded, and the mixture was resuspended in 3 mL of the same buffer and added to a 15 mL centrifuge tube. Nile red was added to a final concentration of 10 μM and then incubated on a shaker (37°C, 140 rpm) for 3 hours. The resulting E. coli suspension was then allowed to stand at room temperature for 60 minutes and centrifuged at 4400 rpm for 5 minutes. The supernatant was discarded, and droplets on the tube wall were removed with absorbent paper. The E. coli was resuspended in 3 mL of PBS to obtain the E. coli suspension to be tested, and then transferred to a cuvette. The fluorescence signal was measured in a fluorescence spectrophotometer (Jasco FP-750) with an excitation wavelength of 544 nm, an emission wavelength of 650 nm, an excitation slit width of 10 nm, and an emission slit width of 5 nm. The fluorescence of the E. coli suspension to be tested was tracked. After 100 s, 50 mM glucose was added to rapidly excite the Nile infrared emission and the fluorescence change after 300 s was observed. The results are shown in Figure 2 .

[0032] Depend on Figure 2 As can be seen, before the addition of glucose, the Nile Red fluorescence intensities of the two glycyrrhizic acid treatment groups were 13.7 au and 12.8 au, respectively, higher than that of the saline treatment group (12.5 au). After the addition of glucose, the efflux pump was activated, and the fluorescence intensity of each group decreased. The high-dose glycyrrhizic acid (6.25 mg / ml) treatment group decreased to 12.1 au, the low-dose glycyrrhizic acid (3.125 mg / ml) treatment group decreased to 10.7 au, and the saline treatment group decreased to 10.4 au. These results indicate that glycyrrhizic acid can inhibit the activity of E. coli efflux pumps and inhibit Nile Red excretion.

[0033] Example 2

[0034] The embodiments of the present invention provide the use of traditional Chinese medicine monomers in the preparation of drugs for improving the activity of antibiotics.

[0035] The minimum inhibitory concentration (MIC) of Chinese medicine monomers and antibiotics was determined using MHB nutrient broth using the microbroth two-fold dilution method.

[0036] The microdilution method was used to conduct the in vitro combined antibacterial test of drugs. Fosfomycin sodium, chloramphenicol, cefotaxime sodium and glycyrrhizic acid were diluted in series in the vertical and horizontal directions of a 96-well sterile microplate with the MIC of the Chinese medicine monomer and antibiotic as the initial concentration. Escherichia coli solution was added to each well to make the final concentration of 5×10 5CFU / mL, 37℃ incubation for 24h, the lowest drug concentration without bacterial growth is the combined MIC.

[0037] The results are determined according to FICI = MIC (combined glycyrrhizin) / MIC (glycyrrhizin alone) + MIC (combined cefotaxime sodium) / MIC (cefotaxime sodium alone): FICI ≤ 0.5 is determined as synergistic effect, 0.5 < FICI ≤ 1 is determined as additive effect, 1 < FICI ≤ 2 is determined as irrelevant effect, and FICI > 2 is determined as antagonistic effect.

[0038] The results are shown in Table 2.

[0039] Table 2: Results of combined antibacterial test of glycyrrhizin and cefotaxime sodium

[0040]

[0041] The MIC of glycyrrhizin is > 25 mg / mL, the MIC of fosfomycin sodium is 512 μg / mL, the MIC of chloramphenicol is 512 μg / mL, and the MIC of cefotaxime sodium is 1024 μg / mL, which are measured by micro-broth double dilution method.

[0042] When glycyrrhizin is combined with fosfomycin sodium for antibacterial inhibition, 3.125 mg / mL of glycyrrhizin can reduce the MIC of fosfomycin sodium to 1 / 8 (64 / 512) of the original, and the FICI calculated according to the combined antibacterial test formula is < 0.25, and the two belong to synergistic effect.

[0043] When glycyrrhizin is combined with cefotaxime sodium for antibacterial inhibition, 3.125 mg / mL of glycyrrhizin can reduce the MIC of cefotaxime sodium to 1 / 8 (128 / 1024) of the original, and the FICI calculated according to the combined antibacterial test formula is < 0.25, and the two belong to synergistic effect.

[0044] When glycyrrhizin is combined with chloramphenicol for antibacterial inhibition, 12.5 mg / mL of glycyrrhizin can reduce the MIC of chloramphenicol to 1 / 2 (256 / 512) of the original, and the FICI calculated according to the combined antibacterial test formula is < 0.1, and the two belong to additive effect.

[0045] Example 3

[0046] The embodiment of the present application provides a combined antibacterial composition containing glycyrrhizin and cefotaxime sodium, and the mass ratio of the two is 1:40.96.

[0047] Example 4

[0048] The embodiment of the present application provides a combined antibacterial composition containing glycyrrhizin and fosfomycin, and the mass ratio of the two is 1:20.48.

[0049] Example 5

[0050] The embodiment of the present application provides a combined bacteriostatic composition containing glycyrrhizinic acid and chloramphenicol, and the mass ratio of the two is 1:20.48.

[0051] The above merely provides the preferred embodiments of the present application but not for limiting the present application, and any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a Chinese medicine monomer in the preparation of a drug for enhancing the antibacterial activity of antibiotics against Enterobacteriaceae, characterized in that: The traditional Chinese medicine monomer is glycyrrhizic acid, and the antibiotic is ceftriaxone sodium. The traditional Chinese medicine monomer and the antibiotic are used together.

2. A combined antibacterial composition, characterized in that: The invention contains a traditional Chinese medicine monomer and an antibiotic, wherein the traditional Chinese medicine monomer is glycyrrhizic acid, the antibiotic is ceftriaxone sodium, and the combined antibacterial composition is a composition for combined inhibition of Enterobacteriaceae bacteria.

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